Microencapsulation

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Methods and Industrial Abblicalions
edited by Simon Benita

The Hebrew University of Jerusalem Jerusalem, Israel
Marcel Dekker, Inc. New York.
Basel Hong Kong
Library of Congress Cataloging-in-Publication Data Microencapsulation : methods and industrial applications/ edited by Simon Benita. p. cm. -(Drugs and the pharmaceutical sciences;v. 73) Includes index ISBN 0-8247-9703-5 (alk. paper) 1. Microencapsulation. 1. Benita, Simon. 11. Series. RS20 1.C3M27 1996 6 15'.1 9 d c 2 0
96- 1569 CIP
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Preface
Research, development, and sales of drug-delivery systems are increasing at a rapid pace throughout the world. This worldwide trend will intensify in the next decade as cuts in public health expenses demand lowercosts and higher efficacy. To meet this demand, many efficient drugs currently in use will be reformulated within delivery systems that can be value-added for optimal molecular activity. In addition to the health sector, the cosmetic, agricultural, chemical, and foodindustries operate in an open marketplace where free and aggressive competition demands novel coating techniques with enhanced effectiveness at the lowest possible cost. Currently, microencapsulation techniques are most widely used in the development and production of improved drug- and food-delivery systems. These techniques frequently result in products containing numerous variably coated particles. The exact number of particles needed to form a single administered dose varies as a function of the final particle size and can lie in either the micro- or nanometer size range for micro- and nanoparticulate delivery systems, respectively. The microparticulate delivery systems include mainly pellets, microcapsules, microspheres, lipospheres, emulsions, and multiple emulsions. The nanoparticulate delivery systemsinclude mainly lipid or polymeric nanoparticles (nanocapsules and nanospheres), microemulsions, liposomes, and nonionic surfactant vesicles (niosomes). Generally, the microparticulate delivery systemsare intended for oral and topical use. Different types of coated particles can be obtained depending on the coating process used. The particles can be embedded within a polymeric or proteinic matrix network in either a solid aggregated state or a molecular dispersion, resulting in the formulation of microspheres. Alternatively, the particles can be coated by a solidified polymeric or proteinic envelope, leading to the formation of microcapsules. The profile and kinetic pattern governing the release rate of the entrapped active substance from the dosage form depend on the nature and morphology of the coated
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Preface
particles, which need to be established irrespective of the manufacturing method used. Microencapsulation techniques are normally used to enhance material stability, reduce adverse or toxic effects, or extend material release for different applications in various fields of manufacturing. Until now, the use of some interesting and promising therapeutic substances has been limited clinically because of their restrictive physicochemical properties, which have required frequent administration. It is possible that these substances may become more widely used in a clinical setting. if appropriate microencapsulation techniques can be designed to overcome theirintrinsic inconveniences. Investigators and pharmacologists have been trying to develop delivery systems that allow the fate of a drug to be controlled and the optimal drug dosage to arrive at the site of action in the body by means of novel microparticulate dosage forms. During the past two decades, researchers have succeeded in part in controlling the drug-absorption process to sustain adequate andeffective plasma drug levels over a prolonged periodof time by designing delayed- or controlled-release microparticulate-delivery systems intended for either oral or parenteral administration. The ultimate objective of microparticulate-delivery systems is to control and extend the release of the active ingredient from the coated particle without attempting to modify the normalbiofate of the active molecules in the body after administration and absorption. The organ distribution and elimination of these molecules will not be modified and will depend only on their physicochemical properties. Ontheotherhand, nanoparticulatedelivery systems are usually intended for oral, parenteral,ocular, and topical use, with the ultimate objective being the alteration of the pharmacokinetic profile of the active molecule. In the past decade, ongoing efforts have been made to develop system or drug carriers capable of delivering the active moleculesspecifically to the intended target organ, while increasing the therapeutic efficacy. This approach involves modifying the pharmacokinetic profile of various therapeutic classes of drugs through their incorporation in colloidal nanoparticulate carriers in the submicron size range such as liposomes and nanoparticles. These site-specific delivery systems allow an effective drug concentration to be maintained for a longer interval in the target tissue and result in decreased side effects associated with lower plasma concentrations in the peripheral blood. Thus, the principle of drug targeting is to reduce the total amount of drug administered, while optimizing its activity. It should be mentioned that the scientific community was skeptical that such goals could be achieved, since huge investments of funds and promising research studies have in many cases resulted in disappointing and nonlucrative results and havealso been
Preface
slow in yielding successfully marketed therapeutic nanoparticulate dosage forms. With the recent approval by health authorities of a few effective nanoparticulate products containing antifungal or cytotoxic drugs, interest in colloidal drug carriers has been renewed. A vast number of studies and reviews as well as several books have been devoted to thedevelopment, characterization, and potential applications of specific microparticulate- and nanoparticulate-deliverysystems. No encapsulation process developed to date has been able to produce the full range of capsules desired by potential capsule users. Few attempts have been made to present anddiscuss in a single book the entire size range of particulate dosage forms covered in this book. The general theme and purpose here are to provide the readerwith a current and general overview of the existing micro- and nanoparticulate-deliverysystems and to emphasize the various methods of preparation, characterization, evaluation, and potential applications in various areas such as medicine, pharmacy, cosmetology, and agriculture. The systematic approach used in presenting the various particulate systems should facilitate the comprehension of this increasingly complex field and clarify the main considerations involved in designing, manufacturing, characterizing, and evaluating a specific particulate-delivery system for a given application or purpose. Thus, the chapters, which have been contributed by leading authorities in the field, are arranged logically according to the methods of preparation, characterization, and applications of the various particulate-delivery systems. The first chapter is by C. Thies, a renowned scientist in the field of microencapsulation techniques. To provide an idea of which process is most appropriate for a specific application, the general principles of several microencapsulation processes are summarized and reviewed. This chapter focuses primarily on processes that have achieved significant commercial use. S . Magdassi and Y. Vinetsky present an interesting technique of oil-inwater emulsion microencapsulation by proteins following adsorption of the . P. Benoit and Drs. H. protein molecules onto the oil-water interface. J Marchais, H. Rolland, and V. Vande Velde have contributed a chapter on advances in the production technology of biodegradable microspheres. This chapter deals mainly with the preparation and use of microspheres. The potential o f the various technologies addressed is also discussed, with an emphasis on marketedproducts or those products currently underclinical evaluation. A. Markus demonstrates in his chapter the importance of applying microencapsulation techniques in the design of controlled-release pesticide formulations to meet the multifaceted demands of efficacy, suitability to mode of application, and minimal damage to the environment. The nanoparticulate-delivery systems are introduced by a chapter, authored by myself, B. Magenheim and P. WehrlC, that explains factorial
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Preface
design in the development of nanoparticulate systems. This chapter illustrates the application of the experim9ntal design technique not only for optimization but also for elucidation of the mechanistic aspects of nanoparticle formation by spontaneous emulsification. The second part of the book, which focuses on the evaluation and characterization of the various particulate-delivery systems, starts with an important chapter on microsphere morphology by J. P. Benoit and C. Thies. The chapter helps to clarify definitions and differences, which are very often confused. In addition, the chapter illustrates how morphology can becharacterized by usingdifferent techniques. C. Washington provides his valuable expertise in the presentation of the various kinetic models used to characterize drug-release profiles fromensembles or populations of microparticulate-delivery systems. It is worth noting that the release mechanism of a drug from multiparticulate systems such as microcapsules or microspheres cannot be identified by a study of global release profiles, since it has been shown that overall or cumulative release profiles from ensembles of microcapsules are entirely different from those of single microcapsules. The discrepancy arises from the heterogeneous distribution of the parameters determiningrelease behavior in individual microcapsules, which isbeyond the scope of the present chapter. The following chapter, by P. Couvreur, G . Couarraze, JrP. Devissaguet, and F. Puisieux, presents a very detailed explanation of the preparation and characterization of nanoparticles. The authors first clearly define the morphology of nanocapsules and nanospheres, providing the background, information, and guidelines for choosing the appropriate methodfor a given drug to be encapsulated. K. Westesen and B. Siekmann have contributed an important chapter on biodegradable colloidal drug carrier systems based onsolid lipids. These new colloidal carriers differ from the otherwell-known and widely investigated lipidic colloidal carriers, including liposomes, lipoproteins, and lipid or submicron oil-in-water emulsions by exhibiting a solid physical state as opposed to theliquid or liquid crystallinestate of the above-mentionedand well-knownlipidiccolloidal carriers. The authors present the different methods of preparation and point out the advantagesof the novel dosage forms such as biodegradability, biocompatibility, ease of manufacture, lack of drug leakage, and sustained drug release. Despite three decades of intensive research on liposomes as drug-delivery systems, the number of systems that have undergone clinical trials and become products on the market is quite modest. Even though there have been few successes with liposomes, the need for drug-delivery systems is as acute as ever, and the potential that liposomeshold, although somewhat tarnished, has not been substantially diminished according to R. Margalit and N. Yerushalmi. An interesting and original approach is presented in their chapter on thephar-
Preface
Vii
maceutical aspects of liposomes. Propositions are presented on how at least in some of the hurdles in research and development can be overcome and furthering the substantial strides that have been made in advancing liposomes from the lab to the clinic. An ingenious solution on how the drawis presented by D. Lasic in the backs of liposomes in vivo can be overcome chapter onstealth liposomes. He explains how the stability of liposomes in liposomicidal environments of biological systems presented a great challenge, which was only recently solved by coupling polyethylene glycol to the lipid molecules. An example of the potential of niosomes (a colloidal vesicular system prepared from nonionic surfactants) for the topical application of estradiol is contributed by D. A. van Hal and J. A. Bouwstra, and H. E. Junginger. Niosomes have beenshown to increase the penetrationof a drug through human stratum corneum by a factor of 50 as compared with estradiol saturated in phosphate buffer solution, making this colloidal carrier promising for thetransdermal delivery of drugs. In thethird part of the book, the potential applications of the various particulate-delivery systems are presented. The methods of preparation of microcapsules by interfacial polymerization and interfacial complexation and their applications are discussedby T. Whateley, a scientist who is extremelyknowledgeable in this field. The fast-growingfield of lipid microparticulate-delivery systems, particularly lipospheres, is explained and discussed by A. J. Domb, L. Bergelson, and S. Amselem. Lipospheres represent a new type of fat-based encapsulation technology developed for the parenteraldelivery of drugs and vaccines and the topical administration of bioactive compounds. In their comprehensive and exhaustive chapter, N. Garti andA. Aserin underline the potential of pharmaceutical applications of emulsions, multiple emulsions, and microemulsions, and emphaof size the progress made in the last 15 years in understanding mechanisms systems that open stabilization of these promising liquid dispersed-delivery new therapeutic possibilities. J.-C. Leroux and E. Doelker and R. Gurny in their chapter on the use of drug-loaded nanoparticles in cancer chemotherapy cover the developments and progress made in the delivery of anticancer drugs coupled to nanoparticles, and the interactions of the latter with neoplastic cells and tissues. This is probably themost promisingand encouraging application of nanoparticles and by far themost advanced in the process of development into a viable commercial pharmaceutical product. G. Redziniak and P. Perrier have contributed a chapter on the cosmetic applications of liposomes that have beensuccessfully exploited over the last decade. To complete thewhole rangeof applications of capsular products, a final chapter, by M. Seiller, M.-C. Martini, and myself, discusses cosmetic uses of vesicular particulate-delivery systems. Cosmetics are definitely the largest mar-
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Preface
ket, asmanufacturers have demonstrated that marketedcosmetic products containing these vesicular carriers and tested by dermatologists improve cutaneous hydration and skin texture, increase skin glow, and decrease wrinkle depth. It is now taken forgranted that liposomes and othervesicular carriers represent a major step in cosmetics formulations. However, this field requires numerous research studies coupled with strict controls. It is my hope that the scientific information contained herein will modestly contribute to a better understanding of the various particulate systems of all sizes that are now available and to an improved comprehension of their current andpotential applications.
Simon Benita
Contents
Preface Contributors
111
...
xiii
Part I: Methods of Encapsulation and Advances in Production Technology

1 . A Survey of Microencapsulation Processes Curt Thies
2. Microencapsulation of Oil-in-Water Emulsions by Proteins Shlomo Magdassi and Yelena Vinetsky

3. Biodegradable Microspheres: Advances in
21
Production Technology Jean-Pierre Benoit, Hervt Marchais, Hervt Rolland, and Vincent Vande Velde Advances in the Technology of Controlled-Release Pesticide Formulations Arie Markus
35
4.
73
5. The Use of Factorial Design in the Development of Nanoparticulate Dosage Forms Baruch Magenheim, Pascal WehrlE, and Simon Benita
93
Part I I : Evaluation and Characterizationof Micro- and Nanoparticulate Drug Delivery Systems 6. Microsphere Morphology Jean-Pierre Benoit and Curt Thies
133
i X
Contents
Drug Release from Microparticulate Systems
155
Clive Washington
Nanoparticles: Preparation and Characterization
183
Patrick Couvreur, Guy Couarraze, Jean-Philippe Devissaguet, and Francis Puisieux

Biodegradable Colloidal Drug Carrier Systems Based on Solid Lipids
213
Kirsten Westesen and Britta Siekmann

Pharmaceutical Aspectsof Liposomes: Perspectives in, and Integration of, Academic and IndustrialResearch & Development
259
Rimona Margalit and Noga Yerushalmi

Stealth Liposomes
297
Danilo D. Lasic
Nonionic Surfactant Vesicles (NSVs) Containing Estradiol for Topical Application Don A. van Hal, Joke A. Bouwstra, and Hans E. Junginger
329
Part IIk Applications of Particulate Delivery Systems

13.
Microcapsules: Preparation by Interfacial Polymerization and Interfacial Complexation and Their Applications Tony L. Whateley Lipospheres for ControlledDelivery of Substances Abraham J. Domb, Lev Bergelson, and Shimon Amselem Pharmaceutical Emulsions, Double Emulsions and Microemulsions
349
14.
377
15.
411
Nissim Garti and Abraham Aserin

16. The Use of Drug-Loaded Nanoparticles in Cancer Chemotherapy
535
Jean-Christophe Leroux, Eric Doelker, and Robert Gurny
Contents
Xi
17. CosmeticApplications of Liposomes G6rard Redziniak and Pierre Perrier

18. Cosmetic Applications of Vesicular Delivery Systems Monique Seiller, Mane-Claude Martini, and Simon Benita
Index
577
587
633
Contributors
Shimon Amselem, Ph.D. Director of Pharmaceutics, Department of Pharmaceutics, Pharmos Ltd., Weizmann Industrial Park, Rehovot, Israel Abraham Aserin, Ph.D. Casali Institute of Applied Chemistry, School of Applied Science and Technology, The Hebrew University of Jerusalem, Jerusalem, Israel Simon Benita, Ph.D.* Professor, Department of Pharmacy,School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel Jean-PierreBenoit,Ph.D. Professor, Laboratoire de Pharmacie GalCnique et Biophysique Pharmaceutique, UniversitC dAngers, and Centre de Microencapsulation, Angers, France Lev Bergelson, Ph.D. Professor, Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel Joke A. Bouwstra, Ph.D. Associate Professor, Department of Pharmaceutical Technology, LeidedAmsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands Guy Couarraze, Ph.D. Professor, Centre dEtudes Pharmaceutiques, URA CNRS 1218, Universitt de Paris-Sud, Chbtenay-Malabry, France PatrickCouvreur,Ph.D. Professor, CentredEtudesPharmaceutiques, URA CNRS 1218, UniversitCde Paris-Sud, Chitenay-Malabry, France
*Affiliated with the David Bloom Center for Pharmacy, Jerusalem, Israel.
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Contributors
Jean-PhilippeDevissaguet,Ph.D. Professor, Centred'EtudesPharmaceutiques, UR4 CNRS 1218, Universite de Paris-Sud, Chtltenay-Malaby, France
EricDoelker,Ph.D. Professor, School of Pharmacy,University of Geneva, Geneva, Switzerland Abraham J. Domb, Ph.D.* Professor, Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Medicine, The HebrewUniversity of Jerusalem, Jerusalem, Israel Nissim Garti,Ph.D. Professor, Casali Institute of Applied Chemistry, School of Applied Science and Technology, The Hebrew University of Jerusalem, Jerusalem, Israel Robert Gurny, Ph.D. Head Professor, Department of Pharmaceutics and Biopharmaceutics, School of Pharmacy, University of Geneva, Geneva, Switzerland Hans E .Junginger, Ph.D. Professor, Pharmaceutical Deparhnent, Leided Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands Danilo D. Lasic, Ph.D.** Department of Research, Liposome Technology, Inc. ,Menlo Park,California Jean-ChristopheLeroux,Ph.D. neva, Geneva, Switzerland
School of Pharmacy, University of Ge-
ShlomoMagdassi,Ph.D. SeniorLecturer, Casali Institute of Applied Chemistry, School of Applied Science and Technology, The Hebrew University of Jerusalem, Jerusalem, Israel BaruchMagenheim,M.Sc.+ Department of Pharmacy, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel Hem6 Marchais, Ph.D. Laboratoire de Pharmacie GalCnique et Biophysique Pharmaceutique,UniversitC #Angers, Angers, France
*Affiliated with the David Bloom Center for Pharmacy, Jerusalem, Israel. **Current affiliation: Senior Scientist, MegaBios Corporation, Burlingame, California +Current affiliation: Agis Industries Ltd., Yeruham, Israel
Contributors
xv
Rimona Margalit, Ph.D. Professor, Department of Biochemistry, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel ArieMarkus,Ph.D. Institute for Chemistry and Chemical Technology, The Institutes for Applied Research, Ben-Gurion University of the Negev, Beer-Sheva, Israel Marie-Claude Martini, Ph.D. Professor, Institut Pharmaceutiques et Biologiques, Lyon, France
,
des
Sciences
Pierre Perrier, Ph.D. Parfums Christian Dior, Saint Jean de Braye, France FrancisPuisieux,Ph.D. Head Professor, Centre dEtudes Pharmaceutiques, URA CNRS 1218, UniversitC de Paris-Sud, Chitenay-Malabry, France G6rard Redziniak, Ph.D. Applied Research Manager, Parfums Christian Dior, Saint Jean de Braye, France Hew6 Rolland,Ph.D. France
Director, Centre de Microencapsulation,Angers,
Monique Seiller, Ph.D. Professor, Unit6 dEnseignement et de Recherche des Sciences Pharmaceutiques, UniversitC de Caen, Caen, France Britta Siekmann, Ph.D. Associate Director, Department of Pharmaceutics, Astra Arcus AB, Sodertalje,Sweden Curt Thies, Ph.D. Professor, Department of Chemical Engineering, Washington University, St. Louis, Missouri Don A. vanHal, Ph.D. LeidedAmsterdamCenter for Drug Research, Leiden University, Leiden,The Netherlands Vincent Vande Velde,Ph.D. Technical Director, Centre de Microencapsulation, Angers, France Yelena Vietsky, M.Sc. Casali Institute of Applied Chemistry, School of Applied Science and Technology, The Hebrew University of Jerusalem, Jerusalem, Israel Clive Washington, Ph.D. Lecturer in Pharmaceutics, Department of Pharmaceutical Sciences, The University of Nottingham, Nottingham, England
m i
Contributors
PascalWehrl6, Ph.D. Professor, Laboratorie de Pharmacotechnie, Centre de Recherches Pharmaceutiques, UniversitC Louis Pasteur, Strasbourg, France Kirsten Westesen, Ph.D. Professor, Department of Pharmaceutical Technology, Institute of Pharmaceutics, Friedrich-Schiller University Jena, Jena, Germany Tony L . Whateley,Ph.D. Reader, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland NogaYerushalmi,Ph.D. Department of Biochemistry, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
1
A Survey o f Microencapsulation Processes
Curt Thies
Wizshington University, St. Louis, Missouri
I. Introduction
11. Processes for Preparing Microcapsules A.GeneralComments B. Type A Encapsulation Processes C. Type B Encapsulation Processes
111. Summary
1
2 2 3 10
17
1.
INTRODUCTION
Microcapsulesare small particles that contain an active agent or core material surrounded by a coating or shell. At present, there is no universally accepted size range that particles must have in order to be classified as microcapsules. However, many workers classify capsules smaller than 1 pm as nanocapsules and capsules larger than 1000 pm as macrocapsules. Commercial microcapsules typicallyhave a diameter between3 and 800 pm and contain 10-90 wt. percent core. A wide range of core materials has been encapsulated, including adhesives, agrochemicals, live cells, active enzymes, flavors, fragrances, pharmaceuticals, and inks. Most capsule shell materials are organic polymers, but fats and waxes are also used.
1
2
Shell Material
Thies
Fig. 1 Schematicdiagrams of tworepresentativetypes of microcapsules: (A) Continuous core/shell microcapsule. (B) Multinuclear microcapsule. (Courtesy of C. Thies.) Microcapsules can havea variety of structures. Some havea spherical geometry with a continuous coreregion surrounded by a continuous shell, as shown in Fig. 1A. Others have an irregular geometry and contain a number of small droplets or particles of core material, as shown in Fig.1B. Microcapsules are usedin a wide range of commercial products. They are thekey component of all carbonless copy papers and are present in several oraland injected drug formulations. Encapsulated adhesive resins coated on automotive fasteners are routinely used to assure that such fasteners are firmly set when installed. Microcapsules also are the basis for a number of long-actingcommercial pesticide and herbicide products. Improvement of these products and developmentof new ones is an ongoing process that involves a large number of development groups globally.
II.
PROCESSES FOR PREPARING MICROCAPSULES
A. General Comments
The concept of preparing small particles that carry a core material trapped within a shell material dates back at least to spray-drying work carried out in the 1930s. However, the first truly significant commercial productthat utilized such particles was carbonless copy paper. The microcapsules for this product were made by a process called complex coacervation. Since then many other methods forpreparing microcapsules have been developed and improved significantly. Some are based exclusivelyon physical phenomena. Some utilize polymerization reactions to produce a capsule shell. Others combine physical and chemical phenomena. Many investigators classify en-
Microencapsulation of A Processes Survey
capsulation processes as either chemical or mechanical. I prefer toclassify them as type A or type B processes, since so-called mechanical processes may actually involve a chemical reaction and so-called chemical processes may rely exclusively on physical phenomena. Table 1 lists representative examples of both types of processes. Capsules produced by type A, or chemical processes, are formed entirely in a liquid-filled stirred tank or tubular reactor. Capsules produced by type B, or mechanical processes, utilize a gas phase at some stage of the encapsulation process. In such processes, capsules can be formed by either spraying droplets of coating material on acorematerial being encapsulated, solidifying liquid droplets sprayed into a gas phase, gelling droplets sprayed into a liquid bath, or carrying out apolymerization reaction at a solid-gas interface. No encapsulation process developed to dateis able to produce the full range of capsules desired by potential capsule users. Some readily produce small, liquid-filled capsules, whereas others produce relatively large capsules with a solid core material. In order to obtain a feeling for which process is most appropriate for a specific application, it is appropriate to summarize the general principles of several types A and B processes. This chapter focuses primarily on processes that have achieved significant commercial use. Several reviews giveadditional details[l-61.
B. Type A Encapsulation Processes
l . Complex Coacervation Because of its original use in carbonless paper, the first type A process considered is complex coacervation. It is based on the ability of cat-
Table 1 List of Types A and B Encapsulation Processes

Type A (chemical) processes Complex coacervation Polymer-polymer incompatibility Interfacial polymerization in liquid media In situ polymerization In-liquid drying Thermal andionic gelation in liquid media Desolvation in liquid media Type B (mechanical) processes Spray drying Spray chilling Fluidized bed Electrostatic deposition Centrifugal extrusion Spinning diskor rotational suspension separation Polymerization at liquid-gas or solid-gas interface Pressure extrusion or spraying into solvent extraction bath
Thies
ionic and anionic water-soluble polymers to interact in water to form a liquid, polymer-rich phase called a complex coacervate. Gelatin is normally the cationic polymer used. A variety of natural and synthetic anionic watersoluble polymers interact with gelatin to form complexcoacervates suitable for encapsulation.When the complex coacervate forms, it is in equilibrium with a dilute solution called the supernatant. In this two-phase system, the supernatant acts as the continuous phase, whereas the complex coacervate acts as the dispersed phase. If a water-insoluble core material is dispersed in the system and the complex coacervate wets this core material, each droplet or particle of dispersed core material is spontaneously coated with a thin film of coacervate. When this liquid film is solidified, capsules are formed. Fig. 2 outlines one version of a complex coacervation encapsulation process [7]. The first step is to disperse the core material in qn aqueous gelatin solution. This is normally done at40-60C, a temperature range at which the gelatin solution is melted and liquid. After a polyanion or negatively charged polymer like gum arabic is added to the system, the pH and
Hyoo (40-60 C)
tielalln
Form Emulsion of Core In Solulion Gelalln (40-60C)
1
Gel Shell (515C)
Core Malerial (Llquicl)
Form Liquid Coacervale by Adjusling pH I Adding Polyanion and Dilulion Waler (40-60'C)
7 Crosslink with Glutaraldehyde
Fig. 2 Schematic f l o w diagram of encapsulation process based on complex coacervation. (Courtesy of C. Thies.)
A Survey of Microencapsulation Processes
concentration of polymer are adjusted so that a liquid complex coacervate forms. The pH at which this occurs is typically between 4.0 and 4.5. Once the liquid coacervate forms, the system is cooled to room temperature. The gelatin in the coacervate gels thereby forming capsules with a very rubbery shell. In order to increase the strength of the water-swollen shell and create a gel structure that is not thermally reversible, the capsules normally are further cooled to approximately 10C and treatedwith glutaraldehyde. The glutaraldehyde crosslinks the gelatin by reacting with amino groups located on the gelatin chain. Glutaraldehyde-treated capsules can be dried to a free-flow powder or coated on a substrate and dried. Complex coacervation has been used to encapsulate many waterimmiscible liquids and is used in a variety of products, including inks for carbonless paper, perfumes for advertising inserts, and liquid crystals for display devices. This technology routinely produces single capsules of 20800 pm diameter that contain 80-90 wt. percent core material. Capsules outside this size range can be made, but considerable know-how may be required. Most coacervate capsules have a continuous core/shell structure although the shell is not of uniform thickness. The mechani(see Fig. lA), cal and barrier propertiesof dry capsules formed by complex coacervation generally are sensitive to moisture. When stored at humidities above 70% or immersed in water, the shell of a complex coacervate capsule swells greatly, thereby facilitating transport of material into or out of the capsule. Postreatment of such capsules with urea and formaldehyde under acidic conditions has been used to reduce their moisture sensitivity. The value of complex coacervation encapsulation technology for food and pharmaceutical applications would be greatly enhanced if such capsules could be isolated commercially without the use of a chemical crosslinking agent.
2. Polymer-Polymer Incompatibility Polymer-polymer incompatibility is a second type A process that has been commercialized. This technology utilizes a polymer phase-separation phenomenon quite different from complex coacervation. In complex coacervation, two oppositely charged polymers, gelatin and a polyanion, join together to form the complex coacervate and both polymers become part of the final capsule shell. In contrast,polymer-polymer incompatibility occurs because two chemically different polymers dissolved in a common solvent are incompatible and do not mix in solution. They essentially repel each other and form two distinct liquid phases. One phase is rich in polymer designed to act as the capsule shell. The other is rich in the second, incompatible polymer. The incompatible polymer is present in the system to cause formation of two phases. It is not designed to be part of the final capsule shell, although small amounts may remain entrapped in the final capsule as
Thies
% l
@ V
0000
Phase Separated Solution of Ethylcellulose+ Polyethylene in Hot (80'C) Cyclohexane
Core Material (Small Particles)
Dispersion of Core Material in Hot, Phase-Separated Polymer Solution
+
Cool to 20-c (Ethylcellulose Coating Solidifies)
Elhylmllulose Shell
Wash and Harvest Capsules
Fig. 3 Schematic flow diagram of encapsulation process based on polymerpolymer phase separation. (Courtesy of C. Thies.)
an impurity. This type of encapsulation process generally does not involve any chemical reaction. phase-separation encapFig. 3 illustrates a specific polymer-polymer sulation process used to produce commercial pharmaceutical grade microcapsules [8]. The first step is to disperse the core material in ahot (80C) solution of ethyl cellulose in cyclohexane. Low molecular weight polyethylene, a polymer soluble in hot cyclohexane and incompatible with ethyl cellulose, is added to the system. This induces phase separation with formation of an ethyl cellulose-rich phase and a polyethylene-rich phase. The
core material, a solid unaffected by 80C cyclohexane, is dispersed in this two-phase system. Since the ethyl cellulose is more polar than polyethylene, it adsorbs preferentially on the surface of the core material and thereby causes a thin coating of shell material solution to engulf the particles of core material. When the system iscooled to room temperature, the ethyl cellulose precipitates, thereby solidifying the ethyl cellulose solution and formingsolid microcapsules that can beharvested. Aspirin and potassium chloride are examples of commercial encapsulated pharmaceutical products producedin this manner [g]. Polymer-polymer phase-separation processes normally are carried out in organic solvents and are used to encapsulate solids with a finite degree of water solubility. Many of the capsules produced commercially have an ethyl cellulose shell, are irregularly shaped 200- to 800-pm particles, and are loaded with solid drug particles. They are used to provide taste masking or prolonged oral drug delivery [g, 101. Smaller capsules (e.g., 20-120 pm in diameter) with a biodegradable poly(d,l-lactideglycolide) shell have been preparedby polymer-polymer phase separation and used asinjectable drug-delivery devices [ll].
3. Interfacial Polymerization Interfacial polymerization (IFP) is a third type A encapsulation process that has been commercialized. A unique featureof this technology is that thecapsule shell is formed at or on the surface of a droplet orparticle by polymerization of reactive monomers. This approach to encapsulation has evolved into a versatile technology able to encapsulate a wide range of core materials, including aqueous solutions, water-immiscible liquids, and solids. Many types of IFP reactions are used. Fig. 4 is a schematic diagram that illustrates how the capsule shell forms when a capsule loaded with a water-immiscible liquid is prepared by IFP. In suchcases, a multifunctional monomer is dissolved in the liquid core material. This monomer often is a multifunctional isocyanate but could be a multifunctional acid chloride or a combination of isocyanates and acid chlorides. In all cases, the resulting solution is dispersed to a desired drop size in an aqueous phase that contains a dispersing agent. A coreactant, usually a multifunctional amine, is then added to the aqueous phase. Thisproduces a rapid polymerization reaction at the interface which generates the capsule shell[12]. A polyurea capsule shellis obtained if the IFP reaction involves an isocyanate and an amine. A polyamide or nylon capsule shell is obtained if the IFP reaction involves an acid chloride and an amine. Reaction of an isocyanate with a hydroxylcontaining monomer dissolved in the aqueous phase produces a polyurethane capsule shell.
8
Polyurea Polyurea Shell
Thies
Core Material ["oil")
'.
H20
EMULSION STABILIZER
Fig. 4 Schematic illustrationof an interfacial reaction frequently usedto prepare microcapsules by interfacial polymerization. (Courtesy of C. Thies.)
If the core material being encapsulated is an aqueous solution, the abovereactantadditionsequence is reversed.Thecoreactant,e.g.,a water-soluble amine,is dissolved in the aqueous phase.The resulting mixture is dispersed in a water-immiscible solvent.unti1the desired drop size is obtained. A solvent-soluble reactant like an isocyanate or acid chloride is added to the organic phase, thereby initiating rapid polymerization at the interface to produce a capsule shell. A variety of core materialsof biological interest (e.g., active enzymes) have been producedin this manner [13]. Solids can also be encapsulated by interfacial polymerization reactions, although the polymerization chemistry typically differs from that used to encapsulate liquids. Vinyl monomers that polymerize by free radical reactions generally are used to encapsulate solids. In one process, the solid to be encapsulated is dispersed in aqueous media along with a dispersing agent. Vinyl monomer is added to thesystem and freeradical polymerization is initiated by a redox initiation system [14]. Success of this type of encapsulation process depends on forcing polymer formation to occur at the solid-liquid interface rather than allowing it to occur throughout the entire aqueous media. Another type of interfacial polymerization reaction involves the polymerization of p-xylylene at a gas-solid interface [15]. In this case,polymerization of vaporized p-xylylene radicals occurs spontaneously on contactwith a solid surface. Although interfacial polymerization can be used to produce large capsules, most commercial uses of this technology have focused on the preparation of small capsules that are coated or sprayed onto a substrate rather thanbeing isolated as a free-flow powder. For example, IFP is routinely used to produce 20- to 30-pm diameter
icroencapsulation of A Processes Survey
capsules loaded with pesticides and herbicides. It also is used to form 3-to 6-pm diameter capsules loaded with carbonless paper ink. Capsules with polyurea or polyamide shells have liquid cores or cores that melt below 60-70C. Because one of the reactantsused to create thecapsule shell is dissolved in the core material and is free to react with any reactive groups located on molecules of core material to create new molecular species, IFP encapsulation technology is viewed suspiciously by workers concerned with the encapsulation of fragrances, drugs, and flavors. Many agrochemicals like pesticides and herbicides are relatively water-insoluble low-melting solids or high-boiling point liquids free of groups that could react with the reagent(s) dissolved in the core material. Thus, many current commercial encapsulated agrochemical formulations are prepared by IFP [12]. Capsules formed by IFP often have a continuous core-shell structure and a spherical geometry. Significantly, the exterior surface of many IFP capsules is smooth and uniform. This is not the case for the interior surface. Capsule fracture studies show that the interior surface of many IFF' capsule shells is cratered and irregular. That is, the capsule shell is not uniformly deposited around the core by an IFP process except for a comparatively thin outer region of the shell. In such cases, the shell of an IFP capsule is not a membrane of uniform thickness. It is a thin skin membrane resting on a thick, porous support.
4.
In Situ Polymerization In situ polymerization is a type A encapsulation technology closely related to IFP. Like IFP, capsule shell formation occurs because of polymerization of monomers added to theencapsulation reactor. However, with in situ encapsulation processes, no reactive agents are added to the core material. Polymerization occurs exclusively in the continuous phase and on the continuous-phase side of the interface formed by the dispersed core material and continuous phase. Polymerization of reagents located there producesa relatively low molecular weight prepolymer. As this prepolymer grows in size, it deposits onto thesurface of the dispersed core material being encapsulated where polymerization with crosslinking continues to occur thereby generating a solid capsule shell. The first example of this technology was the encapsulation of various water-immiscibleliquids with shells formed by the reaction at acid pH of urea with formaldehyde in aqueous media [14]. Similar encapsulation reactions occur between melamine and formaldehyde in aqueous media. Addition of anionic polymers to the aqueous phase can have a significant effect on thein situ encapsulation process [16]. In situ polymerization is used extensively to produce small(3- to 6-pm diameter) capsules loaded with carbonless paper inks or perfume for scented strips. Larger capsules loaded with mineral oil are used for cos-
10
Thies
metic applications, whereas capsules filled with epoxy resin are used for fasteners. In all of these cases, the capsules have a continuous core-shell structure. Thetechnology can also be adapted to encapsulate solids.
5. Centrifugal Force and Submerged Nozzle Processes Several interesting type A processes use centrifugal force or two-fluid
submerged nozzles to form microcapsules. In one process, a cup perforated with a series of fine holes is immersed in an oil bath. It is rotated while immersed in the oil, thereby extruding into the oil phase a stream of droplets of an oil-in-water emulsion [17]. The water phase of this emulsion is a concentrated solution of a water-soluble polymer that gels on cooling. Gelatin is a specific example. By controlling the temperature of the oil bath, the external phase of the extruded emulsion droplets is gelled to create oil-loaded gel beadlets that can be isolated and dried. When isolated, the capsules consist of a number of small oil droplets dispersed throughout a matrix of shell material. This process was developed in 1942 in order to produce capsules that improved the oxidative stability of vitamins and fish oils [17]. Capsules can also be produced by coextruding an aqueous gelatin solution and an oil to be encapsulated through a two-fluid nozzle into a moving fluidstream of an oil solution. Fig. 5 schematically illustrates such a nozzle. The gelatin solution surrounds the oil drop to be encapsulated at the moment of extrusion and is gelled thermally before the particles are harvested and dried. In the case shown in Fig. 5 , continuous shell-core capsules are obtained.
C. Type B EncapsulationProcesses
Centrifugal force, extrusion,coextrusion, and formation of sprays are the principal means by which type B capsules are made. Type B methods of encapsulation predate type A encapsulation processes, since spray drying, a type B process, was developed in the 1930s. Because spray drying is the oldest type B encapsulation process, it is appropriate to discuss it first.
1. Spray Drying The first step in a spray-dry encapsulation processes is to emulsify or disperse the core material in a concentrated (40-60 wt. percent solids) solution of shell material. The core material generally is a water-immiscible oil such as a fragrance, flavor, or vitamin. It is emulsified in a solution of shell material until 1-to 3-pmoil droplets are obtained.The shell material normally is a water-soluble polymer like gum arabic or a modified starch. These materials do not form high-viscosity solutions at thehigh-shell material concentrations favored for spray drying. Mixtures of these shell materi-

Core Material (e.g., Vitamin O i l )
@ @
+
2-Fluid2-Fluid. Nozzle
Shell Material (e.g.. Warm Gelatin Solution)
Warm Carrier Phase (e.g.,Vegetable Oil)
l =l
Coil
@
/
Gel Beads that Core can be harvested Material and dried
Fig. 5 Schematicdiagram of a submerged two-fluid nozzle used microcapsules. (Courtesyof C. Thies.)
to prepare
als with hydrolyzed starches (maltodextrins) or hydrolyzed gelatins have been used. Water is the preferred solvent for most spray-drying encapsulations. Concerns about solvent flammability and toxicity severely restrict the use of organic solvents for conventional spray-drying encapsulation applications. However, several groups are exploring the use of spray drying from organic media to produce pharmaceutical capsules from biodegradable polymers. Once a suitable dispersion of core material in a solution of shell material has been prepared, the resulting emulsion is fed as droplets into the heated chamber of a spray drier. The droplets may be sprayed into this chamber or spunoff a rotating disk. In either case, they are rapidly dehy-
72
Thies
drated in a heated chamber, thereby producing dry capsules. The capsules fall to the bottom of the spray-drying chamber where they are harvested. Capsules produced in this manner typically fall between 10 and 300 pm in diameter. They tend to have an irregular geometry and may be aggregates of a number of small particles. Each spray-dried capsule has a number of small droplets of core material dispersed throughout it. Spray-dry encapsulation has a number of advantages. It is a wellestablished technology, involves readily available equipment, andis able to produce large amounts of capsules. Many shell materials preferred for spray drying are approved for food use. Furthermore, these materials are water soluble and notchemically crosslinked. Thus, capsules prepared from them dissolve in water andrelease core material without leaving any capsule shell debris. In contrast, the shell of complex coacervate capsules crosslinked with glutaraldehyde swells greatly in water but does not dissolve. Such capsules can be ruptured, thereby releasing their contents, but capsule shell debris is left behind. For some applications, this is objectionable. Spray-dry encapsulation has several problems and limitations. For example, if water is the preferred solvent, spray-dry encapsulation is limited to shell materials soluble or dispersible in water. The list of candidate water-soluble shell materials is limited, because many candidate materials form aqueous solutions that are too viscous to spray in spray-dry encapsulation protocols. Another limitation is the 20-30% core loading carried by most spray-dried capsules. Spray-drying protocols that allow core loading up to 50-60% have been reported [18], but current spray-dried capsules have lower loadings. A persistent problem with spray-dried capsules is free or surface core material. Because water evaporation from a capsule in the chamber of a spray drier occurs rapidly, it is not uncommon to harvest spray-dried capsules that have a finite amount of free or unencapsulated oil. The higher the core loading, the more pronounced this problem can become. This is undesirable, since free fragrance or flavor oil is susceptible to oxidation and the development of an off-odor or off-taste. Brenner [l81 noted that maximizing core loading and minimizing free core content involves a judicious choice of coating material, emulsifying agent, andspraydryeroperating conditions. Finally, it is important to stress that lowboiling point compounds with a finite degree of water solubility have posed a persistent problem to spray-dry encapsulation. Such compounds volatilize from the capsules in the spray-drying chamber. In summary, spray drying is a viable commercial method of forming microcapsules. It is an established, comparatively low-cost encapsulation technology that continues to develop. To date, spray drying has primarily been used to encapsulate fragrances and flavors.
13
2. Fluidized Bed Coaters Fluidized bed coaters are another type B encapsulation technology. They are limited to encapsulating solid particles or porous particles into which a liquid has been absorbed. Nevertheless, they are used extensively to encapsulate many different solids. Fluidized bed coaters function by suspending a bed or column of solid particles in a moving gas stream, usually air. A liquid coating formulation is sprayed onto the individual particles, and the freshly coated particles are cycled into a zone where the coating formulation is dried either by solvent evaporation or cooling. This coating and drying sequence is repeated until a desired coating thickness has been applied. Many variables affect the fluidized coating process, including dew point of the incoming air. Lehmann [l91 reviewed the operation of fluidized bed coaters. They represent a major capital investment, but are widely usedto produce encapsulated solids, especially for the pharmaceutical industry. A major advantage of fluidized bed coaters is their ability to handle an extremely wide range of coating formulations. They have been used to apply hot melts, aqueous latex dispersions, organic solvent solutions of shell material, and aqueous solutions of shell material. Enteric polymer solutions have been of particular interest, since they are used by the pharmaceutical industry to produce drug-dosage formulations that survive passage through the stomach. Enteric polymers are insoluble in gastric fluid (pH 2) and soluble in intestinal fluid (pH 7), so capsules or tablets coated with them can pass intact through the stomach and not disintegrate until they reach the intestine. Three types of fluidized beds are available: top-spray, tangentialspray, and bottom-spray. These units differ in location of the nozzle or nozzles used to apply the coating formulation. Fig. 6 offers schematic diagrams of a top-spray and a bottom-spray unit. In the top-spray unit (Fig. 6A), the coating formulation is sprayed into the fluidized bed. The droplets of spray leaving the nozzle move countercurrent to the gas stream until they impact the particles being coated. If the spray formulation contains a volatile solvent, evaporation of this solvent from the spray droplets occurs, thereby increasing the solids content, perhaps to such a degree that the spray droplets cannot spread on the particles being coated. This spraydrying effect has been used to explain why solvent-based coating formulations applied in top-spray fluidized bed coaters oftenyield coated particles with a degree of internal void volume and porous coatings. Enteric coatings applied as aqueous latex dispersions are an exception. Such coatings applied in a top-spray fluidized bed coater form a continuous coating analogous to that obtained with tangential-spray and bottom-spray units. Fats
14
Thies
Being Coated
Gas Flow
Expansion Chamber
Core Material Being Coated Formulation Gas Flow
Fig. 6 Schematic diagrams of two types of fludized bed coaters: (A) Top-spray unit. (B) Bottom-spray, or Wurster unit. (Courtesy of C. Thies.)
are also applied by top-spray fluidized bed coaters. Top-spray coaters have limitations, but they are more simple to operate and have higher production capacity than the othertypes of units. In tangential-spray and bottom-spray units, the droplets of coating formulation move in the same direction as the particles being coated. The droplets of coating formulation travel a shorterdistance before theyimpact
15
the particles being coated. Solvent evaporation is minimized, and a more uniform film of coating material is deposited. The bottom-spray, or Wurster coater, shown in Fig.6B has becomea standard unit used to produce encapsulated solids, especially solidpharmaceuticals. The partition and gas-distribution plates of such units are important components, becausethey direct the particles being coated in a cyclic path past the nozzle that supplies the coating formulation. Particles move past this nozzle, receive a spray of coating material, and move up into the upper section of the unit. Here the coating is solidified either by solvent evaporation or cooling of a hot melt. The coated particles then fall back down to thebottom of the unit where a fresh coating is applied. This cyclic process is repeated until the desired weight of shell material has been applied. Application of the coating in a series of cyclic steps is both an advantage anda disadvantage. The disadvantage is that stepwise deposition takes time. A one-step coating process is much quicker, but with a multistep coating process, defects can beminimized or perhaps eveneliminated. This makes it possible to use the Wurster process to produce capsules with a shell that approaches the same bamer properties as a free-standing solvent-cast film. In a one-step coating process, capsule shell defects are not healed and, hence, can havea profound effect on capsule release behavior.
3. Centrifugal Extrusion Fig. 7 is a schematic diagram of a centrifugal extrusion process [20].

The core and shell material, two mutually immiscible liquids, are pumped through a spinning two-fluid nozzle. This produces a continuous two-fluid column or rod of liquid that spontaneously breaks up into a stream of spherical droplets immediately after it emerges from the nozzle. Each droplet contains a continuous core region surrounded by a liquid shell. How these droplets are convertedinto capsules is determined by the nature of the shell material. If the shell material is a relatively low-viscosity hot melt that crystallizes rapidly on cooling (e.g., a wax or wax toughened with a polymer), the droplets are converted into solid particles as they fall away from the nozzle. Suitable core materials typically are polar liquids like water or aqueous solutions, since they are immiscible with a range of hot melt shell materials like waxes. Alternatively, droplets emerging from the nozzle may have a shell that is an aqueous polymer solution able to be gelled rapidly. In this case, the droplets fall into a gelling bath wherethey are converted into gel beads. A specific example is the gelatin of an aqueous sodium alginate shell by an aqueous CaCl, gelling bath. The gel beads produced can be dried to give capsules with a solid shell. Core materials suitable for this type of capsules shell are water-immiscible oils.
16
Core Material Shell Shell
Thies
AIR
,Liquid Core
Shell gelled b y immersion in gelling bath
Shell solidifies
by cooling in air
to
Fig. 7 Schematicdiagram of centrifugaltwo-fluidnozzleused crocapsules. (Courtesy of C. Thies.)
produce rni-
Capsules prepared by centrifugal extrusion tend to be large with diameters typically ranging from over 250 p up to several millimeters.
4. Rotational Suspension Separation Fig. 8 is a schematic diagram of the rotational suspension separation process for preparing microcapsules. In this process, core material dispersed in a liquid shell formulation is fed onto a rotating disk. A flat disk is shown, but conical or bowl-shaped disks can be used. Individual core particles coated with a film of shell formulation are flung off the edge of the rotating disk along with droplets of pure coating material. When the shell

Coating Core Phase (e.g.. molten lot) "Spherical" Material
17
!
Spinning Disc
Rotate
0
h
Free Coating Material
Capsules
Fig. 8 Schematic diagram

process.
of rotational suspension separation encapsulation
formulation is solidified, e.g., by cooling, discrete microcapsules are produced. The droplets of pure coating material also solidify, but they are said to collect in a discrete zone away from the capsules, as shown in Fig. 8. This technology is claimed to be a fast, low-cost, high-volume method of encapsulating a variety of materials that act like solids on the rotating disk, including particles below 150 pm in diameter [21]. In order toobtain optimal results, the core material must have a spherical geometry. Formation of this geometry may require a granulation step before encapsulation is attempted. A variety of hot-melt shell materials can be applied, butmelt viscosities below 5000 CP are favored. Capsule shell formulations that do not solidify rapidly are said to pose problems. Since solidification of the coating formmulation occurs rapidly and the coating formulations applied are crystalline materials susceptible to polymorphic transformations or changes in percent crystallinity on storage, it is reasonable to suggest that capsule shells produced by rotational suspension separation are not at thermodynamic equilibrium immediately after capsule formation is complete.
111.
SUMMARY
Most microcapsules of commercial significance are produced by the encapsulation processes described in this chapter. The commercialization of products that contain microcapsules is progressing as is the development of encapsulation processes. Today, a number of capsule-based products exist.
18
Thies
Several have significant sales. The biggest application, carbonless paper, Such capsules are consumes thousands of tons of capsules per year [5]. produced by complex coacervation, IFP, or in situ polymerization. Large volumes of commercial capsules loaded with agrochemicals, especially herbicides, are madeby IFF. Although a numberof encapsulated food ingredi2 2 , 2 3 ] , the volume of such capsules remains small ents arebeing marketed [ relative to the huge potential that exists. The development of encapsulation technology and microcapsule-based products continues tobe pursued actively by a number of groups globally. Accordingly, it is reasonable to project that this field will experience steady growth for the foreseeable future. It will be interesting to see when the next high-volume capsulebased product appears and what it will be.
REFERENCES
1 . P. B. Deasy, Microencapsulation and Related Drug Processes, Marcel Dekker, New York, 1984. 2. A. Kondo, Microcapsule Processing and Technology, Marcel Dekker, New York, 1979. 3. C. Thies, Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 16, 4th ed., Wiley, 1995, pp. 628-651. 4. W. Slwika, Angew. Chem. Internat Edit., 14539-550 (1975). 5. C. A. Finch, Ullmanns Encyclopedia of Industrial Chemistry, Vol. A16, 5th ed., VCH Publishers, New York, 1990, pp. 575-588. 6. I. C. Jacobs, and N. S. Mason, in Polymeric Delivery Systems, ACS Sympo-
sium Series520 (M. A. El-Nokaly, D. M. Piatt, and B. A. Charpentier,eds.), American Chemical Society, Washington, DC,1993, pp. 1-17. 7. B. K. Green, and L. Schleicher,U.S. Patent 2,800,457,1957. 8. J. L. Anderson, G. L. Gardner, and N. H. Yoshida, U.S. Patent 3,341,416,
9. 1967.
J. A. Bakan, in The Theory and Practice of Industrial Pharmacy, 2nd ed. (L. Lachman, H. A. Lieberman, and J. L. Kanig, eds.), Marcel Dekker, New York, 1986. 10. C. Thies, CRC Crit. Rev. Biomed. Eng., 1983,8:335-383 (1983). 1 1 . B. J. Floy, G . C. Visor, and L. M. Sanders in Polymeric Delivery Systems, ACSSymposiumSeries 520 (M. A. El-Nokaly, D. M. Piatt,and B. A. Charpentier, eds.), American Chemical Society, Washington, DC, 1993, pp.
154-167. 12. H. B. Scher, in Pesticide Chemistry-Human Welfare and the Environment, Vol. 4 (J. Miyamoto and P. C. Kearny, eds.), Pergamon Press, Oxford, UK, 1983, pp. 295-300. 13. T. M. S. Chang, Artificial Kidney, Artificial Liver, and Artificial Cells, Plenum Press, New York,1978.
19
14. M. Cakhshaee, R. A. Pethrick, H. Rashid, and D. C. Shemngton, Polymer Commun., 26:185-192 (1985). E. 15. W. D. Jayne, in Microencapsulation: Processes and Applications (J. Vandegaer, ed.), Plenum Press, New York, 1974, p. 103. 16. K. Dietrich, H. Herma, R. Nastke, E. Bonatz, and W. Tiege, Acta Polymerica, 40:243-251 (1989). Patent 2,299,929, 1942. 17. J. A. Reynolds, U.S. 8:40-44 (1983). 18. J. Brenner, Perfumer and Flavorist, 19. K.Lehmann, in Microcapsules and Nanoparticles in Medicine and Pharmacy (M. Donbrow, ed.), CRC Press, Boca Raton, FL, 1992, pp. 73-97. 20. J. T. Goodwin, and G . R. Somerville, Chemtech, 4:623 (1974). 21. R. E. Sparks, I. C. Jacobs, andN. S. Mason, in Polymeric Delivery Systems, ACSSymposiumSeries520(M. A. El-Nokaly,D.M. Piatt,andB.C. Charpentier, eds.), American Chemical Society, Washington, DC, 1993, pp. 145-153. 22. S. Hagenbart, Food Product Design, April:28-38 (1993). 23. R. Arshady, J. Microencapsulation, 10:413-436 (1993).
2
Microencapsulation of Oil-in-Water Emulsions by Proteins
Shlomo Magdassi and Yelena Vinetsky
Casali Institute ofApplied Chemisty, School ofApplied Science and Technology, The Hebrew University of Jerusalem, Jerusalem, Israel
Introduction I.
21 23 23 25 25 25 27
11. Methods of Encapsulation A. Phase Separation B. Spray Drying C. Heat Denaturation

1 1 1 . Surface Activity of Proteins
IV.
PossibleFilm-Forming Proteins
1.
INTRODUCTION
The formation of microcapsules is a widely used process that combines both basic chemical aspects and technological aspects. This process is usually very complex, since it involves various reactions and steps which take place atdifferent phases. For example, the initial step of a simple coacervation-based process isbased on adsorption of one of the wallforming components onto the core material-continuous phase interface. Therefore, the migration of the molecule from the bulk continuous phase to theinterface and the kinetics and reversibility of the adsorption process are of great importance.The second step is usuallybased on conversion of
21
22
and
Magdassi
Vinetsky
the adsorbed layer into a semisolid wall, with a defined and measurable thickness and permeation properties. This process involves the addition of other molecules, which lead to precipitation or coacervation onto the core material. Usually, this process is performed by adding an electrolyte (simple coacervation) or an oppositely charged polymer (complex coacervation). Since the first wall component may also be present in the bulk phase, and not only adsorbed at the core-continuous phase interface, it could also participate in the coacervation or precipitation process. Itis clear that such a situation is undesirable, as it results in waste of material, which could otherwise be used for the formation of the wall. Moreover, the free molecules in the continuous phase may also lead to aggregation of the individually dispersed particles or droplets of the core material, leading to formation of very large particles, which may sediment or float, and which are often unsuitable for most applications. The next step is usually based on hardening of the wall material by various crosslinking agents, which are added to the continuous phase. It is obvious that such a process should be carefully controlled, because, in addition to the hardening of the wall material of each microcapsule, it may also lead to crosslinking between several microcapsules, thus changing the physicochemical properties of the microcapsules. In most applications, it is desirable to obtain, a dry, microcapsules powder as a final product, which can be further redispersed in various liquid phases. Therefore, removal of the liquid continuous phaseis oftenrequiredand is achieved by a process such as spray drying or lyophylization. Such a process could often lead to agglomeration of the microcapsules, which should be prevented if a redispersion of the microcapsules is desired. These problems are often solved by including various additives in the liquid phase in a way that the removal of the liquid phase would cause the formation of an additional barrier between the individual microcapsules, which will disappear on redispersion. Obtaining dry, redispersible microcapsules is still not the end process. A caking phenomena that occurs as a result of agglomeration caused by the pressure gradient present in the storage containers may also be observed. This problem usually requires the use of anticaking agents, which can be added to the dry powder as well as to the final steps of removal of the liquid phase. The complexity of the process is more severe if the core material is also a liquid: In such cases, the microencapsulation process should also include an emulsificationstep in which a stable oil-in-water or water-in-oil emulsion could be obtained. The emulsification process is based on various parameters, such as interfacial tension, adsorption of the surfactant, or homogenization equipment. The use of a liquid core may also lead to manufacturing problems at the endof the process, during the removal of the continuous
Microencapsulation of Oil-inEmulsions Water
23
liquid phase, as in the case of encapsulation of volatile oils (such as orange oil), which are widely used as fragrance and flavor additives. This chapter deals with the formation of microcapsules in which the core material is a water immiscible oil and the continuous phase is an aqueous solution. The wall material described here is composed of various proteins and, in particular, gelatin. The principles for obtaining the microcapsules are similar, and therefore we will discuss in detail the various steps of the microencapsulation process with emphasis on the proteinbased microcapsules.
II. METHODS OF ENCAPSULATION
Many different techniques have been proposed for the production of microcapsules, and it was suggested(1) that more than 200 methods could be identified in the patent literature. Three methods which are significantly relevant to microcapsules that are based on proteins or polysaccharides, especially when the core material is an oil, are briefly discussed here.
A. Phase Separation
As described in a publication by Finch [l], in the phase separation-based process, the core contents, in a solvent, are first suspended in a solution of the wall material. The wall polymer is induced to separate as a new, viscous, polymerrich phase by adding a nonsolvent, by lowering the temperature,by changing the pH,by adding a second polymer, or by changing other environmental conditions. It is essential that such changes would cause the polymer to come out of the solution and to aggregate around a core dropletto form a continuous encapsulating wall [2]. This process is shown schematically in Fig. 1, which includes the hardening of the coacervate layer, usually by a crosslinking agent. The coacervation of the polymer can be recognized bythe appearance of turbidity, droplets, or separation of the solution into two layers, which contain high and low concentrations of the polymer. Coacervation may be simple or complex: Simple coacervation is obtained by adding a water-miscible nonsolvent to theaqueous polymersolution (e.g., ethanol) or by adding an electrolyte, which causes polymer separation due to a salting-out mechanism. A typical example is the addition of sodium sulfate to an oil-in-water ( O M ) emulsion, whichis formed by using gelatin [3,4]. The system may be stabilized by altering the pH or the temperature by or addition of a crosslinking agent. Complex coacervation occurs with the mutual neutralization of two oppositely charged polymers. A widely used method is based on coacer-
24
Magdassi and Vinetsky
dissolved in water
0
Emulsification
7I
Film forming material, oppositely charged
Hardening of coacervate-capsule wall by chemical or physical means
Removal of water
Fig. 1 Schematic presentation of a formation of microcapsules of oil droplets in water by complex coacervation.
Microencapsulation of Oil-inEmulsions Water
25
vation of cationically charged gelatin (at pH below 8) by a negatively charged polymer such as gum arabic. This process was originallydeveloped for the formation of carbonless copying paper [5].
B. Spray Drying
Microencapsulationby spray drying is based on two steps: emulsification of the coreoil (or dispersion of particles) in the polymer solution followed by removal of the solvent by a hot stream of air. This method, in spite its limitations, is the most prevalent process used to encapsulate flavors [6]. The main problems relatedto this process are the nonuniformed size of the microcapsules (due to coalescence of oil droplets at high temperature and aggregation of microcapsules) and incomplete encapsulation of the core component, which is often a volatile oil. The wall materials which can be used in such a process can be various polysaccharides (starch, gum arabic) or proteins (gelatin, albumin, casein) [7,8]. It is obvious that while choosing the wall material, two important parameters should be taken into consideration: the ability of the polymer to emulsify the oil phase and the behaviorof the polymer at high temperature. The latter has a uniquemeaning regarding theuse of proteins, which can be denaturated specific at temperatures and hence form unsuitable wall material. C. Heat Denaturation Heat denaturation of proteins can also be used for encapsulation, as described recentlyin a patentby Janda et al. [g]. Briefly, the method is based on thefollowing steps:
1. Dispersionor emulsification of thecorematerialinasoluble protein slurry 2. Heating the slurry to form a protein melt followed by denaturation of the protein to render it insoluble in water in such a way that the insoluble layer coats the core droplets or particles
The denaturation process can be achieved by heating only, by pH adjustment, or by using proteolyticenzymes which cause coagulation of the protein. It seems that this method could be suitable with various proteins, such as casein, albumin, corn and soy protein, wheat and rice gluten, and gelatin.
111.
SURFACE-ACTIVITY OF PROTEINS
All three methods described above have a common first step during the process of microencapsulation: adsorption of the proteinmolecule onto the
26
and
Magdassi
Vinetsky
oil-water interface. This adsorption may affect the emulsification of the oil phase (and hencethe droplet size distribution), the natureof the encapsulating wall (viapacking andinteractions between adsorbedmolecules), and the colloidal properties of the newly formed microcapsules (e.g., flocculation, zeta potential). Therefore, it is essential to understand the adsorption of proteins at oil-water interfaces, as well as the chemical parameters which may affect their surface activity. The proteins are macromolecules composed of a mixture of polar and nonpolar side chains and may have positive and negative electrical charges. These molecules may adsorb at the oil-water interface, through their hydrophobic side chains, and hencedecrease the interfacial tension of the emulsion system. The adsorption process is, in general, composed of three consecutive steps [lo]:(1) diffusion of the protein molecule fromthe bulk to the interface and attachment ontothe interface; (2) penetration of new (3) protein molecules, which have to overcome an energy barrier; and molecular rearrangement of the adsorbed molecules. It is interesting to note that the adsorption, or attachment at the interface, is achieved by small segments of protein having a unit surface area of 100-200 A2. The adsorption process obviously can lead to a drastic unfolding of the protein at theinterface, a phenomenon termedsurface denaturation. Under certain conditions, the protein present at the interface may coagulate; a result which may have implications on the formation of microcapsules by the protein-coated oil droplets. an emulsifiSince the first step in forming O/W microcapsules involves cation step, it is necessary to obtain an emulsion which has the required droplet size distribution and is stable, at least for the period of time required to complete the encapsulation process. Basically, it is preferred to use a protein which has some surface activity in terms of the initial adsorption and reduction of interfacial tension. Moreover, the proteins should be good emulsifiers, a property which can be obtained byusing nonrigid, charged protein that causes emulsion stabilization in both electrical and steric mechanisms. Such proteins are gelatin and casein, which are frequently used inprotein-based microencapsulation processes. If the emulsification step is achieved by using surfactants other than protein, the main property required of the coating protein is that it adsorbs onto the previously prepared emulsion droplets. Inthe case of simple or complex coacervation, it is required that the oil coacervate phase have suitable for gelatin coacervate interfacial tensions, as shown by Arneodo etal. [ll], phases and citrus oils. The surface activity of proteins is dependent on many factors [lo] such as the molecular parameters (MW, hydrophobicity, charge density and
Microencapsulation Emulsions Water of Oil-in-
27
isoelectricpoint,and rigidity) andthesolutionconditions(pH,ionic strength, temperature). Therefore, when forming protein-based microcapsules,oneshouldconsiderthe overall effectonboththeadsorption/ emulsification step and the formation of a wall around the oil droplets.
IV. POSSIBLEFILM-FORMINGPROTEINS
As stated earlier, the of use certain proteins depends on the method used to prepare the microcapsules. From various publications, it appears thatthe number of proteins which can beused for microencapsulation is huge. In principle, any protein capable of forming a film is suitable for microencapsulationprovided the film can also be formed on the surface of the oil droplets. In a recent publication, Gennadios et al. [l21 review various edible coatings and films based on proteins. As shown, proteins such as zein, wheat gluten, soy protein, peanut protein, keratin, collagen, gelatin, milk proteins, andcasein can be used as film formers. Microcapsules were formed by emulsification and heat denaturation of egg albumin [13,14] around oil droplets; wheat gluten was used to encapsulate fat-soluble, edible flavors by a spray-drying technique [15]; zein was used by dispersing the oil in alkaline protein solution, in which its pH was lowered to cause phase separation [16]; and casein was usedto encapsulate polyunsaturated fatsby spray drying [17-191. Qpical examples for the use of various proteins to encapsulate oil droplets are given in Table 1. Of particular interestis the gelatin molecule, used either by itself or incombination with other componentsto form microcapsules [20]. Gelatin is a very abundant protein that is produced from collagen of various origins. Gelatin is usually manufactured by two processes, acid and basic, which yield gelatin type A (positively charged below pH S) and type B (negatively charged above pH 5). The molecular weight of the proteins varies from 100,000 to 20,000, a property that is reflected in the bloom number, whichisindicative ofgel strength. Gelatin has unique surface activity and is a good emulsifier for various oil phases. Therefore, the various types of gelatin are often considered ideal candidates for the encapsulation process. For example, gelatinmay form capsules by the simple coacervation process, obtainedby addition of sodium sulfate [32], or by complex coacervation with gum arabic [7], sodium alginate [S], or even by using gelatin type A with gelatin type B. We have recently reported [33] on the formation of gelatin-based microcapsules by formation of an insoluble complex of sodium dodecyl sulfate (SDS) with a positively charged gelatin. It was found that gelatin has the ability to bind anionic surfactants,such as SDS, and various soap molecules via electrostatic and hydrophobic interactions. At a certain mo-
28
v1
1"
i %
.m
a 0
v)
t -
.m
Y
E 0
2 5
c 0 a
Y
Microencapsulation of Oil-in- Water Emulsions

29
. I
c)
B
. I
c)
B
. I
E
V
I;
V
8
I;
I;
3
3
N
. I
c 1
M
h
30
Gelatin in water pH=6.1

Soybean oil
Emulsification
non-adsorbed gelatin
Aqueous solution of SDS, pH=6.1
Mix
Crosslinking agents
V
Microcapsules
Removal of water (lyophilization, evaporation)
Fig. 2 Schematic presentation of formation of microcapsules of oil droplets in water by interaction of anionic surfactant with a cationically chargedgelatin.
Microencapsulation Emulsions Water Oil-inof
31
lar ratio of surfactant to protein, the protein becomes uncharged and, therefore, is insoluble in water. Further addition of SDS leads to redissolving of the gelatin-SDS inwater, probably due tohigh charge (negative) density. It was therefore assumed that if the precipitation of the insoluble gelatin-SDS complex could occur at the surface of the oil droplets, we could obtain microcapsule walls. Since both gelatin and SDS have surface activity, this process may combine the emulsification ability of the two components, with their binding properties. The formation of microcapsules by this method is schematically presented in Fig. 2. As shown,the process is based on threesteps:
1. Emulsification of the oil by usinggelatin type A as theemulsifier. The emulsions which are formed by simple homogenizer are stable for at least several days, having a mean droplet size of several micrometers. The pH adjustment is crucial, since the SDS binding is strongly dependent on the charges of the protein molecule. After theemulsion is formed, the free gelatin should be removed simplybyrinsing the emulsion with water during filtration or centrifugation. 2. The second step is addition of SDS solution at a specific concentration, which is predetermined according to the optimal surfactant-protein ratio which causes precipitation. Fig. 3 gives an
!
-1
$I
Fig. 3 Microcapsules of soybean oil prepared by interaction of SDS with gelatin type A.
32
Magdassi andVinetsky
example of such microcapsules which are obtained without further crosslinking. 3. The last step is the addition of a crosslinking agent such as glutaraldehyde. The hardened wallof the microcapsules allows simple separation of the microcapsules from the continuous phase, and if the continuous aqueous phase is completely removed (by lyophilization or evaporation), a dry powder of microcapsules is obtained. From the description of this process, it is clear that formation of microcapsules of oil droplets can beachieved by various protein-surfactant combinations provided that either the protein or the surfactant has some emulsifying activity, and the interaction between the two can lead to precipitation of a suitable film. REFERENCES
1. C. A. Finch, Spec. Pub1.-R. SOC. Chem. 138(Encapsulation and Controlled
2. 3. 4. 5. 6. 7. 8. 9. 10.
11.
12. 13. 14. 15. 16. 17. 18.
Release):35 (1993). B. H. Kaye, Kona (Hirakata, Japan) 10:65 (1992). P. Spiegl, Austrian Patent DE 244, 1890 (1975). D. Fredj and F. Dietlin, French Patent 84-14653 840925, 1986. Y. Okumura, H. Takai, M. Shiozawa, and K. Kaneko, Japanese Patent 84143268 840712,1986. H. C. Greenblatt, M. Dombroski, W. Klishevich, et al., Spec. Pub1.-R. Soc. Chem. 138(Encapsulation and Controlled Release): 148 (1993). L. Y. Sheen, S. Y. Lin, S. J. Tsai, Zhangguo Nangye Huaxue Huizhi 30(3):307 (1992), C. Arneodo, J. P. Benoit, C. Thies,S. T. P .Pharma 2(15):303 (1986). J. Janda, D. Bernacchi, S. Frieders, U.S. PatentWO-91-US7278911004, 1992. S. Magdassi and N. Garti, in Interfacial Phenomena in Biological Systems (M. Bender, ed.), Marcel Dekker, New York, 1991, pp. 289-299. C. Arneodo, A. Baszkin,J.P. Benoit, and C. Theis, in Flavor Encapsulation (S. J. Risch and G . A. Reineccius, eds.), American Chemical Society, Washington, DC, 1988, pp. 132-147. A Gennadios, T. H. McHugh, C. L. Weller, et al., in Edible Coatings and Films to Improve Food Quality (J. M. Krochta,et al., ed.), Technomic Publishing, Basel, 1994, pp. 201-277. S. Soloway, U.S. Patent 3137631, 1964. J. Kosari and G. M. Atkins, U.S. Patent 3406119,1968. P. P. Noznick and C.W. Tatter, U.S. Patent 3351531,1967. C. Brynko and J. A. Bakan, U.S. Patent 3116206,1963. T. W. Scott, L. J. Cook, K. A. Ferguson et al., Austr. J. Sci 32:291 (1970). T. W. Scott, and G. D.L. Hills, U.S. Patent 4073960,1978.
Microencapsulation Emulsions Water Oil-inof
33
19. G. J. Faichney, H. L.Davies,T.W.Scott et al., Austr. J. Biol. Sci 25:205 (1972). 20. L. S. Jackson and K. Zee, Lebensm. Wiss. Technol.24:289 (1992). . W . Harlowe, andH. W . Schlamens, U.S. Patent 87-129503 21. D. J. Mangold, W 871207, 1987. 82-28757 820226,1983. 22. Q.P. Corporation, Japanese Patent 23. T. Kobayashi, French Patent 1568500 690523, 1969. 24. J. C. Soper, U.S. Patent 89401189,1991. 25. E. Bonatz, K.Golz, R. Nastke, German Patent 90-342045 900625, 1991. 26. D. M. Whitaker, U.S. Patent 88-292495 881230,1991. 84-197776,1986. 27. S. Shimizu and H. Kato, Japanese Patent 1086127 800923, 1980. 28. R. M. Rawlings and D. Procter, Canadian Patent 29. R. Shekerdzhiiski, S. Titeva, K. Mitikov et al., Formatsiya 35(4):23 (1985). 30. B. Sivik, J. Gruvmark, and M. Jakobsson, in Proc.-Scand. symp. Lipids,16th (G. Lambertsen, ed.), Lipidforum, Bergen, 1991, pp. 147-152. 31. H. Jizomoto, E. Kanaoka, K. Sugita et al., Pharm. Res. 10(8):1115(1993). 32. J. Rosenblat, S. Magdassi, and N. Garti, J. Microencaps. 6515 (1989). 33. S. Magdassi andY. Vinetsky, J. Microencaps., accepted for publication.
Biodegradable Microspheres: Advancesin Production Techno

Jean-Pierre Benoit
Pharmaceutique, Laboratoire de Phannacie Galbniqlue et Biophysique cE Microencapsulation, Angers, France Universitt? $Angers,and Centre d
Hew6 Marchais
Laboratoire de Pharmacie GaUnique et Biophysique Phannaceutique, Universitt? $Angers,Angers, France
Hew6 Rolland and Vincent Vande Velde

Centre de Microencapsulation, Angers, France
I. Introduction 11. Polymer Phase-SeparationMethods
36 36 43 43 45 49 50 52 53 53 5 5 57 58 59 60 60 62
111. Solvent Evaporation and Solvent Extraction Methods A. General Description of the Process B. Solvent Evaporation (Emulsification-Evaporation) C. Solvent Extraction (Emulsification-Extraction) IV. Spray-Drying Methods A. The NebulizingSystem B. Apparatus C. Applications D. AdvantagesandInconveniences V. Methods Using Fluids Under Supercritical Conditions A. Solvation of Compounds B. Precipitation of Pure Compounds C. Crystallization of Pure Compounds D. Formation of Microparticles VI. Milling Methods
35
36
1.
Benoit et al. INTRODUCTION
Major advances in the therapeutic sciences and substantial developments in the field of biotechnology have taken place over the past few years which have allowed the syntheses of peptides and proteins on industrial scales. The much valued pharmacological importance of these molecules has been somewhatoffset by their poor ability to penetrate the gastrointestinal barrier. Likewise, their high sensitivities and fragilities together with their very short biological half-lives seriously limit their therapeutic applications. The delivery of proteins and peptides for therapeutic purposes thus presents a major challenge to pharmaceutical scientists. Different strategies have been proposedto overcome these problems. Various modes of administration, such as the nasal or transdermal routes, have been investigated to facilitate the resorption of some proteins and peptides. However, the parenteral route remains the dominant means of delivery of peptides and proteins. From a formulation point of view, different drug-delivery systems involving, e.g., liposomes, mixed micelles, and nanoparticles, have shown much promise, as have controlled-release implants based on the use of biodegradable polymers. The success of the latter technique has contributed to the renaissance of the microencapsulation field. A large number of microencapsulation processes, and modifications of processes, have been reported in the literature and in various patents [l]. In this chapter, emphasis is placed on the description of selected processes leading to microparticle formation from a particular series of polymers used as coating materials; namely, poly(a-hydroxy-carboxylic acids) (PHCA). This category of polymers covers polymers and copolymers of lactic and glycolic acids (PLA, PLAGA),which are the sole excipients presently approved for use by international health authorities for designing biodegradable parenteralimplants. In referring to thevarious pharmaceutical forms discussed herein, the authors define microparticles as polymeric entities falling in the range of 1 1000 pm, covering two types of forms: Microcapsules, micrometric reservoir systems Microspheres, micrometric matrix systems This chapter deals solelywith the preparation and use of microspheres. The potentials of the various technologies addressed hereafterare also discussed.
II. POLYMERPHASE-SEPARATIONMETHODS
Polymer phase-separation, in nonaqueous media, by nonsolvents or polymer addition, also referred to as coacervation, is an excellent technique for
Biodegradable
37
the entrapment of water-soluble drugs such as peptides, proteins, or vaccines to beused as microencapsulatedpreparations. This technique has beenknown sincethe 1960s [2]. Since then numerous textbooks and articles have appeared describing the principles and formapreparation methods which, if adhered to, lead systematically to the tion of microcapsules [l-61. Under certain circumstances, it has been demonstrated that this procedure leads to the production of microspheres. Thus, the coacervation of a polymer suchas poly(d,l-lactic acid-coglycolic acid) (PLAGA) dissolved in methylene chloride with a second polymer such as silicone oil allows the formation of matricial systems[7]. If crystals of active principles are placed in suspension at thebeginning of this process, they will be captured in these matrices after the desolvation of PHCA. The coacervation process of a PHCA, as shown in Fig. 1, can be divided into four steps [7]:
1. In step 1 (Fig. lA), the amount of phase inducer added to the Silicone oil appearsto form a solution is low (l-5% v/v). pseudoemulsion in the organic phase. 2. In step 2 (Fig. lB), for a greater amountof silicone oil introduced in the medium, the beginning of a phase separation is noticeable. The droplets of coacervate are unstable and merge together to give larger structures which expand andfinally rupture. 3. In step 3 (Fig. IC), the quantity of polymer added is sufficient to allow a stabledispersion of PLAGA coacervate droplets. 4. In step 4 (Fig. l D ) , extensive aggregation of coacervate droplets occurs which leads to their precipitation. To prepare wellindividualized microspheres, step 3 must be attained by carefully controlling the addition of the phase inducer to avoid massive aggregation.
This sequenceof events can be conveniently schematized by a phase diagram. On such a diagram, each point corresponds to a defined weight percentage of methylene chloride, PLAGA, and silicone oil. Fig. 2 shows four ternary diagrams resulting from different PLAGA batches having very close mean molecular weights (Mgpc ranging from 35,000 to 55,000) but differing in the percentage of oligomers present: 2.0, 3.1, 4.5, and 40.0% for polymers 1, 2, 3, and 4, respectively. These ternary diagrams express the influence of the proportions of the three compounds on the sequence of events describing the PLAGA desolvation. In thefollowing discussion, we have designated step 3 as the stability window. Significant differences in the areas of stability windows are shown in Fig. 2. PLAGA 1 leads tothe largest zone, corresponding to step 3, whereas PLAGA 3 and 4 exhibit narrower windows. Increasing amounts of
38
Benoit et a/.
Fig. 1 Sequence of events occumng ina 5% PLAGA solution following the addition of 350 CSsilicone oil.
silicone oil were required to be added to PLAGA solutions to induce the formation of stable coacervate droplets according to the sequence: polymers 4, 3,2 and 1, with polymer 1requiring the greatest quantity. These results concerning the influence of the physicochemical nature of PLAGA on the stability window indicate that a relationship exists between theamount of silicone oil added to induce the formation of stable coacervate droplets and the ratio of oligomers in polymer batches involved in the phase separation. The more monodisperse the copolymer, the more methylene chloride acts as a good solvent for the coating material. Consequently, increased quantities of silicone oil are needed to desolvate the copolymer. Thus, polymer 4, which contains the highest percentage of oligomers among all of the copolymers studied, does not readily dissolve in methylene chloride. In other words, it requires low amounts of silicone oil to be precipitated. Under such conditions, the formation of well-individualized microspheres is very difficult to control (i.e., small stability window).
Biodegradable Microspheres
39
SILICONE OIL
SILICONE
2c$Em
20
l
. c
METHYLENE CHLORIDE
MTEKLE~CHLORIDE
SILICONE
350 CS
METHYLENE CHLORIDE
Phase diagramsfor the coacervationof different PLAGA batches. step 2; step 3 (stability window); step 4.
step
From a formulation point of view, the width of the stability window can also be modified by changing the viscosity of the silicone oil. Fig. 3 illustrates the role of the viscosity of the silicone oil on the formation of stable coacervate droplets of polymer 1. For a low-viscosity-grade silicone oil (i.e., 20 CS), no stability window could be detected. By increasing the viscosity of the phase inducer up to a value of 12,500 CS,it was possible to enlarge the stability window. Above this limit, the silicone oil could not be handled for microencapsulation purposes. The evolution of the stability window versus the viscosity of the phase inducer is common for all the polymers studied. A phase inducer of 20 CS leads either to a medium of low viscosity that is unable to stabilize coacervate droplets or to its too rapid solvation by methylene chloride, which in turn induces uncontrolled precipitation of the wall material. These two phenomena may occur simultaneously, although in practice this is verydifficult to demonstrate. Silicone oils of higher viscosities produce the reverse effect and allow one to increase the area of the stability window.
40
Benoit et al.
METHYLENE CHLORIDE
METHYLENTCHL
M-NE
CHLORIDE
Fig. 3 Phase diagrams for the coacervation of pol mer 1. Influence of the phase inducer viscosity on the stability window. step 1; step 2; step 3 (stability window); step 4. It must be also noted that, as expected, the average molecular weight of the coating material also determines the existence, width, and displacement of the stability window within phase diagrams[8]. Fig. 4 summarizes the two main characteristics of a PLAGA batch which may influence the stability window and therefore the formulation of microspheres. Whenthe weight averagemolecular weight decreases, solvation in methylene chloride is good, and the amount of silicone oil necessary to coacervate the polymer is high. A displacement to the left of the stability window is noted. Conversely, when the copolymer batchcontains low molecular weight compounds, i.e., oligomers, the overall hydroof more numerphobicity of the coating material decreases and the presence ous carboxyl and hydroxyl groups necessitates the use of smaller quantities of silicone oil. This induces a displacement to theright of the stability window often accompanied by a decrease in its area. The PHCA composition also exerts a strong effect on the stability window [g]. d,l-PLA appears to be less prone to desolvation induced by silicone oil addition than PLAGA copolymers of comparable molecular weights. The stability window which appears during the coacervation of d,l-PLA is therefore broadened compared with those of the copolymers.
41
METHYLEECHLORIDE
eflicient polymer dissolution
" c -
bad polymer dissolution
WHEN
kw DECREASES WHEN
"-
HYDROPHOBICITY DECREASES
molecular weight compounds)
( /low
m : STABILITY
WINDOW
Fig. 4 Influence of polymer Mw and hydrophobicityon displacement of stability window. As a practical consequence,the isolation of well-individualizedmicrospheres is facilitated when d,l-PLA is used. The authors attribute this phenomenon to a nonrandom distribution of glycolic acid in the copolymer chains, since the solubility parameters of d,l-PLA and PLA25GA50 units are quite similar: 16.4 and 16.8 (J m-3)112, respectively. Glycolic acid tend to form microcrystalline domains which are more sensitive to desolvation and precipitation. Numerous microparticular systems resulting from thecoacervation of a PHCA previously dissolved inhalogenated solvents have been described in the literature. A summary of the main operating conditions is given in Table 1. One must bear in mind that the investigations cited in Table 1 speak of "microcapsules," whereas in reality,matrix-type systems are more likely to be obtained. DCcapeptyl LP, 3.75 mg, a speciality polymer-drug preparation commercialized in Europe byIpsenA3iotech (France) and by Ferring (Germany), is prepared following this fabrication process. It consists ofmicrospheres of PLAGA 25 : 50 (25% l-lactic unit, 25% d-lactic unit, and 50% glycolic unit) containing an analogue of luteinizing hormone-releasing hormone (LHRH), triptoreline, used in the treatment of prostate cancer and endometriosis [19]. Formulation parameters have been adjusted to allow a constant release of triptoreline in vivo over 1month. This kinetic profile was extremely delicate to obtain. It is known, for instance, that a compromise
Benoit et al.
8
e 4 3
%
8 I
0
A A e4
m
r=! 0
R 8
Biodegradable
43
must be made between the intensity of the initial peptide loss and the time interval over which the sustained release occurs [20]. Again, the residual oligomer ratioin a PLAGA batch is one of the variables which can drastically change the release profile of the peptide. Lanreotide, which is a somatostatine analogue, has been approved and is in use in France for the treatment of acromegaly [21]. This drug is delivered from the same type of microsphere prepared according to the aforementioned technology. In conclusion, compared withspray-drying or solvent-evaporation methods, the polymer phase-separationtechnique may protect active principles from beingaltered by exposure to heat or from their partitioning out into dispersing phases. Residual solvent concentrations in resulting microspheres, however, can be very high, especially when heptane is chosen as the hardening agent [15,22].
111.
SOLVENTEVAPORATIONANDSOLVENTEXTRACTION METHODS
The solvent evaporation technique was fully developed at the end of the 1970s [23]. Since then numerousstudies have been carried out on the basis of this method.
A. General Description of the Process
This technique is based on the evaporation of the internal phase of an emulsion by agitation. Initially, the polymeric supporting material is dissolved in a volatile organic solvent. The active medicinal principle to be encapsulated is then dispersed or dissolved inthe organic solution to form a suspension, an emulsion, or a solution. In the following step, the organic phase is emulsified under agitation in a dispersing phase consisting of a nonsolvent of the polymer, which is immiscible with the organic solvent, which contains an appropriate tensioactive additive. Once the emulsionis stabilized, agitation is maintained and the solvent evaporates after diffusing through the continuous phase. The result is the creation of solid microspheres. On the completion of the solvent evaporation process, the microspheres held in suspension in the continuous phaseare recovered by filtration or centrifugation and are washed and dried (Fig. 5) [24]. Although the concept of the solvent evaporation technique is relatively simple, the physicochemical phenomena governing this process are very complex. This system is characterized by the existence of several interfaces through which mass transfer occurs during particle formation (Fig. 6) [25]. The formation of solid microspheres is brought about by the
44
Benoit et al.
Organic solution o f polymer with active principle dissolvcd o r dispcrsed with tensioactive agent
pq
0 0
Dispersing medium
4)
Formation of emulsion under mechanical stirring
Evaporation of solvent
Formation of solid microspheres
Fig. 5 The principle of the preparation of microspheres following the solvent evaporation technique.
evaporation of the volatile solvent L1 at interface L2/G. During the course of solvent evaporation, a partitioning is produced across the interface L1/ L2 from the dispersed phase to thecontinuous phase leading to theformation of solid microspheres. The partitioning across the interface L l L 2 is, however, not limited to the organic solvent; the active principle may also partition to some extent at this interface. The rapidity and importance of
45
Fig. 6 Schematic representationof the different interfaces in the solvent evaporation process: (1) Simple emulsion; (2) multiple emulsion.
Fig. 7 Differentmodes of incorporation of active principles in an organic solution of polymers. (a) Active principle dissolved. (b) Active principle dispersed. (c)
Active principle emulsified. these different transfer processes have a direct effect on microparticle formation. Thus, by studying the various governing parameters, the establishment of optimal conditions for theformulation of individual polymer/active principle couples can be achieved. Several systems may be envisaged based on the nature of the external phase (aqueous or nonaqueous), the incorporation mode of the active principle in the organic solution of the polymer (dissolved, dispersed, or emulsified) and the elimination procedure of the organic solvent (evaporation or extraction). (Fig. 7). The classification proposed by Aftabrouchad and Doelker [26] is in part adopted in the following discussion.
'
B. SolventEvaporation(Emulsification-Evaporation)
1. Oil-in-WaterEmulsion Techniques calling on water as nonsolvent to thepolymer are in general preferred.In fact, these processes are extremely economical and negate
46
Benoit et al.
the recycling of the external phase, which inturn results in the formation of well-individualized microparticles [27-291. In this system, the polymer is dissolved in an organic solvent such as methylene chloride or chloroform. The active principle is dissolvedor dispersed in the same medium, and then the entire mixture is emulsified in an aqueoussolution containing an appropriate surfactant. Beck et al. were thefirst to propose this procedure for the encapsulation of progesterone in PLA microparticles [23]. This technique, particularly suitable for theencapsulation of lipophilic active principles, has since been widely used for the encapsulation of different classes of therapeutic agents such as steroidal hormones [30-351, cytostatics [36-401, antiinflammatories [41-461, and neuroleptics [47,48]. Nevertheless, the microencapsulation of hydrophilic active principles by this process can pose problems. In actual fact, a partitioning phenomenon operates betweenthe dispersed and the dispersing phases which contributes to a substantial lowering of microencapsulation yields. The physicochemical characteristics of the active principle, such as its partition coefficient, its degree of ionization, or its surfactant character will play animportant role in its localization in one or other of thetwo phases of the starting emulsion. Strategies permitting a reduction in the loss of water-soluble active principles have been proposed to increase microencapsulation yields. The solvation of medicinal substances in organic solutions of polymers can be achieved by the addition of hydrophilic cosolvents [49,50]. The advantage of solubilizing the active principle is related to the flexibility of producing particles of extremely small sizes(i.e., bordering onthe micrometer scale) whose internal structures are more homogeneous irrespective of the initial drug particle size. The water solubility of the active principle can be reduced by chemically modifying it prior its incorporation in the organic phase. This modification is simple to perform for water-soluble salts, as they are commonly lipophilic in their nonionized forms. Many medicaments have been encapsulated in this manner [51,52]. The chemical synthesis of lipophilic 5-fluoro-2-deoxyuridine[53] or prodrugs from water-soluble species such as cephradine [54] has likewise permitted higher microencapsulationyields. In this instance, however, structural modifications of the medicine may give rise to toxicological problems. One may also modifythe dispersing phase of the emulsion to reduce leakage of the active principle from the oily droplets. Several investigators have therefore suggested saturating the continuous phase with the medicine [37,38,55-571, adjusting the pHof this same phase, or even addingelectrolytes [40]. It must be noted however, that thevalidity and effectiveness of these strategies vary from case to case. For example,chemical transformations only concern a limited number of active substances, and the saturation of external phases is onlyof value for substances with lowwater solubilities
Biodegradable
47
or inexpensive compounds. All the same, if the adjustment of the pH can prove advantageous for ionizable medicines, this procedure generally accelerates thedegradation of polymers suchas poly(a-hydroxycarboxylic acid) by the hydrolysis of the ester bonds [55,58].
2. MultipleEmulsions:Water-in-Oil-in-Water Another approach has been proposed for the efficient encapsulation of water-soluble active principles by the solvent evaporation technique in aqueous continuous phases. In these systems, active principles to beencapsulated are incorporated in an aqueous solution, which is poured into a casting organic solution of the polymer to form an emulsion of the type water-in-oil (W/O). This primary emulsionisitselfemulsifiedin an external aqueous phase leading to a multiple emulsion of the type water-in-oilin-water (WlOnV). The organic phase acts as a barrier between the two aqueous compartments preventing the diffusion of the medicine toward the external aqueous phase (Fig. 8 ) [29]. This technique was first proposed by Ogawa et al. [59-611 for the encapsulation of a peptide analogue of LHRH. This process proves much more effective when the watersolubility of the medicine is high(>900 mgl mL) [60] and partitioning between the organic phase is disfavorable. The same investigators have demonstrated thecrucial role of the viscosity of the primary emulsion (W/O) in the prevention of the diffusion of the active principle toward theexternal aqueous phase[59]. To this effect, they incorporated an agent (gelatine) capable of holding the active principle in the aqueous internal phase, whereas increasing this phase's viscosity. The effectiveness of such a formulation may nevertheless be compromised by a clash of compatibility between theviscosity-enhancing agent (e.g., gelatine, pectine, agarose) and the active principle which limits its field of application. This process is particularly suitable for the encapsulation of active principles inweak doses, which are strongly water soluble (e.g., hormones, trophic factors) [62-651, and antigens [66-731. Using this technique, the Takeda Laboratories (Japan) havemarketedbiodegradable microspheres of PLA37.5GA.25 (37.5% 1-lactic unit, 37.5% d-lactic unit, and 25% glycolic unit) containing an analogue of LHRH, leuprolide acetate. In France, this speciality is marketed under the name of Enantone LPand is prescribed in thetreatment of prostate cancer along with DCcapeptylLP. In an identical manner, the release of the peptide is effected over a period of 1month [74]. 3. Nonaqueous Emulsions This technique, also known under the name of oil-in-oil emulsion, is replaced by can berepresented by Fig. 5 if the continuous aqueous phase an oily phase. Naturally the dispersed phase (also of an oily nature) needs to betotally immiscible withthe continuousphase. The dispersing medium
'
48
et
Benoit
al.
Formation o f an crnuisic (water-in-oil type)
o f active principle
. ..
Aqueous solution
Organic solution of polymer
Formation of cmulsion undcr mcchanical stirring (watcr-in-oil-in-water type)
Evaporation o f solvent
Formation of solid microspheres
Fig. 8 Preparation principle of microspheres according to the multiple emulsion technique (water/oiYwater). can by constituted by a mineral or vegetable oil or a nonvolatile organic solvent [57,75-771. Although this process essentially permits one to avoid the loss of water-soluble active principles, it has only been used for the encapsulation of a very limited number of active principles, including cytostatics [49,78-801, anti-inflammatories [81,82], antimalarials [83], and
Biodegradable
49
serum albumin [84,85]. A very interesting innovation was proposed by Iwata and McGinity [86]: multiphase microspheres of PHCA containing water-soluble drugs and proteins have been prepared using the multiple emulsion-solvent evaporation technique. A multiple emulsion of the W/O/ 0/0type was utilizedto first provide a protective barrier between the drug and the polymer-solvent phase and secondly to prevent the highly watersoluble drugs from partitioning out of the microspheres. To illustrate this, a drug was dissolved in an internal aqueous phase containing gelatin and Tween 80. This solution was then emulsifiedin soybean oil containing appropriate tensioactive agents. This primary emulsion was in turn dispersed in acetonitrile in which a PHCA was previously dissolved and a W/ 0/0emulsion was obtained. This emulsion was finallyadded to a hardening solution consisting of Span 80 and light mineral oil. It must be stressed that the protective oil phase and the external dispersing phase need to be immiscible withthe organic solution containing the polymer. In certain processes, solvent evaporation may be replaced by sublimation by the implementation of lyophilization after the emulsification step. Thus, DeLuca et al. dissolved an active principle as well as poly(glyco1ic acid) in hexafluoroacetone sesquihydrate and emulsified the mixture in The resulting medium was immersed in dry ice/ carbon tetrachloride [87]. methanolandcooled to a temperature which froze the drug-polymersolvent phase and not the continuous phase. The suspension was then subjected to freeze drying, allowing the removal of the continuous phase solvent and solvent entrapped in embryonic microspheres. In addition to giving elevated microencapsulated yields for watersoluble components, this procedure canhelp prevent the eventual hydrolysis of the medicine or polymer. However, by comparison with aqueous emulsions, this technique exhibits a number of disadvantages related to the use of the nonaqueous solvents: these are often expensive and need to be recycled. Trace residues are oftendifficult to eliminate, which pose technological problems.
C. SolventExtraction (Emulsification-Extraction)
In the emulsification-evaporation method, the organic solvent of the dispersed phaseof the emulsion iseliminated in two stages:
1. Diffusion of the solvent in the dispersingphase (solvent extraction) 2. Elimination of the solvent at the dispersing phase-air interface (solvent evaporation).
In theory, if one uses a continuous phase whichwill immediately extract the solvent(s) of the dispersed phase, the evaporation stage is
50
Benoit er al.
no longer necessary in the formation of microspheres. In practice, this can be achieved by using large volumes of dispersing phase with respect to the dispersed phase [88,89] or by choosing a dispersed phase consisting of cosolvents, of which at least one has a great affinity forthe dispersing phase. One may even formulate a dispersing phase with two solvents in which one acts as a solvent extractor of the dispersed phase
[68,90].
A hybrid technology may also be envisaged where emulsificationevaporation is initialized then interrupted before the volatile solvent is totally eliminated. At this point, the native microparticles are transferred into a large volume of the continuous phase(consisting mostoften of highpurity water) where remainingsolvent is eliminated by extraction. This method was advocated by Benita et al. [31] for the prevention of the development of active principle crystals at the microparticle surface during the solvent evaporation stage or in the continuous aqueous phase, leading to decreased efficiencies in the microencapsulation of medicines. In fact, it has been suggested that the transfer of embryonic microparticles in a large volume of water leads to rapid extraction of organic solvent which is found at the periphery of dispersed globules which still contain some solvent. This leads to the formation of a polymeric barrier at the organic solvent-continuous phase interface, which slows downthe diffusion of the active principle from theorganic phase towardthe aqueous external phase and prevents crystal formation of the active principle [32,91]. An interesting modification of this technology was proposed by Leelarasamee et al. [92]. If the solvent extraction method is retained, the formation of dispersed globules is assured by the injection of a polymeric support solution containing the active principle in a solvent-extracting medium. The entire mixture is placed in a tubular system with reservoirs designed to permit the total extraction of the solvent from the polymer. This system replaces the mechanical stirring blade which assures emulsification in the preceding step. The concept behind this system is to enable the eventual production of microspheres ona continual basis.
IV. SPRAY-DRYING METHODS

Nebulization, or spray drying, is widely used in the chemical, pharmaceutical, and foodindustries [93]. The principle of spray drying bynebulization rests on theatomization of a solution (containing the product to be dried) by compressed air or nitrogen through a desiccating chamber and drying across a current of warm air (Fig. 9).
I
8
I
Fig. 9 Laboratory spray dryer: (1) Drying air inlet + filtration; (2) heating; (3) desiccation chamber; (4) cyclone; ( 5 ) collector for drying powder-microspheres; (6) filtration + air outlet.(A) solution, suspension, emulsionto spray. (B) compressed or nitrogen air. (C) Spray nozzle (e.g., pneumatic, ultrasonic) Four separate phases may be distinguished:
1. Nebulization of the solution in the form of an aerosol 2. Contact of the nebulized solution with the warm air 3. Drying of the aerosol 4. Separation of thedriedproductandtheaircharged solvent
with the
This technique can be used to protect sensitive substances against oxidation (e.g., fish oils, essential oils, vitamins, colorants) [94]. When most often emvolatile substancesare encapsulated, the coating materials ployed are maltodextrine and arabic gum.
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Benoit et al.
A more recent application of this mode of drying has been adapted for the creation of matricial systems of the microspherical type starting from complex liquid mixtures comprising an active principle dissolved (or dispersed) with a polymer in an organic solvent. This type of process permits the isolation of microspheresor microcapsules depending on the initial formulation, albeit in the form of a solution, a suspension, or anemulsion (Fig. 10).
A. TheNebulizingSystem
WO principal nebulizing systems exist[95]: 1. lbrbine method: nebulization by a rotating disk or rotary atomization 2. Atomizer method: nebulization by compressed air or pneumatic nozzle atomization
In the atomizer method, the internal diameter of the atomizer is often between 0.5 and 1.0 pm. Compressed air (or nitrogen) is then mixed directly in the central core along with the solution; nebulization thus being provoked simultaneously by the pressure applied to the interior of the liquid vein and by its ejection velocity from the nozzle. Compressed air subsequently arrives at the exit of the atomizer along with the liquid, inducing the shearing of the liquid vein. This system is convenient for the isolation of extremely fine particles ( 4 0 pm) from nonviscous solutions. In the turbine method, the pressure of the compressedair turns a disc with calibrated orifices at high speeds (25,000-35,000 rpm), provoking the
EMULSION SUSPENSION SOLUTION Liquid drug Polymer Solvent Solid drug Polymer Solvent Liquid drug Polymer Solvent
MICROSPHERE MICROCAPSULE
of the active principle-polymer mixture.
Fig. 10 '&pes of microparticlesobtained by spray drying accordingto the nature
Biodegradable
53
shearing of the solution from the moment of its passage through these orifices. The turbine methodis thus complementary tothe first technique, being adaptedfor very viscous solutions, and for the formation of particle sizes in the range of > l 0 pm. The turbine equipped with its nebulizers must havea desiccation chamber of >l m in diameter.
B. Apparatus
A vast array of spray-drying equipment exists ranging from laboratory nebulizers (Minispray Dryer 190; Biichi,Switzerland) to pilot scale apparatus (model Minor Mobile; Niro Atomizer, Denmark) the first to productionscale equipment (Minor Productions; Niro Atomizer) and ultimately to drying towers of several meters in height. This process is thus industrializable, with users having at their disposal a range of accessories in function with the quantities of products to be fabricated. Changes in particle sizes can thus be effected in a progressive manner. Various factors, such as thermal exchanges and losses, variable yields, and the geometries of atomizers or turbines make it difficult to transform the laboratory-scale equipment to theproduction scale. Most reports in the literature are based on results obtained from laboratory-scale equipment (Minispray Dryer190; Biichi). Being of reduced sizes, these models have limited evaporation capacities. Flow rates are typically of the orderof 15-20 mL min". The entrance temperature of the air (40-200C) allows the user the choice of working with aqueous or organic solvents of low boilingpoints. Only a few industrial companies operate using equipment of greater dimensions [96,97], but notably none employs the atomization tower for the drying of powders.
C. Applications
The formation of biodegradable microspheresby this technology, permitting the liberation of medicines over periods of months or longer from one unique injection, is a relatively recent development [96-1001. Polymers and copolymers of lactic and glycolic acids are used in the formation of some of these systems. To date, only bromocriptine (Parlodel LAR; Sandoz, Switzerland) is commercialized in the form of injectable microspheres, which originates from the spray drying method. This formulation is composed of PLAGA branched to D-glucose [96]. Other patents (busereline [loll, mifepristone [102]) make use of this process for the encapsulation of hydrophilic or lipophilic molecules, allowing their liberation over a period of 1 month after one injection only. The principal applicationsoriginating from this technology are detailed in Table 2.
54 Benoit et al.
m
m
I
SE:
..
E :
Y
..
8
c
41
Biodegradable Microspheres D. AdvantagesandInconveniences
55
The interest in this technique lies, among other things, on the following factors: It can be likened to a continuous process, whereas, in the pharmaceutical industry, the preparationof batches is encountered. Certain models are able to discharge their material contentsin a dry, continual manner (during which thermal treatment processes for the drying of powders must be steadily maintained over the whole process and closely monitored). The residence times of products to be dried at elevated temperatures (>lOO"C) from aqueous solvents are very short. In the case of organic solvents, evaporation temperatures can be even lower. For example, aworking temperature of 30-40C is sufficient to evaporate methylene chloride. These parameters are important when considering the drying of thermally sensitive products. The majority of authors evaporate polymer-methylene chloride solutions at temperatures ranging between 50 and 70C [90,98,105,108]. Particle size distributions areusually monodispersed with a Gaussian distribution more or less depending on the pulverizing head employed (e.g., pneumatic atomizer, disc) and the chosen nebulization parameters such as, for example, the pressure of compressed air, the internal diameter be nebulized. of the atomizer, the viscosity, and flow rate of the solution to In the literature,particle sizes are commonly described as being less than 10 pm, since the geometries of atomizers and the viscosities of nebulized solutions are almost uniform. In published articles for certain polymers, for example, dl-PLA (209,000 MW) or PLAGA (22,000 M W ) [105], the obtention of microspheres is impossible when the Kquid vein cannot be sheared into fine droplets. In these cases, the final result is the formation of filaments. This phenomenonalso arises when the concentration of the polymer is increased in the organic phase, hence increasing the viscosity of the solution [98]. It also appears that the production of these filaments is more readily brought about employing polymers composed of linear chains [98]. The formation of these filaments at this point is also strongly influenced by the performance of the apparatus. The influence of the pressure of the nebulizing air, the geometry of the atomizer (the diameter of its orifice), and the flow rate of the nebulizing solution are important for filament formation. Nebulization of fine particles permits, under certain conditions, to keep active principles at the heart of polymeric matrices in amorphous forms.
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Benoit et al.
On the other hand, Bodmeier and Chen [98]have observeda change in the crystalline form of progesterone nebulized on its own or in the presence of d,l-PLA. These state changes imposekinetic modifications of dissolutions, which may be exploited in pharmacokinetics. Spray drying thus permits the incorporation of lipophilic molecules in these types of copolymersin concentrations between 30 [l051 and 50% [98], withouttheappearance of crystals at particle surfaces. On theotherhand, for concentrations greaterthan 50%, or for hydrophilic molecules such as theophylline [98], the burst effect is more marked in the in vitro release kinetics. This can be explained for the former case by surface crystallizations and in the latter by weak interactions with hydrophobic polymers, which are not sufficient to prevent crystallizations. Residual levels of organic solvents are less than those from the emulsification and evaporation techniques. Levels of 0.1-0.2% can be found quoted in the literature [97]. Certain investigators have managed to arrive at concentrations of 0.05% and below [106]. For a pharmaceutical application, the concentration of organic solvent, by specified unit mass, must be inferior to thelevel set by the country of commercialization. For example, the standard set by the European Pharmacopaefor methylene chloride is less than 5OOppm (0.05%). Inferior residual solvent levels can be obtained by lyophilization of microspheres, and residual humidity levels are typically very low (<l%), starting from solutions of organic solvents. Provided the use of sterile starting materials, this type of process can beadapted for sterile productions. Certain companies suggestspecific equipment that operates at positive pressures in combination with a collecting zone forthe product in a sterile atmosphere. Others radiosterilize their final products, which can induce molecular weight lossesby the scissions of polymeric chains, resulting in modifications of release kinetics. In conclusion, the majority of studies reported in the literatureoriginate from experiments performedlaboratory-scale on equipment. This merits several remarks: The spray nozzle has more or less the same dimensions, producing specific particle sizes lessthan 10 pm. There are little or no complete studies to determine whether a relationship exists between the varying of operating conditions (e.g., flow rates, pressures and temperatures of the compressed air, flow rates of solutions to be nebulized) and the sizes and structural arrangements of particles. These modifications can lead to variations in the release kinetics.
Biodegradable
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In our experience, no kinetic variations could be observed from variations of these above parameters. The sole influence on release characteristics arose frommodifications of starting formulations: Variations in molecular weights of polymers or thetypes of polymers (L-PLA, d,l-PLA, and PLAGA) Variations of the viscosities of mixtures Variations of active principle/polymer ratios It would be of great value to have some knowledge of these variables (sizes, structural arrangements) for the transition from laboratory to industrial scales. These changes in scales entail modifications on the geometries of atomizers and their evaporation capacities and the flow rates of nebulizer solutions. From actual knowledge, the development of spray drying for the formation of microspheres based onPLA and PLAGA is interesting for the fabrication of sizable quantities of microspheres (<l00 g). This is borne out by results obtained with laboratory equipment; in other words, the percentage of dry product compared with the amount of organic solvent must be often <2% in order to obtain microparticles. This remains to beverified with equipment and atomizers of greater sizes. Quantities of organic solvents employed on industrial scales is very expensive (prices being quoted for high-purity solvents, recovery costs, and solvent destructions). It must be stressed that the majority of industrial atomization towers are not equipped to handle chlorinated solvents. Finally, this technology addresses the actual state of development of polymeric preparations of very active medicines at extremely low concentrations, which are rapidly becoming much sought after vehicles of delivery.
V.
METHODS USING FLUIDS UNDER SUPERCRITICAL CONDITIONS
The technique of placing a fluid at a temperature and pressure exceeding its critical point and using it asan extractant is now welldocumented [109]. Increasing applications of this technique are being discovered, with particularly promising ones being found in the field of material transformations (changing physical properties of material). Thus, in the pharmaceutical sciences, where modifications of the physical properties of drugs are necessary, several benefits can also be achieved by taking advantageof supercritical conditions. As described earlier in this chapter, drug-loaded microspheres are often formedby dissolving at least one of their two major components. In practice, one of three procedures can be followed:
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Benoit et al.
The drug can bedissolved and combinedwith the coating substance. Both the drugand coating substances are dissolved. Only the coating substances are dissolved. Thus, we have examined successively how fluids under supercritical conditions are able to dissolve the drug or the container material itself. In a second step, we have studied how the drug (dissolved or not) can be embedded in the microsphere matrix.
A. Solvation of Compounds
An important step in all the techniques presented hereafter is the solubilization of the active principles. Compared with the classic use of solvents, supercritical fluids offer a larger scope of choice in terms of solvents performances (Table 3). Also, the possibility of altering their abilities to solvate solute molecules existsby varying pressure and temperatureconditions. These two parameters are key features in supercritical fluid techniques. In the vicinity of the critical point especially, minute changes in temperature or pressure generate substantial density variations. Solvating properties can be related to thedensities of supercritical phases. Thus, the ability of supercritical fluids to penetrate a substance increases with their densities.
Table 3 Critical Pressure for Selected Fluids with Critical Temperature Below 150C
Fluids Nitrogen Carbon monoxide Argon Oxygen Methane Krypton Ethylene Xenon Carbon dioxide Ethane Hexafluoro-sulfur Propylene Propane Ammonia Isobutane
T (c)
P (bars)
34 34 48 50 46 5 5 50 58 73 48 37 45 42 1 1 1 36
-147 -140 -123
-119 -83 -64 9 16 31 32 45 92 96 132 134
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The literatureprovides basic data concerning the solvating properties of an array of pure solvents under their supercritical conditions [109-1121. In most cases, however, the precise conditions described in the literature are not encountered, as multicomponent mixtures are more commonplace in the laboratory. Added to this, for several good reasons (e.g., like the sensibility of products to high temperatures or forreasons of toxicity), the scientist chooses the wrong solvent. An example of this situation is encountered in the use of carbon dioxide, a good choice for food applications but chemically difficultfor the solvation of polar substances. Under supercritical conditions, however, carbon dioxide exhibits more or less the same solubility as heptane. Thechemist, on the other hand, may avoidthis poorsolubility problem by adding cosolvents to the mixture (usually polar substances). Needless to say, this chemical mixture no longer acts according to the literaturewhen exposed to supercritical conditions. Its behavioris nevertheless independent on each pure component constituting the mixture. The operatorshould bear in mind that supercritical conditions can often be lost in complex mixtures and that two-phase systems may develop instead. Thus, performing practical tests using small-scalelaboratory installations is the only reliable way to observe these phenomena and to obtain the data needed forlarge-scale project development. Another method to obtain relevant information, but somewhat exhaustive, is to search for suitable data for each particular project in the large amountof literature dealing with extraction processes exploiting supercritical conditions [113,114]. Since carbon dioxide is often employed, the solubility data provided by IUPAC is extremely useful [115]. In general, low molecular weight hydrocarbons and lipophilic substancesare good candidates tobe dissolved with supercritical carbon dioxide. Now that the desired starting material is in solution, several treatments can be applied, bearing in mind that the final step will be to recover the dissolved compound possessing the now new and interesting properties so that it can be called the product.
B. Precipitation of Pure Compounds
How can the dissolved compound be recovered? Supposing that a substance A is placed in a classic solvent, S1, and that a solution of A is obtained. At the molecular level, this implies that molecules of A are far away from one another and are surrounded only by solvent molecules S1. Supposing we have a gas, G1, under supercritical conditions which exhibits a lower solubility for compound A than does S1, but which is miscible and soluble in S1. Then when the solution of A in S1 is placed in the presenceof G1, the supercritical conditions will probably disappear and a two-phase
60
Benoit et al.
system will be generated. The lighter phase, predominantly composed of G1, contains a small proportion of S1. The heavier phase, comprising mainly by A and S1, also contains some amount of G1. The presence of G1 in S1 decreases the solvating properties of the mixture, so A precipitates out of the liquid medium in its amorphous state. By precipitation, we mean the formation of solid entities which can be seen by the human eye, having granulometric sizes in the micrometric domain. Recovering A, after supercritical processing, gives the product AGlS1, which does notnecessarily exhibit the same crystallinity, morphology, porosity, surface properties, or solubility properties as the product AS1 generated by a classic chemical methods [116-1211. The principle is helpful in explaining the processes discussed in Section V.D. C. Crystallization of Pure Compounds
A variation of the aforementioned precipitation method is when conditions are adjusted to induce crystallization rather than precipitation. A solution of compound B in a concentration close to its saturation in a supercritical fluid G2 (considered as the solvent of B) can be transported to an areaof the system where the temperature is lower. In doing this, the system becomes saturated in B and crystallization occurs. Highly pure monocrystals can be obtained by this process. Later on, placed back in normal atmospheric pressure and temperature, it becomes very difficultto detect traces of the solvent G2 in the sample [122]. It should be pointed out that the retrograde solubility phenomenon (decreasing solubility with increasing temperature) can be observed [123,124]. Here also, before considering the formation of microparticles, the scientist should remember that the crystallographic arrangements of compounds may be modified by the nature of the crystallizing solvent. Thus, a compound embedded in a microsphere may very well exhibit a crystallographic lattice which differs from the starting bulk material. This is not necessarily due to the close proximity of the host microsphere matrix molecules, but it can be due to the nature of the solvent used during the process.
D. Formation of Microparticles
If an excipient is added to a solution of the active principle, the basic precipitation operation as described above can be applied. Moreover, if the starting solution is fashioned before precipitation occurs, a final product can be obtained in one step. For example, if an organic solution of an active principle is sprayed through a nozzle into a column containing supercritical fluid, the organic solvent is extracted by the fluid from each finely divided droplet of the solution [125,126]. Under supercritical condi-
61
tions, the solute precipitates immediately to form a microparticle powder. An interesting example is the one where biodegradable polyesters are dissolved (PLA, PLAGA). Depending on the degree of crystallinity and on the molecular weights of polymers employed, different types of microparticle formations have been observed[127]. The case where the drug N-butylscopolammonium bromideis added to poly-L-lactide can be cited [128]. Another interesting example is the formation of biodegradable microparticles of enzymes such as catalase and lysozyme and proteins such as insulin from dimethylsulfoxide (DMSO)-water or dimethylformamide (DMF)-water solutions [129,130]. The use of peptidelike materials raises the question of the solubility of DMSO, DMF, and waterin the supercritical fluid. Can solute precipitation be related to polarity changes occurring in the mixtureso that it can be described as a "gas antisolvent precipitation," or is the organic solvent extracted first by the supercritical fluid, so that theextraction is playingthe major role? Related to this question is the interesting study reported by Lokensgard [131]. Following this protocol, methylene chloride can be extracted from PLAGAmicrocapsules downto undetectable levels by supercritical carbon dioxide, whereas residual heptane is only poorly removed. Residual heptane can nevertheless be removed by the use of propane instead of carbon dioxide. In spite of these advantages, the destruction of microspheres has been reported in some instances while operating under supercritical conditions. In actual fact, the extraction properties of supercritical fluids can be tailored to extract residual solvents in pharmaceutical compounds. The purification of glycyrrhizin is an example [132]. A 20,000-ppm sample in ethanol treated with supercritical carbon dioxide at 40C and under350 kg cm-' can be reduced to 455 ppm. In some cases, this operation leads to the formation of porous spongelike matters [133]. Atomization techniques (rapid expansions from supercritical solutions) can be applied to a solution under supercritical conditions. In this process, material is dissolved inthe supercritical fluid and expanded into a lower pressure environment. This results in the nucleation of the solute.By nucleation, we mean that solute molecules gather together to form nanometric entities. Following this event, heat energy needs to be given to the system, since the supercritical phase cools as it decompresses. Akin to standard atomization techniques, where the solvent undergoes a phase change from a liquid to a gas, energy needs to be supplied. This event occurs, however, at much lower temperature ranges during a typical supercritical process; e.g., k200"C in an atomization tower for water compared with 5 5 C for carbon dioxide [134]. A review of this particular field has been published [121,135]. Addition of a polymeric substance in the pre-
62
Benoit et a/.
expansion solution can beof particular interest in order to obtain matrices for controlled drug-delivery devices [136-1411. VI. MILLING METHODS
International health authorities and scientific bodies have recently focused their attention toward the investigation of toxic residual solvents in active ingredients and pharmaceutical formulations, and they have imposed maximum limits on particular solvent [142]. Confronted with these new directives, microparticle manufacturers are trying to concentrate their efforts on solvent-free technologies. One of them is based on the milling of PHCA particles. This technique is preceded by the fusion of the polymer powder and can be completed by a spheronization step. Thegrinding technique by itselfwasfirst introduced byBoswell and Scribner in 1973 [lo]. They proposed melting a PHCA and thoroughly dispersing a non-heat labile drug in the melt. The melt was then congealed in a mass and ground into small particles 1-200 pm in size. Similarly, they advocated spray congealing as an alternative technique for the processing of microparticles. In actual fact, milling of a glassy solid in order to produce microparticulate implants can be performed: After cooling of molten PHCAin which appropriate amounts of drug are dispersed [143,1441 After compression of PHCA particles [1451 After extrusion of PHCA [146,147] Cooling, compression, or extrusion followed by milling culminates in the formation of irregular external particle surfaces. Hence, microparticles formed thereafter arenot truly spheroidal and display broad size distributions. This was the reason why Wichert and Rohdewald proposed complementary steps enabling the spheronization of particles [144]. Particles resulting from the milling of the cold melt (a mixture of d,l-PLA and the neurotropic agentvinpocetine) were added to a buffered 0.05% Ween 80 solution heated to 180C. After emulsification, the microparticles were isolated either by pouring them onto ice water or by spray drying. Although the microparticles were not as smooth as those prepared by the solvent evaporation process, they looked rather spherical. Ruiz has used a similar concept to formulate LHRH- and somatostatin analogue-loadedmicroparticles [146]. The microparticles, called microballs by Ruiz, were obtained according to the following steps. In the first stage, the cryogenic grinding of the extruded polymer, containing a homogeneously dispersed peptide, led to the formation of heterogeneous and porous particles. In a second stage, after sieving, the microparticles
Biodegradable
63
were suspended in a gel under energetic stirring. The gel was heated and the microparticles fused, becoming spherical. When spheronization was achieved, the suspension was rapidly cooled and the gel was dissociated by the addition of an appropriatewashing agent. It should be noted that several investigators describe a reduction in the molecular weight of PHCA during heattreatment [144]. This point must be taken into account when designing biodegradable microparticles.
ACKNOWLEDGMENTS
The authorswish to thankDr. Michael Lynch for his assistance in preparing this chapter.
REFERENCES
1. P. Deasy, Microencapsulation and Related Drug Processes, Marcel Dekker, New York, 1984. 2. J. A. Bakan, Microencapsulation via coacervation-phase separation, National W, June 1966. Industrial Research Conference, Land OLakes, 3. J. E. Vandergaer, Microencapsulation, Processes and Applications, Plenum Press, New York, 1974. 1976. 4. J. R. Nixon, Microencapsulation, Marcel Dekker, New York, 5. A. Kondo, Microcapsule Processing and Technology, Marcel Dekker, New York, 1979. 6. M. Donbrow, Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton,FL, 1992. 7. J. M. Ruiz, B. Tissier, and J. P. Benoit, Microencapsulation of peptides: A
study ofthe phase separation of poly (D,Llactic acid-co-glycolic acid) copolymers 50/50 by silicone oil, Int.J. Pharm. 49:69-77 (1989). J. P. Benoit,Influenceofaveragemolecular 8. J.M.Ruiz,J.P.Busnel,and weights of poly (D,L-lactic acid-m-glycolic acid) copolymers 50/50 on phase separation and in vitro drug release from microspheres, Pharm. Res. 7(9):
9. 928-934 (1990).
3773,919, 1973. 1 1 . J. S. Kent, L. M. Sanders, D. H. Lewis, and T. R. Tice, Microencapsulation 0052 510,1986. of water soluble polypeptides, Eur. Patent 12. J. S. Kent, L. M. Sanders, D. H. Lewis, and T. R. Tice, Microencapsulation of water soluble polypeptides, U.S. Patent 207,864, 1980. ,
C. Thomasin, B. Gander, and H. P. MerMe, Coacervation of biodegradable polyesters for microencapsulation: A physicochemical approach, Proceedings of 20th Intern. Symp. Control. Rel. Bioact. Mater., Washington, DC, 1993, pp. 358-359. U.S. Patent 10. G. A. Boswell and R. M. Scribner, Polylactide-drug mixtures,
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13. J. S. Kent, L. M. Sanders, D. H. Lewis, andT. R. Tice, Microencapsulation of water soluble active polypeptides,U.S. Patent 4,675,189, 1987. 14. J. Komen and J. W . Groenendaal, Process for microencapsulation, Eur. Patent 0377 477,1990. 15. P. Orsolini, R. Y . Mauvernay,andR.Deghenghi,Prockdkpourlamicroencapsulation par skparation de phases de substances mkdicamenteuses hydrosolubles, Swiss Patent 665 558 A5, 1988. 16. B. Gander, C. Thomasin, H. P. Merkle, Y. Men, and G. Corradia, Pulsed tetanus toxoid release from PLGA microspheres and its relevance for imm genicityinmice,Proceedingsof20th Intern. Symp. Control. Rel. Bioact. Mater., Washington DC, 1993, pp. 65-66. 17. C. Thomasin, B. Gander, and P. H.Merkle, Physicochemical characterization of the coacervation process of poly (l-lactic acid) and poly (d,l-lactic acid) by silicon oil, Proceedings of 18th Intern. Symp. Control. Rel. Bioact. Mater., Amsterdam, 1991, pp. 646-647. 18. D. H. Lewis, T. 0. Dappert, W. E. Meyers, G. Pritchett, and W. J. Suling, Sustained release of antibiotics from biodegradable microcapsules, Proceedings of 7th Intern. Symp. Control. Rel. Bioact. Mater., Fort Lauderdale, FL, 1980, pp. 129-131. 19. H. Parmar, S. L. Lightman, L. Allen,R. H. Phillips, L. Edwards, and A.V. Schally, Randomised controlled study of orchidectomy vs long-acting DTrp6LHRHmicrocapsulesinadvancedprostaticcarcinoma,Lancet I1 (8466): 1201-1205 (1985). 20. J. M. Ruiz andJ. P. Benoit, in vivo peptide release from poly (DL-lactic acidco-glycolicacid)copolymer 50/50 microspheres, J. Contr.Rel.16:177-186 (1991). 21. I. Heron, F. Thomas, M. Dero, J. R. Poutrain, S. Henane, F, Catus, andJ .M. Kuhn, Traitement de lacromtgalie par une forme liberation prolongke du lanrkotide, un nouvel analogue de la somatostatine, Presse Med. 22 (11):526531 (1993). 22. B. W. Muller, J. Bleich, and B. Wagenaar, Microparticle production without organic solvent, Proceedings of 9th International Symposium on Microencapsulation, Ankara,1993, pp. 29-40. 23. L. R. Beck, D. R. Cowsar, D. H. Lewis, R. J. Cosgrove, C. T. Riddle, S. L. Lowry, andT. Epperly, A new long-acting injectable microcapsule system for the administration of progesterone, Fertil. Steril. 31(5):545-551 (1979). 24. P. J. Watts,M.C.Davies,and C. D. Melia,Microencapsulationusing emulsificatiodsolvent evaporation: an overview of techniques and applications, Crit. Rev. Ther. DrugCamer Syst. 7(3):235-259 (1990). 25. C. Thies, Formation of degradable drug-loaded microparticles by in-liquid drying processes, in Microcapsules and Nanoparticles in Medicine and Pharmacy (M. Donbrow ed.), CRC Press, Boca Raton, FL, 1992, pp. 47-71. 26. C. Aftabrouchad and E. Doelker, Mkthodes de prkparation des microparticules biodkgradables chargkes en principes actifs hydrosolubles, S.T.P. Pharma Sci. 2(5):365-380 (1992).
Biodegradable
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27. J. W. Fong, Microencapsulation by solvent evaporation and organic phase separationprocesses,inControlledReleaseSystems(D.S.T.Hsieh,ed.), CRC Press, Boca Raton,FL, 1988, pp. 81-108. 28. R. Alex and R. Bodmeier, Encapsulation of water-soluble drugs by a modified solvent evaporation method. I. Effect of process and formulation variables on drug entrapment, J. Microencaps. 7(3):347-355 (1990). 29. R.Bodmeier, H. Chen, P. Tyle,and P. Jarosz,PseudoephedrineHCImicrospheres formulated into an oral suspension dosage form, J. Control. Rel. 15~65-77 (1991). 30. L. R. Beck, V. Z . Pope, C. E. Flowers, D. R. Cowsar, T. R. Tice, D. H. Lewis, R. L. Dunn, A. B. Moore, and R. M.Gilley,Poly(D,L-lactide-cog1ycolide)lnorethisterone microcapsules: an injectable biodegradable contraceptive, Biol. Reprod. 28:186-195 (1983). 31. S. Benita, J.P. Benoit, F. Puisieux, and C. Thies, Characterization of drugloaded poly(D,L-lactide) microspheres, J. Pharm. Sci. 73(12):1721-1724 (1984). 32. D.R. Cowsar, T. R. T i e , R. M. Gilley, and J. P. English, Poly(1actide-coglycolide)microcapsulesforcontrolledreleaseofsteroids,inMethodsin (K. J. Widder and R. Green eds.), Enzymology: Drug and Enzyme Targeting Academic Press, Orlando, FL, 1985, pp. 101-116. 33. T. R. TiceandR. M. Gilley,Preparation of injectablecontrolled-release microcapsules by a solvent-evaporation process, J. Control. Rel. 2:343-352 (1985). P. Benoit, F. Courteille,andC.Thies,Aphysicochemicalstudyofthe 34. J. morphology of progesterone-loaded poly(D,L-lactide) microspheres, Int. J. Pharm. 29:95-102 (1986). 35. V. Rosilio, J. P. Benoit, M. Deyme, C. Thies, and G. Madelmont, A physicochemical study ofthe morphology of progesterone-loaded microspheres fabricated from poly(D,L-lactide-co-glycolide), J. Biomed Mat. Res. 25:667-682 (1991). 36. J.P. Benoit, Preparation et caracttrisation de microsphtres biodkgradables pour chimioembolisation, Ph.D. thesis, UniversitC de Paris Sud, September 30,1983. 37. G. Spenlehauer,M. Veillard and J. P. Benoit, Formation and characterization of cisplatin-loaded poly(d,l-lactide) microspheres for chemoembolization, J. Pharm. Sci. 75(8):750-755 (1986). 38. G. Spenlehauer,M. Vert, J. P. Benoit, F. Chabot, and M. Veillard, Biodegradable cisplatin microspheres prepared by the solvent evaporation method: morphology and release characteristics, J. Control. Rel. 7:217-229 (1988). P. Benoit,Percolationandrelease of 39. M.Deyme,G.SpenlehauerandJ. cisplatin-loaded in poly(1actide-co-glycolide)microspheres for chemoembolization, J. of Bioactive and Compatible Polymers 7:150-160 (1992). of 40. M. Mestiri, F. Puisieux and J. P. Benoit, Preparation and characterization cisplatin-loaded polymethyl methacrylate microspheres, Int. J. Pharm. 89:229234 (1993).
66
41.
Benoit et al.
M. Cavalier, J. P. Benoit, and C. Thies, The formation and characterization of hydrocortisone-loaded poly( +)-lactide microspheres, J. Pharm. Pharmacol.
38~249-253 (1986).
42.
43.
44.
45.
46.
47. 48.
49.
50.
5 1 .
L. A. Luzzi, J.F. Hogan, S. J. N. Leelarasamee,S. A. Howard, C. J. Malanga, Kandzari,andJ.K.H.Ma,Kinetics of drugreleasefrompolylactic-acid hydrocortisone microspheres, J. Microencaps. 3:171-179 (1986). J. H. Ratcliffe, I. M. Hunneyball, A. Smith, C. G. Wilson, and S. S. Davis, Preparation and evaluation of biodegradable polymeric systems for the intraarticular deliveryof drugs, J. Pharm. Pharmacol. 36;431-436 (1984). R. K. Chang, J. C. Price, and C. W . Whitworth, control of drug release by use ofmixturesofpolycaprolactoneandcellulose acetatebutyratepolymers. Drug Dev. Ind. Pharm. 13:1119-1135 (1987). R. Bodmeier and H. Chen, Preparation and characterization of microspheres containing the anti-inflammatory agents indomethacin, ibuprofen and ketoprofen, J. Control. Rel. 10:167-175 (1989). J. K.Lalla andK. Sapna, Biodegradable microspheres of poly(DG1actic acid) containing piroxicam as a model drug for controlled release via the parenteral route, J. Microencaps. 10(4):449-460 (1993). J. W. Fong,Processforpreparation ofmicrospheresandmodificationof release rate of core material, U.S. Patent4,479,911, 1984. J. W. Fong, J. P. Nazareno, J. E. Pearson, and H. V. Maulding, Evaluationof biodegradable microspheres prepared by a solvent evaporation process using sodium oleate as emulsifier, J. Control. Rel. 3:119-130 (1986). R.Wada, S. H.Hyon,and Y. Ikada,Lacticacidoligomermicrospheres containing hydrophilic drugs, J. Pharm. Sci. 79(10):919-924 (1990). G . E. Hardee, G . W. R.Davidson,H.Chen,andR.Bodmeier,Microencapsulation of antimicrobial agents for extended release after injection, Proceedings of 18th Int. Symp. Control. Rel. Bioact. Mater., Amsterdam, 1991, pp. 203-204. vitro N. Wakiyama,K. Juni, and M. Nakano, Preparation and evaluation inof polylactic acid microspheres containing local anesthetics, Chem. Pharm. Bull.
29~3363-3368 (1981).
W. Whitworth, Dissolution characteristics of 52. R. K. Chang, J. C. Price, and C.

polycaprolactone-polylactide microspheres of chlorpromazine,DrugDev.
Ind. Pharm. 12:2355-2380 (1986). 53. T. Seki, T. Kawaguchi, H. Endoh, K. Ishikawa, K. Juni, and
M. Nakano, Controlled release of 3,5-diester prodrugs of 5-fluoro-2-deoxyuridine from poly-L-lactic acid microspheres, J. Pharm.Sci. 79 (11):985-987 (1990). 54. C. Ustariz, Conception et Btude dun nouveau mtdicament anti-infectieux? I action prolongke adapt6 aux traitement des animaux de rente par la cBphradine, Ph.D. thesis, UniversitB de Montpellier I, France, June2 , 1993. 55. R.BodmeierandJ. W. McGinity,Polylacticacidmicrospherescontaining quinidinebaseandquinidinesulfatepreparedbythesolventevaporation technique. I. Methods and morphology, J. Microencaps.4:279-288 (1987). W. McGinity,Polylacticacidmicrospherescontaining 56. R.BodmeierandJ.
Biodegradable
67
57.
58.
59.
60.
61. 62. 63.
64.
65. 66.
67. 68. 69.
quinidinebaseandquinidinesulfatepreparedby the solvent evaporation technique. 11. Some process parameters influencing the preparation and properties of microspheres, J. Microencaps. 4:289-297 (1987). R. JalilandJ. R. Nixon,Microencapsulationusingpoly(L4acticacid). I. Microcapsulepropertiesaffectedbythepreparativetechnique, J. Microencaps. 6:473-484 (1989). R. Bodmeier and J. W . McGinity, The preparation and evaluation of drugcontaining poly(D,L-lactide) microspheres formed by the solvent evaporation method, Pharm. Res. 4:465-471 (1987). Y. Ogawa, M. Yamamoto, H. Okada, T. Yashiki, and T. Shimamoto. A new technique to efficiently entrap leuprolide acetate into microcapsules of polylacticacid or copoly(lactidglyco1ic)acid,Chem.Pharm.Bull.36(3):10951103 (1988). Y. Ogawa, M. Yamamoto, S. Takada, H. Okada, and T. Shimamoto, Controlled release of leuprolideacetatefrompolylacticacid or copoly(1actid glycolic)acidmicrocapsules:influenceofmolecularweightandcopolymer ratio of polymer, Chem. Pharm. Bull. 36:1502-1507 (1988). H. Okada, Y. Ogawa, andT. Yashiki, Prolonged release microcapsule and its production, U.S. Patent 4,652,441,1987. T. Heya, H. Okada, Y. Ogawa and H. Toguchi, Factors influencingthe profiles of TRH release fromcopoly(D,L-lactic/glycolicacid) microspheres, Int. J. Pharm. 72:199-205 (1991). T. Heya, H. Okada, Y.Tanigawara,Y.Ogawa,and H. Toguchi,Effectof counteranion of TRH and loading amount on control of TRH release from copoly(D,L-lactidglycolic acid)microspherespreparedbyin-waterdrying method, Int. J. Pharm. 69:69-75 (1991). D. Bodmer and T. Kissel, Sustained release of the somatostatin analogue octreotide from microspheres, Proceedings of 18th Int. Symp. Control Rel. Bioact. Mater., Amsterdam, 1991, pp. 597-598. P.J. Camarata, R. Suryanarayanan, D. A. Turner, R. G. Parker, and T. J. Ebner, Sustained release of nerve growth factor from biodegradable polymer microspheres, Neurosurgery 30(3):313-319 (1992). J. H. Eldridge, C. J. Hammond, J. A. Meulbroek, J. K. Staas, R. M. Gilley, and T. R. Tice, Controlled vaccine release in the gut-associated lymphoid tissues. I. Orally administered biodegradable microspheres target the Peyers patches, J. Control. Rel. 11:205-214 (1990). S. Cohen, T. Yoshioka, M. Lucarelli, L. H. Hwang,and R. Langer, Controlled delivery systems for proteins based on poly(lactidglyco1ic acid) microspheres, Pharm. Res. 8(6):713-720 (1991). G . P. Talwar, Controlled delivery of diphtheria toxoid M. Singh, A. Singh and using biodegradable poly(D,L-lactide) microcapsules, Pharm. Res. 8(7):958961 (1991). H. T. Wang, E. Schmitt, D. R. Flanagan, and R. J. Linhardt. Influence of on the in vitro controlled release of protein from poly(esformulation methods ter) microspheres, J. Control. Rel. 17:23-32 (1991).
68
Benoit et a/.
70. D. T. OHagan, J. P. McGee, J. Holmgren, A. Mowat, A. M. Donachie, K. H. G. Mills W. Gaisford, D. Rahman, andS. J. Challacombe, Biodegradable microparticles for oral immunization, Vaccine 11(2):149-154 (1993). 71. H. Jeffery, S. S. Davis, andD. T. OHagan, The preparation and characterization of poly(1actide-co-glycolide) microparticles. 11. Theentrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique, Pharm. Res. 10:362-368 (1993). 72. M.J.Alonso, S. Cohen, T. G.Park,R.K.Gupta, G. R.Siber,andR. Langer, Determinants of release rate of tetanus vaccine from polyester microspheres, Pharm. Res. 10(7):945-953 (1993). 73. H. K. Sah and Y. W . Chien, Evaluation of a microreservoir-type biodegradable microcapsule for controlled releaseof proteins, Drug Dev. Ind. Pharm. 19(11):1243-1263 (1993). 74. N. Lahlou, H. Navratil, Y.Lanson, B. Lardennois, M. Joubert-Collin, and M. Roger, Pharmacocinetique de la leuprortline retard et freinage gonadotrope chez des patients traitts pour cancerde la prostate, Lett. Pharmacol. 7(7):2-7 (1993). 75. R. JalilandJ. R. Nixon,Microencapsulationusing poly(llactic acid). 11. Preparative variables affecting microcapsule properties, J. Microencaps. 7(1): 25-39 (1990). 76. R. Jalil and J. R. Nixon, Microencapsulation using poly(L-lactic acid). 111. Effect of polymer molecular weight on the microcapsule properties, J. Microencaps. 7(1):41-52 (1990). 77. R. Jalil and J. R. Nixon, Microencapsulation using poly(l1actic acid). IV. Release properties of microcapsulescontainingphenobarbitone,J.Microencaps. 7(1):53-66 (1990). 78. D. C. Tsai, S. A. Howard, T. F. Hogan, C. J. Malanga,S. J. Kandzari, and J. K. H. Ma, Preparation and in vitro evaluationof polylactic acid mitomycin C microcapsules, J. Microencaps. 3:181-193 (1986). 79. A. M. Hazrati and P. P. Deluca, 5-Fluorouracil in biodegradable polymeric microspheres, Proceedings of 16th Int. Symp. Control. Rel. Bioact. Mater., Chicago, 1989, pp. 79-80. R. Wada, S. H. Hyon,and Y. Ikada,Controlled 80. 0. Ike, Y. Shimizu, cisplatin delivery system using poly(D,L-lactic acid), Biomaterials 13(4):230234 (1992). 81. S. Goto, M. Kawata, M. Nakamura, K. Maekawa, and T. Aoyama, Eudragit RSandRL(acrylicresins)microcapsulesaspHinsensitiveandsustained release preparations of ketoprofene, J. Microencaps. 3(4):293-304 (1986). 82. T. Sato, M. Kanke, H. G. Schroeder, and P.P. DeLuca, Porous biodegradable microspheres for controlled drug delivery. I. Assessment of processing conditions and solvent removal techniques, Pharm. Res. 5:21-30 (1988). 83. J. Bontemps, P. Pirson, J. B. Falmagne, R. Jerome, P. Teyssit, L. Delattre, and B. Evrard, Microparticules comportantun polymtre biodegradablecontrblant la liberation dun actif antimalarique, compositions pharmaceutiques en comprenantet proc6dC depreparation, Eur. Patent 0301969,1989. 8 4 . D. L. Gardner,Process ofpreparingmicrocapsules of lactidesorlactide
Biodegradable
69
copolymerswithglycolides
1987.
andor ecaprolactone, U.S. Patent 4,637,905,
85. G. Spenlehauer,F. Spenlehauer-Bonthonneau, and C. Thies, Biodegradable
microparticles for delivery of polypeptides and proteins, in Biological and SyntheticMembranes (D. A. Butterworth, ed.), Alan R. Liss, Inc., New York, 1989, pp. 283-291. W. McGinity, Preparation of multi-phase microspheres of 86. M.IwataandJ. poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolicacid) containing a w/o emulsionby amultipleemulsionsolventevaporationtechnique,J.Microencaps. 9(2):201-214 (1992). 87. P.P. DeLuca, M. Kanke, T. Sato, and H. G. Schroeder, Porous microspheres for drug delivery and methods for making the same, U.S. Patent 4,818,542,
1989. de implantables par 88. M. Boisdron-Celle, Preparation de microparticules 5-FU 89.
90.
91. 92. 93. 94. 95. 96.
97.
98.
757 (1988). 99. B. Conti, F . Pavanetto, and I. Genta, Use of polylactic acid for the preparation of microparticulate drug delivery systems, J. Microencaps. 9(2):153-166 (1992).
sttrtotaxie et tvaluation thtrapeutique chez le rat porteur dun gliome malin Ph. D. thesis, Universitt de Paris-Sud, January 31, 1994. M. Boisdron-Celle, P. Menei and J. P. Benoit, Preparation and characterization of 5-fluorouracil-loaded microparticles as biodegradable anticancer drug carriers, J. Pharm. Pharrnacol.47:108-114 (1995). H. T.Wang, H. Palmer, R. J. Linhardt, D. R. Flanagan, and E. Schmitt, 11:679-685 (1990). Degradation of poly(ester) microspheres, Biomaterials A. K. Kwong, S. Chou, A. M. Sun, M. V. Sefton, and M. F. A. Goosen, In vitroandinvivoreleaseofinsulinfrompoly(1acticacid)microbeadsand pellets, J. Control. Rel. 4:47-62 (1986). N. Leelarasamee, S. A. Howard, C. J. Malanga, and J. K. H. Ma, A method for preparationof polylactic and microcapsules of controlled particle size and drug loading, J. Microencaps. 5147-157 (1988). F. Nielsen, Spray drying pharmaceuticals, Manuf. Chem. 53(7):38-39 (1982). J. Broadhead,S. K. E. Rouan, and C. T,Rhodes, The spray-drying of pharmaceuticals, Drug Dev. Ind. Pharm.18(11-12):1169-1206 (1992). K. Masters, Spray Drying Handbook, 4th ed. Halsted Press, New York, 1985. M. Montini, A. Pedroncelli, F. Tengattini, M. Pagani, D, Gianola, L. Cortesi, G . Pagani, andI. Lancranjan, Medical applications of intramuscularly administered bromocriptine microspheres in Pharmaceutical Particulate Camers, Therapeutic Applications (A. Rolland, ed.), Marcel Dekker, New York,1993, pp. 227-274. A. Rummelt, T. Kissel, P. Koci and L. Mazzoni, Ketotifen-microspheres: A novel parenteral depot formulation for asthma and allergy prophylaxis, Proceedingsof 18th Intern. Sym. Control.Rel.Bioact.Mater.,Amsterdam, 1991, pp. 279-280. R. Bodmeier and H. Chen, Preparation of biodegradable poly(+,-)lactide microparticles using a spray-drying technique, J. Pharm. Pharrnacol. 40:754-
70
Benoit et a/.
100. R. Jalil and J. R. Nixon, Biodegradable poly(lactid acid) and poly(1actide-coglycolide) microcapsules: problems associated with preparative techniques and release properties, J. Microencaps. 7(3):297-325 (1990). 101. R. Schmiedel and J. K. Sandow, Microcapsule production containingsoluble protein or peptide usingmixtureofpo1y:hydroxy-butyricacidandpoly: lactide-co-glycolide, Eur. Patent 315875A, 1989. 102. G. Cohen and J. L. Dubois, Injectable microspheres containing antiestrogenic and antiprogestomimetic steroids, German Patent 4 036 425, 1991. 103. D.Wise,G.J.MCCormick,andG. P. Willet,Sustainedrelease of an antimalarial drug using a copolymer of glycolicflactic acid, Life Sci. 19:867874 (1976). 104. D. Wise, J. D. Gresser, and G. J. MC Cormick, Sustained release of a dual antimalarial system, J. Pharm. Pharmacol. 31:201-204 (1979). 105. F. Pavanetto,I.Genta, P. Giunchedi,andB.Conti,Spray-dryingasa method for biodegradable microspheres preparation, Proceedings of 18th Intern. Sym. Control. Rel. Bioact. Mater., Amsterdam, 1991, pp. 676-677. . Muller,Piroxicamreleasefromspraydried 106. B. W. WagenaarandB. W biodegradable microspheres, Biomaterials 15:49-54 (1994). . Bruhn, B. W. Muller, Preparation and characterization of spray-dried 107. B. W poly (DL-lactide)microspheres, Proceedings of 18th Intern. Sym. Control. Rel. Bioact. Mater., Amsterdam, 1991, pp. 668-669. 108. P. Giunchedi, B. Conti, L. Maggi, and U. Conte, Spray dried ketoprofen microspheres as potential oral or intra articular delivery systems, Proceedings of 20th Intern. Symp. Control. Rel. Bioact. Mater., Washington, DC, 1993, pp. 386-387. 109. M. E. Paulaitis, V. J. Krukonis, R. T. Kurnik, and R. C. Reid, Supercritical fluid extraction, Rev. Chem. Eng. 1(2):179-250 (1983). Permt, Les FluidesSupercritiques,CaracttristiquesetApplications 110. M. Actes du colloque de Pont-li-Mousson, May 20-21, 1987, Institut National Polytechnique de Lorraine, France, 1987. 111. M. Permt, Proceedings of the International Symposium on Supercritical Fluids, Vol. 1 and 2, October 17-19, 1988, Institut National Polytechnique de Lorraine, France,1988. Les Fluides Supercritiques, Caracttrisation et 112. M. Permt, 2kme Colloque sur applications, Paris, October 16-17,1991, Institut National Polytechnique de Lorraine, France,1991. Williams, Extraction with Supercritical Gazes, Chem. Eng. Sci. 113. D. F. 36(11):1769-1788 (1981). of dense (supercritical) gas-extraction and 114. L. G. Randal, The present status densegaschromatography:Impetusfor DGCMS,Separation Sci. and Technol. 17(1):1-l18 (1982). 115. P. G. T. Fogg, Carbon dioxide in non-aqueous solvents at pressures less than 200KPA, in Solubility Data Series,Vol. 50, Pergamon Press, 1992. 116. C. J. Chang, A. D. Randolph, and N. E. Craft, Separation of p-carotene mixturesprecipitatedfromliquidsolventswithhigh-pressure C02, Biotechnol. Prog. 7:275-278 (1991).
Biodegradable
71
117. T. W. Randolph, A. D. Randolph, M. Mebes, and S. Yeung, Sub-micrometersized biodegradable particles of poly (L-lactic acid) via the gas antisolvent spray precipitation process, Biotechnol. Prog. 9 (4):429-435 (1993). 118. A. Glatz, Thesis, Extraction von steroiden mit verdichteten Gasen, Ph.D. thesis, Universitat des Saarlandes, Germany, 1985. of drug micronized 119. H. Loth and E. Hemgesberg, Properties and dissolution by crytallization from supercritical gazes, Int. J. Pharm. 32:265-267 (1986). D. Randolph, Precipitation of microsize organic particles 120. C. J. Chang and A. from supercritical fluids, AlChE J. 35(11):1876-1882 (1989). 121. G.Donsi and E. Reverchon, Micronization by means of supercritical fluids possibility of application to pharmaceutical field, Pharm. Acta Helv. 66(56):170-173 (1991). 122. A. Tavana-Roudsari, Diss. Abstr. Int. B51(9):4487 (1991). 123. P. G. Debenedetti and S. K. Kumar, The molecular basis of temperature effects in supercritical extraction, AlChE34:645-647 J. (1988). 124. J. W. Tom and P. G.Debenedetti,Particlesformationwithsupercritical fluids. A Review, J. Aerosol Sci.22555-584 (1991). 125. B.W. Muller and W . Fischer, Verfahren zur Herstellung einer mindestens einen Wirkstoff unf einen Trager umfassendenZubereitung, German Patent DE 3744329 A l , 1989. 126. T. Tauchi,T. Aki, T. Murakami,H.Tashiroand H. Ito, Granulation of organic substances using supercritical fluids, Jpn. Kokai Tokkyo Koho JP 01.176.437 [89.176.437], July 12, 1989, Chem. Abstr. 112:237587r (1990). . Wassmus, Aerosol solvent extraction system, 127. J. Bleich, B.W. Muller andW A new microparticle production technique, Int. J. Pharm. 97(1-3):lll-117 (1993). . Muller, K. H.NagelsandH.M.Wolff,Influenceof two 128. J. BleichB. W production parameters on microparticle size in aerosol solvent extraction system(ASES),Proceedingsof20thIntern.Symp.Control.Rel.Bioact. Mater., Washington, DC, 1993,pp. 456-457. 129. P. G. Debenedetti,J. W. Tom and S. Yeo, Supercritical fluids: a new medium for the formation of particles of biomedicalinterest of protein microparticles by antisolvent precipitation, Proceedings of 20th Intern. Symp. Control. Rel. Bioact. Mater., Washington, DC,1993,pp. 141-142. 130. P. G. Debenedetti,G. B. Lim, and R. K. Prudhomme, Formation of protein 542,314, 1993. microparticles by antisolvent precipitation, Eur. Patent 131. D. M. Lokensgard, Solvent extraction process, U.S. Patent, 5,232,707,1993. 132. A. Mori, K. Morikawa, T. Matsuva, S. Onaka, and Y. Nakanishi, Removal oforganicsolventsfrompharmaceuticalsbyextractionwithsupercritical 02.292.216 [90.292.216], Decemcarbon dioxide, Jpn. Kokai Tokkyo JP Koho Chem. Abstr. 1143214422k (1991). ber 3, 1990, 133. R. De Ponti, C. Torricelli, A. Martin, and E. Lardini, Use of supercritical pharmaceutical implants or drug delivery systems, PCT Int. Appl. WO 91 09.079, 1991, Chem. Abstr. 115:263520v (1991). 134. W . Worthy, Chem. Eng. News 59(31):16-17 (1981).
72
Benoit et a/.
135. E. M.Phillips and V. J. Stella, Rapid expansion from supercritical solutions: application to pharmaceutical processes, Int. J. Pharm. 94:l-10 1993. 136. J. W. Tom and P. G . Debenedetti, Formation ofbioerodiblepolymermi137. 138. 139. 140. 141. 142.
crospheres and microparticles by rapid expansion of supercritical solution, Biotechnol. Prog. 7:403-411 (1991). J. W. Tom and P. G . Debenedetti, Precipitation of poly(hydroxy acid) and coprecipitation of polymerldrug particles by rapid expansion of supercritical solutions, Polym. Prep.33(2):104-105 (1992). p. G . Debenedetti, J. W. Tom, X. Kwauk, andS. D. Yeo, Rapid expansionof supercritical solution (RESS) fundamentals and applications, Fluid Phase Equilib. 82:311-321 (1993). P. G . Debenedetti, J. W. Tom, S. D. Yeo,and G . B. Lim, Application of supercritical fluids for the production of sustained delivery devices, J. Control. Rel. 24(1-3):27-44 (1993). S. D. Yeo, G . B. Lim, P. G . Debenedetti, and H. Bernstein, Formation of microparticulateproteinpowdersusingasupercriticalfluidantisolvent. Biotechnol. Bioeng. 41(3):341-346 (1993). J. W.Tom, G . B. Lim, P. G. Debenedetti, and R. K. Prudhomme, applications of supercritical fluids in the controlled release of drugs, A.C.S. Symp. Ser. 514:238-257 (1993). M.Gachon, Limites des solvants rtsiduels, STP-Pharma Pratiques 1(5):531-
536 (1991). 143. A. Smith and I. M. Hunneyball, Evaluation of poly(1actic acid) as a biodegradable drug delivery system for parenteral administration, Int. J. Pharm. 30~215-220 (1986). 144. B. Wichert and P. Rohdewald, A new method for the preparation of drug
containing polylactic acid microparticles without using organic solvents, J. Control. Rel. 14:269-283 (1990). 145. F. Hutchinson, Continuous release pharmaceutical Compositions, h r . patent 0058481B1,1986. 146. J. M. Ruiz, Sustained release particles preparation, U.K.Patent GB 2 246 514 A, 1992. 147. V. J. Csernus, B. Szende and A. V. Schally, Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo, Int. J. Peptide Protein Res.35557-565 (1990).
Advances in the Technology of ControlledRelease PesticideFormulations

Arie Markus
The Institutes for Applied Research, Ben-Gurwn University of the Negev, Beer-Sheva, Israel
Introduction I.
73 77 81 82 82 82 83 85 86 87
11. Interfacial Polymerization

111. Phase Separation
IV. Quality Control A. Storage Stability B. ReleaseRate of Pesticide C. Toxicity to Fish D. Acute Oral Toxicity to Mice E. Efficacy Case Study-Antifly
V.
1.
INTRODUCTION
The science of pesticide formulation covers a very broad field, since it deals with the development, production, and storage of the formulations [l-41, as well as with the interactionof pesticides withthe environment, including plants, insects, animals, soil, air, and water [5]. Pesticide formulations can be classified into thefollowing types:
73
74
Markus
Aqueous solutions Emulsifiable concentrates Dispersion concentrates (aqueous and nonaqueous flowables) Wettable powders Dry flowables (water-dispersible granules) Controlled-release formulations Others (e.g., dusts, aerosols)
The choice of formulation is influenced by the following factors: the physical properties of the pesticide (melting point, solubility, volatility); the chemical properties of the pesticide (hydrolytic stability, thermal stability, irradiation stability); the mode of application of the formulation (soil vs. foliar); the crop to be treated and agricultural the practices; the biological properties of the pesticide (crop selectivity, transport, LD,, for mammals and nonmammalian species); and economicconsiderations. This chapter covers controlled-release techniques. Pesticides are conventionally applied to crops by periodic broadcasting or spraying. Very high, and possiblytoxic, concentrations are applied initially, and these often decrease rapidly in the field to concentrations that fall below the minimum effective level. As a result, repeated applications are needed to maintain control[6]. The formulation of a pesticide must thus be designed to meet the multifaceted demands of efficacy, suitability to mode of application, and minimization of damage to the environment. Controlled-release formulations meet these demands in that they enable smaller quantities of pesticide to be used more effectively over a given time interval and in that their design enables them to resist the severe environmental processes that act to eliminate conventionally applied pesticides; i.e., leaching, evaporation, and photolytic, hydrolytic, and microbial degradation [6]. In most instances, the rate of removal of a conventionally formulated pesticide follows first-order kinetics [6-91. The time, t,, that an effective level of pesticide is maintained after a single application is given by:
where M, is the minimum effective level, Mm is the amount of agent applied initially, and k, is the rate constant [6]. It follows from Eq. (1) that an increase in the effective duration of action of a conventionally applied pesticide would require than an exponentially greater quantity of the pesticide to beapplied. If, however, the pesticide could be maintained at the minimum effective level by a continuous supply from a controlled-release system, then the optimum performance of the insecticide would be realized, and this duration of action, to, would be given by:
Controlled-Release Pesticide Formulations

to
75
(2) where kd is the rate constant for pesticide delivery from the controlledrelease device [6]. The relationship between the level of application and the durationof action of a pesticide is shown Fig.1 for a conventional formulation and for a controlled-release formulation [6]. The area between the two curves represents the amount of pesticide that is wasted. It is apparent that fora short duration of effectiveness, e.g., 1 week or less, the efficiency of the conventional method is adequate. As theduration of effectiveness increases, the efficiency of the conventional system decreases exponentially (as Fig. 1indicates). In addition to the advantages described above, controlled-release pesticides have other important advantages over conventional formulations [6]:
=
(Mm
- Me)(KdMe)
Reduction of mammalian toxicity for highly toxic substances Extension of duration of activity at an equallevel of active agent Reduction of evaporative losses and of flammability of liquids
1,000
CONVENTIONAL PESTICIDE
loot
Fig. 1 Relationship between the level of application and the duration of action
for conventional and controlled-release formulations. The graphs are plotted using equations 1 and 2 and assuming that the half-life of the pesticide is 15 days and the minimum effective level is 1 g/acre. (Reprinted from Ref. 6.)
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Markus Reduction of phytotoxicity Protection against environmental degradation Reduction of leaching into the earth and transportation into streams Reduction of contamination of the environment Increased convenience of use by conversion of liquid materials into solids and flowable powders Separation of reactive components Control of the release of active agents Decreased costs because less active material is needed Convenience of handling.
Although the advantages of controlled release are impressive, the disadvantages of the technology must not be forgotten. Each application has to be examined individually and the positive and negative aspects weighed carefully [ 6 ] .Some of the following aspects of controlled-release formulations may be deleterious and thus require careful appraisal: (1) costs of the materials and processing of the controlled-release preparation, which may be substantially higher than standard formulations; (2) fate of the polymer matrix (see below) and its effect on the environment; (3) fate of polymer additives, such as plasticizers, stabilizers, antioxidants, and fillers; (4) the environmental impact of the degradation products of the polymer matrix produced in response to heat, hydrolysis, oxidation, solar radiation, or biological agents; (5) cost, time, and probability of successin securing government registration of the product,if this is required [6]. Various techniques have been proposed for the production ofmicrocapsules [lo-131; one source suggests that hundreds of methods are to be found in the scientific and patent literature. In general, these methods for microencapsulation can beclassified as: Separation from an aqueous solution Formation by polymer-polymer incompatibility Interfacial polymerization Polymerization in situ Drying froma liquid state Solvent evaporation from anemulsion Gelation in the liquid state by cooling Desolvation Physical techniques, such as spray drying or fluidized bed reactions, are not usually suitable for encapsulation of pesticides. Similarly, addition polymerization is not suitable for pesticides, since many pesticides contain phosphorus, nitrogen, or sulfur atoms, which are known to be radical scavengers, or since the compounds may not be stable in acid or alkali media. Interfacial
Formulations Controlled-Release Pesticide
77
polymerization is the method of choice for highly toxic insecticides, since the active ingredient is completely enveloped by the polymer and in most of cases release takes place via diffusion. This technique facilitates a dramatic decrease in toxicity. Both phase separation and interfacial polymerization techniques may be used for nontoxic pesticides. The only technique suitable forbiological pesticides is phase separation: With any other technique, the active ingredient, which in this case is a biological microorganism, would not be able to cross the envelope surrounding it.
II. INTERFACIAL POLYMERIZATION
The technique for the microencapsulation of pesticides by interfacial polymerization comprises two stages. First, a liquid, molten, or dissolved pesticide containing a dissolved monomer is agitated at a high speed in an aqueous solution. The pesticide in which the monomer is dissolved constitutes the organic phase. Interfacial polymerization at the dropletsurface is then completed by the addition of a second water-soluble monomer to the continuous aqueous phase. The resulting aqueous slurry may be used as such or the pesticide-containing capsules may be recovered as a dry powder. When the pesticide is water miscible, it is also possible to carry out interfacial polymerization by making a dispersion of the liquid or molten pesticide in an organic phase, such as high-boiling petroleum ether. The following factors influence the performance of the capsules:
1. Composition of the polymeric capsule wall: The monomers can be chosen so as toproduce a variety of interfacial polycondensation products as wall materials; e.g., polyamides, polyesters, polyureas, polyurethanes, or polycarbonates. Within each of these categories, a range of polymers may be produced, depending on the starting monomers. It is also possible to vary the wall composition by forming copolymers in a simple process that is based on a mixture of oil-soluble or water-soluble monomers. 2. Degree of crosslinking: For cases in which a higher degree of wall integrity is essential, multifunctional monomers are used. 3. Capsule wall thickness: The thickness of the capsule wallis a function of the concentration of the monomers. In theproduction of the capsule, the wall continues to thicken until all of the available monomer is consumed. 4. Capsule size: The size of capsules is determined by the degree of agitation and by the type of emulsifying agent used in the continuous phase. Capsule size can be varied from an average diameter of a few micrometers to a millimeter. If there is a requirement for
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a formulation that must be sprayed, the diameter of the capsule must beless than 100 pm. 5 . Physical form of product: The final product may take the form of an aqueous slurry or of microcapsules that can be filtered off, washed, and dried to a free-flowing powder. Aqueous slurries have been found to be useful in the formulation of pesticides, since they need only to bediluted with water in order to be ready for field application as a sprayable product. In addition, they are cheaper, being more economical to manufacture. 6. Additives: A range of additives, such as ultraviolet light absorbers, antioxidants, and synergists, may be dissolved inthe oil phase (pesticide) being encapsulated. It should be remembered that these additives must be soluble in the oil and must not react with the monomers.
To date, a wide range of insecticides, herbicides, and fungicides have been encapsulated within variety of polymeric shells. At present, polyamide shells, especially polyurea andpolyurethane, are particularly popular. The company that introduced encapsulated pesticidaI formulations to the market is Pennwalt Corporation. Their first product was Penncap M(encapsulated methylparathion), and this was followed by KNOX OUT 2FM (encapsulated diazinon), Penncaptrin (encapsulated permethrin), and Pennphos (encapsulated chlorpyriphos) and later by the herbicide trifluralin [17]. Polyurea was used for the envelope, and the method of encapsulation is described in patents [14-171. The Pennwalt envelope was synthesized by the reaction of polyisocyanate with amines andacid chlorides. Another company active in the market is Stauffer Chemical Company, whose interest lies mainly in encapsulated herbicides. The Stauffer process for the production of the polyurea envelope differs from that of Pennwalt in that the isocyanate also serves as a source of amine. In the reaction process, some of the isocyanate is converted intothe amine, which then reacts with the remaining isocyanate [B-211. A number of other companiesare also involved inthe production of encapsulated biocides and herbicides. Monsanto Chemical Company markets encapsulated Alachlor under the trade name of Lasso. The envelope for the Alachlor formulation is also based on polyurea andpolyurethane, and the method andchemicals-the isocyanates, amines, and the emulsifiersused byMonsanto aredescribed in their patents [22-261. Kedem Chemicals Ltd. (Israel) produces NO-ROACH (encapsulated diazinon) [27] and Antifly (encapsulated pyrethroids), and Dow manufactures Empire (encapsulated chlorpyriphos). In all the products mentioned above,the toxicity of
79
the formulation is at least one tenth that of the emulsifiable concentrate (EC), and the efficacy lasts longer without a reductionof insecticidal activity. Several other companies [28-321 have used polyurethane or polyurea shells as envelopes for pesticides; among the biggest of these, we can mention Hoechst Bayer AG [33], and Ciba-Geigy [34]. The process for the production of encapsulated pesticides-in this case, diazinon and pyrethroids (Figs. 2-4)is described schematically below. So far, we have discussed the interfacial polymerization technique in which polyamides are used as the envelop for the pesticides. However, experiments have been performed with envelopes made from a variety of polymers, including urea-formaldehyde and melamine-formaldehyde polymers, polyesters, epoxy, and polysilane. The formulations based on these polymers as envelopes have, so far, not been successfully commercialized.
Fig. 2 Encapsulated diazinon.
F
Fig. 3 Encapsulated diazinon.
cI
Fig. 4 Encapsulatedpyrethroids.

Production of encapsulated D e s e
81
..
withemulsifier
water-soluble pesticide +
Emulsification
Aqueous solution of amines Polymerization
111.
PHASE SEPARATION
The second technique used in pesticide encapsulation is phase separation, and the capsule obtained in this case is generally of the matrix type. This technique is suitable only for nontoxic and biological pesticides, since encapsulation into matrix-type capsules does not considerably reduce thetoxicity .of the product.An advantage of this mode of encapsulation is that, should the need arise, is it possibleto grind the formulation, since in most cases, the active ingredient is absorbed onto andwithin the polymer matrix. In the phase-separation technique, the active ingredient is suspended in a solution of the wall material. The wall polymer is then induced to separate out as a viscous liquid phase by coacervation (i.e., by adding a nonsolvent), by lowering the temperature, by adding a second polymer, or by a combination of these methods. Coacervation, which maybe simple or complex or of the salting-out type, is manifested as turbidity, droplet formation, or theseparation of liquid layers as described below. Simple coacervation occurs when a water-miscible nonsolvent (e.g., ethanol) is added to an aqueouspolymer solution, causing the formation of a separate polymer-rich phase, A typical example of a simple coacervation system is water-gelatin dissolved in water: this system is, however, difficult to control, so it is little used in practice. Complex coacervation takes place by the mutual neutralization of two oppositely charged colloids in aqueous solution. One of the most
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widely used methods of microencapsulation by this process is the use of a solution of positively charged gelatin (pH <S), which forms a complex coacervate with negatively charged gum arabic. Other polymer systems may be employed and other electrolytes may be used with gelatin. Complex coacervation is closely related to the precipitation of colloidal material from solution: It immediately precedes precipitation. The process was originally developed in the 1950s for the coating used in the manufacture of carbonless copying paper, with gelatin and gum arabic as the two colloids. In salt coacervation, the polymer is separated out from the aqueous solution by salting out, typically by adding an electrolyte to an aqueous polymer solution. The method may be used to encapsulate water-insoluble oils or dispersed solid particles, but it is difficult to control the microcapsule size and the agglomeration of particles. The system may be stabilized by altering the pH or temperature. The polymers thatcan be used in the phase-separation technique are [6]. summarized in Table 1 Biological pesticides, such as Bacillus thuringiensis isruelensis (Bti) [36] and Trichoderma harzianum [37-391, have beenencapsulated in matrix-type capsules in which envelopes were produced from two types of polymers-natural polymers, such as alginates, or synthetic polymers, such as polyethylene. The methodsof polymerization are shown below.
W. QUALITY CONTROL
The following parameters must be determinedfor any biocide formulation: the amount of active ingredient, size distribution, shelf life, biological release rate, toxicity, and efficacy. Since the first two types of tests are well documented, they will not be described here. The other tests will be described in detail below.
A.
Storage Stability
A sample of the microencapsulatedpesticide formulation is maintained in a closed vessel in an oven at 54C for 2 weeks or at 40C for 6 weeks. The amount of the active ingredient in the productis analyzed before and after heating, and the formulation is considered to be stable if the amount of active ingredient does not change.
B. Release Rate of Pesticlde

The apparatus used for this test is illustrated in Fig.5. One gram of encapsulated pesticide is placed on a plate, which is then sealed into a cylindrical glass vessel.The vessel isprovided with a digital thermometer and inlet and

Table 1 Polymers that Can Be Used in the Phase-Separation Technique
83
Natural Polymers Carboxymethylcellulose Cellulose acetatephthalate Ethylcellulose Propylhydroxycellulose Gelatin Gum arabic Starch Bark Methylcellulose Galacturonicacid salt Polybutadiene Polyisoprene Neoprene Polysiloxane Styrene-butadiene rubber Silicone rubber Zein Cellulose nitrate Shellac Alginate Waxes-paraffin Proteins Kraft lignin Natural rubber Methocel Synthetic Elastomers Hydrin rubber Chloroprene Butyl rubber Ethylene-propylene-diene terpolymer
Synthetic Polymers Polyhydroxyethyl methacrylate Polyvinyl alcohol Polyacrylate Polyethylene Polyacrylonitrile Polypropylene Chlorinated polyethylene Polystyrene Polyvinylpyrrolidone Polvether Ethylenevinylacetate copolymer Poly@-xylylene) Polyvinylidene chloride Polymethylmethacrylate Polyvinyl acetate Polyvinyl chloride
outlet tubes. The inlet tube is equipped with a flowmeter to control the volume of air introduced into the vessel. The air stream carries the released pesticide out of the vessel into a beaker containing ethanol. The temperature of the system is maintained at a predetermined value by an external heater. The amount of pesticide in the ethanol is determined, and the release rate calculated as a function of time.
C. Toxicity to Fish
The fish (10per tank) are kept in 16-L aquaria under thefollowing optimal conditions: water quality as close as possible to pH 7 (neutral); water temperature 23-25C; and good strong light for least 12 h a day (morelight makes them grow too fast). The test is performed in a room free of insecticidal contamination as follows. Adult fish of either sex are supplied
84
Production of cncausnlated
Markus
of part of the
Drying of the
of capsules
Filtration and washing

thermocouple
85
Fig. 5 Apparatus for the determination of release rate of a pesticide from an encapsulated formulation. with adequate standardized food (Europet Basic food) before and after the experiment. Food is withheld for 2 days before the experiment. A solution of the pesticide formulation is obtained by placing the formulation in water in a high-shear mixer for 5 min. After addition of the pesticide to the fish tanks, mortality is checked after 3, 6 , 24, 48, 72, and 96 h. The times for 50% and 95% mortality (LTso and LT,,) are then determined f r o m the results.
D. Acute Oral Toxicity to Mice
It is preferable to use adult male animals (2.0-2.5 months) weighing 25-30 g. Standardized mouse food is given throughout theexperiment. A solution of the formulation is obtained by placing it in water in a Vortex mixer for 5 min. The quantity of the solution depends on theweight of mouse-l mL of solution is givenper 20 g body weight.The pesticide solution is placed in a 2-mL syringe and introduced into the stomach of mouse via the mouth. and 168 h, andLD, is Mortality is checked at 0,5,24,48,72,96,120,144, determined.
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E. Efficacy
In this method, which measures the susceptibility of a population of cockroaches (BlutfeZZugermunicu) to a given insecticide, the cockroaches are exposed to standard insecticide residues in a Petri dish, and mortality is determined. The cockroaches should be obtained, as far as possible, from the same area, andkept in a suitable container until required. It is preferaif it is not possible to obtainenough ble to use adult males for the test, but males, females may be used. The test should becarried out in a room free of insecticidal contamination. The cockroaches are given adequate standardized food before the experiment. The cockroaches are first anaesthetized with carbon dioxide before being put into the insecticide-containing Petri dish. The Petri dish is then placed in an experimental vessel, which is maintained at25-30C and relative humidity >25%. Solutions of the insecticide formulation are obtained by using a high-shear mixerfor 5 min. From the results, the times for 50% and 95% knockdown (LT,, and LTg5)can be
Fig. 6 Cockroach.
Formulations Controlled-Release Pesticide
87
determined. (A cockroach is considered knocked down if it fails to move on being returned to its normal posture.) Owing to thefact that thecapsules become attached to the cockroach (or ant) leg, they are introduced into the nest, where they then exert their action on all the insects in the nest.Figs. 6 and 7 show the capsule attached to theleg of a cockroach.
V. CASE STUDY-ANTIFLY
Antifly (encapsulatedpyrethroids) is manufactured anddistributed by Kedem Chemicals Ltd. under license from Ben-Gurion University of the Negev, Beer-Sheva, Israel. TheLD,, of Antifly for mice, determined by the test described above, is 12,500 mg/kg and the LD50 for golden orfe fish 2200 pg/l. The biological efficacy of this encapsulated formulation versus that of the EC against cockroaches, flour beetles, house flies, and fleas, and ticks is shown in Fig. 8, and Tables 2,3, and 4, respectively.
Fig. 7 Closeup of cockroach leg showing diazinon capsule stuck to the leg.
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110 '20
.
50
40
Emusified concentration
30 20 10 0
Blattellagermanica exposedto Fig. 8 % mortalityasafunctionoftimeof pyrethroids. a, emulsified concentration; U, Antifly.

Table 2 Biological Efficacy After 2 4 h of Antifly Against the Flour Beetle (Tribolium castaneum)
Pyrethroid formulation Antifly EC
% mortality at a concentration (ppm) of

0
250
500
750 95
75
50
i t
80 68
74
EC, emulsifiable concentrate. 0, control (without pesticide-water only) in the case the of capsules and solvent in the caseof EC product. Table 3 Biological Efficacy After 24 h of Antifly Against the Housefly (Musca dornestica) Pyrethroid formulation Antifly EC
% mortality at a concentration (ppm) of
0
25 100
75 250
125
500 1 0 0 68
750
1 0 0 95
1 0 0
60
60 80
100 60
100
EC, emulsifiable concentrate 0, control (without pesticide-water only) in the case the of capsules and solvent in the case of EC product.

Table 4 Biological Efficacy of Antifly Against Fleas and Ticks8
89
Parasite Dogs
~ ~
of
level
Presence of parasites after days

~
type
sex First spraying Mongrel M Mongrel Ridgeback Mongrel Mongrel M Mongrel F M Pointer German M shepherd German M shepherd F Belgium shepherd Second spraying Spitz M F Spitz F Spitz F Terrier F Terrier German M shepherd F German shepherd
F F F
14
21 0 0 0 0 0 0 0 0 0 0
28 0 0 0 0 0 0 0 0 0 0
60
+ ticks
l1 ,l l1 l 1
Fleas
High
II l1
+ o o + o o + o o
0 0
n
, l , l
I,
0 0
s
s
l1
+ o o + o o
0
0 0
I,
+ o o
II
+ o o
0 0 0 0 0 0
0
Fleas
n
, ,
I,
II ,I
2 2 1-2 2 2 2 2
0 0 0 0 0 0
0
0 0 0 0 0 0
0
0 0 0 0 0 0
0
0 0 0 0 0 0
0
0 0 0 1 0 0
0
T h e experiment was performed in an animal shelter in Tel-Aviv, Israel under the supervision of the Chief Veterinarian. The spraying was done directly on the dogs.No untoward side effects were observed. From this table we see that spraying the dogs with Antifly gave excellent results: 28 for After the first spraying, the dogs remained without fleas and ticks days; before the spraying, they were heavily infested with ticks and fleas. After the second spraying, they wereof free fleas and ticks for 60 days. It is well known that the commercial product keeps the dogs free of ticks and fleas for a very short time (i.e., a few days).
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REFERENCES
pp. 717-826. 2. J. Miyamoto and P. C. Keierney, Pesticide Chemistry-Human Welfare and the Environment,Vol. 4, Pergamon Press, 1983, pp. 241-400. 3. W. Van Valkenburg, Pesticide Formulations. Marcel Dekker, New York, 1973. 4. K. C. Seymour, Pesticide Formulation and Application Systems, 2nd Conference. ASTM, Philadelphia, 1983. 5. H. B. Scher, Advances in Pesticide Formulation Technology, A.C.S., Washington, DC, 1984. 6. A. F . Kydonieus,ControledReleaseTechnologies:Methods,Theoryand Applications, Vols. 1and 2, CRC Press, Boca Raton, F L , 1980. 7. G . 0.Fanger, General Background and history of controlled release. Pesticide Symposium (N. F. Cardarelli, ed.), University of Akron, Ohio, 1974. . Baker andH. K. Lonsdale, Principles of controlled release, in Proceed8. R. W ings of Controlled Release Pesticide Symposium (F. W. Hams, ed.), Wright State University, Dayton, Ohio, 1975. 9. D. H. Lewis and D. R. Cowsar, Principles of Controlled Release Pesticides. A.C.S. Symposium Series 53, A.C.S., Washington, DC, 1977. 10. R. Arshdy, Preparation of nano and microspheres by polycondensation, J. Microencaps. 6:l-12 (1989). 11. R. Arshdy, Preparationof nano and microspheres by interfacial polycondensation, J. Microencaps. 6:13-18 (1989). 12. R. Arshdy, Preparationof albumin microspheres and microcapsules, J. Controll. Rel. 14:111-131 (1991). 13. C. A. Finch, in Encapsulation and Controlled Release.(D. R. Korsa andR. A. Stephenson, eds.), Royal Society of Chemistry, 1993. 14. J. E. Vandegaer and N.J. Wayne, U.S. Patent 3,577,515. 15. J. Shimkin, U.S. Patent 4,497,793,1985. 16. P. A. Cantilli, German Patent 2,722,973, 1977. 17. R. C. Koestler, U.S. Patent 4,360,376, 1982. 18. K. B. Scher, German Patent 2,312,059,1973. 19. H. B. Scher, German Patent 2,648,562. 20. H. B. Scher, German Patent 2,706,329,1977. 21. H. B. Scher, Belgian Patent 867646,1978. 22. G. B. Beestman, Eur. Patent 17409,1980. 23. P. J. Allert, German Patent 2726539,1977. 24. G. B. Beestman, Eur. Patent 148169,1985. . Magin, U.S. Patent 4,563,212,1986. 25. Z . B. Becher andR. W 26. G. B. Beestmanb, U.S. Patent 4,640709,1987. 27. A. Markus andZ.Pelah, U.S. Patent 4,851,271,1990. 28. M. N. Hitsu and K. Kabushiki, U.K. Patent 1,091077, 1967. 29. H. P. Crodts andJ. E. Karloske, U.S. Patent 4,517,326, 1983. 30. K. Riecke,U.S.Patent4,622,267,1986. 31. G. Garber, B. Chatenet, and P. Pellenard, U.S. Patent 4,309,213,1982.
1 . Geissbuhler, H. Advances in Pesticide Science, Part 3. Pergamon Press, 1979,
91
32.C. H. Nemeth, U.S.Patent 4,107,292,1978. 33.Bayer,Eur.Patent50,264,1981. 34. Ciba-Geigy AG, Eur. Patent 214936,1986. 35. A. Markus, Z .Pelah, and N. Aharonson, Israeli Patent 84219, 84910, 1992. 36. J. Margalit, A. Markus, and Z. Pelah. Effect of encapsulation on the presistence of Bacillus thuringiensis var. israelensis serotype H-14, Appl. Microbiol. Biotechnol. 19:1982, (1984). 37. J. R. Connick, W . R. Walker, and W . R. Goynes, Jr., Sustained release of mycoherbicides from granular formulations. 10th International Symposium on Controlled Release Bioactive Materials. San Francisco, 1983, p. 283. 38. H. L.Walker and J. R. Connick, Sodium alginate for production and formulation of mycoherbicides, WeedSci. 31:333 (1983). . J. Lumsden, and J. Connick, Encapsulation of 39. D. R. Farvel, R. D. Marois, W potential biocontrol agents in an alginate-clay matrix, Phytopathology 75:775, 1985.
5
The Useof Factorial Designin the Development of Nanoparticulate Dosage Forms
Baruch Magenheim,* and Simon Benita
School ofPharmacy, The Hebrew University of Jerusalem, Jerusalem, I s r a e l
Pascal Wehrl6
Laboratoire de Pharmacotechnie, Centredes Recherches Pharmaceutiques, Universitk Louis Pasteur, Strasbourg, France
I. Introduction
11. The Principle of Experimental 97 Design 111. Illustration of Experimental Design Application
IV. 100 Statistical Methodology 100 A. Preliminary Study B. Sequential Experimental Design Building 100 Modelsof the C.106 Validation V. Effects of the Controlled Variables on Nanoparticle Formation Properties 109 System A. PLA-Acetone-Dichloromethane B. Dichloromethane-Indomethacin-Poloxamer188 System C. Conclusion on the Application of Experimental Design to Nanoparticle Formation Process VI. Summary VII. Symbols
94
98
and
107
120 121 124
*Currentafliliation: Agis Industries Ltd., Yeruham, Israel.

93
94 1.
Magenheim et al.
INTRODUCTION
In the last two decades, the international scientific community has expended enormous efforts to create colloidal carriers with improved drug initial developdelivery that are appropriate for systemic use 11-91, and the ment of nanoparticulate dosage forms should be viewed in this context. Colloidai drug carriers mainlyinvolve submicrometer emulsions, nanoparticles, liposomes, and lipid complexes. The body distribution of the active molecules depends on their physicochemical properties. Therefore, the molecules reach the target organ but also diffuse and distribute throughout the entire body, sometimes exercising harmful and even toxic effects to healthy organs and tissues. This is the case with various anticancer drugs [l] and some antifungal drugs such as amphotericin B [lo]. Attempts have been made to modify the pharmacokinetic profile of various therapeutic classes of drugs through their incorporation in colloidal carriers [3,11-191. These references are not intended to represent anexhaustive or up-to-date citation of the literature but rather to reflect the efforts made to evaluate the drugbiodistribution of these loaded colloidal carriers following intravenous administration. The potential applications of nanoparticles as colloidal drug carriers by different ways of administration have been extensively reviewed [20291. Evidence has been reported by various investigators [30-361 that, following oral administration, nanoparticles may cross the intestinal mucosa or traverse the mesentery lymph vessels toward the lymph nodes. Nanoparticles may consistently improve the bioavailability of poorly absorbed drugs [36]. Some success has been achieved in enhancing the efficacy and reducing the toxicity of anticancer drugs loaded in nanoparticles, as reported in some reviews [21-231. These encouraging results have focused more andmore interest on nanoparticles as promising colloidal drugs carriers. Recently attempts have been made to deliver drug-loaded nanoparticles by means of novel routes of administration (e.g., transdermal, lymphatic) [37-401. Furthermore, new strategies are now emerging in nanoparticle research to allow the delivery of antisense oligonucleotides and DNA fragments, with incursion in the expanding field of gene therapy [41,42]. The potential applications of nanoparticles in AIDS treatment are also currently under investigation [43-451. Nanoparticles have been well defined by Couvreur [22] as a solid submicrometer drug carrier of a polymeric nature in the nanometer size. The nanoparticles are divided into two main groups: nanospheresand nanocapsules. Nanospheres are polymer matrices in which the drug is dissolved or dispersed, whereas nanocapsules consist of a polymer wall entrapping an oily core in which the drug is dissolved [46,47]. Since the term
Factorial Design of Nanoparticulate Dosage Forms
95
nunoparticles refers mainly to nanospheres in the literature, this terminology is used throughout this chapter. A large number of processes for the preparation of nanoparticulate drug carriers have beendescribed in the literature and have been reviewed by some authors in the past [48], as well as in this book. The physicochemical characterization of nanoparticles has been reviewed as well [49]. The readers are referred to the above-cited references which present the current status of nanosphere and nanocapsule manufacturing processes and characterization. Nanoparticles are usually intended for parenteral administration. The polymers for parenteral use have to fulfill a series of strict requirements. They must be nontoxic, nonimmunogenic, biodegradable into nontoxic metabolites, and free of impurities. It is also desirable that they be easy to process and possible to prepareeconomically in large quantities [50]. Polyalkylcyanoacrylates (PACA)and polyesters arethe polymers most extensively used to form thewall or matrix of biodegradable nanocapsules or nanospheres, respectively. The research group lead by Couvreur exhaustively investigated the PACA nanoparticles, and further information should be sought in their numerous publications [22,51]. Polyesters are biodegradable polymers with long-standing safe use and acceptance as surgical sutures. They are easily prepared as homo- and copolymers in a wide range of molecular weights. These biodegradable polymers undergo simple hydrolysis forming naturally occurring nontoxic metabolites. Their degradation pathway is byhomogeneous, bulk degradation [50]. The family of the poly(a-hydroxy acids), mainlypolyglycolic and polylactic acids, and copolymers wasextensively studied in the field of drug-delivery systems development [50]. The preparation of polylactic acid (PLA) nanocapsules was first reported in the literature by Fessi et al. [52] using an interfacial deposition process following solvent displacement. The PLA nanocapsules were thoroughly investigated and exhibited marked advantages over previously studied colloidal carriers [53]. Despite promising experimental results, it was reported that these nanocapsules were not able to retain the drug in their oily internal core more thana few minutes under perfect sink conditions [54]. Therefore, PLAn'anoparticles in which the drugwas entrapped in the polymeric matrix in the absenceof any oily core material were prepared on the basis of the method reported by Fessi et al. [52] with some modifications [55581. Other investigators have also recently adopted a similar method for investigating the incorporation of drugs in PLA nanospheres [59,60]. The authors of this chapter prepared nanoparticles able to retain drugs over longer periods of time following their immersion in the release
96
Magenheim et al.
medium under perfect sink conditions which usually prevail in physiological conditions. We also reported theinfluence of the manufacturing process variables and formulation parameters on nanoparticle characteristics and drug release profile [55-581. The materials and methods utilized were reported in detail in the various cited publications. Therefore, only a schematic representation of the nanoparticle manufacturing process is shown in Fig. 1. The most dominant variables in the search for optimal nanoparticle formulation conditions were the concentrations of PLA, acetone, dichloromethane, indomethacin, and Poloxamer 188. Between these different parameters responsible for nanoparticle formation some interactions occurred. A faster and more accurate identification of these interactions was carried out by a factorial experimental design allowing modelization
Fig. 1
Schematic representation of the nanoparticle manufacturing (From ref. 58.)
process.
Factorial Design
of Nanoparticulate Dosage Forms
97
using multiple regression. At the same time, an attempt was made to achieve optimization of the formulation. Experimental design is the application of statistical principles for data collection [61]. A designed experiment introduces systematic changes into the variables of a system with the objective of discovering which variables are responsible for the observed changes in the output. In a designed experiment more informative data can be obtained from much fewer experiments than from traditional experimental techniques. This cost-saving approach allows researchers to collect useful data from every point of the experiment. This methodology has been introduced in the last few years and is being used more frequently with high success rates in the pharmaceutical sciences [62-641. Some recent examples of the fields of application include analytical chemistry [65-701, granulation and formulation of tablets [71-741, the preparation of beads [75], microcapsules [76,77], and microspheres [78,79]. In fact, some investigators use experimental design in the formulation of nanoparticles [SO-821. Experimental design was usually utilized only for formulationoptimization purposes. By this method, few experiments allowed the selection of the optimum conditions of product manufacturing in a multivariable environment. The objective of this chapter is to illustrate the application of the experimental design technique not only for optimization but also for elucidation of the mechanistic aspects of nanoparticle formation by spontaneous emulsification, as depicted in Fig. 1. The technique enables a huge body of information to be gathered and statisticallyvalidated regarding the simultaneous influence of all the various controlled variables on nanoparticle characteristics and on the interactions between these variables. The information achieved while performing a relatively reduced amount of experiments has been exploited to gain physicochemical insights of the underlying complex phenomena involved in nanoparticle formation.
II. THE PRINCIPLE OF EXPERIMENTAL DESIGN

Theoretically, all the parameters that can affect the physicochemicalproperties of a final product may be included in the experimental design. However, in pharmaceutical manufacturing processes, there may be numerous important parameters due to the large number of ingredients usually required for formulation. Even the equipment used in the multiple processing steps provides additional sources of variability. If all the variables are included in the experimental design, the search process may become fastidious and lead to a prohibitive number of experiments even if a screening approach such as the Hadamard matrix is initially applied. Therefore, ex-
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perimental design can be an important tool and asset if it is exploited by an investigator who has previous knowledge on the behavior of the system. The investigators must first identify the potentially dominant parameters that should be subjected to the screening process in order to limit to a reasonable extent the number of experiments needed. Some of the variables are initially fixed for technological, regulatory, or economical reasons. Other variables are found in preliminary experiments to be critical and therefore cannot be subjected to any variation without affecting fundamentally the desired final product features. A delicate equilibrium is established between what is and is not worthinvestigating. The researcher must not neglect important factors which may lead to misleading interpretations of the system behavior. Furthermore, factors with a priori negligible or in contrast vital effects have to be excluded inorder to simplify the experimental design. Investigator skills, knowledge, and experience play a crucial role in selecting the appropriatevariables. It should be pointed outthat in cases where all dominant variables are not included in the experimental design, even the most sophisticated models will usually not be useful. In these cases, the researcher should consider the inclusion of additional variables previously omitted. The sequential building nature of the experimental designs enables the goal to be achieved with an addition of only a minimal number of experiments.
111.
ILLUSTRATION OF EXPERIMENTAL DESIGN APPLICATION
To illustrate the previous statements, a practical example of the application of experimental design to the study of nanoparticle formation process is given here. In preliminary investigative work, dozensof variables were examined in an attempt to select the most important variables affecting the nanoparticle properties. This obviously leads to a huge numberof experiments. However, initial results and experience gained in disperse systems allowed i x the nature and concentration values of most of the preeminent us to f variables. Biocompatibility, biodegradation, and regulatory acceptability motivated the selection of polyesters as nanoparticle-forming polymers. Among these polymers, the fast release of the drug from nanoparticles prepared with different copolymers of polylactic and polyglycolic acids resulted in the choice of d,l-PLA. Technological drawbacks excluded the use of other PLA stereocopolymers. Some organic solvents like chloroform were rejected owing to potential toxicity and carcinogenicity. The semipolar solvent acetonitrile was found to have a strong interaction with dichloromethane, preventing nanoparticle formation. Regulatory require-
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ments favored the selection of Poloxamer 188and human serum albumin as hydrophilic surfactants. However, very lowdrug entrapment was observed, with the latterapparently due to its protein-binding capacity. Phospholipids promoted the simultaneous formation of liposomes, leading to a mixed undefined nanoparticulate-liposomal system that was not desired. The temperature of the organic and aqueous phases andtheir stirring rate did not show any effect in the range studied. On the other hand, some of the variables such as pH and thesolvents evaporation rate were foundto beso critical that any variation would result in a drug entrapment yield too low to be pharmaceutically acceptable. The controlled variables initially considered worth studying were the amounts of PLA, acetone, and dichloromethane used inthe manufacturing process (PAD system). This systemallows a priori different aspects of nanoparticle formation to beclarified. It permits the evaluation of possible interactions between acetone and dichloromethane during the migration of the formerfrom the organic phase to theaqueous phase and the degreeby which both solvents affect PLA precipitation. The consequent changes in the ratio of the PLA to thesolvents may highlight the potential impactof the organic phase viscosity on nanoparticle characteristics. Additionally, by selecting only three controlled variables, it is possible to readily obtain descriptive graphs which allow an easy visualization of the effects, thereby facilitating their evaluation. The general statistical methodology based on multiple regression using the generalized least squares theory and which allows plotting this graphs is calledthe response surface methodology [65]. Many fundamental aspects of the nanoparticle formation mechanism were elucidated from these series of experiments [58-591. The drugpartitioning between the organic and aqueous phases was shown to play an important role. Thus, a second set of controlled variables was investigated in order exhaustively to assess the partitioning contribution to drug entrapment in the nanoparticles. This set consisted of the amounts of dichloromethane, indomethacin, and Poloxamer 188 used inthe manufacturing process (DIP system). They wereselected for the following reasons: (1) The solubility of indomethacin in dichloromethane and in the aqueous phase ultimately determines its partitioning behavior; (2) the solubility inthe aqueous phaseobviously is dependent on the surfactant (Poloxamer 188) concentration; and (3) variations in poloxamer concentrations may help to estimate the effect of the surfactant on incipient nanoparticle stabilization. The most appropriate way to carry out the research would be to add these threenew controlled variables (DIP) to the experimental design used to evaluate the first three variables (PAD). Indeed, dichloromethane was common to both systems. However, the results obtained with the PAD system demonstrated that a level of less than 0.5 mL of dichloromethane
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led to the formation of nanoparticles with unacceptable properties. Thus, the PAD experimentaldesign couldnot serve as a base to evaluate the DIP effects with a complementary design. Nevertheless, it did allow for the concentrations of acetone and PLA to be set preferential for use in the DIP experimental design. Before presenting a detailed explanation of the experimental design results, an introduction to the statistical methodology is presented for a better comprehension of the subject.
I V . STATISTICAL METHODOLOGY
Until recently, formulation and processes were investigated and developed simply by trial and error while varyingone factor at a time. Needless to say, this approach is both time and energyconsuming as well as costly. Furthermore, in the worst case scenario, misleading conclusions can be drawn, particularly when different variables interact. Owing to the multiple requirements, the modem designer can no longer be satisfied with a method that leads to uncertain and nonvalidated results. The use of a statistical approach is necessary. The response surface methodology [64,83-861 using an experimental designisaneconomical method aimed at collecting a maximum of information with a minimum of experiments. Another important advantage of this general method is that the experimentaldesigns can be changedprogressively until an appropriately efficient model is found to describe the studied phenomenon.
A. Preliminary Study
Before building a factorial design, the experimental field must be defined precisely. If it is too large, it may lead to nonrealistic experiments and result in microenvironments inwhichsystem behavior is different (like peaks or valleys on a topographical map) and may be not detected.If it is too small, it may be far from the optimal region. Results yielded in preliminary work allowed the experimental fields to be defined corresponding to the inner volume of the cubes described in Figs. 2 and 3 for the PAD and DIP systems, respectively.
B. SequentialExperimentalDesignBuilding 1. Hadamard Matrix
In formulation or process optimization tasks, a great number of controlled variables such as amountof excipients or process parameters may have an influence on the studied characteristics (defined as response variables).
Factorial Designof Nanoparticulate Dosage Forms
101
0.50
S
Y
@
(D
0
25
io
I
0
@
15
5 0 D
,W'
0
0.00
PLA (mg)
Fig. 2 Description of the experimental field and the Hadamard matrix, 23,and 33 experimental designs of nanoparticle formulation for the PAD system (see details in text). (From ref. 38.)
INDOMETHACIN (%)
Fig. 3 Description of the experimental field and the Hadamard matrix, 23, and Box andWilsonexperimentaldesignsofnanoparticleformulationfor the DIP system (see details in text).
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The analysis of all these factors generally leads to a prohibitive number of experiments to be performed. Ascreening experimental design is the most convenient way to reduce this number. Such an experimentaldesign allows (by multiple regression based on the generalized least squares theory) the calculation of a very simplemathematical model.The polynomial equation (Eq. 1) includes only the main effects (terms of first order) of the different controlled factors (X,) on theresponse variable (Y):
I=I
For thepresent experimental field:

Y =k
+ ax, + bX, + cX,
(2)
where k is the constant term (corresponding to boin Eq. 1) and a, b, and c are the regression coefficients (corresponding to b, in Eq. 1) of the dichloroequation. X,, X,, and X, correspond toPLA,acetone,and methane for the PADsystem and to dichloromethane, indomethacin, and Poloxamer 188 for the DIP system, respectively. Hadamard [87] developed an original matrix allowing the evaluation of such a model, based on the following equation:
=
l+Hn+Hn I
+Hn-H,
So that, for example, matrix H, (for n = 2) can be established by
recurrence calculation:
+l +l +l +l +l -1 +l -1 H . , = +l +l -1 -1 +l -1 -1 +l
As for any experimental design, this matrix gives incoded values -1, +l, the levels of the controlled factors that are to be set for the experiments (Table 1). For thefactors studied in this example, theactual values are reported are defined by any setof four in Table1and the corresponding experiments nonconsecutive vertices of the cubesdefined in Figs.2 and 3. One can see that only four experiments are needed to estimate the linear effects of three different parameters. The four experiments are performed and the response measured. If A is the matrix column containing the constant and the regression coefficients of the model to be evalu-

Table 1 Matrix of Experiments for the Hadamard Experimental Design
103
no.
1 2 3 4
Experiment
" iirzzTG
k
x 1
x 2
x 3
Real values, PAD Real values, DIP system system L (mg) (mL) (mu (%l (%)
1 5 5 15 5
0.50 0.00 0.00 0.50
+l +l +l +l
+l
-1 +l -1
+l
+l
-1 -1 +l
+l
-1 -1
75 75 25 25
0.75 0.50 0.75 0.50
8.75 8.75 6.25 6.25
2.00 0.50 0.50 2.00
ated (Eq.5 ) , if X is the Hadamardmatrix (H4) (Eq. 4) and if Y is the matrix column containing the four measured responses (Eq. 6 ) ,
the theory of the least squares allows usto write:
Y=A*X
so that
A = X"
The inversion operation (-l) is generally a complex calculation for which a pocket calculator or a computer is needed. But here, because the Hadamard matrix is of size n.n, symmetrical, orthogonal, and contains only +l and -1 values, the multiple regression calculation can be done very easily:
(where nis the number of experiments and tis the transposing operation). Finally,
The model is validated by performing an additional central experiment. If the experimental result value for this point is different from the
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expected value, the model is rejected. Other models which take into consideration the interactions between thefactors can beapplied by adding only a few experiments tothe previous design.
2. Experimental Design of z3Type The extreme vertices of the experimental fields shown in Figs. 2 and3 constitute a typical 2kexperimental design, withk the numberof controlled variables, 3 in this example. The generalized least squares theory is applied again in order to evaluate a model including the interaction Xi Xj:
3. Models for the Analysis of Quadratic Effects If the model including the first-order terms of main effects aswell as the second-order term of interaction does not fit the data, the response surface is probably not plane. Thus, this potentially important curvature can be described by a more complex model taking into account the quadratic effects (X,') of each of the controlled variables as reflected by Eq. (12):
Y =C bo b+ , .C Xb ,+ u * X , .Z Xb j+ u-x (12)
a. Box and Wilson composite design. The Box and Wilson method [84,85] is the most economical way of evaluating the model described by Eq. (12). This design is defined by the addition of six experiments at an axial distance of & CY from the center of the cube in directions passing through the center of each of the six faces, to the previous design (points outside the cube; see Fig. 3). The CY value is selected according to orthogonality and rotatability criteria and depends also on the number of central point replicates and controlled variables. The equations applied for CY selection are beyond the scope of this presentation and can be found in textbooks dealing with statistics and experimental design. Orthogonality implies that theestimates of the regression coefficients of the model are uncorrelated. This property is verified for the Hadamard and 23 experimental designs. For a composite design, only quasiorthogonality can be achieved. The rotatability property leads to a variance function which is constant on circles centered at the origin. The level of this function is dependent on the distance to the origin and not on thedirection from this origin. Eq. (12) can be translated for the DIPsystem as:
=k
+ d.DCM.IND + e.DCM.POL + f.IND.POL +g DCM' + h.INDZ + i.POL'
+ a.DCM + b.IND + c.POL
(13)
Factorial Design
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where k is the constant term and a-i are the regression coefficients. DCM representsthedichloromethanevolume,andINDand POL the indomethacin and Poloxamer 188 concentrations in the formulation, respectively. It should be noted that the term (DCM.IND.POL) for the triple interaction between these three controlled variables is not included in Eq. (13). This is dueto constraints associatedwith the Box and Wilson quasiorthogonality condition which excludes any third-order term in the derived mathematical model.
b. Experimentaldesign of 33 type. The efficiencyof the experimental designs is defined as the ratio between the number of terms in the mathematical equation describing the model and the number of experiments needed to perform the appropriate experimental design. For three controlled variables, the efficiency increases from 34.5% for a 33experimental design up to 58.8% for the corresponding composite design using three center replicates in both cases. However, the Box and Wilson composite design described above cannot be applied to the PAD system, because it leads to negative values for some of the controlled variables (DCM CO). Therefore, a complete 33 type design must be applied by adding 18 experiments to the23 design, 1 at themiddle of each of the 12 edges and 1at the center of each of the six facesof the cube defined in Fig. 2.Eq. (12) can be translated for thepresent system as:
Y =k
+ a.PLA + b.ACE + c.DCM + d.PLA.ACE + e.PLA.DCM + f.ACE.DCM + g.PLA.ACE.DCM + h.PLA2 + i.ACE2 + j.DCM2
(14)
where k is the constant term and a-j are theregression coefficients. DCM represents the dichloromethane volume,PLA the weight of polymer, and ACE the acetonevolume in the formulation. In contrast to the composite design, in the 33-type design, there are much more experiments than the number of terms of the derived matheso matical equation. Therefore, orthogonality can never be reached that the third-order term (PLA.ACE.DCM) can be included as shown in Eq. (14).
c. Reducingapproach-precise analysis. Having the 33experimental design utilized inthe study of the PAD system three controlled variables and one response variable at a time, a four-dimension space is defined, which cannot be graphically represented. Hence, further analysis keeping one of the threeresponse variables constant at onelevel each timewas performed (32models). This allowed the calculation and validation of models for each described in Fig.2 and the drawing of surface face of the experimental cube response and contour plots which permited an easy visualization of the
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different effects. For example, acetone and PLA were precisely analyzed for the different constant amounts of DCM. An identical procedure was used where PLA and acetone were kept constant at different amount levels as for DCM. Applying the same concept to the Box and Wilson design utilized in the study of the DIP system results in the definition of 22 models. This occurs because, differently than in the 33design, in the present case, the experimental points added to the 23experimental design are located outside the cube defined in Fig. 3, at an a distance from its center.However, these 2* models do not allow the detection of quadratic effects. Therefore, to allow the drawing of surface response and contour plots, the equations obtained with Box and Wilson design-derived models were recalculated while keeping the value of one of the variables constant each time. Being that the models derived from Box and Wilson experimental design validated, the recalculated equations are validated as well. Only illustrative graphs obtained at fixedlevels of indomethacin of 7.5% w/v are presented.
C. Validation of the Models

The above models used to evaluate the different response variables were validated by means of the following statistical tests:
1. Analysis of variance (ANOVA) of which the principle is given in Table 2. The experimental F-ratio was compared with the theoretical value:For(p-l,n -p), where a ! is the chosen risk set in this study at 0.10.
Table 2 Principle of the Analysis of Variance Source
ss
DF
MS
F ratio
Residual Total
i= 1
n-l
Factorial Design
of Nanoparticulate Dosage
Forms
107
2. Multiple correlation coefficient (Rz). It gives the part of the variation explained by the model. It is calculated by using Eq. (15).
3. The lack of fit test which indicates if the lack of fit between the experimental values and the values calculated according to model equations can be explained by the experimental error. This test was performed exclusively on the Box and Wilson and the 33 experimental designs, because they were the only designs which needed to be subjected to this test according to the acceptability criteria described in Table 3. To perform this test, three additional nanoparticle batches with a formulation determined by the central point of the experimental designs were manufactured. The principle of this test is briefly described in Table 4. The experimental F-ratio obtained is compared with the theoretical value: Fa(n-p-c+l, c-l), where a is the chosen risk set in this study at 0.01. Further details should be sought in statistics textbooks.
The Student t-test, was further applied for each term of the polynomial equations to evaluate theirsignificance.
V. EFFECTS OF THE CONTROLLED VARIABLES ON NANOPARTICLE FORMATION AND PROPERTIES
The controlled variables studied in this particular example were the amounIts of PLA, acetone, dichloromethane, indomethacin, and Poloxamer 188 used in the manufacturing process. As explained above, two sets of controlled variables were studied separately. The first set was composed of PLA, acetone, and dichloroTable 3 Acceptability Criteria
Analysis of variance Correlation coefficient Lack of fit
P P
5 2
Model validation
9
R*2 0.9
0.01 0.10
YES YES
0.7 zs Rz <0.9
P <0.01
R <0.7
P >0.10
NO NO NO
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Table 4 Principle of the Lack of Fit Test

Source
ss
DF
MS
F ratio
SSresidual-SSpurecmr
Lack of fit
sslack of fit
iEB
n-p-c+l
n-p-c+l
I-
MSlackoffit
MSpureemr
methane (PAD; see Fig. 2), and the secondset included dichloromethane, indomethacin, and Poloxamer188 (DIP; see Fig. 3). To better understand the experimental design approach, the results are presented and discussed separately for each set of data with a comprehensive evaluation in the conclusion section. The response variables evaluated included drug entrapment yield (yield), percentage of free drug (FD), mean particle size (MPS), standard deviation (SD), and coefficient of variation (CV). Since SD andCV provide similarly useful information regarding the polydispersity of the system, only thedataon CVis presented. Drugentrapment yieldis defined as the ratio between theincorporated and the total initial amount of drug. A drug entrapment yield of 100% corresponds to a drug content in nanoparticles of 5.88%. Particle size distribution was measured by photon correlation spectroscopy. In this example,the objective was to analyze the effects of the controlled variables on the parameters that characterize the nanoparticle colloidal system and to elucidate the nanoparticle formation mechanism.
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109
The importance of the drug entrapment yield for nanoparticlecharacterization is obvious and needs no further comment. From the percentage of free drug, itcan be deduced if low drug entrapmentyield is due to poor formation of nanoparticles or toineffective drug incorporation. The particle size distribution of the nanoparticles is one of the most important physical properties of a colloidal dispersion. Indeed, it is explicitly mentioned in the definition of nanoparticle. Sizing of particles in the suboptical range is associated with considerable difficulties, because the particle size value measured by the various sizing methodologies may be different. Thus, one single sizing procedure should be used throughout the entire set of experiments to avoid any misleading interpretation associated with the lack of consistency of the data obtained. Therefore, photon correlation spectroscopy (PCS), which is a convenient, fast, and well-established sizing technique in the submicrometer range,was carefully applied in this example while looking at the polydispersity value of the measurements. Moreover, to rule out the possibility that small amounts of large particles might contaminate thecolloidal dispersion without being detected by PCS, all the batches were further examined by optical microscopy. From the overall results obtained during the preliminary investigation of nanoparticle formation process, the following underlying mechanism was proposed [58]. According to this mechanism, the mixing of the organic and the aqueous phases results in the diffusion of acetone from the organic to the aqueous phase. Part of the PLA which migrates together with the acetone on contact with the aqueousphase precipitates at theinterface owing to its insolubility in water. The remaining PLA isdissolvedin the dichloromethane. Following evaporation of organic solvents, the previously dissolved PLA precipitates thus forming the matrix of the nanoparticle in which the drug is entrapped. Such an hypothesis has also been proposed by other investigators [60,61].
A.
PLA-Acetone-Dichloromethane System
l. General Analysis
In this example, the intent in the first part o f the research was to find a generalmodel able to describe the complete system while varying all the parameters at thesame time. The datadid not fit the Hadamard matrix model (Table 5 ) , suggesting that quadratic effects and/or interactions between the controlled variables, undetectable by this model, may occur.
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Table 5 Statistical Validation ParametersValues Obtained for the PAD System

Hadamard
23
3 3
LOF RZ
0.732 0.672 0.799 0.350
P
Yield
R 2
0.867 0.737 0.937 0.943
P
0.499 0.755 0.136 0.209
R2
0.933 0.712 0.958 0.935
P
0.005 0.017 0.000 0.584
P
0.030 0.409 0.267 0.157
FD
MPS CV
0.454 0.623 0.316 0.303
The 23designwas then applied by adding only four experimental points to the Hadamard matrix. This design permits the postulation of an improved model which has the possibility of detecting interactions between the controlled variables. The data did not fit this model as well (see n b l e 5 ) , reflecting the possible occurrence of quadratic effects unidentified by this model. In thelight of the potential presence of quadratic effects and interactions, a 33design, which allowsfor the postulation of a second-order model able to detect these phenomena, was applied. All the results obtained with the previous design form part of the new model emphasizingthe ability of saving experiments by the factorial design approach. The datafor the drug entrapment yield and the MPS fit this model with high significance (see Table 5). Owing to the numerous experimentalpoints forming the design, R2values were not very high, but the lack of fit test ascribes that to the experimental error,thus validating the model. PLA have a significant influence on It was observed that acetone and drug entrapment yieldwhich increases as the amounts of these factors increase (Table 6). The amount of acetone may affect its migration velocity during the initial formation process of the nanodroplets and subsequently the kinetics of PLA precipitation. PLA precipitation rate is concentration dependent. When more polymer is present in the medium, the free volume available for it is reduced. Therefore, the possible conformationsthatthepolymermoleculescan adopt decrease, whereas the precipitation due to solubility constraints occur more readily. The deposition of the initial thin wall of PLA around the emulsified organic phase nanodroplets prevents further diffusion of the drug toward the aqueous medium. Hence, a faster PLA precipitation driven by higher amounts of acetone or PLA results in higher drug entrapment yield. The density of the polymer matrix formed increased with higher PLA concentrations resulting in an even higher yield.
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Table 6 Regression Coefficients and StatisticalValidation of the
Polynomial Individual Terms Obtained with the 33 Model for thePAD System Drug entrapPercentage drugfreeyield ment Term RC
-16.27 1.87 9.49 103.69 -0.04 -2.61 -1.60 -0.01 -0.29 -131.37 0.18
of
Mean particle size RC

664.37 -6.59 -79.02 321.20 .0.19 6.22 -36.23 0.05 3.20 382.76 -0.44
P
0.681 0.089 0.084 0.312 0.399 0.133 0.848 0.211 0.201 0.156 0.267
RC
7.77 -0.13 -0.53 -3.33 0.00 0.05 -0.26 0.00 0.01 16.03 -0.01
P
0.028 0.142 0.233 0.690 0.519 0.741 0.709 0.145 0.415 0.043 0.696
P
0.008 0.273 0.015 0.574 0.505 0.513 0.447 0.342 0.020 0.452 0.615
k PLA ACE DCM PLA*ACE PLA*DCM ACE*DCM PLA~
ACE^
DCM~ PLA*ACE*DCM
The model describing the influence of the controlled variables on the percentage of free drug was validated by ANOVA (see Table 5), but the R2 value (0.672) was below the value set in the acceptability criteria (0.700; see Table 3). However, since the experimental R2 was so close to the preset value and the lack of fit test reached statistically acceptable levels, the effectof the controlled variables emerging from this model can be analyzed as a trend even though the model is not strictly validated. The quadraticinfluence of the amountof dichloromethane on the percentage of free drug (seeTable 6) is due toa more favorable drug partitioning into the organic phase when higher amounts of dichloromethane are present. The slight trend of effect of PLA (linear and quadratic; see Table 6) is related to the density of the polymer matrix formed, which affects the further partitioning of the drug between the nanoparticles and the aqueous phase. Acetone exhibits the greatest influence on the mean particle size, which decreases as the amountsof acetone increase (seeTable 6). A large concentration gradient of acetone acceleratesits migration to the aqueous phase during the initial formation process of the nanodroplets, and hence PLA precipitation occurs earlier, leading to smaller nanoparticles. It was not possible to describe the simultaneous effect of the controlled variables on the standarddeviation or thecoefficient of variation (see Table 5). This is apparently dueto thelack of accuracy of these specific measurements, as ' values. reflected by the low R
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Magenheim et al.
The factors that affect the drug entrapmentyield (acetone and PLA) are different from those affecting the percentage of free drug (dichloromethane). This difference may be attributed to thedifferent mechanisms governing eachaspect of the overall process. In the former case, the precipitation of the PLA promotes the formation of the embryonic nanoparticles. The extentof this precipitation, which is affected by the amounts of acetone and PLA, determines the amount of drug that will be entrapped. In the latter case, the drugpartition between the dichloromethane and the aqueous phase determines the extentof free drug in the aqueous phase.
2. ReducingApproach-PreciseAnalysis a. PLA and acetone variation at constant DCM levels. Drug entrapment yield dependsontheamount of acetoneat a level of 0.5 mL of dichloromethane and on PLA in the absence of dichloromethane (Table 8 and Fig. 4a and b, respectively). It appearsthat the mechanism of nanoparticle formation is different in both cases. In thefirst case, a typical emulsion of dichloromethane in the aqueous phase is formed. In the second case, probably an organic acetonic phase rich in PLA was emulsifiedin the aqueous phase despite the good aqueous solubility of acetone. Therefore, in the first case, the rapid migration of the acetone is vital to the development of the first nanoemulsion, whereas in the second case, the rapid precipitation of the PLAis the key factor. No fitting to the drug entrapment yield and the percentage of free drug was observed for 0.25 mL of dichloromethane (Table 7). It appears that atthis intermediate dichloromethane level, various indefinite complex systems are obtained which are difficult tobe analyzedby one single model, and thus no model is validated for these response variables. The data of the percentage of free drug did not fit any model (see Table 7). Such behavior may be attributed tothe fixation of the 'dichloromethane level, which was the main factor affecting the free drug response variable. The models describing the effects of PLA and acetone on the mean particle size were validated for all the dichloromethanelevels (see Table 7). For thelevels of 0.00 and 0.25 mL, no terms of the mathematical equations were statistically significant (Table8). Therefore, nodefinitive conclusions . could be derived in these cases. The significant linear and quadraticeffects of acetone and the interaction between this solvent and PLA observed in the case of 0.50 mL of dichloromethane (Table 8) are due to the reasons 33model. explained above for the It should be noted that the mean particle size of the nanoparticles obtained using 0.50 mL of dichloromethane were higher than those ob-
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_. -
- l\
(W
Fig. 4 Drug entrapmentyield as afunction of PLA and acetone amountin batches prepared using(a) 0.50 mL and (b) without dichloromethane.
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Table 7 Statistical Validation ParametersValues Obtained for the 3 Models

Corresponding to the Various ConstantLevels of Each Controlled Variable of the PAD System
~ ~~ ~~
Fixed variable Dichloromethane
Level
0.00 mL
Test
Yield
0.062 0.927 0.552 0.614 0.014 0.974 0.212 0.824 0.165 0.854 0.992 0.110 0.361 0.733 0.132 0.876 0.226 0.815
FD
0.121 0.884 0.801 0.424 0.107 0.894 0.304 0.767 0.112 0.890 0.059 0.930 0.234 0.811 0.158 0.859 0.513 0.639
MPS
0.031 0.955 0.081 0.912 0.003 0.990 0.001 0.996 0.042 0.945 0.100 0.899 0.111 0.891 0.145 0.867 0.109 0.941
CV
0.380 0.722 0.003 0.992 0.479 0.661 0.542 0.621 0.891 0.327 0.701 0.508 0.404 0.707 0.350 0.740 0.285 0.780
0.25 mL
0.50 mL
Acetone
5 mL
10 mL
15 mL
P R P R P R P R P
R2
P
R2
PLA
25 mg
50 mg
75 mg
P R P R P R
tained without dichloromethane (Fig. 5c and a, respectively). Mean particle size depends strongly on the amount of acetone at a level of 0.5 mL of dichloromethane and is almost independent from it in the absence of dichloromethane(see Table S). This difference is attributed to different mechanisms of nanoparticle formationas described above. The profiles of the graphs depicting the influence of acetone andPLA on the mean particle size of the nanoparticles obtained using 0.25 mL of dichloromethane are almost identical to those obtained without dichloromethane (Fig. 5b and a,respectively). This is mainlydue to thesolubility of dichloromethane in water at a low concentration.
b. DCM and acetone variation at constant P L A levels and P L A and DCM variation at constant acetone levels. PLAandacetone play primordial roles in the formation process of nanoparticles. Therefore, with a single exception, novalidated models for thedrug entrapment yield and percentage of free drug were achieved when either PLA or acetone were kept constant while the remaining parameters were varied (see Table 7). Furthermore, as previously shown, at intermediate DCM concentrationscomplex colloidal systems were obtained. Thus, in anycase where the DCM concen-
115
el
a,
2
U d
el
a,
2 "d
cl
a,
8 " d
cl
0
E
p!
2 c
cl
8
0
116
Magenheim et al.
Fig. 5 Meannanoparticlesizeasafunction
of PLA and acetone amountin batches prepared using (a) 0.00, (b) 0.25, and (c) 0.50 mL of dichloromethane. (From ref. 58.)
tration is varied, as required when PLA or acetone are kept constant, the experimental field will include a range of concentrations leading to indefinite colloidal systems which are unlikely to be fully characterized. With acetone as the major factor affecting the particle size when its value is fixed at some level, the variation of the other factors is unable to alter the particle size distribution that remained almost unchanged over a reasonable concentration variation range (Table 9 and Fig. 6a-c). At high PLA and low acetone concentrations, a dramatic increase in MPS was noted (Figure 6a) which can be attributed to thehuge increase in viscosity of the organic phase and thus preventing the formationof optimal nanoparticles. It was not possible to describe the simultaneous effect of the controlled variables on the standard deviation or the coefficient of variation (see Table 7). This was apparently due to the lack of accuracy of these specific measurements, as reflected by the low R values. No model could be validated in any of the levels of PLA for the particle size (see Table 7).
c.
Interbatch variability. The nanoparticles prepared using 0.25 mL particle size DCM, 25 mg PLA, and 5 mL acetone, exhibited poor interbatch measurements reproducibility. Some of the batches obtained were within the range of data values previously described, whereas others exhibited a
117
118
et
Magenheim
al.
Table 9 Regression Coefficients and Statistical Validation of the Polynomial Individual Terms Obtained in the Mean Particle Size Equation for the Dichloromethane and PLA Variation at Constant Acetone Levels in the PAD System
5 mL
10 mL
15 mL
RC
Term
k DCM PLA DCM*PLA DCM* PLA~
212.57 -805.20 -2.72 4.86 2361.60 0.04
P
0.025 0.008 0.296 0.047 0.001 0.158
RC
128.06 -95.33 -0.28 0.12 314.67 0.01
P
0.016 0.253 0.818 0.884 0.061 0.426
RC
100.39 -124.00 0.19 0.44 274.67 0.01
P
0.069 0.278 0.909 0.705 0.163 0.685
Factorial .Designof Nanoparticulate Dosage Forms
119
120al.
et
Magenheim
much larger mean particle size and polydispersed population distribution. Therefore, two independent analyses were carried out, one for each of the nanoparticle populations. The results reported are for the reproducible type of populations. For the larger and heterogeneous populations,no general models could be validated, and therefore the results are not presented. Apparently theconcentration of the formulation components in this special combination led to complex instability problems which resulted in insufficient control of the nanoparticle manufacturing process.
B. Dlchloromethane-Indomethacin-Poloxamer 188 System
The data did not fit the Hadamard matrix and the 23model for any of the responses variables (Table 10). Only the Box and Wilson composite design was able todescribe the behavior of the system, accounting for the complex nature of the physicochemical phenomena involved in the formation of nanoparticles (Table 10). The decreasein the drug entrapmentyield noted while increasing the DCM concentration at low Poloxamer 188 levels (see Fig. 7a) can be attributed to therelative decrease of the PLAconcentration in the DCM phase. This resulted in a slower PLA precipitation and the formation of a less dense matrix, which allowed for a larger diffusion of the drug toward the aqueous phase. The decrease in drug entrapment yield along with the Poloxamer 188 concentration increase at low DCM levels (see Fig. 7a) is apparently due to an increase in drug partitioning. This is supported by the increase in the free drug along with the increase in Poloxamer 188 concentration (see Fig. 7b). The synergistic effect of the simultaneous increase of DCM and Poloxamer 188 on drug entrapment yield is clearly observed in Fig. 7a, accounting for the validation of the interaction term. The moderate increase in the drug entrapment yield observed while increasing the Poloxamer 188 concentration at high DCM levels is speculated to be caused by the migration of part of the excess DCM toward the Table 10 Statistical Validation ParameterValues Obtained for the DIP System
Hadamard
P
R2
2 3
Box and Wilson

R2
0.881 0.984 0.920 0.996
LOF -
P
0.636 0.257 0.538 0.131
P
0.062 0.012 0.114 0.299
R2
0.812 0.890 0.767 0.661
P
0.894 0.090 0.020 0.994
Yield
FD
MPS CV
0.484 0.297 0.401 0.134
0.848 0.944 0.897 0.989
121
aqueous phase promotedby the solubilizing power of high Poloxamer 188 concentrations. In this case, the actual PLA concentration in the DCM increases, resulting in better drug entrapment. The extentof free drug is controlled by the solubilization of the drug in aqueous phase, which increases as the Poloxamer 188 concentration increases (Table 11 and see Fig. 7b). The mean particle size increases at higher DCM concentrations because of the increase in the incipient nanoparticle volume (Tablell and see Fig. 7c). Poloxamer 188 does notaffect the particle size, indicating that low concentrations are enoughto stabilize the system (Table11and see Fig. 7c).
C. Conclusion on the Application of Experimental Design to Nanoparticle Formation Process
The overall results presented in this example are in agreement with the mechanism of nanoparticle formation process that has been previously suggested (see Fig. 8). It was clearly observed throughthe factorial design approach that nanoparticle formation process is very fast, and hence the manufacturing conditions immediately after the aqueous and organic phase mixing determine thephysicochemical properties of the nanoparticles. Only the second-order mathematical models derived from the Box and Wilson and 33experimental designs were able to describe the behavior of the system and account for the complex nature of the physicochemical phenomena involved in the formationof nanoparticles. The migration velocity of the acetone and the kinetics of PLA precipitation are the most significant parameters in nanoparticle formation. The former particularly affects the mean particle size, whereas both have an important influence on the drug entrapment yield. The mean particle size is also affected by the amount of dichloromethane, since it determines the volume of the incipient nanoparticles. The extentof free drugis controlled by a phase partition phenomena which depends fundamentally on the concentrations of dichloromethane and Poloxamer188 in the organic and aqueousphases, respectively. The rapidity of the nanoparticle formation process is also supported by other independent observations demonstrating that no difference was observed in particle diameter before and after evaporation of acetone. This may reflectthe fast and complete precipitation of the polymerin the course of nanoparticle manufacturing process. The kinetics of PLA precipitation are highly dependent onits ratio to dichloromethane. The lack of significance of the interactions between acetone and dichloromethane suggests that both solvents behave independently andthat acetone is not retained by the dichloromethanein its migration toward the aqueous phase.
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Table 11 Regression Coefficients and Statistical Validationof the Polynomial Box and Wilson Composite Design for the Individual Terms Obtained with the DIP System Drug entrapment Percentage yield size particle Mean drug free Term k DCM
IND
of
P
0.887 0.258 0.491 0.056 0.517 0.290 0.230 0.322 0.447 0.018
RC
56.71 180.59 0.53 -10.70 -24.33 46.12 -1.57 -65.29 -3.92 0.97
P
0.558 0.300 0.978 0.581 0.049 0.031 0.389 0.578 0.247 0.417
RC
4.62 67.64 -4.49 -14.41 -2.38 6.65 0.76 40.57 0.31 3.26
RC
353.38 -1421.61 52.32 60.00 -8.80 -57.33 -0.67 1331.75 -2.88 -9.89
P
0.312 0.041 0.440 0.387 0.814 0.372 0.915 0.012 0.490 0.396
POL DCM*IND DCM*POL IND*POL

DCM~ IND2
POL2
Fig. 7 (a) Drug entrapment yield, (b) percentage of free drug, and (c) mean particle sue as a function of poloxamer188 and dichloromethane concentration in batches prepared using indomethacin 7.5% wlv.
Factorial Design of Nanopatticulate Dosage Forms
123
' .
124
Dichloromethane Acetone PLA Drug Poloxamer Water
Magenheim et al.
Dichloromethane PLA Acetone Drug
IC
n
Drug
Drug
Poloxamer
POIOXBIIWH
Dichloromethane
Dichloromethane
Polo
Poloramer
Water
Acetone Drug Acetone

Poloramer
Water
Poloramer
Fig. 8 Schematic descriptionof the proposed nanoparticle manufacturing mechanism. (From ref. 58.)
The nanoparticle systems obtained at the various levels of dichloromethane and their mechanism of formation appear to be different. At a level of 0.5 mL of dichloromethane, a typical emulsion of dichloromethane in the aqueous phase is initially formed, whereas in the absence of dichloromethane, an acetonic phase rich in PLA, insoluble in the aqueous phase, constitutes the internal dispersed phase of the emulsion. In the former case, the rapid migration of the acetone is critical for the development of the first nanoemulsion, whereas in the latter case, the rapid precipitation of the PLAfrom the acetonic phase is the key factor. VI. SUMMARY The use of factorial design in investigating the nanoparticle formation process clearly demonstrates the advantage of this efficient statistical technique in characterizing complex physical systems containing various controlled variables with only a minimal number of experients needing to be performed. Time and fastidious work are saved by this valuable tool which uses the sequential approach to identify the potentially dominant parameters of the studied system. It should be emphasized that the experimental design not only offers the potential of reducing the number of experiments needed to optimize a manufacturing process but may also contribute to a better physicochemical understanding of the phenomena involved in the
125
construction of a specific formulation aimed at resolving a pharmaceutical formulative problem. It is well know that in the pharmaceutical manufacturing processes many important parameters exist owing to the large number of excipients usually required for stable and viable formulations. Thus, investigators cannot andshould not bypass, avoid, or neglect the optimization process of the formulations. Failure to optimize the process of the formulations can lead to problems with safety, regulatory bodies, therapeutic use, and economics. Modern pharmaceutical formulation design requires the use of efficient, comprehensive, informative, and validated techniques that can be exploited to gain physicochemical insights into the underlying complex phenomena generally involved in the formulation of any pharmaceutical dosage form. The factorial design presented in this chapter and applied to optimize the nanoparticle manufacturing process as well as elucidate the mechanism of nanoparticle formation appears to meet the various and complex requirements needed for a potent technique to achieve a global and comprehensive characterization of the drugformulation and manufacturing process.
VII. SYMBOLS
PAD DIP PLA ACE DCM IND POL RC
PLA-acetone-dichloromethane dichloromethane-indomethacinAPoloxamer 188 amount of polylactic acid
amount of acetone amount of dichloromethane percentage of indomethacin percentage of Poloxamer 188 regression coefficient ss sum of squares DF degrees of freedom MS mean of squares n total numberof experiments P number of terms of the model (constant included) Y i measured response Y i estimated response Y mean response B group of the experimental points, central points excluded C group of the central points LOF lack of fit Yield drug entrapmentyield
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Magenheim et al.
F D percentage of free drug

MPS CV
mean particle size coefficient of variation
REFERENCES
1. R. K. Zee-Cheng and C. C. Cheng, Delivery of anticancer drugs, Methods
Find. Exp. Clin. Pharmacol., 11:439 (1989). 2. L. C. Collins-Gold, R. T. Lyons, and L. C. Bartholow, Parenteral emulsions for drug delivery, Adv. Drug Deliv. Rev., 5:189 (1990). 3. G. Gregoriadis (ed.), Liposomes as Drug Carriers, Wiley, London, 1988. 4. P. C. De Smidt and T. J. C.Van Berkel, LDL-mediated drug targeting, Crit. Rev. Ther. Drug Carrier Syst. 7:99 (1990). 5 . M. J. Poznansky and R. L. Juliano, Biological approaches to the controlled delivery of drugs: a critical review, Pharmacol. Rev., 36:277 (1990). 6. M.K. Bijsterbosch andT. J. C. Van Berkel, Native and modified lipoproteins as drug delivery systems, Adv. Drug Deliv. Rev., 5:231 (1990). 7. S. S. Davis, C. Washington, P. West, L. Illum, G. Liversidge, L. Sternson, and R. Kirsh, Lipid emulsions as drug delivery systems, Ann. N.Y. Acad. Sci., 507:75 (1987). 8. M. Singh and L. Ravin, Parenteral emulsions as drug carrier systems, J. Parenter. Sci. Technol., 40:34 (1986). 9. R. J. Prankerd andV. J. Stella, The use of oil-in-water emulsions as a vehicle for parenteral drug administration, J. Parenter. Sci. Technol., 44:139 (1990). 10. J. Brajtburg, W. G. Powderly, G. S. Kobayashi,and G. Medoff,Amphoof mechanism ofaction, Antimicrob. Agents tericin B: Current understanding Chemother., 34:183 (1990). Chiannilkulchai, N. Ammoury, B. Caillou, 11. N. J. P. Devissaguet, and P. of doxorubicin-loaded nanoparticles afCouvreur, Hepatic tissue distribution ter i.v.administrationinreticulosarcomaM5076metastasis-bearingmice, Cancer Chemother. Pharmacol., 26:122 (1990). 12. C. Verdun, F. Brasseur, H. Vranckx, P. Couvreur, and M. Roland, Tissue distribution of doxorubicin associated with polyisohexylcyanoacrylate nanoparticles, Cancer Chemother. Pharmacol., 26:13 (1990). 13. V. Andrieu, H. Fessi, M. Dubrasquet, J.P. Devissaguet, F. Puisieux, and S. Benita,Pharmacokineticevaluation ofindomethacinnanocapsules,Drug Des. Deliv., 4:295 (1989). 14. Y. Akasaka, H. Ueda, K. Takayama, Y. Machida, and T. Nagai, Preparation and evaluation of bovine serum albumin nanospheres coated with monoclonal antibodies, Drug Des. Deliv., 3:85 (1988). P. Speiser,Distributionof 15. E. M. Gipps, P. Groscurth, J. Kreuter,and P. polyhexylcyanoacrylate nanoparticles in nude mice over extended times and after repeated injection, J. Pharm. Sci., 77:208 (1988). 16. E. M. Gipps, R. Arshady, J. Kreuter, P. Groscurth, andP.P. Speiser, Distribu-
Factorial Design
127
17.
18.
19. 20. 21. 22. 23. 24. 25. 26. 27.

28.
29. 30. 31.
32.
tion of polyhexyl cyanoacrylate nanoparticles in nude mice bearing human osteosarcoma, J. Pharm. Sci., 75:256 (1986). V. Lenaerts, J. F. Nagelkerke, B. T. Van, P. Couvreur, L. Grislain, M. Roland,and P. Speiser,Invivouptakeofpolyisobutylcyanoacrylatenanoparticles by rat liver Kupffer, endothelial, and parenchymal cells, J. Pharm. Sci., 73:980 (1984). L. Illum, P. D. Jones, R. W. Baldwin, and S. S. Davis, Tissue distribution of poly(hexyl2-cyanoacrylate) nanoparticles coated with monoclonal antibodies in mice bearing human tumor xenografts, J. Pharmacol. Exp. Ther., 230:733 (1984). B. Skeie, J. Askanazi,M.Rothkopf, S. Rosenbaum, V. Kvetan,andB. Thomashow, Intravenous fat emulsions and lung function: A review, Crit. Care Med., 16:183 (1988). P. Speiser, Drug targeting by drug entrapment into ultrafine compartments as carriers, Appl. Biochem. Biotechnol., 10:221 (1984). S. J. Douglas, S. S. Davis, and L. Illum, Nanoparticles in drug delivery, Crit. Rev. Ther. Drug Carrier Syst., 3:233 (1987). P. Couvreur,Polyalkylcyanoacrylatesascolloidaldrugcarriers,Crit.Rev. Ther. Drug Carrier Syst., 5:l (1988). P. Couvreur, L. Roblot-Treupel, M. F. Poupon, F. Brasseur, and F. Puisieux, Nanoparticles as microcarriers for anticancer drugs, Adv. Drug Deliv. Rev., 5:209 (1990). M. R. Violante, Potential of microparticles for diagnostic tracer imaging, Acta Radiol. 374(Suppl.):153 (1990). P. Couvreur, E. Fattal, and A. Andremont, Liposomes and nanoparticles in the treatment of intracellular bacterial infections, Pharm. Res., 8:1079 (1991). J. Kreuter, Peroral Administrationof Nanoparticles, Adv. Drug Deliv. Rev., 7:71 (1991). P.P. Speiser, Nanoparticles and liposomes: a state of theart, Methods Find. Exp. Clin. Pharmacol., 13:337 (1991). P. Couvreur andF. Puisieux, Nanoparticles and microparticles the for delivery of polypeptides and proteins, Adv. Drug. Delivery, Rev., 10:141 (1993). A. Joshi, Microparticulates for ophthalmic drug delivery, J. Ocul. Pharmacol., 10:29 (1994). P. Jani, G . W. Halbert, J. Langridge, and A. T. Florence, The uptake and translocation of latex nanospheres and microspheres after oral administration to rats,J. Pharm. Pharmacol., 41:809 (1989). T. M. Kukan, S. Bezek, V. Koprda, J. Labsky, J. Kalal, K. Bauerova, and Tmovec, Fate of 14C-terpolymer (methylmethacrylate-l4C,2-hydroxyethylmethacrylate, butylacrylate) nanoparticles after peroral administration to rats, Pharmazie, 44:339 (1989). C. Damge, C. Michel, M. Aprahamian, and P. Couvreur, New approach for oraladministrationofinsulinwithpolyalkylcyanoacrylatenanocapsulesas drug carrier, Diabetes 37:246 (1988).
128
Magenheim et al.
33. M. Aprahamian, C. Michel, W. Humbert, J.P. Devissaguet, and C. Damge,
Transmucosal passage of polyalkylcyanoacrylate nanocapsules as a new drug carrier in the small intestine, Biol. Cell., 61:69 (1987). 34. C. Damge, C. Michel, M. Aprahamian, P. Couvreur, and J. P. Devissaguet, Nanocapsules as carriers for oral peptide delivery, J. Control. Rd., 13233
(1990). 35. D. T. O'Hagan, Intestinal translocation of particulates-implicationsfor drug 5:265 (1990). and antigen'delivery, Adv. Drug Deliv. Rev., 36. P. Jani, G.W. Halbert, J. Langridge, and A. T. Florence, Nanoparticle uptake
by the rat gastrointestinal mucosa: quantitation and particle size dependency, J. Pharm. Pharmacol., 422321 (1990). 37. J. C. Verhoef, H.E. Bodde, A. G. de Boer, J. A. Bouwstra, E. H.Junginger, F. W. Merkus, and D. D. Breimer, Transport of peptide and protein drugs 15233 acrossbiologicalmembranes,Eur.J.DrugMetab.Pharmacokinet.,
38. (1990).
M.J.CappelandJ. Kreuter, Effect of nanoparticles on transdermal drug delivery, J. Microencaps., 8:369 (1991). 39. P. Maincent, P. Thouvenot, C. Amicabile,M. Hoffman, J. Kreuter, P. Couvreur, and J. P. Devissaguet, Lymphatic targeting of polymeric nanoparticles after intraperitoneal administration in rats, Pharm. Res., 9:1534 (1992). 40. S. M. Moghimi, A. E. Hawley, N. M. Christy, T. Gray, L. Illum, and S. S. Davis, Surface engineered nanospheres with enhanced drainage into lymphatics and uptake by macrophages of the regional lymph nodes, FEBS Lett.,
41.
C. Chavany, T. Le Doan, P. Couvreur, F. Puisieux, and C. HClkne, Polyalkylcyanoacrylate nanoparticles as polymeric camers for antisense oligonucleotides, Pharm. Res.,9:441(1992). B. E. Saison, An ap42. G. Schwab, I. Duroux, C. Chavany, C. HCBne, and proach for new anticancer drugs: oncogene-targeted antisense DNA, Ann. Oncol., 455 (1994). 43. F. Stieneker, J. Kreuter,andJ.Lower,Highantibodytitresinmicewith polymethylmethacrylate nanoparticles as adjuvant for HIV vaccines, AIDS,
A. M. Steffan, C. Royer, S. 44. V. Schafer,H.vonBriessen,R.Andreesen, Troster, J. Kreuter,andH.Rubsamen-Waigmann,Phagocytosisofnanoparticles by human immunodeficiency virus (H1V)-infected macrophages: a possibility for antiviral drug targeting, Pharm. Res., 9541(1992). 45. A. Bender, V. Schafer, A. M. Steffan, C. Royer, J. Kreuter, H. RubsamenWaigmann, and H. von Briesen, Inhibition of HIVin vitro by antiviral drug145:215 (1994). targeting using nanoparticles, Res. Virol., P. Speiser, In vitro studies of poly(methy1) methacrylate) 46. J. Kreuter and P. adjuvants, J. Pharm. Sci., 651624 (1976). 47. H. Kopf, R. K. Joshi, M. Soliva, and P. Speiser, Study of micelle polymerization in the presence of low molecular weight drugs. Part 2 : Mode of binding of incorporatedlow molecular weight model substances to polyacrylamide-based nanoparticles, Pharm. Ind.,39:993 (1977).
5431(1991).
344:25 (1994).
129
48. C. Vauthier-Holtzscherer, S. Benabbou, G. Spenlehauer, M. Veillard, and P. Courvreur, Methodology for the preparation of ultra-dispersed polymer systems, S.T.P. Pharma Sci., 1:109 (1991). S. Benita, Nanoparticle characterization: a comprehensive 49. B. Magenheim and physicochemical approach, S.T.P. Pharma Sci., 1:221 (1991). 50. P. P. DeLuca, R. C. Mehta, A. G. Hausberger, and B. C. Thanoo, Biodegradable polyesters for drug and polypeptide delivery, in Polymeric Delivery Sys(M. A. El-Nokaly, D. M. Piatt, and B. A. tems, Properties and Applications Charpentier, eds.), American Chemical Society, Washington, DC, 1993, p. 53. 51. P. Couvreur, and C. Vauthier, Polyalkylcyanoacrylate nanoparticles as drug camer: present state and perspectives, J. Control. Rel., 17:187 (1991). 52. H. Fessi, F. Puisieux, J. P. Devissaguet, N. Ammoury, and S. Benita, Nanocapsule formation by interfacial polymer deposition following solvent displacement, Int. J. Pharm., 55:Rl (1989). 53. N.Ammoury,H.Fessi, J. P. Devissaguet, M. Dubrasquet, and S. Benita, Jejunal absorption, pharmacological activity, and pharmacokinetic evaluation of indomethacin-loaded poly(d,l-lactide) and poly(isobuty1-cyanoacrylate) nanocapsules in rats, Pharm. Res.,8:101 (1991). 54. N. Ammoury, M. Dubrasquet, H. Fessi, J. P. Devissaguet, F. Puisieux, andS. Benita, Indomethacin-loaded poly(d,l-lacti'de) nanocapsules: Protection from gastrointestinal ulcerations and anti-inflammatory activity evaluation in rats, Clin. Mater., 13:121 (1993). 55. B. Magenheim, and S. Benita, Elucidation of the indomethacin in vitro release mechanism from polylactic acid nanoparticles, Pharm. Res., 10:S187 (1993). 56. B. Magenheim, M. Y. Levy, and S. Benita, A new invitro technique for the evaluation of drug release profile from colloidal camers-Ultrafiltration technique at low pressure,Int. J. Pharm., 94:115 (1993). 57. P.WehrlC, B. Magenheim, and S. Benita, Optimization of a Colloidal Drug DeliverySystemBased on PolyesterNanoparticlesbyMeansofFactorial Design, 13th Pharmaceutical Technology Conference and Exhibition, Strasbourg, France, 1994, pp. 429-453. 58. P. WehrlC, B. Magenheim, and S. Benita, The influence of process parameters on the PLA nanoparticle size distribution, evaluated by means of factorial design, Eur.J. Pharm. Biopharm., 41:19 (1995). 59. T. Niwa, H.Takeuchi, T. Hino, N. Kunou, and Y. Kawashima, Preparations of biodegradable nanospheres of water-soluble and insoluble drugs with D,Llactide/glycolidecopolymerbyanovelspontaneousemulsificationsolvent J. Control.Rel., 25239 diffusionmethod,andthedrugreleasebehavior, (1993). 60. T. Niwa, H. Takeuchi,T. Hino, N. Kunou, and Y. Kawashima, In vitro drug releasebehaviorofD,L-lactidelglycolidecopolymer(PLGA)nanospheres with nafarelin acetate prepared by a novel spontaneous emulsification solvent diffusion method,J. Pharm. Sci., 83:727 (1994). 61. C. D. Potter, Experiment design software-Better data, less work, Scientist 8:18 (1994).
130
Magenheim et al.
62. G. A. Lewis and M. Chariot, Non classical experimental designs in pharmaceutical formulation, Drug Dev. Ind. Pharm., 17:1551 (1991). 63. A. Gustavo GonzBlez, Optimization of pharmaceutical formulations based on response-surface experimental designs, Int. J. Pharm., 97:149 (1993). 6 4 . P. Wehrlt, P. Nobelis, A. CuinC, and A. Stamm, Response surface methodology, an interesting statistical tool for process optimization and validation, Drug Dev. Ind. Pharm., 9:1637 (1993). 65. C. Altesor, P. Corbi, I. Dol, and M. Knochen, Application of experimental design to the developmentofamulticomponentderivativespectrophotometricmethod-Simultaneousdeterminationofsulfamethoxazoleandtrimethoprim, Analyst, 118:1549 (1993). 66. H. C . Qiu and F. H. Walters, The use of a 3-factor statistical mixture design and a 2-factor chromatographic response function to the RP-HPLC separation of several free alpha-amino acids, Anal. Lett., 26:1799 (1993). P. Kaufmann, Lipid class analysis by normal phase high 67. K. C. Arnoldsson and performanceliquidchromatography,developmentandoptimizationusing multivariate methods, Chromatographia, 38:317 (1994). ofexperimentaldesigntooptimize the 68. G. Contarini and R. Leardi, Use analysis of volatile compounds by dynamic headspace extraction followed by coldtrappingandcapillaryGC,HRC-J.High.Res.Chromatogr.,17:91 (1994). 69. M. M. Rogan, K.D. Altria, and D. M. Goodall, Plackett-Burman experimental design in chiral analysis using capillary electrophoresis, Chromatographia, 38:723 (1994). 70. C. J. Weatherell andE. P. C. Lai, Factorial experimental design and principal component analysis of the interaction of animal glues with polymeric and silica-based stationary phases in size exclusion chromatography, J. Chrornatogr. A, 669:31 (1994). 71. P.WehrlC,P.Nobelis,A.CuinC,andA.Stamm,IntCr&t des analyses factorielles et des plans d'exp6riences pour l'ttude, l'optimisation et la validation desproc6dCs.11.Optimisation et validationdelagranulationhumideen mtlangeur-granulateur-stcheur haute vitesse, S.T.P Pharma Pratiques, 2:173 (1992). 72. B. Iskandarani, J. H. Clair, P. Patel, P. K. Shiromani, and R. E. Dempski, Simultaneous optimization of capsule and tablet formulation using response surface methodology, Drug. Dev. Ind. Pharm., 19:2089 (1993). 73. P. Wehrlt, P. Nobelis, A. Cuin6, and A. Stamm, Scaling-Up of wet granulation-A statistical methodology, Drug. Dev. Ind. Pharm., 19:1983 (1993). 74. S. Ogawa, T. Kamijima, Y. Miyamoto, M. Miyajima, H. Sato, K: Takayama, and T. Nagai, A new attempt to solve the scale-up problem for granulation using response surface methodology, J. Pharm. Sci., 83:439 (1994). 75. S. R. Goskonda, G. A. Hileman, and S. M. Upadrashta, Development of matrix controlled release beads by extrusion-spheronization technology using a statistical screening design, Drug. Dev. Ind. Pharm., 20:279 (1994). 76. M. Farivar, H. S. Kas, L. Oner, and A. A. Hincal, Factorial design-based
Factorial Design
of Nanoparticulate Dosage
Forms
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77. 78.
79. 80. 81. 82.
83. 84. 85. 86. 87.
isosorbide-5-mononitratemicrocapsules, J. optimization of the formulation of Microencaps. 10:309 (1993). J. M. Pernot, H. Brun, B. Pouyet, M. Sergent, and R. Phan-Tan-Luu, Influence of experimental parameters on the microencapsulation of a photopolymerizable phase, J. Microencaps. 10:323 (1993). X.M. Zeng, G. P. Martin, and C. Marriott, Albumin microspheres as a means of drug delivery to the lung: analysis of the effects of process variables on particlesizesusingfactorialdesignmethodology,Int.J.Pharm.,107:205 (1994). X. M. Zeng, G . P. Martin, and C. Marriott, Tetrandrine delivery to the lung: The optimisation of albumin microsphere preparation by central composite design, Int. J. Pharm., 109:135 (1994). M. J. Alonso, A. Sanchez, D. Torres, B. Seijo, and J. J. Vila, Joint effects of on physico-chemical characteristics of monomer and stabilizer concentrations poly(butyl2-cyanoacrylate)nanoparticles, J. Microencaps.,7517 (1990). R. Carpignano, M. R. Gasco, and S. Morel, Optimization of doxorubicine incorporation and of the yield of polybutylcyanacrylate nanoparticles, Pharm. Acta Helv., 66:28 (1991). M. C. Julienne, M. J. Alonso, J. L. Gomez Amoza, and J.P. Benoit, Preparation of poly (D,L-Lactide/glycolide) nanoparticles of controlled particle size distribution: Application of experimental designs, Drug Dev. Ind. Pharm., 18:1063 (1992). G. E. BoxandN.R. Draper, in Empirical Model Building and Response Surface, ( G . E. Box and N. R. Draper, eds.),Wiley, New York, 1987, p. 205. G. E. Box, W. G . Hunter, and Hunter. J. S., Response surface methods, in Statistics for Experimenters (G. E. Box, W. G. Hunter, and Hunter. J. S., eds.), Wiley, New York, 1978, p. 510. 0. L. Davies, The determinationof optimum conditions, in The Design and (0. L. Davies,eds.),Longman, New AnalysisofIndustrialExperiments York, 1978, p. 495. WehrlC (1990) Aspects des analyses multifactorielles et des plans dexperiences appliquCs h loptimisation et h lavalidationdeformes et deprocedes galeniques. Ph.D. thesis No. 149, University of Paris XI. G. Sad0 and M. C. Sado, Notions de plan optimal et critkres doptimalitk, in Les plans dexperiences, AFNOR, Paris, 1991, p. 10.
Microsphere Morphology
Jean-Pierre Benoit
Laboratoire de Pharmacie Galdnique et Biophysique Pharmaceutique, Universitd &Angers,and Centre de Microencapsulation, Angers, France
Curt Thies
Washington University, St. Louis, Missouri
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I. Introduction
11. Progesterone-Loaded Poly(d,l-Lactide) Microspheres
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IV. Hydrocortisone-Loaded Poly(d,l-Lactide) Microspheres V. VI. Lomustine-Loaded Poly(d,l-Lactide) Microspheres Cisplatin-Loaded Poly(d,l-Lactide) Microspheres
VI1. Summary of Microsphere Morphological Studies
1.
INTRODUCTION
Many drug-loaded microspheres are prepared by processes that involve intimate mixing of a polymeric carrier with a drug. This creates a situation where a variety of physicochemical interactions occur, and these interactions can influence therapeutic performance of the final device. Consequently, the morphology of drug-loaded polymer microspheres warrants careful study. The purpose of this chapter is to illustrate how the morphology of biodegradable microspheres prepared by removal of avolatile
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solvent from oil-in-water or water-in-oil-in-water emulsions canbe characterized. In such processes [1,2], a polymer carrier is dissolved ina volatile solvent like methylene chloride (CH,Cl,). A drug is then either dissolved or suspended in the solution and the resulting mixture is emulsified in an aqueousphasethat contains an emulsifier. The solvent is allowed to evaporate, thereby giving drug-loaded microspheres. Although the procedure is conceptually simple, it is not in practice. A number of facton include solubility of affect the natureof the microspheres obtained. These the drug in the casting solvent and aqueous phase, drug-polymer ratio, nature of the polymer used as the carrier, and aqueous phase emulsifying agent. Crystalline drugs that are freely soluble in the casting solvent may exist in the final microsphere as a molecular dispersion, or they may form well-defined crystal domains in the polymer matrix. What state a drug assumes in a microsphere is defined by the drug-polymer interaction and percent loading of drug in the final microsphere. Residual casting solvent is another factor, since it could prevent drug crystallization if present in the final microsphere. When a crystalline drug is soluble in the casting solvent, its ability to crystallize in a microsphere is repressed at low drug loadings because of the nature of the solvent evaporation process. The drug-polymer-solvent solution used to form the microspheres is initiallya dilute solution of relatively low viscosity. When it is emulsified in an aqueous phase, the volatile solvent begins to partition into the aqueous phase from which it is removed by evaporation. As this process progresses, the viscosity of the organic phase increases steadily. For polymers with a glass transition temperature (Tg) above the temperature at which evaporation occurs, the viscosity of the polymer-containing organic phase increases to infinity as the volatile solvent concentration decreases toward zero. If the drug-polymer-solvent phase is sufficiently viscous when the saturation solubility of drug in the solvent is reached, and if the rate at which the remaining solvent evaporates is sufficientlyhigh, drug crystallization in the viscous polymer matrix is effectively prevented. In this manner, drug that has little mutual miscibility with the polymer can be molecularly dispersed in the polymer. The dispersion is a metastable state formed, because the drug does not have time to crystallize before the polymer phasesets up as an organic glass. If the drug and the polymer are mutually miscible, a true molecular solution of drug in the polymer is formed, like a solution of a plasticizer in a polymer. Cases of partial drug-polymer miscibility fall between these extremes. With drugs that are weakly soluble in the volatile solvent, formation of well-defined crystalline domains occurs readily. The above list of possibilities illustrates why a drug incorporated in a
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polymeric microspheremay exist there in a wide variety of physical states. It is appropriate toillustrate some of these possibilities with specific examples of drug-loadedmicrospheres fabricated from lactide (PLA)and lactide-glycolide (PLAGA) polymers.
II. PROGESTERONE-LOADED POLY(d,l-LACTIDE) MICROSPHERES
Fig. 1contains SEM photomicrographs that show howthe surface structure of 100-300 pm PLAmicrospheres varies with progesterone loading [3]. At 23 w t . percent loading, the microsphere surface is perfectly smooth. At 35 wt. percent loading, a few crystals are embedded in the surface. At 68 wt. percent loading, the microspheres are coated by a continuous layer of progesterone crystals. The formation of free drugcrystals on thesurface of microspheres or in the aqueous phase as solvent evaporation progresses is a problem often encountered in the solvent evaporation process when the drug used is soluble in the casting solvent. Free crystal formation can be repressed if the aqueous phase emulsifier is removed from the system before solvent evaporation is complete [3,4]. Solvent evaporation is then taken tocompletion in emulsifier-free water. Fig. 2, obtained by differential thermal analysis (DTA), illustrates the thermal behaviorof a PLA microsphere sample loaded with 23wt. percent progesterone [3]. From these scans, a number of conclusions can be made. First, there is no endothermic peak at 120-132C characteristic of the a or p polymorphs of progesterone. The progesterone is molecularly dispersed in the microsphere sample and does not form macroscopic progesterone of crystal domains that can be detected by DTA. The x-ray powder diagram this sample is a diffuse halo, thereby confirming that well-defined progesterone crystal domains are not present. Since no crystalline progesterone is present, a key question is whether or not the progesterone and PLA form a molecular solution much like a plasticizer and polymer. This question can be answered by prolonged annealing experiments carried out well above the Tgof PLA but well below the melting point (Tm) of progesterone. The logic for this experiment is as follows. Drug molecules trapped as a molecular dispersion in a polymer matrix at temperatures below the polymers Tg are locked in a polymer glass and cannot diffuse in a finite time to nucleate and/or grow finite domains of crystalline drug. When the polymer is heated above its Tg, chain mobility is greatly increased, thereby greatly increasing diffusivity of the drug in the polymer matrix and enabling it to crystallize if it wishes. Crystallization will occur if the polymer and drug are mutually immiscible. If they are mutually miscible, there is no crystallization. It is assumed that
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Fig. 1 Scanning electron micrographs of poly(d,l-lactide) microspheres containing (A) 23%, (B) 35%, (C) 68% progesterone.
mutual miscibility of the drug and polymer does not change measurably with temperature over the temperature range of interest. It also is assumed that the drugis not degraded measurablyduring the annealing process. In order tominimize the possibility of degradation, all annealing experiments are carried out in vacuo. PLA Fig. 3 contains DTA scans for an annealed progesterone-loaded microsphere sample [3]. Annealing was done for 22 h at 110C. The annealed samples were quench cooled in liquid nitrogen and their thermal behavior was evaluated after a known aging cycle.All of the DTA scans in Fig. 3 have a well-defined endotherm at 130C due to the melting of CYprogesterone crystal domains. This is evidence that PLA and progesterone are immiscible with each other at a progesterone loading of 23%. The progesterone is initially present as a molecular dispersion. This metastable dispersion existsin thePLA glass atroomtemperature,becausethe
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Fig. 2 Aging of PLA Tg for PLA microspheres loaded with 23 wt. percent progesterone and stored at 22C. (From Ref. 3.)
progesterone molecules did not have time to crystallize in the polymer matrix as CH,Cl, evaporation progressed to completion and the PLA set up to a glassy state. When given the opportunity, it crystallized. Most of the crystal domainsformed in PLA by annealing at 110C were the apolymorph (130C Tm). Occasionally, a melting event at 120C was observed. In this case, P-progesterone domains formed. A DSC scan of the freshly annealed sample used to generate Fig. 3 showed that an estimated 80% of the progesterone carried by the PLAmatrix had crystallized during the 110C annealing process. The melting endotherm had a maximum at 130C but was broad and skewed toward 120C. After 3 and 7 days of storage at 22"C, the endotherm became much sharper and was still centered at130C. This change in the endotherm on aging caused the apparent progesterone crystallinity calculated from the DSC scan to fall to 55% and suggests that at least some p-progesterone crystal domains were initially present in the PLA matrix. They either were transformed on storage into a-progesterone crystal domains or simply dispersed in the PLA matrix. Fig.3shows that the nature of the PLA Tg event for the 110C annealed microsphere sample also changes on storage at 22C. Initially, it is a rather diffuse event located at 49C. After 3 days at 22"C, it becomes more visible and shifts upward to 51C. After 14 days at 22"C, it is a welldefined event located at 53C with an endothermic peak. The type of aging shown by the PLATg event in Fig. 3 is commonly observed with amorphous glasses stored close to but below their Tg [5]. Some workers attribute this to development of molecular order in the
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polymer glass, whereas others argue that this phenomenon is due to relaxation of stresses and strains arising from nonequilibrium cooling. Petrie [5] gives a good overview of the subject from the latter viewpoint. Regardless of theorigin of this aging behavior, it is relevant to note from Fig. 2that the unannealed progesterone-loaded PLA microsphere sample retained a comparatively diffuse Tg event at 37C with no endothermic peak when aged 83 days at 22C. In contrast, theannealed sample gave a Tg event that rapidly developed an endothermic peak on storage at 22C and fell at 49-53"C, close to theTg of drug-free PLA. The difference in aging of the PLA Tg event of drug-free and drugloaded microspheres is related to residual solvent content of the microspheres and/or degree of molecular dispersion of progesterone in the PLA microspheres before heat treatment.Although all PLA microspheres appeared to bethoroughly dry after vacuum drying at 22C for atleast 20 h, they actually still contained between 1 and 5% CH,Cl,, the solvent from which the PLA microspheres were cast [3]. The low PLA Tg values shown in Fig. 2 could be caused solely by residual solvent trapped in the microspheres that acts as a plasticizer, thereby lowering the Tg of PLA and making it more diffuse. However, drug-free PLA microspheres develop a well-defined 57CTg eventwith an endothermic peak within 70 days of22C aging. This suggests that the diffuse and low PLA Tg event shown in Fig. 2 is not due solely to residual
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CH2Cl, but is caused, at least in part, by interference of molecularly dispersed progesterone with the relaxation or ordering phenomenon responsible for developmentof a well-defined PLA Tg event by progesterone. The effect of molecularly dispersed drug on the behavior of a polymers Tg event merits further investigation. When progesterone is molecularly dispersed in PLA, it appears tobe vulnerable to degradation. The progesterone carried by two PLA samples loaded with 23% progesterone and two PLA samples loaded with 31.7% progesterone experienced 24-47% degradation when stored 16months in a desiccator at 25C [6]. All four samples contained only molecularly dispersed progesterone. The samples loaded with 31.7% progesterone turned yellow during the aging process. The nature of the degradation products was not determinednor was the origin of the degradation process. Residual CH,CI, could have beenacontributing factor. The key point is that progesterone molecules present as a molecular dispersion in a PLA matrix are more susceptible to degradative attack than if they are located in crystalline progesterone domains.
111.
PROGESTERONE-LOADED85/15 POLY(d,l-LACTIDEGLYCOLIDE) COPOLYMER MICROSPHERES
A series of 85/15 (w/w) PLAGA copolymer microspheres (25-50 wm in diameter) was prepared by the solvent evaporation process with CH,Cl, as the casting solvent [7]. Progesterone loading ranged from 0 to 64.6 wt. percent. As in the case of progesterone-loaded PLA microspheres, the external appearanceof the copolymer microspheres varied with drug loading; however, thevariation was morepronounced.Copolymer microspheres with 10 wt. percent progesterone loading had a smooth external surface. Increasing theloading to 28 wt. percent caused the surface of the microspheres to become serated and some free crystals embedded in the surface were visible. At 50 wt. percent loading, the microspheres were porous with progesterone crystals visible inthe surface. lists DSCthermal event temperatures for progesterone-loaded Table 1 PLAGA copolymer microspheres [7]. These data illustrate a number of features of such microspheres. Column 7 shows that samples with 22.8-64.6 wt. percent progesterone contain crystalline progesterone domains regardless of their thermalhistory. A sample loaded with 10.9% progesterone did not developcrystalline progesterone domains until it was annealed at80C for 24 h. Although some freshly made samples contain a mixture of aprogesterone (130C Tm) and P-progesterone (120C Tm), the p form always predominates.This is interesting because progesteronesolutions evaporated to dryness from CH,C12 always yield only a-progesterone. In
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most cases, crystalline progesterone domains that form in a PLA matrix also are the a-polymorph. Crystallization in the copolymer matrix favors formation of the p-polymorph. Samples with 22.8 and 34.4 wt. percent progesterone develop nearly all of their crystallinityspontaneously during the DSC heating cycle. Before heating,most of the progesterone is molecularly dispersed in the copolymer matrix. This crystal growth creates the exothermic peak at 80-115C (column 6 ) . Muramatsu et al. [8], reported that a molten progesterone specimen cooled at 10"C/min was cooled so fast that it remained supercooled throughout the cooling process. Crystallization in the p form always occurred between 37 and 120C when the supercooled specimen was reheated. Accordingly, the behavior of microsphere samples with22.8 and 34.4% progesterone parallels that of supercooled progesterone when it is reheated. In the case of the microspheres, the equivalent to a supercooled specimen was achieved at essentially constant temperatureby carrying out solvent evaporation in the copolymer matrix. As CH,Cl, evaporation progressed to completion, the viscosity of the copolymer phase increased dramatically, thereby effectively enabling the progesterone to act as a supercooled specimen and hence remain molecularly dispersed. On heating above Tg of the copolymer, the diffusivity of progesterone in the PLAGA matrix increases greatly. It is free to crystallize and does so in essentially the p-form like a supercooled progesterone sample. Another interesting observation is the change in relative proportion of a- and P-progesteronethat occurs on aging. Theproportion of aprogesterone increases when the copolymer samples are aged at 4C or annealed at 80C. This is not surprising, since the a-polymorphis the more stable form.Nevertheless, it is interesting thatthis transition proceedsto a measurable extentduring 9-12 months of storage at 4"C, well below the Tg of the copolymer. Muramatsu et al. [ 8 ] , reported that crystals of a-and pprogesterone seemed tobe stable atroom temperature for several months or longer. As noted in Section 11, the natureof the crystalline progesterone event in an annealed progesterone-loaded (23%) PLAsample also changed on storage. In this case, the change appeared to be complete in 7 days at 22C. These data are evidence that the p-ta transition of progesterone located in a solvent-cast polymer matrix proceeds more easily than in progesterone crystals grown in the absence of a polymer. The consistent development of crystalline progesterone domainsin a range of PLAGA copolymer microsphere samples is evidence that progesterone and the copolymer have at best a limited degree of mutual miscibility. When given an opportunity to crystallize, progesterone that initially matrix crystallizes. This behavexists as a molecular dispersion in a PLAGA ior is analogous to that observed with PLA homopolymer. For both poly-
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mers, the solvent evaporation process can be manipulated to produce microspheres that contain molecularly dispersed progesterone, provided the loading is low (e.g., below 30%), but this is a metastable state. When the samples areheated, progesterone crystallization occurs spontaneously. Why crystallization of progesterone in the lactide-glycolide copolymer matrix yields primarily the p polymorph and crystallization in a PLA matrix yields primarily the a polymorph remainsunresolved. However, it is clear that the polymer matrix affects what crystalpolymorph forms. The copolymer Tg data in column 5 of Table 1 provide further evidence that progesterone and lactide-glycolide copolymer are essentially immiscible [7]. If they were mutually miscible, a change in copolymer Tg proportional to progesterone loading would be expected. This does not occur. Several progesterone-loaded copolymer samples stored at 4C for a prolonged period have a copolymer Tg of 48-49"C, which is essentially the same as that of the copolymer before it was fabricated into microspheres. All samples annealedat 80C for 24 h havea 47-48C copolymer Tg. Most of the samples with low copolymer Tg values listed in Table 1were evaluated soon after manufacture. Such samples often had an endotherm at 6580C (column 4) associated with volatilization of residual CH,Cl, from the microspheres as they were heated. When heated, these samples lose 2.54.6% of their weight owingto CH,Cl, volatilization. When the samples are aged 9-12 months at4"C, the endotherm due to CH,Cl, volatilization disappears (column 4), sincemuch of the residual CH,Cl, diffuses from the microspheres during storage. Column 3 of Table 1 shows that the residual 1%. Annealing CH,Cl, content of three samples aged at 4C was well below for 24 h at 80C also removes residual CH,Cl, from the polymer matrix. Thus, the low copolymer Tg values recorded in Table 1 are attributed primarily to residual CH,Cl,. Low PLAGA Tg values stored 12 months at 4C are attributed to slow equilibration of the polymer matrix. The boiling point of CH,Cl, is 4O-4l0C, whereas the CH,Cl, evaporation endotherm for the microspheresis located between 64 and 80C. This suggests that CH,Cl, interacts strongly withthe lactide-glycolide copolymer and is difficult to remove completely from a matrix of such a copolymer. Persistent retention ofCH,Cl,by PLA microspheres also has been observed. The question of solvent retention by microspheres formed by the solvent evaporation process and intended for injectable drug formulations warrants much closer scrutiny. A numberof papers have been published on the in vivo performance of injected lactide and PLAGA microsphere samples, but the residual solvent content of the injected microspheres is never given. Although such microspheres may appear to be "dry" after anextensive drying process, they still may contain sufficient residual casting solvent (e.g., CH,Cl,) to have a meaningful effect on microsphere morphology,
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drug stability in the microsphere, and drug-release rate from the microsphere. The matter of how residual solvent affects acute and chronic toxicology of the injectable microspheres must alsobe considered. Because of the tenacity with which lactide and lactide-glycolide copolymers hold solvent, published studies of the in vivobehavior of microspheres prepared from such polymers should specify the residual solvent content carried by the microspheres and what the solvent@)is. Before concluding this section, it is relevant to mention that the Tg event of the PLAGA experiences an agingeffect analogous to that of PLA. The Tg event becomes more well defined on aging and develops a significant endothermic peak. The rateof development of this peak increases as the aging temperature approaches Tg of the copolymer[7].
IV.
HYDROCORTISONE-LOADED POLY(d,l-LACTIDE) MICROSPHERES
A series of 50- to 150-pm diameter PLA microspheres loaded with 4.247.0 wt. percent hydrocortisone was prepared by continuous evaporation ofCH,Cl,[9]. Hydrocortisone has finite solubility in water and is only partially soluble in CH,Cl,. Up to hydrocortisone loadings of 32 wt. percent, partially hydrolyzed poly(viny1 alcohol) (PVA) gave stable emulsions from which spherical microspheres were prepared. At higher hydrocorwhich microspheres tisone loadings, PVA did not give stable emulsion from could be made, whereasmethylcellulose did. Accordingly, allmicrospheres loaded with 32 or more percent of hydrocortisone weremade with . methylcellulose as the aqueousphase emulsifier. A PLA microsphere sample loaded with 12.6% hydrocortisone stored at 22C for 14 weeks had one well-defined endotherm at 56Cand a second at 208-209C [9]. The 56C event is characteristic of drug-free PLA and is due to the glass-transition event of PLA. This event did not have an endothermic peak when the microspheres were freshly made. The peak developed on storage of the microspheres at 22C and appears to be sharper and larger than similar endothermicpeaksdeveloped by aged PLA and lactideglycolide copolymer samples loaded with progesterone. The 208-209C endotherm that appears in the DSC scan of PLA microspheres loaded with 12.6% hydrocortisone issmall. It disappears when the microspheres are heated to 250"C, quench cooled, and reheated. The x-ray powder diagram of such microspheres taken before they were heated had d-spacings characteristic of crystalline hydrocortisone. Thus, the event is due to the melting of crystalline hydrocortisone domains located in the microspheres. The 208-209C fusion temperature is well below the 228C fusion temperature found for well-defined hydrocortisone crys-
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tals. This suggests that thecrystal domains in the microspheres are not well defined. Since the event is so small, much of the hydrocortisone exists in the polymer matrix as a molecular dispersion. This observation was unexpected because the initial PLA-solvent-steroid mixture used to form the microspheres was turbid and contained undissolved steroid crystals. It was thought that these would be trapped in the final microspheres to give a significant fusion event at 228C. Direct microscopic observation throughout the solvent evaporation process revealed that this did not occur, because the steroid crystals initially present in the CH,Cl, phase migrated spontaneously into the aqueous phase on emulsification. The crystalline steroid domains in the isolated PLA microspheres were formed by crystallization of solubilized drug that occurred as solvent evaporation progressed. Since the viscosity of the PLA-steroid-solvent solution increased steadily as solvent evaporation progressed, the steroid crystal domains nucleated and grew in a progressively more viscous medium. This was assumed to hinder the development of defect-free crystal domains and the extent of steroid crystallization. Microspheres with larger steroid contents would undoubtedly have a more pronounced steroid fusion event which would fall closer to 228C. In such cases, the crystallization would occur sooner in the process owing to thelarger amount of drug present. The study of hydrocortisone-loaded PLA microspheres providedanother illustration of how tenaciouslyPLA binds CH,Cl,. The sample loaded with 12.6% hydrocortisone had a 2.74% chlorine content after it was dried in vacuo at 22C for 24 h. Extending the drying time to 72 h reduced the chlorine content to 2.56%. Assuming these chlorine contents are due exclusively to residual CH,Cl,, it is calculated that the microspheres contained 3.8% solvent after 22 h drying and 3.3% after 72 h drying. Increasing the drying time from 22 to 72 h had relatively little effect on residual CH,Cl, content. The hydrocortisone in PLA microspheres does not appearto degrade rapidly. Samples stored for 55 days at 25 or 37C experienced no meaningful degradation [9].
V. LOMUSTINE-LOADED POLY(d,l-LACTIDE) MICROSPHERES
Lomustine, 1-(2-chloroethyl l-3-cyclohexyl-l-nitrosourea (CCNU), is well known for its efficacy on tumors of the central nervous system. A promising way of optimizing the action of CCNU is to incorporate it into microspheres and target the microspheresto tissue sites via selective arterial catheterization. For this reason, an effort has been madeto develop injectable CCNUloaded microspheres. Although the development process is complicated by the reactivity and comparative instability of CCNU, CCNU-loaded mi-
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crospheres have been prepared from poly(d,l-lactide) (PLA) [4,6,10] and poly(@-hydroxybutyrate)(PHB) [11,12]. The PLA microspheres were cast from CH,Cl,; the PHB microspheres were cast from chloroform. In both cases, free CCNU formed in the aqueous phase as solvent evaporation progressed to completion unless evaporation was interrupted and the aqueous phase emulsifier was removed from the system before solvent evaporation was complete. If no free crystals formed, the surface of PLA microspheres loaded with 23%CCNU were smooth and free of defects [6]. If free crystals formed, thesurface of the microspheres was rippled and rough.The surface of 50-250 pm PHB microspheres is filled withmacroscopic pores. PHB is a relatively crystalline polymer that crystallizes during the solvent evaporation fabrication process, thereby generating a porous structure. Juni and Nakano [l31 show a SEM photomicrograph of50-400 pm PHB microspheres cast from CH,Cl, that illustrates their surface porosity. These large pores are notvisible in the surface of 15 pm PHB microspheres [ll]. If free CCNU crystals formed during solvent evaporation, the amount of CCNU incorporated in the microspheres was reduced as one would expect. However, the efficiency of CCNU incorporation in 50-250 pm PLA or PHB microspheres was always high whether or not free crystals formed. Large amounts of CCNU were not partitioned into the aqueous phase or degraded during the fabrication process. This is significant in view of the sensitivity of CCNU to water. When efforts were made to prepare 15-pm diameter CCNU-loaded microspheres, the problem of free CCNU crystal formation increased markedly [12]. The high CH,Cl,-water interfacial area promoted development of free crystals, whereas the small size of the microspheres being formed made it difficult to minimize free crystal growth by removing the aqueous phase emulsifier before solvent evaporation was complete. Accordingly, smallPLA orPHB microspheres prepared to datehave CCNU loadings below 10% [12]. Fig. 4 is a DTA/TGA scan for a freshly formed 50-250 p m CCNU-loaded (23%) PLA microsphere sample [lo]. 93"C, the crystalline The DTA scanhas no event at 57"C, the Tg of PLA, or melting temperature of CCNU. The broad exothermic event extending from approximately 115 to 140C and the accompanying TGA weight loss are due to thermal degradation products. The absence of a crystalline CCNU melting point indicates that there are no crystalline CCNU domainsin this microsphere sample. The CCNU is molecularly dispersed in the PLA glass. The absence of a PLA Tg event suggests that CCNU plasticizes the PLA. formed Since the DTA scan began at 30"C, a DSC scan of a duplicate freshly CCNU-loaded PLA sample was made from 0 to 70C. It revealed a broad Tg event centeredat 25C. The x-ray diffraction pattern of this second CCNUPLA sample was completely amorphous. The 25C PLA Tg eventcoupled with the experimental observation that CCNU-loaded PLA microspheres
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Fig. 4 Simultaneous DTAlTGA scan (10"Umin) of freshly formed CCNUloaded (23 wt. percent) PLA microspheres.
sinter together on prolonged 22C storage prompted the conclusion that PLA CCNU acts as a PLA plasticizer andismolecularlydissolvedin microspheres. The discovery that freshly formed progesterone-loaded PLA and lactide-glycolidecopolymer microspheres subjected to the same drying procedure as CCNU-loaded PLA microspheres retain 2-5 wt. percent CH+& could be due led to the realization that the reduced PLA Tg reported above in part to residual CH,Cl, and not plasticization of PLA by CCNU. The observation that CCNU tends to crystallize spontaneously in the aqueous phase as CH,Cl, evaporation proceeds is an indication that the mutual miscibility of PLA and CCNU is low. If they were highly miscible, the CCNU would not tend to form free crystals. In order togain more insight into how CCNU and PLA interact in CH,Cl,cast microspheres, the thermal behavior of a number of aged CCNU-PLA microsphere samples was evaluated. Table 2 summarizes the thermal data obtained [lo]. Question marks (?)in the table designate cases where the event could not be characterized reliably either because it was too diffuse or the thermal analysis did not extend to a sufficiently low temperature. PLA Tg values for the samples characterized fall at 21 f 4C or 49 2 3C. SamplesA , C, J, and E have a 21 rt 4C Tg event which does not develop a well-defined endothermic peak.
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These samples do not have a Tm event attributable to the melting of CCNU crystal domains. Fig. 5 illustrates the nature of the PLA Tg event for sample A after 35 months of storage at -20C followed by varying periods of storage at 20C. There is no majorchange over the 20C storage times examined. The small spike at 43C for the sample stored 14 days at 20C is believed to be an artifact. Fig. 6 also shows the 70C CCNU fusion event characteristic of samples B and D. This peak is attributed to the melting of imperfect CCNU crystal domains. X-ray analysis confirms that crystalline CCNU is present in these microspheres. The crystal domains formed because the aqueous phase emulsifier used in the solvent evaporation process was not removed soon enough. The data presented above show that several samples with widely 4C. In these different thermal histories have a PLA Tg event at 21 cases, the thermal history is not the key factor controlling behavior of the PLA Tg event. The absence of crystalline CCNU in the PLA microspheres when formed is the controlling factor. When the CCNU is molecularly
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Fig. 6 DSC scans of CCNU-loaded (20.4 wt. percent) PLA microspheres after 34
mo storage at -20C followed by designated storage time at 20C (From Ref. 10.)
dispersed, it appears to act in effectas a PLA plasticizer. When the CCNU forms crystal domains in the PLA matrix during solvent evaporation, the observed PLA Tg approaches that of CCNU-free PLA and behaves on aging like CCNU-free PLA. The contribution of residual CH,Cl, to the PLA Tg event of microspheres in which CCNU is molecularly dispersed remains to bedefined. PLA microspheresinitially loaded with 22 wt. percent CCNU stored in a desiccator in the dark show no change in CCNU content during 8 weeks at
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3C [6]. Significant degradation occurred when the storage temperature was 22-37C. After 8 weeks at 37C approximately 46% had degraded. Free CCNU crystals stored at 22C in a desiccator in the dark showed no detectable change in 8 weeks. Thus, CCNU molecularly dispersed in the PLA microspheres was more unstable than free CCNUcrystals. Whether this is due to residual CH,Cl, in the microspheres, CCNU-polymer interactions, oxygen, or moisture has not been determined. However, it is clear that the molecular dispersion of CCNU in the PLA microsphere is more unstable at 22 and 37C than free CCNU crystals.
VI. CISPLATIN-LOADED POLY(d,l-LACTIDE) MICROSPHERES
Cis-diamminedichloroplatinum (cisplatin) is one of the most potent anticancer agents known. Accordingly, it was incorporated in poly(d,llactide) (PLA) microspheres [14]. Spherical, unaggregated PLA microspheres were obtained by dispersing cisplatin in a CH,Cl, solution of PLA and subsequently emulsifying this dispersion in a pH 2 aqueous phase that was saturated with cisplatin and contained a mixture of 88% hydrolyzed poly(viny1 alcohol) and methylcellulose. Cisplatin loadings ranged from 4.5 to 45.5 wt. percent. At all cisplatin loadings examined, the microspheres isolated contained macroscopic pores. The higher the cisplatin loading, the more porous themicrospheres. Because of this porosity, such microspheres did not retain CH,Cl, as tenaciously as microspheres free of macroscopic surface pores. Cisplatin is insoluble in CH,Cl,, so it was present in the PLA microspheres as small, crystalline particles believed to be similar in size to the 1- to 10-pm particles originally dispersed in CH,Cl,. X-ray microprobe analysis showed that the cross section of microspheres loaded with 45.5wt. percent cisplatin contained a uniform distribution of cisplatin domains (Fig. 7). There was no preferential partitioning of the drug particles toward the outer edges of the microspheres or toward the interior.
VII. SUMMARY OF MICROSPHEREMORPHOLOGICAL STUDIES
The microsphere examples discussed in Sections 11-VI illustrate that a diverse range of microsphere morphologiesexist. Factors that affect what morphology is obtained with a specific drug-polymer combinationinclude the nature of the drug, the nature of the polymer, the amount of drug in the microsphere, and the nature of the emulsifier in the aqueous phase. Microspheres prepared from biodegradable lactide polymers were discussed
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Fig. 7 X-ray microprobe photograph of cross sections of cisplatin-loaded (45 wt. percent) poly-d,l-lactide microspheres. (From Ref. 14.)
exclusively in this chapter, because they are actively being developed for parenteral formulations. Essentially an infinite number of potential drugpolymer combinations could be used to prepare anessentially infinite number of different microspheres. Many would have morphologies analogous to those described in this chapter. However, many should differ significantly. Morphology studies of lactide microspheres containing drugs other than those discussed here indicate this is the case [2]. One way to cause significant morphological changes is to alter the manner in which solvent is removed from the polymer-drug phase [15]. Another approach is to form microspheres from drug-polymer combinations with a high degree of mutual miscibility or mutual affinity. In such cases, drug crystallization may not occur even at relatively high loadings and polymer properties may be altered dramatically. A specific example is progesterone-loaded polystyrene microspheres [16]. Further studies such as those described in this
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chapter will provide valuable insight into thestructure of various polymerdrug combinations and suggest ways to control encapsulation process parameters so that unique microspheres are produced. REFERENCES
1. P. J. Watts, M. C. Davies, and C. D. Melia, Microencapsulation using emulsifi-
2. 3. 4.
5.
6. 7.
8. 9. 10.
11.
12.
cation solvent evaporation: an overview of techniques and applications, Crit. Rev. Ther. Drug Carrier Syst., 7(3):235-259 (1990). C. Thies, Formation of degradable drug-loaded microparticles by in-liquid drying processes, in Microcapsules and Nanoparticles in Medicine and Pharmacy (M. Donbrow, ed.), CRC Press, Boca Raton,FL, 1992, pp. 47-71. J. P. Benoit, F. Courteille,andC.Thies,Aphysicochemicalstudyof the morphology of progesterone-loaded poly (D,L-lactide) microspheres, Int. J. Pharm., 29:95-102 (1986). J. P. Benoit, S. Benita, F. Puisieux, and C. Thies, Stability and release kinetics of drugs incorporated within microspheres, in Microspheres and Drug Therapy, Pharmaceutical, Immunological and Medical Aspects (S. S. Davis, L. Illum, J. G. McVie, and E. T. Tomlinson, eds.), Elsevier, Amsterdam, 1984, pp. 91-102. S. E. B. Petrie, Thermal behavior of annealed organic glasses, J. Polymer Sci. A2,10:1255-1272 (1972). S. Benita, J. P. Benoit, F. Puisieux, and C. Thies, Characterizationof drugloaded poly(d,l-lactide) microspheres, J. Pharm. Sci., 73:1721-1724 (1984). V. Rosilio, J. P. Benoit, M. Deyme, C. Thies, andG. Madelmont, A physicochemical study ofthe morphology of progesterone-loaded microspheres fabricated from poly(D,L-lactide-co-glycolide),J. Biomed. Mat. Res., 25:667-682 (1991). M. Muramatsu, M. Iwahashi, and U. Takeuchi, Thermodynamic relationship J. Pharm. Sci., between alpha and beta forms of crystalline progesterone, 68~175-177 (1979). M. Cavalier, J. P. Benoit, and C. Thies, The formation and characterization of hydrocortisone-loaded poly((&)-lactide) microspheres, J. Pharm. PharmaCOL, 38:249-253 (1986). J. P. Benoit, G. Madelmont, F. Puisieux, and C. Thies, The storage behavior of CCNU-loaded poly (d,l-lactide) microspheres, Polymer Preprints 27(1):2728 (1986). M. C. Bissery,F. Puisieux, and C. Thies, A study of process parameters in the making of microspheres by the solvent evaporation procedure, Proceedings of 3rd APGI Congress, Paris, 1983,233-239. M. C. Bissery, F. Valeriote, and C. Thies, In vitro and in vivo evaluationof 2) lactideandpoly(& CCNU-loadedmicrospherespreparedfrompoly( hydroxybutyrate), in Microspheres and Drug Therapy, Pharmaceutical, Immunological and Medical Aspects (S. S. Davis, L. Illum, J . G. McVie, and E. Tomlinson, eds.), Elsevier, Amsterdam, 1984, pp. 217-227.
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13. K.Juni and M. Nakano, Poly(hydroxyacids) in drug delivery, Crit. Rev. Ther. Drug Carrier Syst., 3:209-232 (1987). 14. G . Spenlehauer, M. Veillard, and J .P. Benoit, Formation and characterization of cisplatin-loaded poly(d,l-lactide) microspheres for chemoembolization, J . Pharm. Sci., 75:750-755 (1986). 15. T. R. Tice and D. H. Lewis, Microencapsulation Process, U.S. Patent 4389,330,1983. 16. F. Courteille, J. P. Benoit, andC. Thies, The morphology of progesteroneloaded polystyrene microspheres,J. Control. Rel., 30:17-26 (1994).
7
D r u g Release from Microparticulate
Systems
Clive Washington
The University o f Nottingham, Nottingham,England
I.
Introduction
156 158 159 160 161 162 163 165 167 168 168 169 169
11. Measurement of Drug Release

111. Equilibrium Dialysis
IV. V. Sampling and Separation Methods Continuous-Flow Methods
VI. In Situ Methods
VI1. Mechanisms of Drug Release

VIII. Diffusion in a Homogeneous Sphere IX. Diffusion Through an Interfacial Bamer X. XI. XI1 Coated Particles Systems with Nonuniform Drug Distribution Heterogeneous ParticleMechanisms A. Dispersed and Phase-Separated Systems B. Erodable and Biodegradable Systems Regulated Drug Release
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172 173
XIII.
XIV. Pulsatile Release Systems
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XV. Simulation Methods for Drug Release
Washington
173 174 175
XVI. XVII.
Empirical Models of Drug Release Summary
1.
INTRODUCTION
Disperse systems for drug delivery have found wide application in pharmacy; initially for external use as creams and ointments and later for oral administration and subcutaneous or intramuscular delivery. A significant number of materials have also been formulated for intravenous administration, and these represent a distinct class of products. Materials intended specifically for intravenous administration must contain an extremely small a loose upper limit) , and load of large particulates (5 pm is often quoted as this sets manufacturing and quality control criteria for these materials which are significantly more stringent than for external or oral formulations, in which the occurrence of the occasional large particle is usually immaterial. Consequently, particulates for intravenous use are normally formulated to a particle size which is well below 1 pm; often this is done simply to ensure thatmuch larger particles are absent. A typical example of such a material is the intravenous triglyceride emulsion Intralipid (Kabi Pharmacia, Sweden), which is emulsifiedto a droplet size of 200-300 nm. In addition, there is much interest in the biopharmaceutics of small particulates for drug-targeting applications. There is an extensive recent literature dealing with the preparation of microparticles for delivery of a wide range of drugs. In many cases, very small particles (those smaller than 100 nm diameter) show particularly interesting behavior; for example,such small particles can escape from the capillaries in certain fenestrated tissues and accumulate at sites of inflammation. There is also an increasing body of evidence that submicrometer particles can be taken up from the gastrointestinal tract into the systemic circulation. . Consequently, there is much interest in the preparation and characterization of submicron particulates containing active materials; for example, antifungals, antibiotics [2-41, antiinflammatory agents anticancer agents [l], [5,6], and peptides [7,8]. Unfortunately, the handling and characterization of such formulations is a good deal more complex than that of similar supramicron systems, a fact which, judging from the earlier literature, is only just being realized by many experimenters. Drug-release data have a number of potential applications. At the simplest level, the data can be used for quality control purposes to ensure
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the constancy of behavior of a manufactured product. It can further be used to try to understand the physicochemical structure of the delivery system and the release mechanism; however, this normally requires the fitting of the data to that computed for a model of the drug-delivery system. Finally, such data can be used in an attempt to predict the likely behavior of the system in vivo. This is a much more complex problem which involves a knowledge of the interaction of many biological components with the particle, and this area is poorly understood at present. The release of a drugfrom a carriersystem isactually a combination of a range of processes transferring drug between a range of sites. Some of these processes are illustrated in Fig. 1. The drug first has to move through the camer,usually by diffusion through a solid or pores; it has to cross the interfacial layer, at which it may be bound if it is surface active; and finally, it must diffuse into the continuous phase. If it has phase separated, the drug may have to dissolve inthe carrierparticle prior to diffusion, and the timevarying hydration of the carrier particle may be important in many systems. The experimental release rate is the result of all these processes, although one is usually considered to be rate limiting and So dominates the kinetics. Some of these processes can be driven in reverse if the concentrationof the solute in the external phase is sufficiently high; indeed, this may form a method for loading microparticles. Under these conditions, the reversible
D b- \+ D
Fig. 1. Some possible sites of distribution of a solute drug(D) or phase-separated drug (shaded regions) in a microparticle.
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k ,
/
kf
[Dlcontinuous
Fig. 2. Equilibrium of drug release between particle and continuous phase.

equilibrium nature of drug diffusion will be evident. The kinetics will depend on the detailed mechanism, but as an example, consider the simplest scheme of Fig. 2, in which the drug in the particle is in first-order equilibrium with that in the aqueous phase, with no interfacial terms being significant. The rate of appearance of drug in the continuous phaseis givenby:
If the concentration in the external phase is zero, this reduces to:
.Consequently, the experimental release rate is only indicative of the release process behavior athigh dilutions; this is termed a perfect sink experiment, and it is generally desirable in order to simplify the interpretation of the experimental data.In concentrated systems, the experimental release kinetics are also influenced by the reverse reassociation reactions, which are represented here by k, (which may include other compartments, such as readsorption to a surface site on the particles). Obviously this model may be complicatedby such factors as surface binding, carrier degradation, and diffusion in the carrier. More complex models involving ionization of the solute and surface binding have also been analyzed[9,10], but in general, it is simpler to understand the behavior of perfect sink systems, so experiments are normally performed under these conditions.
II. MEASUREMENT OF DRUG RELEASE
The measurement of drug release from submicronparticulates poses many difficulties which are not encountered for larger particulate systems. The
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main difficulty arises when an attempt is made to separate the released drug in the continuous phase from the suspended colloidal camer; this becomes increasingly difficult and slow as the particles become smaller; since the release rates increase with decreasing particle size, a size is reached at which the separation time is the same order as the drug-release time. Under such circumstances, the time resolution of the experiment is inadequate anduseful data cannot beobtained. The size limitat which this occurs obviously depends on the camer and solute, butmost for purposes, one can expect to encounter difficulties for particles smaller than approximately 1pm. Emulsion systems may prove particularly troublesome owing to extremely rapid drug diffusion coupled with their polydisperse nature. Consequently, it ispreferable for many systemsto use analytical techniques which do not depend on separation of camer and solute. The methods available fall into four diffuse groups: (1) equilibrium dialysis, (2) sample and separate, (3) continuous flow, and (4) in situ methods.
111.
EQUILIBRIUM DIALYSIS
In equilibrium dialysis, a small volume of the concentrated drug-particle suspension is contained in a dialysis bag, which is immersed in a larger volume of continuous-phase acceptor fluid [ll-141. Preferably both compartments are stirred, and the drug then diffuses out of the particulate carrier into its local continuous phase andthen through the dialysis membrane into theacceptor phase, which is periodically sampled and assayed. This technique is highly misleading, since the particulate system is not diluted into a sink phase, and so sink conditions will not beachieved; under these circumstances, it will be difficult to interpret the release data. A simple analysis of this experiment (15) demonstrates thatthe measured rate of drug appearance in the external sink does not depend significantly on the particle release rate; instead it depends largely on thepartition coefficient of drug between its carrier and local acceptor phase. If we consider the model shown in Fig. 3, it can be shown that the concentration (A,)of drug in the external acceptor phase is given by:
where Kp is the partition coefficient and k, the dialysis membrane diffusion rate. This is independent of the particle release rate, k,, whichwe are seeking to measure. This partition-rate correlation is easily discernible in some published data; for example, the data of Sasaki et al. [l21for the release of a series of mitomycin C prodrugs from a triglyceride emulsion.
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DIALYSIS BAG
SINK
VC
Fig. 3. Kinetics of nonsink membrane dialysis technique.
Levy and Benita [l61 confirmed the disadvantages of this method in studies of an emulsion containing diazepam in an Intralipid-like base; they also demonstrated a reverse dialysis technique, in which the emulsion was diluted into asink and small preequilibrated dialysis bagscontaining buffer were used to sample the continuous phase. This method has the advantage that a large sink can be used, but it was still found to be rather slow owing to the diffusion rate through the bags (about 1 hr), and the drug release rate from the emulsion could not be distinguished from the release of a true solution preparation using this method. The investigators concluded that emulsion carriers released their drug very rapidly, and they suggested that this could be studied if smaller dialysis microcapsules were available.
W.
SAMPLING AND SEPARATION METHODS
Examples of this method normally start by preparing a concentrated suspension of the particulate carrierand rapidly diluting it into amuch larger bulk of acceptor phase [17-191. Samples of this diluted system are then removed at intervals, and the particles are separated so that the continuous phase may be assayed for released drug. This technique is perfectly adequate for larger microparticles, in which the separationcan be effected in seconds or minutes by filtration or centrifugation, and the release times are measured in hours. However, as the particle size decreases, the difficulty of separa-
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tion increases. Centrifugation times increase dramatically for thecomplete sedimentation of submicron particulates, and if the particles are polydisperse, extended centrifugation may be needed to sediment the smallest component. Filtration through membrane filters is an attractive alternative; however, the possibility of released drug binding to thefilters must be considered. Ultrafiltration membranes withlow binding properties are available, but these still adsorb small amounts of many drugs [20]. Even mildly hydrophobic materials may be strongly adsorbed to many filtration materials; in addition, the particles mayblock the filters, so that only limited sample volumes may be obtained. A rapid and useful variant of this technique used in our laboratories involves diluting a small amount (approximately 10 mg) of the particles in suspension into 100 mL of continuous phase ina 100-mLsyringe equipped with a 0.2-pm encapsulated membrane filter, so that samples of continuous phase may be expressed at suitable intervals; a time resolution of approximately 30 S may be obtained in this manner, but blockage of the filter normally limits the number of samples which can be obtained. A more technically sophisticated version of this method was described by Magenheim et al. [20], who used low-pressure ultrafiltration to separate samples of the released drug from a relatively large volume of sink fluid. This apparatus had a time response of a few minutes, which made itsuitable for studying microparticle formulations but too slow for emulsion release studies. The ultrafiltration step can be accomplished rapidly by centrifugal filtration [21], and simple centrifugal ultrafilters are available which make this a most convenient method. A particularly useful device in this respect is the Sartorius@ Centrisart (Epsom, UK) which uses an (initially empty) ultrafilter tube that floats on the top surface of the sample to becentrifuged inside the main centrifuge tube, and the filtrate enters this inner tube via centrifugal force. The advantage of this method is that the particles in the sample are forced downward away from the ultrafilter, thus avoiding filter clogging. An interesting final example of sampling and separation measurements was described by Widder et al. [22], who studied drug release from magnetite-albumin microspheres; these could rapidly be separated from the continuous phase using a magnet.
V.
CONTINUOUS-FLOW METHODS
The continuous-flow method most commonly uses an ultrafiltration cell to recover samples of clean continuous phase; this is fed from a pressurized reservoir and flows continuously through a detector (usually ultraviolet [UV]) for a direct continuous measurement of drug release [23,24a]. This technique is conceptually simple but presents a number of sources of error,
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with the most significant being variations (usually a decrease) in the flow rate through the ultrafiltration membrane as the experiment proceeds, thus distorting the release profile. The response time of such an experiment is approximately the time taken to replace the continuous phase in the ultrafiltration cell; consequently, a thin flat cell (i.e., one having a large ultrafiltration membrane area but a small total volume) is preferable. Qpical flow rates of 1 mL min" in a 10-mL cell suggesta 10-min time resolution; if the drug release rate is comparable to this, the time resolution can be improvedby deconvoluting the cell response function from the observed release function as we have described previously [25]. We suggest (but have not yet had time to implement) a significant improvement to this experiment. If the ultrafiltrate analysis is performed using a multichannel diode array spectrometer with multicomponent analysis software, it should bepossible to identify multiple components simultaneously in the filtrate. Consequently, the drug-carrier system in the cell could be spiked with an inert nonabsorbing marker dye in the continuous phase and the concentration of both dye and released drug measuredin the ultrafiltrate. This would allow variations in flow rate to bemeasured and, more importantly, provide a simultaneous instrument response function for deconvolution of the cell response time. Scholsky and Warner (26) have described an ultrafiltration technique in which the diluted colloid was circulated around one side of an Amicon (Mass.) microfiber ultrafiltration cartridge, and the release buffer was circulated over the opposite side. The appearance of the drug on the acceptor side was monitored by continuous UV spectrometry. The time resolution of this instrument was not assessed but appears to be of the order of hours rather than minutes, presumably owing to the membrane diffusion rate.
VI. IN SITU METHODS

In situ methods depend onthe use of analytical techniques which are only sensitive to the drug in solution and not to that in the solid particles or bound to their surface, so that theassay of released drug may be performed without any separation step, The difficulties discussed for many of the alternative methods would suggestthat in situ techniques would be particularly valuable; however, they have not found widespread use, largely on account of the difficulty of identifying suitable assay methods formost drug substances of interest. The most obvious method is to use optical absorption, since many molecules will have different spectroscopic properties depending on whether they are boundto theparticulate or freein solution. This methodwas used by Illumet al. [27] to study the release of rose bengal
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from cyanoacrylate microspheres. Scattering from the microspheres is a significant problem, and themethod can only normally be performedif the drug is strongly colored; the carrier particles often absorb or scatter light too strongly for measurements to be made in the UV region of the spectrum. It,is possible that these difficulties could be minimized by the use of multichannel spectrometers with multicomponent analysis, but this has apparently not been explored. A novel in situ sampling method was described by Minagawa et al. [28], who attempted to measure the release of isocarbacyclin methyl ester from an emulsion formulation using silanized glass beads to adsorb the free hydrophobic drug. This technique resulted in a time resolution of approxirelease time of a few minutes from a typical mately 15-30 S and an apparent triglyceride emulsion. As discussed below,techniques of this type are really too slow to study dynamic distribution processes in submicron emulsions, but they could prove to bevery useful for solid microparticle studies. Electrochemical techniques would appear to be valuable, but again have been little used. Polarography and ion-selective detection would have a response time of seconds, but very few drugs are amenable to detection using these methods. However, an attractive alternative is to use pH as an indicator of drug release. Many drugs are weak acids or bases, and their release can be monitored by measuring the neutralization of a suitable medium as the drug diffuses into it.We have used this method to study the release of model organic acids and the basic drug chlorpromazine from triglyceride emulsions [29]. This experiment has a time resolution of approximately 1S, and this is sufficiently rapid to measure drug release from emulsions, which occurs over a period of tens to hundreds of seconds. The results demonstrate thatthe release of drug is limited by interfacial transport, andso is sensitive to thenature of the emulsifier as well as the drug. The rapidity of drug release in these emulsion systems casts doubt on many earlier measurements in this area.
VII. MECHANISMS OF DRUG RELEASE

It is rarely possible to make any interpretation of drug release dataunless the experimenter has some preconceived model concerning the structure and composition of the system under study. Unfortunately, many workers give the impression that their systems consist wholly of monosized spherical particles uniformly loaded with drug; this is rarely the case, and it is useful to discuss some of the ways in which real formulations may depart from this ideal. First, the particles can depart from a monosized distribution of spheres. They usually have a distribution of sizes, with the occasional large
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particle having a significant fraction of the total drugmass. Without going into details of release mechanisms, this will imply a range of release rates, with the smaller particles showing rapid release and the larger ones a slower component. In theory it is straightforward to integrate the release rate expression over the particle size distribution if this is known. The particles may also be nonspherical; many papers show electron micrographs of spherical particles which show minor or gross distortions; these can be inducedby freeze-drying, so that the dried formulation shows very different release profiles to thesuspension formulation. Also, the structure of the particle may not be uniform; it could display core-shell behavior, with the drug being nonuniformly distributed across the particle section, or multiple phases of drug and carrier. It is evident that if the particles are porous or have a fractal interior structure, an even greater range of uncertainty exists concerning the drug distribution and release behavior. Finally, most polymer particles have varying degrees of crystallinity, which can be related to thedrug release rate from them. Second, we must consider all the possible locations for the drugload in the formulation. Since smallparticles have relatively rapid release rates, the drug will probably be in thermodynamic equilibrium between the carrier andits continuous phase. To ascertain the position of this equilibrium, it is usually helpful to isolate the continuous phase by ultracentrifugation and assay for unentrapped drug. The drug in the continuous phasemay be present in true solution or more likely in micellar solution if a significant amount of excess surfactant, from the particle preparation, is present. Removal of this surfactant can be troublesome; if it is dialyzed out, the drug it carried can be precipitated in the sample, so that two particle populations, one of carrier and one of pure drug, are present. If lecithin surfactants are used, an extensive liposomal phase may be present (30). The effects of this liposomal material on release of isocarbacyclin methyl ester froma triglyceride emulsion was highlighted by Minagawa et al. (28); over a third of the drug was present in the liposomes, and this caused a surprisingly long release profile for this fraction of the drug. Finally, if the drug is surface active, it will adsorb to theoutside of the particles and may provide a burst effect on dilution. Consequently, it will be appreciated that release data can only be unequivocally modeled if great effort is taken to check allof these possibilities prior to measuring the release profile. Despite all these difficulties, many experimenters still try to draw conclusions about the particle structure andrelease mechanism from therelease kinetics. As we will see, there is one furtherhurdle; that is, many of the model release mechanisms from particles of different types lead to very similar release kinetics, normally exponential or exponential-sum. As a result, it is rarely possible to draw
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conclusions about the structure of the particle from the release profile unless a considerable amount of supporting evidence is available.
VIII. DIFFUSION IN A HOMOGENEOUS SPHERE

This is the simplest model of a drug-releasing particle; it assumes that the drug is initially uniformly distributed in a sphere made of material with a uniform diffusion coefficient. Because of its simplicity, it can be modeled analytically; the mathematicsfor this process are well known [31] and were applied to the drug-release problem by Guy et al. [32]. The expected release-time profile for this model is
where M, is the amount of drug in the particle at time t and the dimensionless time, T, is given by:
D is the diffusion coefficient of the drug in the oil droplet and r is the droplet radius. This sum-of-exponentials series provides a decay rate that initially is steeper than a single or best-fit exponential, and at long times is slower than a single or best-fit exponential; this emphasizes the difficulty in obtaining a sustained release effect from a monodisperse sphere formulation of this type, and indeedthe rapid initial release may be indistinguishable from the burst effect observed in many systems. In order to simplify a discussion of this model, simplified forms of this equation have been used. Expansion and truncation of the series yields:
which suggests that the initial release rate is inversely dependent on the particle size and proportional tothesquareroot of time. This latter relationship is often termed the Higuchi law; this is in fact an incorrect use of the term, since Higuchi derived this relationship for a completely different model system. Truncated series of this type show an increasing error at later times, and they should only be used for analysis of the release of the first 5-10% of the drug or unacceptable errors will result.
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Despite this unavoidable consequence of the model, many investigators emphasize that their data obey the square root law over much longer intervals; much of theliterature in this area is clouded by the blind application of this law. Addition of a further termin the series yields [33]
which provides a fit for data atlonger short (!) release times. An approximation for drug release at long times (i.e., the release of the last few percent of the drugload) is given by:
This expression can be rearranged into the linear form:
Thus, a graph of ln(1 - M j M o ) against time will have a limiting slope of (-$D)/? at long times, enabling the diffusion coefficientof the drug in the oil droplet to be calculated. The simple uniform diffusion model is applicable to those systemsin which the drug is uniformly dissolved in the particle, and the matrix does not degrade during the experiment or present an interfacial barrier. In emulsions, the most obvioussystem, release is controlled by the interfacial transfer characteristics, and it is only in solid systems that bulk diffusion becomes limiting. Some swelling noneroding polymersmay release by this mechanism; normally these contain pores of solute dimensions through which the drug can diffuse; if the pores are of larger dimension than the drug molecules, then a heterogeneous model (see below) would be more appropriate [34]. An interesting variant of the diffusion controlled model was discussed by Cremers et al. 1351 to describe the release of drugs from ion-exchange microspheres. These are prepared from combinations of proteins (normally albumin) and negatively charged polyelectrolytes such as heparin [36,37] or poly (L-glutamate) [38]. In such systems, the drug is retained in the particle by ionic interaction with the polyelectrolyte and is released by an ion-exchange process when ions from the release medium diffuse into the particle. Cremers et al. [35] modeled this process by assuming that the ion transport into theparticle was rapid and in equilibrium with the rapid
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solvent swelling of the particle. Under these conditions, diffusion the of the drug in the matrix is rate limiting, and so the release kinetics is similar to that of the simple diffusion model. However, there is one important difference; theactivity of free drug (i.e., not bound to the polyelectrolyte) in the microsphere is controlled by the ionic strength of the release medium. Thus, the fraction of drug extracted increases as the ionic strength of the medium is increased, although the release rate is constant and controlled by the diffusion coefficientof the drug in the microsphere. One oft-suggested method of circumventing the problem of nonlinear release and obtaining a constant release profile is to use a collection of phases spheres of varying sizeso that small spheres contribute to the initial of drug release, whereas larger spheres contribute to release at longer times. A few moments thought will reveal that this argument is flawed; a collection of spheres with varying sum-of exponential release profiles, however weighted, can only yield an overall sum-of-exponentials release profile; addition of a wider range of particle sizes will just make the initial slope steeper and the tail longer. A flat release profile cannot be decomposed into a sum of exponentials. However, we should note that this apof the individual particles was not proach may be feasible if the release rate identical to this theoreticalspherical particle releaseprofile. IX. DIFFUSION THROUGHAN INTERFACIAL BARRIER The previous model assumed that the release of drug was limited by the rate at which it diffused through the carrier particle. Although this model may be valid for particles of larger sizes or those madeof solid materials, it is unlikely to be limiting for smaller liquid droplets such as those found in parenteral emulsions. In such cases, the expected diffusion coefficients would lead to drug release times of a few tens of microseconds; we have measured the release rates from model emulsions to be of the orderof tens must beof much greater relevance of seconds; and so the interfacial barrier in this case. The model for releaseof drug from a spherical particle in this situation is more complex than that for the situation in which a barrier is not present. In fact Guy et al. [32] state that no complete solution exists and present approximatesolutions for shortand long times. At short times, the release is zero-order andis given by:
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where K is a reduced interfacial rate constant related to the true interfacial rate constant k, by:
It is difficultto verify this regimen for real systems, since the presence of a burst release component often overlaps on this timescale. At long times, where T >> 1, the release is given by a single exponential decay:
-3kf M O-l-exp(T) This is a useful method for obtaining k,, since it is straightforward to linearize:
"
M,
Thus, a graph of ln(1 - M,/M,) against time will have a limiting slope at long times of -(3k,)/$ enabling the interfacial transport rate constant of the drug betweenthe particle and the release medium to befound.
X. COATED PARTICLES
The coated spherical particle is a rather fundamental type of delivery system, in a range of macroscopic controlled-release devices, as and it has been used well as microspheresin which the outer coating or membrane is distinct in composition from the inner core. Liposomes may be classified as coated particles, and Pekarek et al. [39] described a method of making two-layer microspheres by the control of surface wetting and interfacial energetics. A full analysis of diffusive release from a coated particle is complex, but it has been fully detailed by Lu and Chen [40]. The full solution can be simplified by taking a number of specific cases; the most useful is that for release into aninfinite sink. Solutions are also givenfor the cases where the diffusion coefficient inthe core or the inner matrix are rapid.
XI. SYSTEMS WITH NONUNIFORM DRUG DISTRIBUTION
It has been suggested that the burst effect observed in many systems is due to weakly bound drug at or near theparticle surface, and if the particles were briefly washed, this would deplete the drug concentration near the particle surface and lead to a more uniform release of drug. Lee [41a,41b,42] studied release from macroscopic devicesof this type, but the
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results are equally applicable to particulate systems. The results suggest that by suitable selection of concentration distribution it ispossible to achieve steps or peaks in the release rate rather than a continuously declining rate, and that,for example, a pseudo-zero-order release may be possible by using a multilayer structure with varying drug concentrations in the layers. Although it is easier to visualize these techniques being applied to relatively large particles, it maybe technologically feasible to prepare colloidal systems withthese characteristics. XII.
HETEROGENEOUSPARTICLEMECHANISMS
The simple uniform spheremodels discussedabove are probably only valid for a small number of systems, including emulsion formulations and some lightly doped nondegradablemicroparticles. The majority of microparticle systems are heterogeneous, andso drug diffusion through themis complex. We could imagine, for example, polymeric microspheres, in whichthe crystallinity varies spatially with polymer blend; microspheres in whichthe drug and carrier are phase separated into distinct domains; and systems where drug diffusion is so slow that degradation of the microsphere was rate limiting. For example, Magenheim et al. [20] reported variations in the release of indomethacinfrom poly-(lactide-co-glycollide) (PLGA)and poly(1actic acid) (PLA) nanoparticles which could be attributed to variations in the solvent permeability of the polymer. Many examples of heterogeneous systems have been studied in the literature to varying degrees of detail.
A. Dispersed and Phase-Separated Systems
A potentially common system is that in whichthe drug has phase separated from theparticle matrix into pure domains and is then released by diffusion through the polymer. Although systems of this type have been widely studied as macroscopic devices for controlled release (e.g., see ref. 43), many microscopic particles also display these characteristics. Baker and Lonsdale [33] studied a system of this type, and they found thatthe release rate was given by:
where C, is the drug solubility in the dissolution medium and A the drug loading per unit volume. The drug particles are assumed to be small compared with the particle radius. Release of drugs from heterogeneoussystems has often been treated using the expression derived by Higuchi [M] for the flux, Q, from a planar slab of polymer containing the drug:
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Q = ~ D C , ( ~C c,)t ,
In this expression, c, is the solubility of the drug in the matrix, c, is the initial drug concentration, and c, is the drug solubility in the sink phase. Unfortunately, this expression refers to an infinitely deep matrix which is not significantly depleted, and so it is rarely applicable if the majority of the particles drug load is released. However, the use of expressions in which release is proportional to the square root of time are so firmly ensconced in pharmaceutics that little thought is normally given to their significance or applicability. As the loading of solid drug in the matrix is increased, ultimately a point will be reached at which the drug particles are in contact with each other; this normally occurs at loadings of around 10-20%. In this case, as the drug diffuses out of the matrix, solvent-filled channels are left, which act as pathways through which the remaining drug is preferentially released. The full expression for release from a spherical matrix of this type is rather complex [44] and involves terms for the porosity and tortuosity of the matrix. These terms can only be accessed with difficulty, and it would appear that such treatments are straining the limits of the pure analytical approach to drug-release modeling. Ultimately numerical simulation may prove to bemore useful for these systems. Examples of pure multiphase systems are difficult to find, since many fall into the category of erodable or degradable systems rather than diffusive systems. However, one important class of multiphase systems ismultiple emulsions, in which the drug is carried in an inner (normally aqueous) phase within an oil droplet. These systems have been extensively studied, although they have not been widely exploited owing mainly to stability problems of the inner aqueous droplets within the oil droplets [45-471. Magdassi and Garti [48, 491 suggested that the Baker and Lonsdale [33] expression should be applicable to release from multiple emulsions, and they found a reasonable agreement with experiment, as did Raynal et al. [50], who studied the release of sodium lactate from a liquid paraffinisopropyl myristate multiple emulsion system. B.ErodableandBiodegradableSystems There is an extensive literature on hydrating and eroding systems, which is largely applicable to macroscopicdevices for sustained-release applications. Eroding devices differ from those previously discussed, because drug release is by definition controlled by erosion of the particle rather than diffusion of drug within the matrix. This represents an increasingly important class of particles owing to the currentinterest in biodegradable particles for drug delivery and particularly for the delivery of macromolecules
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such aspolypeptides and DNAfractions. The most commonlyused materials for such applications are poly(1actic acid) (PLA) andpoly(glyco1ic acid) (PLG) and their copolymers (PLGA); polyanhydrides are also receiving much attention [51,52]. These polymers can be made to degradein vivo on a timescale of hours to weeks, depending on the device size and polymer blend, and can easily be made intoparticles by solvent evaporation methods. Allemann [53a,53b] described a salting-out method which appeared to reduce the burst effect from the resulting microspheres. These polymers degrade by backbone cleavage by simple hydrolysis, also known as type 3 erosion [54]. It is fairly certain that both macroscopic devices and microsphere formulations degrade uniformly in the bulk rather thanprogressively from the surface. The erosion rates are largely controlled by the crystallinity of the polymer, with amorphous polymers showing the most rapid degradation, and polymers degrading more rapidly above the glass transition temperature [55,56]. The older literature on these systems has been reviewed by Heller [57]. More recent studies have provided a detailed description of the effects of polymer chain length and copolymer mix on release; for example, the work of Le Corre et al. [58] demonstrated that release rates of bupivacaine from pure PLA microparticles varied with polymer chain length, with the shortest polymers showing the most rapid release rate, and an increased proportion of glycolide also causing an increase in release rate. The drug release from a surface eroding particle, in contrast toa bulk eroding particle, is given by:
where k, is an erosion rate constant, r is the initial radius of the sphere, and c, the concentration of drug in the sphere [59]. There is little modeling work on the release from bulk eroding particles, although these appear to lose no mass until they have reached a critical chain length (m.w. 15,000), after which breakdown is rapid [55]. In general, however, it is inappropriate to use equations of this type to describe release, because the particles are rarely homogeneous. For example, the PLAPLGA particles produced by Le Corre [58] referred to above showed a porous cross section; the origin of the poresis uncertain, but may be due to phase separationof oily droplets of the drug (bupivacaine base) within the polymer as the dichloromethane solvent was lost. This complex structure led to a rapid initial release rate, presumably from pores close to thesurface, followed by a slower rate which the authors suggested was due to the onset of polymer degradation. These polymers appear to behave similarly when prepared as spray-dried particles [60];such particles are normally hollow microcap-
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sules. Particle degradation appeared to be localised at specific pore sites, which presumablywere also responsible for drug release. Iwataand McGinty (61) described multiphase PLGAparticles prepared by a multiple emulsion method, which degraded internally over several weeks in the release experiment despite maintaining a uniform wall structure. It is evident thata detailed modeling of the release kinetics from such increasingly complex systems will be demanding, to say the least. However, there is much interest in these systems because it may be possible to use them to provide delayedpulsatile release of drugs (see below). The other majorcandidates for the production of biodegradable microspheres are naturally occurring proteins and polysaccharides. Albumin microspheres have been studied for many years in this regard (e.g., see ref. 62), as have a number of other proteins. Gelatin [63] and hyaluronic acid [64] are among a range of polysaccharides studied for this purpose. These systems are normally made by a water-in-oil emulsion technique, leading to microsphere sizes of a few micrometers down to hundreds of nanometers if high shear dispersion is used. There is some difficulty in the interpretation of release data from these systems, since they are normally fairly stable in sterile aqueous media, andso proteases are normally added to the release medium in order to bettersimulate their degradation in vivo. It may thus appear that surface erosion modelswould be applicable; however, the actual behavior of the particles is more complex, since the microspheres normally swell prior to being digested, and so one must assume that some enzyme is transported into the core of the particle during this initial swelling phase. This behavior was demonstrated by Magee et al. [65], who studied the size of albumin microspheresby laser diffraction as they were digested in a trypsin-containing medium; the microspheres swelled to several times their initial diameter over 20-30 mins prior to being degraded over a further30-60 min by the enzyme. Control of release rates from protein or polysaccharide particles can be achieved by varying the particle size or the degree of crosslinking. For example, Jayakrishnan et al. [66] demonstrated that the release of methotrexate from casein microspheres was influenced by the degree of crosslinking as a result of varying the amount of glutaraldehyde crosslinker used in the particle preparation; similar results were found by Rubino [67] and by Dilova andShishkova [68]for chemically and thermally denatured albumin microspheres.
XIII. REGULATEDDRUG RELEASE

The most sophisticated drug-delivery systems now attempt to control or regulate the release of drug according to some local chemical signal or
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biological need in the tissue. Such systems are called regulated-release devices, and they evidently pose significant difficulties for in vitro characterization, since it is necessary to confirm that the release rate responds to the external stimulus. Although the study of such devices is in its infancy, a number of systems have been developed; these have largely been macroscopic rather than particulate systems. It seems inevitable, however, that this technology will be applied to particulates in the near future. For example, Yui et al. [69] demonstrated that crosslinked hyaluronic acid was degraded by hydroxyl radicals, and that this could be used to trigger degradation of the matrix at inflammation sites. Release can also be triggered by an external stimulus. The best known of these systems are the magnetic microspheres, in which the application of an external magnetic field causes both local accumulation and modifies the drug-release rate. Saslawski et al. [70] demonstrated that this technology could be used to deliver insulin from ferrite-containing alginate microspheres, with an oscillating magnetic field causing a 50-fold increase in the insulin release rate.
XIV. PULSATILE RELEASE SYSTEMS

There is much interest in producing systems which release their payload in one or more pulses over a controlled time period. Such a system would be useful for the delivery of multiple-challenge antigens and peptide hormones. Pulsed systems are generally produced by engineering a time delay into the particle-degradation mechanism; for example, Kibat et al. [24b] demonstrated that liposomes coencapsulated with phospholipase would release their drug payload after a delay, corresponding to the time taken for the phospholipase to destroy the integrity of the liposomes. Delayed release can also be achieved with some of the larger polylactide/glycolide microsphere systems [71], since these normally show a lag phase prior to release; this corresponds to thetime taken forsolvent swelling and internal polymer degradation. Other pulsed-release systems under study include polyiminocarbonates [72] and poly(ortho esters) [73]. XV. SIMULATION METHODS FOR DRUG RELEASE The gradual increase in complexity of drug-release models ultimately causes them to become mathematically intractable or incapable of analytic solutions. In such cases, it is possible to tackle the problem by simulation techniques, in which a particle is set up on a lattice, and Ficks laws applied to calculate the diffusion of the drug as a function of space and time. Models of this type can take intoaccount processes such as the hydration of the parti-
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cle, and the consequent spatial variation in the diffusion coefficient, variable drug distributions, and drugbinding at interfaces, depending mainly on the amount of computing power available. The use of such methods is in its infancy, but a number of simple deviceshave beenstudied in this way. Armand etal. [74] used this technique to study release from Eudragit matrix formulations, in whichthe primary release mechanism wasthe diffusion of solvent into the polymer, followed by dissolution of drug and its diffusion out of the device. Both experiment and data demonstrated that drug release follows a square root law for small release fractions after a lag phase. Liu et al. [75] used simulation techniques to study the release from a loaded sphere coated with a further layer of polymer, in which solvent penetration was taken intoaccount. Their experimental data was obtained from macroscopic systems (several millimeters in diameter), and good correlation with the calculations was found for the thinner polymer coatings.
XVI. EMPIRICAL MODELS OF DRUG RELEASE

Many investigators have regarded the analytic modeling of drug release as unjustifiably complex and have used more empirical techniques for data analysis. These approaches vary from those which provide useful data about the system dynamics to those which one is convinced have been adopted simply to minimize the size of error bars in a graph. Probably the most useful of the former group is the diffusional exponent approach described by Peppas and colleagues [76,77],which goes some way to explainning the behavior of hydrating or eroding systems. In such systems, the diffusion coefficient is not constant, and the term anomalous d i m i o n is often used to indicate that a constant value of the diffusion coefficient does not satisfactorily fit the data. The termsFickian and non-Fickian are also used to indicate whether or not a material is diffusing with a temporally and spatially constant diffusion coefficient; these are somewhat misleading, since, at a particular instant and point in space, diffusion always occurs according to Fick's law. The diffusional exponent method proposesa power law relationship for drugrelease:
The constant n is termed the diffusional exponent, and it should equal 0.5 for diffusional (Fickian) release from a planar slab. Values greater than0.5 indicate anomalous diffusion, -and are generally indicative of a system which swells in the solvent prior to diffusional release. Analysis of the
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model and comparison to the exact solutions demonstrates thatn is equal to 0.5 for a flat slab and 0.432 for a sphere. Since simple diffusive release from spheresis often adequately fitted when n = 0.5, it is evident that this approach requiresprecise data toallow the extractionof a useful value for n. A corollary to this is that many of the experimental techniques in the literature require improvement before they can be used to discriminate between the various mechanisms of drug release. One of the morepopular empirical relations is to describe release as a biexponential process:
of the two lifetime components into Where k, and k, are the rate constants which the decay function is being decomposed. The exponentials usually consist of a rapidand a slow function, being assigned to burstphase and sustained release,respectively. The decomposition of a monotonically decreasing function into exponential componentsshould be performed with some caution,since it is an ill-posed problem. Lanczos [78]demonstrated that a sum of three exponentials waswell fitted by a sum of two exponentials with distorted time constants and amplitudes, and further thatrecovery of exponential components requires data of an extremely high degree of accuracy, far beyond that observed for most drug release experiments.Attempts to obtain more than two release components from experimental data must often be treated with scepticism. A corollary of this problem is that it is extremely difficult to extract true multiple exponentials, so that when these exist, a double exponential function provides an apparently adequate model. This has further led many experimenters into the misconception that the exponential components correspond to two distinct physical processes (which may be true in some cases), and further that monodisperse homogeneous microspheres display a single exponential releaseprofile (which we have seen is completely false). The use of empirical functions to model release data has been taken to an extremeby Lin [79], who tried 10 separate simple functions as fitting equations forthe release of theophylline microcapsules. Although a best-fit equation was found (lly = A/x + B ) , this approach does not significantly increase our understanding of the physical chemistry of the system. XVII. SUMMARY Our understanding of microparticle systems has developed considerably over the past 10 years. In themid 1980s, a typical drug-microparticlepubli-
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cation was characterized by a rather fuzzy electron micrograph of vaguely spherical adhering objects, with possibly a laser sizer size measurement that did not agree with the microscopic dimensions. Fortunately, we now have much more well-characterized systems, with details of internal morphology and accurate size distributions, and we have gained experience in the reproducible preparation of high-quality dispersed systems. Despite these achievements, the two central problems still remain. First, how do we design a microsphere system that will have specific desired properties? Second, evenif we know exactly what is required, how do we manufacture it? The second question is still largely a black art, with pockets of knowledge centered around specific drug-particle combinations and only rather vague ground rules being known. It ispossible that the application of expert systems may assist in the correlation of the enormous amount of scattered information and experience that at present is poorly accessible. The first question is more tractable; as we have seen, models of many particle types can be created and understood. However, many details can only be explained qualitatively after the event. As a result, microparticle development is still an iterative process, with developers cycling round a make-try-modify loop which in many cases never reaches a usable conclusion. It would save many hours of experimentation with expensive materials (not to mention the lives of many animals) if such systems could be tested by simulation prior to production. Ultimately, a full understanding of the behaviorin vitro and in vivo of drug release from a complexsystem like a microparticle suspension is probably beyond the possibility of a rigorous analytic solution. Systems may ultimately be evaluated using numerical simulation of dissolution and diffusion in three dimensions, with full details of their heterogeneous in vivo environment. This would require a massive computing effort,but it is well withinthe capabilities of modem parallel processors. It would then be possible to study, for example, the effect of changing the polymer blend or drug partition coefficienton the pharmacokinetics of an intramuscular particle depot. Under these circumstances, it finally would become possible to design a microparticle system rationally by computational methods, by much the same philosophy that new chemical entities are developed at present. REFERENCES
1. D. J .Kerrand S. B. Kaye, Chemoembolism in cancer chemotherapy, Crit. Rev. mer. Drug Camer Syst., 819-37 (1991). 2. S. S. Davis, C. Washington, P. West, et al., Lipid emulsions as drug delivery systems, Ann. N.Y. Acad. Sci., 507:75-88 (1987).
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3. K. A. Lamb, C. Washington, and S. S. Davis, Toxicity of amphotericin B emulsion to cultured canine kidney cell monolayers, J. Pharm. Pharmacol., 43~522-524 (1991). S. S. Davis, Toxicityof amphotericin B emul4. C. Washington, M. Lance, and sion formulations, J. Antimicrob. Chemother., 31:806-808 (1993). 5. K. Yokoyama, H. Okamoto, M. Wantanabe, et al., Development of a corticosteroidincorporatedinlipidmicrospheres,DrugsExp.Clin.Res., 11~611-620 (1985). 6. Y. Shoji, Y. Mizushima, A. Yanagawa, et al., Enhancement of antiinflammatory effects of biphenylacetic acid by its incorporation into lipid microspheres. J. Pharm. Pharmacol., 38:118-121 (1986). 7. S. S. Davis, L. Illum, andE. Tomlinson (eds.), Delivery Systems for Peptide Drugs. NATO Advanced Science Institutes series, Vol. A125, Plenum Press, New York, 1986. for delivery 8. P. Couvreur andF. Puisieux, Nanoparticles and microparticles the of polypeptides and proteins, Adv. Drug Deliv. Rev., 10:141-162 (1993). S. L. Silvestri, Theoretical considerations of drug 9. R. T. Lostritto, L. Goei and release from submicron oil in water emulsions, J . Parent. Sci. Techno]., 41: 214-224 (1987). 10. S. Silvestri, L. L. Wu, and B. Bowser, Release of polyionizable compounds J. Pharm. Sci., 81:413-418 from submicrometer oil in water emulsions, (1992). 11. M. Hashida, T. Yoshioka, S. Muranishi, and H. Sezaki, Dosage form charac1: Stability and drug release, Chem. teristics of microsphere in oil emulsions. Pharm. Bull., 28:1009-1015 (1980). 12. H. Sasaki, Y. Takakura, M. Hashida, et al., Antitumour activityof lipophilic prodrugs of mitomycin C entrapped in liposome or o/w emulsion, J. Pharm. Dyn., 7:120-130 (1984). 13. S. Benita, D. Friedman, and M. Weinstock, Pharmacological evaluation of an injectable prolonged release emulsion of physostigmine in rabbits, J. Pharm. Pharmacol., 38:653-658 (1986). 14. S. Miyazaki, N. Hashiguchi,W. M. Hou, et al., Preparation and evaluation in vitro and in vivo of fibrinogen microspheres containing adriamycin, Chem. Pharm. Bull., 34:3384-3393 (1986). for the measurement 15. C. Washington, Evaluations of non-sink dialysis methods of drug release from colloids: effects of drug partition, Int. J. Pharmaceut., 56~71-74 (1989). 16. M. Y. Levy and S. Benita, Drug release from submicronized olw emulsion: a new in vitro kinetic evaluation model., Int. J. Pharmaceut., 66:29-37 (1990). 17. D.C.Tsai, S. A.Howard,T. F. Hogan, et al., Preparation and in vitro evaluation of polylacticacid-mitomycinCmicrocapsules, J. Microencaps., 3~181-193 (1986). . Bouzon, M. Rollet, et al., Dry emulsion-A sustained release 18. N. Farah, J form: Modelling of drug transfers in liquids, Int. J. Pharmaceut., 36:81-88 (1987).
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19. S. Henry-Mitchelland, M. J. Alonson, A. Andremont, et al., Attachment of antibiotics to nanoparticles: Preparation, drug release and antimicrobial activity in vitro, Int.J. Pharmaceut, 35:121-127 (1987). 20. B. Magenheim, M. Y. Levy, and S. Benita, A new in vitro technique for the evaluation of drug release from colloidal carriers-Ultrafiltration technique at low pressure, Int.J. Pharmaceut., 94:115-123 (1993). 21. N. Ammouri, H.Fessi, J. P. Deuissaquet,F. Puiseurc, and S. Benita, In vitro release kinetic patternof indomethacin from poly(D,L-lactide) nanocapsules. J. Pharm. Sci., 79:763-767 (1990). 22. K. Widder, G. Flouret, and A. Senyai, Magnetic Microspheres: Synthesis of a J. Pharm. Sci., 68:79-81 (1979). novel parenteral drug carrier, 23. D. J. Burgess, S . S. Davis, and E. Tomlinson, Potential use of albumin microspheres as a drug delivery system. I. Preparation and in vitro release of steroids, Int.J. Pharmaceut., 39:129-136 (1987). 24a. F. Koosha, R. H. Muller, and S. S. Davis, A continuous flow system for invitro evaluation of drug-loaded biodegradable colloidal carriers, J. Pharm. Pharmacol., 40:1310 (1988). 24b. P. G. Kibat, Y. Igari, M. A. Wheatley, et al., Enzymatically activated microencapsulated liposomes can provide pulsatile drug release, FASEB 4:25332539 (1990). F. Koosha, Drug release from microparticulates; deconvo25. C. Washington and lution of measurement errors, Int.J. Pharmaceut., 59:79-82 (1990). J. Warner,Automateddeviceforanalysingdrug 26. K.M.ScholskyandR. releasekineticsfromcolloidalparticles,Rev.Sci.Instrum.,58:1545-1547 (1987). E. Mak, andS. S. Davis, Evaluation of carrier capacity 27. L. Illum, M. A. Khan, and release characteristics for poly(buty1-2-cyanoacrylate) nanoparticles, Int. J. Pharmaceut., 30:17-28 (1986). T. Suwa, and A. Tsuji, Entrapping efficiency 28. T. Minagawa, Y. Kohno, and drug release profile of an oil-in-water (o/w) emulsion formulation using a polydimethylsiloxane-coated glassbeadassay.Pharm.Res.,11503-507 (1994). 29. C. Washington and K. Evans, Release rate measurements of model hydroJ. Control Rel., 33:383phobic solutes from submicron triglyceride emulsions, 390 (1995). 30. K.Westesenand T. Wehler,Physicochemicalcharacterizationofamodel intravenous oil-in-water emulsion, J. Pharm. Sci., 81:777-786 (1992). 31. H. S. Carlslaw and J. C. Jaeger, Conduction of Heat in Solids, Clarendon Press, Oxford, UK, 1959. 32. R. H. Guy, J. Hadgraft, I. W . Kellaway,andM. J. Taylor, Calculations of drug release rates from spherical particles, Int. J. Pharmaceut., 11:199-207 (1982). 33. R. W. Baker andH. K. Lonsdale, Controlled release: mechanisms and rates, In Controlled Release of Biologically Active Agents (A. C. Tanquary and R. E. Lacey, eds.), Plenum Press, New York, 1974, pp. 15-71.
Drug Release from Micropatticulate Systems
179
34. R. S. Langerand N. A.Peppas,Presentandfutureapplicationsofbiomaterials in controlled drug delivery systems, Biomaterials, 2:201 (1981). 35. H. F . M.Cremers,R.Vemjk, H. P. J. M. Noteborn, et al., Adriamycin loading and release characteristics of albumin-heparin conjugate microspheres, J. Control. Rel., 29:143-155 (1994). 36. W. E. Hennink, J. Feijen, C. D. Ebert, and S. W. Kim, Covalently bound conjugates of albumin and heparin: synthesis, fractionation and characterization, Thromb. Res., 29:l-13 (1983). 37. H. F. M. Cremers, G. Kwon, Y. H. Bae, et al., Preparation and characterization of albumin-heparin microspheres, Biomaterials, 15:38-48 (1994). 38. W. E. Longo, H. Iwata, T. Lindheimer, and E. P. Goldberg, Preparation and drugreleaseproperties of albumin-polyglutamic acid adriamycin microspheres, Polymer Prep., 2456-57 (1983). 39. K. J. Pekarek, J. S. Jacob, and E. Mathiowitz, Double-walled polymer microspheres for controlled drug release, Nature, 367:258-260 (1994). 40. S. M. Lu andS. R. Chen, Mathematical analysis of drug release from a coated particle. J. Control. Rel., 23:105-121 (1993). .I. Lee, Diffusion release of a solute from a polymeric matrix: Approximate 41a. P analytic solutions,J. Membrane Sci., 7:255-275 (1980). 41b. P. I. Lee, Kinetics of drug release from hydrogen matrices, J. Control. Rel., 2~277-288 (1985). 42. P. I. Lee, Initial concentration distribution as a mechanism for regulating drug release from diffusion controlled and surface erosion controlled matrix systems, J. Control. Rel., 4:l-7 (1986). 43. J. R. Cardinal, Matrix systems, in Medical Applications of Controlled Release FL,1984, pp. (R. S. Langer and D. L. Wise, eds.), CRC Press, Boca Raton, 86-97. 44. T. Higuchi, Mechanism of sustained-action medication: theoretical analysis of rate of release ofsoliddrugsdispersedinsolidmatrices,J.Pharm.Sci., 52~1145-1149 (1963). 45. A. T. Florence andD. Whitehill, Some features of breakdown in water-in-oilin-water multiple emulsions, J. Coll. Interface Sci., 79:243-256 (1981). 46. A. T. Florence and D. Whitehill, The forulation and stability ofmultiple emulsions, Int. J. Pharmaceut., 11:277-308 (1982). 47. J. A. Omatosho, T. L. Whateley, T. K. Law, and A. T. Florence, The nature of the oil phase and the release of solutes from multiple W/O/W emulsions, J. Pharm. Pharmacol., 38:865-870 (1986). 48. S. Magdassi and N. Garti, Release of electrolytes in multiple emulsions: Coalescence and breakdown or diffusion through oil phase, Colloids Surfaces, 12:367-373 (1984). 49. S. Magdassi and N. Garti, A. kinetic model for release of electrolytes from W/O/W emulsions, J. Control. Rel., 3:273-277 (1986). 50. S. Raynal, J. L. Grossiord, M. Seiller, and D. Clausse, A topical W/O/W multiple emulsion containing several active substances: formulation, characterization, and study of release, J. Control. Rel., 26:129-140 (1993).
180
Washington
51. Y. TabataandR.Langer,Polyanhydridemicrospheresthatdisplaynearconstantreleaseofwater-solublemodeldrugcompounds,Pharm.Res., 10:391-399 (1993). 52. Y. Tabata, S. Gutta, and R. Langer, Controlled delivery systems for proteins using polyanhydride microspheres, Pharm. Res. 10:487-496 (1993). E. Doelker,Invitroextended 53a. E. Alleman, J. C.Leroux,R.Gurny,and release properties of drug loaded poly (DL-lactic acid) nanoparticles produced by a salting-out procedure, Pharm. Res., 10:1732-1737 (1993). 53b. E. Allemann, R. Gurny, and E. Doelker,Drug-loadednanoparticlesJ. Pharmaceut.BioPreparationmethodsanddrugtargetingissues,Eur. pharmaceut., 39:173-191 (1993). 54. c. G. Pitt, T. A.Marks,andA.Schindler,Biodegradabledrugdelivery systems based on aliphatic polyesters: Applications to contraceptives and narcotic antagonists, in Controlled Release of Bioactive Materials (R. W. Baker, ed.), Academic Press, New York, 1980, p. 36. 55. C. G. Pitt, M. M. Gratzl, G. L. Kimmel, et al., Aliphatic polyesters11. The degradationof poly (DL-lactide), poly (e-caprolactone) and their copolymers in vivo, Biomaterials, 2:215 (1981). 56. S. Izumikawa, S. Yoshioka, Y. Aso, and Y. Takeda, Preparations of poly (1lactide) microspheresof different crystalline morphology and effect of crystalline morphology on drug release rate, J. Control. Rel., 15:133-140 (1991). 57. J. Heller, Bioerodible systems, in Medical Applications of Controlled Release (R. S. Langer and D. J. Wise, eds.), CRC Press,Boca Raton, FL, 1984, pp. 1-23. 58. P. Le Corre, P. Le Guevello, V. Gajan, et al., Preparation and characterization ofbupivacaine-loadedpolylactideand polylactide-co-glycolide microspheres, Int.J. Pharmaceut., 107:41-49 (1994). 59. H. B. Hopfenberg, Controlled release from erodible slabs, cylinders, and spheres,inControlledReleasePolymericFormulations(D.R.Pauland F. W. Harris, eds.), American Chemical Society, Washington, DC, 1976, pp. 182-194. 60. B. W. Wagenaar andB. W. Muller, Piroxicam release from spray-dried biodegradable microspheres, Biomaterials, 15:49-54 (1994). J. W. McGinty, Dissolution, stability, and morphological proper61. M. Iwata and ties of conventional multiphasepoly(DL-lactic-co-glycolicacid) microspheres containing water soluble compounds, Pharm. Res., 10:1219-1227 (1993). 62. J. J. Burger, E. Tomlinson, E. M. A. Mulder,and J. G. McVie,Albumin microspheres for intra-arterial tumour targeting: I. Pharmaceutical aspects, Int. J. Pharm., 23:333-344 (1985). 62. R. Narayani and K. P. Rao, Controlled release of anticancer drug methotrexate from biodegradable gelatin microspheres, J. Microencaps., 11:69-77 (1994). as 64. L. Illum,N.F, Farraj, A.N. Fisher, et al., Hyaluronic ester microspheres a nasal delivery system for insulin, J. Control. Rel., 29:133-141 (1994). 65, G. A. Magee, N. Willmott, and G. W. Halbert, Development of a repro-
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66.
67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79.
duceableinvitromethodforassessingthebiodegradation of protein microspheres,J. Control. Rei., 25:241-248 (1993). A. Jayakrishnan, W. A. Knepp, and E. P. Goldberg, Casein microspheres: preparation and evaluation as a camer for controlled drug delivery, Int. J. Pharmaceut., 106:221-228 (1994). 0 .P. Rubino, R. Kowalsky, and J. Swarbrick, Albumin microspheres as a drug delivery system: relation among turbidity ratio, degree of crosslinking, and drug release, Pharm. Res., 10:1059-1065 (1993). V. Dilova and V. Shishkova, Albumin microspheres as a drug delivery system for dexamethasone, Pharmaceutical and pharmacokinetic aspects, J. Pharm. Pharmacol., 45:987-989 (1993). N. Yui, J. Nihira, T. Okana, and Y. Sakurai, Regulated release of drug microspheres from inflammation responsive degradable matrices of crosslinked hyaluronic acid, J. Control. Rel., 25:133-143 (1993). 0.Saslawski,C.Weingarten, J. P. Benoit,and P. Couvreur,Magnetically responsive microspheres for the pulsed delivery of insulin, Life Sci., 42:15211528 (1988). et al.,BiodegradablemiJ. H.Eldridge, J. K. Staas, J.A.Muelbroek, crospheres as a vaccine delivery system, Mol. Immunol., 28:287-294 (1991). S. Pulapura,C.Li,and J.Kohn, Structure-property relationships for the design of polyiminocarbonates, Biomaterials, 11:666-678 (1990). P. Wuthrich, S. Y.Ng,K.V.Roskos,andJ.Heller,Pulsatileanddelayed release of lysozyme from ointment-like poly-(ortho esters), J. Control. Rel., 21:191-200 (1992). J. Y. Armand, F. Magnard, J. Bouzon, et al., Modelling of drug release in gastric liquid from spheric galenic forms with Eudragit matrix, Int. J. Pharm., 40~33-41 (1987). H. Liu, P. Magron, J. Bouzon, and J. M. Vergnaud, Spherical dosage form with a core and shell. Experiments and modelling, Int. J. Pharmceut., 45:217227 (1988). G. W. Sinclair and N. A. Peppas, Analysis of non-Fickian transport in polymers using simplified exponential expression, J. Membrane Sci., 17:329-331 (1984). P. L. Ritger and N. A. Peppas, A simple equation for descriptionof solute release. 11. Fickian and anomalous release from swellable devices, J. Control. Rel., 2:37-42 (1987). C. Lanczos, Applied Analysis. Prentice-Hall, Englewood Cliffs, NJ, 1957, pp. 272-280. S. Y. Lin, In vitroreleasebehaviour oftheophyllinefromPIB-induced ethylcellulose microcapsules interpreted by simple mathematical functions, J. Microencam.. 4213-216 (1987).
8
Patrick Couvreur, Guy Couarraze, Jean-Philippe Devissaguet, and Francis Puisieux
Universitk de Paris-Sud, URA CNRS 1218, Chdtenq-Malaby, France
I. Introduction-Definitions
11. Nanoparticles Obtained by Polymerization of a Monomer
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191 197 197
A. Nanospheres B. Nanocapsules
111. Nanoparticles Obtained by Dispersion of Preformed
Macromolecules A. Nanospheres B. Nanocapsules C. Drug Loading and Release
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IV. Summary
1.
INTRODUCTION-DEFINITIONS
Nanoparticles as drug colloidal carriers have attracted more and more interest in the last decade, since these systems were found to be able to increase efficacy and/or to reduce toxicity of potent drugs [1,2]. More recently, they have even been found to be efficient vectors to deliver synthetic fragmentsof DNA into cells [3].
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Nanoparticles may be defined as submicron (<l pm) colloidal systems, generally, but not necessarily, made of polymers (biodegradable or not). Thus, this term is somewhat general, since it does not take into account the morphological and structural organization of the system. According to the process used for the preparation of nanoparticles, nanospheres or nanocapsules can beobtained. Nanocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a unique polymeric membrane; nanospheres are matrix systems in which the drugis nanoparticle dispersed throughout the particles. In the literature, the term is, however, often improperly used to designate ultradispersed matrix systems less than 1pm in size. Several methods have been developpedfor preparing nanoparticles. They can be classified in two main categories according to whether the formation of nanoparticles requires a polymerization reaction or whether it is achieved directly from a macromolecule or a performed polymer. It is the primary objective of this chapter to describe the methodologies available for nanoparticle preparation. Some of these methods are related to the manufacture of latex found in polymer chemistry. However, the application to drugs imposes a number of constraints in selecting the materials (biocompatible and biodegradable macromolecules) and for the size of the particles to be prepared (below 1 pm). Thus, the methods developed in polymer chemistry have accordingly been adapted to meet these requirements. This chapter provides the backgroundinformation and guidelines for choosing the more appropriate method for a given drug to be encapsulated. Further, the mechanisms of nanoparticles formation are also discussed from a physicochemical point of view. Data concerning drug attachment and drugrelease are also given.
11. NANOPARTICLES OBTAINED BY POLYMERIZATION OF A MONOMER
A. Nanospheres
Most of the methods for preparing nanospheres based on the polymerization of monomers consist of introduciog a monomer intothe dispersed phase of an emulsion, an inverse microemulsion, or dissolved ina nonsolvent of the polymer. The polymerization reactions in these systems take place in two phases: a nucleation phase followed by a growth phase(propagation).
I . Poly (methylmethacrylate) and Polyacrylamide Nanoparticles Poly(methylmethacry1ate)nanospheres have been produced by a radical dispersion polymerization in whichthe monomer is dissolved in an aque-
185
ous phase [4]. The polymerization is initiated by y-irradiation from amCo source (500 Krad yrays) by heating at elevated temperature or by using a chemical intitiator (potassium peroxodisulfate) [5]. The oligomers formed precipitate to form aggregates which may be stabilized by surfactant molecules. The final polymer particles are obtained by the growthof aggregates. In this process, the molecular weight as well as the particle size increase with increasing monomer concentration, decreasing initiator concentration, and decreasing temperature [6]. Polycrylamide nanospheres were proposed by Birrenbach andSpeiser [7] in the 1970s. They were prepared by an inverse microemulsionpolymerization mechanism. Acrylamide was used as a monomer and NN-methylene bis-acrylamide as a crosslinking agent. In thksystem, microemulsions consisted of monodispersed spherical swollen aqueous micelles stabilized by a layer of surfactant molecules and dispersed in an organic phase. The nucleation process is continuous, and it occurs throughout the duration of the of polymer particles formed polymerization reaction [8]. Thus, the number increases steadly with time but their size remains constant. The polymer particles result from the fusion of several micelles by collision, which occurs during the growth state of the nucelated micelles [9]. By this method, Birrenbach and Speiser [7] succeeded in preparing polyacrylamide nanospheres in the presence of antigens for the development of vaccine adjuvant. Like for poly(methylmethacry1ate) nanospheres, the polymerization was of unstable initiated by y-irradiation which is a drawback for the entrapment drugs that are easily degraded under a needed dose of 0.5 Mrad. But the main disadvantage of those nanoparticulate systems was that they could not be used for intravascular administration because of the nonbiodegradability of the polymers used.
2. Polyalkylcyanoacrylate Nanospheres To open wider prospects for nanoparticles, nanospheres consisting of polyalkylcyanoacrylates (PACA) were developed by Couvreur et al. [10,11]. These polymers, which have beenused for several years as surgical glues, are bioresorbable.
a. Preparationand characterization. Preparation of PACA nanospheres can be achieved by emulsion polymerization, in which droplets of water[1 2 1 . Anionic polyinsoluble monomers are emulsified inan aqueous phase merization takes place in micelles after diffusion of monomer molecules through the water phase and is initiated by negatively charged compounds. Classically, the cyanoacrylic monomer is added to an aqueous solution containing both dextran 70 (1%)as a protectant of colloids and glucose (5%) for isotonicityin MHCl.The polymerization of the alkylcyanoacrylate monomers occurs spontaneously at room temperature with stir-
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ring, leading to the formation of 180 nm (220 nm) colloidal nanospheres. Acid polymerization medium is necessaryto avoid too quick a polymerization of the monomer, which can lead to unwanted agglomerates. Drugs can be combined with nanoparticles after dissolution in the polymerization medium either before the introduction of the monomer or after its polymerization. The pH of the final suspension is then adjusted to pH 7, and nanospheres can be freeze-dried with their drug content. Various studies have shown that after the freeze-drying process, PACA nanospheres can be easily resuspended in water [13]. Furthermore, comparative particle size dimensions measurements showed no significant modification of the carrier after freeze-drying. It has been shown that such a formulation was very reproducible regarding s u e and drug adsorption rate even after preparation at a semi-industrial level [13]. Moreover, when prepared under sterile conditions in an aseptic roomunder laminar flow,cyanoacrylic nanospheres met the usual requirements neededfor intravenous administration, such assterility and lack of bacterial endotoxins. Concerning the morphological characterization of PACA nanospheres, scanning electron microscopy (SEM) generally shows spherical particles of a diameter of about 200 nm [14]. After freeze-fracturing, it was observed that these particles consisted of a solid core, whose inner structureappeared highly porous [15]. The polymer which constitutes the nanospheres doesnot appear as a hollow envelope but rather asa ball with a dense polymeric network. The specific surface area of polybutylcyanoacrylate (PBCA) nanospheres was calculated by Kreuter [l61 to be 106.5 mVg. Other physicochemical parameters of cyanoacrylic nanospheres were measured [161. Polycyanoacrylates had a slight negative charge with a tendency for a decrease in charge with increasing ester side chain length. Furthermore, serum yielded an even more pronounced decrease in surface charge, which was caused by the adsorption of the serum contents, demonstrating a significant interaction of these components with nanospheres. The water contact angle decreased with decrasing hydrophobicity in the following order: PBCA > polyethylcyanoacrylate (PECA) > polymethylcyanoacrylate(PMCA) [16], Using a gel-permeation chromatographic method, it was shown that PACA nanospheres appearto be built by an entanglement of numerous small oligomeric subunits rather than by the rolling up of one or a few long polymerchains [17]. Chromatographic profiles suggested an uniform distribution of molecules with unexpected low molecular weights ranging between 500 and 1000. Furthermore, no monomeric residues were found. The low molecular weight of oligomeric subunits is consistent with later-discussed observations concerning excretion and metabolization of nanoparticles. The most significantadvantage of
Nanoparticles: Characterization Preparation and
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alkylcyanoacrylate over other acrylic derivatives previously used for preparing nanospheres is in the polymerization step. In contrast to other acrylic derivatives, requiring an energy input for this process that can affect the stability of the adsorbed drug, alkylcyanoacrylates can be polymerized easily without suchcontribution.
b. Drug incorporation and adsorption. A successful nanoparticle system may be one which has a high loading capacity to reduce the quantity of carrier required for administration. Two theoretical curves can be proposed to describe the adsorption of drugs onto nanoparticles: Langmuirian-type and constant partitioning-type-isotherms. In fact, it was found that nanoparticles can entrap a drug according to a Langmuirian adsorption mechanism, because of their large specific area [18]. The drug can either be incorporated into nanospheres during the polymerization process or be adsorbed ontothe surface of preformed particles. In the former case, the drug-polymer interaction may result in the covalent linkage of the drug with the polymer. This was observed with vidarabine, an antiviral agent, whose nucleophile N in positions 3 and 7 may playa role of initiator for the anionic polymerization mechanism of the cyanoacrylic monomer [19]. To a lesser extent, a similar interaction was described with peptidic compounds such as growth hormone-releasingfactor (GRF) [20]. In fact, with GRF, it was shownthat if the drugwas added very early after the beginning of the polymerization process (5 min), 50% of the peptide was found covalently linked to the polymer. On thecontrary, when the peptide was added later on (i.e., 60 min after the beginning of the polymerization), it was not chemically modified, but the loading capacity was poor. Thus, it was found that thereis a narrow window of time forthe addition of GRF to thepolymerization medium which results in both the preservation of the chemical structure of the peptide and a satisfactory drug-loading capacity [20]. In contrast, surface pressure and surface potential studies of polyisobutylcyanoacrylate monolayers spread at the air-water interface in the presence of ampicillin revealed that theassociation which forms ampicillin with the polymer was weak and could be easily disrupted [21]. Based on the surface potential and surface pressure experiments, models of polyisobutylcyanoacrylate-ampicillin arrangements in the interfacial region have been proposed for the low- and high-polymer surface densities [22]. Concerning loading capacity of cyanoacrylic nanospheres, it was found that both the nature and quantities of the monomer used influenced the adsorption capacity of the carrier. Generally, the longer the alkyl chain length, the higher is the affinityof the drug. Taken in its entirety, the capacity of adsorption is related to the hydrophobicity of the polymer and
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to thespecific area of the carrier. Moreover, the percentage of drug adsorption generally decreases with the quantity of drug dissolved inthe polymerization medium, according to theLangmuirian isotherm[23]. Special attempts have been made for the association of synthetic fragments of DNA to PACA nanospheres [3]. In fact, the association of antisens oligonucleotides with nanoparticles was possible only inthe presence of a hydrophobic cation, such as triphenylphosphonium (Fig. 1) or quaternary ammoniumsalts. The poor yield of oligonucleotide association without cations could be explained by the hydrophilic character of nucleic acid chains that areknown to be soluble in water. In the proposed method, oligonucleotide adsorption on the nanosphereswas mediated by the formation of ion pairs between the negatively charged phosphate groupsof the nucleic acid chain and the hydrophobic cations [3]. The adsorption efficiency of oligonucleotide-cation complexes on nanospheres was found to be highly dependent on several parameters: oligonucleotide chain length, nature of the cyanoacrylic polymer, hydrophobicity of the cations used as ion-pairing agents, and ionic concentration of the medium [3]. When adsorbed onto nanospheres, oligonucleotides were found to be protected from the degradationby a 3'-exonuclease in vitro [3].
50
100
150
Hydrophobic ion
(PM)
Fig. 1 . Adsorption of oligothymidylate to polyalkylcyanoacrylate nanoparticles. Oligothymidylate (2 mM) complexed with increasing concentrations of positively charged tetraphenylphosphonium chloride (PP)was added to PIBCA (W) or PIHCA (+) nanoparticles (10 mg/mL). Adsorption of oligothymidylate to PIHCA nanoparticles in the presence of the negatively charged tetraphenylboron sodium (0) was measured under the same experimental conditions, as a control. (From ref. 3.)
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c. Biodegradation and drug
release. Another characteristic of cyanoacrylate polymers is that their rate of degradation is dependent on the length of their alkyl chain. The dominating mechanism of particle degradation was found to be a surface erosion process [24]. This process occurs through the hydrolysis by enzymes of the ester side chain of the polymer [25]. By this mechanism, the polymeric chain remains intact, butit gradually becomes more and more hydrophilic until it is water soluble. This degradation pathway is consistent with the productionof alcohol during the bioerosion of PACA nanospheres in vitro in the presenceof esterases [25]. Indeed, the action of rat liver microsomes and tritosomes on the ester hydrolysis of polyisobutylcyanoacrylate nanospheres was clearly demonstrated [25]. A relationship between isobutanol production (i.e., nanosphere bioerosion) and esterase concentration was found. Therefore, we consider that the production of formaldehyde via chemical degradation (reverse Knoevenagel reaction) as proposed by several investigators [26] plays a marginal role in the overall degradation of PACA nanospheres under physiological conditions. Indeed, the amount of formaldehyde produced after nanosphere degradation was found to be5% of the theoretical quantity that would have been producedif the polymer had been entirely degrated by this pathway [25]. The suggestion of Vezin and Florence [26] that esterases do not affect the degradation rate is therefore contested. Since the biodegradability of polyalkylcyanoacrylate depends on the nature of the alkyl chain, it is possible to choose a monomer whose polymerized form has a biodegradability corresponding to theestablished program for drug release [14]. Indeed, by using a double radiolabeled technique (14Clabeled nanospheres loaded with [3H]actinomycin), it was found that drug was released from nanospheres as a direct ,consequence of the polymers bioerosion [25]. This was confirmed using GRF, another drug model[20]. With this peptidic compound, in the absence of esterases, no drugrelease was observed, whereas in the nanospheres suspension turbidity remained unchanged, indicating no polymerbioerosion. Drug release appeared to be dependent on the esterase content of the incubation medium. The liberation of GRF was quicker in rat plasma (content of esterases around 150 @mL) than in Ringer lactate medium containing esterases (100 pg/mL). At the same time, the turbidity (i.e., the expression of nanosphere bioerosion) decreased with the release of the peptide (Fig. 2). That the GRF release directly resulted from the polymers bioerosion was confirmed by the fact polyisohexylcyanoacrylateyielded that themore slowly bioeroded polymer the slowest decrease of turbidity and the most progressive release of the peptide. Finally, the fact that the drugrelease was related to the turbidity decrease suggested that GRF was homogeneouslydispersed throughout the polymeric matrix rather than adsorbed atthe surface of the polymer.
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GRF
GRF
f
" +
GiRF
f
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400
8 v
W
80
300 60
200
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IT U
40
1oo
m
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l-
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Fig. 2. Release of GRF from PIHCA nanoparticles is the consequence of polymer's bioerosion (decrease of the turbidity of the suspension). (From ref. 20.)
With rose bengal, a biphasic release from PACA nanospheres was observed [18]. The release profile of this compound was described by using a simple biexponential function:
Pt = Ake-a
+ Be-b'
where Pt is the concentration of compound remaining in the nanosphere at time, t; the concentrations Aand B and the two rate constantsa and b can
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be used to characterize the release of the compound. The initial release rate was faster when the marker was adsorbed only at the surface of the carrier, whereas it was reduced when the compound was incorporated into the polymeric network of the nanospheres. By employing dextran sulfate as the stabilizer in the preparation of PACA nanospheres, a slower release rate was obtained for the model drug rose bengal [27]. Thus, with this compound, the release was not only dependent on the kinetic of bioerosion of the polymer.
B. Nanocapsules
After the development of nanospheres obtained by emulsion polymerization of alkylcyanoacrylates as described in the preceding section, it has been possible using the same monomer to manufacture particles of nanometric size containing an oil-filled cavity. This procedure, which was first developed by Al Khoury et al. [28], has beenapplied for insulin delivery by Damge et al. [29].The characterization of polyalkylcyanoacrylate nanocapsules has also been performed by different investigators [30-321.
I . PreparationandCharacterization Nanocapsules of PACA are obtainedvia an interfacial polymerization process in emulsion. Nanocapsules are formed by mixing an organic phase with an aqueous phase. The organic phase is classically an ethanolic solution of the monomer mixed together with the oily core material and the lipophilic drug to be encapsulated and to which, in some cases, soya bean lecithin is added as an additional surfactant. Oils which have been used to prepare nanocapsules include Miglyol, benzylbenzoate, ethyl oleate, and Lipiodol. The encapsulation efficiency of a lipophilic drug generally deso pends on its partition coefficient between the oil and the aqueous phase, the oil must be chosenaccordingly. The aqueous phase is a solution of a nonionic surfactant, usually Synperonic PE-F68 at 0.5% at a pH between 4 and 10. To form nanocapsules, the organic phase is allowedto runslowly-for example, through a wide-bore syringe needle or a micropipette tip-into the aqueousphase, which is magnetically stirred. The mixture immediately becomes opalescent. After stirring for 15-30 mins, the ethanolis removed by evaporation under reduced pressure. If required, the nanocapsules canbe furtherconcentrated by evaporation; this allows them to be rediluted in a physiological buffer for injection. 200 Nanocapsules formed in this way have a mean diameter between and 300 nm with a narrow polydispersity. The speed of the magnetic stirring has no influence on the particle size, which depends solely on the nature and
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the volumeof the oil and on the volume of the diffusing organic phase. The proportion of oil to monomer must be correctly chosen to avoid simultaneous formation of either flakes of polymer or of a single oily emulsion. The presence of a surfactant in the aqueous phase is, in fact, not necessary for thesuccessful formation of nanocapsules, but it does prevent them from aggregating on storage to a cake which is difficult to disperse. Nanocapsules formedin this way are physically stable for several years at ambient temperature andmay be sterilized by autoclaving at 120C for 20 min (the effect of such a procedure on any encapsulated drug must, of course, be considered.) However, as a result of their vesicular character, nanocapsules are not easily lyophilized, since they tend to collapse releasing the oily core. In fact, many factors could influence the type of colloid formed by anionic interfacial polymerization ofPACA: the nature of the aqueous phase, the pH, the composition of the organic phase (monomer, oil, ethanol), the ratio of monomer to the aqueous phase, and the emulsification conditions. The degree of polymerization and, therefore, the molecularweight, depend ona balance between initiation, propagation, and termination [33]. The number of growing chains depends on the concentration of initiators (OH-, CH,O-, CH,COO-, CN-). For the same quantity of monomer, when the numberof live chains is high, the degreeof polymerization (DP) is low. Similarly, DP is reduced when the concentration of terminating agents (H+) is high [34,35]. The concentration of the initiating and the terminating agents available for the monomer depends on the physicochemical nature of the system in which the componentsare dispersed at themoment of the formation of the colloid. In thecase of a system composed of two immiscible phases, such as an emulsion, the presence and the concentration of the solutes in the different phases is a function of the polarity of the solute molecules and of the dielectric constant of the medium. In contrast, at an interface between an aqueous and an organic medium, there can be large local variations in properties which can themselves cause changes in the interfacial properties in a dynamic system. For example, spontaneous emulsification depends on the interfacial tension. Thus, although the degree of polymerization depends on the propagation reaction, other characteristics of the colloid formed such as particle size and morphology depend on interfacial phenomena inducted by the dynamic mixing of an organic phase with an aqueous phase. Thus, when the quantity of ethanol was increased, there was a tendency to form a suspension of polymeric flakes. However, with an increase in the volume of oil, an oily layer appeared onthe surface of the suspensions.
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For systems having an oiVethano1ratio of some percentage, the macroscopic aspect is markedly more homogeneouswith the presence of a majority of nanocapsules. Since the preparation of nanocapsules of PACA is rather similar to the preparation of PACA nanospheres, it is necessary to verify that the colloidal suspension does not consist of a simple mixture of an oil-in-water emulsion containing polymeric nanospheres of similar size. Several different experiments confirmed that the preparation was indeed composed of oil-filled nanocapsules [32]. First, turbidity measurements showed a clear difference between nanocapsules and a mixture of nanospheres and the emulsion containing the same amount of polymer and oil. Second, the vesicular nature of nanocapsules prepared in this way was confirmed by electron microscopy. Scanning electron microscopy showed a smooth exterior to thenanocapsules,whereasthethincapsulewallandtheinternal cavity could be visualized by transmission electron microscopy [28]. Fig. 3 shows a polyisobutylcyanoacrylate nanocapsule after cryofracture andobservation by transmissionelectronicmicroscopy. The membrane thickness ranges from 5 to 10 nm.
2.
Mechanism of Formation Theoretically, nanocapsules result from the interfacial polymerization of the cyanoacrylic monomersat the surface of the oil droplets dispersed in the aqueous phase. A drugcan be encapsulated in the internalcavity of the polymerized membrane during the formation of the system provided that its oil-water partition coefficient is in favor of the oily phase. Nevertheless, a simple emulsification of an organic phase, containing alkylcyanoacryrate monomers and oil, in an aqueous phase does not allow nanocapsule formation. As a matter of fact, because of the affinity of alkylcyanoacrylates for the oily phase, a bulk polymerization of the monomers in the oil droplets occurs preferentially. Polymeric matrix systems, which can be compared with nanospheres in which the oil is dispersed throughout the polymeric network,result. To obtain nanocapsules, a dynamic process must be created which brings the monomer to the oil-water interface. This transfer is performed by the diffusion of a cosolvent from the organic phase to the aqueous phase. This cosolvent must be a solvent for the monomer and for the oil on one hand and miscible with the aqueous phase on the other hand. Usually, ethanol can be used as diffusing solvent. So, nanocapsules are prepared by emulsificationof an organic phase containing the monomer in an aqueous phase. The main point is the compositionof the organic phase in which the oil and the diffusing solvent are mixed together. In the case of nanocapsules, the natureof the emulsification process is spontaneous.
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Fig. 3 . Polyisobutylcyanoacrylate nanocapsulesobserved by transmission electron microscopy after cryofracture. (From ref. 32.)
In addition to their vesicular structure, the nanocapsules made from alkylcyanoacrylates differ from nanospheres by their size, the molecular mass of the polymer, and the characteristics of the drug release. Usually, nanocapsules are larger than the corresponding nanospheres and the degree of polymerization of the polymer is also significantly higher. These differences are theresult of the specific mechanismof nanocapsule formation. This mechanism is complex, because it results from boththe chemical characteristics of the polymerization and the physical characteristics of the polymerization medium (viscosity, interfacial tension, and cosolvent diffusion from the organic phase to theaqueous phase). These processes usually lead to the formation of mixed systems where nanocapsules coexist with other polymeric particles. Figure 4 shows photographs takenby transmission electronic microscopy of a nanocapsule preparation after separation by ultracentrifugation. Particles of lower density are true nanocapsules containing an oily cavity;
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(b)
Fig. 4. Transmission electron micrographs of a nanocapsule preparation after separation by ultracentrifugation. (a) Floating layer. (b) Pellet. particles of higher density are in fact nanospheres with a size comparable to that of nanospheres obtained by emulsion polymerization. Nevertheless, the mechanism of formation of these nanospheres is not anemulsion polymerization, because the molecular mass of the polymer of these particles is high, similar to that associated with nanocapsules (about 100,000 versus about 1000 for nanospheres) [31]. Since the nanospheres obtained during the formation of nanocapsules have the same polymeric molecular mass as the nanocapsules, it is possible a chemito conclude that thepolymerization mechanism is the same. From cal point ofview, the anionic polymerization mechanism of alkylcyanoduring the formation of nanocapsules as acrylate is not very much modified
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compared with nanospheres. It could be possible to consider that the nucleophilic initiation of alkylcyanoacrylate polymerization, which is normally performed by the hydroxyl ions of water, could be performed by ethoxy ions, although the dissociation constant of ethanol is very low. In fact, polymerization of alkylcyanoacrylates in the presence of ethanol is very restricted and leads to the formation of small oligomers with low molecular mass. Although the presence of ethanol seems not to modify the polymerization of the monomer from the chemical point ofview, the diffusion of ethanol is the major physical factor which imposes the conditions of polymerization and, thus, those of nanocapsules formation. Indeed, when the total volume of ethanol is kept constant, but is initially distributed differently between the organic phase and the aqueous phase, it appears thatthe molecular mass of thecreated polymer doesnotdependon the total amount of ethanol but only on that part of the ethanol diffusing from the organic phase to the aqueous phase. The very high molecular mass of the polymer making up the nanocapsules, is only obtained when the diffusing fraction of ethanol is more than half the total.The role of ethanol diffusion in the formationof nanocapsules is illustrated in Fin. - 5 . The rapid diffusion of ethanol from the organic phase to the aqueous phase leads to primary
Aqueous phase
/
0
Nanocapsules
fragmentation of thepolymeric film
\
6 9
@ @
Nanoparticles
Marangoni effect:
interfacialpolymerization of monomer
Fig. 5. Mechanism of formation of nanocapsules
by interfacial polymerization.
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convection currents, which are perpendicular to the interface, yielding an higher concentration of monomers on the peak of the surface undulations of the interface between the organic and the aqueous phases. Since alkylcyanoacrylate monomers have tensioactive properties, interfacial tension gradients appear on the interface, leading to secondary convection currents at a tangent to the interface (Marangoni effect). So primary and secondary convection currents promote spreading of the monomer at the interface where an interfacial polymerization will be able to propagate. This kind of polymerization leads to a higher molecular mass than ina single emulsion polymerization where the oligomers, being widelydispersed in the aqueousphase or in the micelles, have a greater probability of meeting cationic species (H,O+) which terminate the polymerization. In contrast, during interfacial polymerization, the oligomers are relatively protected from termination agents and the polymerization can develop owing to the monomer reservoir set up within the organic phase. The interfacial turbulences due to the secondary convection currents of the Marangoni effect initiate the fragmentation of the interfacial polymeric film and initiate the spontaneousemulsification of the system. During this emulsification, fragments of films fold back on themselves at the,interface around the oil droplets to form the nanocapsules, whereas the remainder of the film fragments lead to the formation of nanospheres of smaller size with a polymer molecular mass retaining the characteristics of interfacial polymerization (high molecular mass). The structure of the mixed systems (nanospheres and nanocapsules) formed during the preparation of nanocapsules is confirmed by the analysis of the drug-release kinetics. Fig. 6 shows the comparison of the in vitro release of tetracaine from polyisobutylcyanoacrylate nanospheres (true nanospheres) and from the two fractions (obtained after separation by ultracentrifugation) of the nanocapsule suspension (true nanocapsules and nanospheres). The similarity of the release kinetics of the two nanosphere systems confirms that they have an identical structure (matrix structure). Analysis of the release kinetics of nanocapsules, compared with those of nanospheres, shows that thetime constant of nanocapsules is superior to that of nanospheres which wouldhave the same diameter. This is coherent with the vesicular structure of the nanocapsules.
111.
NANOPARTICLES OBTAINED BY DISPERSIONOF PREFORMED MACROMOLECULES
Nanospheres A.
As presented above, nanoparticles can be obtained by inducing the polymerization of a monomer in certain conditions. However, many polymeric
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nanwpsulcs obtained
0 nanosphcres obldncd
by inlerfacirl polymubtlon by emulsion polymerization
*
0
2
h u e nannosphcrcs obtained
10
time (h)
Fig. 6. In vitro release kinetics nanoparticles.
of tetracain from polyisobutylcyanoacrlate
materials not capable of being prepared by emulsion polymerization, such as polyurethane, epoxy, polyester, and others, including semisynthetic polymers such as cellulose derivatives, remained unavailable for aqueous dispersion. This problem has led to the development of pseudolatexes (artificial latexes) obtained by dispersion of preformed polymers. For drug delivery purposes, following an intravenous injection, the polymeric material needs to meet physicochemical and biological requirements adapted and optimized for this specific application. Among these requirements, submicron size to avoid the risk of embolization following intravenous injection and biodegradability to nontoxic metabolites are of crucial importance, thus limiting the number of available polymers. If the method of preparation implies the use of nonsafe ingredients (solvents, stabilizers), steps of purification should be added in order to assure the required qualities of the final product. Most of the synthetic and artificial latexes preparedfor industrial applications did notmeetthese requirements, and various approaches were progressively developed
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in order to obtain polymeric nanospheres fulfilling the pharmaceutical criteiia. Polymeric nanospheres prepared by emulsion polymerization may encounter somedrawbacks: With the exception of alkylcyanoacrylate, most of the monomers suitable for a micellar polymerization in an aqueous phaselead to slowly or nonbiodegradable polymers. The polymerization process is mainly limited to the vinyl addition reaction, and the molecular weight of the polymeric material cannot be fully controlled. Residues in the polymerization medium (monomer, oligomer, organic solvent, surfactant, initiator of polymerization) can be more or less toxic and may necessitate further purification of the colloidal material. During the polymerization process, activated monomer molecules may interact with the drug molecules, thus leading to their inactivation (20). In order to avoid some of these limitations, the methods of obtaining nanospheres from various preformed macromolecular materials, whose physicochemical and biological properties can be well controlled, have been developed. Two approaches using the emulsification of a solution of the macromolecular material or using a controlled desolvation of this material have beensuccessively considered.
1. NanospheresPrepared by Emulsification The methods of preparing nanospheres by emulsification were adapted from the industrial techniques available for obtaining the artificial latexes used in surface-coating applications (e.g., paints, adhesives, textilesizing, paper coating). Initially, artificial latexes were developed as waterbased dispersions in order to avoid economical and environmental problems encountered with the use of organic solvent-based coatings. They present the major advantage that any water-insoluble polymeric material can be used and all the industrial techniques for obtaining artificial latexes rely on oneof the threefollowing processes:
1 . Solution-emulsification [36]: The polymer is dissolved ina volatile solvent immiscible with water. This organic phase is dispersed in an aqueous phase by conventional emulsification techniques and appropriate emulsifiers. The polymeric particles are formed from the oil-in-water ( O W ) droplets by removing the organic solvent following its vaporization.
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2. Phuse-inversion [37]: The polymer is first ground with a fatty acid. An alkaline aqueous solution is then added and mixed to form a water-in-polymer dispersion. Further addition of water leads to the inversion of phases and to a dispersion of the polymer in the aqueous solution. In this case, the emulsifier is the soap formed from the reaction between thefatty acid and the alkali. 3. Self-emulsification [38]: Some polar groups such as amine or quaternary ammonium, when attached to polymer molecules, may confer autoemulsioning properties and remove the need for any added emulsifier for the dispersion of the polymerin an aqueous phase. However, the presence of polar groups may also modify the water sensitivity, more specifically the pH sensitivity, of the colloid.
If pharmaceutical applications are considered, the emulsification techniques may present some limitations:
To obtain small (<lpm) and monodisperse nanospheres, the starting material, that is, the O/W emulsion, must be very fineand homogeneous. Large amounts of emulsifiers and time- and energyconsuming techniques to reach high shearing forces are therefore necessary,
Most of the organic solvents and surfactants used are not compatible with biologicalapplications, thus demanding furtherpurification of the colloidal material, Thesemethodswere only capable of preparing polymericnanospheres and needed further adaptations to allow the use of nonpolymeric macromolecules. Artificial latexes, preparedfrom ethylcellulose [39]andfrom cellulose by using emulsification techniques, have been developed acetophtalate [40] for filmcoating of tablets and othersolid dosage forms administered via the oral route. However, size requirements andemulsifier biocompatibility are less restrictive for this pharmaceutical application than they are for drug delivery following an intravenous injection. The preparation of injectable nanospheres requires the use of safe, well-tolerated, water-insoluble, macromolecular material. Among many other polymers, poly(esters) such as poly(&-caprolactone), poly(1actide), poly(glycolide), and poly(1actide-co-glycolide)meet the biological requirements of safety and biodegradability to nontoxic metabolites. On the other hand, some macromolecular materials of biological origin, such as proteins, might be considered as safe and biodegradable, but with the excep-
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tion of some vegetal proteins such as zein or gluten, they need physical or chemical modifications to become water insoluble.
U.
Nunospheres of synthetic polymers. Gurny et al. [41] and Krause et al. [42] have prepared poly(1actide) nanospheres according to thesolutionemulsification technique. The polyester is dissolved in an organic volatile solvent immiscible withwater, such as chloroform, and the organic solution is dispersed in an aqueous phase to form an O N emulsion. Continuous emulsification under mixing prevents coalescence of organic droplets and allows the spontaneous evaporation of the solvent at room temperature and the formation of the colloidal particles. Residual organic solvent is removed under reduced pressure. The emulsifiers can, if necessary, be removed by dialysisof the suspension or by washings following the separation of nanospheres by ultracentrifugation. Ibrahim et al. [43] and Allemann et al. [ M ] have proposed replacing water-immiscible solvents, such as chloroform (all trace of which must be eliminated for an injectable dosage-form) by a miscible one such as acetone, which can easily be removed by evaporation. The method consists of adding under continuous stirring to an acetone solution of polymer an electrolyte or a nonelectrolyte-saturated aqueous solution containing poly(viny1 alcohol) (PVA) as a viscosity agent and stabilizer. The saturated aqueous solution prevents acetone from mixing with water by a salting-out process and a W/O emulsion is obtained. Addition of water to this emulsion allows complete diffusion of acetone in theaqueous phace inducing the formation of nanospheres. Subsequent washings by ultracentrifugation or dialysis allow the elimination of the salt or of the nonelectrolyte used to trigger the saltingout process. One of the investigators claim is that surfactants are not necessary to obtain nanospheres, since a protective colloid is used. However, when injectable surfactants are available the presence of PVAremains questionable if the dispersion is intended to beinjected. For that reason, poly(1actide) and poly(1actide-co-glycolide) nanospheres were developed by solvent evaporation using serum albumin as a colloidal stabilizer [45]. The characterization of the albumin coating has been studied [46].
b. Nunospheres ofnuturulpolymers. Kramer [47] hasprepared albumin nanospheres according to the emulsification technique. Albumin is first dissolved in water andthis solution is dispersed in cotton oil to form a W/O emulsion. This emulsion is then poured into oil heated to morethan 100C, thus resulting in an heat-denaturation of the protein and, simultaneously, in the vaporization of the aqueous phase. Subsequent washings and purifications are necessary to yield an aqueous suspension of albumin nanospheres, which can finally behardened by crosslinkage by aldehydes.
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Yoshioka et al. [48] also suggested a method leading to the formation of protein nanospheres at room temperature following the direct crosslinkage of gelatin by an aldehyde added to a W/O emulsion. Schroder and Stahl [49] proposed a similar method (W/O emulsion) to prepare nanospheres from polysaccharides such as dextran and its derivatives.
2. Nanospheres Prepared by Desolvatation As previously mentioned, one drawback of the emulsification methods is the use of emulsifiers and eitherchlorinated solvents or oily phase or saturated salt solutions, which implies more or less tedious purifications to obtain the final aqueous dispersion. Another limitation is the size and the homogeneity of the droplets containing the macromolecularmaterial, and it is almost impossible to get a monodisperse population of nanospheres. Attempts have been made to omit this step of preparation and have led to the development of methods using a controlled desolvation to obtain the colloidal aqueous dispersion of the macromolecularmaterial. Desolvation is a well-known method for isolating macromolecules from liquid media. Thus,polymers can beprecipitated from theirsolutions following the addition of a third component or of a nonsolvent miscible with their solvent. The sameeffect is obtained with an aqueous solution of proteins when neutral salts or alcohol are added. However, the process generally leads to a bulk precipitate of polymeric or protein material rather than colloidal particles.
a. Nanospheres of synthetic polymers. Fessi et al. [50] proposed a new, simple, and mild method yielding nanometric and monodisperse polymeric particles without the use of any preliminary emulsification. Briefly, an organic solution of the polymer is prepared and added under moderate stirring to a nonsolvent liquid phase. Both solvent and nonsolvent must havelow viscosity and high mixing capacity all in proportions. As an example, acetone and water meet these conditions. The only complementary operation followvolatile solvent by vaporizaing the mixing of the two phases is to remove the tion under reducedpressure. Further concentration of the aqueoussuspension can becarried out under the same conditions or by freeze-drying. The mean diameter of the particles obtained is about 200 nmwith a low dispersity. It was observed that the size of the particles was mainlyrelated to the type of polymer used and less to the experimental conditions. This method has been successfully applied to various biodegradable preformed polymers such as poly(1actide) ,poly(glyco1ide),poly(1actide-co-glycolide), poly(&-caprolactone), poly(alkylcyanoacrylate), and other nonbiodegradable preformed polymers such as poly(acrylic), poly(vinylch1oride-coacetate), ethylcellulose, cellulose-acetophtalate, and poly(styrene). Small
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amounts of surfactants, either lipophilic or hydrophilic, can be added to the solvent or to the nonsolvent (for an injectable suspension, phospholipids and Synperonic PEF68 can used). be However, it was observed that surfactants were not necessary to obtain the colloidal dispersion (which forms spontaneously) but only to stabilize it and to facilitate its redispersion following caking by sedimentation. It was alsonoteworthy thatthe sense and the rate of mixing of the two phases had no influence, the organic solution of the or vice versa, with polymer being slowly or rapidly added to the nonsolvent most critical conditions or without the aid of additional stirring. In fact, the for obtaining the spontaneous formation of colloidal particles alone, avoiding any bulk precipitation of the material, were the low concentration of the polymer in the organic phase and the 1/2 ratio of solvent to nonsolvent volumes. One of the most interesting and practical aspect of this method is its capacity to bescaled up fromlaboratory to industrial amounts. Chang et al. [51] and Bodmeieret al. [52] described a method, similar to that of Fessi et al. [50], to prepare nanospheres without any surfactant byusingacrylic copolymers bearing quaternary ammonium groups. As previously mentioned, these autoemulsioning polymers are water sensitive, more specifically pH sensitive, and they may become soluble, thus limiting both the applications and the choice of the starting material. Byusing carboxylated polymers such as Eudragit S, Fessi et al. [50] were able to obtain colloidal particles by the simple addition of an aqueous alkaline solution of the polymer to an acidic medium. However, subsequent neutralization of the suspension to physiological pH led to a clear solution.
b. Nanospheres of Natural Polymers. Marty et al. [53] have proposed a method of preparing gelatin nanospheres by controlled desolvation from an aqueoussolution. The slow addition of a desolvating agent (neutral salt, alcohol) to a gelatin solution containing a surfactant induces progressive modifications of the proteins tertiary structure to give a more and more hydrophobic material. The initially clear solution gradually turns opalescent asthe gelatin becomes less soluble in waterandtends to form nanometric aggregates of the desolvated protein, leading to a milky suspension. If salts or alcohol are added in excess, bulk precipitation occurs and the supernatant becomes limpid once more. Thus, Marty et al. [53] recommended the monitoring of the desolvation process by continuous nephelometric measurements. Gelatin nanospheres formedin this way need to be hardened by crosslinkage with an aldehyde, the excess of which is neutralized with a sulfite. Finally, purification to remove desolvating agent, surfactant, and other reactants is carried out by dialysis or by washings following ultracentrifugation. The elimination of the desolvating agent can be by vaporization if alcohol is used. This method presents some draw-
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backs, such as the monitoring of the desolvating process and the need for purification to eliminate various added materials. Stainmesse et al. [54] proposed a simpler, single-step technique to prepare protein nanospheres. Adapted from the method developed for polymeric nanospheres[50], the technique consists of pouring an aqueous solution of the protein (i.e., albumin) into anonsolvent. In particular, the nonsolvent can be ethanol or boiling water, thus leading to heat denaturation of albumin and to the formation of colloidal particles as small as 80 nm. Further strengthening of albumin nanospheres can be obtained either by heat sterilization (121C) or by crosslinkage. It is noteworthy that no additive (surfactant, protective colloid) was necessary to obtain a stable dispersion, owing to the small and monodisperse size of albumin nanospheres, even under theconditions of heat sterilization. The sameprocess was usedto obtain an aqueousdispersion of a water-insoluble protein, such as zein, by dissolving the proteinaceous material in an organic solvent, such as ethanol.
B. Nanocapsules
Nanocapsules present the unique characteristic of being a solid vesicular system. The polymeric wall renders nanocapsules much more stable than nanoemulsions andliposomes, whereas the inner core allows a large loading capacity according to the solubility of the drug moleculein this core. It might be considered that nanocapsules could be obtained by adapting some of the various and well-known techniques for microencapsulation to yield smaller particles. However, all attempts to reduce the size of microcapsules to below 1 pm, essentially based on conventional emulsification techniques, were unsuccessful. Two methods of preparing nanocapsules have been successively developed in our group:
1. The first one, which have been previously considered in this chapter, was the method of Al Khoury et al. [28], using an interfacial polymerization of alkylcyanoacrylate monomer. When prepared by interfacial polymerization of alkylcyanoacrylate monomer, nanocapsules may have similar limitation to the one previously mentioned for nanospheres; thatis, the possible chemicalinteraction between the monomer and the drug molecules [20]. 2. The second one was the method of Fessi et al. [55], using an interfacial deposition of a preformed polymer. The advantages of using well-characterized performed polymers have been previously emphasized and is particularly interesting when perfectly safe and biodegradable polymers mustbe used, such as the polyes-
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ters already used for surgical sutures, implants, and other biomedical applications. Fessi et al. [S51 were the first to propose a method for obtaining nanocapsules from preformed polymers, and their approach was to apply the principle of spontaneous emulsification. Spontaneous emulsification [56] can occur when a solution of a liquid L1 in a liquid L2 is carefully added at the surface of a third liquid L3, L2 being soluble simultaneously in L1 and L3, whereas L1 is insoluble in L3. Molecules of L1 are progressively transferred to L3, where they are notsoluble, from their solution in L2, while reciprocal diffusion occurs between L2 and L3. This process, called diffusion and stranding, allows the formation of droplets of L1 in L3 to give an ultrafine emulsion. Various explanations of this phenomenon, beyond the scope of this chapter, have been given, including localvariations of the interfacial tension between L1 and L3, due to the reciprocal diffusion between L2 and L3, or the Marangoni effect. The method of obtaining nanocapsules [55] was adapted from the Briefly, the preformed polymer, method used to preparenanospheres [50]. the phase intended to become the core of the nanocapsule, and the drug molecule to be loaded in the carrier are dissolved in a volatile solvent perfectly miscible with the nonsolvent. This solution is then poured in the nonsolvent of both the polymer and the core, and the resulting mixture immediately turns milky owing to the spontaneous formation of nanocapsules. The solvent can be removed by evaporation at room temperature under reduced pressure. The size of nanocapsules is about 200 nm with a low dispersity ( 5 0 nm). The loading capacity of nanocapsules is related to the solubility of the drug in the core and the maximum loading could be obtainedwhen the core is the pure drug molecule. Smallamounts of surfactants, either lipophilic or hydrophilic, can be addedto thesolvent or to the nonsolvent. However, it was observed that surfactants were not necessary to obtain nanocapsules. The same critical conditions as previously cited for thepreparation of nanospheres by desolvation of preformed polymer apply to the preparation of nanocapsules, that is, the concentrations of both the polymer and the core in the solvent and the solvent to nonsolvent ratio. It should be mentioned here that, in contrast to nanospheres, nanocapsulesoffer the major advantage of a low ratio of polymer to othercomponents. Moreover, nanocapsules can be obtained with either an oily or an aqueous core according to a careful selection of the polymer, the solvent and the nonsolvent. The feasibility and reliability of this method have beenstudied for either laboratory or industrial batches. One of the very intriguing aspects of the method of Fessi et al. [55] is why the mixing step does not lead to the simultaneous formationof poly-
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meric nanospheres on one hand and of a core emulsion on the other. Moreover, the mechanism of formation of nanocapsulesis not totally understood, but a partial explanation could be as follows: When the solution of the preformed polymer and the core is poured in the nonsolvent, core droplets are formed by spontaneous emulsification as the solvent diffuses in the nonsolvent. The polymer, insoluble in both the core and the nonsolvent, could be desolvated at the interface of these two immiscible components. This interfacial polymer deposition following solvent displacement leads the polymer to act as a barrier and to stabilize the emulsion formed by the corematerial. It can be hypothesized that the polymer might playa role analogous to the role of a surfactant in emulsions.
C. Drug Loading and Release

The loading capacity of colloidal delivery systems and their release kinetics, which make the drugmolecule available, are important criteria. Depending on both thepreparation process and the physicochemical properties of the drug molecule and the carrier, the drug entrapment can be either by inclusion within the carrier and/or by superficial adsorbtion onto this carrier. Electrical charges on both the drug molecule and thecarrier may influence the loading capacity.
1. Drug Loading Whatever their process of preparation, polymerization of monomers or dispersion of preformed polymer, entrapment within nonporous nanoin the macromolecular spheres requires the solubility of the drug molecule material, whereas porous nanospheres may entrap the drug molecule by adsorption either superficially or within the macromolecular network, The described following the adsorption of drugs ontonanospherescanbe Langmuir-type or the constant partitioning-type isotherms. In fact, nanospheres generally entrap drugmolecules according to a Langmuir adsorption mechanism owing to their large specific surface area. Entrapment within the core of nanocapsules implies the solubility of the drug molecule in the oily phase. It should be mentioned that the drug to polymer ratio can be as large as 500% in nanocapsules (inner core madeof the drugitself) when this ratio is usually under 10% in nanospheres. 2. Drug Release Drug release from colloidal carriers is dependent on boththe type of carrier and the loading mechanisminvolved. As the specific area of a nanometric dispersion is very large, the release rate may be more rapid than from other larger structures (microspheres and microcapsules) and colloidal carriers are generally not intended to act as sustained-release
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delivery systems. It has to be mentioned that drugrelase data from colloidal delivery systems are highly dependent on the experimental conditions of the study [57]. More specifically, precautions have to be taken toavoid any artefactual rate-limiting step, particularly the presence of a membrane in a diffusion study or theabsence of sink conditions.
Nunospheres. Releasefromnanospheres may be different according to the drug-entrapment mechanism involved. When the drug is super!% cially adsorbed, the release mechanism can be described as a partitioning process (rapid and total release if sink conditions are met). When the drug is entrapped within the matrix, diffusion plus bioerosion will be involved with a biodegradable carrier, whereas diffusion will be the only mechanism if the carrier is not biodegradable. From this, it can be inferred that entrapment within the matrix of nanospheres may lead to sustained release, the rate of which may be related to the rate of biodegradation of the polymer. b. Nanocapsules. Releasefromnanocapsules is related to partitioning processes within immiscible phases. The equilibrium between the carrier (loaded drug) and the dispersing aqueous medium (free drug) is dependent both on the partition coefficient of the molecule betweenthe oily and the aqueous phases and on the volume ratio of these two phases. This means that the amount released is directly related to thedilution of the camerand that the release is practically instantaneous when sink conditions exist. Diffusion of the drug through the polymeric wall of nanocapsules does not seem to be a rate-limiting step [58]. Drug leakage from nanocapsules can be advantageously reduced by coating the polymeric wall with an outer layer of phospholipids.
IV. SUMMARY
a.
From this chapter, it emerges that a number of methods are available today for the preparation of nanoparticles. These methods are based on a wide variety of principles, but theyare generally concerned with either the polymerization of a monomer into an homogeneous or an heterogeneous phase or with the precipitation of a preformed polymer. Some of these processes present drawbacks from technological standpoint, whereas others are probably unsuitable for intravascular administration owing to the solvents or surfactants used in the method of preparation. The methods available for the entrapment or encapsulation of hydrophilic compounds are few innumber anddelicate, since they often require a step involving the transfer of the nanoparticles from an oily preparation medium to an aqueousmedium more suitable for pharmaceutical purposes.
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In the future, efforts should be made tohave a better understanding concerning the mechanism of formulation of these colloidal systems at the molecular and supramolecular level. This could lead to new formulation process and could open new prospects in the area of drug delivery by means of nanoparticle systems.
REFERENCES
1. N.Chiannilkulchai,
2. 3. 4.
5.
6. 7. 8. 9. 10.
11.
12: 13. 14.

15.
Z . Driouich, J. P. Benoit, et al.,Doxorubicin-loaded nanoparticles: Increased efficiency in murine hepatic metastases, Select. Cancer Therapeut., 5:l(1989). E. Fattal, M. Youssef, P. Couvreur, and A. Andremont, Treatment of experimental salmonellosis in mice with ampicillin-bound nanoparticles, Antimicrob. Agents Chemother., 33:1540 (1989). C. Chavany, T. LE Doan, P. Couvreur, et al., Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides, Pharm. Res., 9:441 (1992). J. Kreuter andP. Speiser,New adjuvantson a polymethyl(methacry1ate)base, Infect. Immun., 13:204 (1976). J. Kreuter and H.J. Zehnder, The use of aCo-y-irradiation for production of vaccines, Radiat. Effects, 35161 (1978). J. Kreuter, Nanoparticles-preparationand applications,in Microcapsules and Nanoparticles in Medicine and Pharmacy (M. Donbrow, ed.), CRC Press, Boca Raton, FL, 1992, p. 125. G . Birrenbach andP . Speiser, Polymerized micelles and their use as adjuvants in immunology,J. Pharm. Sci., 65:1763 (1976). M. T. Carver, E. Hirsch, J. C.Wittmann,et al., Percolationandparticle nucleation in inverse microemulsion polymerization, J. Physical. Chem., 93:4867 (1989). F. Candau, Z . Zekhnini, and F. Heatley, 13C NMR study of the sequence distribution of poly(acry1amide-co-sodium acrylates) prepared in inverse microemulsions, Macromolecules, 19:1895 (1986). P. Couvreur,M. Roland, andP. Speiser, U.S.Patent, 4,329,332,1982. P. Couvreur, M. Roland, and P. Speiser, U.S. Patent 4,489,055, 1984. P. Couvreur, B. Kante,M. Roland, et al., Polycyanoacrylate nanocapsules as potential lysosomotropic camers: Preparation, morphological, and sorptive properties, J. Pharm. Pharmacol., 31:331 (1979). C. Verdun, P. Couvreur, H. Vranckx, et al., Development of a nanopartiJ. Control. Rel., 3:205 clecontrolledreleaseformulationforhumanuse, (1986). P. Couvreur,B.Kante, M. Roland,and P. Speiser,Adsorption of antineoplasticdrugs to polyalkylcyanoacrylatenanoparticlesandtheirrelease J. Pharm. Sci., 68:1521 (1979). characteristics in a calf serum medium, B. Kante, P. Couvreur, V. Lenaerts, et al., Tissue distribution of 3H-acti-
209
16. 17. 18. 19. 20. 21. 22.
23.
24. 25. 26. 27.
28.
29. 30. 31. 32. 33.
nomycin D adsorbed on polybutylcyanoacrylate nanoparticles, Int. J. Pharm., 7:45 (1980). J. Kreuter, Physicochemical characterization of polyacrylic nanoparticles, Int. J. Pharm., 14:43 (1983). L.Van Snick, P. Couvreur, D. Christiaens-Leyh, and M. Roland, Molecular weights of free and drug-loaded nanoparticles, Pharm. Res., 1:36 (1985). E. Mak, andS. S. Davis, Evaluation of carrier capacity L. Illum, M. A. Khan, poly(buty1-2-cyanoacrylate) nanoparticles, Int. and release characteristics for J. Pharm., 30:17 (1986). V. Guise, J. Drouin, J. Benoit, et al., Vidarabine loaded nanoparticles: A physico-chemical study, Pharm. Res., 7:736 (1990). J. L. Grangier, M. Puygrenier, J. C. Gauthier, and P. Couvreur, Nanoparticles J. Control. Rel., 15:3 as carriers for growth hormone releasing factor (GRF), (1991). A. Baszkin, P. Couvreur, M. Deyme, et al., Monolayer studies on poly(is0Pharmacol., 39:973 butylcyanoacrylate)-ampicillin association, J. Pharm. (1987). A. Baszkin, M. Deyme, P. Couvreur, and G. Albrecht, Surface pressure and surface potential studies of poly(isobuty1-cyanoacrylate)-ampicillin interacJ. Bioact. Comp. Polymers, 4:llO (1989). tions at the water-air interface. S. Michelland, M. J. Alonso, A. Andremont, et al., Nanoparticles as carrier of antibiotics for intracellular antibiotherapy, Int. J. Pharm., 35:121 (1987). R.H. Muller, C. Lherm,J. Herbort, and P. Couvreur, In vitro model for the degradationof alkylcyanoacrylate nanoparticles, Biomaterials, 11590 (1990). V. Lenaerts, P. Couvreur, D. Christiaens-Leyh, et al., Degradation of polyisobutylcyanoacrylate nanoparticles, Biomaterials, 565 (1984). W . R. Vezin and A. T. Florence, In vitro degradation rates of biodegradable poly-n-alkylcyanoacrylates, J. Pharm. Pharmacol., 305 (1978). S. Douglas, L. Illum, and S. S. Davis, Poly (butyl-2-cyanoacrylate) nanoparticles with differing surface charges, J. Control. Rel., 3:15 (1986). N.AI Khoury Fallouh, L. Roblot-Treupel, H. Fessi,et al., Developmentof a new process for the manufacture of polyisobutylcyanoacrylatenanocapsules, Int. J. Pharm., 28:125 (1986). C. Damge, C. Michel, M. Aprahamian, and P. Couvreur, New approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier, Diabetes, 37:46 (1991). F. Chouinard, F.W. Kan, J. C. Leroux, et al., Preparation and purification of isohexylcyanoacrylate nanocapsules, Int. J. Phann., 72:211 (1991). M. Gallardo, G. Couarraze, B. Denizot,et al., Study of the mechanisms of formation of nanoparticles and nanocapsules of Polyisobutyl-2-cyanoacrylate, Int. J. Pharm., 100:55 (1993). J. M. Rollot, P. Couvreur, L. Roblot-Treupel, andF. Puisieux, Physicochemical and morphological characterization of polyisobutyl cyanoacrylate nanocapsules, J. Pharm. Sci., 75:4, 361 (1986). I. C. Eromosele, D. C. Pepper, and B. Ryan, Water Effects on the Zwit-
210
Couwreur et a/. terionic Polymerisation of Cyanoacrylates, Makromol. Chem., 190:1613 (1989). E. F. Donnelly, Ionic and Zwitterionc Polymerisation of n-Alkyl 2-Cyanoacrylates, Polymer Let. Ed., 15:399 (1977). D. C. Pepper, Anionic and Zwitterionic Polymerisation of a-Cyanoacrylates, J. Polymer Sci.: Polymer Symp., 62:65 (1978). D. Aleony and H. Wittcoff,U.S. Patent 2,899,397,1959. W. Cooper, U.S. Patent 3,009,891, 1961. P. Judd, Brit. Patent 1,142,375, 1969. G. S. Banker, U.S. Patent 4,330,338, 1982. J. W. Van Der Hoff, U.S. Patent 867,031,1977. R. Gurny, N. A. Peppas, D. D. Hamngton, and G. S. Banker, Development of biodegradable and injectable latices for controlled release of potent drugs, Drug Dev. Ind. Pharm.,7:l (1981). H. J. Krause, A. Schwartz, and P. Rohdewald, Polylactic acid nanoparticles, a colloidaldrugdeliverysystemforlipophilicdrugs,Int.J.Pharm.,27:145 (1985). H. Ibrahim, C. Bindschaedler, E. Doelker, et al., Aqueous nanodispersions by a salting-out process, Int. J. Pharm., 87:239 (1992). E. Allemann, R. Gurny, and E. Doelker, Preparationof aqueous polymeric nanodispersions by a reversible salting-out process: Influence of process parameters on particle sue, Int. J. Pharm., 87:247 (1992). Rhone-PoulencRorer,Microsphtres,leurprocCd6 de prtparation et leur utilisation, Fr. Patent 2660556, 1990. T. Verrechia, P. Huve, D. Bazile, et al., Adsorption / desorption of human serum albumin at the surface of poly(1actic acid) nanoparticles prepared by a solvent evaporation process, J. Biomed. Mater. Res., 27:1019 (1993). P. A. Kramer, Albumin microspheres as vehicles for achieving specificity in drug delivery, J. Pharm. Sci., 63:1646 (1974). T. Yoshioka, M. Hashida, S. Muranishi, and H. Sezaki, Specific delivery of mitomycin C to the liver, spleen and lung: Nano- and microspherical camers of gelatin, Int.J. Pharm., 8:131 (1981). U.Schroeder and A. Stahl, Crystallized dextran nanospheres with entrapped antigen and their use as adjuvants, J. Immunol. Meth., 70:127 (1984). H. Fessi, J-P. Devissaguet, F. Puisieux, and C. Thies, ProcCdC de prCparation de systkmes collo'idaux dispersibles d'une substance sous forme de nanoparticules, Fr. Patent 8618446, 1986. R. K. Chang, J. C. Price, and C. Hsiao, Preparation and preliminary evaluation of Eudragit RL and RS pseudolatices for controlled drug release, Drug Dev. Ind. Pharm., 15:361(1989). R. Bodmeier, H. Chen, P. Qle, and P. Jarosz, Spontaneous formation of drug8: 161 (1991). containing acrylic nanoparticles, J. Microencaps., J. J. Marty, R, C. Oppenheim, and P.P. Speiser, Nanoparticles-a new colloidal drug delivery system, Pharm. Acta Helv., 53:17 (1978). S. Stainmesse, H. Fessi, J.-P. Devissaguet, and F. Puisieux,ProddB deprtpa-
34. 35. 36. 37. 38. 39. 40. 41. 42. 43.
44.
45. 46. 47. 48. 49.
50.
51. 52. 53.

54.
Nanoparticles: Preparation Characterization and
21 1
rationdesystbmescolloidauxdispersiblesduneprotkinesousforme de nanoparticules, 1st Add. to Fr. Patent 8618446, 1988. 55. H. Fessi, J-P. Devissaguet, and F. Puisieux, Procede de preparation de systbmes colloidaux dispersibles dune substance sous forme de nanocapsules, Fr. Patent 8618444,1986. 56. C. V. Sterling andL. E. Scriven, Interfacial turbulenc: hydrodynamic instability and Marangoni effect, A. I. Ch. E.J., 5514 (1959). 57. C. Washington, Evaluation of non-sink dialysis methods for the measurement of drug release from colloids: Effects of drug partition, Int. J. Pharm., 56:71 (1989). 58. N.Ammoury, M. Dubrasquet, H. Fessi, et al., Indomethacin-loaded poly(d,llactide) nanocapsules : protection from gastrointestinal ulcerations and antiinflammatory activity evaluation in rats, Clin. Mat., 13:121 (1993).
Biodegradable Colloidal D r u g Carrier Systems Based o n Solid Lipids

Kirsten Westesen
Friedrich-Schiller University Jena, Jena, Germany
Britta Siekmann
Astra Arcus A B , Sodertalje, Sweden
I. Colloidal Carriers in Drug Delivery and the Use of Biodegradable Lipids A. Polymeric Carrier Systems B. Lipid-Based Carrier Systems
11. Lipid Suspensions as an Alternative Delivery System for the Administration of Poorly Water-Soluble Drugs A. Lipid Nanopellets for Persorption B. Fatty Acid Nanoparticles Prepared from Oil-in-Water Microemulsions C. Lipospheres-Lipid Particles Used for Sustained Drug Release D. Colloidal Lipid Suspensions Prepared by Precipitation in Emulsified Organic Solvents E. Colloidal Lipid Suspensions Prepared by MeltEmulsification
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111. Physicochemical Characterization of Melt-Emulsified Colloidal Lipid Suspensions A. Investigations of the Physical State of Colloidal Lipid Dispersions Prepared from Crystalline Glycerides B. Investigations on the Crystallinity and the Recrystallization Process of Melt-Emulsified Glyceride Nanoparticles
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C . Mechanism of Gel Formation in PhospholipidTripalmitate Stabilized Suspensions into Solid Lipid Nanoparticles D. Incorporation of Drugs
IV. Prospects Summary Future and
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1. COLLOIDAL CARRIERSIN DRUG DELIVERY AND THE USE OF BIODEGRADABLE LIPIDS
Many drug substances are characterized by poor solubility in aqueous media and thus cause formulation problems with regard to parenteral administration. Besides the use of cosolvents, drug complexation and solubilization in surfactant micelles, incorporation in colloidal carrier systems represents an alternative way to render poorly water-soluble drugs applicable by the parenteral and in particular the intravenous route. Futhermore, incorporation of drugs in particulate carriers provides a possibility to maif controlled or sustained release is desired. In recent nipulate drug release years, colloidal carrier systems have also attracted growing interest concerning drug delivery to site-specific targets, especially incancer chemotherapy [1,2]. The objective of drug targeting is an increase of drug concentration at the desired site of action with a simultaneous decreaseat nontarget sites, with the rationale being the enhancement of therapeutic efficacy with a simultaneous reductionof unwanted side effects. During the last decades, several approaches have beeninvestigated to develop submicron-sized drug-delivery systems. Based on the carrier material, the conventional vehicles used as drug carriers can generally be divided into two groups, polymeric andlipidic systems.
A.PolymericCarrierSystems
Polymeric nanoparticles are (amorphous) solid colloidal particles consisting of nonbiodegradable synthetic polymers or of biodegradable macromolecular materials of synthetic, semisynthetic, or natural origin. They usually exhibit a longshelf life and a good stability on storage. Drug release from polymeric nanoparticles is in many cases found to be biphasic with an initial burst phase followed by a sustained release [3]. The burst effect can be related to drug adsorbed to the camer surface. The methods for the pr ration of polymeric nanoparticles such as emulsion polymerization and solvent evaporation techniques often involve the use of toxicologically harmfulexcipients and additives,forexample,organicsolvents,cancerogenic monomers, and reactive crosslinking agents, the complete re-
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moval of which from the productis hardly possible from a technical point of view [4]. Moreover, the carrier material itself can represent a potential toxicological risk. Apart from polymer accumulation on repeated administration owing to slow biodegradation, toxic metabolites may be formed during the biotransformation of polymericcamers; for example, formaldehyde as a metaboliteof polycyanoacrylates [5]. B.Lipid-Based
Carrier Systems
In order to avoid potential toxicological problems associated with polymeric nanoparticles, a great deal of interest focuses on lipid-based carrier systems. Lipidic camers comprise, among others, liposomes, lipoproteins, andlipid oil-in-water emulsions. These vehicles are composed of physiological lipids such as phospholipids, cholesterol, cholesterolesters, and triglycerides, and owing to the biological origin of the carrier material, the toxicological risk is much lower than thatof polymeric particles. There are, however, a number of drawbacks inherent in conventional lipid carriers which are basically related to physicochemical instabilities. Liposomes, for example, tend to fuse, therebyreleasing drug from the vesicles. In particular,small unilamellar vesicles (SUV) are in a thermodynamicallyunfavorable state owing to the high curvature of the phospholipid bilayer [6]. The problem is even more pronounced when drugs are incorporatedin the liquid crystalline phospholipid bilayer. The storage stability of liposomesis therefore limited. Owing to lipid exchange with lipoproteins, liposomes are often also unstable in the vascular system [7]. Moreover, the reproducibility of liposome manufacture in terms of vesicle sizeand properties is limited and thus complicates largescale production. These technological problems have so far impeded the widespread clinical useof liposome formulations. Lipoproteins are the physiological carriers of water-insoluble lipids in the vascular system. Since lipoproteins exhibit a considerable circulation time in blood and are not taken up by the reticuloendothelial system (RES), they have been considered as drug-delivery systems [8,9]. Lowdensity lipoproteins (LDL) have attracted particular interest in this respect, because they areableto leave the vascular system by specific receptor-mediated uptake into cells. Tumor cells exhibit an increased uptake of LDL owing to their high cholesterol need, which forms thebasis of the LDL drug-targeting concept in cancer chemotherapy [lo]. As with liposomes, the shelf life of drug-loaded LDL is, however, only very limited of LDL on account of physicochemical instabilities. Furthermore, isolation from human blood is a time-consuming procedure, and large-scale production is limited by the access to human blood. The isolation procedure also bears the potential infectious riskwith respect to hepatitis and human
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immunodeficiency virus (HIV). A circumvention of these problems might be the preparation of artificial LDL [ll]. Production of artificial LDL containing the apolipoprotein compound which mediates receptorrecognition has, however, not yet been reported so far. Submicron-sized vegetable oil-in-water ( O N ) emulsions have been used as a calorie source in parenteral nutrition for decades [12,13]. These systems are manufactured in large scale and display an acceptable longterm stability. Lipidemulsions have therefore been extensively investigated as drug-carrier systems [14,15]. However, despite these efforts and the available production technology, there have been remarkably few drugcontaining lipid emulsions on the market until now, whichpoints to formulation problems caused by the susceptibility of the carrier toward incorporation of drug. This can be attributed to drug crystallization within the oil droplets and perturbations of the stabilizing emulsifierfilm by drug crystals or diffusing drug molecules which have a high mobility in the liquid oil phase. These perturbations may induce instabilities of either mechanical (reduction of film elasticity, film ruption) or electrochemical nature (influence on the zetapotential) causing coalescence, particle growth, and drug leakage. Drug leakage is the unwanted loss of drug from the carrier on storage. The high mobility of incorporated drug molecules also causes a fast release of drug fromthe carrier in biological fluids, so that it is hardly possible to achieve sustained release by an emulsionformulation [161. LIPID SUSPENSIONS AS AN ALTERNATIVE DELIVERY SYSTEM FOR THE ADMINISTRATIONOF POORLY WATER-SOLUBLE DRUGS
As outlined above, most drawbacks associated with conventional lipid drug-carrier systems can be attributed to the liquid or liquid crystalline state of the dispersed lipid phase. It was therefore obviousto combine the superiorities of colloidal lipid carriers, such as the biodegradability and biocompatibility of the carrier material as well as the easeof manufacture, with the advantages of the solid physical state of polymeric nanoparticles with respect to size stability, drug leakage, and sustained drug release. One approach was the idea to prepare aqueous suspensions of lipid nanoparticles by using solid lipids and thus circumventing the toxicological problems associated with polymer particles. Possible advantages of the solid physical state of the dispersed lipid phase are summarized in Table 1. By definition, solid particles cannot coalesce, and they may therefore exhibit a better physical stability than liquid or liquid crystalline carriers. Since the mobility of incorporated drug molecules is drastically reduced in a solid phase, drug leakage from the camer and drugprotrusion into theemulsi-
II.
Systems
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Table 1. Possible Advantagesof the Solid Physical State of Lipid Carrier Systems
1. Avoidance of coalescence enhanced physical stability 2. Reduced mobility of incorporated drug molecules reduction of drug leakage circumvention of instabilities due to interactions between diffusing drug mole-
cules and emulsifierfilm sustained drug release 3. Static interface solidlliquid facilitated surface modification
fier film would be counteracted. In addition, drug release from the carrier is not a diffusion-controlled process but is matrix-dependent provided the drug is distributed in the matrix and the matrix does not melt at body temperature. Thedegradation rate of the carrier lipids is assumed to determine drugrelease, which inthat case could be controlled to a certain extent by the choice of matrix constituents. Moreover, the presence of a static interface might facilitate a surface modification of the carrierparticles after solidification of the lipid matrix; for example,by subsequent adsorption of nonionic surfactants. The latter might be of relevance to reduce carrier uptake by the reticuloendothelial system, which is known to be related to surface properties. Surface modification is therefore one approach to drug targeting using colloidalcarriers [171. It is known to the authors that first attempts to develop lipid-based colloidal suspensions already date back to decadesagowhen the first parenteral lipid emulsions becamecommercially available. The early concepts considered to transfer the production principles of submicron-sized phospholipid stabilized O W emulsions tothepreparation of colloidal lipid suspensions by substituting the dispersed liquid oil phase by solid triglycerides. However, the attempts to formulate long-chain triglycerides into phospholipid-stabilized aqueous suspensions failed because of stability problemssuchas the formation of semisolid gels, whichwas also observed by the authors [l81 and will be discussed later (cf. Section 1II.C). Until the beginning of this decade, the development of colloidal lipid suspensions which exhibit a satisfactory long-term stability had not been reported in the pharmaceutical literature. Despite obvious stability problems, a patent application on the production of lipid nanopellets for persorption (cf. Section 1I.A) by melt-emulsification was filed by Speiser in 1985 [19]. Product stability is, however, not taken into consideration in the application.
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In contrast to submicron-sized lipid suspensions, the production of solid lipid matrices in the micrometersize range has already been reported in the late 1950s and the beginning of the 1960s [20,21]. Different preparation techniques have been described, such as milling of drug-containing lipid phase [20]; melt dispersion, for example, by stirring an emulsifier-free wax emulsion in water [21,22]; solvent methods in which the wax is dissolved in an organic solvent that is evaporated [22]; spray-drying of lipids dissolved in a volatile solvent [23]; and spray-congealing of the lipid melts [20,23]. Lipid microspheres are predominantly used as sustained-release oral solid-dosage formulations. Stability problems with respect to sedimentation or gel formation have not been described. The microparticles are generally separatedfromtheaqueous dispersion medium.These microparticulate systems are, however, not suitable as intravenous drug carriers. Moreover, the long-term stability of lipid microparticles has been rarely investigated. Most investigators focus their interest on product paThe few studies rameters and drug release of freshly prepared systems only. published on polymorphic behavior of spray-dried and spray-congealed lipid micropellets reveal that the micropellets are obtained in an unstable polymorphic formwhich transforms gradually into the stable modification on storage [24]. This polymorphic transition is accompanied by changes in the surface structure of the micropellets. A possible influence of particle aging on drugrelease is commonlynot discussed inthe literature, although physicochemical alterations during storage such as polymorphic transitions of the lipids, changes in particle size and shape, and increased crystallinity might be of relevance. In the beginninngof the 1990s, a number of research groups focused their activities on the development of solid lipid-based colloidal carrier systems, reflecting the pressing importance of alternative parenteral carriers. The different approaches are briefly summarized in the following sections. It has to be mentioned that the prevailing number of these studies are still at an early stage of development, and up to now most systems under investigation are only poorly characterized from a physicochemical point of view. In particular, stability data of these lipid carriers are lacking, and some investigators seem to disregard or underestimate this important aspect.
A.
Lipid Nanopellets for Persorption
In a patent application, Speiser [l91 discloses lipid nanopellets as an oral drug-delivery system with a particle size small enough for persorption. Persorption is definedas the transport of intact particles through the intestinal cell layer into thevascular or lymphatic system under avoidanceof the
Biodegradable Systems Carrier Colloidal Drug
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first-pass effect in the liver. Persorbed lipid nanopellets are believed to be concentrated in adipose tissues to form a sustained-release depot of incorporated drugs [19]. This theoretical mechanism is, however, not substantiated by experimental data. Aqueous suspensions of lipid nanopellets are described to exhibit a particle size of predominantly 80-800 nm. The carriers consist of waterinsoluble solid lipids such as long-chain glycerides, fatty acids, and fatty alcohols, and surface-active agents, preferably physiological emulsifiers. Colloidal suspensions are obtained by melting the lipids and adding them to the warm aqueous phase. The lipid melt is dispersed in the aqueous phase by high-speed stirring and sonication. Drugs can be incorporated into the carrier matrix by dissolution or dispersion in the melt or by addition of a solution of the drug in a volatile organic solvent to the melt and subsequent solvent evaporation. Data regarding the storage stability of these lipid nanopellets are not specified in the patent application. Based on Speisers patent application [191, Kecht-Wyrsch investigated the preparation of aqueous suspensions of lipid nanopellets [25]. However, Kecht-Wyrsch succeeded only in preparing lipid suspensions with a lipid concentration as low as 0.2 to 1.0% (w/w). These suspensions exhibited a documented storage stability of 3 months, whereas higher concentrated suspensions displayed a considerable particle growth and tended to form semisolid gels [25]. This investigator reports furthermore that preparation of lipid nanopellets by high-pressure homogenization was not feasible and yielded particles in the micrometer size range only [25]. Comprehensive physicochemical studies on lipid nanopellet suspensions of the type described by Speiser [l91 are yet outstanding. However, the limited set of published data demonstratedalready some of the physicochemical difficulties and problems associated with the suspension state compared with the emulsion state.
B. Fatty Acid Nanoparticles Prepared from Oil-ln-Water Microemulsions
Gasco et al. have developed a method to prepare solid lipospheres from O/W microemulsions [26-281. Microemulsions were prepared from mixtures of melted fatty acids which may contain a drug, a surfactant, and possibly a cosurfactant in warm water by stirring. Typical formulations comprise stearic acid, m e e n 20, taurodeoxycholate, and butanol [27,28]. The warm microemulsion is diluted and cooled down by dispersion in a cold aqueous phase. In a subsequent step, the liposphere dispersion is washed by diaultrafiltration. Lipophilic drugs can be incorporated in the melted stearic acid prior to formation of the microemulsion. The method
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typically yields lipid particles with an average size of around 200-300 nm depending on production variables such as mixing rate, temperature protocol, and pH of the aqueous medium[28]. In a study of critical formulation parameters for the formation of stearic acid lipospheres from diluted microemulsions using butanol as a cosurfactant, it was found that the growth kinetics typically displayed a biphasic pattern with an initial, rapid size increase up to 10h after dispersion followed by a slower equilibration phase [D]. Incorporation of decanoic acid in the lipid phase was observed to impede submicron particle formation at concentrations about 25% and to disrupt lipid phase equilibrium resulting in continuous and accelerated particle growth. Below around pH4, it was found that minimal variation of acidity could strongly modify growthkinetics. Moreover, differences in butanol amount strongly affected both initial particle growth and equilibrium stability, and at optimum butanol concentration, maximum liposphere stability was observed. The investigators hypothized that the observed effects might be attributed to interfacial interactions in the microemulsion or to differentcrystallization pathways of stearic acid [29]. Studies on the crystallization of washed and dried stearic acid lipospheres by x-ray powder diffraction and differential scanning calorimetry indicated that polymorph C is always present in the lipospheres and that additional crystallization of polymorph B from the microemulsion system is favored in the presence of small amounts of butanol, whereas this polymorph is not present in mixtures of stearic acid, butanol, and Tween 20, which had undergone the same thermal cycle and had the same composition as the dried lipospheres [30]. At present, it is not clear whether polymorph B has its own liposphere population separate from that of polymorph C or whether the two polymorphs cocrystallize in the same liposphere [30]. It has to beconsidered that thecrystallization studies were conducted ondried lipospheres which had been separated from the dispersion medium. The crystallographic state of lipospheres in aqueous dispersion was not investigated but might be different from the findings on dried lipospheres, as crystallization is generally affected by the crystallization medium. Moreover, the composition of lipospheres in dispersion is different from dried lipospheres, since butanol is evaporated from the lipospheres during the drying procedure as indicated by gas chromatographic results on dried lipospheres [30]. In a modification of the microemulsion method developed by Gasco et al. [27,28], ?keen 20 and the organic solvent were substituted by purified egg lecithin [31]. The obtained average diameters of drug-loaded lipospheres ranged between 70 and 180nm. The shape of the stearic acid particles was described to be spherical [31]. The stability on storage of
Biodegradable Colloidal Carrier Systems Drug
22 7
stearic acid liposphere suspensions with respect to properties which might be relevant for drug distribution and release such as particle size, particle shape, and degree of crystallinity was not discussed by the investigators. The fact that the lipospheres were freeze-dried for storage [31] points, however, to stability problems of these lipid suspensions. Results from our own laboratory indicate that stearic acid liposphere suspensions prepared according to Gasco et al. [31] display a significant particle growth within a couple of hours or days after preparation necessitating their lyophilization for pharmaceuticalapplications [32]. Timolol-containing lipospheres that comprisedpalmitic and decanoic acids as carrier material were suggested as an ocular delivery system by Gasco et al. [33]. Timolol was mainly present as ion pairs in the lipospheres in order to increase its lipophilicity. Differences in the incorporation capacity were attributed to the different lipophilicity of the ion pairs [33]. It has not yet been investigated how far themicroemulsion method can betransferred to the use of carrier lipids other than fatty acids. Although the patent by Gasco [26] claims that triglycerides and fatty alcohols also can be employed as lipid components, no evidence thereof is provided in the patent examples, which represent exclusively fatty acid lipospheres. Moreover, long-term stability neither of the aqueous dispersions nor of the dried or freeze-dried storage formulations have been published yet. This is, however, of importance, since even freeze-dried suspensions might undergo particle fusion on storage (cf. Section 111). In addition, studies on possible changes during freeze-drying or storage with respect to particle shape, degree of crystallinity, or polymorphic transitions of the stearicacid which might influence drug distribution within the carrier and drugrelease have not yet been reported.
C. Lipospheres-Lipid Particles Used for Sustained Drug Release
An injectable lipid-delivery system for controlled release, referred to as [34]. The liposphere Lipospheres, has been reported by Domb and Maniar system is described to represent a particulate dispersion of solid spherical particles consisting of a hydrophobic core compoundsuch as triglycerides or fatty acid derivates surrounded by a layer of phospholipids. Preparation of Lipospheres can beaccomplished either by a melt or by a solvent technique in which the core compound is melted or dissolved in an organic solvent and is agitated in an aqueous medium at a temperature above the melting point of the core compound until a homogeneous dispersion is obtained. Lipospheres are formed when the dispersion is rapidly cooled down or the solvent is evaporated, respectively [34]. Both production meth-
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ods are basically known from the preparation of wax microspheres [e.g., see ref. 221. The storage stability of Lipospheres seemsto be very limited. Domb and Maniar [34] designate a 7-day storage stability of submicronsized tristearin lipospheres (based on particle size analysis) as exceptionally stable. Lipospheres in the micrometer size range Seem to be more stable. The particle sizes and distributions were found to be similar to the original formulation after 30 days [34], and for one formulation, Domb and Maniar [34] observed no signs of aggregation for 10 months stored at 4C. Results from our own laboratory indicate, however, that such liposphere formulations tend to sedimentation (unpublished data). Considering the physicochemical properties of the matrix material used for theexamples in the Lipospheres patent application [34], the spherical shape of the particles, in particular those in the nanometer size range, might not be preserved on storage. Additional changes in particle properties such as the modification of the lipids on storage as has been reported for sprayed micropellets [24] or the degree of crystallinity of the matrix have not yet been discussed for Lipospheres by the investigators despite their possible relevance for drugrelease. In a strict sense, liposphere preparations do not represent colloidal carrier systems owing to their micron size. The average particle size of a and Lipotypical liposphere formulation is between 1 and 20pm[35], spheres are thus suitable for intramuscular or subcutaneous injection but not for intravenous administration. Since there are, however, a number of in vivo studies performed using these solid lipid-based drug carriers, the results will be briefly reported below to illustrate the potential of the use of solid lipids in sustained drug release and drugdelivery. It has to beconsidered, however, that the properties of the colloidal solid lipid particles might be significantly different from those of microparticles. It is therefore not unambiguouslypossible to conclude drug release behavior of colloidal lipid carriers from results obtained on microparticulate systems. The extended release of drugs from microparticulate liposphere formulations has been demonstrated by in vitro release studies. Drug release was observed over several days, although there is an initial burst of drug related to the first few hours [34], indicating that parts of the drug are not incorporated in the particle core but areassociated with the carriersurface; for example, in the lecithin layer, from where the drug is fast released. The in vitro data on sustained drug release could be substantiated by in vivo studies, and sustained delivery of local anesthetics, antiinflammatory agents, insect repellents, and antibiotics was observed in different animal models by monitoring the therapeutic effect over time after liposphere administration [34,35]. The use of liposphere preparations as vaccines in immunization by incorporating antigens in the lipid carrier has also been
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demonstrated. Immunization with malaria antigen encapsulated in Lipospheres was shown to increase the IgG antibody titer in rabbits [34]. No significant differences were observed whether the antigen was incorporated in the Lipospheres from an aqueous buffer solution or added as a lyophilized powder to the lipid components in the first step of liposphere preparation [34]. Comparison of the animal immune responseusing Lipospheres or liposomes as carriers of the same antigen revealed that the antibody titers in animals injected with Lipospheres were much higher than in those injected with liposomes, whereas antigen alone or in the presence of adjuvants produced virtually no titer [34]. The drug loading into theliposphere core is highly dependent on the method of preparation. In contrast to melt-dispersed Lipospheres, formulations prepared by the solvent method showed that a significant amount of the drugis trapped in the phospholipid layer [36]. These results might be of relevance to drugrelease from the particles.
D. Colloidal Lipid Suspensions Prepared by Precipitation in Emulsified Organic Solvents
Sjostrom andBergenstdhl have presented a method to prepare submicronsized particles of cholesteryl acetate by precipitation in lecithin-stabilized O W emulsions [37]. Cholesteryl acetate and lecithin were dissolved in an organic solvent, and the organic solution was emulsified in an aqueous phase containing a cosurfactant by high-pressure homogenization to yield a submicron-sized OiW emulsion. On removal of the organic solvent by evaporation under reducedpressure, cholesteryl acetate nanoparticles precipitated in the emulsion droplets. Inspired by our laboratory, Sjostrom and BergenstHhl employed, among others, a blend of phospholipids and bile salts as emulsifiers. It turned out that only with a blend of phosphatidylcholine and sodium glycocholate particles as small as 25 nm could be obtained. The storage stability as monitored over a period of 30 days by repeated size measurements depended onthe lecithidbile salt ratio. With increasing ratio, the stability was found to be reduced. It was not possible to prepare stable emulsions of the cholesteryl acetate containing organic solvent in water using pure phosphatidylcholine without the addition of cosurfactant. Transmission electron microscopy (TEM) of freeze-fractured replicas of a selection of cholesteryl acetate nanoparticles freshly prepared by precipitation in solvent emulsions revealed that the majority of particles had a smooth surface and were spherical [38]. However, larger particles, in particular those above 80-100 nmin size, displayed a nonspherical particle shape and a layered structure already in fresh preparations (Fig.
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Fig. 1. TEM pictureoffreeze-fracturedreplica of cholesteryl acetate particles prepared by precipitation in a phospholipidlsodium glycocholate-stabilized cyclohexane-in-wateremulsion. The bar corresponds to 128 nm.
1). In a subsequent investigation, the morphology of cholesteryl acetate particles was identified as disk-shaped platelets of uniform thickness by cryotransmission electron microscopy [39]. The conclusions drawn from TEM observations of freeze-fractured replicas and from cryopreparations are controvertial, and they might result from particle aging, polymorphism of particle shape, limited number of sample preparations, or misinterpretation of observations. For particles as small as 10 nm, it is, for example, difficult to distinguish between a disk shape and a platelet shape of a particle. TEM studies on freeze-fractured replicas of aged cholesteryl acetate particles are still outstanding. Although cholesteryl acetate particles were designed as a model of drug nanoparticles, itis possible to use these kinds of colloidal dispersions as drug-camersystems as well as by incorporating lipophilic drugs intothe cholesteryl acetate coreor by substituting cholesteryl acetate on account its artherogenic potential by other camer materials such as triglycerides. In an attempt to transfer the precipitation method described by Sjostrom and Bergenstihl [37] to the manufacture of tripalmitate nanoparticles, we succeeded in preparing aqueous tripalmitate suspensions with mean particle sizes ranging from 25 to 120 nm depending on the lecithin-cosurfactant blend, with sodium glycocholate beingthe most effective coemulsifier [40].
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Physicochemical characterization of these tripalmitate suspensions by TEM revealed that, in contrast to cholesteryl acetate, tripalmitate precipitates as anisometrical nanoparticles of predominantly plateletlike shape with a layered structure (Fig. 2). The particles resemble those prepared by meltemulsification (cf. Section III.C), although they are smaller. According to investigations byx-ray diffraction and differential scanning calorimetry (DSc), the recrystallization tendency of precipitated tripalmitate nanoparticles is similar tothat of melt-emulsified suspensions (cf. Section 1II.B). These results indicate that the physicochemical properties of lipid nanoparticles seem to depend predominantly on the matrix material with less importance being placedon the production method. The storage stability of precipitated tripalmitate nanoparticles is, however, poorer than that of melt-emulsified systems. Precipitated tripalmitate suspensions tend to grow in particle sizewithin several weeks or a few months.Residual amounts of organic solvent could be detected by 'H nuclear magnetic responance spectroscopy and DSc. Apart from toxicological problems arising from residues of organic solvents, a clear limitation of the precipitation method is the low lipidconcentration in the suspension after evaporationof the solvent. In the case of tripalmitate precipitated in chloroform emul2.5% sions, the maximum lipidconcentration achievable was no more than (w/w)owing to the limitedsolubility of the triglyceride in the organic solvent. Much higher triglyceride concentrations can be achieved by the melt-emulsification method described below (cf. Section 1I.E). E. ColloidalLipidSuspensionsPrepared by Melt-Emulsification Inthe beginning of the 199Os, two patent applications on solidlipid nanoparticles emerged from the laboratories of Muller [41] and Westesen [42]. Both patent applications are based on a melt-homogenization technique favoring the use of high-pressure homogenizers. Whereasthe patent [41] does not give any precise details with application by Muller and Lucks respect to thestability and the physicochemical properties of the disclosed colloidal lipid particles, that by Westesen and Siekmann [42] provides a detailed description of the novel systems, in particular regarding the stabilization and the long-term stability. Siekmann and Westesen were the first to report on melt-emulsified aqueous lipid suspensions with a storage stability of more than 12 months [H]. The reported melt-homogenization method circumvents the use of organic solvents. Solid lipids, for example, long-chain glycerides, are melted, and the melt is dispersed in an aqueous phase by sonication or preferably by high-pressure homogenization with the aid of emulsifying
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Fig. 2. TEM pictures of freeze-fractured replica of tripalmitate particles prepared by precipitation in a phospholipidsodium glycocholate-stabilized chloroform-inwater emulsion. (a) The bar corresponds to 150 nm. (b)The bar corresponds to 185 nm.
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agents. Emulsification by high-pressure homogenization is more effective and yields more uniformlysized particles than sonication. Thelatter method also bears the disadvantage of considerable metal contamination from the sonication probe. Metal contamination can be avoidedusing highpressure homogenizers with ceramic valves. Moreover, high-pressure homogenization can be used for virtually any batch size from small- to largescale production. The mean particle size of melt-emulsified lipidsuspensions is typically in the range between 50 and 300 nm determined by photon correlation spectroscopy (PCS), and it depends on the homogenization parameters as well as on the composition of the suspensions; for example, on the matrix constituents, the volume fraction of the dispersed phase, and the type and amount of emulsifying agents. It was observed that the mean particle size of lipid nanoparticles increased with increasing melting point of the matrix constituent, indicating an effect of the melt viscosity [NI. At a constant matridemulsifier ratio, the mean particle size increases with increasing concentration of the dispersed phase. With increasing energy input, for example, by increased homogenizationpressure or by prolonged homogenization times, the meanparticle size isgenerally decreasing until it reaches a minimum and then levelsoff. In some cases, a reverse effect, that is, increasing particle size, can be observed with too high-energy input, a phenomenon referred to as overemulsification. In summary, the effects of composition and homogenization parameters are basically similar to those found for oil-in-water emulsions [43,44], and they thus reflect the emulsionlike state of the dispersed molten lipids during emulsification. The effect of the type and amount of emulsifying agents is very pronounced with respect to both the particle size distribution and the storage stability of melt-emulsified solid lipidnanoparticles [45]. Glyceride dispersions stabilized by phospholipids only in concentrations analogously to the stabilization of commercial parenteral emulsions, that is 12 W% lecithin related to the fat phase, display a broad and bimodal size distribution with relatively high mean particle sizes. Moreover, these systems were observed to exhibit a consistency change during storage and form semisolid, ointmentlike gels. Increasing the amountof lecithin to 60 W%(related to the fat phase) results in a decrease of the meanparticle size and in a more homogeneous size distribution, but it cannot effectively prevent gel formation. The prevention of gel formation is, however, possible by the addition of highly of phospholipid-stabilized mobile coemulsifying agents to the aqueous phase glyceride dispersions. These coemulsifiers also decrease the mean particle glyceride size of the lipid nanoparticles, and lecithin-coemulsifier-stabilized dispersions generally display monomodal, homogeneous size distributions with mean sizes well below those of commercial lipid emulsions (Fig. 3).
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40

"
ss
30
"
PL (12O/o)/SGC PL (60%)
PL (6O%)ISGC
lntralipid 100/0
20
? C
x i *
a2
10
0 0
100
200
300 400 Particle size (nm)
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Fig. 3. PCS particle size distribution (by number) of differently composed glyceride nanoparticledispersionsusing the suppository base Witepsol W35 as matrix constituent, compared to a commercial lipid emulsion for parenteral nutrition (Intralipid 10%).PL, phospholipids, SGC, sodium glycocholate.
Both ionic and nonionic surfactants such as the anionic bile salt glycocholate [l81 or thenonionic polymer tyloxapol[46] proved to be efficient coemulsifiers with regard to particlesize distribution andlong-term storage stability (>l8 months), indicating that electrostatic or steric barriers can counteract gel formation. The use of steric stabilizers such as tyloxapol or thenonionic block copolymers of polyoxyethylene and polyoxypropylene, that is, poloxamers, require relatively high amountsof surfactants for effective stabilization, which can be attributed to theirspecific stabilization mechanism. Steric stabilization requires that thestabilizer molecules form a sufficiently thick layer on the particle surface and adsorb to the interfacein a specific steric arrangementwith the ethyleneoxide chains protruding into the continuous phase so as to build up an effective steric barrier toward particle attraction. The number of these high molecular weight molecules required for effective stabilization is therefore relatively high as expressed on a weight basis. The efficiency of lecithin-nonionic polymer blends was shown to depend on the phospholipid/surfactant ratio in the case of tyloxapol [46]. Nonionic polymers can be used as stabilizers by themselves without being combined with lecithin, but it was observed that glyceride dispersions stabilized by poloxamers only tend to form gels when beingsubjected to shear forces; for example, by pressing the dispersion through the needle of a syringe. The same phenomenon was observed when the dispersions were
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stabilized by highly negatively charged phospholipids [45]. Steric or electrostatic barriers can thus not be the only prerequisite for preventing gel formation. A model of the mechanism of gel formation and its prevention based on further experimental data which suggestthat the phenomenonis related to the crystallization of the dispersed phase is discussedin Section 1II.C. After publication of the first paper on the preparation, stability, and physicochemical characterization of melt-emulsified colloidalglyceride dispersions by Siekmann and Westesen [M], Muller et al. disclosed the preparation of solid lipid nanoparticles with particle sizes between 200 and 400 nm by high-pressure homogenization of a preemulsified lipidmelt in a conference abstract. However, no information concerning the long-term stability and physicochemical properties of these systems were given [47]. In a recent publication, optimization of homogenization parameters with the aim to obtain formulations suitable for injection according to the United States Pharmacopeia (USP) XXII monograph entitled Particulate Matter was reported [48]. These investigators also came to the conclusion that highpressure homogenization is superior to sonication, and solid lipid nanoparticle dispersions produced under optimized homogenization conditions were regarded as suitable for intravenous injection, since their content of particles >5 pm is similaror below that of commercial parenteralfat emulsions [48]. The study was conducted on melt-homogenized dispersions of trilaurate (Dynasan 112) stabilized by either soya lecithin or poloxamer using an emulsifier concentration of 12 or50 W% related to the fatphase. The number of large particles depended on homogenization parameters and was considerably affected by the type and amount of emulsifier. Qpical stability problems described for dispersions stabilized by phospholipid mixtures rich inphosphatidylcholine such as gel formation were not reported by the investigators. It has to be considered, however, that melt-emulsified trilaurate displays a pronounced supercooling effect, as was found by thermoanalytical and x-ray investigations from our laboratory[49]. We ob3 months served that trilaurate nanoparticle dispersions stored for more than did not display any thermal transition in differential scanning calorimetry (Fig. 4), indicating that the melt-dispersed lipid does not recrystallize but remains in thestate of a supercooled melt. Recrystallization of meltemulsified trilaurate nanoparticles requires cooling to below 0 C. It can therefore be argued that trilaurate nanoparticles are liquid at room temperature and thus represent O W emulsionsaccording to the International Union of Pure and Applied Chemistry (IUPAC) definition [50] instead of solid lipid particles as claimed by Muller et al. [48]. Since stability problems of lipid suspensions seem to be predominantly related to the crystallization of the dispersed phase (cf.Section III.C), gel formation of supercooled trilaurate dispersions cannotbeexpectedand may explain the good stability of
230 Siekmann
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295
300
305
310
315
320
325 330 TEMPERATURE ( K 1
Fig. 4. DSC thermogram of (a) a 10% trilaurate (Dynasan 112) dispersion stored for 3 months at room temperature and (b) the thermally untreated raw material Dynasan 112. Heating rate: 10 Wmin. phospholipid-stabilized trilaurate dispersions. It thus has to be considered that in a strict sense such trilaurate nanoparticles do not represent solid lipid nanoparticles. In a conference contribution, an 8-monthlong-term stability study on melt-homogenized solid lipid nanoparticles was reported by the group of Muller [51]. The composition of the investigated systems was, however, not described, impeding the readers evaluation of the physical state of the colloidal lipid particles. The investigators confirmed our previous results that the use of a combination of surfactants (a complex of emulsifiers) proved to be optimal for long-term storage [Sl]. They also reported that certain solid lipid nanoparticle dispersions may acquire a gel-like consistency during storage, resulting in solidification of the systems [52]. It was found that especially high temperatures and light exposure caused solidification within a few days. In conference contributions, spray drying [53] and lyophilization [54] were suggested to generatea dry storage formulation of
Biodegradable Colloidal Drug Carrier Systems
231
solid lipidnanoparticles to circumvent storage problems. However,data on the stability of the lyophilizates on long-term storage with respect to initial particle size were not reported. Results from our own laboratory indicate that lyophilization is not necessarily a suitable technique for improvement of long-term stability, since it turned out that the lyophilized powders of melt-homogenized triglyceride nanoparticles displayed an impaired redispersibility and exhibited a pronounced particle growth after storage for 12 months, which can tentatively be attributed to sintering processes during storage [S]. Surface modification of colloidal drug carrier systems represents a promising approach to reduce carrier uptake by the reticuloendothelial system (RES). In order to divert colloidal particles away from theRES, the surface of the particles should be hydrophilic and noncharged [17]. In preliminary experiments, we established that melt-emulsified glyceride nanoparticles can be surface modified by adsorption of nonionic block copolymers [45]. Lecithin-stabilized tripalmitate nanoparticles were incubated directly after preparation with poloxamer 407 and poloxamine 908 solutions. Polymer adsorption wasconfirmedby measurements of the zetapotential employing laser Doppler anemometry (Table 2). The surface potential of tripalmitate nanoparticles is considerably reduced by the adsorption of the polymers as compared with the phospholipid-stabilized dispersion. In a conference abstract MaaBen et al. [56] described that thein vitro uptake of surface-modified solid lipid nanoparticles into human granulocytes was significantly reduced compared with surface-modified polystyrene nanoparticles. The investigators concluded from the distinct reduction of phagocytic uptake in vitro that surface-modifiedsolid lipidnanoparticles possess the potential to prevent RES uptakein vivo.
Table 2. Zetapotential of Surface-Modified Solid Lipid Nanoparticles After Incubation (24 h) with Nonionic Block Copolymers
Composition (% [w/w])
l
5 5 5
PL (%) 2 2 2
Polymer
Zetapotential (mV) -29.6 -1.9 -2.9
3% P4W 3% P 908
Abbreviations: l, tripalmitate (Dynasan 116); PL, phospholipids (Lipoid S 100); P 407, poloxamer 407 (Phonic F127); P 908, poloxamine 908 (Tetronic 908).
232 Siekmann
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111. PHYSICOCHEMICALCHARACTERIZATIONOF MELT-EMULSIFIED COLLOIDAL LIPID SUSPENSIONS
The idea behind the use of solid lipids as a carrier material is that the emulsified molten lipids resolidify on cooling, creating a solid matrix in which the mobility of incorporated drug is drastically reduced. The advantages of the solid state of the carrier have already been outlined in Table 1. Although solid lipid-based carrier systems are produced from crystalline raw materials, it has to be considered that recrystallization from the dispersed lipid melt might be different from that of the bulk phase. The high dispersity and the small particle size and the presence of emulsifying agents and drugs as well as the high surface-to-volume ratio of melt-emulsified lipids may account for differences in the crystallization behavior, the degree of crystallinity, and the polymorphism of the nanoparticulate lipids compared with the bulk materials. Investigations by Skoda and van den Tempe1 [57] revealed that the crystallization temperature of a tristearate solution in paraffin oil which wasemulsified in anaqueousphase waslower thanthat of the bulk triglyceride. Crystallization in the dispersed state thus requires a higher degree of supercooling, which can onthe one hand be attributed to fact the that crystallization can only take place in small, discrete, and limited volumes. Whereas crystallization in the bulk can proceed quickly over the whole material once it has started in one location, crystallization in dispersed systems thus has to start individually in each droplet independently of the state of crystallization inother droplets. Such a crystallization behavior can be easilyvisualized by polarizingmicroscopy for lipid-in-water dispersions with droplet sizes inthe micrometer size range (Fig. 5). On the other hand,it has to be considered that crystallization in the dispersed state may be affectedby emulsifying agents. It was shown before that thepresence of emulsifiers has an effect on the polymorphism and the crystal structure of bulk triglycerides [58]. Crystal nucleation in crudely emulsified triglyceride solutions was observed to depend onthe molecular structureof the employed emulsifying agents [57]. Owing to these effects,alterations of the crystallization behavior in the dispersed state can be expected and may lead to liquid, amorphous, or only partially crystallized systems.The physicochemical characterization of glyceride nanoparticles was therefore first of all focused on the question whether melt-emulsified glycerides recrystallize in the dispersed state. In addition to the studies on the crystallization behavior, it should be investigated why solid lipid nanoparticles stabilized by phospholipids only, that is, analogously to the preparation of submicron-sized lipid O N emulsions,
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Fig. 5. Polarized light microscopy picture of a crude dispersion of tripalmitate stabilized by phospholipids and sodium glycocholate (magnification: X 160): The dispersed lipidmelt forms spherical, isotropic droplets. The excess of phospholipids in the system forms large multilamellar vesicles which exhibit maltese cross-like structures in the polarized light microscope. Crystallizationstarts from the particle to surface and proceeds over the whole particle volume. Larger droplets tend recrystallize first.
tend to form semisolid gels and how gel formation can be prevented by certain stabilizers and coemulsifying agents (see above).
A. Investigations on the Physical Stateof Colloidal Lipid Dispersions Preparedfrom Crystalline Glycerides
Long-chain triglycerides which are solid at room temperature in the bulk exhibit a complex polymorphic behavior and can crystallize in three basic polymorphic forms, termed alpha(a),beta prime (g'), and beta (g). Polymorphic transformationsare monotropic and takeplace from a to p' to p, with the latter being the thermodynamically stable polymorph [59]. For complex glyceride mixtures such as the hard fat suppository base Witepsol W35, an intermediatecrystal form (pi) between p'and p has been described
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[60,61]. The different polymorphic forms can be unambiguously distinguished by their x-ray spacings, and x-ray diffraction should thus provide a direct method to characterize the physical state of glyceride nanoparticles. The glyceride carrier systems under investigation represent, however, dilute dispersions with lipid concentrations typically of approximately 10 W%. Furthermore, it has to be taken into account that dispersed the glycerides are present as nanoparticles with sizes as small as 50 nm. Considering thatthe long spacings of the investigated glycerides typically range from approxif only 10-15 crystal mately 3.5 to 5 nm, such small particles can consist o layers. In view of the low glyceride concentration and the low number of repeating units in the nanoparticles, only broad low-intensity diffraction peaks characteristic of poorly ordered systems can be obtained using a conventional x-ray source, ashas been demonstrated by investigations using a Debye-Scherrer camera [62]. The necessarily long exposure times of several hours furthermore preclude a systematic investigation, and time-resolved xray diffraction measurements during temperature scans are not possible. In contrast, synchrotron radiation x-ray diffraction provides an efficient way of performing such measurements even in dilute dispersions of submicronsized glyceride particles [62], as is illustrated below. Synchrotron radiation small and wide-angle x-ray diffraction measurements were performed on melt-homogenized glyceridedispersions employing tripalmitate and hard fat (Witepsol W35) as carrier matrices and lecithin, bile salts, and nonionic polymers (poloxamer, tyloxapol) as emulsifiers. The dispersions had beenstored for several weeks prior to x-ray measurements. Systems stabilized by poloxamers tended to form semisolid gels. Small-angle x-ray (SAX) diffraction revealed that all investigated carrier systems were crystalline at room temperature. The reflections in SAX diffraction patterns of the lipid dispersions were generally broader than those in the corresponding raw materials. This can be attributed to a particle size effect. The SAX spacings of tripalmitate dispersions correspond to thep modification of the raw material (Fig. 6). Lecithin-bile salt-stabilized hard fat dispersions (Fig. modification of 7b) did not display the double peakcharacteristic of the pi hard fat(Fig. 7a). Whereas for the semisolid hard fat gels this characteristic double peak could be found (Fig. 7c), the broad SAX diffraction peaks of the hard fat dispersions did not permit a clear distinction between p'and pi forms. In contrast, the crystal modifications of all carrier systems could be unambiguously identified by wide-angle x-ray (WAX)diffraction and indicated that bothtripalmitate and hard fat nanoparticles are in the p modification at room temperature (Fig. 8a and sa). Hard fat-based carriers displayed particularly weak and broadmaxima at wide angles. Obviously,the crystal lattice of hard fatnanoparticles is highlyperturbed, since the complex com-
235
0.1 I
0.2 I
0.3
s (m")
Fig. 6. Synchrotron radiation small angle x-ray diffraction patterns (intensity Z vs scattering vector S): (a) bulk tripalmitate (raw material) and (b) 10 W% colloidal dispersion of tripalmitate stabilized by 1.2 W% soya lecithin and 0.4 W% sodium glycocholate (mean PCS particle size: 167 nm). The intensities of raw material and dispersion are not on the same scale.
0.1 I
0.2
9.3
s (nrn-1)
Fig. 7. Synchrotron radiation small angle x-ray diffraction patterns (intensity Z vs scattering vector S): (a) bulk hard fat (raw material); (b) 10 W% colloidal dispersio 0.4 W% sodium glycocholate of hard fat stabilized by 1.2 W% soya lecithin and (mean PCS particle size: 92 nm); and (c) l 0 W% colloidal dispersion of hard fat stabilized by 1.8 W% Pluronic F-68 (semisolid gel). The intensities of raw material and dispersionsare not on the same scale.
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position of hard fatprecludes the formationof a true lattice. Moreover, the accumulation of lattice imperfections may be attributed to the small crystallite size in the dispersions. WAX diffraction at body temperature indicated that tripalmitate nanoparticles are still crystalline (Fig. 8b), whereas hard fatcarriers do not give any diffraction peaks (Fig. 9b), suggesting that the carrier material is melted at body temperature. The different physical state of the carriers at body temperature may influence their biopharmaceutical properties. The liquid hard fat nanoparticles give rise to fast drug release, whereas tripalmitate carriers may be suitable as sustained-release delivery systems. Being crystalline at room temperature, both carriersystems may provide an enhanced in vitro stability and a decreased risk of drug leakage onstorage as well as directly on administration owing to adsorption of drug to the wall material of syringes, containers with infusion media, or tubes. Preliminary investigations on the recrystallization behavior of melthomogenized glyceride dispersions were performedby time-resolved x-ray diffraction studies during temperature scans [62]. Hard fat nanoparticle dispersions were heated above the melting point of the carriermatrix, and the molten camer was cooled down stepwisemonitoring the WAX diffraction pattern. The raw material was exposed to a similar temperature program. The measurements indicated that recrystallization of the hard fat nanoparticles is different from that of the bulk material. Whereas the dispersed glycerides recrystallizein the a form, thebulk lipids recrystallize in the pmodification and transform rapidly into the piform. Furthermore, recrystallization of the bulk lipids starts at a higher temperature than the dispersed glycerides. The results indicate that hard fat can represent a supercooled melt when dispersed as nanoparticles dependent on storage temperature and storage time (cf. Section 1II.B).
B. Investigations on the Crystallinity and the Recrystallization Processof Melt-Emulsified Glyceride Nanoparticles
Preliminary time-resolved x-ray studies already indicated differences regarding recrystallization from the melt of dispersed lipids compared with their bulk materials. In a next step, the recrystallization process should be systematically investigated. Owing to the limited access to synchrotron radiation x-ray sources, differential scanning calorimetry (DSC) was established as an alternative method to study the recrystallization behavior, the time course of polymorphic transitions, and the degree of crystallinity of melt-homogenized glyceride dispersions [63]. Knowledge of the crystallinity isof special relevance to evaluate the incorporability of lipophilic drugs

NS)
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a)
0
-11
US)
x10
Channel
d
.
1
2
-11
x10 2
Channel
Fig. 8. Synchrotron radiation wide-angle x-ray diffraction patterns of a 10 W% tripalmitatedispersionstabilizedbylecithinkodiumglycocholate(intensity Z vs detectorchannel): (a) recorded atroomtemperatureand (b) recordedatbody temperature.
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x1 0
Channel
Fig. 9. Synchrotron radiation wide angle x-ray diffraction patterns of a 10 W%

hard fat dispersion stabilizedby lecithidsodium glycocholate (intensity Zvs. detector channel): (a) recorded at room temperature and (b) recorded at bodytemperature.
239
into the glyceride matrix. The chances to include foreign molecules, in particular those with a structurecompletely different from the matrix glycerides, are expected todecrease with the increasing order of the crystal lattice of the fatmatrix. Therefore, the time course of recrystallization and polymorphic transitions may correlate with the time course of expulsion of incorporated drug fromthe crystal lattice. The degreeof crystallinity of colloidal glyceride crystals is influenced by two factors:
1. The coexistence of recrystallized and amorphous (supercooled) particles, possibly as a result of a size effect as well as the crystallization probability 2. distortion of the crystal lattice by lattice imperfections owing to the high surface-to-volume ratio and the low periodicity within the nanoparticles
An estimate of the crystallinity index can be obtained from thermoanalytical data by relating the heatof fusion of the glyceride nanoparticles to that of the bulk glycerides. DSC heating scans of tripalmitate nanoparticle suspensions indicate an effect of the emulsifying agent on the thermograms. Tripalmitate dispersions stabilized by phospholipids only form ointmentlike semisolid gels directly after preparation. Thesegels display a sharp endothermal peak at around 56-57C. In the presence of a coemulsifier such as the bile salt sodium glycocholate, tyloxapol, or poloxamers, tripalmitate dispersions do not gel. In thecorresponding thermograms of freshly prepared systems, the p melting peak is at 54C. The melting temperatures found for the dispersed triglycerides correspond to the melting point of the ppolymorph of bulk tripalmitate (Table 3). X-ray studies demonstrated, however, that thedispersed systems were in the p modification. In freshly dispersed, coemulsifier-stabilized dispersions, an additional endothermalpeakat around 40C was detected which can be assigned to the a modification. In general, the a phase is transformed intohigher melting polymorphs within days or weeks. Obviously,phospholipids accelerate these polymorphic tranof cosurfactants. A similar effect of sitions, which is retarded in the presence the coemulsifying agent was observed for hard fatsuspensions. Tentatively, this phenomenoncan beinterpretated as an epitaxical effect of the phospholipids due to structural similarities of the hydrocarbon chains of phospholipids and triglycerides. It was generally observed that the melting point of dispersed glycerides was decreased by up to 13C compared with the bulk material. This excludes unambiguous interpretation of DSC thermograms. As indicated by Table 3 and already outlined above, thermoanalytical data suggest
240
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Westesen and
Table 3 . Melting Temperaturesof Glyceride Polymorphs and Melt-Emulsified Glyceride Dispersions

Form Hard Fat (C)
20-22 32-33 38-39
Tripalmitate (C)
44-45 56-58
ppolymorph
54-56 Dispersion (fresh) 56-59 Dispersion (stored)
a polymorph
P ipolymorph P polymorph
12-32 31-33
65-67
that the dispersed glycerides are present in the ppolymorph. Synchrotron radiation x-ray diffraction clearly demonstrated, however, that the lipids modification, respectively. Exact interpretation of were in the stablep or pi DSC thermograms and unambiguous assignment of polymorphic forms thus require a priori knowledge about the crystal modifications, or else interpretation of DSC transition peaks might lead to erroneous assignment of polymorphic forms. Knowledge of the existing polymorph is, however, the basis for deriving the crystallinityindex of solid lipid nanoparticles from thermoanalytical data, since different polymorphs have a different heat of fusion. This aspect has not been taken into consideration by Muller et al. [52], who calculated the crystallinity index of glyceride nanoparticles from thermoanalytic data directly without assuring correct assignment of crystal modifications. The melting point depression observed with melt-emulsifiedlipid suspensions can be explained by the colloidal dimensions of the glyceride nanoparticles and their high surface-to-volume ratio. The excess pressure or solubility, respectively, experienced by the colloidal solid as predicted by increases its chemical potential. As a consethe Kelvin equation [M] quence, small crystals melt at a lower temperature than the bulk. The melting point, T, of small crystals of radius, r, can be derived from the modified Thomson equation [64], where To is the melting point o f the bulk material, y represents the interfacial energy, V the molar volume, and AHrus is the molar heat of fusion:
Furthermore, the thermoanalytical studies revealed that induction of recrystallization of both hard fat andtripalmitate dispersions requires more supercooling than in bulk. The supercooling amounts to approximately 20C below that of the bulk glycerides inboth cases; that is recrystallization
24 l
I I
I I
200
290
300
310
320
TEMPERANR (K)
Fig. 10. DSC thermograms of a 10 W% hard fat dispersion stabilized by lecithin/ sodium glycocholate: (a) day of preparation, recorded from 20 to 60C; (b) day of preparation, recorded from5 to 60C; and (c) stored for 2 weeks at 4"C, recorded from 20 to 60C. Heating rate: 10 Wmin.
from the melt starts at a much lower temperature in dispersed fat nanoparticles than in the bulk glycerides. As a result of the higher supercooling required for nucleation, isothermal recrystallization of hardfatnanoparticles is considerably retardedatroomtemperature (Fig. 10). DSC scans from room temperature to 60C on the day of preparation indicate that hard fat nanoparticles have not yet recrystallized. Recrystallization can be induced by recording the thermograms from5C onward. A broad peak with several maxima is then obtained pointing to the presence of different polymorphs. After 2 weeks of storage at 4"C, the thermogram recorded from room temperature displays a relatively sharp peak correpolymorph. The peak shoulderindicates the presence of sponding to thepi the residual p'form. Investigations on the time course of polymorphic transitions indicate thatpolymorphic transitions generally proceed faster in the dispersed glycerides than in bulk. Recrystallization of phospholipid-cosurfactant-
242 Siekmann
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stabilized tripalmitate nanoparticles can already be detected 2 hs after preparation. Most of the triglyceride is present in the p modification with minor amountsof the CY form. After several days to a few weeks of storage, the CY polymorph has been completely transformed into the stablep modification. Incontrast, this transformation takes several monthsin meltcrystallized bulktripalmitate. In the nanoparticles, the heat of fusion of the p modification increases continuously until a plateau around 20 J/g is reached after approximately 2 months of storage (Fig. 11). The plateau value corresponds approximately to10% of the heat of fusion of the bulk material, indicating that the crystallinity of stored tripalmitate nanoparticles is similar to thatof the bulk. The initial increase during storage can be interpreted as a progressive annealing of the crystal lattice of the nanoparticles. The crystallinity index of glyceride suspensions calculated as the ratio of the heat of fusion of the nanoparticles compared with that of their bulk materials thus depends on the storage time and canincrease to up to 100%. Apart from the storage time, the crystallinity index of glyceride nanoparticles is affected by the type and amountof emulsifier, the presence of foreign compounds, andthe matrix material [63]. The crystallinity index of hard fat suspensions is generally lower than that of similarly composed tripalmitate systems. This difference is also reflected in the corresponding
24 22
1,
.A
IU
1 .
I
100
200
300
400
Storage time (days)
Fig. 11. Heat of fusion determined by DSC at different time intervals of a 10 W% tripalmitatedispersionstabilized by 2.4 W% soyalecithinand 0.4 W% sodium glycocholate.
243
x-ray diffraction patterns and can be attributed to thelower crystallinity of the complex crystal lattice of hard fat which is more distorted than that of tripalmitate. Blending tripalmitate with monostearate (5% related to tripalmitate) results in a decrease of the crystallinity index compared with pure tripalmitate suspensions. Moreover, the monostearate apparently exhibits an a phase-preserving effect. X-ray data point to theinclusion of monostearate in the tripalmitate lattice without changes in the lattice dimensions. The creaYed lattice imperfections reduce the crystallinity of tripalmitate. The a polymorph is stabilized in these systems, because the presence of monostearate impairs the configurational changes of the tripalmitate molecules associated with the polymorphic transition. This observation is also described in the literaturefor bulk triglycerides [65]. The differences regarding the recrystallization behavior, the time course of polymorphic transitions, and the degree of crystallinity of melthomogenized glyceride nanoparticles and their bulk materials as revealed by thermoanalysis can be attributed to thedispersed state, thepresence of emulsifiers or otherforeign compounds, and thehigh surfaceholume ratio of the nanoparticles [63]. The results are of relevance to evaluate the the incorporability of drugs into the (storage) stability of a composition and glyceride camer matrix, and to explain the possible expulsion of drugs from the crystal lattice.
C. Mechanism of GelFormationinPhospholipid-Stabilized Tripalmitate Suspensions

Colloidal lipid suspensions display striking similarities to submicron-sized lipid O M emulsions concerning the chemical composition of these dispersions. Furthermore, suspensions of solid lipidnanoparticles can be manufactured by a melt-homogenizationprocess which is in principle similar to the production of parenterallipid emulsions. The obvious similarities between lipid emulsions andlipid suspensions with respect to chemical composition and production method might lead to theconclusion that lipid suspensions could be regarded aslipid emulsions with solidified droplets. However, in contrast to lecithin-stabilized lipid emulsions, the preparation of similarly composed lipid suspensions, that is, by employing phospholipids as the only [18,52]. The observed stabilizer, can result in the formation of semisolid gels instability of lipid suspensions containing the same type and amount of emulsifiers as parenteral lipid emulsions thus indicates basic physicochemical differences of these twotypes of colloidal dispersions. As already outlined above, the prevention of gel formation is possible by the addition of highly mobile cosurfactants such as bile salts [B]or the nonionic polymer
t.
Systems
245
tyloxapol[46], but results obtained with different types, amounts, and combinations of emulsifiers indicated that electrostatic repulsion or steric stabilization do not represent the exclusive prerequisites to prepare stablesolid lipid nanoparticles [45]. Results from the production of glyceride nanoparticles further point to that gel formation might be attributedto recrystallization of the dispersed lipid, since gel formation was generally not observed above the melting temperature of the glycerides [66]. X-ray studies did, however, not display basic differences regarding the crystalline state of the semisolid gels and theliquid suspensions [62]. In order to study the physicochemical processes underlying the gel formation phenomena, structuralinvestigations of tripalmitate nanoparticle dispersions and gels were initiated by transmission electron microscopy of freeze-fractured specimens (FF-TEM) and of frozenhydrated specimens (cryo-TEM) [67]. FF-TEM of lecithidcosurfactant-stabilized tripalmitate supensions revealed that the emulsified glycerides recrystallize as anisometrical particles of predominantly plateletlike shape (Fig. 12a). Larger particles clearly display a layered structure with terraces, steps, and kinks (Fig. 12b). The prevailing existence of platelets indicates the preferential formation of certain crystal faces. The crystal shape of tripalmitate nanoparticles resembles melt-crystallizedmicro- and macroscopic tripalmitate crystals of the /3 modification [68,69]. Owing to these similarities, the prevailing crystal face of tripalmitate nanoparticles could be identified as the (001)-face. As a consequence of the characteristic order of molecules in the crystal lattice, the different crystal faces exhibit different physicochemical properties such as polarity and surface tension. In melt-crystallized macroscopic tripalmitate crystals, the (001)-face, which contains the methyl end groupsof the hydrocarbon chains, is nonpolar. However, owing to its preferential formation in the aqueous medium, it can be deduced that the (OOl)-face of tripalmitate nanocrystals is hydrophilic. This was confirmed by decoration of the fractures with water, which condenses preferably on the large (001)-face [67]. The detected hydrophilicity of the (001)-face points to the association of surfactant molecules with this nanocrystal face. FF-TEM of tripalmitate suspensions stabilized by phospholipids only which form semisolid, ointmentlike gels displays a coherent network of
Fig. 12. Transmissionelectronmicrographs of melt-homogenizedtripalmitate. (a) Top: 10 W% tripalmitate suspension stabilized by 1.2 W% soya lecithin and0.4 W% sodium glycocholate (the bar represents 230 nm). (b) Middle: 10 W% tripalmitate suspension stabilized by 1.2 W% soya lecithin and 0.4 W% sodium glycocholate(thebarrepresents 150 nm).(c)Bottom:10 W% tripalmitate gel containing 1.2W% soya lecithin (the bar represents 250 nm).
246 Siekmann
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lamellar crystal platelets with a thickness corresponding to that of tripalmitate nanocrystals in suspension [67]. From the view onto the network plane it seems that the gel structure is formed by individual platelets which have agglomeratedto a cardhouselike structure (see Fig. 12c). It is striking that aggregation of the platelets occurs via the lateral crystal faces and not via the (001)-face, which also represents the prevailing crystal face in the gels. Owing to its large expansion, it can be deduced that the (001)-face exhibits a low surface tension, pointing to preferential phospholipid adsorption on the (001)-face. From the electron microscopic pictures, the tripalmitate gels can be characterized as ultrafine three-dimensional networks formed by aggregated tripalmitate crystals. The aqueous phase interpenetrating thenetwork is completely immobilized inthese structures. The physicochemical processes during cooling can be directly monitored by polarized light microscopy of crude lecithin-bile salt-stabilized tripalmitate dispersions [45]. The dispersed lipid melt formsspherical, isotropic droplets which transform into anisometrical, optically anisotropic particles when the melt recrystallizes (Figs. 5 and 13). Crystallization starts from the particle surface, pointing to an epitaxical effect of the emulsifier molecules present at the droplet interface. Beside the fat particles, the typical Maltese cross-like structures representing multilamellar vesicles (MLV) can beobserved. The high number of MLV indicate that a substantial portion of phospholipids is bound in vesicles despite the fact that the emulsifier was dispersed in the melted lipid phase prior to the emulsification process. The existence of small unilamellar vesicles in colloidal tripalmitate suspensions was confirmed by cryo-TEM [67]. The numberof vesicle structures in tripalmitate suspensions is, however, smaller than in commercial parenteral O/W emulsions, althought the lecithin concentration was higher in the suspensions. The microscopic observations oncrudeand submicron-sized tripalmitate dispersions clearly demonstrate that recrystallization of meltemulsified tripalmitate is associated with the formation of anisometrical particles, thereby inducing a substantial increase in particle surface. The newly generated surfaces need to be stabilized immediately in order to avoid particle aggregation and the formationof two-phase gel systems. Phospholipids belong to the group of insoluble swelling amphiphilic lipids. In aqueous media, they spontaneously form vesicles. Sincephospholipid molecules do not tend to leave the bilayer, their concentration in the is very low. Moreover, phospholipids only exhibit a moderate aqueous phase mobility inaqueous media. Owing to this phase behavior, lecithin molecules are not expected to be sufficiently mobile to stabilize newly generated tripalmitate surfaces immediately. The use of phospholipids as the only emulsifier, even in excessamounts, thus results in gel formation. In contrast,
247
Fig. 13. A polarized light microscopy picture of a crude dispersion of tripalmitate stabilized by phospholipids and sodium glycocholate after termination of the recrystallization process (magnification: X 160). The suspended particles are optically anisotropic and exhibit anisometricalshapes.
the employed cosurfactants are micelle-forming agents. Micelles are highly dynamic structures and representa reservoir of surfactant molecules from which the emulsifier can be recruited when the emulsion droplets turn into anisometrical suspension particles. Owing to the high solubility and mobility of the cosurfactants in the aqueous phase, they are able to stabilize the freshly generated surfaces immediately. Based on the experimental results from electron microscopy and on theoretical considerations regarding crystal structure andmolecular mobility, a model as illustrated in Fig. 14 has been developed to describe the instability phenomena of colloidal tripalmitate suspensions. The recrystallization of the colloidally emulsified tripalmitate results in an increase in particle surface area due to the change in the overall particle shape and a sudden local lack of emulsifier in the particle surfaces. The time, cl, corresponds to the time which the recrystallization process takes. During the emulsification process, the excess of phospholipids has formed small vesicles (SUV) which are randomly distributed in the continuous phase and
248
Suspension
Emulsion
Bile salt micelles
Fig. 14. Model of the gel formation mechanism in phospholipid stabilized triglyceride dispersions(SUV, small unilamellar vesicle). which exist in a metastable state after finalization of the emulsification which the phospholipid process. The time, t2, corresponds to the time molecules would need to leave the vesicles and to diffuse to the newly created insufficiently stabilized particle surfaces. The time, tz, is, however, significantly longer than the time, C,,resulting in an instability of the dispersed state andgelation via the lateral faces of the crystals. In contrast, dispersions which contain an admixture of a soluble, micelle-forming component such as bile salts are metastable; that is, the dispersed state can be maintained over a considerably long timeof storage. The soluble, micelle-forming component is able to cover thenewly created tz, for the surfaces in a time, t3, which is significantlyshorter than the time, phospholipid molecules and which is short enough that no gelation of the dispersed particles, that is aggregation via unprotected crystal faces, can occur (see Fig. 14). Furthermore, it has to be considered that phospholipids and cosurfactants adsorb to different nanocrystal faces. The TEM results suggested that phospholipids are preferentially adsorbed to the (001)-face, so that the lateral faces are insufficiently stabilized and aggregation can occur via these faces. It can be assumed that the lateral faces are covered to a greater extent by the cosurfactants. Moreover, it seems likely that phospholipids-
Systems
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in contrast to thehydrophilic cosurfactants-are at least partially incorporated into thetripalmitate crystal lattice owing to similarities in molecular geometry. Crystal growth can thus proceed over adsorbed phospholipid moleculesbut is interrupted where cosurfactants are adsorbed tothe nanocrystal surface. From the results outlined above, it can be concluded that lipid suspensions do not simply represent lipid emulsions with solidified droplets, since the solidification of the dispersed phase is associated with basicdifferences in the physicochemical behavior of lipid emulsions and suspensions as illustrated by the more complex and complicated stability aspects of the latter. It should be noted that the particle shape of melt-homogenized lipid nanoparticles might be considerably different from thatof microparticles of the same lipid matrix, which may display a predominantly spherical particle shape. The differences can be attributed, among others, to the different production processes. Lipid microparticles are generally prepared by methods which include a rapid cooling step forcing the lipid to solidify immediately in the amorphous state or in an unstable polymorphic modification of low crystalline order with a high concentration of lattice imperfections. In this case, the low-ordered, polycrystallinic crystal lattice does not exert a strain on the particle shape, and surface minimized; that is, almost spherical particles can be formed. In contrast, tripalmitate nanoparticles represent extremelysmall single crystals. Sincethe production process does not include a rapid cooling step, the colloidal lipid particles can crystallize slowly, so that the stable polymorph can be formed usually exhibiting a highly ordered crystal lattice which is reflected by the characteristic anisometrical crystal shape. It is thus not possible to transfer results obtained on lipid microparticles to theproperties of lipid nanoparticles. It has to be taken into consideration, however, that not alllipid materials will form colloidal particles with anisometrical shape as for tripalmitate. Colloidal suspension particles of complex lipid mixtures, such as WitepsolW35, exhibit a spherical shape [l81 in the dispersed state, and of the the structure of the particle cores is significantly different from that tripalmitate nanoplatelets. Whereas the internal structure of colloidal tripalmitate particles resembles a stack of cards or lamellae, the core of colloidally dispersed Witepsol W35 particles seems to be polycrystalline with low order within each crystallite. D. incorporation of Drugs into Solid Lipid Nanoparticles Solid lipid nanoparticles have been demonstrated to represent crystalline colloidal particles [62,63]. Colloidal systems with the dispersed lipid existing
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in the solid amorphous state have not yet been described in the literature. The crystalline state of the particle matrix has to be taken into account when estimating the potential of incorporating drugs into these camer systems, since the inclusion of foreign compounds into a crystalline lattice is generally limited. To a certain extent, foreign molecules can be included in lattice imperfections. It has to be considered, however, that on the one hand the the inclusion capacity is number of lattice defects is limited and consequently low. This inclusion mechanism can thus only be taken advantage of with highly active compounds which do not require administration of high therapeutic doses. On the other hand, it was shown that the crystallinity of glyceride nanoparticles is increasing with storage time owing to annealing of the crystal lattice [63]. As with the kinetics of polymorphic transitions, annealing is accelerated in the colloidal state and drug molecules located in lattice imperfections can be expelled from the lattice with increasing annealing, leading to stability problems onstorage. Results from our laboratory [45] suggest that the incorporability of lipophilic compounds into melt-homogenized glyceride nanoparticles depends predominantly on whether the drug can be included in the crystal glyceride lattice without strongly disturbing its structure, that is, that the molecular structureof the drug fits into thelattice, or whether the drug and the carriermatrix can forma crystal lattice together. If this is not possible, the drug is expelled from the camer matrix on recrystallization or, respectively, polymorphic transitions of the glycerides, and it precipitates in the aqueous phase as was found, for instance, for menadione and cortisone. There are, however, a number of drugs which have been demonstrated to be incorporable in lipid suspensions. In a conference contribution, Schwarz et al. [70]reported that they succeeded in incorporating tetracaine and etomidate in solid lipidnanoparticles without severe reduction of the physical stability. We were able to show that, in contrast to cortisone, the structurally related compound prednisolone could be incorporated into tripalmitate nanoparticles, and these drug-loaded camers proved to be stable for This observation indicates that even small structural several months [45]. changes, for example, the steric arrangement of functional groups, can be of relevance for incorporation in the glyceride lattice. The incorporability of retinol and ubidecarenone suggests that long, apolar side chains of the drug compound might be beneficial for inclusion in the glyceride matrix [45]. The use of lipophilic prodrugs, for example, esters of long-chain fatty acids, might thus represent an approach to improve the incorporability of drugs. Another approach to increase the drug-incorporation capacity is controlled solidification of melt-homogenized glyceride nanoparticles as lowordered crystals of the a phase or even in amorphous form. Because of
Biodegradable Colloidal Carrier Systems Drug
25 1
their completely disordered state, amorphous solids exhibit a high incorporation capacity for foreign compounds. It has to be considered, however, that thestabilization of amorphously solidified triglycerides is energetically unfavored because of the extremely low activation energy required to transform amorphous triglycerides into thea modification. It might therefore be more promising to concentrate effortson the stabilization of the a phase. Owing to thelower crystalline order in the metastablea polymorph as well as to the rotational motion of the hydrocarbons which reduce the crystal packaging in the a form, theincorporability of foreign compounds intothe a phase is increased compared with the stable p modification. An attempt a phase by the use of asymmetrical glycerides or cocrystallizto stabilize the ing mixtures of different matrix lipids is currently being investigated in our laboratory. Considering the observed acceleration of polymorphic transitions for colloidal dispersions of tripalmitate as well as of hard fat, longterm stabilization of the a modification in colloidal dispersions might be extremely difficult to achieve. Recent results demonstrate that on the other hand the drug can affect the recrystallization of thecamer lipids. Recrystallization studies on ubidecarenone-loaded tripalmitate nanoparticles by DSC and time-resolved x-ray diffraction indicated that incorporation of the lipophilic drug can cause a significant reduction of the recrystallization temperature of the camer [71]. Moreover, in drug-loaded camer systems, anaccelerated polymorphic transition into the stable p modification was observed, since the a polymorph of the camer matrix could not be detected in ubidecarenonecontaining tripalmitate nanoparticles. These results furthermore demonstrate the increasing complexity of solid lipid nanoparticles when loaded with drugs. The situation is even more complicated by the fact that drugs differ in their physicochemical properties. In addition, the therapeutic dose depends on the drug requiring different payloads. Therefore, drugconcentration has to be considered as an additional complicating factor.
IV. SUMMARY AND FUTURE PROSPECTS
Colloidal dispersions based on biodegradable solid lipids have been found to be potential drug-carrier systems of complex physicochemicalproperties as expressed by their recrystallization behavior and their tendencyto form semisolid gels, which have prevented theirsuccessful establishment as drug carriers for several decades. The results regarding the gel-formation mechanism reported here emphasize the importance of physicochemical characterizationforproduct development. Despitetheir complexity, solid lipid nanoparticles represent an interesting alternative to conventional drugcarrier systems such as polymer nanoparticles, liposomes, and lipid emul-
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sions, since it has been demonstrated that stability problems are associated with the crystalline state of the dispersed lipids and can be circumvented by a proper choice of matrix constituents, emulsifying agents, and production parameters. Manufacturing of solid lipid nanoparticles can avoid the use of potentially harmful additives, so that toxic residues from the production process as often associated with the preparation of polymeric nanoparticles can be circumvented. Owing to the biodegradability of physiological lipids, accumulation of the camer material on repeatedadministration is not expected. In a comparative in vitro cytotoxicity study using human granulocytes, it was found thatpolyester nanoparticles exhibited severe cytotoxic effects at 0.5% concentration, whereas no cytotoxic effects were observed with solid lipid nanoparticles [72]. In vivo toxicitystudies of solid lipid nanoparticles, however, have not yet been published. The possible advantages of solid lipid nanoparticles compared with lipid emulsions or liposomes is their stability against coalescence and the reduced mobility of incorporated drug molecules preventing drug leakage from the carrier. As outlined, careful physicochemical characterization is, however, required to ensure thesolid physical state of the lipid in the colloidal state. A satisfactory long-term stability of lipid suspensions can be achieved considering the results obtained regarding the mechanism of gel formation (cf. Section 1II.C). A general limitation of crystalline solid lipid nanoparticles is, however, represented by the limited inclusion capacity for most drugs, whereas others can be incorporated in high amounts [71]. Drug incorporability might be increased by chemical modificationsof the drug, by controlled crystallization and stabilization of low-ordered crystal polymorphs of the carrier material, or by solidification of the lipid melt in amorphous form, as has already been discussed (cf. Section4II.D). Colloidal solid lipid dispersions represent complex colloidal systems which may contain different types of coexisting colloidal structures such as solid lipid particles, nonsolidified particles of supercooled melt, micelles, mixed micelles, and/or vesicles. Therefore, it has to be considered that the distribution of drug molecules might be influenced by the type and concentration of coexisting structures but also by the incorporation process, physicochemicalcharacteristics of the drug, and theemulsifier. The extremely small particle size of solid lipid nanoparticles, which can be less than 50 nm, might be beneficial with respect to drug targeting. Small carrier size generally favors reduced uptake by the RES. Moreover, intravenously injected particles smaller thanthefenestrations of the endothelial wall, that is, below 150nm, might be ableto leave the vascular compartment through these fenestraein the sinusoids of the liver, spleen, andbone marrow orat locations where the basal membrane of the
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endothelium is damaged; for example, at sites of inflammation or in tumor tissues. Drug targeting might also be possible by surface modification of solid lipid nanoparticles (cf. Section 1I.E). The drugrelease from solid lipid nanoparticles is probably not diffusion controlled as in lipid emulsions owing to thereduced mobility of drug molecules incorporated in the solid matrix. It can be expected that the ratelimiting step of drug release is the degradation of the lipid carrier matrix. Drug release from lipid suspensions should therefore sustained be compared with lipid emulsions. In contrast to polymeric nanoparticles, lipid nanoparticles can be degraded by lipases in the blood. Enzymatic degradation by lipases depends, among others, on the hydrocarbon chain length of the lipids. Hence the degradation rate and drug release can be controlled to a certain extent by the use of differently composed carrier matrices as well as by the choice of emulsifier. Moreover, as has been demonstrated by x-ray diffraction studies [62], it is possible to compose the carrier matrix so it is solid at storage temperature but liquid at body temperature, giving rise to fast drugrelease similar to thatfrom lipid emulsions. Besides parenteral application, solid lipid nanoparticles are also suitable for other routes of administration and might be an interesting carrier system forthe peroral administration of poorly water-soluble drugs with a low peroral bioavailability. An advantage of the emulsified lipid particles might be their improved wettability in gastrointestinal fluids. Whereas the poor absorption of certain drugs can be related to their poor wettability, incorporation into solid lipid nanoparticles provides completely wettable carriers. The lipid particles will probably be digested similarly to food lipids. Because of their high dispersivity, solid lipid nanoparticles exhibit a high specific surface area for enzymatic attack by intestinal lipases. Enzymatic degradation of the lipids leads to the release of incorporated drugs in molecularly dispersed form or solubilized in micelles formed by degradation products and bile salts facilitating their solubilization in the intestine and subsequent absorption. As a concluding remark, it should be emphasized that the use of colloidal drug carriers based on solid lipids is at present-compared with other colloidal systemssuch as polymer nanoparticles, liposomes, and lipid emulsions-at its early phase of the development, although the conceptional idea is not new. The authors have tried their best to present a comprehensive review of what is state of the art regarding solid lipid delivery systems based on own experimental data and experiences as well as on published material of other research groups working inthe field. We would like to draw the readers attention to the fact that much data have unfortunately been published exclusively as conference abstracts. These abstracts generally lack substantial scientific data, since they often only contain a
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brief introduction to the field of research, hypothetical advantages of the proposed delivery systems, and a short statement of what the conference contribution is dealing with. It is very difficult to interpret results from a conference abstract if not even the composition of the studied systems is disclosed. Nevertheless, we have tried to take into account the results extractable from conference proceedings as far as possible. Since in many cases comprehensive scientific publications are yet outstanding, conference abstracts represented the only source of information. Itis hoped that these data will soon be available to thescientific communityas full-length papers in order toelucidate further thepossibilities and limitations of solid lipidbased drug-delivery systems. REFERENCES
1. K. J. Widder, A. E. Senyei, and B. Sears, Experimental methods in cancer therapeutics,J. Pharm. Sci., 71:379 (1982). 2. P. K. Gupta, Drug targeting in cancer chemotherapy: A clinical perspective J. Pharm. Sci.,79:949 (1990). 3. E. Tomlinson, Microsphere delivery systems for drug targeting and controlled release, Int.J. Pharm. Tech. Prod. Mfg., 4:49 (1983). AWmann, R. Gurny, and E. Doelker, Drug-loaded nanoparticles4 . E. Preparation methods and drug targeting issues, Eur. J. Pharm. Biopharm., 39:173 (1993). 5. B. Kante, P. Couvreur, D. Dubois-Krack, et al., Toxicity of polyalkycyanoacrylate nanoparticles. I. Free nanoparticles, J. Pharm. Sci., 71:786 (1982). J. Carpenter,andD.R.Alford,Spontanousfusionof 6. B.R.Lentz,T.
7. 8. 9. 10.
11.
12. 13.
phosphatidylcholine small unilamellar vesicles in the fluid phase, Biochemistry, 265389 (1987). T. M. Allen, Astudy of phospholipid interactions between high-densitylipop teins and small unilamellar vesicles, Biochim. Biophys. Acta, 640:385 (1981). R. E. Counsel1and R. C.Pohland,Lipoproteinsaspotentialside-specific J. Med. Chem., delivery systems for diagnostic and therapeutic agents, 25:1115 (1982). J. M. Shaw,K. V. Shaw, S. Yanovich, et al., Delivery of lipophilic drugs using lipoproteins, Ann. N.Y. Acad. Sci., 74937 (1987). P. C.De Smidt andT. J. C. van Berkel, LDL-mediated drug targeting, Crit. Rev. Ther. Drug Carrier Sys., 7:99 (1990). K. Westesen, A. Gerke, and M.H. J. Koch, Physiochemical characterization of protein-free low density lipoprotein models and influence of drug loading, Pharm. Res., in press (1995). 0 . Schuberth andA.Wretlind,Intravenousinfusions of fatemulsions, phosphatides and emulsifymg agents, Acta Chir. Scand., 278(Suppl.):1(1961). A. Wretlind, Development of fat emulsions, J. Parent. Enteral Nutr., 5:230 (1981).
255
14. L. C. Collins-Gold, R. T. Lyons, L. C. Bartholow, Parenteral emulsionsfor drug delivery, Adv. Drug Deliv. Rev., 5:189 (1990). of oil-in-water emulsions as a vehicle 15. R. J. Prankerd andV. J. Stella, The use for parenteral drug administration, J. Parent. Sci. Technol., 44:139 (1990). 16. B.Magenheim,M. Y. Levy, and S. Benita, A newin vitro technique for evaluation of drug release profile from colloidal carriers-Ultrafiltration technique at low pressure, Int. J. Pharm., 94:115 (1993). 17. R. H. Muller, Colloidal Carriers for Controlled Drug Delivery and Targeting Wissenschaftliche Verlagsgesellschaft, Stuttgart, 1991. 18. B. Siekmann and K. Westesen, Submicron-sized parenteral carrier systems based on solid lipids, Pharm. Pharmacol. Lett., 1:123 (1992). 19. P. Speiser, Lipidnanopellets als Tragersystemfir Arzneimittel zur peroralen 0 167 825 (15.01.86). Anwendung, European Patent Application EP 20. M. J. Robinson, A. Bondi, and J. Swintosky, Sulfamethylthiadiazole: Human blood concentration and urinary excretion data following oral doses, J. Am. Pharm. Assoc., Sci. Ed. 17:814 (1958). 21. C. R. Kowarski, B. Volberger, J. Versanno, and A. Kowarski, A method of preparing sustained release sulfamethazine in small size batches, Am. J. Hosp. Pharm., 21:409 (1964). 22. Y. Kawashima, H. Ohno, and H. Takenaka, Preparation of spherical matrixes ofprolonged-releasedrugsfromliquidsuspension, J. Pharm.Sci.,70:913 (1981). 23. T. Eldem, P . Speiser, and A. Hincal, Optimization of spray-dried and -congealed lipid micropellets and characterization of their surface morphology by scanning electron microscopy, Pharm. Res., 8:47 (1991). 24. T. Eldem, P. Speiser, and A. Hincal, Polymorphic behaviour of splayed lipid micropellets and its evaluation by differential scanning calorimetry and scanning electron microscopy, Pharm. Res., 8:178 (1991). 25. P. Kecht-Wyrsch,HochdisperseGlycerid-MikropartikelalsperoralesArzneitragersystem, Ph.D. thesis, Eidgenossische Technische Hochschule, Zurich, 1987. 26. M. R. Gasco, Method for producing lipid microspheres having a narrow size distribution, U.S. Patent 5,250,236 (05.10.93). 27. M. R. Gasco and S. Morel, Lipospheres from microemulsions, I1 Farmaco, 45:1127 (1990). 28. M. R. Gasco, S. Morel, and R. Carpignano, Optimization of the incorporaJ. Pharm. Biopharm. tion of deoxycorticosterone acetate in lipospheres, Eur. 38:7 (1992). 29. L. Boltri, T. Canal, P. A. Esposito, and F. Carli, Lipid nanoparticles: evaluation of some critical formulation parameters1, Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 20:346 (1993). 30. D. Aquilano, R. Cavalli, and M. R. Gascoj Solid lipospheres obtained from hotmicroemulsionsinthepresenceofdifferentconcentrationsofcosurfactant: The crystallization of stearic acid polymorphs, Thermochim. Acta, 230:29 (1993).
256
31. R. Cavalli, 0. Caputo, and M. R. Gasco, Solid lipospheres of doxorubicin and idarubicin, Int.J. Pharm., 89:R9 (1993). 32. H. Bunjes and K. Westesen, Preparation of solid lipoid nanoparticles by two different methods-A comparison, Eur. J. Pharm. Biopharm., 40 (Suppl.):39 S (1994). 33. M. R. Gasco, R. Cavalli, and M. E. Carlotti, Timolol in lipospheres, Pharmazie, 47:119 (1992). 34. A. J. Domb andM. Maniar, Lipospheres for controlled delivery of substances PCT Patent Application WO 91l07171 (30.05.91). 35. A. J. Domb, Liposphere parenteral delivery system., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 20:121 (1993). S. Anselem, et al.,Characterization of Lipo36. M.Maniar,D.Hannibal, I: Effect of carrier and phospholipid on the loading of drug into the spheresTM lipospheres, Pharm. Res.8 (Suppl.), S-l85 (1991). 37. B. Sjostrom and B. Bergensthhl, Preparation of submicron drug particles in lecithin-stabilizedolwemulsions. I. Modelstudiesof theprecipitation of cholesteryl acetate, Int.J. Pharm., 8853 (1992). of submicron 38. B. Sjostrom, K.Westesen,andB.Bergenstlhl,Preparation drugparticlesinlecithin-stabilized olw emulsions.11. Characterization of cholesteryl acetate particles, Int. J. Pharm., 94:89 (1993). 39. B. Sjostrom, A. Kaplun, Y. Talmon, and B. Cabane, Structures of nanoparticles prepared from oil-in-water emulsions, Pharm. Res., 12:39 (1995). 40. B.SiekmannandK.Westesen,Investigations on solidlipidnanoparticles J., Pharm. Biopharm., in prepared by precipitation in olw emulsions, Eur. press (1995). 41. R. H. Mullerand J. S. Lucks,Dispersionsofsolidlipidnanospheresas sustained-release drug carriers, German Patent Application DE 4131562 (25.03.93). of Bioactive 42. K. Westesen and B. Siekmann, Solid Lipid Particles, Particles AgentsandMethodsfortheManufactureandUseThereof,PCTPatent Application WO 94/20072 (15.09.94). 43. L. W. Phipps, The high pressure dairy homogenizer, College of Estate Management, Reading, UK, 1985, pp. 46-78. 44. P. Walstra, Formationof Emulsions, in Encyclopedia of Emulsion Technology (P. Becher, ed.), Vol. 1, Marcel Dekker, New York, 1983, pp. 57-127. 45. B. Siekmann, Untersuchungen zur Herstellung und zum Rekristallisationsverhalten schmelzemulgierter intravenos appliziebarer Glyceridnanopartikel, Ph.D. thesis, Universityof Braunschweig, Germany, 1995. 46. B. Siekmann and K. Westesen, Melt-homogenized solid lipid nanoparticles I. Preparation and particle size stabilized by the nonionic surfactant tyloxapol. determination, Pharm. Pharmacol. Lett., 3:194 (1994). 47 J. S. Lucks, R. H. Muller, and B. Konig, Solid lipid nanoparticles (SLN)-An alternative parenteral drug delivery system, Eur. J. Pharm. Biopharm., 38(Suppl.):33S (1992). 48. C. Schwartz, W . Mehnert, J. S. Lucks, and R. H. Muller, Solid lipid
257
49.
50.
51. 52. 53. 54.

55.
56. 57.
58.
59.
60.
61. 62. 63.
64.
65.
nanoparticles (SLN) for controlled drug delivery. I. Production characterization and sterilization,J. Control. Rel.,30:83 (1994). K. Westesen and H. Bunjes, Do nanoparticles prepared from lipids solidat roomtemperaturesalwayspossessasolidlipidmatrix?, Int. J. Pharm., 115:129 (1995). D. H. Everettt, International Unionof Pure and Applied Chemistry, Manual on Colloids and Surface Science, Pure Appl. Chem., 31577 (1972). A. zur Muhlen, J. S. Lucks, W. Mehnert, and R.H. Muller, Longterm stabilJ. ity of solid lipid nanoparticles (SLN) for parenteral application, Eur. Pharm. Biopharm., 4O(Suppl.):53S (1994). .S. Lucks, and R. H. Muller, Effect of storage conditions on longC. Freitas,J term stabilityof solid lipid nanoparticles (SLN) in aqueous dispersion, Eur. J. Pharm. Sci., 2:178 (1994). H. Muller, Stabilizationofsolidlipid C.Freitas, W . Mehnert,andR. nanoparticles (SLN) by spray drying, Eur. J. Pharm. Biopharm., 4O(Suppl.):29S (1994). C.Schwarz, W. Mehnert, and R. H. Muller,Lyophilizationofsolidlipid nanoparticles (SLN), Eur.J. Pharm. Sci., 2:177 (1994). B. Siekmann and K. Westesen, Melt-homogenized solid lipid nanoparticles stabilized by the nonionic surfactant tyloxapol. 11. Physicochemical characterization and lyophilisation, Pharm. Pharmacol. Lett., 3:225 (1994). S. MaaBen, H. Weyhers, W. Mehnert, and R. H. Muller, Surface-modified solid lipid nanoparticles (SLN)-In vitro affinity to human granulocytes, Eur. J. Pharm. Biopharm., 4O(Suppl.):41S (1994). W. Skoda and M. van den Tempel, Crystallization of emulsified triglycerides, J. Colloid Sci., 18568 (1963). N. Garti, E. Wellner, andS. Sang, Crystal structure modifications of tristearin by food emulsifiers, J. Am. Oil Chem. Soc., 59:181 (1982). N. Garti and K. Sato, Crystallization and Polymorphism of Fats and Fatty Acids., Marcel Dekker, New York, 1991. K. Larsson,Classificationofglyceridecrystalforms,ActaChem.Scand., 20:2255 (1966). K. Thoma, P. Serno, and D. Precht, RontgendiffraktometrischerNachweis der Polymorphie von Hartfett, Pharm. Ind., 45:420 (1983). K. Westesen, B. Siekmann, and M. H. J. Koch, Investigationson the physical J. state of lipid nanoparticles by synchrotron radiation X-ray diffraction, Int. Pharm., 93:189 (1993). B. Siekmann and K. Westesen, Thermoanalysis of the recrystallization proB: Biocess of melt-homogenized glyceride nanoparticles, Colloids Surfaces interfaces, 3:159 (1994). Vol. 1, OxfordUniversity R. J. Hunter,Foundations ofColloidScience, Press, Oxford, UK, 1986. J. S. Aronhime, S. Sarig,and N. Garti,Dynamiccontrolofpolymorphic transformation in triglycerides by surfactants: The button syndrome, J. Am. Oil Chem. Soc., 651144 (1988).
258 Siekmann
66.
and
Westesen
67. 68. 69. 70.
71. 7 2 .
K. Westesen and B. Siekmann, Investigation on the gel formation phenomenon of phospholipid stabilized solid lipid nanoparticles, submitted to Int. J . Pharm . (1995). B. Siekmann and K. Westesen, Elecron-microscopic characterization of melthomogenized solid lipid nanoparticles, Eur. J. Pharm. Sci., 2:190 (1994). W. Skoda and M. van den Tempel, Growth kinetics of triglyceride crystals, J. Crystal Growth, 1:207 (1967). W. Skoda, L. L. Hoekstra, T. C. van Soest, et al., Structure and morphology of p crystals of glyceryl tristearate, Kolloid Z.Z.Polym., 219:149 (1967). C. Schwarz, W. Mehnert, and R. H. Muller, Influence of drug incorporation and lyophilisation on the physical stabilityof solid lipid nanoparticles(SLN), Eur. J .Pharm. Biopharm., 4O(Suppl.):52S (1994). H. Bunjes, K. Westesen, and M. H. J. Koch, Incorporation of ubidecarenone in solid lipid nanoparticles, Eur. J. Pharm. Sci., 2:177 (1994). S. MaaBen, C. Schwarz, W. Mehnert, et al., Comparison of cytotoxicity between polyester nanoparticles and solid lipid nanoparticles (SLN), Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 20:490 (1993).
l0
Pharmaceutical Aspects of Liposomes: Perspectives in, and Integrationof, Academic andIndustrial Research and Development
Rimona Mat'galit and Noga Yerushalmi
Tel Aviv University, Tel Aviv, Israel
I.
Introduction
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11. Liposomes: Definition, Needs for, and Outline of Their Advantages and Drawbacks A. Liposomes: A Family of Structurally Related Microparticles B. Deficiencies of Treatment with Free Drugs that Justify the Pursuit of Drug Delivery Systems and Consideration of Liposomes for the Task 1 1 1 . Selection of Liposome me/Species: Views and Criteria from Academic and Industrial Research and Their Proposed Integration A. Liposome m e , Size, and Production Issues B. Lipid Composition
IV. Targetedmodified Liposomes: An Interesting and Exciting Scientific Tool, But Can They Be Made into Products (Especially Immunoliposomes)? A. Targeting: Definition, Stages, and Status B. The Means Through Which Liposome Targeting Has Been Attempted and Their Feasibility for Liposomes as Pharmaceutical Products
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Margalit and Yerushalmi
V. Liposomes as a Sterile, Pyrogen-Free System with Pharmaceutically Acceptable Shelf Life, Stability, and Dosage Sterile, A. Pyrogen-Free 277 Liposomes B. Shelf Life, Stability, and Dosage Forms
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VI. Liposome Characteristics (Percentage of Encapsulation, Kinetics of Release, Biological Activity)-in Basic Research and in Quality Assurance A. Efficiency of Drug Encapsulation B. Kinetics Release of Drug C. Biological Activities of Liposome-Encapsulated Drugs
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VII. Summary and Future Prospects
1.
INTRODUCTION
The scientific literature is rich withcomprehensive reviews of liposomes as drug-delivery systems (DDS) (see, e.g., refs. 1-11). Each review presents a unique and specific point of view, whichcan range from the strictly physicochemical, through various biological levels, to the clinical. Furthermore, stemming from the application for which theyare reviewed, the prospectof liposomes as pharmaceutical products is inherently presentin these reviews even when not specifically identified and addressed. Onthis background, it was deemed essential to first address aquestion that might be raised by the reader, which is why yet another manuscript on liposomes as pharmaceutical products. Liposomes as DDS are among research topics that are being vigorously investigated in both academic and industrial laboratories, with different outlooksbut common goals and endproducts.Addressing fundamental questions while being continuously innovative are preceived to be among the responsibilities of the academician in this field. Addressing developmental issues such asscale-up, shelf life, dosage forms, long-term stability, and cost-effective production are perceived to be among the responsibilities of the industrial scientists. Yet the R&D of liposomes as DDS encompass industry-oriented issues that should already be taken into account at the basic academic stage, as well as questions that arise at the industrial stage that have distinct basic science components. Stemming from this premise, the objectives of this chapter are to address those issues and questions and attempt to provide an integrated view that would (1)be of use and contribute to the mutual understanding of
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the topics most critical to investigators in both industry and universities and (2) serve as guidelines for newcomers to thefield. It is hoped that, through this, this chapter will become a modest addition to, rather than a repetition of, the existing literature. In order to lay out a common groundfor the discussion of liposomes as DDS, the first part of this chapter provides a brief definition of liposomes and discusses the unmet therapeutic needs that justify their pursuit The subsequent partsof this as DDS and their advantages and drawbacks. chapter attempts toprovide the proposed integrated approach, addressing the following issues: (1) types of liposomes, with respect to scale-up, production, and cost; (2) preparation and production of sterile and pyrogenfree liposomes; (3) shelf life, stability, anddosage forms; (4) targeted (mostly surface-modified) liposomes as exciting research tools and as pharmaceutical products; and (5) characterization of liposome-drug systems (such as encapsulation efficiencies, drug-release kinetics, and biological activity) for academic studies and for industrial quality assurance. The final part will offer conclusions, taken from theposition of an academician, but hopefully relevant for all liposome investigators. The authors are well aware thatthe line of division between academic and industrial liposomal R&D is quite often indistinct. Investigators in academics research could be engaged in questions that bear on liposomes as pharmaceutical products, whereas, more so, investigators in industry could be engaged in basic liposome research. In order toavoid any confusion or resultant grievances (however unintentional), it is stressed here that throughout this chapter the termsacademic and industrial research will be used to indicate the nature and tendency of the research (i.e., basic or applied) and not the physical definition of the environment or community where it is performed.
II. LIPOSOMES: DEFINITION, NEEDS FOR, AND OUTLINE OF THEIR ADVANTAGES AND DRAWBACKS A. Liposomes: A Family of Structurally Related Microparticles
Liposomes, frontline microparticulate carriers investigated and developed for the task of drug delivery, are artificial microscopic and submicroscopic particles made of lipids and water alone that were developed thirty years ago. [l].A variety of liposome species with respect to shape, type, size, and composition have been developed in the course of the last three decades, extending this type of particle into a family of structurally related delivery systems. For the novice in the field, it is worth noting that in the
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early days of liposomes, there was an ongoing debate between the terms vesicle and liposome, the legacy of which ispresent in the names, abbreviations, and acronyms used for various liposome types. Multilamellar vesicles (MLV) are the oldest liposome species, and they have been described frequently enough [1,2,5-7,12, and references therein] that only a brief summary is merited here. They are composed of concentric shells of lipid bilayers, with water between shells and an inner aqueous core. Liposomes of this type form spontaneously on the proper interaction of lipids with water, provided the right choice of lipids and technical conditions have been met. A typical MLV will have 8-15 concentric shells, and run to sizes that can range from (roughly) 0.5 to several micrometers in diameter. Consequently, a typical MLV preparation will be quite heterogeneousin terms of liposome sizes. Unilamellar vesicles (ULV) are composed, as their name indicates, of a single lipid bilayer and an inner aqueous core [1,2,6,7,8,10,11,13,14, and references therein]. Depending on the method of preparation, ULV can be made in various size ranges, from as small as 20 nm to several micrometers in diameter, with a significantly smaller size distribution within a preparation comparedwith MLV. Both MLV and ULV can be made from a wide (although not infinite) range of lipid and lipid mixtures [l-161 and can accommodate hydrophilic and hydrophobic drugs in their aqueous and lipid compartments, respectively. Furthermore, with careful selection of liposome type, encapsulation of matter that is as small as the lithium ion up to macromolecules as large as genetic material (of several hundred thousand daltons) can be achieved. Scanning the threedecades of liposome literature makes it quite clear that besides the abbreviationsMLV and ULV, there aremany more names and types of liposomes the distinction among and the classification of which are frequently based onmethodsand/or devices of preparation with/ without contribution of the resultant size-related properties. In fact, this horn of plenty can be a sourceof confusion to thenovice in the field and a burden on both veterans and newcomers, as exemplifiedby the two cases outlined below. The first case concerns liposomes that have been classified as large unilamellar vesicles (LUV). Oneof the major methods forproducing such liposomes is the reverse-phase evaporation technique[13]; such liposomes have been abbreviated REV and are usually classified as LUV. A REV preparation can have a relatively wide size distribution reaching the range of 600 nm in diameter. Subjected, as frequently recommended, to postprep aration fractionation steps, the size distribution of an REV preparation can be quite narrow together with a reduction in the dominant size. Yet, regard-
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less of whether such fractionation has or has notbeensubjected to postpreparation steps, these sytems will all be referred to as REV and considered to be LUV. Other approaches for producing unilamellar liposomes are based on extrusion devices, such as the LIPEX extruder [18]. Liposomes produced with this device have been named LUVET and are also referred to as LUV; typical preparations of this type have a relatively 30 to several hundred narrow size distribution. Sizewise, they can run from nanometers in diameter, depending on the pore sizes of the filters used. The second case concerns SUV, which is the the acronym for small unilamellar vesicles as well as for sonicated unilamellar vesicles. SUV with a size range of 20-nmdiameters are usually obtained through the use of probe sonicators and are one of the oldest types of liposomes. One also finds the acronym SUV in usefor unilamellar liposomes that are in sizeranges of 40, 60, and even up to 100 that have been made by a variety of methods other than probesonicators. Yet another device for the production of unilamellar liposomes is the Microfluidizer [19], which yields (depending on operating conditions) liposome preparations with a narrow size distribution that (de0 0to pending onoperating conditions) can range in diameter from under1 several hundred nanometers.Named MEL, for microemulsified liposomes, such liposomes can qualify as either SUV or LUV. Besides the multiplicity in the field with respect to liposome names and classifications that these few examples illustrate, there is also an inherent weakness in using methods and devices for liposome preparation as major criteria for classification. Clearly some methods and devices can become obsolete and othershave not yet been invented. In an attempt to ease the current liposome namehype situation, and atthe sametime offer an informative classification, a simplified and straightforward two-tier approach thateliminates references to preparation methods and/or devices ishereby proposed.This two-tier approach is used throughout this chapter with the hope thatit will find favor with and be of use to both veterans and novices in the field. The first tier, which is the primary classification, is based on a major structural difference between liposomespecies which is simplythe number f division is set between one and more than one of lamellae. The line o lamellae. On this premise, there areonly two major types of liposomes, for which there is no need to use names other thanthe traditional terms MLV and ULV. Liposome preparations, whether MLV or ULV, might also contain a (usually small) share of liposomes denoted oligolamellar that have few, such as two to three, lamellae. These are considered to be a minor type of liposomes, as to date there has been neither reports on applications for which such liposomes are specifically sought, nor procedures for their preparation as the major dominant particle.
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The second tier, which will be illustrated below by several examples, is a subclassification according to the dominant size range and/or size distribution, whichever is the more relevant. For example,in a reported study, the investigated liposome type would be simply classified as ULV 80 f 30 nm. These details would allow the reader infer to (1) that this preparation is quite homogeneous and truly unilamellar and (2) that the majority of these so that, if endowed by proper receptor affinliposomes are sufficiently small ity, they canalso be endocytosed by nonphagocyticcells through the coated pit mechanism [7].Identifying an investigated liposome preparation ULV as 650 f 200 nm would be a clear indication to the reader that this preparation probably contains not only unilamellar but also olgiomallelar liposomes (i.e., liposomeswith more than one lamellae but less that the number designated todefine MLV). A preparation identified as MLV 0.5-2.5 pm would clearly indicate that this is a rather heterogeneous liposome preparation tha has probably not beensubjected to any fractionation procedures and contains particles that candiffer fivefold in diameter. Besides offering simplification and being informative, it is argued that this classification will leave descriptive terms such as small or large to the personal inclination of the investigators and the readers, and it will help clarify differences between liposomes and other lipid-based particles that are under development as delivery systems. The latter are, for the most, proprietary technologies such as plurilamellar liposomes that are reported 0 0pm [20]; to contain well over 100 lamellae and to range in size up to 1 multivesicular liposomes (DepoFoam), also referred to as MLV, each particle of which consists of several wall-sharing large unilamellar liposomes, with a size that can reach 100 pm [21]; solvent dilution microcamers (SDMC) [22-231; and transferosomes [24-251 developed specifically for the transdermal administration to intact skin.
B. Deficiencies of Treatment with Free Drugs that Justify the Pursuit of Drug Delivery Systems and Consideration of Liposomes for the Task
Whether viewed from the clinical, the physiological, the financial, or any other of the many aspects involved, treatment with drug-loaded delivery systems is substantially more complex that with free drug. Therefore, investment in research and developmentof liposomes* has to be justified, on one hand, on the existence of therapeutic needs that are truly unmet with
*Although most of the issues discussed with respect to the needs for liposomes apply to otherdrug-deliverysystem also, the discussion will refer to liposomes alone, asthey are the topicof this chapter.
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the free drug and, on the other hand, by the ability of the liposomes to provide significant improvements in clinical outcomes that can be developed into established treatment modalities. Tracing the fate of a drug administered in its free form, dissolved or dispersed in an appropriatevehicle, can provide a glimpse of those unmet needs thatjustify the pursuit of liposomes as DDS. Exampleswill be given below for two cases, from which the general picture can be inferred, not only for systemic but also for nonsystemic (such as topical and regional) administrations. The first example is taken from tumor treatment by chemotherapy, where the deficiences of treatment with free drug come from the effects of the drug on the biological environment and vice versa. It is well known that conventional chemotherapeutic drugs, such as doxorubicin, vinblastine, vincristiine, 5-flourouracil (5-FU), and many others [26] are virulent poisonous substances, a property which is an asset with respect to drug effects on malignant cells but is disastrous when the drug interacts with any other (healthy) cells. Most frequently, such drugs are administred in their free form systemically. Lack of targeting to the tumor sites results in the indiscriminate distribution of the drug over the whole body, reducing the efficacy of the treatment and at the same time leading to undesirable side effects and toxicity. Free-form administration of drugs exposes themto the elements of the biological environment, and they are often prematurely cleared, which is especially critical for drugs that act at a specific stage of the cell cycle. The drugs (in free form) are also susceptible to inactivation and/or degradation and to scavenging by endogenous carriers such as serum albumin and serum lipoproteins, which can aid as well as abet drug access to the target zones. The second example concerns wound healing, where growth factors, such as epidermal growth factor (EGF), fibroblast growth factors a and b (FGF-a), (FGF-b), platelet-derived growth (PDGF), transforming growth TGF-P), andmany others, are under development factors (U and P (TGF-(U, as topical therapeutic agents for the acceleration and stimulation of the self-healing process in wounds and bums [27-351. Current dosage forms used in both basic and developmental growth factor studies that are appropriate for the future treatment of patients center on free growth factor in vehicles such as solutions of saline or other physiological buffers (poured onto thewound or soaked into a gauze dressing), cellulose gels, and collagen sponges[27-381. The use of such vehiclesand procedures corresponds to theimmediate (or almost immediate) exposure of the total growth factor dose to the wound. As in the previous example, the mutual effects of the drug and of the biological environment underminesuccessful therapy with the freedrug.
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Growth factors are especially vulnerable to the biological environment, which is enzymatically hostile to polypeptides. In addition to the enzyme-catalyzeddegradation, growth factors that are free in the wound are subject to continuous clearance from the wound area and can be scavenge through binding to thevarious particulate and soluble wound fluid components. Owing to their susceptibilityto the proteolysis, the growth factors are also prevented (for the most part) from exerting any self-targeting within the wound area toward their sites of action (i.e., their specific receptors). The small share of the dose that doesreach the target form is often too low to affect a significant difference, especially as growth factors are agents that act at a specific stage of the cell cycle [39-411. Hence, effective therapy also requires a continuous supply of active growth factor near enoughto its sites of action for a sufficient duration [29,31,40]. The other side of the coin, namely, the detrimental effects of growth factors on the biological system, are seen on attempts to increase the bioavailability at the target by dose escalation. It is well known, and not only for growth factors but for hormones in general, that substantially high (local) concentrations, even if transient, can result in adverse effects and toxicity [31,39]. In order towrest some benefit from the application of growth factors despite the deficiencies described above, current treatment regimens use multiple dosing at frequent intervals; for example, twice daily for 70-100 days [41]. Such regimens prevent the implementation of state of the art (1)minimal interferapproaches to good wound management that advocate ence with the wound; (2) maintenance of a moise wound environment, which wasfound to be most conducive to the self healing; and (3) the use of occlusive dressings that are changed once every few days [43-501. Obviously, treatment regimens that interfere with the healing will detract from any benefit that a medication for heating can provide. Although the deficiencies of free-drug administration, defined above, clearly call out for ways and means to overcome them and in principle a delivery system is among the most viable solutions, such systems need to meet particular attributes in order toqualify for the task. These attributes can be divided into three categories. The first two categories have been amply discussed, are considered to be within the realmof basic science, and comprise qualities that the carrier needs in order to function (denoted category I) and in order to avoid the situation of replacing one set of problems by another (denoted category 11). The third (denoted category 111) comprises qualities required in order to develop the drug-carrier systems into pharmaceutical products that can then be implementedestabinto lished treatment modalities. Properties included in category I are (1) the ability to provide mutual biological environment; (2) effective protection of the carried drug and the drug targeting, including the ability to reach and access the targetzone; (3)
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stability on route from the site of administration to the site(s) of drug action; and (4) the ability to perform as a sustained-release drug depot. In order to meet the attributes make that upcategory 11, the carrier should be biocompatible, biodegradable, nonimmunogenic, and nontoxic. A major share of the qualities that are dominant in category I11 have to dowith the production of the drug-carrier systems. The carrier should be of the type that its preparation prccedures (including drug loading) can be scaled up, yield a sterile and pyrogen-free product, and meet the requirements of quality assurance. Another major part of category I11 properties concerns postproduction requirements, such as dosage forms that can provide longterm shelf life and high stability. Needless to say, in order to ensure that as wide as possible patient population could have access to this new therapy, all cost-effective issues should be considered within category 111, be they the cost of production itself, sources and cost of raw materials, shelf life length, expenses of storage, and others. Among drug-delivery systems that can address a major share of the unmet needs encountered when treatments are with free drug, liposomes are frontline candidates. Their advantages for the task have been well documented and include the following: Liposomes can meet that required mutual drughiological environment protection. Liposomes are biocompatible and biodegradable and, to a significant extent, are also nonimmunogenic and nontoxic. Their versatility in terms of type, size, and lipid compositions allows the encapsulation and delivery of a wide range of drug species, and liposomes can act as sustained-release depots. Despite all these qualities, significant problems in the implementation of therapies with liposomes still exist, particularly with respect to targeting; theresponses of the biological system to liposomes in the blood stream; the stability of both particle and drug in vivo; the in vivo rates of drug release; shifts in rather than prevention of drug toxicity; and long-range effects of chronic liposome administration. The advantages of liposomes as well as these unresolved issues are addressed in the following sections, taking into consideration not only these category I 1 1that are critical for the evolution and I1 aspects but also those of category 1 of liposomes from entities under research and development into entities that are pharmaceutical products.
SELECTION OF THE LIPOSOME TYPUSPECIES: VIEWS AND CRITERIAFROM ACADEMIC AND INDUSTRIAL RESEARCH AND THEIR PROPOSED INTEGRATION
111.
A. Liposome Type, Size, and Production Issues
One of the initial decisions that need to be made on venturing into the development of a liposomal drug-delivery system for a specific therapeutic
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case centers on the type or types of liposomes that will be explored. Whetherthat selection will bemadefrom the abundance of existing liposome species or theinvention of new liposome species is contemplated, the criteria and considerations that are used to make that decision constiof the majorpoints of diversion between investigators in academic tute one and industrial research. The selection of liposome types in industry will follow, in general, guidelines for pharmaceutical products, and would be relatively free from the limitations of accessibility to devices for liposome production. The main concerns would be production and product issues. As discussed under category I11 (see Section II.B), a critical criterion would be a liposome production method/procedure thatcould be scaled up and executed under GMP rules and regulations. Other critical criteria would stem from the requirements for a product that would have batch-to-batch reproducibility andbestable, sterile, and pyrogen free. As also discussed above, the liposome production method had to be cost effective, also taking into consideration sources, supplies, and cost of quality raw materials. Cost and environmental considerations would also take into account the issue of waste disposal, in particular for those methods of liposome production that involve the use of organic solvents. Needless to say, the selection of the liposome type would also take into consideration the need for acceptable shelf life and dosageforms. The academician is perceived to have a considerably higher level of freedom, being able to choose, in principle, from all types of liposomes for which procedures of preparation are in existence. Physiological and clinical aspects the drive selection criteria. These include the specific therapy in question, drug properties, the route of liposome administration with all its inherent issues, and the mutual effects of the drug and the biological system. However, as exemplified by the following case, other criteria often become deciding factors. Liposomes as delivery systems have become common andprevalent enough that the situation in which an investigator with expertise and a primary interest in using a particular drug-for example, a 6000-d polypeptide-seeks liposomes asa delivery system for it. Screening the horn of plenty with respect to liposome types and species, certain dogmas in the field might drive that investigator to exclude from consideration MLV, although these liposomes might well fit his therapeutic objectives and the other requirements. Concerns of stability and retention of the native active conformation could lead that investigator to also exclude ULV made by methods that bring the encapsulated polypeptide in contact with organic solvents or detergents. If costly devices for making ULV of sufficiently large size range are unavailable because of financial limitations, the investigator might end up selecting small ULV (20 nm in diameter) made with a
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probe sonicator. This selection can posesevere limitations on theefficiency of encapsulation and on other properties of the system that can result in low efficacy of treatment that would discourage the investigator fromany further pursuit of the liposomal approach. Even if those small ULV would prove to be satisfactory with respect to physicochemical properties and successfully functional in vitro and/or in vivo, the questionof whether they could be developed into apharmaceutical product would stillbe faced. The same situationwould have to be faced withnumerous otherliposome types that are currently used in academic research, such asthose madeby reverse phase evaporation, ethanol injection, detergent dialysis, French press, detergent removal through gel exclusion chromotography, and others [2,3,57,12-14,18-191. It is stressed that for objectives other than drug delivery, especially those where liposomes serve as modelsof cell membranes or as a solubilization media for lipophylic matter, most of the limitations discussed in this section are of no consequence. However, cases in which efforts and resources have been invested in developing liposomal drug systems that prove to be successful in animal model studies and even in clinical trials, with liposome types that cannot be produced as pharmaceutical products, are not far fetched. For such cases, the progress of that system into an established treatment modality would be (at best) significantly delayed until similar positive results would be obtained with another, productsuitable liposome type. Needless to say, a major share of the already done studies would have to be repeated. The clear conclusion that emerges from the issues discussed in this section is that, in the selection of liposome type, the burden of change in current considerations is mostly on the shoulder of the academician. It is proposed that the academician integrate the industrial criteria into his or her initial selection process, even if the requirements of a planned study could be accomplished withthe use of small-scale, nonsterile, preparations that would be discarded at the end of a days work. A few examples of implementing the integrated view are offered below. A comprehensive list of selected liposome types is deemed to be beyond the scope of this chapter. Thisis due not only to the abundance of liposome production methods but also to the realization (already introduced in the previous section) that this is a dynamic field where new species and/or production methodologies are expected tobe continuously invented. The first example concerns MLV,* that have fallen out of favor with
*Other properties that might favor MLV will be discussed in subsequent sections of this chapter, as will be the already mentioned drawback due to the sue heterogeneity and others issuesin this vein.
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many investigators. Granted, there definitely are treatments and applications for which MLV would not do. For treatments of tumors andinfectious diseases in which the targets are outside the RES and where intravenous administration with long-term retention of the liposomes in circulation is required. On the other hand, MLV can be eminently suitable for some therapies that require nonsystemic administration as well as for therapies that requireintravenous administration and fast uptake of the liposomes by phagocytic cells in circulation. MLV can be easily made in the laboratory and canalso meet thecriteria for pharmaceutical products. The latter is not meant to imply that all industrial issues with respect to MLV production have beenresolved, but thattheir resolution is feasible. A pertinent example of a current problem that needs resolution is the already mentioned issue of organic solvents, such as methanol,chloroform, dichloromethane, Freon, and similar ones, that are routinely used in MLV production. The use of such organic solvents is currently critical for proper MLV production. They are applied for the dissolution and optimal mixing of the different lipids that make up the formulation and of those drugs that are introduced through the lipid phase. They also make a major contribution to the attainment of the form and dispersion of dry lipids for optimal contact with the swelling solution. On the other hand, the specific solvents used present a problem ontheir own, both in the industrial and the academic laboratory level, as regulatory authorities phase more and morepossible solvents out of use. Owing to the environmental and economic issues involved, waste disposal of these solvents, which could accumulate to substantial quantities in industrial-scale MLV production, is another cause for concern. Clearly, if MLV or any of the many types of ULV.for which MLV are the source material are to be used, other meanswould be required to still produce the MLV, but with an altogether elimination of the need for organic solvents or their replacementby lesshazardous materials that could be used with much lower quantities. Based on the issues discussed above, we propose that MLV should berevisited. The second example is the case in which, based on the therapeutic objectives, MLV are ruled out and it has been determined that the optimal ULV should have a dominant size of 200 nm diameter. The many ways by which such liposomes are produced can be divided into two categories: The first is device oriented, using as source material MLV that are subject to extrusion, homogenization, or microemulsification.devices in order to transform them into thedesired ULV. The second category includes methods such as reverse phase evaporation, ethanol injection, detergent dialysis, detergent removal through gel exclusion chromotography, and others (2,3,5-7,12-'14,18-19). In this category, the start materials are usually multicomponent systems that include lipids, drug, water, and an organic
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solvent or detergent.Despite the relatively ease and distinct lower cost of producing liposomes by methods of the second category, even assuming the contact between drug and organic solvent or detergent is not a limiting factor, it is argued here that the first category is the better choice. The ability to scale-up procedures of the second category is questionable and,at best, might need investment for itsdevelopment while scaled-up models of the first category devices are already in existence. The issue of organic solvents and waste has already been discussed above for MLV, but unlike the case of MLV, it is questionable if the critical roles of the organic solvents/detergents in methods of the second category can be eliminated or even reduced in quantity (especially relative to the other components of the system), The recommendation here for the selection of ULV made by the device-oriented methods is not meant to ignore the cost limitations. Rather it is argued that ULV of a specified size range that are producedby either type device should be sufficiently similar, especially as all use the same source material (i.e., MLV). With the right choice, this should make it possible to perform the basic stages of a study with one device and proceed to theindustrial R&Dlevels withanother without significant changes in the liposomal properties. For example, it should be possible for the academician to use small relatively inexpensive extrusion devices (as well as design and build one) and use for the industrial stage a microemulsifier operated under conditions that would give the same size liposomes under similar temperature, buffer, and other experimental parameters.
B. Lipid Composition
The source and cost of lipids have been concerns for liposomologists in both academic and industrial research.For the major run-of-the-mill lipids usedinmost liposome formulations, such as phosphatidyl choline, the increase in available sources has somewhat eased those concerns. This trend can be expected to continueand grow as more liposomes will successfully complete clinical trials and move into the arena of products, making the supply of high-purity lipids a viable business venture. The situation is different when it comes to the more rare lipids. The motivation for incorporating relatively rare lipids into a liposome formulation comes from the need to endow the liposomes with unique properties that will take into account the natureof the drug in question and address specific demands of the intended therapy. The perceived freedom of the academician in the selection of liposome types might also be extended into selection of lipid composition, but in reality, the paucity of source materials for uncommon lipids together with their high cost might negate theiruse at the pharmaceutical product stage. Thus,even at the basic stage, a balancewould have to
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be struck between the liposome formulation and the properties to be endowed by specific lipids.In general, the integrated approach would dictate giving up that singular lipid and finding alternative means to endow the it should be liposomes with the samedesired attribute. On the other hand, borne in mind and taken into consideration that basic studies might offer unanticipated solutions to particularly difficult pathological situations that would justify even exceptionally high costs. The evolution within the Stealth (a registered trade name of Liposome Technology, Inc., Menlo Park, CA)liposomes is an instructive example. The unique feature of these liposomes is their ability to delay/evade uptake by the reticuloendothelial system (RES) for longer periods than regular liposomes of similar size range. This allows for prolonged retention in circulation and through that higher shares of the dose can reach the non-RES targets. The first generation of Stealth liposomes required a specific ganglioside in their formulation [51]. Subsequent generations of Stealth liposomes have seen the move from that lipid to the relatively less rare regular and hydrogenated phosphatidyl inositol and specific hydrogenated phosphatidyl choline-cholesterol mixtures [52], ending up in formulations that do not require rareand/or modified lipids at all. In the current version of these liposomes, the stealth property is achieved through surface modification by polyethylene glycols [53]. The extent to which elimination of the dependence on rare lipids has been a driving force in this evolution has not been specified byits developers. Yet, even if considerations of lipid raw materials were not a major driving force for this particular evolution, this case shows that such considerations can be accommodated without giving up on desirable formulation-dependent liposomal features.
IV. TARGETED/MODIFIEDLIPOSOMES:ANINTERESTING AND EXCITING SCIENTIFIC TOOL, BUT CAN THEY BE MADE INTO PRODUCTS (ESPECIALLY IMMUNOLIPOSOMES)?
The ability to be targeted to the sites of drug action, and to them alone, is among the critical properties required of liposomes. It is well known that despite enormous investigative efforts, the achievement of targeting that would be highly effective in vivoand applicable to a wide range of therapeutic objectives is still an elusive goal. As will be discussed below, complete targeting has not been achieved even in some of those cases defined as passive targeting, where the targets are within the RES [54]. Despite the frequent appearanceof the term targeting in liposome research and literature, its very prolific use has somewhat blurred it into meaning different
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things to different people. Owing to this current state of affairs, it was deemed advantageous to offer here a brief discussion and definition of targeting prior to a discussion of the means by which liposome targeting is attempted, and their evaluation from both the academic and industrial points of view.
A. Targeting: Definition, Stages, and Status

It is offered here that liposome (or any other drug-delivery system) targeting can beviewed as a two-tier process, the first of which takes place on the introduction of liposomes into a living system, and thus is termed here targeting at theorgan level. The objectives and efforts within this tier are focused on getting the drug-loaded liposomes from site(s) the of administration to the orgadtissuehicinity where the state(s) of disease reside. The second tier, termed targeting at the cellular level, is a form of fine tuning. It centers on pinpointing the drug-loaded liposomes, on their arrival at thatlocation, as close as possible to the actual sites of drug action. It should be emphasized that in order toavoid interference with the therapeutic activity, the liposomes might need to be placed close to-but not at-the sites of drug action. Thus, targeting at thecellular level can involve two distinct types of sites: sites at which the liposomes reside, which willbe termed camer sites, and those at which the drugbinds in order to initiate its therapeutic effect, which will be termed drugsites. The targeting capabilities demanded of liposomes for therapies that require systemic administration depend to a large extent on the locations of the sites of drug action. For ultimate targeting to locations outside the RES, the liposomes need capabilities to address the needs of both targeting tiers. For targeting to locations within the RES, the liposomes need to provide the second tier alone, as thefirst one is provided forby the biological system itself. For therapies thatdonot require or are preferably achieved by nonsystemic drug administration, the situation is somewhat similar to RES targets; that is, the liposomes haveto provide only for the second tier of targeting. In contrast to the previous case, in the nonsystemic therapies, the first tier is achieved (or one could say eliminated) by the mere selection of the route of administration. Included in such therapies would be topical drug administration in the treatment of wounds, bums, and ocular conditions and regional drug administrations such as intraperitoneal infusions and aerosol inhalations for treatment of disorders in the peritoneal cavity and in the lungs, respectively [55-581. Liposomal specifications for the achievement of the first tier of targeting in systemic applications for non-RES targets have been comprehensively and extensively reviewed together with the inherentphysiological and ana-
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tomical obstacles [2-12,51-54, and references therein]. In an effort to refrain from redundancy, the reader is referred to these referenced sources and the discussion below is focused on the less-addressed specifications for achievement of the second tier. The basic premise is that once the liposome arrives at, oris introduced into, theorgadtissueAocation where the state(s) of disease reside, effective targeting at the cellular level requires that the liposome adhere with high affinity to its designated carrier sites and be retained there, despite cellular and fluid dynamics,for a sufficient duration in order torelease an effective drug dosefor the time spandictated by the specifics ofdrug and disease. Ideally, the liposome should also be instrumental in aiding drug access to sites of drug action. Tailoring liposomes to this end should be done with care andis rational for those cases where detailed knowledge of the cellular or tissue location of the drug site is available. Obviously, for intracellular sites of drug action, liposome internalization would be beneficial, but it is not an absolute necessity. For sites of drug action that are extracellular, such as certain membrane-embedded receptors or matrix-residing bacterial colonies, liposome internalization would defeat the purpose.
B. The Means Through Which Liposome Targeting Has Been Attempted and Their Feasibility for Liposomes as Pharmaceutical Products
The main efforts for endowing liposomes withtargeting abilities have been focused on liposomal features. Other avenues that explore treatment regimens such as liposome dose size and frequency of administration or pretreatment with blank liposomes have enjoyed less attention. With respect to liposomal features, the two main avenues explored were liposome specifications such as size, type, and lipid composition and liposome surface modification. The attempts to confer a targeting ability on liposomes through manipulation of their specifications, provided they do notstray from the integrated criteria for selection of liposome types discussed in the previous section, will continue to result in liposomes that can be developed into pharmaceutical products. Some success has been found in this avenue of exploration, as shown recently by preferential accumulation of small daunomycin-encapsulatingliposomes in solid tumors, althoughthe operating mechanism has yet been elucidated. [59]. It has been recently shown that small stealth liposomes accumulate, apparently by entrapment, in the intracellular spaces within solid tumors [60]and are retained within those spaces for a sufficient duration to allow drug to diffuse from the liposomes .into the tumor cells.
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The attempts to confer targeting ability through liposome surface modification are an entirely different matter. Antibodies are, by far, the most extensively investigated and attempted class of targeting agents that have been attached to the liposomal surface [2,3,7-8,12,61-641 for both tiers of targeting. A comprehensive discussion of these liposomes, for which the termimmunoliposomes has been universally accepted, is beyond the scope of this chapter and the reader is referred elsewhere [e.g., see refs. 1-12,51-54-55, and those cited therein]. The discussion here will touch briefly onthestate in the fieldwith respect to targeting with immunoliposomes andtheir feasibility as pharmaceutical products. A fair share of the studies pursuing targeting with immunoliposomes have been conducted in cellcultures. Taken as studies aimed at thefirst tier of targeting, cell cultures are not the best choice for a model system, especially for systemic applications. Taken as studies aimed at the second tier of targeting, cell cultures are quite suitable models even if the goal of the investigators was to test the first tier rather than the second. Animal model studies have shown, to date, that despite being such an elegant approach, successfulinvivo targeting withsystematically administered immunoliposomesis quite limited and still far from beingthe overall solution to liposomal targeting. Moreover, the question of whethere it might ever be a wide-range solution is still open. On the other hand, in vitro studies with immunoliposomes, which have shown specificity in binding and/or improved a biological response that was restricted to cells carrying the appropriate antigen, clearly demonstrate that it should be possible to achieve the second tier of targeting with this approach. Obviously, this speaks fora realistic potential of immunoliposomesin nonsystemic applications where, as already discussed, the first tier of targeting is achieved (or the need for it eliminated) by the selection of the administration route. Theoretically, targeting of intravenously administered immunoliposomes could also become possible for a wide range of applications if the means could be found to endow the immunoliposomeswith the ability to provide the first, without compromising their ability to provide the second, tier of targeting. Because they have a future with at least some targeting applications, the current situation with immunoliposomes makes the question of their feasibility as a pharmaceutical productrelevant and valid. This is a rather complex question comprising issues, to be discussed below, that concern the antibodies themselves as well as the antibody-liposome systems. It is stressed that thediscussion isfocused only on theissues that are related to the question at hand;namely, the feasibility of immunoliposomes aspharmaceutical products. Physiological and clinical advantages and drawbacks of immunoliposomes, are not addressed here.
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Obviously, to be a pharmaceutical product, immunoliposomes are required to meet all criteria that bear on antibody production, stability, retention of biological activity (for targeting, at least with respect to the antigen-antibody interaction), and appropriate shelf life. Another major factor in the making of an antibody into a pharmaceutical product (or part of) is the combination of specificity and antigen abundance. The antibody has to be specific enough for its designated use (whether as a targeting or as a therapeutic agent) yet the antigen shouldbe general andabundant enough so that theproduct would be applicable to a wide enough range of the patientpopulation. Viewed as future pharmaceutical products, the process of modifying a liposome into an immunoliposome is wrought with obstacles that start from the need to meet industrial criteria of the type previously defined for the into selection of the liposome type and thelipid composition and continue the modification process. The most frequently reported surface modification approaches employing agents such as SPDP and similar ones [64-651 are multistep processes. First, the antibody and a lipid undergo, each separately, chemical modification and purification procedures, including masking of active residues for molecular protection. Next come liposome formation (incorporating that modified lipid into theformulation) and the unmasking of an active residue on the antibody, after which the antibody-liposome association process takes place. The process is completed by further purification steps. Performed on a a large scale, as would be neededfor a product, it is easy to see thatthis could turn into a monumental endeavor. Suffice to consider the many steps along the way where significant liposome, drug, and antibody losses might occur and the issue of sterility in the final product. Considerations of raw materials, waste disposal, stability, retention of encapsulated drug, and drug stability would all add to the difficulty, as wouldthe need for an overall cost-effective process. On this basis, it seems clear that although immunoliposomes will continue to bea useful research tool, their development into pharmaceutical products, even for cases where they can provide targeting, will have to be decided case by case, and taking into account not only the obstacles listed above, but also those that could arise from physiological and clinical aspects. Other types of liposome surface modifications for the purpose targeting that are notbased on theimmune system might be more feasible as future pharmaceutical products. The case of polyethylene glycolated stealth liposomes for systemic administrations has already been discussed. Other potential opportunities are bioadhesive liposomes, which have beendeveloped in recent years for nonsystemic topical and regional applications [56581. These arebased on theuse of ubiquitous surface-bound agents such as collagen, hyaluronic acid and growth factors, and on surface modification procedures thatare less complexthan those discussed above forantibodies.
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With respect to targeting, the first concern of an investigator developing a drug-liposomesystem for a specific therapeutic objective is to find the means to achieve targeting. Together with this concern, it is proposed that the investigator should weigh his or her options along the lines discussed above. If the therapy in question requires addressing both tiers, then, unless the investigator intends to embark on the development of new targeting agents, at present he or she might be better off attempting targeting with nonmodified liposomes, exploring liposome specifications. If the therapy requires the second tier alone, targeting opportunities are offered by surface-modified liposomes, with current ornewly developed agents. In all cases, regardless of whether targeting is pursued with current or new surface-modifying agents, the feasibility for future pharmaceutical products, as exemplified above for immunoliposomes, should be taken into account. In closing this section on targeting, two additional points are offered. It is stressed that thediscussion above should not be taken as a proposal to discount immunoliposomestotally. Rather it is meant to propose extreme caution in the pursuit of antibodies as liposomal targeting agents. Recent studies with immunoliposomescarrying an antimyosin antibody are a pertinent example [66].These liposomes have been shown to. be capable of targeting in vivo, and this antibody meetsthe combined criteria of specificity and antigen abundance for sufficiently large patient populations, as discussed above. For such a case, overcoming other hurdles with respect to immunoliposomes as pharmaceutical products, could be well worth the efforts. Finally, despite the heat of the battle, it is imperative not to lose sight of the critical objective of targeting: It is the drug which should be targeted, with the liposome a means to that end, even though it is often experimentally wiser to first pursue targeting with drug-free liposomes. Even if successful targeting of a given liposome system has been achieved, and theresultant liposomes can meet the criteria of becoming pharmaceutical products, the work is not yet done. Completion requires experimental verification that those liposomes canreach the target while still carrying a drug load that is sufficient for effective therapy.
V. LIPOSOMES AS A STERILE, PYROGEN-FREE SYSTEM WITH PHARMACEUTICALLY ACCEPTABLE SHELF LIFE, STABILITY, AND DOSAGE FORMS A.Sterile,Pyrogen-FreeLiposomes
In basic research, an investigator can forego the need for sterile, pyrogenfree liposomes provided the experiments conducted are &ch that the lipo-
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somes do not come into contact with living matter at all or that such contacts, bethey in vitro or in vivo,are of short duration. For a pharmaceutical product, such qualities are critical. The steps thatshould be takenin order to obtain pyrogen-free liposomes are not essentially different from those implemented for other pharmaceutical products [5,67].Securing the sterility of liposomal products isan entirely different matter and has the makings of a major obstacle [67]. The major approaches in use, such asheat sterilization and y-irradiation, are not suitable the for end product [5,67].The two feasible approaches are sterilization by filtration and an aseptic production process. Of the two,the lattercan potentially be applied to all species and types of liposomes, regular as well assurface modified. The sterile fill is restricted, obviously, to liposomes small enough to pass the selected pore size of the filter, usually 0.2 pm, provided obstructions such as filter clogging and filter adsorption (especially for surface-modified liposomes) do not turn into insurmountable complications or lead to significant losses in matter. Consequently, the issues surrounding the production of sterile liposomes, by being of mutual interest and need to liposome investigators in both academic and industrial research, constitute an area where cooperation will be neededin order to arrive atacceptable solutions.
B. Shelf Life, Stability, and Dosage Forms

It is well known that the formation of liposomes from dry lipids suspended in an aqueous media is driven by thermodynamic stability [1,68].Hence, to survive and function as liposomes, these particles need to be surrounded by of a water, particularly at the start and end of the road; namely, at the end liposome production process and when called to action within the biological milieu. During thetime interval between production and in vivoperformance, the liposomes have to be stored for an acceptable period that, for pharmaceutical products, usually extends beyond 1 year. For refrigerated (but not frozen) aqueous suspensions and lyophilized powders, defining the majorrisks of long-term storage and reviewing, from the point of view of the academician, the advantages and drawbacks of each form of storage and for administration, are the topics of this section.
1. Shelf Life and Long-Term Stability

Breakdown in sterility, chemical destablization, formation of undesirable degradants and loss of therapeutic activity are among the major risks run in long-term storage of any pharmaceutical entity. When it comes to liposomes, guarding against and prevention of such risks are inherently more difficult. More than one chemical entity is involved: lipids, drugs, and for modified liposomes, the surface-anchored agents. It also involves the pres-
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ervation of particle integrity and the retention of the encapsulated drug within the liposome. The major advantages of liposomal storage in the form of aqueous suspensions are (1) the retention of the original type and specifications of the liposomes selected and produced for the designated therapeutic task and (2) their ready to use mode. The former is of particular importance when unilamellar liposomes are concerned, especially when substantial efforts have been invested in their production and where the effective therapy is dependent on a specific size and narrow size distribution of the liposomal preparation. A critical component of the ready to use mode involves the distribution between encapsulated and unencapsulated drug. Obviously, effective therapy in which the liposomes will make a substantial difference compared with free drug requires that a sufficiently highshare of the drug in the administered dose be encapsulated. This is particularly important fortoxic drugs (such as chemotherapicagents) where protection of the biological environment from the drug is a major motivation for liposomal delivery. In such cases, even a relatively small share of unencapsulated drug could defeat that objective. The considerations outlined above favor the storage of liposome preparations that have been cleaned from unencapsulated drugs together with the retention of this state throughout the storage. Removal of unencapsulated drug, which constitutes a displacement of the equilibrium distribution of thedrugbetweenthe liposomes and the external aqueous phase, will inevitably create anelectroof the liposomes [56-58,691. As will chemical gradient, driving the drug out be discussed in greater detail in a following section, the rate of this diffusion will depend on several factors, with drug properties being foremost among them. Evaluation of the release of kinetics of the drugin question and of the effects of already recognized tools for implementationof satisfactory storage conditions, as well as the development of new tools for the task, start at the basic research level. The risks of loss of encapsulated drug on long-term storage of liposomes thathave undergone separation from unencapsulated drug and are therefore in a nonequilibrium state are particularly high for small molecular weight drugs stored under those nonequilibrium conditions. For suchcases, storage at high lipidconcentrations [69], the introduction of additional intraliposomal barriers such as theammonium sulfate gel [67], and utilization of remote-loading procedures [70] could be used to reduce therisk of that loss. For othercases, even though it might take away from the ready to use advantage, the only recourse would be to store the preparation underequilibrium conditions (i.e., without the removal of the unencapsulated share) and use it as it is or institute a separation procedure immediately prior to administration.
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Besides being economically more attractive, storage of liposome-drug systemsin dry form, as freeze-dried powders, seems the better choice, since it leads to significant reduction in the risks discussed above. On the other hand, excepting selected situations (to be discussed in the next section), the dry powder is not the ready to use form, and liposomal reconstitution through rehydration would be required prior to use. Needless to say, reconstitution has to be performed under sterile and pyrogen-free conditions. The main concerns and drawbackswith this dosage form center on the nature of the liposomal species and on thesituation of drug encapsulation that are obtained on reconstitution. Studies aimed at these issues have shown that unless specific steps-mainly the introduction of cryoprotectants into the liposomal formulation-are taken prior to lyophilization, the reconstituted systemswill revert to MLV, independent of the original liposome species [71-731. Although the feasibility of this approach has been proven and these exipients are considered safe, potential adverse effects of those cryoprotectants on the properties of the drug in question and (where relevant) on the surface-bound agent cannot be ignored. Independent of liposome species, less is known about the fate of drug distribution in the reconstituted system between the liposome and the (new) aqueous medium. If the share of unencapsulated drug is beyond the level of acceptance for the system in question, this will require correction through actions such as revisions in conditions of lyophilization and/or reconstitution and postreconstitution purification steps. For those liposomes thatare surface modified, the distribution of that agent should be also considered. If big enough, steric hindrance might favor its relocation on reconstitution to the liposomal surface, but the situation could arise where the share of reconstituted liposomes having that agent inside is not negligible. This could result in problems that range from the mere loss in the fraction of targeted liposomes to the hazards of liposomes carrying toxic drugs circulating indiscriminately in the body. The characteristics of the reconstituted liposome, including preservation of original structures through the use of cryoprotectants, the possible adverse effects due tothose additives, and the distributions of the drug and the surface-bound agent in the reconstituted systems, have been explored [71-731. However, it is argued here that whether such investigations have been conducted with drug models or with specific drugs, they need to be done anew for eachspecific liposome-drug system investigated and developed for a particular therapeutic objective. Undoubtedly, such questions have strong components that put them within the domainof basic research.
2.
Dosage Forms for Administration Suspended in an aqueous suspension form, regardless of the original storage form, the liposomes are ready for application utilizing not only the
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intravenous route but other routes of administration as well. They couldbe applied topically to the eyes, to wounds, and to burns. They could be injected subcutaneously or intramuscularly or introduced into the peritoneal cavity through injection or infusion. Other routes of administration requirefurther processing of the aqueousliposome suspension, which touch on basic and industrial issues that havenot been addressedyet in this chapter. A most prominent case isprocessing into aerosols of dru'gencapsulating liposomes (regular and surface modified) that would be useful for pulmonary applications. The making of liposomal aerosols for drug delivery opens new questions such as theeffects of the aerosolization process (where relevant) aerosolization components on (1) all aspects of stability (drug, lipids, surfacebound agents), (2) the retention of encapsulated drug, (3) the retention of (liposome) particle integrity and size, and (4) the retention of therapeutic activities, without which success inthe retention of the other propertiesis immaterial. Liposome aerosols have been prepared and characterized with respect to some of these properties [74-821, and the results attest to both the feasibility of this dosage form and to the strong dependence of its success on the drug and the liposome species explored. Consequently, the data that has accumulated using drug models will not save an investigator developing a drug-liposomesystem aerosol form from the need to invest in basic research of the stability and retention issues listed above. The physical features of the aerosol and their optimization with respect to the designated therapeutic goal would also be required, utilizing cascade impingers and similar devices [74,77-781, as well as a means for aerosol collection and analysis.
VI. LIPOSOME CHARACTERISTICS (PERCENTAGE OF ENCAPSULATION, KINETICSOF RELEASE, BIOLOGICAL ACTIVITY)-IN BASIC RESEARCH AND IN QUALITY ASSURANCE
In this chapter, much has already been said about basic liposomal traits such asthe efficiency of encapsulation, kinetics of drug release, and retention of biological activity.The investigation of these traits is inthe realm of basic research, and among other objectives is intended to serve in system optimization and in the development of in vitro models for pursuit of the liposomal invivo fate. Once these encapsulation, release, and activity specifications are set for a given liposome-drug system, they can be used within the industrial environment in the evaluation of the retentionof the liposome properties in the scaled-up production and as part of the battery of criteria for quality assurance. In consideration of the common ground these properties constitute for investigators from both academia and industry, several comments andproposals are offered below.
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A.
Efficiency of Drug Encapsulation
Throughout the three decades of liposome research, several definitions have been introduced as measures for this property. For example, the total in the system, the ratioof drug fraction of encapsulated drug from the to lipid, and the volume (per given lipid quantity) of the internal aqueous of encapsulated matter, such asCF phase. For moleculesserving as models (carboxyfluorescein) and similar derivatives, elegant procedures have been devised in order todetermine the efficiency of encapsulation [2,6,12]. One of their most favorable features is the elimination of the need to separate the drug-encapsulating liposomes from the media containing the unencapsulated drug. For most real drugs, irrespective of the efficiency parameter pursued, the experimental process for determination of encapsulated matter will require such a separation step. Although simple in concept, in Section V.B.l abovewith respect to cleanscontinuatibn of the discussion in ing from unencapsulated drug, most methodologies contain steps that can lead to overestimation or underestimation of the quantity of liposomeencapsulated drug, especially for small molecular weight drugs. Separations that makeuse of semipermeable matrices such as exhaustive dialysis, various filtration devices, or gel-exclusion column chromatograpies all run the risk of dilution of the external medium in the course of separation. This creates a driving force for efflux of the encapsulated matter, which would be most pronounced for small molecular weight drugs. The encapsulation level determined at the end of such proceduresmight be an underestimation of the initial level, with the deviation depending not only on drug properties but also on the specific experimental conditions used for the separation. This could become a problem notonly withrespect to accurate evaluation of encapsulation efficiencies. Batch-to-batch reproducibility and implementation of encapsulation efficiency as a quality assurance criterion would also be adversely affected, unless extensive care is taken in duplicating the separation process from batch-to-batch. Separation by centrifugation is supposedly free from the risks of external medium dilution. However, if a single cycle is used, there could be overestimation of the encapsulated fraction due to adsorption of unencapsulated drug to the liposomal pellet. Additional cycles of washing drug-free in buffer could reduce that error, but they need to be used withcaution in order to avoid the risk of dilution, which can lead (as discussed above) to underestimation. How then can the level of encapsulation be accurately assessed without the interferences that occur as a result of the separation steps? One. approach exemplified by the aforementioned CF is to develop assays that can distinguish (quantitatively) between encapsulated and unencapsulated matter in the same system. Such assays would be strongly dependent on
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drug properties and probably would be useful for a limited number of cases. Another possibility, to be discussed in the following section, is the determination of encapsulation efficiencies through studiesof drug release.
B. Kinetics of Drug Release
Irrespective of the level of organization at which they are performed, studies of drug release/diffusion from liposomal systems are directed toward issues that are relevant to the in vivo as well as to the non-in vivo arenas. For liposomes in the in vivo arena, the drug-release studies are expected to yield data and understanding that will lead to (1) minimizing the loss of encapsulated drug on route from the site of administration to the site of drug action and (2) the ability to match the rate of release (once the liposomes arrive at the target) to the requirements of the therapy. The objectives of drug release studies that concern the non-in vivo arena are (1) physicochemical characterization of the systems, including liposomes processed into aerosols or reconstituted from freeze-dried powders; (2) various aspects of system optimization such as the selection of liposome type, lipid composition, and parameters of shelf life; and (3) criteria for quality assurance. In order to derive relevant data from such studies, the experimental conditions should be set to fit the specific objectives, especially with respect to the extent of liposomes and drug (each, separately) dilutions that the system is anticipated to undergo. Detailed discussed of the in vivo objectives are outside the theme of this chapter, anddiscussion of the impact of such dilutions has been discussed elsewhere [55]. The nonin vivo objectives are within the subject of the present discussion and will be addressed below. Drug release from intact liposomes into the media within which the liposomes are placed (or suspended) is essentially a process of diffusion in a heterogeneous system; the latter made of several phases of water andlipid bilayers, the number of which willdepend on the liposome type andon drug solubility properties. At thevery least, two phases are involved: a lipophylic drug embedded within the lipid bilayer of a unilamellar liposome. In principle, initiation of drug diffusion simply requires setting the driving force (i.e., an electrochemical gradient of the drug from its liposomal location to the external medium) afterwhich the process will proceed until equilibriumis established (or reestablished). When the starting material is a drug-liposome system at equilibrium with respect to drug distribution, reduction in the drug concentration at the external medium suffices to trigger the releaseprocess. Several means can be used togeneratethatreduction:precipitation, enzyme-catalyzed degradation, and dilution of the liposomal system into the medium of choice such asbuffer or body fluid(real or simulated). The latter
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is, by far, the easiest and most general means to generate such a drug concentration reduction that is suitable for a wide range of the objectives listed above. If, as discussed above with respect to encapsulation efficiencies, the liposomal system has been previously subjected to cleaning procedures in order to rid it of unencapsulated drug, the electrochemical gradient is already in existence and the diffusion process already operative prior to the specific kinetic experiment. Independent of the state of the system on initiation of a particular experiment (i.e., equilibrium or nonequilibrium of drug distribution), it is proposed that sustaining a unidirectional flux of the drug from the liposomal system into the bulk medium during the entire experiment is a key element. Data from experiments done under such conditions represent the worst-case scenario for drug loss from the liposomes. This knowledge can then be used to design shelf life conditions under which drug loss is minimized [56-58,691. Data of this type can also be used to set up,in the process of liposome surface modification, the conditions that will prevent depletion of the encapsulated drug. Once the diffusion has been initiated (or is already in progress), the experiment itself consists of quantitative evaluation of drug accumulation in the medium and/or drug loss from the liposomes at designated periods. Critical to the successful execution of such studies is the quantitative separation of liposomes and the external medium in aliquots withdrawn fromthe reaction mixture at designated time points. Classic means forthe quantitative separations of particles from their suspension medium, such as centrifugation and filtration, are often compromised by drug loss to the filtration matrix or by centrifugation procedures that are too slow withrespect to the time that has elapsed between samplings. Additional experimental constraints might be encountered in drug and liposome assays. The dilution which is essential for the onset and maintenanceof the gradient can result in lipid and druglevels in the separated fractions of the withdrawn aliquots that fall below the limits of detection and/or quantitation. for separation together with signifiElimination altogether of the need cant reduction of the risks of falling belowdetection limits is offered by the use of appropriate dialysis set-ups [56-58,691. In this approach, a liposome preparation is enclosed within a dialysis sac that is immersed in a drug-free medium. The wide variety among available semipermeable dialysis membranes makesit possible to select, according to specifications of the investigated system, a membrane that will not adsorb the drug and will not be a barrier to the drug but at the same time be a complete barrier to the liposomes. Analysis of the raw data, rather than remaining at the phenomenological level, can not only give insights into the mechanism(s) but can also
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aid in identifying factors that are instrumental in gaining control of the release. Among the theoretical frameworks available for the analysis of diffusion data, two properties make the theoretical approach developed by Eyring particularly suitable for drug diffusion from liposomes. It allows drug release from homogeneous (unencapsulated drug) and heterogeneous (liposome-associated and liposome-encapsulated drug) systems to be dealt with simultaneously. It yields parameters that allow the direct determination of the fraction of encapsulated drug and of the half-life of drug release. In the long run, such parameters are especially useful for systems that are destined to serve as therapeutic entities, in defining optimization criteria, andfor designing dose ranges and treatmentregimens. Studying the kinetics of drug release from liposomal (regular and surface-modified) systems, using theexperimentaland data processing approaches discussed above, the authors were able to sortseveral underlying principles that are general to these release processes [56-58,691: It was found that the release kinetics can be described as a series of parallel first-order processes, each representing a drugpool that exists in the system at time = 0. One pool represents the drug that is unencapsulated and all others drug thatis liposome associated. The mathematical expression for this type of mechanism is given by [see refer. 69 for additional details]:
f=
j= 1
2 &<I-
e+>
(1)
where t represents time, the experimental independent parameter, and f represents the dependent experimental parameter, which is the cumulative release, normalized to the totaldrug in the system, at time = 0. The total number of independent drug pools is given by n, fi is the fraction of the total drug occupying the jth pool at time = 0, and kj is the rate constant of the diffusion of the drug from the jth pool. For data derived from liposome-drugsystems in whichthe drug was at equilibrium distribution at the onset of the experiment, this form of data analysis will yield the percentage of encapsulation through the magnitude of fi for the encapsulated pool. It is proposed that, althought extracted from kinetic data, this is the most accurate evaluation of this thermodynamic parameter, since it is free of the limitations imposed by separation procedures (see Section VI.A above). Being diffusion processes, it was anticipated and verified experimentally [56-58,691 that theproperties of the drug are the primary parameters dictating the specifics of drug release. For a given drug, liposomal properties such as liposome type, lipid composition, and liposome concentration
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constitute means for some modulation drug release. Among those liposomal parameters, liposome concentration was found to be the most useful constant of the tool for thetask [56-58,691. It has been shown that the rate release of the encapsulated drug generally decreases with the increase in and that the phenomena and trend are indep liposome concentration [69] dent of drug and of liposome species, whereas the actual magnitudes are system-specific. Besides providing an understandingof the liposomal system of interest, how then can such studies be used for the attainment of those non-in vivo objectives that are among the concerns of the industrial scientist? With respect to shelf life, if the selected dosage form is liposome suspensions, storage should obviously be at high liposome concentrations. Furthermore, the kinetic parameters determinedfor the system of interest become product specifications (or fingerprints) that will constitute critiwith cal input into the data base on which the decision of whether to store or without unencapsulated drug will rest. If the selected dosage form is a freeze-dried powder of drug-encapsulating liposomes, these fingerprints can be useful in defining the optimal conditions for reconstitution and in verifying that the reconstituted system has retained its original properties. Regardless of the dosage form selected for storage, retention of the same , magnitudes of the kinetic parameters can be included within the batteryof quality assurance tests. When it comes to surface-modified liposomes, the processes of drug release add some concerns that are of interest to both the liposome investigators in both academic and industrial research. A particular concern is the risk of drug (encapsulated) loss that can occur in the course of the modification itself, as well as in the subsequent procedures of separation andpurification. Kinetic studies of the type discussed in this section can be used to determine whether such losses are significant at all and to evaluate their extent. For thesystems at risk, inclusion of drug in the wash buffers could eliminate the problem. Whether the modification is done on preformed drug-encapsulating liposomes oron a singlelipid component prior to liposome formation, such studies can also address the extentto which (if at all) the modification interferes with drug release and the optimal conditions for minimizing that interference. In conclusion, studies of drug release from liposomes that can be categorized as basic research and therefore inherently within the domain of the academician are also an essential part of many aspects and needs of product development and maintenance that are delegated to thedomain of the industrial liposomologist. Such studies are needed anew for each drugliposome system, conducted with the specific drug of interest rather than with models.
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C. Biological Activities of Liposome-Encapsulated Drugs

The obviousness of the need to retain the therapeutic activity of the liposome-encapsulated drug is so clear and unambiguous that it does not necessitate, besides this statement, any further discussion. The context in which this issue is discussed here centers on basic and product-oriented pre-in vivo studies that are deemed critical for theevaluation and application of liposome-drug systems. Monitoringand ensuring the biologicalactivity of the liposomeencapsulated drug has the highest priority whether the studies are conducted at the basic research level, at the stage of produce development, or in the course of routine production of a pharmaceutical product. The span of systems studied varies among these objectives, and are the most extensive at the basic stage, where it is attempted to best understand the system, optimize it, and elucidate the operating mechanisms. Even for productswherethe finaldecisionmight favor the administration of a liposomal system that contains both encapsulated andunencapsulated drug, the basic studies should explore both the complete system and a preparation free of unencapsulated drug. This is to verify that the biological activity is indeed that of the encapsulated drug, since if not, it defeats the objective of using liposomes. In light of the discussions in the previous sections, the time span between separation and initiation of the activity experiment should be selected according to the properties of the system at hand, with the goal of minimizing the loss of the encapsulated drug to thenew drug-free medium. In addition to the obvious controls of free drug and vehicle, further verification that it is the encapsulated drug whichis responsible for all (or at the least most) of thetherapeutic activity, drug-free liposomes suspended in the vehicle and in free drug should also be tested. Where modified liposomes are concerned, at least part of those studies should be duplicated for the (control) unmodified liposomes also. For those cases in which the basic studies have ensured that the designated product (i.e., the encapsulated drug in the specified liposomes) has retained a satisfactory level of biological activity,the product development stage can forego controls of drug-free liposomes suspended in vehicle and in free drug. Similarly, if theproduct ismodified liposome, nonmodified liposomes can be abandoned. Yet the weight of separately evaluating, the contributions of the system at equilibrium (i.e., containing both encapsulated and unencapsulated drug) and at nonequilibrium (i.e., containing encapsulated drug alone) statescannotbe ignored. Such data should beespecially sought for the final storage and administration dosage forms, be those aqueous suspensions originally, freeze-dried powders that
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are rehydrated, or aerosols. Evidently, once the liposomal system reaches the stage of an established pharmaceutical product, the biological studies can be limited to continuous monitoring of that system alone. It is indisputable that the ultimate preclinical level of organization at which the therapeuticactivity of the liposome-encapsulated drug shouldbe studied is that of the whole animal. Nevertheless, for the objectives listed above, it is well worth the effort to precede with in vitro studies. If done properly in relevant systems, such studies can significantly reduce the investments (such as time, efforts, and resources) needed at the in vivo stage without compromising the quality and significance of the results and their conclusions. Furthermore, for quality assurance of established products, the in vitro studies might suffice. As will be illustrated by several examples below, selecting and implementing in vitro systems for evaluation of the in vivo designated therapeutic activity is an attainable task. In vitro testing of liposome-encapsulating chemotherapeutic drugs for tumor treatment has been extensively studied and reported, and a newcomer to thefield can scan the liposome literature to select the systems suitable to his or her objectives. A similar situation exists with respect to liposomes encapsulating antibiotics for the treatment of intracellular infections of phagocytic cells.l b o additional examples that have not been as extensively reported, and therefore are addressed here in more detail, concern wound healing and the treatment of extracellular bacterial infections. The wound-healingeffects of several growth factors is exerted mainly (if not solely) through their stimulation of cell proliferation. This opens the doorto in vitro testing of the biological activity of suchliposomeencapsulated polypeptides through the selection of suitable cell lines. Basically, two types of cell lines can be used as thetest system: those that have been specifically developed to have absolute dependence on a particular growth factor and those that can be made to have such dependence through the use of specific experimental conditions. For EGF, which is in the first line of growth factors in development for wound healing, the first type is represented by the cell line BALBMK, whichis the classic for EGF bioassay[83]. The well-known NIH3T3 line, growninverylow serum (0.125%), is a representative of the second type [Yerushalmi and Margalit, in preparation]. For both cases, the first task is to determine the doseresponse curves to free EGF, in order to determine the range in which stimulation of proliferation is measurable yet stillfree from adverse effects [83, Okon, Nunez and Margalit, in preparation; Yerushalmi and Margalit, in preparation]. Next, the liposomal systems including the various partial and control groups discussed above can be tested. Implementing such studies with both lines, it was found that EGF does indeedretain its biological
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activity when encapsulated in regular as well as in surface-modified liposomes. Cultures of the relevant test organism can serve as the in vitro systems for testing liposomes that are designated for infection treatment of extracellular organisms, irrespective of the route of administration or anatomical locations. Adapting the paper disk version of the growthinhibition assay of free [84] to liposome-encapsulated, antibiotics, this approach was satisfactorily implemented for the following cases: liposome-encapsulated ampicillin[G-861 and liposome-encapsulated cefazolin [I. Schumacher and R. Margalit, unpublished data]using Micrococcw Zuteus and Staphylococcus aureus as the respective test organisms. Furthermore the adaptation to liposomal systems is general enough that it can be easily extended to other antibiotic-liposome systems and additional test organisms. In all cases in which the in vivo designated therapeutic activity is tested in vitro, it is imperative to distinguish quantitatively between the total drug dose in the liposomal preparation and the actual dose that has become available to the cells during the time course of the experiment. Treatment with equal total doses of free and of liposome-encapsulated drug where the latter system yields lower response need not be interpreted as liposome-associated loss in drug activity. Nor should similar levels of response to freeand to liposome-encapsulated drug be taken to indicate that the liposomal system has no potential to improve clinical outcomes. In both cases, it might simply be that in actuality the comparison was between unequal doses, with the liposomal one beingthe lower. This situation could arise when not all of the encapsulated drug is released and thus made available to the cells during the experiment. Two approaches can be taken in order todistinguish between cases in whichthe equal or lower response of the liposomal treatment is an artifact of comparisons at unequal doses andwhen it is truly due to the loss of activity and/or inadequacy of liposomes for that specific treatment. Both approaches require previous data and analysis of the drug-release profile, especially at the level of dilution used in the bioassay. For the question of whether the liposomeencapsulated drug has retained its activity, such data can then be used to design bioassay conditions under which all of the encapsulated drug 'is made available. For the question of whether the liposomal systems have the potential to improve clinical outcomes, such data can be used to estimate the actual dose to which the cells have been exposed. Obviously, caution should be exercised in making quantitative use of drug-release profiles derived under conditions devoid of living matter. Determination of the kinetics of drug release in the cellular system tested under the conditions of bioassay, when possible, would augment the available data and increase the accuracy with whichthe dose-response curve of the liposomal
290Yerushalmi
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system can beshifted to its true place. Finally, in closing,it is noted that a similar awareness of differences between total and actual liposomal doses is needed for dose-response comparisons in vivo, especially whenthe objective is to evaluate if liposomes can improvethe clinical outcomes.
VII. SUMMARY AND FUTURE PROSPECTS

It is the opinion of the authors that any communication on liposomes as delivery systems cannot be concluded without a reminder of the current reality. Awareness of the substantial hurdles to the implementation of liposomes as drug-delivery systems inestablished treatment modalities cannot be escaped. Nor can the current state of affairs where, despite three decades of research, the number of systems that have matured intoclinical trials and/or have becomeproducts on themarket is quite modest. Yet the need for drug-delivery systems is as acute as ever and the potential that liposomes hold, although somewhat tarnished, has not been substantially diminished. On this background, it is proposed thatat least some of those hurdles can be overcome and substantial strides be made in advancing liposomes from the laboratory to theclinic through attention and mutual awareness of the issues faced by liposome researchers in the academic and industrial environments and through increased collaborations. It is hoped that this chapter will make a contribution, however modest, in these directions.
REFERENCES
1. A. D. Bangham, Liposomes: The Babraham connection, Chem. Phys. Lipids,
64:249 (1993). 2. G. Gregoriadis, Liposomes as Drug Carriers, Recent Trends and Progress, Wiley, New York, 1988. of liposomes in biotech3. T. Sat0 and J. Sunamoto, Recent aspects in the use nology and medicine, Prog. Lipid Res., 31:345 (1992). 4. D. C. Litzinger and L. Huang, Phosphatidylethanolamine liposomes: Drug delivery,genetransferandimmunodiagnosticapplications,Biochim.Biophys. Acta, 1113:201 (1992). D. D.Lasic,Stericallystabilizedliposomes,Biochim. 5. M.C.Woodleand Biophys. Acta, 1113:171 (1992). 6. J. H. Senior, Fate and behavior of liposomes in vivo: A Review of controlling factors, CRC Crit. Rev. Therapeutic DrugCamer Sys., 3:123 (1987). 7. P. Machy and L. Lesserman, Liposomes in Cell Biology and Pharmacology, Libbey, London, 1987. 8. G. Gregoriadis, Overviewof liposomes, J. Antimicob. Chem., 28:39 (1991). 9. J. A. Karlowski and G . G. Zhanel,Concepts on the use of liposomal
29 l
10.
11.
12. 13. 14.
16. 17. 18. 19. 20. 21. 22.
23.
24.
25. 26. 27.

28.
antimicrobialagents:Applicationsforaminoglycosides,Clin.Infect.Dis. 16:654 (1992). G. Gregoriadis and A. T. Florence, Liposomes in drug delivery, Drugs 45:15 (1993). A. Gabizon, Tailoring liposomes for cancer drug delivery: From the bench to the clinic, Ann. Biol. Clin., 51:811 (1993). G. Gregoriadis, Liposome Technology, Vols. 1-111, CRC Press, Boca Raton, FL, 1984. F. Szoka, Jr., and D. Papahadjopoulos, Comparative properties and methods of preparation of lipid vesicles, Ann. Rev. Biophys. Bioeng., 9:467 (1980). M.J.Hope,M.B.Bally, G. Webb,andP.R.Cullis,Productionoflarge unilamellar vesicles by rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential, Biochim. Biophys. Acta, 81255 (1985). S. Carnie, J. N. Israelachvili,andB.A.Pailthorpe,Lipidpackingand transbilayer asymmetries ofmixedlipidvesicles,Biochim.Biophys. Acta, 554:340 (1979). V .V. Kuman, Complementary molecular shapes and additivity of the packing parameters of lipids, PNAS, 88:444 (1991). M. J. Hope, and C. N. Kitson. Liposomes. A perspective for dermotologists, Dermatol. Clin., 11:143 (1993). E. Mayhew, R. Lazo, W.J.Vail, et al., Characterization of liposomes prepared using a microemulsifier, Biochim. Biophys. Acta, 755:169 (1984). Ostro. Novel multilayered lipid vesicles: Comparison of A. S. Janoff and M. J. physical characteristics of multilamellar liposomes and stable plurilamellar vesicles, Biochemistry, 24:2833 (1985). S. Kim and G. M. Martin, Preparation of cell-size unilamellar liposomes with high captured volume and defined size distribution, Biochim. Biophys. Acta, 646:l (1981). J .. Horton, and C. R. Baxter, Topical liposomal delivery of C. I. Price,W antibiotics in soft tissue infection, J. Surg. Res., 49:174 (1990). of aminoglyC. I. Price, J. W. Horton, and R. B. Charles. Liposome delivery cosides in bum wounds, Surg. Gynecol. Obstet., 174:414 (1992). M. E. Planas, P. Gonzalez, L. Rodriguez, et al., Noninvasive percutaneous induction of topical analgesia by a new type of drug camer, and prolongation oflocalpaininsensitivityby anesthetic liposomes, Anesth. Analg., 75:615 (1992). G. Cevc andG. Blume, Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force, Biochim. Biophys. Acta, 1104:226 (1992). Physicians Desk Reference, 48th ed., Medical Economics Data Production Co., Montvale, NJ, 1994. G. L. Brown, L. B. Nanney, J. Griffen, et al., Enhancementof wound healing by topical treatment with epidermal growth,N. Engl. J. Med., 321:76 (1989). J. M. Davidson, M. Klagsbrun, K. E. Hill, et al., Accelerated wound repair,
292
and
Margalit
Yerushalmi
29. 30. 31. 32. 33. 34. 35* 36. 37. 38. 39. 40. 41. 42. 43.
44.
45.
4 6 .
47. 48.
cellproliferation,andcollagenaccumulation are producedbyacartilageJ. Cell Biol., 100:1219 (1985). derived growth factor, T. K. Hunt, Basic principles of wound healing, J. Trauma, 30:S122 (1990). A. N. Kingsnorth and J. Slavin, Peptide growth factors and wound healing, Br. J. Surg., 78:1286 (1991). D. R. Knighton andV. D. Fiegel, Regulation of cutaneous wound healing by growth factors and the microenvironment Invest. Radiol., 26604 (1991). G. Schultz, D. S. Rotatori, and W. Clark. EGF and TGF-alpha in wound J. Cell. Biochem., 45346 (1991). healing and repair, R. A. Yates, L. B. Nanney, R. E. Gates, and L. E. King, Epidermal growth factor and related growth factors, Int. J. Dermatol., 30:687 (1991). D. T. Graves and D. L. Cochran, Mesenchymal cell growth factors, Crit. Rev. Oral Biol. and Med., 1:17 (1990). S. T. Feldman,Theeffect of epidermalgrowthfactor on corneal wound healing: Practical considerations for therapeutic use, Refractive Corneal Surg., 7:232 (1991). D. R. Knighton, K. Ciresi, V. D. Fiegel, et al., Classification and treatment of chronic nonhealing wounds. Successful treatment with autologous plateletderived wound healing factors, Surg. Gynecol. Obstet., 17056 (1990). D. H. Herndon, P. G. Hayward, R. L. Rutan, and R. E. Barrow, Growth hormones and factors in surgical patients, Adv. Surg., 2565 (1992). J. C. Pastor and M. D. Calonge, Epidermal growth factor and corneal wound healing. A Multicenter study, Cornea, 11:311 (1992). S. A. Servold, Growth factor impacton wound healing. Clin. Podiatr. Med. Surg., 8:937 (1991). M. H. Gartner, J. D. Shearer, M.F. Bereiter, et al., Wound h i d amino acid concentrations regulate the effect of epidermal growth factor on fibroblast replication, Surgery, 110:448 (1991). P. A. Falcone and M. D. Caldwell, Wound metabolism, Clin. Plast. Surg., 17:443 (1990). D. L. Steed, Randomized prospective double-blind trial in healing chronic diabetic foot ulcers. Diabetes Care, 151598 (1992). 0.M. Alvarez, P.M. Mertz, and W. H. Eaglestein, The effects of occlusive J. dressingson collagen synthesis and re-epithelization in superficial wounds, Surg. Res., 35:142 (1983). P. M. Mertz and W. H. Eaglestein, The effect of a semiocclusive dressingon the microbial population in superficial wounds, Arch. Surg., 119:287 (1984). W. H.Eaglestein, P.M. Mertz, and V. F. Falanga, Occlusive dresings, AFP, 34:211 (1987). S. F. Swain,Bandagesandtopicalagents.Plast.Reconstr.Surg.,20:47 (1990). G. C. Xakellis and M. A. Chrischilles, Hydrocolloid versus saline-gauze dressings in treating pressure ulcers: a cost-effective analysis, Arch. Phys. Med. Rehab., 72:436 (1992). L. Hulten, Dressings for surgical wounds, Am. J. Surg., 1763428 (1994).
of Liposomes
293
49. C. K. Field,andM.D.Kerstein,Overviewofwoundhealinginamoist environment,Am. J. Surg., 176:2S (1994). 50. C. S. Burton, Venus ulcers, Am.J. Surg., 176:37S (1994). into 51. T. M. Allen and A. Chonn, Large unilamellar liposomes with low uptake the reticuloendothelial system, FEBS Lett., 223:42 (1987). 52. A. Gabizon and D. Papahadjopoulos, Liposome formulations with prolonged circulation timein blood and enhanced uptake by tumors, Proc. Natl. Acad. Sci. USA, 85:6949 (1988). 53. T. M. Allen, C. Hansen, F. Martin, et al., Liposomes containing synthetic lipid derivatives of poly(ethy1ene glycol) show prolonged circulation half-lives in vivo, Biochim. Biophys. Acta, 1066:29 (1991). 54. G. Poste, Liposome targeting in vivo: Problems and opportunities, Biol. Cell, 47:19 (1983). 55. R. Margalit, Vesibes as topical drug delivery systems, in Vesicles, Surfactant Science Series (M. Rossof, ed.), Marcel Dekker, New York, 1994 pp. 527-5 E. Avidor, Bioadhesive liposomes 56. R. Margalit, M. Okon, N. Yerushalmi, and fortopicaldrugdelivery:Molecularandcellularstudies, J. Control. Rel. 19:275 (1992). 57. N. Yerushalmi and R. Margalit, Bioadhesive, collagen-modified liposomes: Molecular and cellular level studies on the kinetics of drug release and on binding to cell monolayers, Biochim. Biophys. Acta, 1189:13 (1994). 58. N. Yerushalmi, A. Arad, and R. Margalit, Molecular and cellular studies of hyaluronic-acid modified liposomes as bioadhesive carriers of growth factors fortopicaldeliveryinwoundhealing,Arch.Biochem.Biophys.313:267 (1994). R. T. Proffitt, Selective in vivo localization 59. E. A. Forssen, D. M. Coulter, and ofDaunorubicinsmallunilamellarvesicelsinsolidtumors,CancerRes. 52:3255 (1992). 60. A. A. Gabizon, Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes, Cancer Res., 52:891 (1992). 61. J. Senior and G. Gregoriadis, Dehydration-rehydration vesicle methodology facilitatesanovelapproachtoantibodybindingtoliposomes,Biochim. Biophys. Acta, 100358 (1989). V. P. Torchilin, and L. Huang, Influence of the steric 62. A. Mori,A. L. Klibanov, barrier activity of amphipatic poly(ethyleneglyco1) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo, FEBS Lett., 284:263 (1991). ofliposomesin 63. S. Toshinoriand J. Sunamoto, Recent aspects in the use biotechnology and medicine, Prog. Lipid Res., 31:345 (1992). P. Machi, in Vesicular 6 4 . L. Lesserman, C. Langlet, A.-M. Schmitt-Verhulst, and M. Tartakoff, ed.), 1989, pp. 447-471. Transport, Part B (A. 65. L. Huang and X. Zhou, Targeted delivery of DNA by liposomes and polymers, J. Control. Rel., 19:269 (1992). 6 6 . A. A. Bogdanov, L. V. Gordeeva, V. P. Torchilin, and L. B. Margolis, Lectin-
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and
Margalit
67.
6 8 .
69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79.
80.
81. 82. 83.
84.
bearing liposomes: differential bindingto normal and to transformed mouse fibroblasts, Exp. Cell. Res., 181:363 (1989). S. Amselem, R. Cohen, and Y. Barenholtz, In vitro tests to predict in vivo performance of liposomal dosage forms, Chem Phys. Lipids, 64:219 (1993). M. K. Jain and R. C. Wagner, Introduction to Biological Membranes, Wiley, New York, 1980. R. Margalit, R. Alon, M. Linenberg, et al., Liposomal drug delivery: therJ. Control.Rel., 17:285 modynamicandchemicalkineticconsiderations. (1991). L. D. Mayer,M. B. Bally,M.J.Hope,andP.R.Cullis,Uptakeof antineoplastic agents into large unilamellar vesicles in response to a membrane potential, Biochim. Biophys. Acta, 816:294 (1985). M. Friede, M. H. V. Van Regenmortel, and F. Schuber,Lyophilizedliposomes as self items for the preparation of immunogenic liposomes-peptide conjugates, Anal. Biochem., 211:117 (1993). L. M. Crowe, C. Womersley, J. H. Crowe, et al., Prevention of fusion and leakage in freeze-dried liposomes by carbohydrates, Biochim. Biophys. Acta, 861:131 (1986). L. M. Crowe, J. H. Crowe, A. Rudolph, et al., Preservation of freeze-dried liposomes by trehalose, Arch. Biochem. Biophys., 242:240 (1985). I. W.Kellawayand S. J.Farr.Liposomesasdrugdeliverysystems to the lungs, Adv. Drug. Del. Rev., 5:149 (1990). P. K. Gupta and A. J .Hickey, Contemporary approachesin aerosolized drug delivery to the lungs, J. Control. Rel.,17:129 (1991). H. Schreier, R. J. Gonzalez-Rothi, and A. A. Stecenko, Pulmonary delivery of liposomes, J. Control. Rel.,24:209 (1992). S. J. Farr, I. W. Kellaway,and B. Carman-Meakin, Comparison of solute partitioning and efflux in liposomes formed by a conventional and aerosolized method, Int. J. Pharmaceut., 51:39 (1989). K. M. G. Taylor, G. Taylor, I. W. Kellaway, and J. Stevens, The stability of liposomes to nebulization, Int.J. Pharmaceut., 5857 (1990). R. W. Niven and H. Scherier, Nebulization of liposomes. I. Effects of lipid composition, Pharmaceut. Res., 7:1127 (1990). R. W. Niven, M. Speer, and H. Scherier, Nebulization of liposomes.11. The effects of size and modeling of solaute release profiles, Pharmaceut. Res., 8:217 (1991). R. W. Niven, T. M. Carvajal, and H. Scherier, Nebulization of liposomes. 111. The effects of operating conditions and local environment, Pharmaceut. Re 9515 (1992). S. B. Debs, R. M. Straubinger, E. N. Brunete, et al., Selective enhancement of pentaminide uptake in the lungs by aerosolization and delivery in liposomes, Am. Rev. Respir. Dis., 135731 (1987). C. Carpenter and J. Zendegui, A biological assay for epidermal growth fa uorogasterone and related polypeptides, Analyt. Biochem., 153:279 (1985). Y. Akimoto, Y. Mochizuki, A. Uda, et al., Concentrations of ampicillin in
,
of Liposomes
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humanserumandmixedsaliva following a single oraladministration of lenampicillin, J. Nihon Univ. Sch. Dent., 32:14 (1990). 85. I. Schumacher and R. Margalit, Chemical andbiological assays for liposomeencapsulated antibiotics using ampicillin as the test drug, submitted (1995). 86. I. Schumacher and R. Margalit, Physicochemical and biological characterization of liposome-encapsulatedampicillin, submitted(1995).
11
Stealth Liposomes
Danilo D. Lasic*
Liposome Technology,Inc., Menlo Park, California
I. Introduction
1 1 . Liposomes in Drug Delivery 1 1 1 . Stability of Liposomes A. Physical Stability B. Chemical Stability C. Colloidal Stability D. Biological Stability
IV. Sterically Stabilized (Stealth) Liposomes A. Theoretical Understanding of Steric Stabilization of Liposomes B. An Extended DLVO Interparticle Potential V. VI. VII. Coupling of Polyethylene Glycol to Lipids Some Experimental Results and Discussion Applications of Sterically Stabilized Liposomes A. Medical Applications
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*Currentaffiliation: MegaBios Corporation, Burlingame, California.
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INTRODUCTION
Liposomes are spheric$, self-closed vesicles of colloidal dimensions with (phospho)lipid bilayers that sequester part of the solvent, in which they are suspended, into theirinterior [l-31. With respect to their size and number of lamellae, one defines small or large unilamellar vesicles or, in the case of many concentric bilayers, large multilamellar vesicles (Fig. 1) [2]. Owing to their structure, chemical composition, and colloidal size, which can be well controlled by preparation, liposomes possess several properties which can be useful in various applications [3]. The most important properties are small, uniform, and controllable size and special membrane and surface characteristics which include bilayer phase behavior, its mechanical properties and permeability, charge density, the presence of surface-bound or grafted polymers, or attachment of special ligands, respectively. Additionally, owing to their amphiphilic character and very large surface to volume ratios, liposomes are a powerful solubilizing system for a wide range of compounds. Apart from these physicochemical properties, liposomes prepared from natural lipids exhibit many special biological characteristics, including biocompatibility,biodegradability, and (specific) interactions with biological membranes and various cells. Because of these properties, liposomes are used in various applications such as solubilizers for difficult-to-dissolve substances, dispersants, sustained-release and delivery systems for microencapsulated substances, stabilizers, protective agents, and microreactors. In addition, because of
unilamellar vesicle
vesicle
Fig. 1. Schematicpresentation of variousamphiphilicaggregatesandphases formed by different amphiphiles at different concentrations in mixtures with wat Open sphere presents hydophilic part of the molecule while the hydrophobic tail is shown as a wavyline.
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their similarity to thecell membrane, they are used in many studies in cell biology and medicine[2,3]. Bilayers can be made entirely from naturally occurring substances and are therefore nontoxic, biodegradable, and practically nonimmunogenic. This makes them ideal candidates for drug-delivery systems. Apart from well-known endeavors in drug delivery, the industrial applications include liposomes asadjuvants in vaccination, signal enhancers/carriers in medical diagnostics, analyticalbiochemistry, and transfection vectors in genetic engineering. Currently, however, thelargest market is incosmetics, in which liposomes serve as dispersants for various substances and/or as a support matrixfor various ingredients and, according to some reports, as a penetration enhancer [4]. Although some of the claims of cosmetic products are not supported by rigorous scientific tests, one can surely say that liposomes definitely represent an improvement simply because of the fact that hydrophobic substances are suspended in an aqueous and biocompatible matrix as opposedto creams andgels, which use skin-irritating oils, alcohols, and surfactants [3].
II. LIPOSOMES IN DRUGDELIVERY

The optimistic expectations of liposomes as drug-delivery systems have not been met mostly because of their stability problems. Later, we shall show that the problems with the shelf lifestability were in mostcases successfully solved, whereas thestability of liposomes in liposomicidalenvironments of biological systemspresented a greater challenge that only recently has been solved [S-lo]. Numerous experiments have shown that intravenously administered liposomes quickly released the encapsulated or membrane-bound molecules. It was found thatthe leakage can begreatly reduced by the inclusion of cholesterol into the membranes of liposomes. But this did not help much in the fast clearance and sequestration of liposomes by the mononuclear phagocytic (MP) cells of the immunesystem located mostly inthe liver and spleen, which is the major drawback in most of their applications. The first attempts to alter their biodistribution by either surface ligands or membrane composition were undertakenin the late 1970s. The results showed that liposome disposition can be altered, but predominantly within the mononuclear phagocytic system, including the intrahepatic uptake itself. In the absence of an undesired increase of blood circulation times by the presaturation, suppresion, and impairment of the MP system, blood circulation times were prolonged using neutral and small liposomes composedof rigid lipids and cholesterol. The first substantial improvements were achieved by the incorporation of ganglioside G,, or phosphatidylinositol at
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Secondary amine.linkage
Fig. 2. Structure of thepoly(ethy1enegy1col)-lipidmoleculeN-(carbamyl",poly-(ethyleneglycolmethyl ether)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine), normally sodium salt. Usually molecular weights of PEG are 750, 1000, 2000,3000,5000, and 12000. Carbamate and secondary amine linkages are shown. (Courtest of S. Zalipsky, Liposome Technology, Inc.)
5-10 mol% into the bilayer. The best results, however were obtained by substituting these two lipids with synthetic polymer-containing lipids. The longest circulation times were achieved when polyethylene glycol covalently bound to thephospholipid was used (Fig.2). It seems that intermedi5 mol% in the bilayer ate molecular weights from 1500 to 5000 da at about give rise to the longest blood circulation times [lo]. Fig. 3 shows bloodclearance profiles of several different formulations. Surface grafting of polymers offers much better control overthe colloidal properties. In the case of liposomes, surface adsorption of polymers normally leads to leakage of the encapsulated molecules or even possible aggregation of liposomes due to bridging interactions. It is believed that theenhanced stability is due to the mrface-attached polymer which represents a steric barrier to the interacting macromolecules. For this reason, they areoften called sterically stabilized [lo] liposomes, whereas commercially they are often referred to as Stealth (a registered trade nameof Liposome Technology, Inc.) liposomes because of their invisibility to the body's immune system (Fig. 4) [ll].
Stealth Liposomes
l
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Fig. 3. Blood clearance profiles of several liposome formulations. From top to bottom: 2000PEG-DSPE/EPC/Cho1, EPC/Chol (2/1), EPG/EPC/Chol. formulations withmolarratio 0.15/1.85/1. Abbreviations: PEG, methoxypoleythyleneglycol; DSPE, distearoylphosphatidylethanolamine;EPC.partiallyhydrogenatedegg phosphatidyl choline (IV 40), Cholesterol; EPG, egg phosphatidyl gylcerol. 100 nm liposomes were labeled with 67Ga-desferal. (From ref. 59.)
In order tounderstand this phenomenon and owing to the confusion in the field, we shall briefly review the stability of liposomes, and on the basis of this understanding, show how to make vesicle preparations with enhanced stability in a particular medium.
111.
STABILITY OF LIPOSOMES
In liposome research and applications, the term stability is used rather vaguely and is often not properly defined. In the broadest sense, liposome stability involves the stability of the particle and its constituents, including the entrappedmolecules in any, including liposomicidal, environment. This general definition of stability contains various aspects which can bedefined separately but oftensynergistically act together [3]. We shall somehow arbitrary define stability as being a sum and a product of physical,chemical and colloidal constituents. In addition to these inherentstability components, applications of various liposome products require us also to define the stability of liposomes on application, which is often in a liposomicidal (liposome deleterious) environment, and the long-term stability of the product. Most of them are of a pharmaceu-
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Stealth Liposome
(b)
Conventional Liposome
Fig. 4. Schematic presentation of (a) stealth and (b) conventional conventional and Stealth liposome. Polymer chains can be attached on one or both sidesof the vesicle. (Courtesyof Phillip Le Roy Carter, Liposome Technology, Inc.)
tical nature and, therefore one can define biological and shelf life stabilities, which is a cumulative property of several particular stabilities, respectively.
A.
Physical Stability
This aspect of liposomes mostly involves preservation of the liposome structure and entrapment; thatis, the stability of liposome size and shape (size distribution of the sample) and 'the barrier properties of the bilayers for the encapsulated or membrane-bound molecules.
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Both the preservation of the size as well as encapsulation depend on the mechanicalproperties of the bilayers and thermodynamics of the system. Phospholipid molecules in the vesicle membrane are in equilibrium with free molecules in solution at concentration of critical micellar concentration (CMC). The values of CMC for phospholipids are very low, norM, and this practically does not affect liposome properties as mally < encountered in laboratory work. Actually, having in mind these low values and being aware of the slowness of these processes, one can in principle also question the concept of CMC at all. From the kinetic point of view, this looks rather more like a lipid solubility than a quick, dynamic process as observedin micellar solutions [12].
l . Thermodynamic Stability Helfrich has shown that all single-componentbilayers and symmetrical bilayers increase their free energy on curving, because the spontaneous curvature of the system, C,, is zero. For large bilayered fragments, one can assume that the bending is elastic and the excess energy due to curvature can be expressed as
1 E = - K (C, + C,- CO)* + k C,C*
2
where C, and C, are the principal curvatures, C, is the spontaneouscurvature, K is the bending modulus, and k is the bending modulus of Gaussian curvature. In the simplest case of bending a plane into a sphere, principal curvatures are equal and reciprocal to the radius of curvature, R. On closure, the radius of curvature equals the liposome radius, and one can write E=81rK+41rk (2) Assuming K > > k , we shall neglect the last term. Eq. (2) shows the excess energy of the self-closed system due to the bending [12-141. Because E exceeds thermal energy, that is, E-10-50 kT, where k is the Boltzmann constant and T is temperature, the entropy, which is, according to the Equipartition principle, 3/2 kT per liposome, cannot stabilize the system. unstable From 81rK < kT, it follows that liposomes are thermodynamically [3,12,13]. Neglecting k , Eq. (2) shows that theoretically there are two possible ways to produce thermodynamically stable liposomes: entropic stabilizaC, in the case of tion requires E = kT, whereas E = 0 for C, = C, asymmetrical bilayers [151. K is proportional to thethickness of the membrane. Membrane thinning due to the interdigitation of bilayers or action of cosurfactants, as in
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microemulsions, can give rise to K = kT, and entropically stabilized vesicles can be formed. Another way to produce thermodinamically stable liposomes is to create nonzero spontaneous curvature. This can bedone by asymmetrical bilayers or by asymmetrical conditions across the bilayer, such as different values of pH, concentrations of counterions, or interacting (macro)molecules. Furthermore, it is likely that some of the novel lyotropic phases, such as cubic sponge phase, can spontaneously disintegrate into liposome solution on dilution owing to the large critical fluctuations and low values of K . Similar intermediate structures may also occur in some liposomepreparation methods, such as in demulsification of particular reverse phases or in some lipid-detergent mixtures during the preparation of vesicles by the detergent-depletion technique. Here, however, the changes are driven by the removalof the nonpolar phase[3]. The entropically stabilized spontaneous vesicles are not interesting for applications, because owing to their existence, they have low values of K,which inherently reduce their bamer and permeability properties. Alternatively, the existence and stability of these vesicles canbebased on transmembrane gradients which can dissipate. In contrast, asymmetrical bilayers, including some novel amphiphiles, such as bipolar lipids with asymmetrical polar heads, or polyethylene glycol (PEG)-lipids, can make very stable and nonpermeableliposomes with C,, = 0. The stability of liposomes against external perturbations such as dilution can be understood by a very low CMC value of lipids and the large energy required to open a liposome. The energy to create a pore, break up the liposome, and causefusion or formation of liquid crystalline phase in excesssolvent is [161
E, = 2 m r - IT r2y
(3)
where y is the surface tension and the edge interaction at the polarnonpolar interface of exposed bilayer. Contamination of bilayers with single-chain surfactants, which can effectivelyshield the interface, may rather large value ( 20reduce E,, [3,12]. Normally, however,it still has a 50 kT) and pores, even if formed, quickly reseal. This can explain why vesicles can be relatively stable. All this indicates that liposomes can be kinetically trapped into their structures, because they are formed ina nonequilibrium irreversible process in conditions of high excesses of energy, and the energy needed to open or break them can also be = 50 kTfor r > R. Moreover, a simple Gedanken (thought) experiment shows the advantage of kinetically trapped systems as opposed to systems at thermodynamic equilibrium for applications such as microencapsulation. A system in
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equilibrium quickly adapts to the external change, such asdilution, concentration, pressure, the presence of other substances, injection into body fluids, or temperature by a phase change. For instance, micelles or microemulsion droplets quickly disintegrate on dilution (i.e., move in phase diagram into different regions), whereas liposomes made from phospholipids with low values of critical vesicle concentration are stable against dilution. This simple analysis can explain many of the unexpected data from the drug delivery by colloidal carriers. Furthermore, it is interesting to note that liposomes can be stabilized against some phase transitions, such as freeze-thawing and freeze-drying, by the addition of water-soluble cryoprotectants, such as sucrose. This enables manufacturing of freeze-dried liposome formulations.
2. Mechanical Stability According to the theory of smectic liquid crystals, there are three different ways to deform a thin membrane: area dilationkondensation, surface extension at constant area (shear), and bending. Each deformation is characterized by the appropriatemodulus and the knowledge of material properties of the bilayer can be used to improve the behavior of membranes under stresses; that is, prepare more stable liposomes. Deformation can be a consequence of osmotic imbalances, adsorption, hydrodynamic shears, and other forcesand results in changes of vesicle shapes, membrane tension, and possibly rupture and closure with reequilibration of internal and external solutes, topological changes, such as the formation of buds, invaginations, or daughter vesicles, or simply liposome disintegration. Obviouslymechanically stronger,that is, more cohesive, bilayers resist external perturbations better. Micropipette manipulation [l71 studies have shown that the strongestbilayers consist of equimolar DSPC and cholesterol. Quantitatively this observation was explained by a theory in which cholesterol is regarded as a crystal beaker of the gel phase and inducer of chain ordering in the fluid phase without rigidification of the overall phase. This gives rise to the liquid-ordered phase, which has a high degree of conformational order and no resistance totheshear forces. It was found that liposomes behave as tiny osmometers. However, they can sustain large osmotic imbalances before they break. Experimentally, it is known that, for instance, DSPC/cholesterol (3/2 mol/mol) can sustain osmotic pressure of several dozens of atmospheres without bursting. This can be explained by the above arguments of high mechanical strength of membranes. Often when the pressureis too high, liposomes can transiently open, reequilibrate their contents, and quickly reseal, which
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explain minimal changes in size distribution after osmotic shocks in some systems. Bilayers can be also mechanically stabilized by polymerization [3] or by using lipids with fluorocarbon chains [18]. Despite reduced van der Waals attraction among carbon chains, these lipids form stronger andless permeable bilayers owing to stronger hydrophobic interaction [3,18]. Permeability of bilayers depends on the natureof permeating species, mostly on their charge, size, hydrophobicity, and hydration, and can be correlated to the phase behavior and mechanical stability (i.e., cohesivity) of the bilayer and structural defects. In addition, it also influences the bilayer stability against chemical degradation. Although the thermodynamic behavior of bilayers and its correlation to permeability has beenknown for quite some time, the strong correlation betweenthe mechanical properties of the bilayers and the stability of liposomes was realized only recently. With several other surface characteristics, this knowledge allows one tocontrol the stability and interactability of liposomes andrationally design their properties.
B. Chemical Stability
Lipids are very sensitive biomolecules and quickly undergo different degradation reactions. The most common degradation pathways are oxidation and hydrolysis. In general, polar heads are much more stable than the hydrocarbon chains. Hydrocarbon chains, especially the unsaturated ones, are subject to oxidation, whereas hydrolysis pinches off a hydrocarbon chain from the backbone molecule. In the case of double-chain phospholipids, it produces a fatty acid and lyso lipid. Ultimately, it produces two fatty acids, glycerol, and a free polar head. Because the reaction is acid and base catalyzed, it exhibits strong pH dependence [19-211. The protective measures include optimization of pH, normallyaround 6.5, and, preferentially, freezing or drying thesample in thepresence of cryoprotectants. Oxidation is a radical reaction which ultimately results in the breakage of chains or, in the case of two adjacent double bonds, the formation of cyclic peroxides. Protective measures which include the use of saturated lipids and the addition of antioxidants, such vitamin E (a-tocopherol) or BHT, results in a substantial reduction of oxidation and samples can be stable (within tight specifications, normally 5% degradation) for years [3,21]. Chemical and physical stability are closely related. Mechanically strong andwell-packed bilayers reduce theaccess of oxidizing or hydrolyzing agents. This in turn reduces the changesin sizedistribution, fusion, and
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mechanical properties of the bilayer owing to thereduction of the concentration of the degradation products, which influncethe mechanicalcharacteristics of bilayers such as bending and stretching elasticity, molecular reorientation rate (flip-flop), and permeability [3]. Several synthetic lipids, including the ones with fatty acids linked to the glycerol backbone via ether linkage, are chemically more stable. However, they are not biodegradable by mammalian enzymes, and this limits their usein parenteral drugdelivery, whereas they are often used in chemical or cosmetic applications. The lytic rate of various degradative enzymes can be reduced by using lipids with unnatural chirality. For instance, almost all naturally occurring lipids are levorotary, that is, left-handed. Obviously, dextrorotary lipids are less susceptible to the attack of various phospholipases and yield chemically more stable liposomes in biological environments. Of course, chemical stability becomes a much more complex issue when degradable substances are encapsulated. In these cases, the optimal stability is a compromise between various contributions, and in some cases, it may not be achieved even in a frozen or freezekpray-dried liposomal product. C. Colloidal Stability Changes in the particle size distribution in colloidal systems occur mostly via two mechanisms: On the molecular level, this can be an asymmetrical molecular exchange, whereas on the particle level, this is mostly aggregation, flocculation, and/or fusion. The first process is the so-called Ostwald ripening and the latter, in the case of liposomes, aggregation and fusion.
1. Ostwald Ripening
In this process, larger particles grow at the expense of smaller ones. Smaller particles have a larger surface to volume ratioand, additionally in the case of vesicles, higher curnature gives rise to a higher chemical potential. Assuming that the concentration of exchangeable monomersis equal to the CMC, we see that this size disproportionation is negligible for most M. Forsome synthetic phospholipids, which have values of CMC < surfactants, however, the CMC can bemuch higher, and this becomes the dominant s u e growth mechanism. In the case of sodium heptylnonyl benzenesulfonate, with a cmc of of 5 mM, it was shown that vesicles grow proportionally to to;that is, they double their size to 40 nm in40 days [22]. UsingthesameequationsandtheCMC =5 x M of lecithin,onegetsthe kinetics of apparent growth < 5 x 10 nm/day.
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2. Aggregationand Fusion In phospholipid liposome preparations, the increase of the particle size is mostly due to particle aggregation, because the size growth due to Ostwald ripening is negligible. Aggregation is the first step in a vesiclevesicle fusion which depends on the thermodynamic stability of the sample, crystal defects, and the presence of intrinsic or exogenous fusogenic substances or vectors. Fusion is a practically irreversible process, whereas aggregation can be reversible: Often liposome systems are only loosely aggregated, and mild swirling of the sample redisperses liposomes. Here we shall discuss only the origin of aggregation. First,we shall define different forces acting betweenliposomes, comment on the DLVO theory, and develop a more general interparticle potential. Later, we shall use this concept to simulate the interaction of bilayers with macromolecules. We shall not be concerned with the kinetics of aggregation and subsequent fusion, which was reviewed extensively [23,24]. There are several attractive andrepulsive forces which act between colloidal particles. Ubiquitous attractive forceis van der Waals attraction, whkh acts between all particles separated by a medium of different polarizing properties. In the case of charged particles, there is a strong electrostatic force. Mixing two samples of liposomes bearing opposite charges results in flocculation.Mostly, however, particles bear the same charge, and this results in electrostatic repulsion,which leads to a strong repulsive force. Theabove-mentioned van der Waals attractionandelectrostatic repulsion forces are the basis of the DLVO theory of the stability of colloidal particles [25]. In many cases, however, this theory could not explain the stability of liposomes against aggregation. In the case of predicting a too stable colloidal suspension, additional attractive forces, such as hydrophobic attraction or ion-ion correlation attraction, were introduced, whereas to explain the stability of uncharged liposomes and liposomes in high ionic strength media, other repulsive forces were recognized. The aggregation of noncharged liposomes is normally prevented by the repulsive hydration force,which is due to the hydration of polar heads. In order to bring two bilayerstogether, onehas to remove water molecules from the polar heads. This results in a repulsive force and so does the vertical motion of polar heads,which causes the so-called protrusion repulsive force. In soft bilayers that can thermally undulate, another repulsive force was recognized. This undulation forceis due to the excluded volume effect two approaching undulating membranes exert on one another. order In to approach, they have rigidify, to This is entropically unfavored and results in a repulsive force.
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These forces can explain the colloidal stability of conventional liposomes and provide a basis for their stabilization. Historically, mostly surface charge density, zetapotential, size distribution and homogeneity,ionic strength, and pH of the medium were found to determine colloidal stability in vitro. In biological systems, however, the behavior is completely different because of the presence of active liposomicidal substances. Also, ionic strength is around 290 mM. Later we shall show additional repulsive interactions originating from the surface-attached molecules. Since Egyptian times it has been known that lyophobic colloids, such as pigments in inks, can be stabilized by natural polymerssuch as arabic gum, starch, or cellulose. This gives rise to the so-called steric repulsion, which willbe discussed in Part IV.
D. Biological Stability
Even the most stable conventional liposomes are rather unstable in the biological milieu. In addition to interactions with various plasma components and enzymes, an active uptake of liposomes by the bodys immune system takes place (Fig. 5 ) . It has been shown that mechanical stability can help reducethe interactions in vitro and reduceleakage in vivo but cannot slowdown appreciably the uptake of liposomes by phagocytic cells on parenteral administration. It was assumed [6] that the predominant mechanisms of liposome clearance from blood were (1) opsonization by immune and nonimmune components in blood, such as immunoglobulins, complement factor proteins, and fibronectin, which trigger subsequent macrophage uptake; and (2) reaction with plasma (lipo)proteins, which can be expressedas
Fig. 5. Interactions of liposome with cells. There are predominantly four possible interactions: adsorption, lipid exchange, fusion and endocytosis. (From ref. 82.)
310
Lasic L+O===>LO L 0 + R-M ===> M-R 0 L E==> M (L)

(4)
(5)
and PLP(m) PLP

U
PLP PLP(n) PLP

U
PLP(1-m-n)
U
L(1) === > L(1-m) ===
L(1-m-n)
===>
(6)
where: L is liposome containing 1 lipid molecules, 0 is opsonin, M is macrophage, R is receptor for 0 ,PLP are plasma lipoproteins which bind m,n lipid molecules. Improved mechanical stability can greatly reduce liposome disintegration as shown by eq. (6). In order for effective opsonization to take place, a macromolecule has to bind to the liposome surface. We believe that this adsorption can be electrostatic, hydorphobic, H bonding, or physisorption due to van der Waals forces; thatis,
=
west
whfo
+ wH~ond + wspec
( 7 )
where the total adsorption energy contains contributions from electrostatic, hydrophobic, van der Waals, hydrogenbonding, and specific interactions. The latter ones occur mostly when some antibodies or haptens are attached. Obviously, an ideal drug targeting would tend to eliminate all with exception of W,,. Probably in some proteins the contributions toW,of adsorption or adhesion requires conformational change to trigger subsequent macrophage uptake. Similar conclusions may be applied to interaction with plasma proteins. Obviously more cohesive bilayers exchange lipid molecules with the surroundings moreslowly. Very cohesive bilayers in the gel state, however, . containtoo many hydrophobic defects to be stable [3]. This model of liposome uptake can explain the above-mentioned results. Clearly, uncharged liposomes should be more stable, because any strong ionic binding is absent (Wuf = 0). Hydrophobic binding, such as penetration of a segment of the macromolecule into thebilayer, should also be reduced in the presenceof more mechanically cohesive bilayers (reduction of Wh,J. Physisorption, however, is not prevented; that is, W,,, and W,,,,,,", are < 0. Later, we shall show that physisorption can be greatly reduced by the presence of a steric shield of an inert, nonimmunogenic (Wspec = 0), nonionic (W-, = 0) polymer. The absence and reduction of chemisorption, that is, electrostatic and hydropohobic binding, does not reduce the H bonding and physical adsorption of macromolecules onto the liposome. This may explain their lower
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31 1
stability in vivo and their macrophage uptake. In contrast, the presence of a steric shield, as exhibited by a noninteractive surface-attached polymer (shown in Fig.4), prevents orreduces all the differenttypes of macromolecular interactions with liposomes. Results show that in the presence of surface-grafted polymer, both fluid and less cohesive or charged liposomes can exhibit prolonged blood circulation times [26]. This property is important in designing liposomes with well-defined leakage characteristics and containing no cholesterol, as well as improving their encapsulation efficiency for hydrophobic molecules. However, the anchoring of the PEG moiety must be optimized; that is, lipid chains should be distearoyl or should match the fluidity of the bilayer. It seems that in contrasttoelectrostaticstabilization, whichwas found to be effective in many in vitro applications, steric stabilization is a ubiquitous way to stabilize particles in biological systems and in media with variable salinity. Of course, stericstabilization can be imposed on charged bilayers, andin these cases, in vitro stability may be increased, whereasthe stability is not additive in the in vivo case. A combination of the two, that is, electrosteric repulsion in the case of surface-attached polyelectrolytes, would probably increase the stability of liposomes because of thicker and more rigid and mutually electrostatically repulsive brushes in vitro. However, as was discussed before, it is unlikely that it would increase biological stability, because W,, # 0. Polyelectrolyte chains bearing opposite charge to thebilayer may, however, disrupt the bilayer integrity. IV. STERICALLY STABILIZED (STEALTH) LIPOSOMES One of the major breakthroughs in the medical applications of liposomes was the realization that neither mechanical nor electrostatic stabilization can substantially improve the stability of liposomes in biological environment, especially in the systemic circulation. In analogy with the stabilization of inorganic and polymeric (latex, polystyrene beads),colloidal particles polymer coating was imposed on the surface of the liposomes. Because adsorption of block copolymers (polyethylene oxide-polypropylene oxide) caused leakage, lipids with covalently attached polymers were introduced, predominantly polyethylene oxide. Before we describe some practical applications of these systems, we briefly describe the theoreticalbackground of steric stabilization and of the polymer behavior at the interfaces. Fig. 4 is a graphic presentation of conventional and polymer-coated liposomes.
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A. Theoretical Understanding of Steric Stabilization of Liposomes The repulsive forces between the two particles can be greatly increased by coating the surface of the particle with appropriate molecules. Natural and synthetic polymers are the most common materials used. They must be inert, well solvated, and compatible with the solvent and have polarizability close to water. Steric stabilization can be induced by surface attachment of various natural or synthetic polymers, either by adsorption, hydrophobic insertion, electrostatic binding or preferentially by grafting via a covalent bond. Nonionic, water-compatible, flexible, and well-hydrated polymers are preferred. The repulsion between surfaces with attached polymers was shown to bedependent on thegrafting density and the degreeof polymerization. In the low-grafting densities, that is, in the region where the polymer chains cannot interact among themselves, the so-called mushroom modelwas defined which yields steric repulsion at distance, h, as 1271
where
h.=..(%)
and D is average distance between adjacent grafting points, U is the size of the segment, and N is the degree of polymerization. At higher densities where polymers start to interact, this causes their extension and the socalled brush model [27] can be applied. The repulsive force is now
F , ?
= - [(h&)
kT
- (h/hc)34]
These equations explain the repulsion in the case of polymers which form both the so-called adsorption and depletion layer. In the case of depletion layer-forming polymer, the conformation probably looks more like an inverse droplet or pear that alters the density profile but leaves the scaling laws unchanged [28]. Theoretical analysis predicted extension of the chains to be40% longer as opposed to the model above[29]; that is 7/5 R, vs R,, where Flory Radius R, = a M.
B . AnExtended DLVO Interparticle Potential

The stability of colloidal particles against aggregation is normally described by the DLVO theory. This theory is based on theinterplay of the attractive
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313
van der Waals and repulsive electrostatic forces, and the minima in the cumulative potential indicate the stability regions. Obviously, the theory cannot explain the stability of an uncharged liposome suspension. This can be corrected by invoking hydration repulsion. For the most general case, however, we propose that the total potential between the particles should include all the above-mentioned forces, including steric
+ v,, ( 1 1 ) I have also included the hydrophobic attractive force despite that its existence is still controversial. A treatment, analogous to the DLVO for two potentials, shoule be performed to find stability of the system. In a first approximation, potentials could be treated as additive. For rigorous treatment, however, one may expect dependence betweed various terms. It is reasonable to expect the interdependence of electrostatic, hydration, and steric interaction in the case of charged surface and covalently bound polymers due todifferent water structure at the surface. Inthe case of grafted polyelectrolyte chains, however, the conformation of chains themselves is a function of electrostatic interactions and surface charge, and obviously different analyses should be performed.
v,, = - v ,
v,,,+ v,, + v,,, +
VU",
V.
COUPLING OF POLYETHYLENE GLYCOL TO LIPIDS
Apart from various adsorption methods, which are prone to cause leakage of the liposome-encapsulated molecules, PEG polymer chains of various lengths can be covalently coupled to lipids using several different coupling agents. Although shorter oligoethyleneoxide spacers have been coupled to many different lipids [3], the only lipid used for the attachment of longer PEG chains was phosphatidylethanolamine with different chain lengths The and degreesof saturation because of the reactivity of the amino group. reactivity of this group is further catalyzed by thedeprotonation by triethylamine. Several researchers also attached PEG chains on cholesterol, butresults in general do not show prolonged blood circulation times. This is not unexpected, because these molecules, such as cholesterol -0(CH,-CH,-O),,-H, are commercially available as surfactants. This means that PEG-lipid molecules can be quickly lost, especially after dilution, from the bilayer owing to their high CMC values. Synthetic chemistry using other phospho- or sphingolipids has not been worked outyet, or the products mayyield rather unstable and hydrolyzable ester bonds. Polyoxyethylene glycols of different chain lengths, including n = 3, 8, 17,45,70, 115, and 270, have been attached. The polydispersity of the polymer depends on thepolydispersity index, p , of the starting material and virtually does not change during the synthesis.
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Commercial-grade material has p , defined as the ratio of weight over number average molecularweight, around 1.1, whereas BioTech grade hasp = 1.05. Different researchers used different coupling strategies, all adopted from the protein-modification field [30].All of them start with methoxyPEG and as a lipid use phosphatidylethanolamine. Three different synthetic routes were employed using succinyl, cyuronic chloride, and carbamate derivatives yielding ester, secondary amine, and uretane linkage, respectively (Fig. 6). Although the rate constant of hydrolysis of the ester linkage produced by the first reaction probably does not contribute significantly to the liposome uptake, itisless stable than the urethane bond
I
mPEG-N-PE H 3
1. Succinic anhydride 2. N-hydroxysuccinirnideI DCC 3. PE I triethylamine
4
0
1 CD1
Tresyl-cl 2. PE
mPEG-OH
1
mPEG-0
&-PE
2. PE I TEA
Fig. 6. Schematic presentation of four different synthetic routes to link polymer to the lipid. Most frequently used synthesisis the one on the right side. (Courtesy of Samuel Zalipsky, Liposome Technology, Inc.)
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produced by the third reaction. The second coupling method, however, uses a toxic reagent. Owing to the above reasons, we have used the following synthetic route: m-PEG (1) and carbonyl diimidazole in a molar ratio 1:l.l were reacted in dried benzene in nitrogen atmosphere at 75C for 16 hs, yielding carbamate of mPEG ether (2). Coupling 2 to PE was achieved by refluxing2:3:triethylaminein molar ratio 1:2:2 in benzene at 95C for 6 hs. The PEG-PE (4) was purified by reverse phase silica gel chromatography by eluting withch1oroform:methanol or water:ethanol mixtures. Because 4 forms micellesin aqueous solutions, this compound can be purified simply by dialyzing micellar solution against water or preferably against saline. PEG-lipids behave as ionic surfactants in aqueous solutions and the addition of electrolytes appreciably reduces their CMC values; in the case of 2mPEG-DSPE, CMC decreases from mM to 30 p M on addition of salt [31]. The chemistry of the heterobifunctional PEG chains for the subsequent attachment of antibodies to the far end of the grafter polymer chain was also established [32]. Because all the above syntheses use unreactive mPEG on one side, a heterobifunctional polymer derivative had to be prepared. On oneside, it bears the reactive succinimydil carbonate for the attachment to the amino group of phosphatidylethanolamine and on the other tert-butyloxycarbonyl-protected hydrazide group. Various ligands, including monoclonal or polyclonal antibodies, can belinked to preformed functionalized liposomes containing this conjugate owing to the chemical versatility of hydrazide groups. Recently, several new polymers which rendered liposomes long circulating were discovered. Polyethyloxazoline and polymethyloxazoline with a degree of polymerization around 50 and attached to lipid were shown to be equivalent to PEG [33], whereas polyacrylamide and poly(viny1)pyrrolidone prolonged the blood circulation of liposomes to a lesser extent, probably because of a single chain lipid anchor [34]. Preliminary results using several others polymers did not match the prolongation of the blood circulation as achieved by PEG. It seems that they do not combine the same hydrophilicity and flexibility behavior as was found true does PEG or the presence of dipolar interactions. The same for oligo- and polysaccharides, such as glucuronic acid, dextrans, cellulose derivatives, and copolymers of glucuronic acid with simple sugars. These polymers can form a stronger and denser steric shield, possibly at an increased density of hydrogen-bondingsites but at compromised mobility of the chains. Furthermore, there are manyspecific receptors for various sugars in the body.
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Liposomes containing PEG-lipid can be prepared by using virtually all the standard preparation methods. After thin-film hydration, better dispersal is observed and liposomes show reduced lamellarity (number of lamellae). Unilamellar liposomes, produced by various techniques, are generally characterized by enhanced stability. VI. SOME EXPERIMENTAL RESULTS AND DISCUSSION The hypothesis of increased repulsive pressure above membrane and reduced adsorption on the blood circulation lifetimes was tested by measuring the repulsive pressure between membranes with and without incorporated polymer-bearing lipid. Fig. 7 demonstrates that bilayers containing PEG lipid show much larger interbilayer spacings, and even on strong compression, the bilayers are still 4 nm apart as compared with surface unmodified bilayers, which show practical collapse tothe hard wall repulsion [35,36]. These data are comparedwith the theory in Fig. 8. The interbilayer repulsion calculated from the repulsive pressure of surface-attached polymer in a mushroom configuration (broken line), that is, from
P=(
5/2) kTN/D2 a(~/(h/2))~
and for N = 4 4 , a = 0.35 nm, D = 3.57 nm, is in nice agreement with experimental data [36]. For distances > h,, the repulsive pressure is zero. Similar results were also obtained by the surface force apparatus [37]. These results also show increased repulsion with an increasing amount of PEG polymer, whereas biological data find optimal circulation times at lower surface coverages or show a saturationlike behavior at grafting densities which assure complete coating of the surface. Whether this is due to some specific interactions of possibly the flexibility of the chains has not been evaluated yet [lo]. Surface force measurements have found reversible repulsive force at all separations, and the thickness of the steric barrier was found to be controlled by the amount af added PEG-lipid. At low coverages, Dolan and Edwards' theory of steric forces was found to describe experimental data satisfactorily, whereas at higher coverages, Alexander de Gennes' theory described the data better[37]. At low coverages, in the mushroom regimen, the Dolan-Edwardsexpression for force between two curvedcylindrical surfaces of radius, R , can be described by
"
Pc@)
- 7 2 1 r A k T e x p - -h
R,
Stealth Liposomes I
31 7
-60
( , , , , I I
-20
20
60
100
140
Distance from Bilayer Center (A)
201%
8
OO
l21 10 A
V .
0 0 0
20
40
60
80
100
l !O
Distance Belween
Bilayer Surfaces dw
(A)
Fig. 7. (A) Electron density profiles of bilayers with (bottom) and without (top) 2mPEGylated lipid as determinedby S A X S measurements of multilamellar vesicles. ?kro unit cells are shown, and it can be clearly seen that the presence of polymer increases the gap between the bilayers. Furthermore, an increased electron density in the aqueous gap may indicate that the polymer, which is known to form the depletion layer, is stretched away from the bilayer. (B) Pressure versus gapbetweentheadjacentlamellae:solidsquares,stearoyloleoylphosphatidyl 4 mol%of cholinelcholesterol (2/1) bilayers;solidcircles,somebilayerswith 2000PEG-lipid in 1 0 0 mM NaCl and in 1 mM NaCl (open circles). For all the applied pressures, the presence of polymer-bearing lipid markedly increases the aqueous gap betweenthe bilayers. (From ref.36).
318
Lasic
20
40
60
80
100
120
Distance Between Bilayer Surfaces dw
(A)
Fig. 8. Theoretical interpretationof the experimental data from Fig. 8. Experimental data of SOPUChol bilayers containing 4 mol% of mPEG-lipid fit into repulsive pressure calculated for the mushroom model of steric repulsion (see Eq. [7]). (From Ref. 71.)
where Rg is the radius of gyration of the polymer in a theta solvent and corresponds to the thickness of extending polymer. A is the surface coverage of the polymer. At higher grafting densities, that is, in the brush regimen, the force could bedescribed by
F,(h) - 16 kT T L 35D3 R
"
[~($T+~(&T-Iz]
where
L = D ( : ~
The expressions for force between two cylindrical surfaces (F,) and repulsive pressure (F)can be calculated using Derjaguin approximation
Conformational changesin monolayers of PEG-lipids and theirmixtures with phospholipids were measured by film-balance experiments and fluorescent microscopy: two phase transitions were observed-at low coverages pancake-mushroom and at higher mushroom-brush. In the pancake state, phase segregation was observed. Only when a depletion layer was
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319
formed in the mushroom regime, could the lipid molecules intermix with PEG-lipids. Composite polymer-lipid films were deposited on solid substrates and studied by ellipsometry. Disjoining pressures as a function of humidity were studied, and it was found that distance versus pressure curves are governed by steric and electrostatic forces and were in good agreement with surface force apparatusand osmotic stress technique measurements [38]. This simple theory states that higher grafting densities of longer chains result in better stabilization, as experimentally verified above, and presumably longer circulation times. In vivo experiments, however, have shown that the best results were obtained at around 5 mol% of relatively short chains, n = 40-130, and it seems preferably 40-70. This is probably due to several factors. First, in these membranes or surfaces,inclusion of PEG-lipids shows saturation behavior. The overall pressureabovethe bilayer can destabilize liposome by creating hydrophobic defects which then act as sites of attachment for opsonins. Their adsorption may also dependonthe mobility of the polymer chain, which decreasesafter mushroom-brush transition. At higher polymer concentration above the bilayer, the polymer may also exhibit phase transition (collapse of polymer brush) [7].The later hypothesis could be tested by using other polymers which do not have solubility gaps in their aqueous solubility but can still lead to enhancedstability in circulation. The vesicle size dependence of the blood circulation times which decrease with increasing size is still not understood. Although the liver uptake is still low,the spleen uptake increases for 2 R > 200 nm. It is possible that the spleen uptake exhibits size dependence, and such filtering may result in the accumulation of the largersterically stabilized liposomes in the spleen.
VII. APPLICATIONS OF STERICALLY STABILIZED LIPOSOMES
Sterically stabilized vesicles present anew model in studies of polymers and scaling laws, colloid stability, and cell biology. It seems that cells function viaspecific attraction and nonspecific repulsion. Therefore, these liposomes offer a much better model for thestudy of cell function and interaction than conventional liposomes, which can exhibit many nonspecific attractive interactions. This property of sterically stabilized liposomes offers several useful applications, mostly in diagnostics and drug delivery. In basic sciences, scaling laws of polymer behaviour near various interfaces can be experimentally verified. This knowledge can be applied to the biomaterialsscience for designing the surfaces which come into contact with body fluids, mostly blood.
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The phase behaviour of various liposomes containing various amounts of PEG-lipids with various chain lengths (well-defined HLB values, HLB = hydrophobic lyophobic balance) can be used to characterize further the liposome formation and stability.
A. Medical Applications
Stealth liposomes provide an entirely new and unique drug-delivery vehicle because of their ability to evade quick clearance by the immune system. Using such liposomes, the goal of specific cell targeting became theoretically possible. Indeed, early studies of Stealth liposomes labeled with antibodies have shown increased delivery of antibody-labeled Stealth liposomes to the targettissue in accessible sites [39]. The accesible sites are in the vascular compartment,at sites of increased vascular permeability (which occurs naturally at the sites of infections, inflammations, tumors, and other traumatic conditions, or it can be induced artificially) on systemic administration and in the peritoneal cavity and in the centralnervous system on localized administration. This was also the case in nontargeted Stealth liposomes, which represent the majority of applications and will be discussed below. A remaining problem is the delivery of the encapsulated drug into the cell, because the majority of cells are not phagocytic and fusion of liposomes and cells is a very rare phenomenon. One approach to overcome this difficulty is to promote fusion by surface-attached fusogenic proteins or polymers, whereas a simpler way is to try to induce leakage of liposomes by changes in temperature or pH or toprepare liposomes with compromised stability. Although this is one of the current hot issues in liposome research, several applications are not affected by the apparent lack of drug bioavailability (see below). These applications include the anthracycline anticancer drugs. The agents can be loaded at very high concentrations (normally 1 g drug/7.5 g lipid; see Fig. 9) and have shown great improvements in therapeuticefficacy on encapsulation into Stealthliposomes. According to current understanding [l,6], the prolonged presence of liposomes in blood allows them to extravasate intosites where the vasculature is leaky. This often happens to be in tumors [ll],and indeed experiments in animals and humans have shown increases in drug concentration in tumors as compared with the administration of the free drug [40-471. In thesecases, it seems that liposomes are trapped in the tissue where they slowly release their contents either by leakage or on degradation by enzymes, such as phospholipases. This coincides with the observation that peak drug levels in tumors are observed 3-4 days after liposome administration as compared with a few hours for the free
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Fig. 9. Cryoelectron micrograph of unilamellar vesicles with encapsulated drug. Doxorubicin can be encapsulated at very high lipid concentrations using various gradients. In our case, we have used ammonium sulfate gradient in which weak base NH, with pK = 9.3 exchanges with weak-base doxorubicin HCl, pK = 8.3. When doxorubicin enters liposome its charged form precipitates with sulfate and of doxorubicin until most this molecular sink keeps the chemical potenial gradient of the drug is precipitated. This results in very high concentrations the liposome in interior,andthedrugsolidificationgivesrise to peculiarchanges of liposome shapes. (Micrograph: Courtesy of P. Frederik, University of Limburg, Maastricht).
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drug [40]. At the peak level, the concentration of the drug can be 10-30 times superior in the case of Stealth liposome-encapsulated drug [ll]. Fig. 10 shows the effect of various treatments on the tumor size in a C26 solid tumor mouse model, which is practically insensitive to treatmentwith free adriamycin and treatments with conventional liposomes containing the controls drug [41]. Placebo liposomes, and their mixture with free drug, as did not have anysignificant effect over the untreated(i.e., saline-injected) animals. The administration of Stealth liposomes containing Adriamycin (SDox), however, resulted in complete remission of tumors in the case of early treatment and in significant improvements in the delayed treatment (Fig. 10). Practically identical results were obtained with the very similar drug epirubicin [42]. Improvements were also achieved in other resistant tumor models, such as mammary carcinoma. The S-Dox formulation was substantially more effective not only in curing mice with recent implants from various tumors but also in reducing the incidence of metastases originating from Fig. 11. these intramammaryimplants [43], as can be seen from Arrest of the growth of human lung tumor cells wasachieved with the S-Dox formulation [44]. The datashow that atequivalent doses, S-Dox can arrest the growth, whereas free drug and in drug conventional liposomes do 1.1 cm3/week in not. Both only decrease the size growth from approximately untreated animals to approximately 0.5 cm3/week, which is in agreement with previous observations that conventional liposomes are effective mostly in the treatment of experimental liver metastasis. Treatment with S-Dox also shows a linear dose response[M]. One hundredpercent of mice survivedto 2 mg/kg weekly for 6 weeks. The week 12 when the formulation was dosed at mice appeared healthy and active with minimalbody weight losses (2-19%) (Fig. 12). The same extravasation mechanism of small liposomes at sites of leaky vasculature also can beused intreatment of inflammations and infections, which are also characterized by enhanced vascular permeability [3,10]. For instance, 10-fold improvements in the liposome concentration lung infected with Klebsiellapneumoniae [49]. Because many fungal, bacterial, and viral infections do not reside in the liver andspleen, longcirculating liposomes loaded with antifungals, antivirals, and antibiotics may also improve the treatment of some disseminated infections [3]. Furthermore, since it seems that many Stealth liposomes end up in the skin, deep tissue macrophages, and lymph nodes, it is possible that they can eradicate parasites in these sites, which are very difficult to target, and on the other hand, often harbor infections, possibly including human immunodeficiency virus (HIV) andMycobacterium avium.
'
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0 L
0 lL
c
L
t i
9
Y)
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0 1 2 3 4 5 6 7 8 9 1 0 WEEKS
6.0
.
-
g 5.0
4.0
WEEKS
6.0
0
C
-
W 4.0
1 2 3
5 6 7
9101112
WEEKS
Fig. 12. Growth of human lung tumor cancer in SCID mice which were engrafted with 2 million "L-1 cells and given weekly injections of nothing (O), doxorubicin in conventional liposomes (A),doxorubicin in Stealth liposomes (m). Open squaresrepresentdelayedtreatmentwithStealthliposomescontaining doxorubicin. The two experiments were performed 10 with and 5 animals (A and B, respectively). The fraction of surviving miceafter the treatment is shown in parenof a dose theses. (C) The same treatment as in (A) and (B) for the determination response: from top to bottom: untreated mice (O), 0.5 mgkg 1 mgkg (A), and 2 mgkg (0)body weight.
(e),
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Although many new drugs are being currently monitored and tested for encapsulation in sterically stabilized liposomes, phase I and 1 1 clinical trials of adriamycin encapsulated in Stealth liposomes in humans have yielded very encouraging results. It was found that blood circulation times in humans, as measured by the plasma concentration of the drug, are around 45 hs. This results in greatly reduced bone marrow supression, neutropenia, and leukopenia, as well asshifted biodistribution of doxorubicin versus tumors as observed in AIDS patients with Kaposis sarcoma [3,11,50,51]. Unfortunately, but notunexpectedly, the altered (and unnatural) biodistribution may give rise to unusual toxicities. In this case, the dose-limiting factor seems to be stomatitis, which was observed in some patients [1l]. Another possibility of long circulating liposomes is their use as a blood substitute. Several hemoglobin-based red blood cell substitutes with desirable physicochemical properties are too toxic and not safe(hemostasis and thrombosis) forin vivo applications. It was shownthat sterically stabilized liposomes with encapsulated hemoglobin showed a significant decrease in immunotoxicity [52]. Future developments include targeting of Stealth liposomes, spleen targeting by large sterically stabilized liposomes, long circulating microreservoirs or platforms fortherapetic activity (prevention of septic shock), and potential development of oral and topical liposome formulations. In conclusion, polymer-grafted liposomes and their applications show an interplay of various sciences, from theoretical polymer physics, colloid and interface science, organic chemistry, to biology, immunology,pharmacology, medicine, anatomy, and oncology [3,10]. As a result, very stable liposomes in biological environmentswere designed and showed remarkable therapeutic potential. The next goals are now to improve spatial and temporal control of the release of the encapsulated drug. Targeting can be improved in physically accessiblesites by the attachmentof antibodies to the of the temporal release of the drug will end of polymer chains. Better control be tackled by three approaches:physical approaches use temperature, pH, or (target) binding-sensitiveinduced phase change; chemical approaches try to destabilize liposome at a particular location by a chemical change of membrane-forming lipid; whereas biological approaches can use surfaceattached fusogenic proteins and polymers or stimulate internalization. We dare to conclude that sterically stabilized vesicles are an example of how complex and elusive problems can be solved by a multidisciplinary approach while for the continuation of this development even broader interdisciplinary work will be required. I believe that other polymer-liposome systems will
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be developed inwhichpolymerswillhaveadifferentfunction,suchas or fusion characteristics. inducing specific binding, permeability,

REFERENCES
W. Home, J. Mol. Biol., 8:660-668 (1964). 1. A. D. Bangham, and R. 2. D. Papahadjopoulos ed., Ann. N.Y. Acad. Sci., 308:l-412 3. D. D. Lasic, Liposomes: From Physics to Applications, Elsevier, Amsterdam, 1993. .Braun Falco, H. C. Korting, H. I. Maibach eds., Liposome Dermatics, 4. 0 Springer-Verlag, 1992. 5. D.Papahadjopoulos, et al.,Proc.Natl.Acad.Sci.USA,88:11460-11464 (1991). 6. D.D.Lasic, F. J. Martin,A.Gabizon, et al.,Biochim.Biophys.Acta, 1070:187-192 (1991). 7. M. C. Woodle andD. D. Lasic, Biochim. Biophys. Acta, 1113:171-199 (1992) 8. L. Huang (ed.),J. Liposome Res., 57:109-430 (1992). 9. T .M. Allen, Adv. Drug Del. Rev. (in press). 10. D. D. Lasic, Angew. Chemie Int. Ed. Engl., 33:1685-1699 (1994). 11. D. D. Lasic and F. Martin eds., Stealth Liposomes, CRC Press, Boca Raton, FL, 1995. 12. P. Mukherjee, personal communication. 13. W. Helfrich, Zeit. Naturforsch., 28c:693-703 (1973). 14. D. D. Lasic, Biochim. Biophys. Acta, 592501-502 (1982). 15. D. D. Lasic, Nature,35279 (1992). 16. K. H. Klotz, M. Winterhalter, and R. Benz, Biochim. Biophys. Acta, 11471161-164 (1993). 17. D. Needham and R. Nunn, Biophys.J., 58:997-1009 (1990). 18. C. Santaella,P. Vierling, andJ. G. Riess, Angew. Chem., 30567-568 (1991). 19. M. Grit, J. H. deSmit, A. Struijke, and D. J. A. Crommelin, Int. J. Pharm., 50~1-6 (1989). 20. M. Grit and D.J. A. Crommelin, Chem. Phys. Lip., 62:113-122 (1992). 21. F. J. Martin, inSpecializedDrugDeliverySystems (E. v l e , ed.), Marcel Dekker, New York, 1990, pp. 267-316. 22. E. W. Kaler,A.K.Murthy,B. E. Rodriguez,and J. A. N.Zaszadinski, Science, 245:1371-1374 (1989). 23. S. Nir, J. Bentz, J. Wilschut,and N. Duzgunes, Prog. Surf. Sci., 13:l-124 (1983). 24. S. Okhi, D. Doyle, S. Hui, and E. Mayhuew eds., Molecular Mechanism of Membrane Fusion, Plenum Press, New York, 1988. 25. J. N. Israelachvili, Intermolecular and Surface Forces, Academic Press, New York, 1985. 26. M. C. Woodle, et al., Biochim. Biophys. Acta, 1105:193-2OOO (1992). 27. P. G. deGennes, Adv. Colloid. Interface Sci., 27:189-209 (1987).
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28. P. G. deGennes, Personal communication, 1992. 29. K. Hristova and D. Needham, in ref.11, chapter 5,35-50. 30. S. ZalipskyandC.Lee,inPoly(ethy1ene)GlycolChemistry, J. M. Hams (ed.), Plenum Press,New York, 1992, pp. 347-370. 31. S. Zalipsky, in ref.11, chapter 9,93-102. 32. S. Zalipsky, Bioconjugate Chem., 4:296-301 (1993). 33. M. C. Woodle, C. Engbergs, and S. Zalipsky Bioconjugate Chem., 5:493-497 (1994). 34. V.P. Torchilin, et al., Biochim. Biophys. Acta 1195,181-185, 1994. 35. D. Needham, T. J. McIntosh,andD. D. Lasic,Biochim.Biophys.Acta, 1108:40-48 (1992). 36. D. Needham,K, Hristova, T.J .McIntosh, et al., J. Liposome Res.;2:411-430 (1992). J. N. Israelachvili,Biophys. J., 37. T.Kuhl, D. Leckband,D.D.Lasic,and 66~1479-1488 (1994). 38. T. R. Baekmark, G. Elander, D. D. Lasic, and E. Sackmann, Laugmuir (in press). 39. H. F. Dvorak, A. J .Nagy, J. T. Dvorak, and A. M. Dvorak, Am. J. Pathol., 133:95-108 (1988). 40. A. Gabizon, CancerRes. 52:891-896 (1992). 41. E. Mayhew, D. D. Lasic, S. Babbar, and F. J. Martin, Int. J. Cancer, 51:302309 (1992). 42. S. K. Huang, et al., Cancer Res., 52:6774-6781 (1992). 43. J. Vaage, E. Mayhew, D. D. Lasic, and F. J. Martin, Int. J. Cancer, 51:942948 (1992). 44. S. Williams, et al., Cancer Res., 53:3964 (1993). 45. P. Working, et al., J. Liposome Res., 4:667 (1994). 46. J. Vaage and E. Barbera, in ref.11, chapter 14,149-172. 47. D. D. Lasic, in ref11, chapter 13,139-148. 48. T. M. Allen, in ref 11, chapter 16,187-196. 49. I. M. K. Baker-Woudenberg and M. C. Woodle,J. Infect. Dis. (in press). 50. J. Boggner andF. Goebel, in ref. 11, chapter 23,267-278. 51. D. Nortfeld,et al. in ref 11, chapter 22,257-268. 52. R. Beissinger, et al., J. Liposome Res., 3575 (1993). 53. D. D. Lasic, Amer. Sci., 80:20-31 (1992). 54. G. R. Haran, R. Cohen, L. K. Bar, and Y.Barenholz, Biochim. Biophys. Acta, 1151:201 (1993). 55. D. D. Lasic, et al., FEBS Lett., 312:255-259 (1992).
12
Nonionic Surfactant Vesicles Containing Estradiol for Topical Application
Don A. van Hal, JokeA. Bouwstra, and Hans E. Junginger
LeidentAmsterdam Center forDrug Research, Leiden University, Leiden, The Netherlands
I. Introduction
11. Nonionic Surfactant Vesicles A. Preparation B. Physical State C. Drug Entrapment
330 330 331 333 333 333 334 335 336 336 337 337 338 338 339
111. Liposomes, Micelles, and Transferosomes

IV. Transdermal Application of Vesicles V. Interactions Between Vesicles and Skin A. Interactions Between NSVs and Skin 1. Transport Studies 2. Thermodynamic Activity 3. Penetration Enhancement by Surfactants 4. Increase in Drug Transport by Vesicles 5 . Physical State
VI. Summary
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INTRODUCTION
About 17 years ago, vesicular systems were added to the armamentarium for delivering drugs to or through the skin: nonionic surfactant vesicles (NSVs) [l] and lipid vesicles (liposomes) [2]. In this chapter, the term vesicles stands for both liposomes as well as NSVs. The application of vesicular systems in cosmeticsand for therapeutic purposes may offer several advantages: The vesicle suspension is a water-based vehicle. This offers high patient compliance in comparison with oily dosage forms. The vesicles allowthe incorporation of hydrophilic, amphiphilic, and lipophilic drugs. The vesicles supply both a lipophilic and an aqueous environment for the encapsulation of drugs. The characteristics of the vesicle formulation are variable and controllable. The vesicle characteristics can be controlled by altering vesicle composition, size, lamellarity, trapped volume, surface charge, and concentration. Biocompatible and biodegradable systems maybe designed. The vesicles may act as a depot, releasing the drug in a controlled manner. The vesicles may protect the drug from degradation. Because liposomes consist mainly of phospholipids, and NSVs are mainly prepared from nonionic surfactants. Differences in characteristics exist, between liposomes and NSVs, especially since NSVs are prepared from unchanged single-chain surfactants and cholesterol, whereas liposomes are prepared from double-chain phospholipids (neutral or charged). One of the advantages in using NSVs is the higher chemical stability, lower costs of the chemicals, and the large number of classes of surfactants available to design these vesicles on demand.
II. NONIONICSURFACTANTVESICLES NSVs consist of one or more nonionic surfactant bilayers (Fig. 1) enclosing an aqueous space. NSVs consisting of one bilayer are designated as small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs). Vesicles with more bilayers are called multilamellar vesicles (MLVs). Since1975[3]vesicleshave been prepared from synthetic surfactants. Various synthetic surfactants that have been used for the preparation of vesicles are listed in Table 1. For vesicles prepared from nonionic surfactants, we prefer to use theterm nonionic surfactant vesicles, or
Estradiol-containing NSVs
..
.
33 1
.
Fig. 1 Structure ofnonionic surfactant aggregates: L W (left), micelle (middle), MLV (right).
NSVs. The NSVs are alreadyused in cosmetics by L'Oreal (Paris, France), and they are still being tested for their therapeutic potential. In our laboratory NSVs, have been prepared from two groups of surfactants [22,30,31]:
1 . Polyoxyethylene alkyl ether surfactants (GEO,,,),in which m'rep-
resents the number of oxyethylene units and n represents the number of carbon atoms in the hydrocarbon chain. q2EQ, C,,EC4, and G=9E0,0 (Brij96) form liquid-statevesicles and G,EQ and q,EQ form gel state vesiclesoat room temperature. 2. . Sucrose ester Wasag-7. Wasag-7. consists of 70% stearate ester and 30% palmitate ester(40% mono, 60% di/tri-ester) and forms gel-state bilayers at room temperature. These surfactants were chosen because they are relatively nontoxic.
.. A .thorough description of the formation and properties of these types of vesicles from nonionic surfactants i s given by Hoflandet al. [22].and Van Hal [31].
A. Preparation
NSVs can be formed using the same methods that are used fur the prepara. .. . tion of liposomes [32].
1 . ." Sohication Method [30,31] The lipophilic components are dissolved in a n apolar solvent,such a s a mixture of chlorofoni and methanol. The solvent evaporates overnight
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Table 1. Vesicle Preparation from Synthetic Surfactants Surfactant

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References
Fatty acids Polyglycerol alkyl ether or esters and polyethoxylated analogues Alkyl glycoside surfactants Sucrose esters Polyoxyethylene alkyl ether surfactants Triethoxylated cholesterol Glyceryl dilaurate or distearate with polyoxyethylene-10-stearyl ether Crown ether surfactants Polyoxyethylene di-alkyl ether surfactants Diphenylazomethine ammonium amphiphiles
2,s-l7 18-20 21 22
23
Ufasomes Nonionic surfactant Vesicles, niosomes AGVs Lipid vesicles Nonionic surfactant Vesicles, NSVs Novasome Niosomes Bilayer membranes, vesicles Bilayer assemblies
24 25,26 27 28,29
under vacuum, resulting in a surfactant film. The film isthen hydratedwith aqueous solution and the mixture is sonicated at 80C. The method results in the formation of both LUVs and MLVs.
2. Ether Injection Method [7] The oil-soluble components are dissolved in diethyl ether and injected intothe aqueous phase at 60C. The method results in the formation of mainly MLVs.
3. HandshakingMethod [7] The apolar ingredients are dissolved in a mixture of chloroform and methanol. The solvent evaporates overnight under vacuum. The resulting surfactant film is hydrated with an aqueous solution and gently shaken at 50-60C to allow the formation of vesicles. This preparation method yields MLVs. 4. Reversed Phase Evaporation [l81 The oil-soluble components are dissolved in a apolar solvent such as chloroform. The aqueous phase is added and the mixture is sonicated to form an emulsion. The solvent evaporates overnight under vacuum. The resulting suspension is shaken to form the vesicles. Mainly MLVs are formed.
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5. Method as Described by Handjani-Vila [5]

The surfactants are added to the aqueous phase and the mixture is agitated to yield a homogeneous lamellar phase. The suspension is then homogenized by means of agitation or ultracentrifugation.
B. Physical State
The bilayers of the vesicles are either in the so-called liquid state orin gel state (Fig. 2), depending on the temperature, the type of lipid or surfactant, and the presence of other components such as cholesterol. In the gel state, thealkyl chains are present in a well-ordered structure, andin the liquid state, the structure of the bilayer is more disordered.The surfactants and lipids are characterized by the gel-liquid transition temperature, Tc [33]. In general, the action of cholesterol is twofold: On the one hand, cholesterol increases the chain order of liquid-state bilayers, and on the other, cholesterol decreases the chain order of gel state bilayers [34]. At a high cholesterol concentration, the gel state is transformed to a liquidordered phase. C. Drug Entrapment The NSVs have proven to be able to entrap several model compounds and active substances, such as carboxyfluorescein [7,22,31], sucrose [M], the DGAVP peptide [35], estradiol [30,31,36], and dithranol [31]. The entrapment efficiencies of NSVs are in the same order of magnitude as in liposomes. The exact efficiency depends on the drug, vesicle composition, and preparation method. The entrapment efficiencies and maximum capacities of lipophilic drugs decrease as the cholesterol content increases [31].
111.
LIPOSOMES,MICELLES,ANDTRANSFERSOMES
Liposomes are vesicles consisting of various types of phospholipids [32]. These amphiphilic molecules can be neutral, ionic, or zwitterionic. Like
Gel state
Liquid state
Fig. 2 Gel state and liquid state of bilayers without cholesterol.
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NSVs, they consist of bilayers,enclosing-anaqueous core and are capable of encapsulating lipophilic, hydrophylic, and amphiphilic drugs. Liposomes can vary in sizebetween 50 nm and several micrometers., A micelle (see Fig. 1) is another surfactant aggregate that is able to entrap lipophilic molecules in an aqueousmedium. They are much smaller than liposomes and contain no aqueous core or lipid bilayers. Transfersomes [37-411 consist of a mixture of a lipidic agent with a surfactant. The bilayers of transfersomes are much more elastic than those of most liposomes. A transfersome is one or two orders of magnitude greater in size than mixed-lipid micelles. Furthermore, transfersomes contain an aqueous core enclosed by bilayers. Although lipophilic and amphiphilic drugs may be incorporated in transfersomes as well, their unique drugs such as, for potential is to act as carrier for encapsulated.hydrophi1ic example, insulin [39].
IV. TRANSDERMAL APPLICATION OF VESICLES The effectiveness of vesicles has been investigated by several research groups (Table 2). Liposomes, in particular, have received a great deal of attention. In several studies the diffusion of a drug was facilitated or achieved certain selectivity into humanor nonhuman skin by vesicleencapsulation. Others studies show that theinfluence of liposomes on drug trans? port is negligible. Our group has investigated the potential of NSVs to human stratum corneum increase .the diffusion of estradiol through [30,31,36,42,43].
Table 2. Effect of Liposomes and NSVs on the Permeation of Drugs Through in the Skin in Vitro and Vivo .. .
Effect Liposomes No or decreased effect of vesicles Superiority of liposomes In vitro In vivo animal In vivo human vitro In studies In vivo animal In vivo human In vivo animal In vitro' human References
3,649 5 0 5 2 53,s ' 24,56-67 1,56,58,68-82 79,83-92 38, .39 31,36,42
,
Transfersomes Superiority of transfersomes NSVs Superiority of NSVs
':
Esriadiol-containing NSVs
335
There are three reasons for the controversy found in the literature data with regard to the effects that vesicular formulation may achieve: The first reason is the natureof the control formulation in the diffusion studies. The effect of the liposome formulation was compared with aqueous solutions of drug or emulsions, ointments, creams, gels, or suspensions. The second reasonis thefact thatvarious drugs have been used,such as steroid hormones, glucocorticoids, anti-infectives, local anesthetics, retinoids, and minoxidil [M]. NSVs have been studied for the topical administration of pyrrolidone-carboxylate [2] and estradiol [36]. A thirdreason is that liposome formulations with completely different chemical and physical properties have been tested. All of these factors influence the thermodynamic activity of the drug in the formulation, a parameter which determines the driving force for thedrug to diffuse from the formulation to the skin [45]. The thermodynamic activities of the drugs in the tested vesicle . . formulations are therefore very likely to differ.
V. INTERACTIONS BETWEEN VESICLES AND SKIN
Increased drug transport through the skin may be achieved by specific interactions between vesicles and skin. Because the vesicles may alter the structure of the skin, a thorough knowledge of the normal structure of the skin is important. The vesicles may alter the structure of the skin in such a way that the diffusion of the drug is facilitated. The interaction can be influenced by the chemical characteristics of the vesicles, such as .the amount and type of surfactant,membranestabilizer, or charged compounds. The physical characteristics include the physical state (gel or liquid state) of the bilayers, the number of bilayers, or the size of the vesicles. The interaction may also be influenced by skin characteristics: hydration of theskin, sweat production, skin microflora, sebum,and cellular debris. Three mechanisms bywhich vesicle-associated effects occur have been described so far: (1) penetration of intact vesicles, (2) adsorption of vesicles onto the skin surface, and (3) penetration of vesicle constituents into the skin (Table 3). The so-called transfersomes are also listed in Table 3. Cevc reports [39] that these .vesicles areelastic enough to move through the constrictions of the skin.. .The transfersomes are thought to exhibit their action owing to hydration forces and osmotic gradients [37,38]..It is claimed that drugs which are applied by transfersomes are able to reach an almost 100% maximum biological or therapeutic effect, which is comparable to . injectioil [39].
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Table 3 . Overview of the Reported Mechanisms on Vesicle Action Mechanism Penetration of intact vesicles 1. Into the stratum corneum and into the deeper layers of the skin The vesiclesare considered to cross the biological bamer and may even target their constituents to specific sites Adsorption of vesicles to the surface of the skin This hypothesis comprises the idea that vesicles adsorb to the surface of the skin. This would: 1. Allow a direct transfer of the drug to the stratum corneum 2. Induce an occlusive effect 3. Result in a gradient of thermodynamic activity Penetration of vesicle constituents This hypothesis comprises the disruption of the vesicles and penetration of molecularly dispersed material or bilayer fragments into the stratum corneum. VesicletReferences Liposomes Pro: 1,78,90,93 Transfersomes Pro: 38-41
Liposomes Pro: 47,50,55,62,67,73,87, 94-97 NSVs Pro: 31,42,43
Liposomes Pro: 55-57,59,60,63,65,67,68, 72,87,88,95,97,.98-103 NSVs Pro: 31,42,43
A.
Interactions Between NSVs and Skin
l . Transport Studies Our transportstudies using estradiol [30,31,36] have revealedthat the effect that NSVs have on the diffusion of the drug through the stratum corneum in vitro depends on the vesicle composition and the thermodynamic activityof the drug. Incorporation of estradiol ingel-state Wasag-7:CHOL:DCP (CHOL, cholesterol; DCP,dicetylphosphate),Wasag-7:CHOL:DCP, and C,,Eq:
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CHOL NSVs resulted in an increase in estradiol steady-state flux by a factor of 2.2,2.4, and1.5, respectively [31]. NSVs of G,EQ did not have a significant effect on the estradiol penetrationas compared with the control [36]. The steady-state flux could be increased by a factorof 4.8 by incorporation of estradiol intoliquid-state C,,EO,:CHOL (1:0.43) NSVs [31]. Decreasing the CHOWSURF molar ratio of G,EO,:CHOL from 0.43 to 0.18 resulted in an increase in steady-state flux. Compared with the control, the flux was increased by a factor of 9.5. The flux increase was even higher in liquid-state Brij96:CHOL and G,EO,:CHOL vesicles, being a factor of approximately 40, respectively [36].
2. Thermodynamic Activity The first important parameter that influences the penetration of a drug is the thermodynamic activity of the drug in the formulation, which is the driving force for diffusion of the drug into the skin. This so-called push-effect [45] is equal in samples at saturation level, where the thermodynamic activity reaches a value of 1. In order to check the differences in thermodynamic activity between the vesicle suspensions and the control solution (estradiolsaturated phosphate buffer saline (PBS)), reversed membrane experiments were carried ou$ The stratum corneum and Silastic sheeting were mounted into diffusion cells with the Silasticsheeting facing the donor chamber. The experiments thatwere carried outwith liquid-state G,EQ:CHOL (1:0.43) vesicles and with gel-state Wasag-7:CHOL:DCP vesicles showed 1.8that the diffusion of estradiol by means of NSV suspensions was a factor 2.2 higher than thediffusion of estradiol applied in estradiol-saturated PBS [31]. A possible explanation for the results is a higher estradiol thermodynamic activity (push-effect) in the NSV suspensions as compared with the PBS solution. This may indicate that theNSV suspensions were supersaturated. This corresponds to the meta-stable state of estradiol incorporation [311.
3. Penetration Enhancement by Surfactants In drug transport studies, one has to consider a second mechanism whichis associated with thesurfactants of the vesicles. Animportant mechanism that may be responsible for this increase in steady-state flux is that theliquid state G,EO,:CHOL [31], G,EO,:CHOL, and Brij96:CHOL vesicles [36]also appeared to have a penetration enhancement effect, which is called the pull-effect [45]. This was confirmed by pretreatment experiments. The stratum corneum was pretreated with empty NSVs prior to application of the drug control solution [31,36]. Pretreatment with liquidstate G,EQ:CHOL (1:0.18) vesicles resulted in an increase of the steadystate estradiol flux by a factor of 2.9 [31], which is clearly lower than the
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factor 9.5 that could be achieved by application of estradiol-loaded vesicles. Pretreatment with liquid-state G,EQ:CHOL and Brij96:CHOL also increased the estradiol flux, but with a clearly lower factor, as compared with the- estradiol-loaded formulations ,[31]. Pretreatment with gel-state G,EQ:CHOL vesicles revealed that.gel-state surfactants did not increase the estradiol flux [36]. the liquid-state G=,EO,,,:CHOL vesiThe increase in f l t q induced by cles is partly caused by4he action of the surfactants with skin lipids of the stratum corneum. The surfactants can act as penetration enhancers. The addition of estradiol in G=,EO, micelles increased the estradiol flux by a factor of 2 [36]. Probably the G=,,EQ, micelles are able to penetrate molecularly dispersed stratum corneum and lower the barrier function.
4. Increase in Drug Transport by the Vesicles . However,'for the G,EQ/CHOL(1.:0.18) formulation, the penetiation of surfactants is not a valid explanation, as could be deduced frdm ,an experiment that involved.the diffusion of estradiol from G,EQ micelles. The addition of estradiol to this formulation had no effect on the diffusion throughstratum ,corneu'm [31]. A. change in thestructure of stratum corneum incubated with G,EQ:CHOL NSVs could only occasionally be visualized using freeze-fracture electron microscopy (FFEM) when' compared with stratum corneum. that was treated with PBS for 48 h '[31]. However, the observed minor changes in stratum corneum structure cannot explain the increased transport rate of estradiol. The vesicular form of G,E07, which is induced' by the addition of a small amount of cholesterol, appears to be crucial for obtaining a strong increase in estradiol flux. This is also confirmed by the fact that occlusive buffer application of estradiol in a G,EO,:CHOL formulation in phosphate (PB), which probably contains micelles instead of vesicles, had noeffect on the flux as comparedwith the control situation [31]. To achieve a maximum effect, estradiol has to be associated with the vesicles. The importance of encapsulation of estradiol in NSVs is confirmed ,by the results of Hofland et al. [36]. The penetration,of bilayer constituents into the stratum corneum probably facilitates the simultaneous diffusion of estradiol when estradiol is . . . associated these to bilayers. . . . 5. Physical State The physical state of the NSVs is another important parameter n i flux enhancement. The results showed that liquid-state .vesicles could.strongly increase the flux [31,36], whereas gel-state vesicles resulted in only a slight flux increase [31] or nomcrease at all [36]. The flexibility.of the' bilayers is decreased by an increase in CHOW .SURF molar ratio andby the use of surfactants forming gel-state bilayers.
' '
'
339
The fact that an increased cholesterol content of C;,EQ:CHOL vesicles decreased the steady-state flux of estradiol confirms this hypothesis. Furthermore, the results show that the increase in estradiol flux issmaller after the application of gel-state bilayers compared with liquid state vesicles [31,36]. The fact that the gel-state vesicles from Wasag-7:CHOL:DCP exert almost the same steady-state flux in the reversed membrane experimentas in the transport studies [31] shows that the main mechanism behind the steady-state flux increase concerning gel-state vesicles is achieved through differences in thermodynamic activity. These vesicles, and probably also the vesicles from the other gel-state vesicles Wasag-15:CHOL:DCP and C,,EO,:CHOL, do not lead to an increase in drug transport when a direct contact between vesicles and skin occurs. The FFEM results showed that the formation of bilayer stacks occasionally plays a role after occlusive application of NSVs [31,42]. We concluded that the formation of bilayers only occasionally explains. the increased fluxes, especially in the case of liquid-state vesicles. Bilayer sheet formation plays a major role after nonocclusive application of NSV suspensions [31]. The ultimate contact between the estradiol-saturated bilayer sheets and the stratum corneum may increase the flux of estradiol, possibly via three mechanisms: first, the contactallows a direct transferof drug and vesicle constituents to the stratum corneum; second, via the formation of a thermodynamic activity gradient across the stratum corneum [42]; and third, the penetration of the vesicle constituents into the stratum corneum.
VI. SUMMARY
Incorporation,of estradiolinto NSVs may lead to. increased penetrationof the drug throughhumanstratum corneum. The results have shown thatit is possible to increase the estradiol flux by a factor of 50 as compared with estradiol-saturated PBS. These results make NSVs promising for transdermal application of drugs, because with penetration enhancers such as h o n e or oleic acid in propylene glycol, onlyan enhancement factor of 1.4 or 3.6 can be reached [104].. The mechanisms by which the increase in steady state flux can be described are thestate ofsupersaturation of the vesicle suspensions and the intrinsic penetration enhancement effects of vesicle bilayers that are in the liquid state at the experimental temperature. Depending on the of surtype factant, a penetration enhancementmay be caused by the diffusion of the surfactants into the stratum corneum. Furthermore, the elasticity of the bilayers may play an important role [38,41]. Decreasing the elasticity of
340
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the bilayers by increasing the cholesterol content or by applying surfactants forming gel-state bilayers results in a decrease in estradiolflux. The formation of bilayer sheets on top of the stratum corneum can only occasionally explain the increased fluxes obtained after occlusive application of the NSVs tested in this investigation. After nonocclusive application, largeregions of bilayer stacks could be visualized on the surface of the stratum corneum using freeze-fracture electron microscopy.
REFERENCES
. Gusalekharam, Liposomes: A selective drug delivery system 1. M. Mezei andV for the topical route of administration: Lotion dosage form, Life Sci., 2 6
1473-1477 (1980). 2. G. Vanlerberghe, R. M. Handjani-Vila, and
3. 4.
5 .
6.
A.Ribier, Les niosomes une nouvelle famille de vesicules a base damphiphiles non ioniques, Colloques Nationaux du CRNS, 938:304 (1978). J. M. Gebicki and M. Hicks, Preparation and properties of vesicles enclosed by fatty acid membranes, Chem. Phys. Lipids, 16:142-160 (1975). M. Hicks andJ. M. Gebicki, Microscopic studies of fatty acid vesicles, Chem. Phys. Lipids, 20:243-252 (1977). R. M. Handjani-Vila, A. Ribier, B. Rondot, and G. Vanlerberghe, Dispersions oflamellarphasesofnon-ioniclipidsincosmeticproducts,Int. J. Cosmet. Sci., 1:303-314 (1979). A. J. Baillie, A. T .Florence, L. Hume, et al., The effect of cholesterolon single chain,non-ionicsurfactantvesicles, J. Pharm.Pharmacol.,36(Suppl.):48P
(1984).
7.
A.J. Baillie, A.T. Florence, L. Hume, et al., The preparation and properties of niosomes-Non-ionic surfactant vesicles, J. Pharm. Pharmacol., 37:863868 (1985).
A.J. Baillie, G. H. Coombs,T. F. Dolan, and J. Laurie, Non-ionic surfactant vesicles, niosomes, as a delivery system for the anti-leishmanial drug, sodium stibogluconate. J. Pharm. Pharmacol., 38502-505 (1986). 9 . J. A.Hunter, T. F. Dolan, G. H. Coombs, and A.J. Baillie, Vesicular systems(niosomesandliposomes)fordeliveryofsodiumstibogluconatein experimental murine visceral leishmaniasis, J. Pharm. Pharmacol., 40: 1618 . 165 10. (1988).
M. N. Azmin, A.T. Florence, R. M. Handjani-Vila,et al., The effect of nonionic surfactant vesicles (niosome) entrapment on the absorption and distribuJ. Pharm. Pharmacol., 37:237-242 (1985). tion of methotrexate in mice, 11. C. Cable and A.T. Florence, Mixed poly(glycero1)-poly(oxyethy1ene) ether niosomes, J. Pharm. Pharmacol., 4O(Suppl.):3OP (1988). 1 2 . C. Cable, J. Cassidy, S. B. Kaye, and A. T. Florence,Doxorubicinin cholesteryl polyoxyethylene modified niosomes: Evidence of enhancement of absorption in mice?J. Pharm. Pharmacol., 4O(Suppl.):31P(1988).
34 1
E. Bardez, and B. Valeur, Bilayer fluidity of 13. A. Ribier, R. M. Handjani-Vila, non-ionic surfactant vesicles. An investigation by differential polarized phase fluorometry, Coll. Surf., 10:155-161 (1984). et al.,Action of 14. S. Lesieur,C.Gabrielle-Madelmont,M.-T.Paternostre, octylglucoside on nonionic monoalkyl amphiphile-cholesterol vesicles: Study of the solubilization mechanism, Chem. Phys. Lipids, 56:109-121 (1990). 15. I. F. Uchegbu, J. A. Bouwstra, and A. T. Florence, Large disk-shaped structures (discomes) in nonionic surfactant vesicle to micelle transitions,J. Phys. Chem., 96:10548-10553 (1992). P .Labrude, et al., Niosomes d'hCmoglobine. I. 16. P. Moser, M. Marchand-Arvier, PrCparation, proprittks physico-chimiques et oxyphoriques, stabilit6, Pharm. Acta Helv., 64:192-202 (1989). . Labrude,andC.Vigneron,Niosomes 17. P. Moser,M.Marchand-Arvier, P n vitro avec les prot6ines plasmatiques et les d'hkmoglobine. 11. Interactionsi phagocytes, Pharm. Acta Helv., 65:82-92 (1990). 18. H. Kiwada, H. Niimura, Y. Fujisaki, et al., Application of synthetic alkyl glycosidevesiclesasdrug carriers. I. Preparation and physical properties, Chem. Pharm. Bull., 33:753-759 (1985). Y. Kato, Tissue distribution and pharmacokinetic 19. H. Kiwada,H. Niimura, and evaluationof thetargetingefficiencyofsyntheticalkylglycosidevesicles, Chem. Pharm. Bull., 33:2475-2482 (1985). 20. H. Kiwada, I. Nakajima, H. Matsura, et al., Application of synthetic alkyl glycoside vesicles as drug carriers. 111. Plasma components affecting stability of the vesicles, Chem. Pharm. Bull., 36:1841-1846 (1988). 21. P. Schenk, M. Ausborn, F. Bendas, et al., The preparation and characterization oflipidvesiclescontainingesters of sucroseandfattyacids, J. Microencaps., 6:95-103 (1989). 22. H. E. J. Hofland, J. A. Bouwstra, G. S. Goons, et al., Nonionic surfactant vesicles: A study of vesicle formation, characterization, and stability, J. Coll. Interf. Sci., 161:366-376 (1993). 23. K. R. Patel, M.P.Schuh,and J. R.Baldeschwieler.Thepharmacological efficacy of a rigid non-phospholipid liposome drug delivery system, Biochim. Biophys. Acta, 797:20-26 (1984). .Hu, C. Ramachandran, D. F. H. Wallach, and N. Weiner, 24. S. M. Dowton, Z Influence of liposomal composition on topical delivery of encapsulatedcyclosporin A. I. An in vitro study using hairless mouse skin, S.T.P. Pharma. Sci., 3:404-407 (1993). 25. L. E. Echegoyen, J. C.Hernandez,A. E. Kaifer, et al.,Aggregationof steroidal lariat ethers: The first example of nonionic liposomes (niosomes) J. Chem. Soc. Chem. formed from neutral crown ether compounds, Commun., 12:836-837 (1988). 26. H. Fasoli, L. E. Echegoyen, J. C. Hernandez, et al., Evidence from ESR studies for virtual immobility in niosomes derived from steroidal lariat ethers J. Chem. Soc. Chem. Commun., 13578-580 (1989). 27. Y. Okahata, S. Tanamachi, M. Nagai, and T. Kunitake, Synthetic bilayer mem
342
van Hal et al.
branes prepared from dialkyl amphiphiles with non-ionic and zwitterionic groups, J. Coll. Interf. Sci., 82:401-417 (1981). 28. T. Kunitake and Y. Okahata, A totally syntheticbilayer membrane, J. Am. Chem. Soc., 99:3860-3861 (1977). . . 29. T. Kunitake andY. Okahata, Formation of stable bilayer assemblies in dilute aqueous solution from ammonium amphiphiles withthe diphenylazomethine segment, J. Am. Chem. Soc., 102549-553 (1980). 30. H. E. J.Hofland,VesiclesasTransdermalDrugDeliverySystems.Ph.D. Thesis, University of Leiden, The Netherlands, 1992. 31. D. A. Van Hal, Nonionic Surfactant Vesicles for Dermal and Transdermal Drug Delivery. Ph.D. Thesis, University of Leiden, The Netherlands,1994. 32. R. R. C. New, Liposomes, A Practical Approach, IRL Press at Oxford Univer1990. , . Press, sity York, New 33. J. R. Silvius, Thermotropic phase transitions of pure lipids in model membranes and their modification by membrane proteins, in Lipid Protein Intera tions (P. C. Jost and0. H. Griffith, eds.), Vol.2, Wiley, New York, 1982, pp.
'
239-281. L. Silver, ed.), 34. B. L. Silver, in The Physical Chemistry of Membranes (B. Alan & Unwin and Salomon Press, New York, pp.209-230,1985. W . Kok, et al., Niosomes for oral delivery of peptide 35. H. Yoshida, C.-M. Lehr, drugs, J. Control. Rel., 21:145-154 (1992). 36. H. E. J. Hofland, R. Van der Geest, H. E. BoddC, et al., Estradiol perme-
ation from non-ionic surfactant vesicles through human stratum corneum in vitro, Pharm. Res., 11:659-664 (1994). 37. M. E. Planas, P. Gonzalez, L..Rodriguez, et al., Noninvasive, percutaneous induction of topical analgesia by a new type of drug carriers and prolongation of the local pain-insensitivity by analgesic liposomes, Anesth. Analg.,95:61438. 621 (1992).
1104~226-232 (1992). 39. G. Cevc, Praparat zur Wirkstoffapplikation in Kleinsttropfchenform, EP 0 475 160 A1 (1992). 40. G. Cevc, Rationale for the production and dermal application of lipid vesi-
G. Cevc and G . Blume, Lipid vesicles penetrate into the skin owing to the transdermal osmotic gradients and hydration force. Biochim. Biophys. Acta,
cles,inLiposome-Dermatics (0. Braun-Falco;H.C.Korting,and H. I. Maibach, eds), Springer, Berlin, 1992, pp. 82-90., 41. G. Cevc, A. Schatzlein, D. Gebauer, and G. Blume, Ultra-high efficiency ofdrugand peptidetransferthroughtheintactskinbymeansofnovel drug-carriers, transfersomes, in Prediction of. Percutaneous Absorption, Walters, eds.), 1993, pp. 226Vol. 38 (K.R. Brain, V. J. James, and K. .A.
236. 42. H. E. J. Hofland,.J. A. Bouwstra, H. E. BoddC, et al., Interactions between
nonionic surfactant vesicles and human skin in vitro: Freeze-fracture electron J. Liposome Res., 5:241microscopy and confocal laser scanning spectroscopy,
263 (1995).
43.
343
44.
45.
4 6 .
47. 48. 49.
H. E. J. Hofland, J. A. Bouwstra, H. E. BoddC, et al., Interactions between in vitro: Freeze fracture electron miliposomes and human stratum corneum croscopicalvisualizationandsmallangleX-rayscatteringstudies,Br. J. Dermatol., 132:853-856 (1995). P. Blecher, Liposome dermaticsto come according to the patent literature, in Liposome Dermatics (0. Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin,1992, pp. 281-287. R. Kadir, D. Stempler, andS. J. Cohen, Delivery of theophylline into excised human skin from alkanoic acid solutions: A push-pull mechanism, J. Pharm Sci., 76:774-779 (1987). N. F. H. Ho, M. G. Ganesan, N. D. Weiner, and G. L. Flynn, Mechanisms of topL T .Control. Rel.,2:61-65 (1985). ical delivery of liposomallyentrapped drugs, H. Komatsu, H. Okamoto, K. Miyagawa, et al., Percutaneous absorption of butylparabene from liposomes in vitro, Chem. Pharm. Bull., 34:3423-3430 V. M. Knepp, R. S. Hinz, F. C.Szoka,and R. H. Guy, Controlled drug release from a novel liposomal delivery system. I. Investigation of transdermal potency, J. Control. Rel., 5211-221(1988). V.M. Knepp, F. C. Szoka, and R. H. Guy, Controlled drug release from a novel liposomal delivery system. 11. Transdermal delivery characteristics, J. Control. Rel., 12:25-30 (1990). H. Komatsu;K. Higaki, H. Okamoto, and H. Sezaki, Preservative activity an in vivo percutaneous penetrationof butylparabene entrapped in liposomes, (1986). . Chem. Pharm. Bull., 34:3415-3422 A. .J. M. Vermorken, M. W. A. C. Hukkelhoven, M. A. M. G. VermeeschMarkslag, et al., The useof liposomes in the topical application of steroids, J. Pharm. Pharmacol.,36:334-336 (1984). H. Hanel, B. Braun, and N. Jovic, Compartive activity of a liposomal and a conventional econazole preparation for topical use according to a guinea pig model, in Liposome Dermatics (0.Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin, 1992, pp. 251-257. L..M. Hansen,F. B. Reynolds,.andM. Foldvari, Topical liposomal tetracaine for IV cannulation, Can. J. Anesth., 37:S64 (1990). M.J.Kerscher,Influence of liposomalencapsulation on theactivityofa herbalnon-steroidalanti-inflammatorydrug,inLiposomeDermatics (0. Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin, 1992, pp. 329-337. J.Laschand W. Wohlrab,Liposomeboundcortisol:Anewapproach to 45:1295-1299 (1986). cutaneous therapy, Biomed. Biochem, Acta, T.Nishinata,K. Kotera, Y. Nakano, and M. Yamazaki, Rat percutaneous transport of diclofenac .and influence. of hydrogenated soya phospholipids, Chem. Pharm. Bull., 35:3807-3812 (1987). A.Kato, Y. Ishibashi,and Y. Miyake, Effect of egg yolk lecithin on transdermaldeliven,ofbunazosin hydrochloride,J.Pharm.Pharmacol.,
I
(1986).
50.
51. 52.
53. 54.
55.
56. 57.
39~399-400 (1987).
344
Hal
van
et al.
58. W. Wohlrab andJ. Lasch, Penetration kinetics of liposomal hydrocortisone in
human skin, Dermatologica, 174:18-22 (1987). and J. Lasch, Penetration of lecithin from 59. W. Wohlrab, U. Lachmann, hydrocortisone-containing liposomes into human skin, Dermatol. Mon.-Schr. 175:344-347 (1989a). 60. W. Wohlrab, J. Lasch, K. M. Taube, and K. D. Wozniak, Hautpermeation von liposomal inkorporiertem Hydrocortison, Pharmazie, 44~333-335 (1989~). 61. K. Egbaria andN. Weiner, Liposomes as a topical drug delivery system, Adv. Drug Deliv. Rev., 5287-300 (1990). 62. K. Egbaria, C. Ramachandran, D. Kittayanond, and N. Weiner, Topical delivery of liposomalinterferon evaluated by invitro diffusion studies, Antimicrob. Agents Chemother., 34:107-110 (1990). 63. K. Egbaria, C.Ramachandran,and N.Weiner,Topicaldeliveryofcyclosporin: evaluation of various formulations using in vitro diffusion studies in hairless mouse skin, Skin Pharmacol., 3:21-28 (1990). 6 4 . V. Gabrijelcic, M. Sentjurc, and J. Kristl, Evaluation of liposomes as drug carriers into the skin by one-dimensional EPR imaging, Int. J. Pharm., 62:7579 (1990). 65. A. Toulon,M. Besnard, B. LeClerc, et al., Modification of estradiol percutaneous absorption by liposomal entrapment, S.T.P. Pharm. Sci., 1:76-82 (1991). 66. A. Meybeck, Comedolytic activity of a liposomal antiacne drug in an experimental model, in Liposome Dermatics(0.Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin, 1992, pp. 235-241. N. Weiner, Influenceof formulation factors on 67. J. Du Plessis, K. Egbaria, and the depositionof liposomal components into the different strata of the skin, J. Soc. Chem., 43:93-100 (1992). V. Gusalekharam, Liposomes: A selective drug delivery system 68. M. Mezei and for the topical route of administration: Gel dosage form. J. Pharm. Pharmacol., 34~473-474 (1982). 69. M. Mezei, Liposomes as a skin drug delivery system, in Topics in Pharma calSciences(D. D. Breimer,and P. Speiser, eds.), Elsevier, Amsterdam, 1985, pp. 345-358. 70. R. Natsuki, S. Tomomichi, R. Matsuo, et al., Absorption and excretion of indomethacin gel ointment containing egg lecithin, J. Pharmacobio-Dyn., 9:s12 (1986). 71. Y . Miyachi, S. Imamura, andY . Niwa, Decreased skin superoxide dismutase activity by a single exposure of ultraviolet radiatin is reduced by liposomal superoxide dismutase pretreatment, J. Invest. Dermatol.,89:111-112 (1987). 72. W. Wohlrab and J. Lasch, The effect of liposomal incorporation of topically appliedhydrocortisone on itsserumconcentrationandurinaryexcretion, Dermatol. MonAchr., 175:348-352 (1989b). 73. N. D. Weiner, N. Williams, G. Birch, et al., Topical delivery of liposomally encapsulated interferon evaluated in a cutaneous herpes guinea pig model, Antimicrob. Agents Chemother., 33:1217-1221 (1989).
345
74. N.Weiner,K. Egbaria, and R. Chandrasekhasan, Topical delivery of liposomally encapsulated interferon evaluated by in vitro diffusion studies and (0.Braun-Falco, cutaneous herpes guinea pig model, in Liposome Dermatics H. C. Korting, and H. 1. Maibach, eds.), Springer, Berlin, 1992, 242-250. 75. V. Masini, F. Bonte, A. Meybeck, and J. Wepierre, In vitro percutaneous absorption and in vivo distribution of retinoic acid in liposomes and in a gel studied in hairless rats, Proc. Int. Symp. Control. Rel. Bioact. Mater., 17~425-426 (1990). 76. C. Artmann, J. Roding, M. Ghyczy, andH. G. Pratzel, Liposomes from soya phospholipids as percutaneous drug carriers. Qualitative in vivo investigatio with antibody-loaded liposomes, Arzneim.-Forsch./Drug Res., 40:1363-1371 (1990). 77. C. Artmann, J. Roding, M. Ghyczy, and H. G. Pratzel, Liposomes from soya phospholipids as percutaneous drug carriers. Quantitative in vivo investigationswithradioactivelylabelledliposomes,Arzneim.-Forsch,lDrugRes., 40~1365-1368 (1990). 78. D. B. Yarosh, Liposome-encapsulated enzymes for DNA repair, in Liposome and H. I. Maibach, eds.), Dermatics (0. Braun-Falco, H. C. Korting, Springer, Berlin, 1992,258-269. on per79. C.Michel,T.Purmann, E. Mentrup, et al.,Effectofliposomes cutaneous penetration of lipophilic materials, Int. J. Pharm., 84:93-105 (1992). 80. C. Michel, N. Groth, T. Herrling, et al., Penetrationof spin-labeled retinoic acid from liposomal preparations into the skin ofSKHl hairless mice. MeaJ. Pharm., 98:131-139 (1993). surement by EPR tomography, Int. 81. H. E. BoddC,L.A. R.-M. Pechtold,M. T. A. Subnel, and F. H. N. De Haanpechtold subnel haan Monitoring in vivo skin hydration by liposomes using infrared spectroscopy in conjunction with tape stripping in Liposome H. C. Korting, and H. I. Maibach, eds.), Dermatics (0. Braun-Falco Springer, Berlin, 1992, pp. 137-149. 82. L.M. Lieb, C. Ramachandran, K. Egbaria, and N. Weiner,Topicaldrug delivery enhancement with multilamellar liposomes into pilosebaceous units: I. In vitro evaluation using fluorescent techniques with the hamster ear model, J. Invest. Dermatol.,99:108-113 (1992). der per83. L.Kr6wczynskiand T. Stozek,LiposomenalsWirkstoffragerin kutanen Therapie, Pharmazie, 39:627-629 (1984). Liposomen-eineneueFormdermatologischerWirkstoffrager, 8 4 . W. Raab, Artzl. Kosmetol., 18:213-224 (1988). skin byliposome85 A.GesztesandM.Mezei,Topicalanesthesiaofthe encapsulated tetracaine, Anesth. Analg., 67:1079-1081 (1988). 86 R. A. Dudley, P. Edwards, R. P. Ekins, et al., Guidelines for immunoassay data processing,Clin. Chem., 31:1264-1271 (1985). 87. M. Jacobs, G. P. Martin, and C. Marriott, Effects of phosphatidylcholine in the topical bioavailability of corticosteroids assessed the human by skin blanching assay, J. Pharm. Pharmacol., 40:829-833 (1988). 88. W . Gehring, M.Ghyczy,M. Gloor, et al., Significance of empty liposomes
I I
346
van Hal et al.

aloneandasdrugcarriersindermatology,Ameim.-Forsch./DrugRes., 40~1368-1371(1990). W . Gehring, M. Klein, and M. Gloor, Influence of various topical liposome preparations with and without active ingredients on the cutaneous blood flow,inLiposomeDermatics (0.Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin, 1992, pp. 315-319. M. Foldvari, A. Gesztes, andM. Mezei, Dermal drug delivery by liposome encapsulation: Clinical and electron microscopic studies, J. Microencaps., 7:479-489 (1990). C. J. N. Oguefior,Topicaldosageform of M. Foldvari,B.Jarvis,and liposomal tetracaine: Effectof additives on'the in vitro release and in vivo efficacy, J. Control. Rel.,27:193-205 (1993). H. C. Korting, Increased activity and tolerability of a conventional glucocorticoid in a liposomal form, in Liposome Dermatics(0. BraunPalco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin, 1992, pp. ,320-328. G. Lasch, R. Laub, and W . Wohlrab, How deep penetrate intact liposomes J. Control. Rel., 18:55-58, 1991. into human skin? M. G. Ganesan, N. D. Weiner, G. L. Flynn, and N. F. H. Ho, Influence of liposomaldrugentrapment on percutaneousabsorption,Int. J.Pharm., 20:139-154 (1984). Siciliano, 1985. Jessberger, 1988; R. Schubert, M. Joos, M.Deicher, et al., Destabilization ofegglecithin liposomes on the skin after topical application measured by perturbed TT angular correlation spectroscopy (PAC) with "'In, Biochim. Biophys. Acta, 1150~162-164 (1993). M. B. Fawzi, U. R. Lyer, and M:Mahjour, U.S. Patent 4,783,450,1988. W. Wohlrab, J. Lasch, R. Laub, et al., Distribution of liposome-encapsulated ingredients in human skin ex vivo, in Liposome Dermatics (0.Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, 'Berlin, 1992, 215-225. M:Mahjour, B. Mauser, Z. Rashidbaigi, andM:B. Fawzi, Effect of egg yolk lecithins and commercial soybean lecithins on in vitro skin permeation of drugs, J. Control. Rel., 14:243-252 (1990). N. Weiner, Topical delivery of liposomally encapsulated ingreK. Egbaria and dients evaluated, in Liposome Dermatics (0.Braun-Falco, H. C. Korting, and H. I. Maibach, eds.), Springer, Berlin, 1992, pp. 172-181. J.RodingandC.Artmann,Thefateofliposomesinanimal skin, in Liposome Dermatics(0. Braun-Falco, H. C. Korting, H.I. Maibach, eds.), Springer, Berlin, 1992, pp. 110-117. on the M.Ghyczy,Chemicalcompositionofliposomesanditsinfluence humidity of normal skin, in Liposome Dermatics (0..Braun-Falco, H. C. : Maibach, eds.), Springer, Berlin, 1992, pp. 308-314. Korting, and H. 1 M. Goodman and B. W . Barry, Action of penetration enhancerson human skin as assessed by thepermeation of model drugs 5-fluorouracil and estradiol. I. Infinite dose technique, J. Invest. Dermatol., 91:323-327 (1988).
89.
90, 91.
92.
93. 94. 95. 96.
9 7 .
98. 99.
100. 101. 102. 103. 104.
ADDITIONAL READINGS
347
A. H. Armin, A. T. Florence, R. M. Handjani-Vila, et al., The effect of niosomes and polysorbate 80 on the metabolism and excretion of methotrexate the inmouse, J. Microencaps., 3:95-100 (1986). G. L. Flynn, S. H. Yalkowsky and T. J. Roseman, Mass transport phenomena and models: theoretical concepts, J. Pharm. Sci., 63:479-510 (1974). R. H. Guy and J. Hadgraft, Mathematical modelsof percutaneous absorption, in Percutaneous Absorption. Mechanisms, Methodology, Drug Delivery(R. L. Bro. Maibach, eds.), Marcel Dekker, New York, 1985, pp. 5-15. naugh and H.I D. J. Kerr, A. Rogerson, G . J. Morrison, et al., Antitumour activity and pharmacokinetics of niosome encapsulated adriamycin in monolayer, spheroid and xenograft, Br. J. Cancer, 58:432-436 (1988). L. Khand, A. Rogerson, G. W. Halbert, et al., The effect of cholesterol on the release of doxorubicin from non-ionic surfactant vesicles, J. Pharm. Pharmacol 39(Suppl.):41P (1987). M. Mezei, Multiphase liposomal drug delivery system, U.S. Patent 4,761,288,1988. L. K. Pershing, L. D. Lambert, and K. Knutson, Mechanism of ethanol-enhanced estradiol permeation across human skin in vivo, Pharm. Res., 7:170-175 (1990). A. Rogerson, J. Cummings, N. Willmott, and A. T. Florence. The distribution of doxorubicin in mice following administration in niosomes, J. Pharm. Pharmacol., 40~337-342 (1988). S. Stafford, A. J. Baillie, and A. T. Florence, Drug effects on the sizeof chemically J. Pharm. Pharmacol., 4O(Suppl.):26P (1988). defined non-ionic surfactant vesicles, U. Tauber, Drug metabolism in the skin: advantages and disadvantages, in Transdermal Drug Delivery (J. Hadgraft, and R. H. Guy, eds.), Marcel Dekker, New York, 1989, pp. 99-112.
1 3
Microcapsules: Preparationby Interfacial Polymerization and Interfacial Complexation and Their Applications
Tony L. Whateley
University ofStrathclyde, Glasgow, Scotland
I. Introduction
349 349
11. Interfacial Polymerization A. Semipermeable, Aqueous Core, and Polyamide Microcapsules B. Nonbiomedical Aspects of Microcapsules with Rigid, Nonpermeable Shells and Liquid Cores
111. Interfacial Addition Polymerization A. Alkylcyanoacrylates
IV. Interfacial Complexation or Interfacial Polyelectrolyte Complexation A. Alginate/Polylysine Microcapsules
351
363 364 364 366 366
1.
INTRODUCTION
The term interfuciulpolymerization is usedhere toinclude interfacial addition polymerization and interfacial condensation polymerization. However, interfacial complexation is a separate process and is generally applied to produce microcapsules with somewhat different properties and specialized applications from those produced from theinterfacial polymerization processes, and these topics will be considered separately.
349
350
MICROSPHERES Monolithic Uniform distribution of particles or a molecular dispersion MICROCAPSULES Liquid contehts, nonpetmeable, rigid.. . membrane .
. .
I .
Whateley
0
y:...
.g:;:.:
.. .....:..-.'
0
I"\
TYPE 2
Aqueous contents, semipetmeable membrane
I ' .,
TYPE I
Solid core, protective or releasecontrolling coating
Fig. 1 'Qpes of microspheres and microcapsules. In general, all of the above processes produce liquid-cored (both aqueous and nonaqueous) microcapsules. Those microcapsules with semipermeable membranes and aqueous cores (type 1 in Fig. 1) are of special interest in the biomedical and pharmaceutical areas. The various types of microcapsules and microspheres are illustrated in Figure 1. The types of microcapsules produced by the processes to be considered in this chapter are types 1 and 2 (in general, only type 1is produced by interfacial complexation). Thus, we are considering microcapsules with liquid cores and either a igid, brittle, nonpermeable shell (type 2) or a for both semipermeable membrane shell (type 1): The preparative methods types have much in common. However, the applications are very different: i t is those with aqueous cores and semipermeable membranes that have most application in the pharmaceutical and biomedical fields. Interfacial polymerization 'and interfacial condensation polymerization are so similar that the topics will be considered together. Interfacial complexation (or interfacial polyelectrolyte complexation) tends to be mainly used to produce a particular type of microcapsule (biocompatible, aqueouscore with a semipermeable membrane) andits prepa. .. ration and applications will be considered separately. Interfacial addition polymerization has been of interest in the medical, biomedical, and Pharmaceutical fields because of the alkylcyanoacylates and their ability to polymerize readily. However, in general, the type of microcapsule (or probably more precisely, microsphere) produced solid, monolithic variety. This topic will also be treated separately. .'is of the An overview of drug delivery by various microencapsulated systems isgivenby Arshady [l]. Systems prepared by a wide range of micro,
Microcapsules Polymerization by
and Complexation
35 l
encapsulation methods are considered. Arshady [2] also reviewed the preparation of microspheresand microcapsulesby interfacial polycondensation techniques. Chang [3] reviewed recent advances. in artificial cells covering especially the biotechnology and medical applications. A number of books covering this area have been published; for example, those by Chang [3], Lim [4], Donbrow [ 5 ] , and Whateley [6].
11.
INTERFACIAL POLYMERIZATION
A. Semipermeable, Aqueous Core, and Polyamide Microcapsules (Type 1 in Fig. 1)
This type of microcapsule was initially developed by Chang [7,8] in Canada in Japan have alsodone extensive work in the 1960s. Kondo and coworkers on these systems. Originally developed by Chang as artificial cells, this type of microcapsule has been used to encapsulate: Proteins Enzymes Antibodies Cells
, .
The important features of the semipermeable encapsulating membrane are: ..
1. Not permeable to large molecules such as proteins, enzymes. 2. Permeable to small moleculessuch as enzyme substrates and products. A pore size of about 2 nm fulfillsthese objectives and can be achieved by the interfacial polymerization process. 3. Nonantigenic surface. 4. Although biodegradability is important in some applications, this .. has been difficult to achieve.
,
A range of reactive monomer-monomer combinations can be used to react at anoil-water interface, as detailed later; theinterface may be planar or at , . the perimeter of a small droplet, asshown in Fig.2. The basic feature of the method to produce microcapsules, which is the same in allcases, is,the formation of a water-in-oil (W/O) emulsion, as illustrated in Fig. 3... Addition of the second monomer, B, to the preformed emulsion initiates the interfacial reaction. Details of preparative procedures are given later. . In this interfacial polymerization process, various combinations of monomers can be used to obtain a range of polymer membranes. Some
352
Whateley
Fig. 2 Microencapsulation by interfacial polymerization method.
Emulsion
Water-in4
Fig. 3 Preparation of microcapsules by interfacial polymerization.
possibilities are illustrated in Figs. 4a-d. Figure 4a shows the polymerization between sabacoyl chloride and hexamethylene diamine (1,6-hexanediamine) to form the polyamide nylon 6,lO: This system was used in the early studies on microcapsule formation by the interfacial polymerization process. Such microcapsules, however, tend to be rather fragile and difficult to separate, wash, and handle. More robust microcapsules can be prepared using terephthalic (p-phthalic) acid dichloride with the diamine (Fig. 4b). Piperazine has frequently been used with terephthaloyl chloride to give a robust poly(terephthaloy1-piperazine) membrane. Substituted
Microcapsules Polymerization by Complexation and
353
diamines, for example, L-lysine (Fig. 4 c )can be used in order to give the microcapsule membrane a charged group. Other classes of polymeric membrane can be formed, for example, polyesters as shown in Fig. 4d. Polyurea and polyurethane membranescan also be produced by the appropriate choice of monomers (see, e.g., ref. g), although most work in the biomedicaYpharmaceutica1 area has been carried out with the polyfunctional halide plus polyamine system to give polyamide membranes, and thesesystems will be considered in more detail. The monograph by Morgan [lo] extensively reviewed the fundamentals of interfacial polymerization, and this review was extended by Millich and Carraher [ll].The complexity of the reaction has been shown by
Ii2N-(CH&N
H2
1,6-hexane diamine
+
cl oc -(CH2)R-COCI
sebacoyl chloride polyamide
HN-(CH,),-NH-~O-O-(CH,)B-OCO
Nylon 6,lO
H ~ N - - ( C H ~ ) ~ - - N H ~ 1,6-hexane diamine
+
C 1W @
CI
terephthaloyl chloride
HN-(CH,),-NH-C
(b)
Fig. 4 Reaction for formation of (a) nylon 6,lO; (b) poly(terephtha1amide); (c) poly(terephthaloy1 L-lysine);(d) polyester.
354
Whateley
COOH
,I
H2N-(CH2)3XIi-NIi2
, .
L-lysine
' .
(c)
poly(terephthaloy1 L-lys'ine)
+
Cl OC-(CHz)a-COCI'
2,2-bis(4-hydroxyphenyl)
propane
.'
sebacoyl
chloride
; & s f
(a
c H
Fig. 4 Continued
Complexation and
355
studies at simple unstirred planar interfaces, and some aspects of this will be considered here.
1. Factors Regarding the Interfacial Polymerization Reaction'
. .. .
a. Reaction rate. The interfacial formation of nylon 6 , l O is rapid, and a ,visible polymeric precipitate can be.seen almost immediately afterthe two components are brought together. The reaction rates.for polyamide,,forma10'-lo6 dm3mol" s-l [lo]. tion have second-order rate constants in the range The reaction of aliphatic glycols with diacid chlorides is too slow to allow the fofmation of high molecular weight polymer, with hydrolysis of the acid chloride being the competingside reaction. In the interfacial polymerization reaction between piperazine and terephthaloyl. chloride; the oligomers react at a ,faster rate with the diacid chloride than piperazine-itself. Thus, atrelatively low concentrations, high molecular weight polymer (1O;OOO-40,000) can be formed.
. tion
b. Precipitation ofpolymer. The site of the reaction affects the precipitaof the polymer: me reaction forming nylon 6 ; l O has usually been assumed to occur onthe organic side of the interface. Precipitation of the polymer prevents diffusion of the monomers and thus stops thepolymerization reaction from.continuing to completion and limits the thickness of the membrane. In this respect, the choice of solvent is important: swollen polymers will allow diffusion of monomers to the reaction site.
c. Impurities. Typical &purities in interfacial reaAions of this type are monofunctional monomers which produce low molecular weight polymer as' a result of chain termination. The presence of ethanol as a stabilizer in chloroform appreciably reduces the molecular weight of poly(piperazine sebacamide) [lo, p. 901.
d. ' Diamine partition coeficient and interfacial transfer rates. The partitio*'coefficient.of the diamine.is easily measured atequilibrium and serires as a guide to exclude monomers which would.be unsuitab1e;forthis type of reaction. However, .transfer rates-become .more important when polymer.ization' is in progress. ,Maintenance of adequate. levels of diamine at. the reaction s i t e k important, and thercfore.the rate of transfer should exceed . . . 'the rate'of removal of diamine by polymerization. .
e. Concentrationratioof reactants. The concentration ratio of the two reactants can be important when highmolecular weight polymer is desired. However, in microencapsulation applications this ratio has not been studied to any great extent; mainly because other. conditions of the reaction place restrictions on the concentrations which can be used. Thus,, in mi-
356
Whateley
croencapsulation, generally one reactant is used in excess. When oil-in water ( O N ) emulsions are used in the microencapsulation of pesticides, the water-soluble monomer can be used in excess, and therefore the microcapsule wall thickness is controlled by the complete diffusion of the oilsoluble monomer to form thewall. Diamine canbe used in excesswhen its partition coefficient is unfavorable for its transfer to the Organic solution, as is found whenlysine isused.
Stirring. Stirring is a critical variable and is found to affect the molecular weight of various types of polymers [lo]. When the interfacial reaction is carried out toproduce membranes around emulsion droplets, thestirring conditions can make the difference between a usable product and a useless mass of polymeric material. The stirring produced by a magnetic follower in a beaker, as would be commonly available in a laboratory, can produce microcapsules down to about 10-20 pm in diameter. More powerful emulsification is generally required to produce smaller microcapsules. Kondo and coworkers (e.g., Wakamatsu et al. [12]) haveprepared small microcapsules (diameter about 1 pm) using a high-speed impeller in a special reaction vessel.
g . Hydrolysis of diacid chloride. Inactivation of diacid chlorides is perhaps one of the most important side reactions, but in many reactions, it causes no problem. The partition of the diacid chloride into the aqueous phase is the principal factor involved, and by restricting the choice of monomer, the problem can be eliminated. The kinetics of hydrolysis of terephthaloyl chloride have been studied in a two-phase system (n-heptane/ water) and found to be first order with respect to the diacid chloride [13]. However, Bradbury et al., [l31 reported that hydrolysis was much slower than polyamidationwith piperazine as diamine. h. Transfer rates of salts. An important consideration is the elimination of HCl formed in the polycondensationreaction. This product is removed by the formation of the hydrochloride salt of the diamine which, being poorly soluble in the organic phase, diffuses to the aqueous phase where the HCl is neutralized by buffer included for this purpose. Hydrochloride salts of diamines are not able to react with diacidchloride and, therefore,if buffer is not included in the aqueousphase, the diamineitself will function as an acid acceptor. In most cases, the transfer of hydrochloride salts will be faster than the transfer, in the opposite direction, of diamine to the reaction site.
i. Solvent: polarity,density,andtoxicity. Chloroform-cyclohexanemixtures in the ratios 1 : 4 or 1 : 3 have been widely used. Morgan [lo] dis-
357
cussed the polymer-solvent interactions of nylon 6,lO and has shown that thick films are expected from chloroform systems, whereas cyclohexane produces thin films. Good solvents tend to producehigh molecular weight polymer when compared with nonsolvents. The density of the organic solvent used in the production of the W/O emulsion in microencapsulation procedures will affect the stability of the emulsion and will determine whether the microcapsules sediment or float in the organic phase. This will affect the isolation technique used to obtain the newly formed microcapsules. In general, when microencapsulation for pharmaceutical applications has been studied, organic solvents have been chosenfrom solvents withlowtoxicity and toxic solvents havebeen avoided.
j . Surfactants. The addition of surfactants in microencapsulation procedures is important for the formation of the emulsion. Surfactant also plays an important role in the transfer of the diamine to the organic phase. Kondo and coworkers have investigated the effect of Span 85 (sorbitan trioleate) on the partition coefficients of various diamines (e.g., Koishi et al. [14], Koishi et al. [15], and Shigeri et al. [16]) and bisphenols [12]. In general, increasing the concentration of surfactant results in an increase in the amount of diamine whichis transferred to the organic phase. For preparation ofpoly(1ysine terephthalamide) microcapsules, a surfactant concentration (sorbitan trioleate) of 20% v/v was successful. The choice of surfactant used in studies where aqueous solutions are encapsulated in semipermeable membranes has been limited mainly to sorbitan trioleate, which has a particularly low HLB (hydrophile-lyophilebalance) value (i.e., 1.8). The main requirement of any surfactant is that it does not react with the diacid chlorides, and it does not have impurities which would interfere with the polymerization reaction.
k. Temperature. The great advantage of interfacial condensation polymerization reactions of this type is that they can be carried out at room temperature. No advantage is found in heating the reaction and many preparations are improved by controlling the temperature rise. The reactions proceed well at 4C. When enzymesand/or proteins are to be microencapsulated, the prowhich enzymes cess can be carried out conveniently at 4"C, a temperature at in solution have favorable stability. Ultrathinmembranes,formedatthe interface betweenaqueous polyethylenimine andhexane solutions of tolylene diisocyanate, were found to be extremelystrong [17]. Such filmsare able to beused in reverse osmosis where pressures of some 100 atm are encountered.
2. Preparation of PolyamideMembrane;Semipermeable, Aqueous-Core Microcdpsules by Interfacial Polymerization . A typical'method to produce nylon 6,lO microcapsules is as follows:
. .
1; Nonaqueous phase: chloroform/cyclohaxane ( l : 4) containing 5% Span 85 (sorbitan trioleate). 2. Aqueous phase: sodium carbonatehicarbonate buffer, pH 9.8 containing,. for example, qnzyme to be microencapsulated plus protective/stabilizing protein (e.g., hemoglobin or bovine serum albumin) and 1,6-hexamethyiene:diamine. . 3. Above phases mixed in about a 1 : 10 volume ratio and stirred while cooling in an ice bath. Stimng rate and method to ,give required microcapsule size. 4. Further nonaqueousphase containing sebacoyLchloride added to . . stirred emulsion. Interfacial polymerization ,reaction allowed to 30 min. . . about for . . proceed 5. Polymerization reaction quenched by addition of excess nonaqueous phase, Microcapsules allowed to sediment and superpatant ,. decanted off. 6. Microcapsules resuspended in 50% %een 20 in saline,sedimented,, decanted, and this,process repeated to remove organic . . solvents. . 7. Microcapsules washed 2 times with saline. and resuspended in saline. .
' '
Some particularaspects of the process: chloroform and cyclohexane do not readily denature proteins. The particolar ratio.'(l : 4).is chosen for 'two . ' reasons: . '
I
1. The density (about 0.91) is'not far-removed from water so,that emulsion formation is not a problem, butstill allows the aqueous core microcapsules to sediment for separation purposes. the diamine between the aqueous and 2. The partition coefficient of. ., organiG,phases is important:.the 1 :.,4..rati,oallows diamine to diffuse into the organic phase, where theinterfacial polymeriiation reaction takes place. AS the interfaciaimembrane, forms, the , . diffusionof the. diamine to the organic interface of the membrane is reduced,and the polymerization becomes self-limiting.
I
,
Thus, about 200-nm thickness microcapsule membranes, which are strong enough for separation; washing, and applications, areformed with equiva. .. . . . lent pore sizes of about 0.2nm. : . ' Interfacial pdymerization .in a high-voltage electric field has been investigated as a means of obtaifiing more monodisperse collections.o f
"
'
and Complexation
359
microcapsules [18]. Kondo and his group have studied this aspect 'of microcapsule formation (e.g., Muramatsu et al. [19]). The proteins human serum albumin (HSA), bovine fibrinogen, and ovalbumin were used [20]. to prepare' microcapsules by interfacial crosslinking using tere-phthaloyl chloride -and subsequent treatment with hydroxylamine to make the microcapsules capable of iron binding. 'Similar polyhydroxamic serum albumin microcapsules had been reported previously by Levy et al. [21].
3. Applications of Semipermeable, and Aqueous "Chang"-Type Microcapsules ,.

'
'
This topic has been well covered'h'the book by Lim [4] and reviewed . . by Chang [3].
a. Artificialred blood cells. Thesehave been,used as asubstitute in blood transfusions [22]. Hemoglobin, crosslinked hemoglobin and,hemoglobin conjugated to polymers have been microencapsulated for this purtype of.microcapsule in obtaining pose. There is a major problem withathis long circulating times, The size mustbe in the rangeof,red blood cells (e.g., 4 0 pm). A more difficult problem is in achievingthe.elasticity.and fluidity of the membrane needed forpassage along small capillaries .(<<l0 pm).
. .
b. Microcapsules containing adsorbentsandimmunoadsorhents. Microencapsulated charcoal is biocompatible, that is, htis no adverse.effect on blood but stilhetains its ability to,absorbsignificant amounts of low molecular ,weighk material (see, e.g.,' 'refs. 23 and 24). Applications Of hemoperfusion with a device containing &out 70 g microencapsulated _.. . . charcoal include: . . .
. . .
I.
. . .
...
Treatment ,of .acute,poisoning .( -Treatment of kidney,failure . . . .

'
. .
. . . .
. . .
......
c;, Microcapsules coniaining h n ig cells'.' : ' topic is 'covered in Sedtion IV. The useof'interfacial polymerization in'this areahas been reviewedby Chang [3].
PS
d. Microcapsules containirzg enzymes .(br.multienzyme systems):

,-.,
..
....
.... Urea removal in. kidney failure; using urease 'which converts' the urea .to.ammonia and carbon dioxide. Co-encapsulation of an-ammonia absorbent caqremove the.ammonia [3]. . . . __ ..
._ .-, .. .
...
..
.....
0:'
Hereditary enzyme deficiency; for example: Phenylketonuria: the 'enzyme phenylalanine ammonia lyase is-defi'.cient and is replaced by the microencapsulated.enzyme [3] ..
360
Whateley
Histidenemia: the enzyme histidase has to be replaced-to avoid the build-up of histidine. Using a histidinemicmouse model, Wood et al. [25] worked on this problem. Histidase from a bacterial source was microencapsulated in poly(terephthaloy1piperazine) microcapsules and administered intraperitoneally. Although blood histidase levels were reduced 50%, there were problems with the toxicity of the microcapsule preparations. The multienzyme artificial cell-type system is covered in Lim [4] and Chang [3]. Enzyme stability: Arginase microencapsulation with a poly(phthaloy1piperazine) membrane was reported by Kondo [26]. He concluded that enzyme inactivation during the process of microencapsulation was due to contact of the arginase with the organic solvent and incorporation of the enzyme into the membrane. Wood and Whateley [27] investigated the loss of enzyme activity during the interfacial polymerization microencapsulation process. The low yields (about 40%) of activity found in polyamide microcapsules for both chymotrypsin and histidase are typical of those reported in the literature. There was some diffusional restriction on the measured enzyme activity, but there was a considerable incorporation of model radiolabeled proteins into the microcapsule membrane during the microencapsulation procedure. Tables 1and 2 show the distribution of labeled albumin and fibrinogen, respectively, following the microencapsulation process. Table 3 shows that fibrinogen is adsorbed to a much greater extent than albumin on poly(piperazine-terephthalamide) microcapsules. Kidokoro et al. [28] looked at theinteraction of fibrinogen with microcapsules of poly(L-lysine&terephthalic acid). They found that fibrinogen increased the disintegration of the microcapsules by adsorbing via a hydrophobic interaction. Hoshino et al. [29] studied the thermostability of glucose oxidase within polyurea microcapsules. Thermostability markedly increased for the microencapsulated enzyme, but this was ascribed to the incorporation of Table 1. Incorporation of ZSI-L,abelled Human Serum Albumin in Poly(piperazine terephthalamide) Microcapsules System
(3.8) 64.1
vity
(%)
6.4,6.8,8.0 28.8 31.6,24.4,30.3 60.9,69.0,62.5
Mean
with s.d. 7.1 (0.8)
100.0
e Organic washingsAqueous Microcapsules (4.3)
total
Microcapsules by Polymerization and Complexation
361
Table 2. Incorporation of '=I-Labelled Fibrinogen in Poly(piperazine

terephthalamide) Microcapsules System Organic phase 10.9 Aqueous washings Microcapsules
12.3,10.5
'%activity (%)
9.9, 15.2,9.5,13.3 65.4,75.8,57.6
total
(1.2) 12.7 (2.9) 66.3 (9.1) 89.9
Mean with s.d.
Table 3. Adsorption of '251-Labelled Proteinsonto Poly(piperazine

terephthalamide) Microcapsules Contact time
5 min
Albumin
%
1.2 1.7 3.2
Fibrinogen %
92
3h 95 24 h
(in 0.9% w/v NaCl, pH 7.4)
the enzyme into the microcapsule wallduring the interfacial polymerization process, as was also reported by Wood and Whateley[27]. Enzyme kinetics: The effect of microencapsulation on enzymekinetics has been investigated by many workers. Urease was microencapsulated at 83% yield with a semipermeable membrane[30]. Only 6% of the microencapsulated enzyme was incorporated into the nylon membrane, and the high activity of the encapsulated enzyme (92%) indicated minimal effects of mass-transfer limitation. Aisina[31]used Chang-type microcapsules with the inert filler of poly(ethy1enimine) to study trypsin and chymotrypsin kinetics. Not only were low molecular weight substrates used, but the effect on the autolysis of trypsin and the autoactivation of trypsinogen were also studied. The Michaelis constant, K,,,, was 0.49mM for thenative chymotrysin but 3.7 mM for the microencapsulated enzyme with acetyltyrosine ethyl ester (ATEE) as substrate. The catalytic constant, k,,, was reduced from 175 to 12 S" for the microencapsulated chymotrypsin. The maximum of the apparent pH-activity curve for chymotrypsin was found to beshifted 1pH unit to more alkaline values when the enzyme was microencapsulated in a polyamide membrane [27]. Fig. 5 shows this I effect. This phenomenon was explained in terms of the hydrogen ion concentration in the microenvironment of the enzyme within the microcapsule being higher than in the bulk solution.
362
Whateley
1 0
PH
Fig. 5 pH-activity curves for microencapsulated a-chymotrypsin chymotrypsin in solution (A) (1 p M A a E , 50 p M NaCl at 25C).
(0) and a-
. . e; Microcapsules containing antibodies; An interesting application was reported by Wallace [32], who developed an assay method for thyroxine . and some steroids by microencapsulating,antibodies in microcapsules (25 ..pmdiameter with membrane pore size permitting entry of hormones of less than 4000 molecular weight) Routine assays were simplifiedby coen-capsulating magnetic iron dextran so that centrifugation could be super. seded by magnetic separation. Other reportsrelating to microcapsules containing antibodies are inciuded in the book by Lim [4] and the review by Chang [3]. .
D$ug delivery. Although interfacial polymerization has not been widely used or the microencapsulation of drugs, Luzzi etai. [33], Florence and Jeikins [34],and McGinity and his group have expanded the method into thisarea [35,36].Interestingly, some water-soluble polyamides for use as drug camers have been synthesized by interfacial polymerization [37]. . . Amine derivatives of poly(ethy1ene glycol) were reacted with succinyl chloride at theinterface of a methylene chloride-water system. . . > . g . Otherapplications of microencapsulationby interfacial polymerization. A novel andinteresting application of semipermeable microcapsules was reviewed by 0Neill[38l1 Magnetic ferric oxide.was.microencapsu1ated in a membrane consisting of a graft copolymer .of polyamide and poly(ethylenimine) (PEI). These were administered to animals together with
363
some known carcinogens, for example, nitrosoureas. The microcapsules were recoveredmagnetically and thespecies bound to thePE1 investigated in order tofollow metabolic changes andtypes of carcinogens occurring in the gut under various dietary situations. The microencapsulation of benzalkonium chloride based on the interfacial polycondensation of isocyanates has been reportedby Pense et al. [39-411. The use of thz HLB approach was shown to be appropriate for deciding the initial formulation [39,40]. An aqueous solution of benzalkonium chloride was poured into an oil phase (e.g., xylene or a blend of xylene and Miglyol) containing a polymeric surfactant (e.g., Hypermer A60). Polycondensation followed hydrolysisof the isocyanate and reaction between the formed amine and further isocyanate. The preparation and properties of polyurea microcapsules was reported by Yan et al. [42,43] usingcyanate and polyamine as the monomers witha nonionic surfactant as the emulsifier. The same group [44] also studied polyamide microcapsules containing oily liquids. The preparation of oil-containing poly(terephtha1amide) microcapsules by interfacial polymerization has also been recently discussed by Alexandridou and Kiparissides [45]. 4. Studies of Microcapsules Prepared by Interfacial Polymerization Lee and Kondo [46] found at 20C for KC1 a permeability of 10.2 X 10-7 cm S-' by following the diffusion though the membrane by an optical method. The same group [47] studied the effect of pH on thepermeability of poly(L-lysine-alt-terephthalic acid)microcapsules to electrolyte ions. A large increase in permeability in going from pH 4 to 6 (and above) was ascribed to an abrupt increase in the microcapsule size. Levy et al. [48,49] used Fourier transform infrared spectroscopy (FT" IR) to study the HSAmicrocapsules. By following the influence of reaction time, they were able to show after 5 min the progressive acylation of the hydroxy and carboxylate groups of HSA. Free amino groups were also determined by a back-titration method [48,50,51]. The interfacial polymerization of m-phenylenediamine andtrimesoyl chloride has been studied by Chai and Krantz [52] using the novel techniques of light reflection and pendant-drop tensiometry allowing rapid real-time assessment of the polyamide membrane formation.
B. Nonbiomedical Aspects of Microcapsules with Rigid, (Type 2 in Fig. 1) Nonpermeable Shells and Liquid Cores
1. The major application of microcapsules with rigid, nonpermeable and brittle membranes has been in the carbonless copy paper industry.
364
Whateley The underside of upper copies is coated with such inkcontaining microcapsules. These are ruptured by the pressure of writing, and the released ink interacts with the lower paper to leave the imprint. However, in general, such microcapsules are not prepared by interfacial polymerization. More specialized applications of this type of microcapsule allow preparation by the interfacial polymerization process, or example, herbicide and pesticide microencapsulation for controlled release. As well as controlling the release, the active ingredient is also kept out of contact with the environment andhandlers. This topic is well covered in Wilkins [g]. Fragrances, for example, are microencapsulated for the scratch n sniff-type application fixed to such articles as paper and tee shirts. Domestic gas samples are microencapsulatedto provide a simple and readily distributed means of indicating the smell of a gas leak. Pigments have been microencapsulated (e.g., see ref. 53). Duel1 and Pendergrass [54] have reported on the use of polyfunctional aziridines for interfacial polymerization for a variety of applications.
2.
3.
4.
5.
111.
INTERFACIALADDITIONPOLYMERIZATION
Interfacial addition polymerization has features similar to interfacial polymerization, but the polymerisusuallycomposed of only one type of monomerand there is nootherproductformed in the reaction. This method, which does not generally require reactants in the aqueous droplets, should be particularly suitable for enzyme microencapsulation. However, in general, solid microspherestend to beproduced ratherthan aqueous-core-type microspheres.
Alkylcyanoacrylates A.
There has been considerable interest in the use of the alkyl-cyanoacrylates to form microcapsules and nanoparticles for drug delivery and targeting by the groups of Couvreur andKreuter. Florence et al. [55]have also reported the preparation of biodegradable microcapsules from poly(alky1-2-cyanoacrylate) monomers. The anionic addition polymerization of analkyl cyanoacrylate is shown in Fig. 6. This process could be initiated, for example, by water in the aqueous phase of an water-in-oil emulsion with the cyanoacrylate dissolved in the oil phase.
Microcapsules by Polymerization and Complexation

CN I CH, = C -COOK
365
CN
CN CN I I HO-CH,- C - CH, - C - ClOOR I COOR
Fig. 6 Reaction for formation of poly(cyanoacry1ate).
Both the groups of Kreuter and Couvreur (e.g., seerefs. 56 and 57) have developedthese systems overanumber of years. Forexample, Kreuter et al. [58] studied the degradation of poly(butylcyanoacry1ate) and poly(hexylcyanoacry1ate) nanoparticles and their association with and toxicity toward isolated hepatocytes. Formaldehyde has been regarded as being the major problem in considering the toxicity of poly(cyanoacry1ate)(PCA) systems. However, the (low) toxicity toward hepatocytes (which did not take up nanoparticles to any significant extent) could not be attributed solely to formaldehyde during degradation. Leonard et al. [59] studied the degradation of the poly(alkylcyanoacry1ates) in detail. Troster and Kreuter [60] studied extensively the body distribution of radiolabeled poly(methyLmethacry1ate) nanoparticles with and without surface surfactants. Poloxamine 1508 was found to be the most effective in reducing liver uptake. The anticancer drug mitoxantrone was loaded into PCA nanoparticles and found to cause a significant volume reduction in the B16 tumor in rats [61]. Harmia et al.[62] and Harmia-Pulkkinen et al. [63] encapsulated pilocarpine in poly(butylcyanoacry1ate) nanoparticles for drug delivery to the eye. The effects of monomer and stabilizer concentrations on the properties of poly(buty1-2-cyanoacrylate) nanoparticles was investigated by -Nonso et al. [M].
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Poly(cyanoacry1ate) nanoparticles have been evaluated as drug carriers by other groups also; for example, Douglas et al. [65] and Puglisi et al. (661Detailed studies of the particle size distribution of poly(cyanoacry1ate) nanoparticles have been reported [67,68].
IV. INTERFACIAL COMPLEXATION OR INTERFACIAL POLYELECTROLYTE COMPLEXATION
A. AlginatelPolylysine Microcapsules This process was introduced by Lim and Sun [69] and has been developed by their groups and Sefton et al. (e.g., see ref. 71). The topic has been reviewed recently by Sun et al. [70] and Chang [3]. The fact that calcium ions crosslink the alginate species (Fig. 7), initially in solution as the sodium salt, allows droplets of gel to be formed in the calcium chloride solution. The addition of an oppositely charged polyelectrolyte (in this case
poly(mannuronic) acid
poly(gu1uronic)
acid
0
poly(mannuronic)
Coolt
- poly(gu1uronic)
COOH
acid
Fig. 7 Structures of alginate components.
and Complexation
367
the polylysine polycation) allows a layer of insoluble polyelectrolyte complex to form at theinterface. The calcium ions remaining in the interiorof the microcapsule are exchanged with an excess of sodium ions, rendering theinterior fluid again. Thus,an aqueous-cored, semipermeable microcapsule is formed under mild conditions (i.e., no extremes of pH, no chemical reactions). The polyelectrolyte complex membrane has proven to be very biocompatible, an aspect covered in more detaillater. l . Preparation of Microencapsulated Cells Cells are suspended in 0.8% sodium alginate (structure shown in Fig. 7) in saline: Droplets are extruded through a syringe into 1.5% calcium chloride solution. The crosslinking of the alginate macromolecules by the calcium ions causes the droplets to become gels (withthe cells entrapped in the gel sphere). The separated gel microspheres are then resuspended in a polylysine solution (0.02%, molecular weight, 35,000) for 5 min. An interfacial macromolecular complexation occurs at the surface of the gel microsphere, forming an insoluble (but semipermeable membrane). The gel microspheres are separated and resuspended in saline at pH 7.4; the calcium ions diffuse out of the gel interior of the microcapsule causing the contents to liquify. These microcapsules tend to be large (>l00 pm)perhaps macrocapsule is a better term? Mannuronic-rich alginate has been shown to link more strongly to the polylysineby Dupuy et al.[72], who used FT-IR to study polylysine/ alginate membranes containing various contents of mannuronic or guluronic acid residues (structures in Fig. 7). A novel method of preparing small alginate/polylysine microcapsules (in the range 5-15 pm) was reported by Kwoket al. [73]. Vaccine-containing microcapsules were preparedusing aTbrbotak air atomizer to spray sodium alginate solution into calcium chloride solution to form temporary microgel capsules which are subsequently crosslinked with polylysine. A different polyelectrolyte complexation reaction was used by Stange et al. [74] to encapsulate liver microsomes. The polyanion cellulose sulfate (sulfate substitution ratio 0.4-0.5)wasusedwith the polycation poly(dimethyldiallylammonium chloride) (molecular weight 40,000)The resulting membrane was semipermeable with an exclusion limit varying from 25,000 to 150,000 molecular weight depending on the preparation conditions. Increased enzymic activity and increased wall strength were claimed for this procedure. The microencapsulation and in vitro performance of various mammalian cells in polyacrylate membranes has been reviewed by the Sefton group at Toronto [71]. The cell suspension is coextruded with the polymer
368
Whateley
solution through a concentric needle assembly and the concept of this novel way for the controlled delivery of therapeutic agents is discussed. Drug encapsulation in alginate microspheres has been reported by by an emulsiWan et al. [75]. Calcium alginate microspheres were produced fication process and hardenedwith isopropyl alcohol or acetone. However, drug loading was found to below.
2. Applications of AlginatelPolylysine Microcapsules Because of the mild conditions in preparation and the biocompatible nature of the formed microcapsules, the vast majority of applications of microcapsules prepared by the interfacial complexation method are in the pharmaceutical, biomedical, and medical fields, and only these applications will be considered. These applications have been reviewed by Sun et al. [70] and Chang [3]. The transplantation of mammalian cells encapsulated in a protective, biocompatible, and semipermeable membrane has greatclinical potential for a range of diseases requiring enzyme- or cell-replacement therapy. The interfacial complexation process has been used to encapsulate islets of Langerhans, the cells which secrete insulin, for the treatment of diabetes. Limited success has been achieved [69,76,77]. Hepatocytes have also been microencapsulated by this process for the treatment of liver failure [78]. Another majorapplication of microencapsulated cells has beenin the production of monoclonal antibodies. The hybridoma cells, producting the monoclonal antibodies, are entrappedin the microcapsules: nutrients, oxygen, and so forth can enter through the semipermeable membrane. The monoclonal antibodies secreted by the encapsulated hybridoma cells remain trapped inside the microcapsules and accumulateto a greater concentration thanin the absenceof encapsulation. To recover the antibodies, the microcapsules are separated from the culture medium and ruptured to release the antibodies. In the absence of encapsulation, the antibodies must be isolated from a large volume of culture medium.
3. Permeability of AlginatelPolylysine Microcapsules CoromiliandChang [79] haveused a heterogeneousmixture of dextrans with molecular weights 10,000-500,000 to study the membrane permeability of alginatelpolylysine microcapsules. Hybrid artificial organs must permit the diffusion of smaller molecules, includingpeptides and proteins, but must exclude leukocytes and immunoglobulins( M W >150,000) Fluorescein isothiocyanate-labeled dextrans were used by Vandenbossche et al. [80] to determine the molecular weight cut-off of similar microcapsules. The molecular weight cutoff of alginatelpolylysinemicrocapsules has
and Complexation
369
been shown to be dependent on preparative conditions [81]. Microcapsules prepared at 40C showed a lower MW cutoff when the concentration of polylysine wasincreased from O.l%(w/v) The ultrastructural characterization of alginatellysine microcapsules was reported by Shimi et al. [82] Poly(1ysine) ofmolecular weight 22,000 was found to be optimum in forming robust microcapsules which were relatively impermeable to high molecular weight species such as immunoglobulins. 4. Biocompatibility of AlginatelPolylysine Microcapsules The topic of biocompatibility is a huge area in its own right: A few recent references are discussed here concerning the biocompatibility of alginate/polylysine microcapsules. The biocompatibility of alginate/polylysine microcapsules was enhanced by Sawhney et al. [83] by photopolymerizing a PEG-based hydrogel onto thesurface of alginate/polylysinemicrocapsules. A less inflammatory response with no fibrotic response was reported. The effect of composition on biocompatibility was examined by Clayton et al. [84], who found that in the peritoneum, high mannuronic acid alginate capsules provoked the weakest response. Islets of Langerhans inmicrocapsules of alginate/polylysine have been proposed as a artificial pancreas (e.g., see ref. 85), who studied the capacity of such microcapsules to activate macrophages in order tounderstand the foreign body reaction withfibrosiswhich has been observed around implanted microcapsules. The host reaction against alginate/polylysine microcapsules has been studied with both empty microcapsules [86] and with microcapsules containing living cells [87]. Histological evaluation showed a significantly higher number of cells stickingto microcapsules containing cells as compared with empty microcapsules.
REFERENCES
1. R. Arshady, Biodegradable microcapsular drug delivery systems: Manufacturing methodology, release control and targeting prospects, J. Bioactive Compatible Polymers, 5:315-342 (1990). 2. R. Arshady,Preparation of microspheresandmicrocapsulesbyinterfacial polycondensation techniques, J. Microencaps., 6:13-28 (1989). 3. T. M. S. Chang, Recent advances in artificial cells based on microencapsulation, in Microcapsules and Nanoparticles in Medicine andPharmacy (M. Donbrow, ed.), CRC Press, Boca Raton, FL, 1992, pp. 323-339. 4. F. Lim, (ed.), Biomedical Applications of Microencapsulation, CRC Press, Boca Raton, FL, 1984.
370
Whafeley
5. M. Donbrow, (ed.), Microcapsules and Nanoparticles in Medicine And Phar-
macy, CRC Press, Boca Raton, FL, 1992. 6. T. L. Whateley (ed.), Microencapsulation of Drugs, Harwood Academic Publishers, Reading, UK, 1992. 7. T. M. S. Chang, Semipermeable microcapsules, Science, 146524-525 (1964). 8. T. M. S. Chang, F. C. Macintosh, andS. G . Mason, Semipermeable aqueous microcapsules, Can.J. Physiol. Pharmacol., 44:115-128 (1966). 9. R. M. Wilkins, (ed.), Controlled Delivery of Crop-Protection Agents, Taylor and Francis, London, 1990. 10. P. W. Morgan,CondensationPolymers:InterfacialandSolutionMethods, NY, PolymerReviewsSeries N0.10, IntersciencePublishers,JohnWiley, (1965). 11. F. Millich and C. E. Carraher, Jr., (eds.), Interfacial Synthesis, Volume 1: Fundamentals, Marcel Dekker,N Y , USA (1977). 12. Y. Wakamatsu, M. Koishi and T. Kondo, Microcapsules 17: Effect of chemical structureof acid chlorides and bisphenols on the formation of polyphenyl ester microcapsules, Chem. Pharm. Bull. 22,1319-1325 (1974). A. N.Hambly,Kineticsof interfacial 13. J. H.Bradbury, P.J.Crawfordand polycondensation reaction, Trans. Far. Soc. 64, 1337-1347 (1968). 14. M.Koishi, N. FukuharaandT.Kondo,Microcapsules2:Preparationof polyphthalamide microcapsules, Chem. Pharm. Bull. 17,804-809 (1969). 15. M. Koishi, N. Fukuhara and T. Kondo, Studies on microcapsules 4: Preparationandsomeproperties ofsulphonatedpolyphthalamidemicrocapsules, Can. J. Chem. 47,3447-3451 (1969). S. Tomioka, Microcapsules 6: 16. Y. Shigeri, M. Koishi, T. Kondo, M. Shiba and on microcapsule size, KolloidEffect of variationsin polymerization conditions Z Z.Polym., 249,1051-1055. Strength of interfacial polymerization films, 17. K. J. Mysels and W. Wrasidlo, Langmuir, 7:3052-3053 (1991). Etuk, andK.R.Murray,Formationofnylon6,lO 18. L.R.Weatherley,B. capsules by interfacial polymerization in a high-voltage electric-field, Powder Technol., 65227-233 (1992). T. Kondo,Preparation of polyamide mi19. N. Muramatsu,K.Shiga,and crocapsuleshavingnarrowsizedistributions,J.Microencaps.11:171-178 (1994). Levy, Polyhydroxamicmicrocapsules 20. D. Hettler, M.C.Andry,andM.C. Mipreparedfromproteins--Anoveltypeofchelatingmicrocapsules,J. croencaps., 11:213-224 (1994). 21. M.C. Levy, D. Hettler, M.C.Andry,and P. Rambourg, Polyhydroxamic serum albumin microcapsules: Preparation and chelating properties, Int. J. Pharm., 69:Rl-R4 (1991). New 22. T. M. S. Chang and R. Geyer (eds.), Blood Substitutes, Marcel Dekker, York, 1989. 23. T. M. S. Chang, Blood compatible coatingsof synthetic immunoadsorbents, Trans. Am. Soc. Intern. Artif. Organs, 26:546 (1980).
Complexation and
371
M. 24. T.
S. Chang, P. Barre, and S. Kuruvilla, Long term reduced time haemoperfusion-haemodialysiscompared to standarddialysis,Trans.Am.
Soc. Intern. Artif. Organs, 31572 (1985). 25. D. A. Wood, T. L.Whateley,andA. T. Florence, Microencapsulation of histidaseforenzymereplacementtherapy,J.Pharm.Pharmacol.,31:79P (1979). 26. T. Kondo, Enzyme inactivation in microencapsulation, in Microencapsulation (J. R. Nixon, ed.), Marcel Dekker, New York, 1976, pp. 67-75. T. L. Whateley,Astudyofenzymeand protein micro27. D.A.Woodand encapsulation-somefactorsaffecting the low apparentenzymicactivity yields, J. Pharm. Pharmacol.,3432-557 (1982). 28. M. Kidokoro, H. Ohshima, and T. Kondo, Interaction of poly(L-lysine-aftterephthalic acid) microcapsules with fibrinogen, J. Microencaps. , 8:63-70 (1991). on the thermostability of 29. K. Hoshino, N. Muramatsu, and T. Kondo, A study microencapsulated glucose oxidase, J. Microencaps. ,6:205-211 (1989). J. Neufeld, Activity and distribution of urease follow30. M. Monshipouri and R. ingmicroencapsulationwithinpolyamidemembranes,EnzymeMicrobial Techn., 31:309-313 (1991). 31. R. B. Aisina, Effectof microencapsulated enzymes, in Microencapsulation of Drugs (T. L. Whateley, ed.), Harwood Academic Press, Reading, UK, 1992, pp. 215-231. 32. A. M. Wallace, Novel immunoassays for clinically important hormones based on microencapsulatedantibodies,inMicroencapsulationofDrugs(T.L. Whateley, ed.), Harwood Academic Press, Reading, UK, 1992, pp. 241-255. 33. L. A. Luzzi, M. A. Zoglio, and H. V. Moulding, Preparation and evaluation J. Pharm. Sci., 59:338of prolonged release properties of nylon microcapsules 341 (1970). . Florenceand A. W. Jenkins,Invitroassessmentofmicroencapsu34. A. T lated drug systems as sustained release parenteral dosage forms, in Microencapsulation,(J. R. Nixon,ed.),MarcelDekker,NewYork,1976,pp. 39-55. . McGinity, Expanded versatility of microcapsules pre35. G. W. Cuff and J. W pared by interfacial polymerisation,J. Microencaps. 1:343-347 (1984). 36. G. W. Cuff, A. B. Combs, and J. W. McGinity, Effect of formulation factors on the matrix pH of nylon microcapsules, J. Microencaps., 1:27-32 (1984). 37. U. Chiba, E. W. Neuse, J. C . Swarts, and G. J. Lamprecht, Water-soluble polyamides as potential-drug carriers. 7. Synthesis of polymers containing intrachain-type or extrachain-type amine ligands by interfacial polymerization, Angewandte Makromolekulare Chemie, 214:137-152 (1994). of carcinogen sources in the 38. I. ONeill. Reactive microcapsules for detection gut, J. Microencaps., 10:283-308 (1993). 39. A. M. Pense, C . Vauthier-Holtzscherer, and J.-P. Benoit, Microencapsulation of benzalkonoium chloride, in Microencapsulation of Drugs (T. L. Whateley, ed.), Harwood Academic Press, Reading, UK, 1-5.
372
Whateley
40. A. M. Pense, C. Vauthier, F. Puisieux, and J.-P. Benoit, Microencapsulation of benzalkonium chloride, Int. J. Pharm.,81:lll-117 (1992). 41. A. M. Pense, C. Vauthier, and J.-P. Benoit, Study of the interfacial polycondensationofisocyanateinthepreparation of benzalkoniumchloride loaded microcapsules, Colloid Polymer Sci., 272:211-219 (1994). Li, Size distribution and zeta-potential of micro42. N. Yan, M. Zhang and P. capsules. J. Membrane Science, 72, 163-169 (1992). of polyurea mi43. N. Yan, P. Ni, and M. Zhang, Preparation and properties crocapsules with non-ionic surfactant as emulsifier. J. Microencaps., 10:375383 (1993). on polyamide microcapsules containing 44. N. Yan, M. Zhang, and P. Ni, Study oily liquids, J. Microencaps., 11:365-372 (1994). 45. S. AlexandridouandC.Kiparissides,Productionofoilcontainingpoly(terephthalamide) microcapsules by interfacial polymerisation: Effectof process variableson the microcapsule sue distribution,J. Microencaps., 11:603614 (1994). 46. K. B. Lee, and T. Kondo,Permeabilitycoefficientofmicrocapsulemembrane, J. Microencaps., 199-356 (1984). on pH of permeabil47. E. Miyauchi, Y.Togawa, K. Makino, et al., Dependence poly(L-lysine-alt-terephthalic acid)microitytowardselectrolyteionsof capsule membranes, J. Microencaps., 9:329-333,1992. 48. M. C. Levy, S. Lefebvre, M. Rahmouni, et al., Fourier-transform infrared spectroscopic studies of human serum-albumin microcapsules prepared by interfacialcross-linkingwith terephthaloylchloride-influence of polycondensation pHon spectra and relation with microcapsule morphology and size J. Pharm. Sci., 80578-585 (1991). 49. M. C. Levy, S. Lefebvre, M. C.Andry, et al., Fourier-transform infrared spectroscopic studies of cross-linked human serum-albumin microcapsules. 2. Influence of reaction-time on spectra and correlation with microcapsule morphology and size, J. Pharm. Sci., 83:419-422 (1994). 50. F. Edwards-Levy, M. C. Andry, and M. C. Levy, Determination of free amino group content of serum-albumin microcapsules using trinitrobenzenesulfonic acid-Effect of variations in polycondensation pH, Int. J. Pharm., 96:85-90 (1993). 51. F. Edwards-Levy, M. C. Andry, and M. C. Levy, Determination of free amino group content ofserum-albuminmicrocapsules.2.Effectof variationsin reaction-timeandinterephthaloylchlorideconcentration,Int.J.Pharm., 103:253-257 (1994). 52. G . Y. Chai and W. B. Krantz, Formation and characterization of polyamide membranes via interfacial polymerization, J. Membr. Sci., 93:175-192 (1994). 53. H. S. Tan, T. H. Ng, and H. K. Mahabadi, Interfacial polymerization encapsulation of a viscous pigment mix-emulsification conditions and particle-size distribution, J. Microencaps., 8525-536 (1991). 54. B.L.Duel1 and D. B. Pendergrass, Microencapsulation by interfacial polyme ization of polyfunctional Aziridines, Abstract Papers Am. Chem. Soc., 208(Pt. 2):336-P0ly (1994).
Microcapsules Polymerization by and

55.
COmpleXatiOn
373
56. 57.
58.
59.
60.
61.
62. 63.
64.
65. 66.
67. 68. 69. 70. 71.
A. T. Florence, T. L. Whateley, and D. A. Wood, Potentially biodegradable poly(alky1-2-cyanoacrylate) membranes, Pharm. J. microcapsules with Pharmacol., 31:422-424 (1979). P. Couvreur, Poly(cyanoacry1ates) as colloidal drug camers, CRC Crit. Rev. Ther. DrugCamer Sys., 51-20 (1988). P. Couvreur and C. Vauthier, Poly(alkylcyanoacry1ate) nanoparticles as drug carriers: Present state and perspectives, J. Controll. Rel., 17:187-198 (1991). J. Kreuter,C. G. Wilson, J. R. Fry, et al., Toxicity and association of poly(cyanoacry1ate) nanoparticles with hepatocytes, J. Microencaps. 1:253257 (1984). of F. Leonard, R.K. Kulkami, G . Brandes, et al., Synthesis and Degradation Sci., 10:259-272 (1966). Poly(alky1 cyanoacrylates) J. Appl. Polymer S. D. Troster and J. Kreuter, Influenceof the surface properties of low contact angle surfactants on the body distribution of C-l4 poly(methy1 methacylate) nanoparticles, J. Microencaps. 9,19-28 (1992). I. Fichtner,Influenceofpoly(buty1P. Beck,J.Kreuter,R.Reszka,and on the efficacy and toxicity of the cyanoacrylate) nanoparticles and liposomes anticancerdrugmitoxantroneinmurinetumourmodels,J.Microencaps., 1O:lOl-114 (1993). T. Harmia, P. Speiser, and J. Kreuter, A solid colloidal drug delivery system for the eye: Encapsulation of pilocarpine in nanoparticles, J. Microencaps., 3~3-12(1986). T. Harmia-Pulkkinen, A. lbomi, and E. Kristoffersson, Manufacture of poly(alkylcyanoacrylate) nanoparticles with pilocarpine andby timolol micelle polymerisation: Factors influencing particle formation, J. Microencaps., 6237-93 (1989). M. J. Alonso, A. Sanchez, D. Torres, et al., Joint effects of monomer and stabiliser concentrations on physico-chemical characteristics of poly(butyl-2cyanoacrylate) nanoparticles, J. Microencaps., 7517-526 (1990). S. J. Douglas, S. S. Davis, and L. Illum. Nanoparticles in drug delivery, CRC Crit. Rev. Ther. Drug Carrier Syst., 3,233-261 (1987). M. Fresta, et al., Evaluation of poly(alky1G. Puglisi, G . Giammona, cyanoacrylate) nanoparticles as a potential drug camer: Preparation, morphologicalcharacterisationandloadingcapacity,J.Microencaps.,10:353-366 (1993). K. Shankland and T. L. Whateley, Particle size distribution of polycyanaoacrylate nanoparticles, J. Pharm. Pharmacol., 41:133P (1989). K. Shanklandand T. L.Whateley,Determinationofrefractiveindicesof polycyanoacrylate particles, J. Coll. Interface Sci., 154:160-165 (1992). F. LimandA.M. Sun, Microencapsulated islets as bioartificial endocrine pancreas, Science, 210:908 (1980). A. M. Sun, I. Vacek, andI. Tai, Microencapsulation of living cells and tissues, in Microcapsules and Nanoparticles in Medicine and Pharmacy (M. Donbrow, ed.), 1992, pp. 315-322, CRC Press, Boca Raton, Florida. H. Uludag, L. Kharlip, and M. V. Sefton,Proteindelivery by microencapsulated cells, Adv. Drug Del. Rev., 10:115-130 (1993).
374
72.
Whateley
B.Dupuy,A.Arien,andA. P. MinnotFT-IRofmembranesmadewith alginate/polylysine complexes-variations with the mannuronic or guluronic 22,71content of the polysaccharides, Artif. Blood Subst. Immobil. Biotech.
82 (1994).
73.
K. K. Kwok, M.J. Groves, and D. J. Burgess, Production of5-15 micrometre diameter alginate-polylysine microcapsules by an air-atomization technique, Pharm. Res., 8:341-344 (1991). et al., A new technique for encapsu74. J. Stange, E. Brugmann, D. Falfenhurst, lation of liver microsomes, in Microencapsulation of Drugs (T. L. Whateley, ed.), Harwood Academic Press, Reading, UK, 1992, pp. 257-262. 75. L. S. C. Wan, P. W. S. Heng, and L.W. Chan, Drug encapsulation in alginate microspheres by emulsification,J. Microencaps., 9:309-316 (1992). et al., An artificial endocrine 76. A.M. Sun, W. Parisius,H.G.Macmorine, pancreas containing cultured islets of Langerhans,Artif.Organs, 4:275
(1980). 77.
M. Y. Fan, Z. P. Lum, X. W. Fu, et al., Reversal of diabetes in BB rats by transplantationof encapsulated pancreatic islets, Diabetes, 39519 (1990). 78. Z .Cai, Z.Shi, M. Sherman, andA.M. Sun, Development and evaluation of asystemofmicroencapsulationofprimaryrathepatocytes,Hepatology, to study the membrane permeability of alginate polylysine microcapsules, Biomat. Artif. Cells Immobil. Biotech.21,427-444 (1993). 80. G . M. R. Vandenbossche, P. Vanoostveldt and J. P. Remon, A fluoroescence method for the determination of the molecular weight cut-off of alginate polylysine microcapsules, J. Pharmac. Pharmacal. 43,275-277 (1991). 81. G. M. R. Vandenbossche, P. Vanoostveldt, J. Demeester and J. P. Remon, The molecular weight cut-off of microcapsules is determined by the reaction 42, 381-386 between alginate and polylysine, Biotechnol. and Bioeng.
(1993). 82. S. M. Shimi, E. L. Newman, D. Hopwood, and A. Cushieri, Semi-permeable microcapsulesforcellculture:Ultra-structuralcharacterisation, J. Microencaps., 8:307-316 (1991). 83. A. S. Sawhney, C. P. Pathak,and J. A.Hubbell,Interfacialphotopoly-
10:855 (1989). 79. V. Coromili andT. M. S. Chang, Polydisperse dextran as a diffusing test solute
merization of poly(ethy1ene glycol)-based hydrogels upon alginate poly(L lysine) microcapsules for enhanced biocompatibility, Biomaterials, 14:10081016 (1993).
H. A. Clayton, N. J. M. London, P. S. Colloby, et al., The effect of capsule J. Micomposition on the biocompatibility of alginate/poly(l-lysine) capsules, croencaps., 8:221-233 (1991). 85. M. E. Pueyo, S. Darquy, F. Capron and G. Reach. In-vitro activation of humanmacrophagesbyalginatepolylysinemicrocapsules, J. Biomat.Sci. Palp. Ed., 5,197-203 (1993). 86. G. M. R. Vandenbossche, M. E. Bracke, C. A. Cuvelier, H. E. Bortier, M. M. Mareel, andJ. P. Remon, Host reaction against empty alginate polylysine
84.
45,115-120 (1993).
and Complexation
375
microcapsules-influence of preparation procedure, J. Pharmac. Pharmacol.,
87. G. M. R. Vandenbossche, M. E. Bracke, C. A. Cuvelier, H. E. Bortier, M. M. Mareel and J .P. Remon, Host reaction against alginate polylysine microcapsules containing living cells, J. Pharmac. Pharmacol., 45, 121-125 (1993).
14
Lipospheres for Controlled Delivery of Substances
Abraham J. Domb* and Lev Bergelson
TheHebrew Universityof Jerusalem, Jerusalem, Israel

Shimon Amselem
Phurmos Ltd., Weizmann IndustrialPark, Rehovot, Israel
Introduction I.
378 379 380 380 382 384 385 386 386 388 389 394 399 403 406 407
11. Preparation of Lipospheres

111. Physical Characterization of Lipospheres A. Morphology B. Structure C. Particle Size D. Viscosity and Drug Loading E. Drug Distribution F. InVitroDrugRelease
IV. Applications of Lipospheres A. Parenteral Delivery of Local Anesthetics B. Parenteral Delivery of Antibiotics C. Parenteral Delivery of Vaccines and Adjuvants D. Topical Delivery of Insect Repellent E. Nanolipospheres for Cell Targeting of Anticancer Drugs
V.
Summary
*Affiliated with the David Bloom Center for Pharmacy, Jerusalem, Israel.
377
378
1.
Domb et al.
INTRODUCTION
Many dispersion systems are currently in use as carriers of biologically active compounds. Dispersion systems used for pharmaceutical and cosmetic formulations can be categorized as either suspensions, emulsions, and dispersions. Suspensions are defined as solid particles ranging in size from a few nanometers up to hundreds of micrometers: dispersed in a liquid medium using suspending agents. Solid particles include microspheres, microcapsules, and nanospheres [l]. Emulsions can be defined as dispersions of one liquid in another, stabilized by an interfacial film of emulsifiers such as surfactants and phospholipids. Despite their long history, emulsions are used less often today than many other dosage forms owing to their inherent instability. Emulsion formulations include water-in-oil and oil-in-water dispersions, multiple water-in-oil-in-water emulsions, and microemulsions [2]. Lipid dispersions such as liposomes are defined as phospholipidvesicles, and they are obtained by dispersing phospholipids in aqueous medium. Liposomes may be unilamellar or multilamellar, depending on the number of lamellae or bilayers, separated each from the other by a water domain. Multilayer liposomes consist of a highly ordered assembly of concentric phospholipid membranes or bilayers entrapping internal aqueous compartments [3]. The rigidity and permeability of phospholipid bilayers can be adjusted by including other water-insoluble components such as sterols and amphiphilesin addition to thephospholipid matrix. Lipospheres represent a new type of fat-based encapsulation system developed forparenteral and topical drug delivery of bioactive compounds [4-lo]. Lipospheres consist of water-dispersible solid microparticles of particle size between 0.2 to 100 pm in diameter composed of a solid hydrophobic fatcore (triglycerides) stabilized by one monolayerof phospholipid molecules embedded in their surface. The internal core contains the bioactive compound dissolved or dispersed in the solid-fat matrix. The liposphere system has beenused for the controlled delivery of various types of drugs, including anti-inflammatory compounds, local anesthetics, antibiotics, and anticancer agents. They have alsobeen used successfullyas carriers of vaccines and adjuvants [7-lo]. Similar systems based on solid fats and phospholipids have beendescribed recently [U-131. Lipospheres have several advantages over other delivery systems, including emulsions, liposomes, and microspheres, forexample, better physical stability, low costof ingredients, ease of preparation and scale-up, high dispersability in an aqueous medium, high entrapment of hydrophobic
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drugs, controlled particle size, and extended release of entrapped drug after a single injection from a few hours to several days. This chapter describes the preparation and physicochemical properties of lipospheres and their use for the parenteral delivery of various drugs and vaccines and the topical administration of active agents.
11.
PREPARATION OF LIPOSPHERES
In contrast to certain oil emulsions, the liposphere approach utilizes naturally occumng biodegradable lipid constituents. The internal hydrophobic core of lipospheres is composed of fats, mainly solid triglycerides, whereas the surface activity of liposphere particles is provided by the surrounding lecithin layer composed of phospholipid molecules. The neutral fats used in the preparation of the hydrophobic core of the liposphere formulations described here include tricaprin, trilaurin, and tristearin, stearic acid, ethyl stearate, and hydrogenated vegetable oil. The polymers used in the preparation of polymeric biodegradablelipospheres were low molecular weight poly(1actic acid) and poly(capro1actone). The phospholipids used to form the surrounding layer of lipospheres were pure-egg phosphatidylcholine, soybean phosphatidylcholine, dimyristoyl phosphatidylglycerol, and phosphatidylethanolamine. Food-grade the preparationof lipospheres lecithin (96% acetone insoluble) was used in for topioal and veterinary applications. Liposphere formulations are prepared by a solvent or melt process. In themelt method, theactive agent is dissolved or dispersed in the melted solid camer; that is, tristearin or polycaprolactone and a hot buffer solution is added at once along with the phospholipid powder. The hot mixture 5 min using a homogenizer or ultrasound is homogenized for about 2 probe, after which a uniform emulsionis obtained. The milky formulation is then rapidly cooled down to about 2 0 C by immersing the formulation flask inan acetone-dry ice bath while homogenizationis continued to yield a uniform dispersion of lipospheres. Alternatively, lipospheres might be prepared by a solvent technique. In this case, the active agent, the solid camer, and phospholipid are dissolved in an organic solvent such as acetone, ethyl acetate, ethanol, or dichloromethane. The solvent is then evaporated and.tberesulting solid is mixed with warm buffer solution and mixing is continued until a homogeneous dispersion of lipospheres is obtained. (200mg), tristearin (400mg), In a typical preparation, dexamethasone and propylparaben (5 mg) are added to a 50-mL round-bottom glass flask. 7 5 C to melt the tristearin-dexamethasone mixture and The flask is heated at
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hot 0.1 M phosphate buffer solution pH 7.4 (75"C, 9 . 3 g) isadded along with egg phosphatidylcholine (200 mg). The mixture is homogenized for 2 min until a uniform milky formulation is obtained. The hot formulation is rapidly cooled to below 20Cby immersing the flask in adry ice-acetone bath with continued mixing to yield a white, thin dispersion.If needed, the pH of the formulation is adjusted to 7.4 with a 1N HC1 solution. The formulation may contain antioxidants such as tocopherol and preservatives such as parabens. Submicron-size lipospheres are prepared by passing (four times) the liposphere formulation by extrusion through a submicron series of filters at a temperature 5C above the melting point of the liposphere core composi200 nm. tion. Particlesize maybe reduced to about Polymeric biodegradable lipospheres can also be prepared by a solvent or melt process. The difference between polymeric lipospheres and the standard liposphere formulations is the composition of the internal core of the particles. Standard lipospheresas those previously described consist of a solid hydrophobic fat core composed of neutral fats like tristearin, whereas in the polymeric lipospheres, biodegradable polymers such as polylactide or polycaprolactone substituted the triglycerides. Both typesof lipospheres are thought to be stabilized by one layer of phospholipid mole111). cules embedded in their surface (see Section Sterile liposphere formulations are prepared by sterile filtration of the dispersion in the hot stage during preparation through a filter at 0.2 pm a temperature5C above themelting point of the liposphere core composition. Heat sterilization using a standard autoclave cycle decomposed the formulation. y-Sterilization of liposphere formulationsdid not affect their physical properties. Lipospheres formulations of 1 : 4 : 2 and 2 : 4 : 2 bupivacaine-tristearin-phospholipid(w/w % ratio) were irradiated with a samples were analyzed forparticlesize, dose of 2.33 Mrad,andthe bupivacaine content, in vitro release characteristics, and in vivo activity. The irradiated formulations had a similar particle size, bupivacaine content, release rate, and anesthetic effectiveness in the rat paw analgesia model to bupivacaine HCl solution (Marcaine). However, a more careful analysis of the formulation ingredients should be performed, since phospholipids may degrade during irradiation [14].
111. PHYSICAL CHARACTERIZATION OF LIPOSPHERES
A. Morphology
Fig. 1 shows a light microscopic picture of a typical preparation of lipospheres composed of tristearin and lecithin consisting of particles having a uniform spherical shape. Microscopic examination of a typical liposphere
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Fig. 1 Light microscope pictureof a typical preparationof lipospheres composed of tristearin and lecithin with average particle size of 10 pm.
Fig. 2 Transmission electron microscopy (TEM) of a single nanoliposphere (200

nm) .
formulation using transmission electron microscopy (TEM) showed spherical particles (Fig. 2).
B. Structure
The phospholipid content on the surfaceof lipospheres was determined by 31Pnuclearmagneticresonance (NMR) before andaftermanogenase
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(Mn) or proseodimium(Prt3) ion complexation [l51 and by trinitrobenzenesulfonic acid (TNBS) labeling using liposphere formulations containing phosphatidylethanolamine (PE) [16]. In both methods, the agents (Mn+2, Prt3, or TNBS) interact specifically with the exposed phosphateor amino groups of the phospholipid. Determination of the surface phospholipid using the TNBS method showed that 70-90% of the phospholipid polar heads are on the surface of the liposphere particles prepared from triglyceride-phospholipid at a 1 : 0.5 to 1 : 0.25 w/w ratio. Increasing the phospholipid content decreases the percentageof surface phospholipid polar heads, which indicates the formation of other phospholipid structures such as liposomesin the formulation. Similar results were obtained by 31P NMR analysis using Mn+and Prf3ions for interaction with the phosphate polar heads [15]. This is illustrated in Fig. 3, which showsP NMR spectra of drug-free liposphere vesicles obtained in the absence (A) and presence after the addition of 5 mM Mn+ (B) of Mn+. The decrease in the peak area represent the relative amount of phospholipid in the outer monolayer avail-
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Fig. 3 31PNMR spectra of lipospheres composed of tricaprin-phospholipid in a weight ratio of 2 : 1 in the absence (A) and presence (B) of Mnt2. 0 ppm corresponds with the resonance position of diacylphosphatidylcholine undergoing isotropic motion.
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able for interaction with Mn+2. For lipospheres composed of tricaprinphospholipid at a weight ratio of 2 : 1 and 5 : 1, 75 and 90% of the phospholipid polar heads wereaccessible to theparamagnetic ions, respectively. For comparison, liposomes of the same composition and size but without tricaprin had only 40% of the phospholipid polar heads on the surface [151. These datasuggest that the proposed structure of a liposphere is that of spherical particle with a monolayer of phospholipid molecules surrounding theinternal solid fatcore,wherethehydrophobic chains of the phospholipids are embedded in the internal triglyceride core containing the active agent, asillustrated in Fig. 4.
C. Particle Size
Analysis of the particle size distribution of lipospheres was performed using a LS 100 Coulter Counter Particle Size Analyzer (Coulter Corp., Hialeah, FL). This instrument can measure particles from 0.4 to 800 pm by particle size-dependent light diffraction patterns. The particle size of submicron lipospheres was estimated from the transmission electron microscope pictures and using a particle size analyzer for 0.01- to 3-pm particle size diameter range. Blank and drug-loaded lipospheres prepared by the melt method without farther treatment had a unimodal shape with an average particle size
Fig. 4 Schematic illustration of proposed structureof a liposphere particle.
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between 5 and 15 pm with less than 2% of particles greater than 100 pm. This particle size formulation is useful for subcutaneous or intramuscular injection or fortopical applications. In an effort to reduce the particle size, lipospheres of 16.9 pm average diameter preparedby the melt method were passed through a laboratory Microfluidizer (Microfluidics, MA, USA). Thereby the average size was reduced to 10.8 pm after8 passes and to 9.4 pm after 40 cycles. Similar results were obtained when using a homogenizer. Lipospheres containing the malaria R32NS1 antigen and differingin their fat composition were prepared by the melt procedure [&lo]. Three groups of neutral fats were used: (1) solid fats such as tristearin and stearic acid with melting points in the rangeof 6570C; (2) semisolid fats such as tricaprin and ethylstearate with melting points in the range of 30-35C; and (3) liquid fats such as olive oil and corn oil. ?kro populations of liposphere particles usually coexisted, one in the size range of 1-10 pm in diameter (population A), and a second population with a diameter between10 and 100 pm (population B). No correlation was found between fat physical state (solid, semisolid, liquid) and particle size distribution of lipospheres formed. Lipospheres made of tristearin were the most homogeneous formulations, with 100%of the particles having an average diameter of about 7 2 3 Pm 181. Biodegradable polymeric lipospheres made of polylactide and lecithin showed a very broad particle size distribution from 2 to 100 pm in diameter, with a mean average size of 30.6 & 25.9 pm and median (% of particles >50pm) equal to 21.6 [lo]. Polycaprolactone lipospheres showed a similar range of particle size distribution, but with a 1.5-fold mean average size (45.6 f 29.5 pm) and a doublemedian value (43.6) compared with polylactide lipospheres. Inclusion of lipid A in the composition of the polymeric lipospheres reduced their mean average particlesize by a factor of 0.25 regardless of the polymer type [lo]. All the liposphere formulations with a polymeric core prepared remained stable. during the3-month period of the study, and no phase separation or appearance of aggregates were observed.
D. Viscosity and Drug Loading
For drugs such as oxytetracycline [17], itraconazole, and dexamethasone, up to20% drug loading into lipospheres was obtained and the formulations were fluid enough to beinjected.Forotheragentslikebupivacaine, lidocaine, and chloramphenicol, drug loading above 10% produced aviscous lotion. Theviscosity of the liposphere formulation is dependent on the drug properties, the ionic strength andofpH the continuous aqueous solu-
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tion, and the ratio and amount of phospholipids and triglycerides. An increase in the contentof the insoluble ingredients (drug and lipids) and in the salt concentration of the aqueousmedium increases the viscosity of the formulation. For topical applications, lotion and paste consistencies are desired, which are achieved by adding NaCl to the water phase and increasing the fatand phospholipid content of the liposphere lipid phase [M].
E. Drug Distribution
The drug distribution in a liposphere formulation was determined by isolating the particles by centrifugation and determiningthe drug content in the isolated cake after extraction of the free drug with, for example, acidic buffer solution for basic drugs. The amount of unencapsulated free drug can be estimated by microscopic analysis(the free drug appears usually as crystals, whereas lipospheres appear as round shapes). A study was conducted to de". Ane bupivacaine distribution in the 2% bupivacaine formulation [19]. It was found that the nonencapsulated ,drug was observed as needle-shaped crystals dispersed among the liposphere particles. These lipospheres anddrug crystals were isolated by centrifugation, thebupivacaine crystals were extracted from the mixturewith acidic buffer solution, and the drug-loaded lipospheres were analyzed for bupivacaine contept after dissolution-extraction of the lipospheres in Triton solution. The drug loading was in the rangeof 70-85 weight% in the core and about20% of the drug was extracted by the acidic solution appearingasnonincorporated drug. About 4% of the drug was soluble in the aqueous solution, which is the solubility of bupivacaine in the buffer solution. In an attempt to determine the form of the unincorporated drug, bupivacaine and phospholipid were dispersed in aqueous mediumin the absence of the tristearin component. A uniform and stable submicron dispersion was obtained. Microscopic examination of this fat-free preparation showed that the dispersed drug microparticles are nonspherical but in the form of long needles. It should be noted thatbupivacaine free base is not dispersible in buffer solution without asurfactant such as phospholipids. Thus, it is apparent that the unincorporated bupivacaine in the tristearin liposphere formulation is in a form of dispersible microparticles composed of the solid drug and phospholipids. Taxol, verapamil, and piridoxamine were incorporated in submicronsize lipospheres up to 90% encapsulation yield.
F. InVitro Drug Release
In vitro release studies were conductedusing a large-pore dialysis tubing of 300,000 molecular weight cut-off (MWCO) to minimize the effect of the
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Fig. 5 Release of bupivacaine from 2 and 5% loaded liposphere formulation.
tubing on the release rate from the formulation (a regular pore size tubing of 12,000 MWCO did affectthe drug release rate). Thecontrol solutions of drugs such as bupivacaine (Marcaine-0.75%in solution for injection) were released through the large-pore tubing within a few hours. Drug releases from 2 and 5% bupivacaine-loaded liposphere formulations are shown in Fig. 5. Both formulations released the drug for 48 h following a first-order kinetics (R2=0.97).These release profiles are expected for forsoluble in the aqueousvehicle mulations that contain 4% of the free drug which are released immediately throughthe dialysis tubing. The release of etoposide, a water-insoluble anticancer agent, from 2% loaded lipospheres placedin a dialysis tubing (300,000 MWCO) is shown in Fig. 6 . Over 90% of the drug was constantly released during a period of 80 h. Lipospheres containing I4C-diazepamwere preparedaccording to the melt technique. The lipospheres showed a very uniform particle size distribution with an average particle size of 8 pm. The lipospheres were evaluated in vitro by placing 0.5 mL of the formulation in a dialysis tubing (300,000 MWCO). The dialysis tubing was placed in a 0.1" phosphate
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Fig. 6 Release of etoposide from a liposphere dispersion. Total amount etoposide in the test specimen was 6.2 mg.
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buffer, and the cumulative release of diazepam was determined by measuring the radioactivity in the release medium. The release medium was changed periodically to provide sink conditions. Sustained release of diazepam was obtained over a period of 3 days. The in vitro release was also determined by mixing a sample of the formulation in excessbuffer solution, specimens of the mixture were taken every few hours, and the drug released into the solution was determined after removalof the lipospheres by ultracentrifugation. The release rate by this method was faster than from dialysistubing. IV. APPLICATIONS OF LIPOSPHERES This section describes the use of lipospheres for parenteral administration of long-acting local anesthetics and oxytetracycline and fortopical application of N,N-diethyl-m-toluamide(DEET) insect repellent.
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Parenteral Delivery of Local Anesthetics
Local anesthetics are preferred over general anesthetics because of the serious complications that can occur during general anesthesia. However, even local anesthetics, which are usually injected as an aqueous solution, are eventually absorbed from the site of application into the circulation. Frequent administration of local anesthetics may result in the development of systemic toxicity. A long-acting formulation that provides extended regional blockade may be useful for pain management following surgery or for chronic pain relief. Lipospheres were used for the delivery of the common local anesthetics bupivacaine and lidocaine for the purpose of extending their effectiveness to a few days after a single injection. Liposphere formulations containing local anesthetics were prepared by the melt and solvent methods as described above. Sterile formulations wereprepared bydissolving bupivacaine (100 g) and egg phospholipid (100 g)in ethanol followed by sterile filtration through a 0.22-pm filter.Tristearin (200 g) was dissolved in hot ethanol and filtered into the same flask and the ethanol was evaporated to dryness. To the remaining semisolid, 5 L of hot (65C) sterile 0.1" phosphate buffer solution containing 0.05% methyl paraben and 0.1% propyl paraben as preservatives was added and the mixture was homogenized for 5 min at high speed. The uniform milky preparation was rapidly cooled down to below 20C by immersing the flask in a dry ice-acetone bath while homogenization continued. The formulation was filled into 10mL vials and stored under aseptic conditions at 4C until used. A variety of models have been used for the evaluation of peripheral analgesic agents. However, only three have been extensively used to evaluate pharmaceutical compositions. There are the Randall-Selitto test, the abdominal construction writhing response to intraperitoneal injection of an irritant, and the pain response of mice after formalin injection [20,21]. The local effects as a function of time of liposphere formulations were tested using the Randall-Selitto experimental animal model. To induce hyperalgesia, animals were first anesthetized and then a yeast or carrageenan solution was injected through the foot pad nearest the first digit. The foot withdrawal score was then measured at the indicated times after the injection, on a scale from 0-25 (0 = could not stand any pressure; 25 = could stand extensive pressure) using the RandallSelitto instrument. Liposphere formulations to be tested were coadministered with the yeast solution. Control animals received an identical volume of water. For animals being tested for more than 24 h, the liposphere formulation was injected at time 0 and yeast was injected 24 h prior to
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liposphere injection, since yeast-induced hyperalgesia is often not measurable at times greater than 24 h postadministration. The hyperalgesic response was developed within the first hour after injection and maintained for 48 or 72 h. The effectiveness of several bupivacaine formulations was investigated. Liposphere formulations containing tristearin as the fat and phosphatidylcholine from eitheregg (PCE) or soybeans (PCS) and loaded with 1 or 2% bupivacaine produced long-lasting analgesia for at least 48 h and for almost 72 h [22]. The blank liposphere formulation did not produce any analgesia, and the Marcainecontrol solution (0.75% in saline) produced a strong analgesic effect which lasted for less than 3 h. A rat sciatic nerve preparation was developed to study prolonged local anesthetic blockade from bupivacaine lipospheres [23,24]. Blockade andor section of the rat sciatic nerve is a time-honored system to study regional anesthesia. The nerve is large and easily seen, and the effects of motor and sympathetic blockade in the foot can be detected. Sensory blockadecan also bemeasuredprovidedthere is no spillover in the saphenousnerve distribution. After anesthesia was induced, bilateral posterolateral incisions were made in the upper thighs, and the sciatic nerves were visualized. Sham vehicle wasinjected around the nerve onone side, and vehicle containing 5 and 10% bupivacaine (0.5-mL dose) was injected on the other side. The facia wasthen closed over the deep compartment to restrict partially egress of drug formulation. Motor blockade was scored on a 4-point scale based on visual observation: 1, normal appearance; 2, impaired ability to splay toes when elevated by the tail;3, toes and foot remained plantar flexedwith no splaying ability; and 4, loss of dorsiflexion, flexion of toes, and impairmentof gait. Both 5 and 10% bupivacaine liposphere formulations showed significant levels of motor blockade through day 3, and in some cases, day 4(Fig. 7). Motor function in all animals returned to normal by day 6. Sympathetic blockade was determined indirectly by foot pad temperature measurements. The foot receives sympathetic innervation largely from the sciatic nerve, although there may be a contribution from the saphenous nerve as well. Skin temperature measurementsof the blockadeside were also monitored as an indication of vascular tone. During the period when motor block was apparent (Fig. 7), the blockaded side was essentially always warmerthan the unblockaded side (Fig. 8). Temperature differences seemed to dissipate within 1 day after motor block resolved. Since sensory blockade measurementsin this experiment are complicated by several factors, it was important to find a method of detecting sensory blockade that is relatively independent of motor responses. Vocalizations in response to defined transcutaneous electrical stimulation of points on the feet were
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TIME (days after injection) Fig. 7 Motor blockade of 5% (n = 6) and 10% (n = 3) liposphere formulations injected near the sciatic nerve of rabbits. The motor rating were:4, clubbed foot; 3, partial clubbing (does not splay); 2, obvious difference in splaying; and 1, normal.
used as a criterion for the measurementof sensory blockade. The threshold to hind paw electric shock and hind paw pad temperature measures of sympathetic block where both increased for 3-4 days. No impairments were observed on the contralateral control side. One week after liposphere administration, sciatic nerves were removed and histologically evaluated. No evidence of nerve damage and very little inflammation of foreign body response wereobserved. . A study was conducted to evaluate the efficacy of 2% bupivacaine liposphere formulation to produce analgesia inthe ratformalin model [24]. The model was designed to assess the antinociceptive ability against chronic pain caused by a test compound. An additional foot flick thermal method was also performed utilizing the sameanimals. The formalin study
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Time (days)
Fig. 8 Vocalizationthreshold to hindpawelectricalstimulationafterbilateral sciatic nerve administration.

compared the effects of administration of 2% loaded bupivacaine lipospheres with blank lipospheres, standard bupivacaine solution (Marcaine, 0.5% i i t h 1 : 200,000 epinephrine), and physiological saline on antinociception in the rat. Rats were pretreated at various times with test or control formulations by infiltration injection into the right popliteal fossa and theninjected with 5% formalin into thedorsal surface of the right hind paw. Nociception wasthen measuredin the form of paw flinches in a 5-min period during both the acute and tonic phases. Both 2% bupivacaine liposphere and Marcaine had an onset of action times within 10 minutes. However, the liposphere formulation was able to maintain significant antinociception for at least 9 h in both acute and tonic phases as compared with less than 3 h for Marcaine. Similar results were obtained in the foot flick thermal stimulus model. Sensory blockade was measured by the time
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required for each rat to withdraw its hind paw from a 56C plate. Latency to withdraw eachhind paw from the hot plate was recorded by alternating the hot plate within 15 S, the trial was paws. If no withdrawal occurred from terminated. Neither the Marcaine nor liposphere formulation caused a change in pawvolume, nordid they significantly influencethe development of formalin-induced edema. The long-acting effect of the bupivacaine liposphere formulation as compared with Marcaine solution was further confirmed bythe rattail flick model [25]. The study compared administration of the 2% bupivacaine liposphere, blank liposphere, standard Marcaine, and saline. After administration of the formulations, tail flick latencies weredetermined.The liposphere formulation exceeded the anesthetic duration of Marcaine by 12-fold. Treatment with blank lipospheres was not significantly different from treatmentwith the saline control. A pilot pharmacokinetic study was performed in rabbits to compare the Cmax andTmax values obtainedafter intramuscular injection of Marcaine HC1 solution and a liposphere formulation (Fig. 9). A total amount equivalent to of 20 mg bupivacaine was injected to rabbits (n = 3) and blood was collected for 72 h. Bupivacaine bloodconcentrations were determined by high pressure liquid chromatography (HPLC) following a United States Pharmacopeia (USP) method. Lidocainewas used as internal standard with a linear calibration curve for bupivacaine between 50 and 1000 ng/mL.Bupivacaine blood levels for bupivacaine solution (Marcaine) and lipospheres after a single injection are given in Fig. 9. The maximal blood concentrations were 681 f 246 and 200 & 55 ng/mL for bupivacaine in solution and in lipospheres, respectively. The toxicity of bupivacaine lipospheres in rats was evaluated. Since the liposphere formulation consists of natural inert components, phospholipids, and triglycerides, they are expected to be biocompatible in vivo [26]. The incidence of microscopic observations after intramuscular injection of liposphere formulations was studied (Table 1). Blank lipospheres, 1% and 5% bupivacaine in lipospheres, 5% dextrose solution, and bupivacaine solution (0.1 mL) were injected in rats followed by histological examination of the site of injection at days 3, 7, and 14. The degree of inflammation, necrosis, and fibrosis was scaledfrom 0 to 4, where 0 means absent and 4 means marked. The degree of inflammation, necrosis, and fibrosiswas similar for all formulations. At day 3, some irritation and inflammation was observed, which was reduced after 14 days. In a second study, the local toxicity of a 2% bupivacaine formulation after daily injections for 2 weeks in dogs was estimated. Minimal local irritation was observed in these studies as determined by histological examination.
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Fig. 9 Bupivacainebloodlevelsafterintramuscularinjection of 2% loaded lipospheres as compared with bupivacaine HCl solution. Drug levels in blood were determined by HPLC. B. Parenteral Delivery of Antibiotics
Several antibiotics, including ofloxacin, norfloxacin, chloramphenicol palmitate, and oxytetracycline, and antigungal agents, such as nystatin and incorporated into lipospheres in highencapsulaamphotericin B, have been tion yield. The use of lipospheres for antibiotic delivery was demonstrated by the development of liposphere oxytetracycline formulations for veterinary use [17]. Parenteral oxytetracycline (OTC) therapy in farm animals requires daily administration of the drug over several days, usually 3-5 days, in order to provide prolongedtherapeutic blood levels. Serum OTC concentrations of potential clinical and therapeutic values in the treatment of OTC-sensitive organisms are estimated in the rangeof 0.15-1.5 mg/mL. The minimum inhibitory concentration (MIC) in milligramsper milliliter for certain patho-
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Table 1. Incidence of Microscopic Observations After Livosvhere Administration
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Formulation Blank lipospheres(0%) inflammation necrosis fibrosis Bupivacaine lipospheres(1%; inflammation necrosis fibrosis Bupivacaine lipospheres(5%) inflammation necrosis fibrosis Dextrose solution inflammation necrosis fibrosis Bupivacaine HC1 solution (0.75%) inflammation necrosis fibrosis
1Day
3 Days 221 010 110 212 313 101 222 212 1 1 1 211 011 1 1 1 222 211 1 1 1
14 Days 212 000 221 211 OOO 110 211 000 200 101 000 101
1 1 1
112 222 000

1 1 1 222 000
112 333 000 1 1 1 212 OOO

1 1 1 223 OOO
OOO
1 1 1
Rats (groups of three) were injected subcutaneously with either formulation and the injection site was histogically examined. The results for eachare ratgiven in the table.Liposphereformulationswerecomposed of bupivacaine-tristearin-phospholipid at a1:2:1 weight ratio. Dextrose used was 5% a solution, and bupivacaine HCI solution was commercial Marcaine solution (0.75% bupivacaine). The degree of inflammation, necrosis, and fibrosis was scored from 0 to 4, where 0 means absent and4 means marked. gens of farm animals are 0.15 for Pasteurella multocida, 0.3 for Staphylococcus and Pasteurella anatipestifer, 0.4 for Haemophyllis paragallinarum, 0.8 for Mycoplasma gallisepticum, and 1.5 for Escherichia coli. Blood levels of above 0.5 mg/mL are required for treatment of most bacterial infections. Several long-acting oxytetracycline formulations have been reported [27-291. These formulationshave been tested in various farm animals and showed adequate blood levels for 72 h after asingle injection at a dose of 20 mg/kg* Oxytetracycline was encapsulated in good yields in solid triglyceride liposphere formulations. The microdispersion containing up to 15 wt% oxytetracycline was injectable through a 20-G needle and was stable for at OTC was released least 1year when stored under refrigerated conditions.
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in vitro from dialysis tubing for 5 days, whereas OTC was released from solution through the tubing in less than 6 h. Blood levels in turkey birds or rabbits were maintainedfor up to4-5 days for the8% loaded formulation. An increase in OTC concentration required a decrease in the content of triglyceride and phospholipid in the formulation resulting in a decrease in the durationof drug release. The formulation degraded in tissue, but remnants of the formulations remained for periods longer than 4 weeks. A study was conducted to determine theeffect of liposphere composition on its physical properties, drug release rate, and injectability through a 22-G needle. The parameters investigated were triglyceride composition, phospholipid type, aqueous phase composition, drug loading, and OTCtriglyceride : phopholipid ratio. Three triglycerides were used, tristearin (mp 75"C), trilaurin (mp 42"C), and tricaprin (mp 33C). Three types of lecithin were utilized, highly pure (99%)egg and soybean phospholipid and foot-grade lecithin (96% acetone-insoluble material). The continuous medium was either 0.1 M phosphate buffer or 5% dextrose solution. The formulations based on tricaprin, regardless of the type of phospholipid or aqueous medium composition, loaded with up to 15% OTC were stable and injectable and provided a prolonged drugrelease rate in vitro. Formulations containing less than 5% phospholipid or more than 15%OTC were less stable and often caused cloggingof the needle, and therefore they were discarded [171. Release studies were conducted in dialysis tubing with a molecular weight cut-off of 300,000 (Spectrum, CA)or without tubing. One milliliter of liposphere formulation was introduced into the prewashed dialysis tubing and placed in a jar containing 800 mL of 0.1 M phosphate buffer pH 4.5. Alternatively, 1mL dispersion was added directly to the buffer solution and placed on anorbital shaker at 37C. Samples were taken at discrete times, centrifuged, and analyzed by ultraviolet (UV) spectrophotometry at 365 nm to determine the drug release rate from the formulations. A commercial OTC solution (Dabecycline, 10% OTC concentration) used as control was placed in the dialysis tubing to determine if the tubing was limitingthe rate of release. The release was slower from thedialysis tubing; however, OTC was released from the formulation for about 3-5 days from both releasing systems. Two animal studies were conducted in order to evaluate the controlledrelease effect of the liposphere formulations by following the OTC blood the injection site. In levels and the elimination of the administered dose from the first study, four OTC-loaded liposphere formulations differing in their compositions were compared with an OTC solution (10% OTC in acidic solution) used as reference and a blank liposphere formulation as control (Table 2). The formulations were injected intramuscularly to groups of six
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Fig. 10 Oxytetracyclineblood concentrations of several liposphere formulations.
OTC blood levelswere determined. The injection sites turkey birds and the were observed forresiduals 7,11, and 28 days postinjection. The average OTC blood levels for the six formulations are given in Fig. 10. All four liposphere formulations show OTC levels above MIC for at least 3 days. Formulation E, composed of trilaurin (80 mg/mL) and phospholipid in a ratio of 2 : 1, showed similar OTC blood levels to the formulations based on tristearin and the more concentrated (120 mg/mL) trilaurin-based formulation. The formulation composed of trilaurin-phospholipid in a 1 : 1 ratio was less effective. These results indicate that a lower phospholipid to triglyceride ratio improves the duration of drug release and that higher drug loading (formulation C) does not affect the formulation effectiveness. Although tristearin showed good results, it is is less susceptible for elimination from the injecnot preferred, because it tion site, as discussed below.
of Substances
399
The residual amountsevaluatedat7 and 1 1 days were maximal (about 90% of the original dose) for tristearin-based formulation (A), about 50% for the trilaurin-based formulations (B, C, and E), and about 20%.for the blank formulation. The deposits contained less than 10% OTC of the original dose. After 28 days, only formulation A had significant amounts of deposits at the injection site, which was mostly tristearin. No OTC was detected in any of the deposits retrieved from theanimals after 28 days. These results indicate that formulations based on trilaurin, a semisolid fat at body temperature (mp = 44C) are more rapidly eliminated from the injection site [17]. Based on these results, second a animal study was conducted in order to identify an optimal formulation which would provide maximal OTC blood levels after a single injection. The composition of formulation E (Table 2) was tested in comparison with a mixture of formulation E and DabecyclineOTC solution in a 4 : 3 volume ratio. Dabecycline-OTC solution was usedas reference. Dabecycline was added to formulation E in order toincrease the initial blood levels, which were too low for the first 5 h after injection. The three formulations were administered intramuscularly to turkey birds (10 birds for each formulation). The OTC solution (Dabecycline 100) resulted in very high serum levels at 3and 6 h, 19.9 and 3.5 mg/mL, respectively, and drug levels of 2.1, 0.4, and <0.1 mg/mL were found at 24, 48, and 72 h, respectively (Fig. 11). The mixture of Dabecycline and the liposphere formulation gave intermediate blood levels during the first day and higher levels 11days than lipospheres alone for 96 h. Evaluation of the injection sites after showed localizedresidues which were estimated as 40-50% and 30-40% of the injected dose for the liposphere and the Dabecycline liposphere formulations, respectively. The residue was mostly the triglyceride and phospholipid of OTC. No residuals were found in the injection and a substantial amount site of OTC solution (Dabecycline). All animals in these studies were healthy and gained weight as the nontreated animals with no pathological signs. In all injection sites there were no signs of damage, swelling, or inflammation. No injection sites showed any necrosis or encapsulation even when precipitates were observed.
C. Parenteral Deliveryof Vaccines and Aduvants
Severalreports describing the improvement of theimmuneresponse achieved by the association of antigens with lipidcamers such as liposomes [30] or microparticles like polymeric biodegradable microcapsules [31,32] have been published. The ability of these delivery systems to enhance
400
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Dabecycline Dabecycbposph 4:3 Liposphere 8%
6-
12
24
36
40
60
72
Time (hours)
Fig. 11 Oxytetracyclinebloodconcentrations of turkeybirdsafteradministration of OTC solution, OTC lipospheres,and a mixture of OTC solutionand liposphere formulations.
immunogenicity was related to the physicochemical characteristics of the particles. Lipospheres represent a new type of fat-based encapsulation that have been also used successfully as carriers of vaccines and adjuvants [8101. The physicochemical properties and immunogenic activity of different liposphere vaccine formulations containing a recombinant malaria antigen, R32NS1, derived from the circumsporozoite protein of Plasmodium falciparum as the model antigen have been described. The manufacture of lipospheres was accomplished by gently melting the neutral fat in the presence of phospholipid and dispersing the mixture in an aqueoussolution of the antigen by vigorous shaking, which results on cooling in the formation of a phospholipid-stabilized solid hydrophobic fat core containing the antigen [9].
Lipospheres for
Controlled Delivery
of Substances
40 1
The effect of the type of fat usedin the preparation of lipospheres on their immune response to encapsulated antigen was tested. Mice were immunized twice at weeks 0 and 4 with lipospheres containing R32NS1 malaria antigen. Although for all liposphere formulations, the first immunization at week 0 caused a very small immune response, however, after the booster injection, a very marked increased of mean immunoglobulin (IgG) antibody levels was observed for most of the liposphere vaccine formulation tested, and the immune response obtained remained very at highlevels of IgG antibodytiters even after the 12-week period of the experiment [8]. The most immunogenic liposphere formulation was the one made of ethylstearate, whereas lipospheres made of stearic acid showed thelowest IgG ELISA titers. The complete order of immunogenic activity (based on fat composition) of the liposphere formulations tested was ethylstearate > olive oil > tristearin > tricaprin > corn oil stearic acid [8]. No correlation between liposphere particle sue or fat chemical characteristics and immunogenicity was found. It is worth noting that the IgG antibody ELISA titers obtained on immunizing rabbits with liposphere R32NS1 were superior to those obtained following similar immunizations with the freeantigen absorbed to alum, which showed no antibody activity at the same antigen concentrations. It was previously shown that this antigen was also poorly immunogenic in humans when injected alone as an aqueous solution or when adsorbed on alum [33]. Incorporation of a negatively charged phospholipid, dimyristoyl phosphatidylglycerol (DMPG) in the liposphere lipid phase, caused asignificant increase in the antibody response to the encapsulated R32NS1 antigen [9]. Enhancement of immunogenicity by inclusion of charged lipids have also beenobserved with certain antigens inliposomes.Negatively charged liposomes produced a better immune response to diphtheria toxoid than positively charged liposomes [34]. However, when liposomes were prepared with other antigens, positively charged liposomes worked equally as well as those bearing a negative charge [34]. Further studies are needed to determine whether negative charges in lipospheres have general abilities to enhance immunogenicity or whether, as with liposomes, charge effects are dependent onindividual antigen composition. An interesting correlation was observed between the liposphere fat to phospholipid (FPL) molar ratio, particle size, and immunogenicity. Low F/PL ratios (50.75) were found to induce the formation of lipospheres of small particle size (70% less than 10 pm in diameter), and this apparently resulted in increased antibody titers [9].Among the ratios tested, a maximal level of IgG antibody production was obtained at a F/PL ratioof 0.75, whereas at larger ratios, decreased antibody production was observed. Although the reason for this phenomenon is unknown, apossible explana-
402
Domb et a/.
tion may be theoccurrence of better antigen orientation and epitope exposures in the small lipospheres because of higher surface curvature. WO populations of particles usually coexist in vaccine-loaded liposphere formulations, one in the size range of 1-10 pm in diameter (population A) and a second population with a diameter between 10 and 80 pm (population B). As mentioned previously, the particle size distribution of lipospheres depends on the fat phospholipid to (FPL) molar ratio, and the immune response to liposphere-encapsulated R32NS1 was also dependent on the F P L ratio. The average size of the particles increases with increasing F/PL molar ratio:Under conditions where theF P L ratio is high (22.5), the large particle population is predominant (approximately 80% of the particles had an average size of 73 pm) [9]. In order to examine the influence of different routes of administration of lipospheres on their immunogenicity, rabbits wereimmunized orally or parenterally (by subcutaneous, intraperitoneal, intramuscular, and intravenous routes) with lipospheres made of tristearin and lecithin (1 : 1 molar ratio) and containing the malaria antigen [S]. The immune response obtained was followed for a period of 12 weeks postimmunization. No antibody activity wasfound after oral immunizationin any of the individual rabbits immunized with the liposphere R32NS1 vaccine formulation. However, rabbit immunization by all parenteral routes tested resulted in enhanced immunogenicity with increased antibody IgG levels over the entire postimmunization period. The individual rabbit immune response shows that. immunization by subcutaneous injection was the most effective vaccination route among all parenteral routes of administration tested [S]. Incorporation of lipid A, the terminal portion of gram-negative bacterial lipopolysaccharide, in lipospheres increased significantly the immune response to R32NS1 malaria antigen resulting in double IgG levels compared with R32NS1 lipospheres lacking the lipid A. The adjuvanteffect of lipid A incorporated in lipospheres was observed evenafter 1600-fold dilution of the rabbit sera [g]. The adjuvant effect of different doses of lipid A in lipospheres was also examined by immunizing rabbits with lipospheres containing R32NS1 and prepared at different final concentrations of lipid A. A gradual increase .in IgG antibody titer with increasing lipid A dose was observed. The strongest antibody activitywas obtained with lipospheres containing 150 pg of lipid Nrabbit. Athigher lipid A dose(200 kg/ rabbit), a decrease in ELISA units was observed [9]. The preparation and use of polymeric biodegradable lipospheres as a studied. The potential vehicle for the controlled release of vaccines was also immunogenicity of polymericlipospheres composed of polylactide (PLD) or polycaprolactone (PCL) and containing the recombinant R32NS1 malaria
of Substances
403
antigen was tested in rabbits after intramuscular injection of the formulain the sera of tions [101. High levels of specificIgG antibodies were observed the immunized rabbits up to 12 weeks after primary immunizationusing a solid phase ELISA assay. PCL lipospheres containing the malaria antigen were ableto induce sustained antibody activity after one single injection in the absence of immunomodulators.PCL lipospheres showedsuperior immunogenicity compared with PLD lipospheres, with the difference being attributed to the different biodegradation rates of the polymers.
D. Topical Delivery of Insect Repellent A common mean for repelling insects consists of applying the compound N,N-diethyl-m-toluamide(DEET) to the skin. The commercially available liquid DEET formulations contain between 15 and 100%DEET, and they are not recommended for use on children. The toxicity of DEET has been extensively reported and related to its high skin absorption after topical that as much as administration [36-381. Previous studies have demonstrated 50% of a topical dose of DEET is systematically absorbed [37,38]. In an effort to develop a new topical formulation for DEET that possesses reduced skin absorption as well as an increase in the duration of repellency, we have encapsulated DEET into lipospheres and studied its skin-absorption dynamWe hypothesized that the encapsulation of ics and duration of action [39,40]. DEET will reduce its contact surface area with the skin and reduce its evaporation rate from theskin surface, resulting in reduced dermal uptake and extended repellent activity. The liposphere microdispersions containing DEET incorporated in solid triglyceride particles were prepared by the melt method using a m mon natural ingredients in one step without the use of solvents. The formulation was preserved by parabens, propylparaben in the oil phase, and methylparaben in the aqueous phase. The average particle size was in the range of 15 pm, and microscopic examination showed spherical particles. The formulations were stable for at least 1 year when stored at 4C and 25C in a closed glass container, and DEET content, particle size, and viscosity remained almost constant. The residual efficacyof liposphere formulations was evaluated on volunteers by applying the formulations to the skin and exposing the subjects to mosquitoes [40]. The time of 100% repellency (zero biting) was the index for determining the effectiveness of a formulation. The formulations were applied on four locations on the arm of volunteers at a 2.5 mg/cm2on a total area of 12-cm2skin surface. Mosquitoes were placed in a screen-bottomed (%mesh netting, 10-cm2exposure area) cylindisplaying hostdrical cup containing 50 5- to 15-day old female mosquitoes
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seeking behavior with access to the skin through the netting. The forearm was placed on the mosquito netting for 10 min every 30 min and the number of biting mosquitoes (evident by a blood meal) were recorded. Prior to any efficacy experiment, the mosquitoes weretested on untreated skin to confirm their host-seeking behavior. W Omosquito species were tested: Aedes aegypti and Anopheles stephemi, both aggressive biters. The results of this experiment is given in Table3.The formulations were repel. 3 h for the6.5, 10, and 20% DEETlent for a minimum of 2.5,3.5, and 6 containing lipospheres, respectively. The DEET-freeformulation (control) and the untreated groupsdid not show any activity against Aedes aegypti, and the 10% DEET solution in alcohol was repellent for about 1.5 h. The blood concentrations after intravenous (IV) dosing and topical administration of 10% DEET in ethanol (group A) and 10% DEET in lipospheres (group B) were measured(Fig. 12). The AUC after dosing was 24,494 DPM/mL for group A (10% alcohol solution) and 8444 DPM/mL for groupB (liposphere formulation). Therefore, theabsolute bioavailabilTable 3 . Repellency Effectiveness of DEET Liposphere Formulationsa Time after application Untreatedb Controlb (min)
2 15 30
No.of biting Aedes aegypti and Anopheles stephemi % DEET in formulation

6 . 20 5
10
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (2) 0 (2) 1(2)
Referencec
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1(1) 0 (1) 1(2) 1(1) 2 (1)
60
90 120 150 180 4 210 240 270 300 380
(5) 5 (4) 3(5) 3(5) 5 (5) 5 (3) 3 (5)

5 (3) 3 (4) 4 (6) 5 (6)
(4)
(4)
4 (0) 5 (0) 5 (3) 5 (3) 5 (5) 5 (5) 4 5 (3) 3 (4) 4 (3) 4 (5) 5 (5)
(2)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1(1) 1(1) 1(2) 2 3 (2)
a Cups with mosquitoes were placed for 10 min every 30 min on the skin of volunteers treated with formulation(2.5 mg/cm2) and the number of bites were recorded The results are average of three independent tests (cup placements), the results in parenthesis are of Anopheles stephensi. Untreated group were without any application and control group was treated DEET-free liposphere formulation. c Reference group received a 10 wt% solution of deet in alcohol.

m -
405
c.
nRHANOL LIPOSPHEFIE
I
I
1
12
24
16
20
Time (hours)
Fig. 12 Bloodconcentrations(DPWmL)aftertopicaladministration of 10% DEET in ethanol or liposphere formulation administeredtopically (1 mL,20 pCi mL) to rabbits (surface area 64 cm). Results represent the average of eight rabbits.
ity of DEET from a 10% ethanol solution was 45%, whereas the bioavailability from DEET lipospheres was only 16%, a threefold reduction in the amount of DEET absorbed. About 74% of the IV administered dose was collected in the urine, and 39 and 19% of the topically administered doses were collected for the alcoholic and liposphere formulations, respectively. Assuming that the error in urine collection issimilar in all experiments, the difference in radioactivity contained in the urine after topical administration of the liposphere dosage form is about 50% of that of the alcoholic dose, which corresponds to theblood bioavailability calculations. The total of residual dose and extracamount of DEET recovered from skin (washing tion from skin) was similar for both formulations, indicating that both formulations were similarly exposed to the skin, and thus the results are comparable. The nonrecovered DEET after topical administration is probably due to evaporation of DEET from the nonocclusive patches to the environment. The lower DEET recovery of the liposphere formulation can be interpreted as more DEETis released to the environment resulting in more repellent activity [39].
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Domb et al.
F. Nanolipospheres for Cell Targeting of Anticancer Drugs
The potential use of lipospheres loaded with paclitakel (Taxol) to overcome tumor cells acquired resistance to the drug was investigated [41]. It was assumed that, if absorbed by the cells, the encapsulation of Taxol willprevent the rapid expulsion of the drug outside the cell, and a sufficient in cell plasma. %o cytotoxic level of drug concentration will be maintained liposphere formulations, one based on tricaprin and the other on polycaprolactone, were comparedwith liposomes of the same compositionbut without the core component(tricaprin or polycaprolactone). The formulations were prepared by the solvent method and extruded through a series of submicron filters to yield nanoparticles of 200-nmsize. The rate and amount determined using lipospheres or liposomes of uptake of particles by cells was containing phosphatidylethanolamine fluorescein. The rate of uptake was of the amount of fluoresfollowed by fluorescence microscopic visualization cence accumulatedin cells or by measuring the amount of fluorescence in each cell usingthe FACS system. The formulations were incubatedwith the wild-type F98 glioma cellline or with the F98 cell lines resistant to 1 X 7.5 X and 8 X mMTaxol. The results indicate that (1) it takesabout 24 h of incubation of particles with cells to reach saturation of particle uptake, (2) cells accumulate higher concentrations of liposomes than lipospheres, and (3) cells which are more resistant to Taxol accumulate higher Taxol concentrations on incubation with Taxol liposomes and Taxol lipospheres than on incubation with the free drug. The cytotoxicity of liposphere- or liposome-encapsulated Taxol on the percentage of cell survivalof two celllines (wild type and F-98/1 X resistant cells) after 6 h of treatment with drug and further incubation of cells up to72 h was studied. F98 cells were incubated with a range of drug concentrations and different preparations for 6 h, then washed with fresh medium, and incubated for additional 66 h. The number of cells in each plate was counted daily. Thedata indicate that Taxol encapsulated in liposomes or lipospheres had a higher cytotoxic effect than free Taxol. The results also demonstrate that although there is no significant difference betweenthe cytotoxic effect of free Taxol or Taxol encapsulated in liposomes on the wild-type cells,there is significantdifference in the effects on resistant cell lines. Taxol encapsulated in liposomes is about 30 and 50% more cytotoxic than free Taxol in cells resistant to and 10 m M Taxol, respectively. These preliminary results indicate that both lipospheres and liposomes were effective to overcome drug resistance, with the liposomes being moreeffective. The blood circulation time of nanolipospheres was compared with that of liposomes [42]. Lipospheres andliposomes of particle size between
407
100 and 200 nm containing radioactive cholesteryl hexadecyl ether were injected to mice and the tissue distribution was determined by following the radioactivity content in blood, liver, and spleen at 1,3,8,and 24 h. The cholesteryl hexadecyl ether in organs was extracted with chloroform and the radioactivity in the extract was determined. The liposphere formulation was excreted faster thanliposomes and was concentrated in the liver rather than in the spleen for liposomes. To increase the retention time of lipospheres in the bloodcirculation, lipospheres were coatedwith polyethylene glycol (PEG) of 5000 molecular weight, which has been shown to increase blood circulation [43]. Liposphere formulation made from phospholipid containing 10% w/w phosphatidylethanolaminewere reacted with aldehyde-terminatedmethoxy-PEG to form PEG-coated lipospheres. The imine-bound PEG was reduced to the amine linkage, which is more stable. Preliminary in vitro experiments indicate that PEG-coatedlipospheres are stable in serum [42].
V . SUMMARY
Lipospheres are solid, water-insoluble microparticles composed of a solid hydrophobic core having a layer of a phospholipid embedded on the surface of the core. The hydrophobic core is made of solid triglycerides, fatty acid esters, or bioerodible polymers containing the active agent. Liposphere formulations were effective in delivering various drugs and biological agents, including local anesthetics, antibiotics, vaccines, and anticancer agents with a prolonged activity of up to 4-5 days. The results presented in this study demonstrate that enhanced immunogenic efficacy can be achievedbyusing liposphere-based antigen formulations indicating the potential usefulness of lipospheres in the formulation of humanand veterinary vaccines. The liposphere approach employs a fat-lipid environment to achieve several goals: to serve as a carrier to protect the antigen, to serve as depot,and to provide a surface interphase necessary for adjuvant activity. It is reasonable to presume that the immunogenic and adjuvant activity of lipospheres may be due toa combination of factors. These factors may include a focused and enhanced delivery of the antigen to an antigenpresenting cell (macrophage) andprotection of the antigen from metabolic destruction at other sites in the body that do no participate in the immune response. The liposphere-delivery system as a fat-based adjuvant formulation may provide both the surface interphase necessary for solubilization and proper orientation of the adjuvant-active material and aspotential carriers for vaccines, which may allowbetter position for processing and presentation of the incorporated antigens resulting in enhanced immunogenicity.
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The feasibility of polymeric biodegradable lipospheres as carriersfor the controlled release of a recombinant malaria antigen was also demonstrated. The microencapsulated peptide induced efficient and potent primary immune responses. Polymeric lipospheres containing R32NS1 malaria antigen were able to induce very high levels of antibody activity after one single injection in the absence of immunomodulators.
REFERENCES
1. M. Donbrow (ed.), Microcapsules and Nanoparticles in Medicine and Phar-
macy, CRC Press, Boca Raton, FL, 1992. 2. P. Becher (ed.), Encyclopediaof Emulsion Technology, Marcel Dekker, New York, 1985. 3. G.Gregoriadis(ed.),LiposomeTechnology, 2nd ed., Vol. 1, CRC Press, Boca Raton, FL, 1993, pp. 527-616. 4. A. J. Domb, Lipospheres for Controlled Delivery of Substances. U.S. Patent 5,188,837, Feb. 1993. 5. A. J. Domb and M. Maniar. Lipospheres for the Delivery of Local Anesthetics. U.S. Patent, Allowed 1993. 6. A. J. Domb, Lipospheres for the Delivery of Insect Repellent. U.S. Patent, Allowed 1993. 7. A. J. Domb, Lipospheres for the Delivery of Vaccines, U.S. Patent Application, 1990, allowed August 1992. 8. S. Amselem, C. Alving, and A. Domb, Lipospheres for the deliveryof vac(S. Cohen andH. Berncines, in Microparticulate Systems for Drug Delivery stein, eds.), Marcel Dekker, New York, 1996 (in press). as a vaccinecamer 9. S. Amselem, A. J. Domb, and C. R. Alving, Lipospheres system: Effects of size, charge, and phospholipid composition, Vaccine Res., 1~383-395 (1992). 10. S. Amselem, C. R. Alving, and A. J. Domb, Polymeric biodegradable lipospheres as vaccine delivery systems, Polym. Adv. Technol., 3:351-357 (1992). 11. S. Amselem, A. Yogev, E. Zawoznik, and D. Friedman, Emulsomes, a novel drug delivery technology, Proceed. Int. Symp. Control. Rel. Bioact. Mater., 21 ~668-669 (1994). 12. S. Amselem, A. Yogev, E. Zawoznik, and D. Friedman, Emulsomes, a New of LipidAssembly,inNonmedicalApplicationsofLiposomes (Y. BarenholzandD.D.Lasic,eds.),CRCPress,BocaRaton, FL, 1995(in press). W. Mehnert, Incorporation of 13. R. H. Muller, C. Schwarz, A. zur Muhlen, lipophilic drugs and drug release profiles of solid lipidnanoparticles, Proceed. Int. Symp. Control. Rel. Bioact. Mater., 21:146-147 (1994). 14. S. lanzini, L. Guidony, P. L. Indovina, et al., y-Irradiation effect on phosphatidyl choline multilayer liposomes: Calorimetric, NMR and spectrofluorimetric studies, Rad. Res., 98:154-166 (1984).
me
409
of vesicles as revealed by 15. D. B. Fenske, Structural and motional properties nuclear magnetic resonance, Chem. Phys. Lipids, 64:143-162 (1993). 16. Y. Barenholz andS. Amselem, Quality control assays in the development and clinical use of liposome-based formulations, in Liposome Technology, 2nd ed., Vol. 1 (G. Gregoriadis, ed.), CRC Press, Boca Raton, FL, 1993, pp. 527-616. oxytetracycline-lipospheresformulations, 17. A. J. Domb, Long acting unjectable Int. J. Pharm., 124:271-278 (1995). . Knowles, DEET Bioavailability from lipo18. D. Hanibbal, M. Rock, and J spheres, Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 19 (1992). pp. 446-447 19. M. Maniar, R. Burch, and A. Domb, In Vitro and In Vivo Evaluation of a Sustained Release Local Anesthetic Formulation, M P S Meeting, Washington, D.C., November 1991. 20. D. Dubuisson and S. G. Dannis, The formalin test: A quantitative study of the analgesic effectsof morphine, mepetidine, and brain stem stimulation in rats and cats, Pain, 4:161-174 (1977). 21. S. Haunskaar, 0.B. Fasmer, and K. Hole, Formalin test in mice, a useful techniqueforevaluatingmildanalgesics, J. Neurosci. Methods 14:69-76 (1985). 22. A. J. Domb,Liposphereparenteraldeliverysystem,Proceed.Int.Symp. Control. Rel. Bioact. Mater., 20:121 (1993). 23. D. Masters and C. Berde, Drug delivery to peripheral nerves, in Polymer Specific Pharmacotherapy (A. J. Domb, ed.), Wiley, Chichester, UK, 1994, pp. 443-455. 24. D. Masters and A. J. Domb, Perinerve timed-release of local anesthetic and prolonged neural block from injectable liposphere formulations (submitted). 25. E. V. Hersh M. Maniar, M. Green, and S. A. Cooper, Anesthetic activity of the lipospheres bupivacaine delivery system in the rat, Anest. Prog., 39:197200 (1993). 26. M. J. Palham, Liposome phospholipid. Toxicological and environmental ad(0. Brown,H.C.Korting,and H. I. vantages,inLiposomeDermatics Maibach, eds.), Springer-Verlag, Berlin, 1992, pp. 57-68. 27. M.F.Landoniand J. 0. Errecalde, Tissue concentrations of a long-acting oxytetracycline formulation after intramuscular administration in cattle, Rev. Sci. Technol., 11:909-915 (1992). 28. M. Oukessou, V. Uccelli-Thomas, and P. L. Toutain, Pharmacokinetics and J. local toleranceof a long-acting oxytetracycline formulation in camels, Am. Vet. Res., 53:1658-1662 (1992). 29. D. A. Adawa, A. Z. Hassan, S. U. Abdullah, et al., Clinical trial of longacting oxytetracycline and peroxicam in the treatment of canine ehrlichiosis, Vet. Q. 14:118-120 (1992). 30. C. R. Alving, Liposomes as carriers of vaccines, in Liposomes: From Biop ics to Therapeutics (M.J. Ostro, ed.), Marcel Dekker, New York, 1987, pp. 195-218.
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Domb et al.
31. J. H. Eldridge, J.K. Staas, J. A. Meulbroek, et al., Biodegradable microspheres as a vaccine delivery system, Mol. Immunol., 28:287 (1991). 32. F. Stieneker, J. Kreuter,andJ.Lower,Highantibodytetersinmicewith polymethylmethacrylate nanoparticlesas adjuvant for HIV vaccines, AIDS, 5:431-435 (1991). 33. L. S. Rickman, D. M. Gordon, R. Wistar, Jr., et al., Use of adjuvant containing mycobacterial cell-wall skeleton, monophosphoryl lipidA, and squalene in malaria circumsporozoite protein vaccine, Lancet, 337:998-1001 (1991). 34. A. C. Allison and G. Gregoriadis, Liposomes as immunological adjuvants, Nature 252252 (1974). 35. T. D., Heath, D. C. Edwards, and B. E. Ryman, The adjuvant properties of liposomes, Biochem. Soc. Trans., 4:129 (1976). 36. J. R. Clem,D. F. Havemann,andM.A.Raebel.Insectrepellent(N,Ndiethyl-m-toluamide) cardiovascular toxicity in an adult, Ann. Pharmacother., 27:289-293 (1993). 37. J. W. Lipscomb, J. E. Kramer, and J. B. Leikin. Seizure following brief exposure to the insect repellent N,N-diethyl-m-toluamide, Ann. Emerg. Med., 21~315-317 (1992). 38. H. L. Snodgrass, D. C. Nelson, and M. H. Weeks, Dermal penetration and potential for placental transfer of the insect repellent N,N-diethyl-m-toluamide, Am. Indust. Hyg. J., 43:747-753 (1982). 39. A. J. Domb,A.Marlinsky,M.Maniar,andL.Teomim,Insectrepellent formulationsofN,N-diethyl-m-touamide(DEET)inlipospheresystem,J. Am. Mosq. Control ASSOC., (1995). P. Patent, 40. A. J. Domb, Lipospheres for the delivery of insect repellent. U. 5,221,535 (1993). 41. A. Gur, Taxol Incorporated in Nanoliposphere Formulations Against Taxol ResistantCells,M.Sc.Thesis,TheHebrewUniversity,Jerusalem,Israel, 1994. 42. L. Lichtman-Teomim, Injectable systems for the delivery of insoluble anticanceragents,M.Sc.Thesis,TheHebrewUniversity,Jerusalem,Israel, 1994. .Martin, Liposome leakage and blood 43. M.C. Woodle,M. S. Newman, andF. J circulation: Comparison of absorbed block copolymers with covalent attachment of PEG, Intern.J. Pharm., 88:327-334 (1992).
1 5
Pharmaceutical Emulsions, Double Emulsions, and Microemulsions
Nissim Garti and Abraham Aserin
Casali Institute of Applied Chemisty, School ofApplied Science and Technology, The Hebrew University of Jerusalem, Jerusalem, Israel
I. Definitions and Introduction

11. Interfacial Phenomena 111. Emulsion Formation IV. Emulsification Techniques V. Emulsion Stability VI. The SurfactantandtheHydrophile-LipophileConcept VII. Phase Inversion
412 418 419 424 427 432 438 440
VIII. Steric Stabilization

IX. Mechanical Stabilization X. Emulsion Characterization XI. Emulsion Applications A. General Considerations B. Parenteral Emulsions C. LipidEmulsionsforDrugDelivery
444
445 448 448 449 453
41 l
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D. E. F. G.
Vaccine Emulsions Emulsions as Adjuvants Oral Emulsions Topical Emulsions H. Perlluoro Emulsions for Artificial Blood
456 457 460 463 466 475 475 477 482 484 494 501 511 514 515 517 518
XII. Double Emulsions A. Definitions and Formation B. Stability and Transport Mechanisms C. New Approaches to Improve Stability D. Naturally Occurring Macromolecular Amphiphiles XIII. Double Emulsion Applications
XIV. Microemulsions XV. Microemulsions Applications A.Oral Administration B.Topical Drug Delivery C. Perfluoro Microemulsions
XVI. Summary
1. DEFINITIONS AND INTRODUCTION
An emulsion is prepared by the dispersion of one immiscible liquid in another, and then it is stabilized by a third component, the emulsifier [l]. The art and science which describe and characterize these systems, in practice and in theory, are known as emulsion technology. The need to bring two immiscible liquids into a prolonged intimate contact with a high surface area has long been a task of emulsion technology. Emulsions.have been widely used in many areas of application-in industry [2], agriculture [3-51, food [6-121, pharmaceuticals [13-151, and cosmetics [16-181. Their industrial uses have an extremely wide range of possible applications such as explosives [19]; petroleum [20], concrete [21,22], rubber, leather,textile [23,24], metal [25], and paper[26] products; and mineral flotation [27]. Pharmaceutical and medical applications are, however, the most studied and are used mainly for drug administration. Emulsions in which the water is the internal dispersed phase are termed water-in-oil emulsions (W/O), whereas emulsions in whichthe oil is the dispersed phase and thewater is the continuous phase are known as O/ W emulsions [28-331. More complex systems, in which one emulsion is further dispersed into another continuous phase, are called double emul-
Emulsions and Microemulsions

multiple (oil) drop internal aqueous drops surfactant films
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external aqueous phase
Fig. 1. Schematic illustration of a double-emulsion droplet. sions, multiple emulsions, or emulsified emulsions [34-421. Two main types of double emulsions were recognized and studied-the W/O/W and the O/ W/O double emulsions (Fig. 1). The droplet-size distribution of emulsion droplets (sometimes also termed macroemulsion) is 0.1-50 pm. A more common averagedroplets size is0.5-5.0 pm. The inner droplet size distriis usually smaller than bution of the W/O emulsion in the double emulsion 0.5 pm, whereas theouter, external double emulsionis quite large and can exceed 10 pm (Fig. 2). Amphiphilic compounds, used to stabilize emulsions (the emulsifier), possess two distinct groups in the same molecule, which differ greatly in their solubility relationships. Such compounds were termed amphipathics(or amphiphiles) by Hartley [43,44] to denote the presence of a lipophilic group having an affinity for the solvent and a second group having an affinity to water. The amphiphile has, using more picturesque terms, an hydrophilic head anda hydrophobic (lipophilic) tail. Amphiphiles tendto concentrate as a monolayer at the water interface and reduce the surface tension (therefore also termed surfactants or surface-active agents). Any excess surfactant that is not accommodated at the surface migrates to the bulk and forms aggregates known as micelles (or reverse micelles, if the bulk is organic solvent or oil) [28,30,31] (Fig. 3). The micelles can accommodate organic molecules intheir hydrophobic core or may be associated only with the amphiphile within the interface layer. The increased solubility of a compound associated with the formation of micelles or inverted micells has been termed by McBain [45] solubilization. The solubilized compounds, when partially hydrophilic, may partition between the continuous phase, the interior of the micelle, and the interfacial region. Thus, the interfacial region
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r
-e
lnlernal droplets
Mulliple droplets
10
20
30
40
50
60
Drop Diameter (pm)

Fig. 2. Size distribution of the internal aqueous (W/O emulsion) and multiple drops (W/O/W emulsion) of a multiple emulsion at the preparation stage.
Adsorption at U L interface Adsorption at

S/L
interface
Fig. 3 . Modes of surfactant action on liquid-air interfaceon solid-liquid interface and in the liquid bulk.
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may not consist entirely of the primary amphiphilic compound. Molecules that exhibit a substantial presence within the interfacial layers are sometimes called cosolvents or cosurfactants. These molecules are typically mediumchain alcohols. By changing the proportions of the surfactant to water and/or oil (constructing a complete phase diamgram), a micellar solution can progressively be converted into various other structures [46-501 (Fig. 4). The microstructures of the intermediate states are not fully understood. Depending on a large number of factors, including the structure of the amphiphile, its relative concentration, the temperature, and the presence orabsence of certain additives, several fascinating structures could be defined and characterized in the solution (Fig. 5). Some are isotropic solutions, amorphous in structure and mostly spherical in shape, called microemulsions, which are defined as S, (or L,) or S2 (or h)(depending on the continuous phase being water or organic solvent) [51-531. Some are isotropic solutions with a fluctuating structure (the continuous phase being oil and water alternatives) called bicontinuous structures or type I11 structures, and some are isotropic but more rigid in their structure and more viscous, appearing as gel-like, and these are called G and M, or M, phases or liquid crystalline phases. The intermediate liquid-crystalline phases may appear in a large variety of mesophases but usually in a given sequence. The most widely occurring liquid crystalline structure hasa lamellar structure [51-55] (Figs. 5 and 6 ) . The less-structured isotropic solutions, microemulsions, which are in essence mixtures of aqueous solutions (sometimes with electrolytes), hy-
Fig. 4. Hypothetical phase regions of microemulsion system composed of oil (0),water (W), andsurfactant (S), showing a, inverted micelles; b, W/O microemulsions; c, cylinders; d, lamellae; e, O N microemulsion;f, normal micelles; and g, macroemulsion phases.
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Long-chain
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LAMELLAII LlOlJlD CRYSTAL
Ionic Surfactant
2_0
SPHERICAL MICELLES
Fig. 5. In a combination of water, a surfactant, and a long-chain alcohol, the areas for solutions of normal and inverse micelles are separated by a micellar liquid crystal, drocarbons (oils) and amphiphilic compounds, have beenthe subject of extensive research (Fig. 7). The name microemulsions was introduced by Hoar and Schulman [56] to describe transparent or translucent systems obtained by titration of an ordinary emulsion havinga milky appearance to clarify it by the addition of medium-chain alcohol. There is muchdebate as to the term microemulsion beingused to describe such systems. Some prefer to call sucha system a swollenmicellar solution or solubilized micellar solution. However, microemulsions greatly differ from macroemulsions in the following characteristics: droplet sizes (the size ofmicroemulsions is less than 0.1 pm whereas the size of emulsions is 0.1 pm); appearance (transparent or translucent for microemulsions and milky for emulsions); thermodynamic stability (emulsions are unstable); and kinetic and time-dependent behavior (emulsion droplet size will grow with time and centrifugation); and modeof separation (microemulsionsare independent in the orderof mixing).
Fig. 6. (a) M, mesophase. Rodlike micelles of indefinite length and arranged in hexagonal array. (b)G phase lamellar structures; sometimes called the Neat phase of cubicpacking of (ModifiedfromRef.[56a])(c)Schematicrepresentation spheres. The spheres are generated by interaction of amphiphilic molecules and water.
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Fig. 7. Schematic structure of water-in-oiland oil-in-water microemulsions.
Although many investigators feel that one must distinguish between swollen micellar solutions and microemulsions in terms of the characteristics of the reservoir (the continuity of the oil) of the solubilized matter, there are no good instrumental methods to distinguish these differences. Thermodynamically speaking, microemulsions are in essence composed of bulk phases of water and oil separated by an interfacial region rich in surfactant, whereas for micellar systems, this is not clearly distinguished and most probably does not exist. However, with the lack of proper instrumentation, it is difficult to use different terms and, therefore, most scientists use the terms attheir convenience.
II. INTERFACIAL PHENOMENA
The most fundamental thermodynamic property of any interface is known as the interfacial tension [31]. The imbalanced perpendicular forces at the liquid-air and liquid-liquid interfaces were considered as the main reasons for two immiscible liquids to separate. The surface cannot be in mechanical equilibrium if there is a net force normal to the interface. The net forces at the interface were called interfacial tensions between liquids or liquid-air [30,57-591. However, many investigators concluded that it is not simple to interpret interfacial tension purely in terms of some net forces on molecules perpendicular to the surfaces, and therefore detailedstatistical mechanical models have been considered to try to describe the interfacial region [60,61]. Other approaches consider the density and the pressure profiles of the interfaces in order toexplain the complexity of the forces at the interface. Such extensive studies resulted in the discovery of various other interfacial forces, which play a significant role in explaining
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the factors affecting the disruption of liquid interfaces and other possible recombinations. The basic macroscopic model of an interface and its properties still show two bulk phaseshaving uniformthermodynamic properties separated by an interfacial region of some thickness and over which the thermodynamic variables may possibly vary. Experimental techniques and theories (distribution functions [62,63], partition functions [59-631, summation of interaction forces in the whole interfacial region [58]) have been developed to quantify the interfacial forces (interfacial tensions). The existing methods fall roughly into two categories [64-661: those in which the properties of the meniscus is measured atequilibrium (e.g., pendant drop shape [58], sessile drop shape [67], and Wilhelmy plate methods [68]) and those measuring properties at nonequilibrium (e.g., the drop volume [69] and du Nouy ring methods [70], which are faster than any of the other methods). One has to bear in mind that often it is difficult to interpret results with regard to rupture (formation) recombination of liquids (coalescence) in terms of interfacial tensions alone and more complex rheological interfacial measurements may be required. Interfacial shear viscosity measurements (the resistance to shear stress of monolayered moleculesin the plane of the surface) and dilatational (compressional) measurements [58] (the interfacial potential [71,72] which results from spreading a changed monolayer) must be measured and considered as a major contributing factor to the formation and/or coalescence of immiscible liquids. The art and science of bringing two mutually insoluble (or only slightly soluble) liquid phases together in the form of droplets (Figs. 8 and 9) is known as emulsion technology[1,31-331. An emulsion is thermodynamically unstable and the tendencyof the two liquids to disrupt and separate is a natural and favorable process. The success of emulsion technology lies in keeping the system in a metastable state by opposing the mutual interfacial once thedroplets approach each flow of liquids of one droplet to the other other asa result of Brownian motionin thesystem. In practice, the emulsion technologists deal with. two separate processes: the formation of the emulsion and thedestruction (rupture) of the droplets and the consequent separation of the droplets into two immiscible liquids. Many technologists will call it the constant battle to keep the emulsion stable.
111.
EMULSION FORMATION
The formation of an emulsion is a fast process and takes place in milliseconds. It is achieved by applying mechanical energy [l].First, the interface between the two phasesis deformed, forming a liquid film between the two liquids, which later deforms to such an extent that droplets form. These
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Oil
Water
Fig. 8. Schematicpresentation of normalemulsiondropletsstabilizedby the adsorption of an emulsifier at its surface in comparison with an oil-in-water microemulsion (emphasizing the size, curvature, and packing differences). droplets are mostly far too large and they are subsequently broken-up or disrupted into smaller ones. Hence, the disruption of droplets is a critical step in the emulsification (Fig. 10). The deformation of the droplets is opposed by the Laplace pressure. The pressure at the concave side of a convex curved interface with interfacial tension, y, is higher than thatat the side by an amount of AP = (2y/r) (where r is the radius of a spherical droplet). Any deformation leads to an increase in AP. The interface be-

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t?
m
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0;
. e .
6
0;
Fig. 10. Processes takingplnceduring the later stage of emulsification. Surfactant is depicted by heavy lines and dots. Not to scale andhighly schematic. (After Ref. 1.)
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tween the liquid deformation requires additional energy known as the viscous forces (velocity gradients) excreted by surrounding liquids, and a viscous stress has to be overcome (same magnitude as Laplace pressure). The pressure gradients(very smallportion of the totalenergy required, (35 kJ m-3) or the velocity gradients needed (approximately 3 mJ * mb3, which dissipates into heat) are mostly supplied by agitation and other external shearforces. However, certain surfactant molecules can be of significant help in reducing these gradient pressures. The molecules that are capable of adsorbing onto the droplets' surfaces and alter their properties and consequently reduce the interfacial tensions and hence stabilize the emulsion are the emulsifiers (Fig. 11). The emulsifier lowers y (e.g., from 40 to 5 mN m-') and thereby the Laplace pressure. The emulsifiers also help with the formation of film of a continuous phase between the droplets (which is a prerequisite for separation of the droplets). The adsorption and surface layer formation is mostly spread by the intense agitationof energy, and .it depends greatly on the nature and concentration of the surfactant. Adding a suitable surfactantmay reduce considerably the agitation energy (factor
(_Liquidphase 2 Adsorbed ions
I
Surfactant stabilizers
Stablllzation by particulate solids
I
Polymeric stabilizer
Fig. 11. Various possible adsorption mechanisms of surfactants on a'liquid dispersed droplet.
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of 10) needed to obtain a certain droplet, since the surfactant reduces the gradient viscosity pressure. One must realize that much of the surfactant is depleted from the interface during the emulsification process owing to the large increase in the interfacial surface area. The formation of emulsion immediately causes the droplets to seek ways to recombine and coalesce again. Luckily the coagulation process is comparatively slow and inpractical systems it takes minutes or months, with the net process resulting in the formation of metastable dispersed liquid droplets in the continuous phase termed emulsion droplets. An alternative way of making metastable emulsion with a large number of droplets is to start from a very tiny nucleus and then allow them to grow to the required size-the condensation method, which is relatively easy with solid particles dispersed in liquid, but is difficult for liquid-inliquid dispersions. The surfactants role in the emulsion formation is critical, since it largely determines which phase is going to be the continuous one: It was experimentally established by Bancrofts rule that the continuous phase will be the one in which the surfactant is soluble. The explanation is presumably that droplets of the phase in which the surfactant is soluble are very unstable to coalesce or that a film devoid of surfactant cannot be made.
IV. EMULSIFICATION TECHNIQUES
Many devices have been designed to produce emulsions. The principles of some of those emulsifying machines are based on construction of vessels with strong flow (with baffles) on which the liquid can impinge (laminar and/or turbulent pipe flow); injection of one liquid into the other; rotorstator machines for vigorous stirring; colloid mills; ball and roller mills; high-pressure homogenizers; ultrasonic techniques; aerosol injection into liquids; and electrical and condensation devices [1,73]. It is beyond the scope of this chapter to elaborate on these various methods (Fig. 12). Emulsions in which water is the internal dispersed phase are termed water-in-oil emulsions (W/O), whereas emulsions in which oil is the dispersed phase and the water is the continuous phase are known as soil-inwater ( O N ) emulsions. More complex systems in which one emulsifier is further dispersed into anothercontinuous phase are called double or multiple emulsions (W/O/W or O/W/O emulsions). The way the substances are added is important. Usually the surfactant, which should be most soluble in the continuous phase, is indeed dissolved in that phase, butit is possible to dissolve it in the dispersed phase, which can result in the formation of smaller droplets. The in situ formation of an emulsifier is also possible (the so-called nascent-soap method). Slow addition of the dispersed phase into
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Uttm T u r n
Colloid mill
HwnogenizePvolve
whistle
Fig. 12. Principle of some emulsifying machines. The slit width in the homogenizer valve is very much exaggerated. Arrows indicate the directions of flow and rotation.
the continuous one during emulsification is often advantageous: It may enable addition of surfactant to the dispersed phase. If both phases are mixed in bulk, the one containing the surfactant usually becomes the continuous phase. Temperature plays a significant role in the emulsion formation. Often, it is required to first dissolve the phases or the emulsifier (if solid). Frequently, the solubility of the emulsifier and its surface tension reaction strongly depend (ethoxylated surfactants) on the temperature, and the hydrophilic water-soluble emulsifier becomes much more lipophilic and more water insoluble as the temperature is raised. Therefore, it is essential to consider the emulsification temperature prior to any emulsification action. One must also remember that the emulsification process produces energy, the dissipation of the viscosity (velocity) gradient energy, which may affect many variables. The emulsification process can be evaluated in terms of emulsion capacity (the maximum amount of dispersed phases that can be emulsified under specified conditions). The emulsifier stability (the amount of phase separation that takes place with time under specified conditions) and the droplets size distribution (which seems to be the most practical way to study emulsification). Much has been written on the critical events that take place at the different stages of emulsion formation. Excellent discussions can be found in the Walstras chapter on Formation of Emulsions in the Encyclopedia of Emulsion Technology [l].The conclusions that should be stressed are (1) any emulsification process goes first through a film formation between the liquids which must exist for a little while; and (2) the film tends to drain rapidly under the influence of gravity. Interfacial forces tend to straighten the interface. The surfactant stabilizes the film. It allows an interfacial tension to exist, causing a considerable resistance to
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drainage of the film. As a result, it implies that slow stirring at the beginning of the emulsification (lower drainage) and high surfactant concentration are recommended. At the second stage, the plane (film) interface is disrupted into droplets. The plane disruption is accelerated by turbulence (only when y is verylow), capillary ripples (surface waves whichcan cause shredding of the droplets), and othereffects. The damping of low-amplitude capillary waves has been thoroughly studied [74]. It turns out that both the viscosity of the liquids and the presence of a surfactant enhance damping. Butif surfactant concentration is locallydifferent, as may occur in practice, the amplitudeof the wave may actually increase at the step of lower y. It has been reasoned thatthis may be a cause of droplet formationduring emulsification. In some cases, an interface is unstable, that is, local perturbation tends to increase, and a Rayleigh-Taylor instability [75,76] occurs mostly when the interface is accelerated perpendicularly and directed from the lighter to the heavier phase (Fig. 13). Kelvin-Helmholtz instability [77] takes place inmany emulsions and arises when two phases move with different velocities parallel to the interfaces.
Fig. 13. Rayleigh-Taylor instability of an interface; the denser phase is below; the interface is accelerated in the downward direction. At the left side, pure Rayleigh-Taylor instability is depicted; at the right side, Kelvin-Helmholtz instability develops at the flanks of the waves. (From refs. 75 and 76.)

V.
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EMULSION STABILITY
Emulsions consisting of microscopic droplets of 0.1-50 pm and dispersed in a continuousphase are thermodynamically unstable and tend to coalesce so that thereis a net reduction in the interfacial area. Hence, coalescence is a thermodynamic spontaneous process. Nevertheless, there are numerous examples of spontaneous, naturally occurring emulsions, which are stable. Milk is a good example. Stable emulsions exist in some areas of application; for example, in food,; (mayonnaise), pharmaceutics (intravenous fat emulsions), cosmetics (some skin creams), and agriculture (some pesticide emulsions). Much has been done in the last decades to understand the factors affecting such unusual stabilities. In order to better understand the concepts of stabilityhtability, it is essential to study the stepsand mechanisms that lead to phase separation of the liquids. Three to four main events can take place either in parallel orin sequence in each emulsion: creaming, flocculation, coalescence, and in some cases also Ostwald ripening (Fig. 14). All the processes lead eventually to an emulsion breaking and phase separation. It is essential to distinguish between the processes and to learn to control them to some extent. Creaming is the rise of dispersed droplets under the action of gravity, with the droplets remaining separated when they touch. Creaming takes place in any dispersion if the phases are not exactly equal in density and if the dispersion medium is truly fluid. It is well known that for small spherical undistorted dropletscreaming will depend strongly on the radius of the droplets, the difference in the density of the two phases, and the viscosity of the continuous phase. The rate of creaming is governed by Stokes equation:
is the creaming rate, pc-pd is the density difference between the two and r is the radiusof the phases, 7 is the viscosity of the continuous phase, droplets. Stokes law assumes that thefollowing conditions are valid [77]: (1) a spherical shape, (2) no droplet deformation, (3) a single particle (dilute conditions), (4) Newtonian fluid of the continuous phase with continuous properties, and (5) no diffusion. Emulsion droplets under rest are spherical, but they can be deformed in the shear field caused by the creaming. With large droplets or droplets stabilized by solid interfacial particles, distortion of the droplets can occur owing to changes in pressure on the different sides of the droplets (top vs bottom) which willlead to an increase in the surface area and result in variation from Stokes equation. The deformation effect
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Coalescence
Breaking
Primary emu1si on
Creaming
Flocculation
Fig. 14. Schematic representation of the instability processes occurring in emulsions.

takes place (as can be easily calculated) only for very large droplets (>l00 pm) andtherefore it is not a severe drawback. However, Stoke's law has an additional limitation, since it is good only for noninteracting droplets, which means it is good only for diluted systems. For more concentratedsystems, the term mass rate of creaming has been adopted (i.e., the motion of the center o f the mass of the dispersed phase) (Greenwald) [ 7 8 ] . Additional complexation is related to the fact that the emulsion droplets vary in size (polydispersity), which also affects the creaming rate, and finally parallel phenomena such as flocculation and coalescence should not be neglected when creaming rates are measured or evaluated. Creaming can be hastened by applying (for charged emulsions) an external electrostatic field across the end of the vessel, centrifugation, or dilution. Creaming rates can be slowed down by decreasing the emulsion droplet sizes, equalizing the densities of the two phases; and increasing the
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viscosity of the continuous phase (by adding viscosity builders with high specific gravitysuch as polysaccharides). Flocculation is the act of stickingtogether of droplets and the formation of three-dimensional clusters. In otherwards, flocculation is a process in which an aggregation of droplets takes place as a result of collisions in combination with adhesive interdroplets forces. It is clear that since in both creaming and flocculation, there is no flow of liquid from one dispersed droplet to another, these two phenomena are reversible. Flocculation is a complex phenomenon caused by Brownian movement, gravity, and shear motions of the droplets and isverydifficult for quantitative evaluation. The free energy change of flocculation, for weakly interacting droplets AG,,, can be positive or negative depending on the droplets concentration, and thus one can assume a critical flocculation concentration belowwhich the emulsionis thermodynamically stable with respect to flocculation but above which reversible flocculation to an equilibrium extent occurs. For strongly interacting droplets, this critical droplet concentration is so low as to be practically insignificant, and the flocculation may be classified as being irreversible. The flocculation process is then sometimes referred to as coagulation. In thermodynamic flocculated unstable systems, flocculation may be effectively prevented if a large enough free energy barrier exists between the flocculated droplets. The free energy barrier is derived from the interfacial forces such as long-range Van der Waals forces and electrostatic forces. The net force of the two sets of interactions will determine the behavior of the emulsion droplets to flocculate and further coalesce or to stay stable in the kinetic sense (Fig. 15). A quantitative treatment of the three different fundamental mechanisms which mayinduce flocculation (Brownian, shear-induced, and, gravity induced) was recently done by Bergenstahl [77]. The Brownian flocculation was first introduced by Smoluchowski [79]and described as the reaction (interaction) between twoparticles colliding and producinga new aggregate. The flocculation rates depend onthe number of particles present in the dispersion (concentration) and on the interaction time. Bergenstahl [77] mentioned two additional factors that affect the Brownian flocculation: the surface forces (surface potential or surface charges) and the hydrodynamic interactions. Fig. 16illustrates the two effects on theemulsion stability to flocculate. The two factorsare verysignificant whenBrownian flocculation takes place and must be strongly addressed if such flocculation has to beminimized. Bergenstahl [77] also found that the shear-induced flocculation (also known as the orthorhombic flocculation) as described by Smoluchowski [81] has similar limitations to those of the Brownian flocculation; that is, surface forces were not included, hydrodynamic interactions were notcon-
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h/n m
Fig. 15. Calculated examples of repulsive (GR), attractive(GA), and total interac(GT) as a function of surface separation distance, h, of two identical tion free energy (DLVO); (b) steric spheres: (a) electrostatic repulsion and van der Waals attraction repulsion and van der Waals attractions.
10
Fig. 16. The stability to flocculation factor including viscous interactions versus the surface potential and the salinity (surface charge) of the emulsion [77,80].
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sidered, and deflocculation was neglected. Gravity-induced flocculation, which is due to settling of particles, was first estimated by Saffman and -mer [82] assuming that every gravity-induced collision leads to attachment of the particles. Bergenstahl added to the basic Stokes law for settling of particles, the hydrodynamic interactions, and the surface interactions (as described by Melik and Fogler [83,84] and was able to predict stability areas for gravity-induced sedimentation as, a function of the droplet size, surface potential, and density difference between the particles. The mathematical expressions are very complicated but allow better evaluation of the possible mechanisms leading to flocculation, better predictions of the factors affecting the emulsions, and good evaluation of the stability to fluctuation that any emulsion can have. Coalescence is joining of small droplets in an emulsion to form large droplets or the process inwhichtwo droplets form one larger droplet leading ultimately to the formation of two separate liquid layers (Fig. 17).
Flocculation
0
Fig. 17. The primary destabilization of an emulsion is the flocculation that leaves aggregates of droplets(A). Subsequent coalescence leaves an emulsion with a wide distribution of droplet size (B).
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Table 1. Factors InfluencingTime for Coalescence of Emulsionsa

Parameter Droplet size Effects References 23,24
Exponential increase increasing with radius Exponential decrease increaswith 23 ing radius Interfacial tension 25 increasing with Increase interfacial tension Decreaseincreasing with interfacial tension.
26
Measured as time for separation rate of droplets size increase or as droplets lifetime
The coalescence process, examined from the point of view ofthe individual droplets in an emulsion, contains four basic steps: (1)The approach of two droplets toward each other achieve to an adhesive contact. (2) the drainage of the film between droplets in contact. The rate of the drainage process determines how rapid the critical thickness of the film is reached. (3) The rupture of the film. This is a stochastic process, the probability of which is determined by the thickness of the film. (4) The merging of the droplets. If the viscosity is low the process is rapid. However, emulsions with high viscosity will merge very slowly and may even be disrupted during the process. Table 1 summarizes the factors influencing time (rate) of coalescence. It can be seen that the droplets size and the interfacial tensions are key factors in the coalescence times. The coalescence process is very difficult to explain in simple mechanical events and the literature is still quite confusing. The main treatments arebased on thekinetics of the individual droplets, as was first suggested byVan den Tempe1 [S51 and later was elaborated by others.The main steps include creaming, consolidation (flocculation) (Fig. 18), film drainage, and film rupture. Bergenstahl [77], in his recent close examination and critical review of the coalescence process, examined a set of principal effects that can influence the coalescence process (Table 2). The large number of parameters affecting the coalescence emphasize the complexity of the process that takes place in the course of the collision of two droplets.
VI. THE SURFACTANT AND THE HYDROPHILE-LIPOPHILE CONCEPT
Surfactants play a significant role in any of the two destabilization phenomena (flocculation and coalescence) by adsorbing onto the oil-water interface and contributing to the repulsive forces and the London dispersion

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Table 2. Principal Effects of Different Variableson the Various Process Steps Involved inthe Phase Separation
upture dation eCreaming Variable
ity Droplet cosity phase Continuous Droplet Volume fraction of dise persed rence Density n Surface ity Surface city Surface Depth of attractive minima nteraction Repulsive esslayer Adsorbed Solubilityof emulsifier in persed the HLB (hydrophilicflipophilic of balance emulsifier) the
0 0
+ +
0
+
0
0 0
+ +
0 0 0 0 0 0 0
+
0 0 0
+
0
+
0
+
-
+ + + 0 + +
0
(+) denotes that the variable has a positive effect; (-) denotes a negative effect; (0) the variable has no effect on the phenomenon (process).
forces. Mixed surfactants are particularly more efficient than single surfactants with respect to the coalescence rate. The enhanced stability is attributed to the formation of intermolecular complexes at the oil-water interface, forming a densely packed layer and reducing the interfacial tension to very low values of about 0.1 mN * m-'. In addition, the interfacial complexes are capable of increasing the interfacial viscosity (although each component giveslowviscosity). Some investigators [86] claim that the synergism is derived from the enhanced adsorption kinetics of the surfactant when such two surfactant mixtures are used. The coherent, strong interfacial film should act as a barrier preventing coalescence by virtue of its rheological properties; for example, its high dilutional viscoelectricity. As a result, most technologists prefer using mixtures of two surfactants in most emulsion applications. The selection of surfactants is still quite difficult and requires much experience and experimental work. Griffin [87,88] has introduced the empirical concept of hydrophile-lipophile balance (HLB) for selecting a surfactant or a blend of surfactants. Surfactants with low HLB will form W/O emulsions and those with high HLB will form O/W emulsions (Table 3). Several methods have been proposed to determine the HLB of some nonionic surfactants (the HLB values are meaningful only for nonionic
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Table 3. Summary of HLB

Ranges and Their Application
HLB range
3-6
7-9
Application agent Wetting OIW emulsifier Detergent Solubilizer
8-18 13-15 15-18
surfactants and practically meaningless for any charged surfactants) and tables with the HLBvalues are available in any textbook. Moore andBell [89] suggested the H L numbers (hydrophile-lipophile ratio) as an improvedcriteria for selection of surfactants. Greenwald [90] suggested a water titration procedure for surfactant in organic solution, but this method is not widely used. Davies [91,92] has developed a method by which he calculated the HLB values of surfactants directly from their chemical formula, using empirically determined group numbers: HLB = 7
+ 2 hydrophilic group number - 2 lipophilic group number
The values of the group numbers are given ina simple HLB table(Table 4). The match betweenthe Davies method for evaluating HLB and the empirical work is quite good. The experimental determination of HLB of a surfactant is also not an easy task. Greenwald [90] developed a titration procedure andRacz and Orban [93] used a calorimetric method (based on the heat of hydration of ethoxylated surfactants) (Fig. 19). Becher and Birkmeier [94] suggested an HLB determination by gas-liquid-chromatography (GLC). In any event, there was a need to find the relationship between the surfactants HLB and the coalescence (stability) of emulsion. As a result, different oils were assigned to different required HLBs in order to obtain maximum stability (Table 5 ) [88,95]. The formulator is asked to match the required HLB of a given oil or mixture of organic solvents to the of surfactants) by varying the nature of the HLB of the surfactant (or mixture surfactants and their relative proportions in the blend. Davies [91,92] has also demonstrated the effect of the partition of the surfactant between the oil and water phases in the emulsion coalescence and related it in an empirical equation to the HLB surfactant. He showed that the HLB group numberis proportional to theenergy barrier to coalescence set up by the water which is firmly bound to the hydroxyl or ester group on the surface-active agent molecule. He also explained the correlation between HLB and the cloud point of nonionic surfactants.
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Table 4. HLB GroupNumbers

Groups Hydrophilic
-S04Na+
38.7 21.2 19.1 9.4 6.8 2.4 2.1 1.3 0.5 0.475
-COO-H+ -COO-Na+ N (tertiary amine) Ester (sorbitan ring) Ester (free) -COOH
0 -
CH (sorbitan ring) Lipophilic -CH"CH, C H , -CHDerived -CHZ-CH2-0-CHZ-CHz-CHZ-OSource: From ref. 91.
0.33 -0.15
One must realize that the HLB concept is based on a simplified picture of the coalescence and the formation process and does not take into account the important role of liquid-film thinning between droplets and the importance of shear and dilational properties of the adsorbed surfactant layer around the droplets. Therefore, it is not surprising that in practice there aremany examples of emulsifications that do not obey the HLBrules and concepts. Many investigators have cast doubt on the validity of the concept [73,75,76,96,97]. In spite of the criticism, the HLB index can be very usefuland practical consideration in the process of making emulsions. In practice, in formulating a given emulsion, one may take any pair of emulsifying agents, which fallat opposite ends of the HLBscale, for example, ' b e e n 80 (sorbitan monooleate, with 20 ethylene oxide units, HLB=5), and use them in various proportions to cover a wide range of HLBs. Emulsions are made in a given way for all combinations with fewpercentages of the emulsifying blend. The stability of the emulsions is then evaluated at each HLB number, and from the rate of droplet coalescence, the most effective HLB value, that is, the HLB value providing optimum stability, can be found. Various other surfactant pairs are compared at these HLB numbers until the most effective pair is found [2].
18 /
16 1L
Wlth ethylene + oxlde e
,
1
/.
1210 m
861-
oxide
Water number
Fig. 19. Correlation of HLB in the water number. (From ref. 90.) Table 5. HLB Values for Various Oil Phases
Application Cream, all-purpose Cream, antiperspirant Cream, cold Cream, stearic acid Creams and lotions Lotions Oil, perfume Oil, mineral Oil, vegetable Oil, vitamin Ointment bases absorption washable Ointment, emollient Polishes type Emulsion
OIW OIW OIW OIW
HLB range
6-8 14-17 7-15 6-15 4-6 6-18 9-16 9-12 7-12 5-10 2-4 10-12 8-14 8-12
W10
OIW OIW OIW OIW OIW
W10
OIW OIW OIW
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VII. PHASE INVERSION
In many cases, when an emulsion is made, a sudden event takes place which implies the inversion of the internal phase of the emulsion into the continuous external phase; for example, O/W emulsion inverts into W/O or vice versa. This phenomenonis related to the HLB value of the surfactant used, or theinternal phase volume. It has been shown that ata given emulsifierconcentration, the viscosity of the emulsion increases as theinternal phase volume(G) increases. At there is a sharp decrease in the viscosity of the emula certain critical GC, sion, which corresponds to the inversion of the emulsion. The @c depends on the emulsifier concentration, and inversion is expected to take place at as theemulsifier concentration increases [75,98]. In theearly days higher GC of emulsion technology, the inversion was attributed toa packing factorof the droplets. Thus, it was assumed that at a phase volume o f db0.74, of the highemulsion droplets cannotaccommodatetogetherbecause density packing, and any attempt to increase the phase volume will cause an inversion or breaking of the emulsion. New theories and recent examples show that an inversion can also take place at low volume phases [99,100] and does not occur at very highlypacked systems (99% by volume) [99]. It seems thatthe inversion isrelated more to the natureof the emulsifier than to theinternal phase volume, andthe emulsion tends to invert if the emulsifier used does not have the proper hydrophilic-lypophilic balances and tendsto migrate from the interface to thecontinuous phaseat a given concentration or temperature. Shinoda [101-1041 has shown that W/O emulsions prepared with nonionic emulsifiers tend to invert if the temperature is raised (since nonionic surfactants become less water soluble in water asthe temperatureincreases and leave the interface). In a series of papers Shinoda and coworkers [96,97,100-1101 have introduced a new concept for emulsifierperformance-the HLB-PIT relationship-and have shown that the phaseinversion temperature (PIT) of nonionic emulsifiers is influenced the by surfactant HLB number (Figs. 20 and 21) [96,97].The following conclusions were drawn:(1) the size of emulsion droplets depends on the temperature and the HLB of emulsifiers; (2) the droplets are less stable toward coalescence close to the PIT; (3) relatively stable O/W emulsions are obtained when the PITof the system is some 206 5 C higher than thestorage temperature; (4) a stable emulsion is obtained by rapid cooling after formation at the PIT; and( 5 ) the optimum stability of an emulsion is relativelyinsensitive to changes of HLB value or PITof the of the system. emulsifier, but instability is very sensitive to the PIT Later, Shinoda et al. [l031 studied the effect of the molar mass and the molar mass distribution of the hydrophilic chain lengths of alkyl or
439
1 6
14
QI
E,
120 ' 0'6
IO
d4
20
40 60 80 100 Phase Inversion Temperature ('C)
120
Fig. 20. Correlation between the HLB numbers of nonionic surfactants and the phase inversion temperatures (PIT, HLB temperature) incyclohexane-wateremulsions stabilized with the surfactants(3 wt% system). 1. Tween 40 9. i-R8C6H40(CH2CH20)8,5H 2. i - ~ C 6 H 4 0 ( C H 2 C H , ~ , 7 , 7 H 10. i-R,2C6H40(CHzCH~)g~7H 3. Tween60 1 1 . i-R,,0(CH,CH20)6,,H 4. i-RgC6H40(CH,CH20)14H 12. i-R,,C,H,O(CH,CH~),,H 5. i-R,,C6H40(CH2CH, O)& 13. i-R9C6H40(CH2CH20)7,4H 6. RL?.0(CH2CH20)10.8H 14. R,20(CH2CH,0)4,2H 7. i-R8C6H40(CH~CH2O),,,H 15. i-R&H4O(CH2CH,0)6H 8. i-R9C6H40(CH,CH20),,,H 16. i-R9C6H4C(CH,CH20)6.2H
alkyl aryl polyoxyethylene ethers and emulsifiers (having the same PIT) on the stability of O/W and W/O emulsions. They found that stability against coalescence increases markedly as the molar mass of the lipophilic and hydrophilic groups increases. Moreover, the emulsions showed maximum stability when the distribution of the hydrophilic groups was fairly broad. It was also found that in those cases where the distribution of the hydrophilic chains is broad, the cloud point is lower and the PIT is higher than in the corresponding case for narrow-size distributions. Thus, the PIT and HLB numbers are directly related parameters provided the distribution of the hydrophilic chains of the emulsifiers is similar. The HLB number of an
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14
12
IO
20
100 Phase Inversion Temperature (C)
60
140
Fig. 21. Correlation between HLB numbers andPITS in various oil-water (1:l) emulsions stabilized with nonionic surfactants(1.5% system). 1. i-%C6H40(CH2CH,03,,,H 4 i-R9C6H,O(CHZCH,O),,,H 2. i-&C6H,0(CH,CH,0),,H 5. i-%C6H,0(CH,CH,0)6,2H 3. i-R9C6H4O(CH,CH,O),,H 6 . i-R9C6H,0(CH,CH,0),,3H
emulsifier of broad distribution is lower than that of the pure emulsifier having the same PIT. A plot of HLB value and PIT forcyclohexane-water emulsions (1 : 1by volume), stabilized by a number of nonionic surfactants [96],is shown in Fig. 22. This figure is useful for the estimation of the change of the HLB value and the optimum hydrophilic chain length from the change of the PIT atvarious temperatures.
VIM. STERIC STABILIZATION
Emulsions can be stabilized also by naturally occurring macromolecular substances such as gums and proteins. Such stabilization takes place quite often in food and pharmaceutical emulsions. In the past, stabilization by amphiphilic macromolecules was attributed to the formation of films atthe interface. The adsorption of
44 7
Fig. 22. Relationshipbetween HLB and PIT forcyclohexane-wateremulsions stabilized with nonionic surfactant (5%).
macromolecules at the liquid-liquid interface is characteristic, for example, protein like casein, bovine serum albumin (BSA), and soy proteins, and strongly depends onthe molar mass distribution of the adsorbedmolecule. The molecules form an adsorbedlayer comprising loops and tails that are anchored in the continuous phase. Thus, when two oil droplets approach each other, through aBrownian encounter, a repulsive force is generated owing to the presence of the adsorbed layers of the amphiphilic polymer. Such stabilization is known as steric stabilization. The steric interaction is effective when droplets approach a distance of b<2d, resulting from the interference of the two adsorbed layers (Fig. 23a). Theories have been
Id
lb)
Fig. 23. Tho limiting(constantadsorption)modelsfor steric stabilization: (a) interpenetration of adsorbed layers without compression; (b) compression without interpenetration.
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designed to explain the contribution of the adsorbed macromolecules to the stability of the emulsion. The two main contributions to the stability/ instability of macromolecular-stabilized emulsions are related to droplets approaching a distance where compression occurs (without interpenetration) (Fig. 23b).
or G , is derived from the reduction in the entropy of polymeric chains (configurational entropy) arising from the reduction in the total volume available to each chain if an interaction takes place (Fig. 24). 2. Mixing term: the mixing term G , comes. f r o m a build-up in the polymeric amphiphile segment concentration in the interaction zone between thedroplets which leads to anincrease in the local osmotic pressure and the free energy (Fig. 24). The G , is alwaysrepulsive in its nature and depends on the , can havea repulsive or nature of the polymers. However, the G attractive effect on the total interaction depending strongly on the nature of the solvent (the continuous phase must be defined as AG, term better than8 solvent for thesurfactant in order for the to be repulsive), nature of the polymer (copolymeris by far bet, and ter), and the molecular mass of the polymer. The roleof G G , in determining the stability-flocculation behavior of dispersions and emulsionsis divided into three sections: (1) high molar
, 1 . Volume restriction: the volume restriction term G
Fig. 24. Total interaction free energy between two flat plates having adsorbed tails or loops: for polymers with the following characteristics:A = lO-OJ; (Y = 1.2; W =2 x gcm-; MW = 6000; (8) = 52 nm;area per chain = 50 nm.
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x
(better
0.5
lhon Q-solvent)
(W
Fig. 25. (a) The interaction free energy-separation curves for two droplets stabilized by high molar mass polymer. (b) The interaction free energy-separation curves for two droplets stabilized bylow molar mass polymer. mass, terminally anchored polymer chains; (2) low molar mass, terminally anchored chains; (3) multipoint anchored chains (e.g;, adsorbed homopolymer having a loop and train-type configuration at the interface). It is, therefore, very difficult in practice to evaluate quantitatively those terms and to predict whether the adsorbed polymer will cause steric stabilization of flocculation (Figs. 24 and 25). In order for dispersion a or emulsion, stabilized by adsorbed polymer, to remain deflocculated, the following criteria must be fulfilled: a. There should be full coverage (by the adsorbed polymer) of the particle or droplet interface. If this is not the case,bridging flocculation may then arise; that is, polymer molecules may become simultaneously adsorbed on the interfaces of both droplets or particles. Alternatively, bare patches on
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the two surfaces may come into direct contact, leading to coagulation (or even coalescence). b.The polymer must be firmly anchored at the interface. Homopolymers are not usually the most effective structures in this respect.For emulsions, AB block copolymers are particularly suitable, where A is soluble in one liquid phase and B is soluble in the other. If the polymer is not strongly adsorbed at the interface, desorption may occur during a droplet collision. c. The polymer layer must be thick enough so that G,, is negligibly small. A fuller discussion of the relationship between stability and G,, is given later in this section. d. The stabilizing moiety (e.g., that part of an AI3 block copolymer which is soluble in the external phase) must be in a good solvent environment. If p 0 . 5 , then Gmhbecomes negative; that is, the steric interaction becomes a net attraction. It is shown schematically in Fig.25 that a significant value of G,, is attained when x>0.5; that is, just beyond conditions for thestabilizing polymer.
close correlation In this way, Napper [l111 was able to demonstrate a between the critical flocculation temperature (CFT) of a dispersion and the corresponding e temperature of the stabilizing polymer, and also between the critical flocculation volume fraction (CFV) of a dispersion and the volume fractionof added nonsolvent required to give a 8 solvent mixture for the stabilizing polymer. With regard to the CFT,some dispersions flocculate on cooling and others onheating. Generally speaking (but not always), the former occurs when a nonaqueoussolvent is the external phase, whereas the latteroccurs when water is the external phase. This behavior mirrors the temperature phase diagrams for thepolymer plus solvent system in question. There has not been a great deal of work carried out to date on the application of these ideas of emulsion systems. Recently, we [l141 have demonstrated that excellent steric stabilization can be obtained in double emulsions in whichthe outeroil droplets are very large (20 pm) and with specially designed block copolymers (with siliconic backbone) that strongly anchor to the oil phase. Similar results have been obtained with BSA, a naturaloccurring amphiphilic protein.
IX. MECHANICALSTABILIZATION
Emulsions can be stabilized also with solid particles which are not amphiphilic in their nature (inorganic compounds) provided that the solids
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Fig. 26. Schematic illustration of solid particles adsorbed at the oil-water interface to provide mechanical stabilization.
adsorb onto theinterface (Fig. 26). Such stabilization is knownas mechanical stabilization, since the solid particles provide a mechanical barrier between the droplets to prevent them from colliding and coalescing. The mechanical stabilization has only limited applications and has not been widely studied. The most impressing attempt was done by Oza and Frank [112,113] to stabilized double emulsions in the presence of a mechanical barrier obtained fromstrong adsorption of colloidal microcrystalline cellulose (CMCC). The double emulsion isa reservoir for a drug thatis strongly entrapped in the inner water phase and does not leach on the shelf (storage conditions). The emulsion is stable, and the entrapped drug is released slowly through the mechanical barrier.
X. EMULSION CHARACTERIZATION
Emulsion are mostly characterized by the size distribution of the droplets and other physical properties such as dielectric properties, optical properties, thermal behavior, rheological properties, and other microscopic and macroscopic observations. The size distribution of the droplets is a very important parameter when characterizing any emulsion. Creaming, flocculation, and coalescence can be evaluated and influenced by the droplet size distribution. Therefore, it is essential to seek adequate methods to determine quantitatively the droplets distribution of an emulsion as a function of temperature, time, pH, and other factors that are responsible for the changes in droplet distribution. Practically speaking, only a limited number of drop-
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lets are ever examined, but the resulting measurements are treated as if they constitute a continuous distribution of sues. Usually, when dealing with multivalued variables, statistical methodswill be utilized to limit the number of variables. Emulsion droplet datacan be obtained in terms of the number of droplets of a specific diameter, D, by means of optical or electron microscopy [115-1161; as diameter versus volume, V, by electrical resistance counting [117], and by diameter, D, versus mass, M, by x-ray coupled with sedimentation [118]. As a result, terms such as number of droplets, diameter (length), area, volume, and mass can be estimated or evaluated. Table 6 demonstrates the parameters that can be calculated. The datacan be presented in of droplets in range, diameter table form (droplets diameter range, number interval, and percentage on a numberof boxes of the range, the less then maximum diameter, and so forth), in histograms and in cumulative plots (of number, surface area,and volume (Fig. 27).
Table 6. Mean Diameter Definitions for Emulsion Droplets

~ ~ _ _ _
) in
Descriptive name found (alternate
Mathematical
Number-length mean (arithmetic Number-surface (surface mean, diameterof average Number-volume mean (volume mean, diameter of average volume) mean Length-surface (linear mean, length diameter mean) mean Length-volume (volume-diameter mean) Surface-volume mean (surface mean, Sauter) Volume-(or weight-) moment mean (volume-(or weight-) mean, weight average particle size, De Brouckere) Could be expressed in terms numbers (AN).
.D ,
(W)
122
(?%F)
D , D , D , D*,
D ,
U3
or D ,
of number percentages (AP) as well as in actual
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12
14
18
18
20
22
24 28 26
30
32
34
36
38
40
42
44
46
Fig. 27. Histogram of dropletwithinrange droplet diameter range (pm).
(% by number) as a function of
Out of all the techniques mentioned above, the most useful information is the droplets size distribution behavior with time (indicating the rates of coalescence and flocculation); with temperature, electrolytes, and the presence or absence of various emulsifiers (as single blends and/or in combination with macromolecules). Much can be learned on the emulsion stability from its rheological properties. Emulsions exhibit a non-Newtonian behavior which is emphasized by its variation in the viscosities as a function of its flow properties (shear rate orshear stress). A creep compliance as a function of small shear stress over a period of time gives important information relevant to emulsion stability. For more detailed information on the rheological properties of emulsion, Technology, the reader is referred tothe Encyclopedia of Emulsion Vol. 1 [l]. A sharp decrease in viscositymeasurements (at high or medium shear rates) of highly concentrated emulsions is clearly associated with the increase in mean drop size. Several such examples exist both with W/O and O N emulsions [119-1211.
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XI. EMULSION APPLICATIONS A. General Considerations
Emulsions are widely used ina range of industrial and foodapplications as well as agricultural, medical, pharmaceutical, and cosmetics. This chapter will obviouslyconcentrate only on the medical andpharmaceutical applications. The use of emulsions in pharmaceuticals was widely spread in the early 1960s and 1970s, but its importance has been diminishing in the past years, mostly because more attractive ways of intake and dosage forms have become available. The main reason for this is the increasing need for formulations that can provide sustained, prolonged, slow, and controlled release of active substances. Furthermore, there has been a rising interest in many solid dosages of active matter such as, tablets, capsules, microspheres, microcapsules, nanoparticulated matter, and powders entrapped on polymeric matrices. Emulsions, microemulsions, and double emulsions also have to compete with various types of vehicles suchas liposomes. They also suffer from a lack of thermodynamic stability and, therefore, their shelf lives are restricted and requirespecial storage conditions. Microemulsions, although thermodynamicallystable, suffer from low-entrapment capacity and require large amounts of emulsifiers, which is not necessarily permitted for drug application. Any change in the emulsions or microemulsions stability (temperature, storage, decomposition of components) will affect the drugrelease, which willimmediately affect the stability. However, since emulsions are very inexpensive and easy to prepare, they will always be attractive to formulators for certain applications. The progress which has occurred in recent years in the area of double emulsions (better stability, smaller droplet size, high capacity, and better controlled release of active matter) has been attracting renewed attention, and new formulations have beensuggested and tried. The droplet size distribution of the emulsion is yet another significant problem. Most emulsions cannot be used for parenteral applications beare small enough in cause of blockage (emboli) problems. Microemulsions size but are made of emulsifiers that do not seem to have health permits. Their applications will be considered according to their delivery methods: by injection (parenteral), by mouth (oral), and by the skin and eyes (topical). It is beyond the scope of this chapter to discuss all the possible applications mentioned in the literature orin patents, and itwould be difficult to list all the constraints related to the use of emulsifiers and oils (health and physical stability, as well as chemical or biological stability). An attempt is
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made to bring some interesting examples of novel systems for controlled release, and discussions are brought only if the example helps in understanding or solving basic and/or critical problems related to the emulsion technology.
B. Parenteral Emulsions
Parenteral emulsions are used as vehicles for carrying lipid-soluble materials, to control the release of drugs, and, if possible, also to targetit. Most of the emulsions mentioned for parenteral use are of O/W or W/O/W type. Intravenous emulsions have very strict requirements regarding droplet size. Large droplets (over 5 pm) cause blockage (emboli) in the body. Toxicity effects have been reported in fat emulsions when meansize rises above 1.5 pm (Table 7) [122].Physicochemical parameters such as the viscosity of the external phase, phase volume ratios, and the partition coefficient of the drug are key factors influencing the release of a drug [123,124]. Variables such as droplet size distribution, surface characteristics, and surface charge are relevant factors in the relationship between physicochemical properties and physiological responses. It has been demonstrated [125,126] that fine particles clear from the blood slower than coarser particle-size emulsions; charged droplets clear quicker than neutral droplets and emulsions stabilized by low molecular emulsifiers are cleared than those stabilized by high molecular weight emulsifiers [127-1291. Emulsions stabilized using phosphatides (phosphatidyl choline) were cleared quickly, whereas those stabilized with thenonionicpoloxamer (Pluronic F108) were cleared slowly. A mixed emulsifier system of phosphatide and Pluronic had intermediate characteristics (Fig. 28) [129]. The addition of drugs to the oil phase had a modifying effect on the clearance. The results support the concept that the emulsifying agent is of major importance for the removal of the emulsion from the bloodstream. Geyer
Table 7. Effect of Particle Size on the Toxicity of Intravenous Lipid Emulsions Administered Intravenouslyto Rats
Mean
Maximum
(pm) size (pm) System size

0.8 Fine
% Greater than 1pm

0 22 84
LDS0 (95% confidence limits) (mJJb)
Medium Coarse
0.35 0.79 1.65
- 112 (103-122) 5.0
(104-124)114
60 (53.5-66.5)
820% fat emulsionof soybean oil stabilized by egg lecithin.
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2 t
0
b
100
200 lime (min)
300
Fig. 28. Effect of emulsification on the elimination of lipid emulsion. A, Pluronic F108:0, egg lecithin; 0 ,mixture of Pluronic F108 and egg lecithin.
[l271 has studied the effect of the emulsifier molecular weight on its clearance and found that clearance of fat emulsionsstabilized by the poloxamers with high molecular weight surfactants were cleared more slowly than from the blood than those emulsified with lowmolecular weight poloxamers. In spite of all those limitations, the administration of essential nutrients via the parenteral route is common practice. Glucose, amino acids, electrolytes, and vitamins are between the water-soluble matters intravenously injected. Fats areimportant sources of energy andmany efforts have been invested in introducing fat-emulsion for parenteralintake. Some commercially available lipid emulsions are listed in Table8. The oils were almost exclusively of vegetable origin, although synthetic glycerides, including simulated human fats and acetoglycerides, have been studied. Soybean oil and safflower oil [l301 appear to give rise to a low incidence of toxic reactions when purifiedand are resistant to oxidative changes (rancidity). Natural and synthetic compounds have both been considered as possible emulsifying agents. The natural agents were various types of lecithin (phospholipids) and the synthetics were nonionic polyoxyethylene-polyoxypropylenederivatives. A wide range of nonionic materials have been investigated as potential emulsifying agents for intravenous fats: polyethylene glycol stearate,
Emulsions and Microemulsions Table 8. Some Commercially Available Lipid Emulsions

Trade phase name Oil
(%)
45 1
Emulsifier (%)
Other components (%) Glycerol 2.5 Xylitol 5.0 Sorbitol 5.0 Glycerol 2.5 Glycerol 2.5
Intralipid Soybean 10 or Egg lecithin 20 1.2 (Kabi-Vitrum) Lipofundin S Soybean 10 or 20 Soybean lecithin (Braun) 0.75 or 1.2 Lipofundin Cottonseed 10 Soybean lecithin 0.75 (Braun) lecithin Egg Liposyn Safflower 10 1.2 (Abbott) 20) (and Travemulsion Soybean 10 or 20 Egg lecithin 1.2 (Travenol)
diacetyltartarateester of monoglyceride (DATEM), partially esterified polyglycerol, polyoxyethylene monostearate (Myrj), polyethylene glycol (Carbowax), nonylphenyl ethers (Tergitol), and polyoxyethylene sorbitan monoesters (Tweens). However, all of these materials gave rise to toxic reactions of one form or another. Only one rangeof nonionics was found to be free from toxic effects: the poloxamers (polyoxyethylene-polyoxypropylene derivatives). These have been widelyused for fat emulsions either ontheir own or as coemulsifiers withlecithin [131,132]. to give guidelines Although Black and Popovich [l331have attempted and recommendations. Burnham and others [l341 have reported that a stable fat emulsion (Intralipid) intravenous-based feeding mixture have been preparedto simplify nutritional support for patients with gastrointestinal disease. Mixing of Intralipid with other nutrients before administration allows a constant infusion through peripheral veins. The physical properties of fat particles in Intralipid formulations were studied before and aftermixing withthree different combinations of amino acids, dextrose, and electrolyte. The effect of storage on lipid particle size was also examined. The mean particle size of the droplets increased slightly over a 48-h storage period at 4"C, but no large droplets were observed. Measurement of the electrophoretic mobility of the droplets indicated that the mixture of a fat emulsionwith an aminoacid, dextrose, and electrolyte did not produce any permanent change in the stabilizingfilm of phospholipid. Total concentrations of divalent cations are greater than 2.5 mmol/L, such as calcium and magnesium, couldlead to aggregation of fat droplets and the separation of the oil as a cream layer. These systems had poor short-term stability, and consequently Burnham et al. [l341 recorn-
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mended that feeding mixtures containing amounts of Ca2' and M e above this limit should beused with care. In similar fashion, Hardy et al. [l351 have shown that mixtures of amino acid, electrolytes, trace elements, glucose, and 10% soybean oil 3-L plastic containlipid emulsions were stable when prepared aseptically in ers and stored at refrigerated temperature for 72 h. The pH-buffering property of the amino acid solution was believed to be an important contributor to the stability of the mixture [136]. The lipid emulsion (mixed with amino acids and glucose for parenteral administration of dogs) was eliminated andmetabolized to triglyceride, free fatty acids, andphospholipids in a fashion similar to that observed during separateadministration of the same nutrients. Clinical data supportthese findings [137]. The clinical use of fat emulsions in parenteral nutrition has been described in detail in various textbooks and reviews [130,138-1411, and a variety of products is available, including those containing amino acids together with fat emulsions [130,142,143]. The possibilities of administering complete intravenous nutrition with fat as one of the sources of energy has been demonstrated in many investigations. Emulsified vegetable oils were used as test systems to explore the characteristics of the reticuloendothelial system. The role of a,-macroglobulin in the phagocytic process (in rats) using a lipid emulsion has been investigated by Molnar et al. [144]. They found thata,-macroglobulin was , an important component, but that this material was not related to the a macroglobulin in humans. They pointed out that plasma contains at least 60 proteins, and this does not include enzymes, clotting factors, complementing components, and hormone carriers. Ashworth and others [l451 examined the uptake of small lipid droplets by hepatic and Kupffercells. The uptakeof small particles was dependent on the size and potencyof the hepatic sinusoids. In addition, the size, composition, structure, and charge on the particles was considered to be f 100 nm was proposed. relevant. A critical particle size o Tonaki et al. [l461 used a gelatin test emulsion to investigate plasma components that promote phagocytosis (opsonins). Particles coated with human serum albumin were also employed. Different uptakes in liver and spleen regions as well as small uptake in the lung were reported. They indicated that there was some specific interaction between reticuloendothelial cells and particles which was dependent on the nature of the particle surface. Intravenous O/W emulsion systems are also employed as diagnostic agents in the form of injectable radiopaques. Kunz et al. [l471 described in detail the preparation,sterilization, and stability of 50% oil-in-water emul-
emulsions Emulsions and
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sions of iodized oil, iophendylate injection, and ethiodized oil (Ethiodol). Three emulsions of each oil injected intraperitoneally into rats gave an excellent radiopaque outline of the peritoneal cavity. Emulsion technology isquite a mature science withregard to pharmaceutical preparations. Most studies were carried out onmicroencapsulation as a release vehicle. Only a limited number of studies are available on improvements or innovations related to emulsions. Some of the key issueswere fat emulsions for parenteral applications:
1. Studies related to the stability assesment and its relation to the nature of the phospholipids [148-1501. Highly negatively charged droplets were prepared by using purified phospholipids enriched with negatively charged phosphatides; use of amino acids, urea, or carboxylic or sulfonic acid to form stable emulsions even after mixing with blood plasma [151]. 2. Attempts to make bettercharacterized parenteral emulsions(by Coulter counter [152,153]) and to improve their production techniques (microfluidizer [1541). 3. Stability studies related to thermal treatments[155,156]. 4. Effect of various nutient ratios on the emulsion stability of total nutrient admixture [157]. 5. Elimination and metabolism of a fat emulsion containing mediumchain triglycerides [1581. 6. Transdermal new formulations for special applications have been also studied. For example, new nicotinic devices and their usein the treatment of withdrawal symptoms associated with smoking cessation [1591.
C. Lipid Emulsions for Drug Delivery
Low water soluble drugs are difficult to administer and, therefore, are good candidates for formulation into O N emulsion. Fat emulsions are known and used in parenteral nutrition (artificialcylomicra have been developed in the form of fat emulsions). The two-phase emulsion system should havean advantage overa solubilized system(use of cosolvents such as polyols) in that the drug contained therein will not be able to precipitate as solid (harmful) particles when diluted. Furthermore, the presence of the bulk of the drug in nonaqueous environment may lead to an increased stability of the drug (e.g., reduced hydrolysis) as well as to a possible controlled-release system. The use of soybean oil emulsionsas carriers for lipid-soluble drugs has
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30.8 LO.0 Dose ( m g I kg I
52.0
Fig. 29. Administration of thiopentone in aqueous and emulsion forms.0 ,emulsion; 0,solution. (After ref. 164.)
been pioneeredby Jeppsson and Ljunberg in Sweden [160-1631. The drugs, dissolved in the oil phase, have included barbituric acids, cyclandelate, diazepam, andlocal anesthetics and the routes used have been intravenous, intra-arterial, subcutaneous, intramuscular, and intraperitoneal. The greatvehicles for the est interest has centered around the use of fat emulsions as intravenous administration of drugs [1601. For thecase of the barbituric acids [MO], a prolongation of anesthesia was observed when the drug was administered in the oil phase of a soybean emulsion comparedwith a solution of the corresponding sodiumsalt (Fig. 29). The results were explained either by a slow release of the drug from the oil particles or thepossibility of a more specific delivery of the drugsto the central nervous system (CNS) when the drug is confined in the oil droplets, the latter being due to the type of close association of droplets and thevascular lining of the CNS. As evidence for the possible close association of lipid particles with vascular linings, Jeppsson and Ljunberg [l641 cited the work of Schoefl and French [165], who demonstrated that in the mammary glands of lactating mice, chylomicra and artificial fat particles (soybean oil emulsion) were concentrated against the luminal surface of the endotheliumof small blood vessels and appeared to adhere closely to it. In some cases, the fatparticles appeared to have sunk deeply into the cytoplasm, or it appeared that thin flaps had encircled them. In contrast, in the lung, the fat particles were freely suspended in the blood plasma. The role of an enzyme, believed to
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be lipoprotein lipase, in this adhesion process and the transport of fat through the vessel wall was discussed. Many more reports exist in the literature on drug delivery by the intravenous administration of fat emulsions containing drugs. Examples are the administration of lignocaine [164,166] (which protects the heart f r o m electrically induced arrhythmias), diazepam [167-1701, hexobarbital [l701 and phenyl butazone[170]. In spite the extensive work invested in stabilizingthe drug-containing fat emulsions with various peimitted emulsifiers, it is not yet clear whether the emulsifiers and the fats decompose in the system and whether they play any role in the release and/or targeting of the drug. Some investigators claim that certain effects (such as sustained release) could well be related to metabolic changes induced by the presence of the emulsion rather than delayed drugrelease or drug targeting [170,171]. Narcotic antagonists are importantcandidates for slow-release action and, therefore, have been tested in various formulations to induce physical dependence in rats following subcutaneousdepot injection [172,173]. Valinomycin [l751 and other anticancer drugs have been introduced into intravenous emulsions [173-1751. Tests in animals showed that the drug contained in an emulsion had effects similar to those of an aqueous suspension, but that the emulsion formulation required a 20-fold lower dose to produce these effects [176]. Artificial intravenous emulsions havealso been tested as on-target delivery systems for drugs that arevery oil soluble. The drugis dissolvedin the oil and the emulsion is injected. The fat particles (chylomicrons) can deposit and close contact between the fat particles, and the endothelium can allow passage of the drug from the oil phase to the tissue without it necessarily passing into theplasma. The excess chylomicrons will be cleared by the liver, adipose tissues, heart muscle, and lactating mammary glands. In this connection, it is of interest to review the work of Meyerson [177], who found a prolonged-release effect after dissolving progesterone in soybean oil and thenemulsifymg it to give an O/W emulsion forintravenous administration. Similarly, Mitushima et al. [l781 have employed a lipid emulsion as a novel carrier for corticosteroids. Their studies in rats have suggested that corticosteroids incorporated inlipid emulsions are taken up by the reticuloendothelial system and certain inflammatory cells to a greater extent than free corticosteroids, thus resulting in stronger antiinflammatory activity [178]. Thus, it was believed that corticosteroids in lipid emulsion could be of value in the treatmentof certain diseases such as rheumatoid arthritis, where phagocytic inflammatory cells play an important role in the pathogenesis.
Aserin 456 D. Vaccine Emulsions
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Adjuvant methods of immunization havethe advantage of achieving both the primary and secondary immune responses with only one injection of antigen and of maintaining the antibody level over a long period of time. Adjuvants have been used recently in tumor immunotherapy [179]. An adjuvant effect is obtained by adsorbing the antigen onto a mineral gel (aluminum hydroxide or phosphate). This is widely usedas an immunizing agent for human use [179]. Water-in-oil emulsions, consisting of anaqueous antigen solution dispersed in a mineral oil phase with the aid of a lipophilic emulsifier (mannide-monooleate), have been prepared to act as efficient immunological adjuvants. These emulsified systems presumable enhance the antibody response (slowly releasing secondary stimulation of antibodies) and the stability of the vaccine on storage [180,181]. Various mechanisms have been suggested to explain the high antibody response which these adjuvants produce, and it has been assumed that the emulsion acts as an inert depot, whichslowly releases antigen over a long period of time [179,182-1841. Steinberg [l831 has suggested that in addition to the depot effect, the oil attracts mononuclear cells about the antigen which can take part in antibody production and give a high antibody level. Freund and McDermott [l851 were some of the first to demonstrate adjuvant activity of mineral oil emulsions. These simple water-in-oil emulsions, subsequently termed incomplete adjuvants, were later modified to give a greater antigenic response by incorporating dried, heat-killed Mycobacterium tuberculosis in the mineral oil phase. Such emulsions are termed Freund's complete adjuvants [186]. The complete adjuvantinitiates a cellular response andis too reactive for immunization in humans, although it is used in experimental work [186-1891. Problems have been encountered in the use of mineral oil emulsion adjuvants, since mineral oil isnot metabolized and will persist at theinjection site [179,187,188]. This has limited the use of mineral oil emulsions in humans. Studies have, therefore, beencarried out in the search for alternative oil phases for adjuvant preparations (hexadecane, squalane, sesame oil, and peanut oil. Stewart-Tu11and others have found that low molecular weight hydrocarbons of chain length C,&,, were goodalternatives to mineral oil). Early studies with vegetable oils .were unsuccessful, as the oils were unstable and gave lessantibody response thanmineral oils [189]. However, further work produced a suitable adjuvant of peanut oil [187]. This adjuvant consists of a water-in-oil emulsion of aqueous antigen together
Emulsions
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with an equal volume of an oil phase. Arlacel A is the emulsifier and aluminum monostearateis the stabilizer. A more recent report by Reynolds [l901 describes the adjuvantactivity of a refined peanut oil base emulsified inaqueous vaccines with glycerol and lecithin. This formulation was found to be metabolizable and caused no granulomasor abscess formation.
E. Emulsions as Adjuvants
Oil-in-water emulsions have been used as adjuvant-type systems in cancer chemotherapy [191].Successful tumor regression has been achieved by intratumoral injections of viable Bacille Calmette-GuCrin (BCG)vaccine, and a potential antitumor preparation has been constructed by combining trehalose dimycolate purified from BCG with aqueous soluble extracts of Re mutants of Salmonella species (Re glycolipid). An essential requirement of activity has been the formulation of an O/W emulsion. A drug injected into the interstitial spaces in muscle tissue is transof it ported away from the injection site by the circulating blood, but some also reaches the regional lymph nodes. The amount of drug so absorbed depends on the injection formulation and the site of the injection. It is known that lipids, fatty acids, and high molecular weight substances are absorbed mainly to the lymph system and lower molecular weight compounds are absorbed into the blood. Many important drugs are water soluble with relatively low molecular weights; therefore, a specific drugdelivery system in the form of an emulsion or other colloidal system is necessary to deliver such agents to thelymphatic system. Sezaki, Hashida and other workers [192-1981 have investigated the use of a water-in-oil emulsion to provide drug-delivery systems using a series of anticancer drugs in a series of formulations; water-in-oil emulsion, water-in-oil emulsion containing gelatin, and also simple oily and aqueous solutions and aqueous intravenous injections for comparison. Injections were given by intramuscular, intraperitoneal, and intragastic routes. Concentrations of both the drug and the oil were measured in the blood, regional lymph, thoracic lymph, and lymph in other parts of the body and also at the injection site following injection at various sites. It was suggested that absorption of a drug from its injection site could be absorbed into the bloodstream or into the regional lymph nodes. It passes to the thoracic duct and thento thebloodstream. It can travel as the isolated drug or in the emulsion formulation. Using mitocycin C and bleomycin, it was noted that the water-in-oil emulsion produceda much greater specific delivery of drug into the lymph system than did an oil-in-water emulsion [195-196]. Sezaki et al. [195],
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therefore, suggested that encapsulating a water-insoluble drug in oil was highly advantageous for the transport of drug fromthe interstitial spaces to the lymphaticcapillaries. They also noted that theconcentration of oil was much higher in the lymph nodes thanin the thoracic lymph and that it rose to a maximum quickly and then decreased in the former, whereas it was relatively constant in the latter. This suggested that theoil was transported to the lymph nodes, where it accumulated and was gradually released downstream. An oil-in-water emulsion containing gelatin as an emulsifying agent was found to be intermediate in its lymphatic targeting ability between the water-in-oil emulsionand the oil-in-water emulsionprepared by using polysorbate 80 [195-1961. The gelatin-containing emulsion was investigated, andit was found that the mitomycin C was partially bound to theoil drops [195]. In subsequent experiments, a water-in-oil emulsion, using a relatively high concentration of gelatin as part of the emulsifying system, was compared with the simple water-in-oil emulsion. The concentration of the gelatin used was20% of the aqueousphase, and the gelatin was thought to form solidified microspheresin the oil. It was noted that a high drug concentration surroundingthe injection site was prolonged with the emulsion formulations, especially the gelatin emulsion. A sustained release of drug into both lymph and blood systems was significantly improved with the two emulsions compared with the aqueoussolutions [194-1981. The time during which the drug concentration in the regional lymph node was higher than that in other lymph nodes (in the orderof several hours with both emulsion formulations as compared with halfan hour for the aqueous solution). Also, the time delay to reach a maximum or peak concentration of drug in both blood and lymph was greater with the emulsion formulations. Microscopical observations made of the muscle fibers and the regional lymph nodes showed that multiple water-in-oil-in-interstitial fluid emulsion was formed in the muscle tissue on injection. With the aqueous gelatin-in-oil emulsion, microspheres (1-2 pm in diameter) were observed to be dispersed in the oil droplets. The investigators [193,194] suggested that this system wasmore stable than the simple water-in-oil emulsion and this reason. This suggests that that itprolonged the release of the drug for the release of drug is due primarily to the breakdownof the emulsion. Measurements of the total amounts of drug absorbed intothe lymph system revealed that the microsphere-in-oil emulsion was superior to the water-in-oil formulation in promoting specific lymphatic absorption. It was concluded [l941 that water-in-oil and microsphere-in-oil emulsions would be advantageous for use in cancer chemotherapy. In the light of these experiments, bleomycin in the form of a microsphere-in-oil emulsion was injected into the appendix of rabbits having a carcinoma on the organ after surfkal removal of the carcinoma [198]. A
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mixture of medium-chain triglycerides (MCTs) was used as theoil phase of the vehicle, since this had a lower viscosity than the sesameoil previously used and, therefore, was easier to inject. The MCT-based emulsion improved the original delivery characteristics of the microsphere-in-oil emulsion with sesame oil as the oil base. It produced a definite improvement in the extent of metastasis. The metastatic cells were seen to be destroyed after 1month (observedmicroscopically) compared with an aqueousformulation which slightly damaged diseased cells, but these recovered within a month. It also delivered little drug IO the lung or liver, where it is especially toxic. Takahaski et al. [l991 investigated the use of water-in-oil emulsions as a vehicle for the injection of anticancer agents (bleomycin and mitomycin of C) directly into tumorsfor antitumor activity and also for the prevention lymphatic metastasis, thereby making use of the sustained-release effect of such a formulation. They found in experimental animals a prolonged level of drug in the tumor using a water-in-oil emulsion, and also a decrease in tumor size of 50% in 16 days and 75% survival rate compared with a 5% decrease in tumor size and a 40% survival rate with an aqueous injection (Fig. 30). Clinical trials on carcinomas produced encouraging results. In some cases, a multiple emulsion was employed as the delivery system.
-1 00
10 14 12
16
Time after injection (davrt
Fig. 30. Changes in mean tumor diameter o f rat carcinoma aftera single 0.5-mg dose of bleomycin in various forms. 0 , untreated control; A, aqueous solution intravenous; V, aqueous solution intratumoral; 0, emulsion intratumoral. (After ref. 199.)
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The chemical composition of a digestible oil may also be critical, as this will determine the physiological mechanisms stimulated during digesof tion. Forexample,thehydrocarbon chain length andthedegree unsaturation of the fatty acids liberated by triglyceride hydrolysis determine the extent of changes in gastrointestinal motility, with the longerchain unsaturated fatty acids havingthe greaterinhibitory effect [200,201]. Incorporation of long-chain fatty acids into the bile salt micelle increases the solubilizing capacity of the micelle for lipophilic materials within the intestinal lumen [202,203], and incorporation of unsaturated long-chain fatty acids may result in an increase in the permeability of the mucosal membrane [204,205]. Short- and medium-chain fatty acids are absorbed via > C , , ) are absorbed into the portal route, whereas long-chain fatty acids ( the lymph, with the unsaturated acids stimulating chylomicron synthesis. Lipophilic compounds selectively absorbed into the lymph are carried in the chylomicron [206], the natural fat particles, and therefore administration of long-chain unsaturated fatty acids may stimulate lymphatic absorption. It is apparent that the chemical composition of the oil phase of the emulsion is a major factor in determining the extent of drug absorption following oral administration.
F. Oral Emulsions
Oral administration of drugs in an emulsion form has been known for over 100 years and is easier to use than intravenous injection. Its main advantage is the morefacile absorption of certain drugs from the gastrointestinal tract. The ONV emulsions arerelatively easy to prepare, since there are no strict requirements on droplet size, nature of surfactant and its purity, nature of its fat, norphysical, chemical and biological properties. The range of emulsifying agents is greater than that available for intravenous emulsions. Materials suchas acacia, tragacanth gums, and methylcellulose as well as various nonionic surface-active agents have been employed in pharmaceutical formulations for oraluse [207,208]. The ionic surfactants are not normally usedfor internal preparations but arereserved for topical and cosmetic applications. The G U S (generally regarded as safe) listings of the U.S. Food and Drug Administration are a useful source of information [209]. Vegetable oils such as cornoil, peanut oil, and soybeanoil are used as emulsion vehicles for drugdelivery. Mineral oils (liquid paraffin) are emulsified to provide a laxative effect but are not used for drug administration unless the drugis intended forthe same pharmacological effect. Many attempts have been madeto entrap various drugs in O N emulsions for oral adsorption.
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Higher levels of drug in the plasma during the first 4 h after oral administration of acetyl sulfisoxazole O/W emulsion were observed in Svenson's early studies [210]. The emulsion was also shown to be three to five times more active against streptococcal and pneumococcal infections in mice. Similar results were reported with sulfonamides [211]. In most formulations, greater advantages were found in drugs with limited water solubility (poor dissolution), such as indoxole [212], griseofulrin [213,214] (Fig. 31), phenytoin [215], and theophylline [216]. Formulation of nitroglycerin in a sesame oil-in-water emulsion yielded lower and later peak plasma levels than the equivalent aqueous solution, butbioavailability was unaffected [217]. The delay in drug absorption from the emulsion was attributed to the time required forthe drug to move from the oil phase into the aqueous phase prior to absorption andto effects of the oil on gastric emptying. Thus, the emulsion provided some sustained-release characteristics to nitroglycerin absorption, whereas not affecting the extent of absorption.
3 r
' 1
Time (hrl
Fig. 31. Administration of griseofulvin in different dosage forms (30 mglkg of micronized griseofulvin in rats). 0, aqueous suspension;A,corn oil suspension; 0 , corn oil-in-water emulsion containing suspended griseofulvin. (After ref.213.)
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Absorption of the fat-soluble vitamin, vitamin A, occurs primarily via the lymphatic pathway [215,218]. Emulsification of vitamin A palmitate and vitamin A alcohol in corn oil has been found to increase the total absorption of vitamin A in normal patients and in those with cysticfibrosis [219,220]. These observations have important implications on both the dietary management of patients with cystic fibrosis and, moregenerally, on the use of the lymphatic system for drugdelivery. Drug absorption into the lymph has twomain advantages overthe portal route. First, the possibility of targeting high concentrations of anticancer agents into the lymph to prevent metastasis along the lymphatic pathway. Second, reduction of firstpass metabolism by delivery of the drug directly into the general blood circulation. The lymph is the primary route of absorption for lipophilic dietary compounds suchas cholesterol, long-chain fatty acids, and the fatsoluble vitamins. As seenwith vitamin A, adsorption may be improved by administration with lipids. Similarly, it has been suggested that the lymphatic absorption of lipophilic drugs may be stimulated by coadministration of lipids [221]. The oral absorption of heparin was shown to be insignificant in rats and gerbils following the intraduodenal administration in aqueous solution in micellar solutions of monoolein or sodium taurocholate or in sodium taurocholate-stabilized mineral oilemulsion[222]. However, significant absorption was achieved by administration in trioctanoin, corn oil,or peanut oil emulsions stabilized with sodiumtaurocholate. Although no mechanism for the enhanced absorption of heparin was proposed, the apparent dependence of this effect on thedigestibility of the oil used in the emulsion was noted. Thiswas seen again in absorption studies with the glycoprotein, urogastrone [223]. Absorption was unaltered following administration in aqueous solution, 0.2% (w/v) Ween 80 solution, liquid paraffin emulsion, and diethylphthalate emulsion but was significantly increased by administration in trioctanoin and in olive oil emulsions. Studies byBloedow and Hayton [224], using lipids rather than emulsions, suggested that generally polar digestible lipids increase the bioavailability of lipophilic, poor watersoluble drugs, whereas nonpolarnondigestible lipids have no effect on the bioavailability but may reduce the rateof absorption [225,226]. The mechanism of intestinal absorption of drugs from oil-in-water emulsion systems has been explored by Kakemi et al. [227-2291 using an in situ large intestinal rat gut loop model. Their results suggested that the partition coefficient of the drug and the absolute volume of the aqueous phase is of importance, with absorption occurring mainly from the aqueous phase. For a drug with a partition coefficient of less than 1, drug transfer into the aqueous phase is rate limiting rather thantransfer from the water
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to theabsorptive membrane. However,Noguchi et al. [230] proposed that for alipid-soluble, water-insoluble drug, the absorption process consists of adsorption of vehicleoils to the absorptive membrane, partitioning of drugs into the membrane lipids with simultaneous partition into hydrolyzed oils, release from the membrane lipids or hydrolyzed oils with movement into the inner compartments, and then transport into the portal or lymphatic systems. The absorption of ephedrinefrom orally administered emulsions given to dogs was investigated byLin et al.[231], whoattempted to rationalize their results using the HLB concept. Correlations of ephedrine present in urine in vivo with the total amount released in vitro using a dialysis method were significant at five different HLB values in the range 10-14. Although emulsions may enhance oral drug absorption and biological availability, the underlying mechanisms causing this effect remain unclear. Both physiochemical and physiological functions appear to be involved; for example, dissolution rate, oil-water partition coefficient, gastrointestinal mobility, bile flow, lymphatic absorption, and membranepermeability. The exact way in which these factors interact to affect drug absorption will depend largely on the nature of the drug and theoil phase. Oral administration of drugs in emulsions, therefore, seems to have great potential as a dosage form provided the problems of stability and drug release can becontrolled.
G. Topical Emulsions
Emulsion systems can be administered to theexternal surfaces of the body and to body cavities as topical formulations in the form of O/W or W/O creams containing drugs. General reviews on the structure of the skin, the absorption of drugs through the skin, and pharmaceutical and cosmetic products for topical administration can be found in the relevant textbooks [232-2341. A good topical formulation should be one in which the dosage form has both physical and chemical stability, has cosmetic acceptability, and also provides the optimum environment for the active ingredient to reach the skin surface [235]. For the corticosteroids, which may be regarded as representative molecules, the topical therapeutic activity may be considered as the result of three interactions: release, penetration, and antiinflammatory activity. These, in turn, result from the interactions between corticosteroid, vehicle, and skin. In the design of topical formulations, all three factors must be considered to optimize drug delivery [233].
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In general, a suitable topical preparation may be produced by:
1. Optimization of the drug concentration in order that all the drug be in the solution 2. Minimizing the amount of solvent used so that the chemical potential of the drug is maximized inthe solution 3. Ensuring that the vehicle components affect the permeability of the stratum corneum in a favorable manner
In emulsion systems, the distribution of the drug between the various phases and the total drug concentration will define the overall concentration gradient that exists across the skin. Also, the relative proportions of the lipid and aqueous phases in the emulsionwill affect the degree of hydration of the stratum corneum and hence drug penetration rates [236]. In general, emulsions will be less occlusivethan greasy materials. The pharmaceutical andcosmetic of a topical product is related toits viscosity, which will affect its consistency, spreadability, and extrudability and will determine the rate which at the active drug candiffuse to the outer layers of the stratum corneum [234,237]. If a thick layer is applied to the skin, the drug in the upper regions of a viscous preparation will not reach the skin during the normal application time. Surfactants present in emulsion systems may also influence the rateof release from the formulation as well as the rate of absorption [238]. Many publications have indicated that the presence of soaps can enhance the penetration rates of poorly absorbed materials [239-2421. The surfactants . may also promote the diffusion from the base itself and hence influence therapeutic performance[236,242]. However, simple relationships between the HLB and surfactant action could not be established, and it was concluded that the surfactants had a direct action on the protein rather than the lipid components of the stratum corneum [243]. The following types of emulsifying agent are used pharmaceutically:
1. Zonic: examples of anionic surfactants commonlyused are the alkali salts of the higher fatty acids and sulfate esters of the higher fatty alcohols; for example, sodium cetyktearyl sulfate. Cationic surfactants employed include the long-chain quaternary ammonium compounds; for example, cetrimide. If ionic surfactants are included in a formulation, care must be taken that they do not interact with the active ingredient [243]. 2. Nonionic: a large number of nonionic emulsifiers existfor stabilizing topical emulsions. The long-chain alcohols (e.g., stearyl) are very hydrophobic and are used to stabilize W/O emulsions. The
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corresponding polyoxyethylene glycol ethers have higher HLB values and therefore better emulsifying capacity and are used in the preparation of O/W emulsions. The overall properties of nonionic surface-active agents depend onthe relative proportions of the constituent hydrophilic and lipophilic moieties of the molecule (HLB value). 3. Mixture of emulsifiers: to obtain a stable emulsion, it is often useful to use mixed-surfactant systems. A combination provides a more rigid film at the lipid-aqueous interface and an increased reduction in the interfacial tension. An example of a complex emulsifiersystemiscetyl alcohol, sodium lauryl sulfate, and glyceryl monostearate. The rheological properties of semisolid emulsions made frommixed emulsifiers have been studied extensively by Barry and Eccleston [2442471. The system comprising oil-alcohol (usually a mixture of cetyl and stearyl alcohols)-surfactant (cetrimide, sodium lauryl sulfate, or Cetomacrogol) was found to producea viscoelastic structurethatgavea bodying effect to the formulation through the formation of a gel network of liquid crystals. The rheological properties were highly dependent on the quantity and natureof the surfactant and the alcohol. Interestingly, the use of pure alcohols of different chain lengths gave rise to the formation of semisolid products with poor stability. Oil-in-water creams are water miscible and hence washable and may be used in a range of active ingredients. After application, the film produced is less occlusivethan a W/O emulsion system.They spread easily and their advantage is that as the water evaporates, they exert a cooling effect at the skin surface. They also produce little irritation, yet their major disadvantage is that despite their fat content,they dry out. Water-in-oil emulsions have some advantages in certain disease conditions, where an emollient effect on the stratum comeurn is required. They also provide some protection barrier for the skin and generally provide occlusion. The occlusion effect increases the hydration of the stratum of some drugs. corneum and hence enhances the permeation percutaneous absorption Examples of the effect of formulation on the of methyl nicotinate are shown in Table 9. The data show that aqueous cream BP releases methyl nicotinate more readily than oily cream BP. The table also showsthe effect of decreasing the drugconcentration on the time of onset of erythema. Simple formulation changes can markedly affect drug release, as shown by comparing the time of onset of erythema after the addition of glycerol to aqueous creamBP.
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Vehicles Concentration of methyl nicotinate
1 4.1 0.5 0.1 0.05 0.01
and
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Table 9. Percutaneous Penetration of Methyl Nicotinate from Four Emulsion

Time of onset of erythema (min) aqueous cream BP
3.3 6.3 4.0 6.3 4.6 16.7 5.9 7.4
oily cream BP
4.5 5.2 6.8 10.5
+ 40% glycerol + 60%

4.6 5.6 6.7 11.7 17.6
aqueous cream
aqueous cream glycerol
Many publications have discussedthe effect of formulation (including emulsion systems) on the activity of topical corticosteroids [248,249]. The factors influencing activity concern the physicochemical characteristics of the steroid and the choice of the vehicle. Simple constants such as the partition coefficient and the solubility of the steroid are important. It appears that the greatest release is obtained from preparations in which the drug is dissolved at its maximal solubility concentration. Other factors, such as the viscosity of the vehicle and the effect of the vehicle on the degree of hydration of the stratum corneum, are also significant. The influence of vehicle on the penetration of individual potent corticosteroids is also very significant. Marked differences in the activity of the same corticosteroid in preparations of different companies could be attributedto vehicle-dependent effects showing the significance of formulations. Many formulations for creams and semisolid products areavailable in the literature and patents. The scientific studies, however, concentrate on the various aspects of the release of drugs or active matter from theemulsion to the skin or to various skins such as membranes and cells [2502521. Most mechanisms treat the release in terms of Ficks second law of diffusion controlled process [250,253,254]. The effect of various nonionic emulsifiers [255-2581 on the release of various antimicrobial agents, various oils and various adjuvants on the rate of release was studied. The release seems to be diffusion controlled [250].
H. Perfluoro Emulsions for Artificial Blood
A formulation for artificial blood (total blood replacement) has been a goal for many investigations [259-2791. Various perfluoro emulsions were tried, including perlluorotributylamine, perfluorobutyltetrahydrofuran,
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perfluoro decalin, perfluoromethyl decalin, and perfluoromethylcyclohexane. The emulsified fluorocarbon provides the necessary oxygenexchange, and the hydroxyethyl starch acts as a plasma expander. The emulsifier isthe polyoxyethylene-polyoxypropylene copolymer (Pluronic F68) [261]. Some encouragingresults were reported by Geyer [261] on complete replacement of blood in rats. The formation of a suitable perfluorochemical emulsion has been difficult, since those oils that provide stable emulsions are not cleared from the body in a satisfactory way and the choice of surfactants is extremely limited [262,263]. Long-term room temperatureshelf stability of ready-for-use concentrated fluorocarbon emulsions is necessary in order fully to exploit their rherapoic potential. The degradation mechanisms of the fluorocarbons, the factors that have impact on their stability and the means of proplonging their shelf life by using fluorinated surfactants or coemulsifiers, have seen carefully studied [259]. Generally, the fluorinated amphiphiles add much to the stability. Davis et al. [262,263] found that mixtures of lecithin and poloxamer were the best systems for emulsion stability. Of interest was the fact that the preferred oil, perfhorodecalin, although originally giving a very fine emulsion onemulsification, had poor stability. The fluorocarbon-emulsifying and emulsion-stabilizing properties of F-alkylated surfactants when used alone or in combination with Pluronic F68 or egg yolk phospholipids (EYP), the two surfactants used in the presently developed fluorocarbon emulsions, were investigated. The stability of these emulsions was evaluated by monitoring the increase in average particle sizes and changes in particle size distribution over time [264,265]. Generally speaking, F-alkylated amphiphiles aremuch more efficient as emulsifiers or coemulsifiers or coemulsifiers for fluorocarbons than their hydrocarbon analogues. Emulsions significantly more stable, than those obtained with egg yolkphospholipids (and a fortiori, Pluronic F68) can be obtained with several of the monodisperse F-alkylated amphiphiles even when used alone, which by itself is remarkable; mixtures of amphiphiles (which includes Pluronic and EYP) are indeed known to be considerably more efficient emulsifiers than any of their constituents taken separately. However, the exact prevision of the behavior of individual surfactants is still uncertain. Some structurally closely related surfactants were found to have widely different emulsion-stabilizing characteristics [266,267]. Examples of various distinct types of behavior aregiven below. Stable, medium concentrated (50% w/v; i.e., 27% v/v) to highly concentrated (up to 100% w/v; i.e., 52% v/v) fluorocarbon emulsions were
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0.8
0.7
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4%
EYP
aJ
0.4
aJ
0 . 3
0.2
0.1
I
I
I
100
200
Days
Fig. 32. Comparison of average particle sue vs time variation for sterilized Foctyl bromideemulsions (90%w/v) prepared(a) with 1% of sodium D-glucose 6-[2(F-octy1)ethyl phosphate]and, (b) with 4%of egg yolk phospholipides(EYP). (After ref. 269.)
obtained with several F-alkylated amphiphiles, including F-alkylated glucosyl-phosphates [268,269], trehalose ester[270], carnitine esters[271], some amino acids of type, amine oxides, phosphocholines, and phosphatidylcholines [272]. Fig. 32 compares, as an example, the average particle size increase over time for a 90% w/v concentrated perfluoro octyl bromide (PFOB) emulsion prepared with the F-alkylated glucosyl-phosphates and Y F ! It is noteworthy that even very low amounts of the fluorinated with E surfactant result in more stable emulsions than when four times larger amounts of EYP are used [268]. F-Alkylated fluorinated surfactant polymers were shown to be more effective emulsifiers than theirnonfluorinated parent compounds [273]. Long-term shelf lives of perfluordecalin (FDC) emulsions (20% w/v) were, however, not achieved. Somewhat better results were obtained with the F-alkylated Tris (hydroxymethyl) acrylaminomethane (THAM) telomers [274,275]. On the other hand, the anionic poly(ethyleneglyco1) phos-
469
phate and theneutral ones were foundto be less efficient emulsifiersthan Pluronic F68 [276]. Further improvement in fluorocarbon emulsion stability is expected when mixtures of surfactants are used, especially if they present complementary attributes. Therefore, F-alkylated amphiphiles havealso been investigated as cosurfactants with Pluronic F68, EYP, or other F-alkylated surfactants. Pluronic F68-type poloxamers are not very effective in reducing the interfacial tension between a fluorocarbon and water, but they exercise considerable steric stabilization. The addition of a fluorinated cosurfactant was expected to compensate for this lack of effectiveness. Polyhydroxylated surfactants were chosen for this purpose, because they could hydrogen bond Fto Pluronic. Considerable stabilization was indeedachievedwhen alkylated maltoside- or xylitol-derived surfactants were usedin conjunction with Pluronic F68 [277]. This synergistic stabilization effect is illustrated in with 5% w/v ofa Fig. 33 for a 50% w/v concentrated FDC emulsion prepared mixture of 2-(F-octyl)ethyl maltoside and Pluronic F68. The stability of the emulsion, which is rather poorwhen Pluronic F68 is used alone, is significantly improved when 20430% of the' poloxamer is replaced by the Falkylated maltoside in the surfactant mixture. It should be noted that this Falkylated surfactant alone (right side of Fig. 33) is totally unable to stabilize the emulsion. The glucosyl-phosphates were found to act synergistically with EYP [268]. Therefore 90% w/v PFOB emulsions were prepared in which the EYP/F-(octy1)ethyl phosphate ratio was decreased stepwise, with the total amount of surfactant being held constant (Fig. 34). The replacement of only one eighth of the EYP by F-(octy1)ethyl phosphate was sufficient to obtain both a definite stabilizing effect and, interestingly, a decrease in particle size which, moreover, is better preserved during sterilization and ageing than in the emulsions prepared with EYP alone. Synergistic stabilization was also observed when mixtures of two Falkylated phosphocholines were used in the formation of 100% w/v FDC emulsions [278]. These concentrated emulsions were significantly more stable than those formulated with EYP. Mixtures of F-alkylated THAM telomers of different length or of these telomers with the F-alkylated xylitol derivative also led to some improvements in FDC or PFOB emulsion stability [275].
1. Biological Evaluation of F-Alkylated Amphiphiles (in Vivo Tests) The acute toxicity of F-alkylated surfactants has been evaluated by intravenous injection in mice of about 0.5 mL (i.e., 25 mWkgbody weight, caudal vein) of their solutions and/or dispersions. The reported maximum
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Garti and Aserin
0 . 04
Maltoside / Pluronic F-68(w/w)
20
40
60
80
100 %
Fig. 33. Average particle sizes initially (0), after 1 month (O), after 1 year (m) at 25"C, in 50% w/v F-decalin emulsion as a function of a ratio of 2-(F-octyl)-ethyl maltoside to Pluronic 68 in thesurfactantmixture, with thetotalamount of surfactant being held constant (5% w/v). tolerated dose (MTD) compatible with the .survival of all injected mice (n=10) and/or lethal dose causing the deathof 50% of the mice (LDS0) are collected in tables [279]. The highest tolerance, by far, is observed for the F-alkylated double-chain glycerophosphocholines. They display considerably higher intravenous tolerance than the correspondingsingle-chain analogues, with MTD values for compounds superior to 8000 mg/kgbodyweightcompared with 25-125 mgkg for compounds. Obviously, the nature of the hydrophilic head is also important. In the single-chain compound series, the following sequence of increasing tolerance was observed: phosphocholine phosphocholine mono-N-ethyl < amino acid < trehalose sucrose
471
n.xn T I 40C
After 6.7 nrollths
x % wlv of 81b
IL !
After 3 m o n t h s
0.00 0
I
1
X%
Fig. 34. Averageparticlesizesin 90% perlluorooctyl bromide emulsions, prepared by microfluidation, as a function of the amount, x%, of sodium D-glucose6[2-(F-octyl)ethyl phosphate], with the remaining (4-x)% consisting o f egg yolk phospholipides.
< maltose < xylitol < poly(ethy1ene-glycol) phosphate < THAM telomer. The high in vivo tolerance of the poly(ethyleneglyco1) phosphate derivatives is seen further to increase with increasing the size of the polar head and its hydrophilic character. These trends indicate that for similar hydrophobic tails, intravenous tolerance improves along the sequence: zwitterionic single-chain < neutral single-chain < zwitterionic double-chain compounds. It also appears that increased hydrophilic character (from neutral to zwitterionic or toanionic compounds) needs to bebalanced by an increase in hydrophobic character or by an increase of the size of its hydrophilic head. No obvious relation is, however, found between hydrophobic chain length an in vivo toxicitywhatever the hydrophilic head. , All in all, F-alkylated glycerophosphocholines, poly(ethy1ene-glycol) 2phosphates and THAM telomers, l-O-[3-(F-octyl)-Zpropenyl]xylitol, (F-octy1)ethyl maltoside, and 6-0-[3'-(F-octyl)propanoyl]trehalose stand out as the most promising candidates for biomedical preparations.
Aserin 472
and
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One of the primary goals for which many of the F-alkylated amphiphiles were synthesized was the obtaining of stable fluorocarbon emulsions to be used as injectable oxygen carriers and in particular as blood substitutes. Consequently, some of the new surfactants were incorporated in emulsions which were tested in isovolemic exchange-perfusion experiments Therein rats. This was the case for 1-0-[3-(F-octyl)-2-propenyl]xylitol. of Pluronic fore, adispersion of 0.5& of the octyl-xylitol in grams per liter F68 was used to prepare a 10% (W/.) emulsion of lY2-bis(F-butyl)ethene (F-ME). This emulsion was tested on a series of 10 conscious rats whose red blood cell count was reduced to about one third of normal (hematocrit 15%) [280]. The behavior of the animals during and after the exchange was comparable to that of control animals, and the survival ratio was 10/10 after the 6-month observation period. This test was also applied to the mixed fluorocarbon-hydrocarbon dowel molecules. One of these compounds, C,Fl,CH=CHCl,H21, was administered intravenously inemulsifiedform at doses 300-600 times larger than those required to stabilize a clinically relevant dose of fluorocarbon emulsion. None of the 33 treated animals died. F-NMR (nuclear magnetic resonance) analysis of the organtissues indicated no metabolism; the halfof the compound in the liver (where fluorocarbon droplets are transiently stored) is about 25 5 days. In conclusion, compared with their hydrogenated analogues, fluorinated surfactants display significantly enhanced surface activity both in terms of effectiveness and efficiency and, in particular, strongly reduced water-fluorocarbon interfacial tensions. Fluorinated amphiphiles also show an increased tendency to form organized supramolecular assemblies, including liposomes, and impart unique properties to these assemblies. There is no doubt that their potential as membrane components and modifiers should be further explored. Their fluorocarbon emulsion-stabilizing capacity, when they are used as surfactants andor as cosurfactants, has been demonstrated butremains unpredictable. The assessment of thebiological properties of fluorinated surfactants has, however, hardly begun. Preliminary evaluation indicates somewhat reduced acute toxicity and definitely lower hemolytic activity compared with their hydrogenated analogues. It is now necessary to establish their fate in the organism and to explore their biological effects both when alone and when incorporated in emulsions, liposomes, or other supramolecular assemblies. The influence of fluorinated surfactants on the recognition of such particulate matterin vivo deserves special attention. The stage now. is set forsuch investigations. Only few detailed studieshave been published so far concerning the biological evaluation of F-alkylated amphiphiles. Some attention has, how-
473
ever, been given to F-alkanoic acids because of their many industrial applications related to their thermal and chemical stability and to their antiwetting and surface activity. Some simple biological tests (in vitro action on cell cultures and on red blood cells) and in vivo tests (acute toxicity in mice after intravenous injection) havebeenreported on a range of other F-alkylated amphiphiles. In a few cases, isovolemic blood exchange-perfusion experiments in rats with emulsions containing such surfactants have been performed. Concerning the F-alkanoic acids, these were found to be toxic with, for example, intraperitoneal LD, (rat) values of 189 and 41 mgkg for Foctanoic and F-decanoicacid, respectively. The major symptoms observed include anorexia, marked bodyweight loss, andhepatomegaly with a concomitant increase in peroxisomesand swelling. But the fluorinated metabolites of these F-acids have not been identified yet. It was reported that F-alkanoic acids covalenty bind to proteins both in vivo and in vitro, but the relevance of this binding to toxicity remains unknown. An increased incidence of Leydig cell adenomas was observed in rats fed with ammonium F-octanoate (300 ppm daily) for 2 years; an endocrine-related mechanism was proposed. The use of diverse F-alkanoic acids was nevertheless deemed acceptable for stabilizing micronized inhalation drug suspensions in 1,1,1,2-tetrafluoroethane7one of the CFC substitutes presently being developed. Studies on F-glucopyranosides also indicate the existence of toxicity due torapid hydrolysis and cleavage. These studies indicate that it is preferable to avoid the direct connection of a F-alkyl chain on a metabolic site which would then give either F-alkanoic acids or unstable F-alcohols. It should be preferable to insert a hydrocarbon spacer between the F-alkyl chain and the possible metabolic sites of the polar head.
2. Effect on the Growth and Viability of Cell Cultures (in Vitro Tests) The growth and viability of cells in the presence of the fluorinated surfactants tested are compared with those of control cultures grown under the same conditions without the surfactant. Cell culture tests were used both to assess direct cell toxicity and as a means of monitoring the final purification steps. Different cell strains were used, including lymphocytes, Hela, Molt-4, hybridoma, K562, or Namalva lymphoblastoid cells. No significant toxic effect of the Namalva cell's growth and viability anionic phosphate esters, the was found at a 1 g L concentration for the three 2-(F-octyl)ethyl D-maltoside, the 6-0-[5'-(F-hexyl)pentanoyl]trehalose, and for the 6-O-[ll'-(F-hexyl)undecanoyl]trehalose in spite of their high surface activity.
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3. Hemolytic Activity Unexpectedly, avery drastic difference in hemolytic potency was found betweensurfactants with hydrocarbon chains and with fluorocarbon chains of similar length. The hemolytic activity is consistently and considerably lower and often totally suppressed for the F-alkylated surfactants as compared with their hydrocarbon analogues. Moreover, the hemolytic activity is seen to decrease with increasing length of the F-alkyl chain, hence with increasing surface activity. The protective effect of the F-alkyl segment against hemolysis takes place despite the definitely higher surface activity if the F-alkylated compounds. The length of the hydrocarbon spacer, or moreexactly its relative length compared with that of the fluorocarbon segment, has also a decisive impact on hemolysis. Thus, the 11-(F-hexyl)-10-undecenyl maltoside, with a long CH==CH(CH,), hydrocarbon spacer, is hemolytic at a lowconcentration (1 g L ) , whereas the 5-(F-hexyl)-4-pentenyl maltoside, with a shorter CH=CH(CH,), spacer, is not hemolytic at 50 g/L. Identical results are found in the carnitine and phosphocholine series, where the compounds witha (CH,),, spacer are significantly more hemolytic than those with shorter spacers. of The natureof the polar head also influences the hemolytic potency fluorinated surfactants. Thus, for the same hydrophobic(F-hexy1)allyl tail, the maltose derivative issignificantlyless hemolytic than the galactose analogue, which is less hemolytic than that of xylitol. This appears to indicate that hemolytic potency is lower for those surfactants with the larger, more hydrophilic heads: It is also noteworthy in this respect, and somewhat unexpected, that the anionic glucose-derived phosphate esters are not more hemolytic than their nonionic F-alkylated sugar or glycoside counterparts. Others have described the partial and total replacement of blood or the use of perfluorochemicals in emulsion form fororgan perfusion
[283-2871.
In conclusion, the most recent work in the area of emulsions for pharmaceutical applications is related to practical improvements of the size of droplets with regard to their toxicity and coalescence and polydispersity [288]. Emulsions are used andcurrently evaluated for parenteraldelivery of nutrients and drug substances [289-2911. Some of the commercial total emulsionsare Emulsan, Intralipid, Lyposyn, and parenteral nutrition (TPN) Lipovenos. All of these emulsions are stabilized with egg lecithin and are based on soybean oil except Liposyn in which a mixture of soybean oil and safflower oil is used. Other TPN emulsions contain cottonsed oil and soybean phosphatides[292,293]. The droplet size and composition of the emul-
475
sion droplets are reminiscent of the endogenously occurring chylomicrons. Chylomicrons are fat globules, 0.5-1.0 pm in size, composed of triglycerides, proteins, cholesterol, and phospholipids. The chylomicrons are generated by the liver and the intestines. The relatively low toxicity'of TPN emulsions has made them an attractive alternative as carriers forlipophilic drugs [294,295]. In addition, much work has been done onusing emulsion forms in the delivery of vaccines [13]. The main efforts are related to artificial blood substitute [296]. The advantages of using the emulsion form in the delivery of drugs are (1) no precipitation of the drug substance will occur upon dilution [297], (2) drug stability may be improved, that is, hydrolysis will be reduced, and (3) a slow release of drug may be obtained. There is a risk that the emulsion will become unstable on injection into the blood stream through, if the emulsifier is too water soluble or if it sensitive to increased electrolyte concentration. There is much scientific literature describing emulsions as a drugdelivery system [297-3011. Examples of commercially occurring products in which emulsions are used for parenteraldelivery of active substances are Diprivan (propofolum), Stesolid Novum (diazepam), and Vitalipid Adult and Vitalipid Infant (vitamins).
XII. A.
Double Emulsions DefinitionsandFormation
Multiple emulsions are complex systems, termed emulsions of emulsions, with the dropletsof the dispersed phase themselves containing even smaller dispersed droplets. Each dispersed globule in the double emulsion forms a vesicular structure with singleor multiple aqueous compartments separated from the aqueous phase by a layer of oil phase compartments [302-3081. Multiple emulsions were first described by Seifriz [306] in 1925, but it was only in the past 20 years that they have been studied in more detail. The two major types of multiple emulsions are the water-in-oil (WIOW) and oil-water-oil (O/W/O) double emulsions. A schematic representation of a W/O/W double-emulsion droplet is shown in Fig. 1. Multiple emulsions have been prepared in two main modes: one-step emulsification and two-step emulsification.There have been several reports on the one-step emulsification for the preparation of W/O/W double emulsions which included strong mechanical agitation of the water phase containing an hydrophilic emulsifier and an oil phase containing large amountsof hydrophobic surfactant. A W/O emulsion is formed, but it tends to invert and form a W/O/W double emulsion [306] (Fig. 35). In addition, double
476
Garti and Aserin
Fig. 35. Possible sequence of events leading to the final formation of an OiW emulsion via a transient (WlOiW)emulsion, when hydrophilic surfactant is initially located in the oil phase.
emulsions can be prepared by forming W/O emulsion with a large excess of relatively hydrophobic emulsifier and small amount of hydrophilic emulsifier followed by heat treating the emulsion until, at least in part, it will invert. At a proper temperature, and with the right HLB of the emulsifiers, W/O/W emulsion can be found in the system. However, there is usuallylittle chance of reproducing these accidental preparations. In most of the recent studies, double emulsions are preparedin a twostep emulsificationprocess by twosets of emulsifiers: a hydrophobic emulsifier I (for thewater-in-oil emulsion) and ahydrophilic emulsifier I1 (for the oil-in-water emulsion) (Fig. 36). The primary W/O emulsion is prepared under high shear conditions (ultrasonification, homogenization), whereas the secondary emulsification step is carried out without any severe mixing (an excess of mixing can rupture the drops resulting in a simple oil-in-water emulsion). The composition of the multiple emulsion is of significant importance, since the different surfactants along with the nature and concentration of the oil phase willaffect the stability of thedouble emulsion [303,308-3101. Much work was done on the nature of the oils and their influence on the manufacturing conditions, as well as on the stability of the double emulsion [309]. Ionic and nonionic surfactants have been used for different applications in accordance with health restrictions. It was, however, well established that combinations of emulsifiers at the outerphase have a beneficial effect on stability, and that the inner hydrophobic emulsifier I must be used
477
hydrophilic surfactant
w/o/w
multiple emulsion
Fig.36. Schematicillustration of a two-stepprocessinformation emulsion.
of double
in great excess (10-30 wt% of the inneremulsion), whereas the hydrophilic emulsifier I1 must be used in low concentration (0.5-5.0 wt%). The inner emulsifier was found to migrate in part to the outer interface and influence the outer emulsifiers (Fig. 37) [310-3121. The HLB of the outer'emulsion was found to be a weighted HLB of the contribution of the two types of emulsifiers [313,314]. In addition, parameters such as the oil phase volume and the nature of the entrapped materials in the inner phase have been discussed and optimized [310-3131.
B. Stability and Transport Mechanisms
Many review articles have been written on the potential practical applications of the multiple emulsions and on the main problems associated with this technology-their inherent thermodynamic instability [304,308,309]. It was concluded that the classic double emulsions prepared with two sets of monomeric emulsifiers; one hydrophobic in nature (to stabilize the inner W/O interface) and the other hydrophilic in nature (to stabilize the
478
100
Garti and Aserin
Tween 80 and POE-oleyl ether POE-lauryl ether

and ester
, , ,
8 8
+
x
Tween 20 POE-nonylphenyl ether
POE
l I
I I
polyoxyethylene
I 1 1 1
Illll
- 1
10
100
\
Weight ratio o f Span 80 to hydrophilicemulsifier
Fig. 37. Weight ratio of Span 80 in the oilphase to hydrophilic emulsifiers inthe aqueoussuspendingfluidaffectingtheformation of water-liquid paraffin-water emulsions due to thetwo separated stepsof emulsification.
outer O N interface) cannot provide long-term stability to the double emulsion. A s a result, relatively large W / O N droplets with short-term stability are obtained,which cannot be used in practice [315]. The complex nature of the double emulsions has caused significant difficulties in the assessment of the stability and in detecting rupture and coalescence phenomena. The main technique is based on measurement of number and size of the double-emulsion drops over a period of time. Such measurements produce ,only limited information on double-emulsion stability, since no information on the coalescence of the inner droplets can be deduced. Similar information is obtained from photomicrography (see Fig. 2). It was and still is very difficult to determine if the internal droplets tend to aggregate or to coalesce. Several more sophisticated techniques have been applied, including freeze-etching, viscosity measurements, and quantitative estimation of addenda transportedfrom the inner phase to the outer phase and vise versa. In addition, engulfment or shrinkage of the double emulsion in the presence of water migration in or out of the droplets has been studied and interpreted in terms of stability (Fig. 38). Several possible mechanisms by which materials may be transported across the oil layer in the W/O/W systems have been proposed and dis-
479
Fig. 38. Possible pathways for the transport of water and entrapped matter from
the inner interface to the outer aqueousphase.
cussed [303,307,310]. The most common is the diffusion-controlled mechanism for ionized lipid-soluble materials. It will be dependent onthe nature of the material (including its dissociation constant) andthe oil, as well as on the pH of the aqueous phase. This system could be used, for example, in the treatment of overdosage of acidic drugs such as aspirin and barbiturates [316]. At low pH values, the barbiturate would exist almost exclusively as the un-ionized form and so would be readily soluble in the oil phase. The drug could therefore easily pass across the oil layer to theinternal aqueous phase containing a basic buffer which wouldionize the addenda that is now insoluble in the oil phase and would become trapped within the internal aqueous phase. This would then be carried out with the emulsion as it is voided from the gastrointestinal tract (Fig. 39). The drug transport was found to follow the first-order kinetics according to Ficks law [316]. Ionized compounds arenotthe only materials to be transported across the oil membrane. It has been demonstrated that both watermolecules and nonelectrolytes can easily migrate through the oil membrane without affecting the double-emulsion stability [317-3191. A mechanism based on micellar transport from one phase to the other has been described and determined. It was also demonstrated that one can alter the diffusion rates through the oil membrane by changing the natureof the oil. This suggests that the diffusion of the addenda through the oil is the ratedetermining step, and that the inner water phase does not have any effect on the determination of release rates. Kita, et al. [20] have suggested two
480
Garti and Aserin
Oil (liquid membrane) phase

ion i zed dr uq
basic
buffer
Un-ionized drug
Fig. 39. W/O/W liquid membrane system for removalof acidic drugs form an aqueous system.
possible mechanisms for the permeation of water and water-soluble materials through the oil phase; the first being via the reverse micellar transport (Fig. 40) and the second by diffusion across a very thin lamella of surfactant formed where the oil layer is very thin (Fig. 41). Our detailed studies on double emulsions stabilized by monomeric nonionic emulsifiers (Span and R e e n ) [317-3191 on the releaseof electrolytes from the inner aqueous phase to the outeraqueous phase have indicated that theosmotic pressure gradient between the two aqueous phases is a strongdriving force for mutual migration of addenda and water fromone phase through the other by both mechanisms. However, it was clearly demonstrated that even if the osmotic pressure of the two phases was equilibrated and no visual coalescence took place (neither of the inner phase droplets nor of the outer phase droplets), electrolytes tend to be transported outmostly through a reverse micellar mechanism controlled by the viscosity and the nature of the oil membrane. The mechanism is similar to the one described by Higuchi for release from polymeric matricesslab into the sink (Fig. 42) [320-3221.
48 1
Fig. 40. Schematic illustration of a model for micellar transport of water from the outer aqueous phase to the inner aqueous phase through the oil layer inWIOIW emulsion.
A modified diffusion-controlled equation was adapted to explain the release results [317-3191. It has been demonstrated that the release factor B,
B =- De-t r,C0 (where F is the release fraction of the marker soluble in the inner phase; De, t, time of release; C,, effective diffusion coefficient;ro,radius of the droplets; initial concentration of marker) can be plotted against l/Co and t, with excellent correlationcoefficients suggestingdiffusion controlled releasemechanism of reverse micellar transport [318,319]. The effective as well as the real diffusion coefficient couldtherefore be calculated. Although the release mechanism was clarified to some extent, it is still very difficult to control the rate of release (to slow it down) mainly B=
3 2[1 - (1 - F)3n]- F
wafer
Fig. 41. Schematic illustration of amodelforwatertransportthroughthis lamellae of surfactant due to fluctuation in the thicknessof the oil layer.
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Garti and Aserin
multiple drop
Fig. 42. -0-dimensional model for diffusion-controlled transport of un-ionized materials from the external aqueous phaseto the internal aqueous droplets in W/ a O W system. owing to thefact that the monomeric emulsifiers that have been used serve both as stabilizing moieties and as transport species. In addition, the fast exchange that those emulsifiers will undergo between the two interfaces and the fact that shearshould be avoided in the second emulsification stage lead to theformation of relatively large double-emulsion droplets with very limited thermodynamic stability.
C. New Approaches to Improve Stability

Fig. 43 demonstrates in part the different processes that can lead to the destabilization of the double emulsion and the processes involved in the release of various molecules (water or entrapped matter) from the inner phase to the outer aqueous phase. One can think of several approaches to overcome instability and release problems in double emulsions. Some of those ideas can be summerized as follows:
1. Stabilizing the inner W/O emulsion by reducing its droplet size
and by forming h-microemulsions or microspheres or by increasing the viscosity of the innerwater.
483
External drop coalescence
growth
/ -
Empty primary contlnuous phase drop
Fig. 43. Various possible mechanisms of emulsion stabilization.
2. Modifying the nature of the oil phase by increasing its viscosity or by adding carriers or complexants to theoil. 3. Stabilizing the inner and/or the outer emulsion by using polymeric emulsifiers, macromolecular amphiphiles (protiens, polysaccharides), or colloidal solid particles to form strong and more rigid film at the interface.
One can consider both naturally occurring macromolecules (gums, proteins) or synthetic grafted block copolymers with surface activities. It should be noted, although it is beyond the scope of this text, that polymerizable nonionic surfactants can form an in situ crosslinked membranes (after adsorption and carrying out a polymerization process). This concept was well documented and tested [323-3251. The polymeric complex that was formed was able to withstand extensive thinning (caused by osmotic driven influx of water) and the resulting swelling of the internal water droplets [326].
Aserin 484
and
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D. Naturally Occurring Macromolecular Amphiphiles
The stability of double emulsions can be improved (as explained above) by forming a polymeric film or macromolecular complexacross the oil-water interfaces. Omotosho and Florence have suggested using macromolecules and nonionic surfactants to form such stabilizing complexes. The film is formed throughan interfacial interaction between macromolecules such as albumin andnonionic surfactants [327,328]. Release rates of methotrexate (MTX) encapsulated in the internal phase of W/O/W emulsions stabilized by a film, formed as a result of an 80), interfacial interaction between albumin and sorbitan monooleate (Span were measured as functions of two formulation variables-the oil phase and the secondary emulsifier composition. The release rate was significantly affected by the natureof the oil phase and decreased in the orderof: isopropyl myristate > octadecane > hexadecane > dodecane > octane which was a reflection of the increasing internal droplet size of the emul4 ) . sion (Fig. 4 The release rate data conforms with first-order kinetics. Comparison of the effective permeability coefficients-calculated from the experimental
80
70
60
h
IPM
octadecane hexadecane dodecane octane
S- 50
2
W
c d
40
30
20
10 0
0 1 2 3 4 5 6 7
Time (hours) Fig. 44. Release of MTX from multiple W/O/W emulsions. The emulsions contained 2.5% Span 80 and 0.2% BSA as primary emulsifiers, with MTX (1 mg/mL) in the internal phase andthe following oil phases: *, octane: A, dodecane; +, hexadecane; 0 , octadecane; and a, isopropylmyristate.
485
apparent first-order rate constants-with the effective permeability coefficient of water in planar oil layers containing nonionic surfactants (determined by a microgravimetric method) supported the hypothesis of diffusion of MTX via loaded inverse micelles. Surfactants with highHLB values, used as the secondary hydrophilic emulsifier, increased the release rates, primarily byincreasing the rate of diffusion of MTX through the nonaqueous liquid membrane (Fig. 45). Omotosho and Florence have also reported use of other macromolecule complexes [327,328]. The formation of multiple W/O/W emulsions with improved stability due to the formation of interfacial complex film between acacia, gelatin, polyvinyl pyrrolidone (PVP), and sorbitan monooleate have been described [327,328]. The long-term stability as assessed by microscopy, showed no significant time changes in droplet size distribution of W/O/W emulsions prepared with acacia, gelatin, or PVP in the internal phase (Fig. 46). Multiple emulsions containing chloroquine phosphate in the internal phase that had been stored for2 weeks surprisingly showed a reduced rate of release of chloroquine phosphate as compared with freshly prepared emulsions, suggesting the release of chloroquine phosphate from these systems occurs by the process of diffusion as opposed to thephysical breakdown of emulsions [328] (Fig. 46). The intramuscular administration of
,
1
*l+
3
S 7 Time (hrs)
24
Fig. 45. Effect of the secondaryemulsifier(emulsifier 11) on the release of MTX from multiple WIOTW emulsions prepared with 2.5% Span 80 and 0.2% BSA as primary emulsifiers with isoprophymyristate as the oil phase.
Aserin 486
and
Garti
L,
..W" A BI
1 0
I 4
Time (hours) Fig. 46. Profile of chloroquine phosphate release from WIONV multiple emulsions. A,, freshly prepared PVP emulsions;A,, PVP emulsions stored for2 weeks; B,, freshly prepared gelatin emulsions; B,, gelatin emulsions stored for 2 weeks; C,, emulsion prepared with gum acacia;G, acacia emulsion storedfor 2 weeks. chloroquine in the form of W/O/W emulsions has a significant advantage, since it could reduce frequency of administration, improve patient compliance, andincrease the therapeuticefficacy of chloroquine. The drug can be formulated as a combination of two types of doses: starting dose which is incorporated into the external phase and a maintenance dose which is encapsulated in the internal phase of the doubleemulsion. Wasan et al. [329,330] have published a novel method for forming stable hemoglobin-oil-in-water (Hb/O/W) multiple emulsion for use as an artificial red cell substitute. A concentrated Hb solution was emulsified in oil to form microdroplets (with Pluronic F101 and Span 80) followed by dispersion of the primary emulsion into an outer aqueous phase containing hydrophilic surfactants (Pluronic F68 or Tween SO). The addition of human serum albumin into both the inner and the outerphases along with added dextran into the outer phase seems to be stabilizing the emulsion. The average diameter of the prepared multiple emulsions after homogenization and filtration was 2-3 pm with good hydrodynamic stability (sensitivity to shear). The formulation showed a very small release of Hb from the primary emulsion to the outer aqueous and good stability of the multiple emulsion during short-term storage (Fig. 47). Frank et al. [331,332] have suggested the use of colloidal microcrystalline cellulose (CMCC) and various monomeric surfactants to stabi-

100 -I
U
W
B D
0
487
90
5
W
80 70 6050
m
B
0 +
-!
U
U)
B
0
40-
0
[ I :
30
U .
20 10
o
B
0
Wilh Albumln
.L
Without Albumln
4
~ l ~ l ' i ~ l ~ l ~
10
12
14
16
18
20
DROPLET DIAMETER (microns)
Fig. 47. Effect of albumin on size distribution of multiple emulsion.
lize double emulsions via a mechanical stabilization mechanism. It was shown that the double emulsions were stable over a period of 1 month (monitored by microscopy). Slow release of certain drugs was achieved by gelling the oil phase (Table 10). Recently [302], the authors of this chapter have usedBSA as a polymeric emulsifier added both to the inner and to the outerinterfaces in the absence and in the presence of conventional monomeric emulsifiers (such as Span 80 and Tween 80). We have tried to evaluate in more detail the mechanism of the release of an electrolyte, NaCl, from the inner phase to the outerphase. Since it was believed that therelease mechanism is associated with a micellar transport which is diffusion controlled, attempts were made to obtain stable double emulsions with relatively small droplets anda minimum oil micellization capacity. Double emulsions were prepared with 10 wt% Span 80, 0-0.5 wt% BSA, and 2 wt% NaCl inthe inner aqueous phase. The outerinterface was stabilized with 5 wt% of Span 80-Tween 80. It should be noted thatafter 25 h of aging, the emulsion oil droplets size did not change significantly. The percentage release plot of NaCl versus wt% of the inner BSA reveals that BSA retards the release of NaCl (Fig. 48). Its maximum effect was obtained at 0.2 wt% BSA. It seems that BSA adsorbs at theinterface together with Span 80 and contributes both to the stability of the double emulsion and to therelease rates.
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Garti and Aserin
Table 10. Comparison of Release of Lidocaine Base From Different Formulations

% Lidocaine Effective incorpobase diffusion rated within coefficient Time for Formulation formulation Dx106 (cm2/s) release (h) after
30%
Release 5 h (%)
C"
2% 25.4 1 dispersion
W/O/Wb
- 6.9
Innermost
0.8
0.03 4.9
186
eous (4:6/1:1), phase CMCC 2% *Release of lidocaine from a 2 wt% CMCC dispersion containing 1 wt% lidocaine base. bRelease of lidocaine from W/O/W a (4:6/1:1) emulsion containing1wt% lidocaine base (25+O.l0C).
0.1
02
03
0.4
05
BSA (wt %) in inner phase
Fig. 48. PercentagereleasewithtimeofNaClfromdoubleemulsionprepared with 10 wt% Span 80 and various BSA concentrations in the inner phase and 5 wt% Span + Tween (1:9) in the outer aqueous phase.
Emulsions
489
In accordance with the previous studies, it was suggested that the polymeric surfactant forms a complex with the monomeric lipophilic surfactant. The complex is probably a thick, strong gelled film that imparts elasticity and resistance to ruptureof the innerdroplets. The film improves the mechanical and steric stability of the double emulsions and slows the coalescence rates. In addition,it appears that it depresses the formation of reverse micelles in the oil phase and slows the transport of the electrolyte via the reverse micelle or mechanism. However, the monomeric emulsifiers covering the outer interface do not prevent the coalescence of the outer droplets in the double emulsions. In fact, after 1 week of aging, a significant increase in droplet size distribution, as well as strongflocculation, was detected. Therefore, BSA was also added to the outer aqueous phase (during the second step of the emulsification) in addition to themonomeric Span 80-%een 80. The Coulter counter measurements (Fig. 49) clearly indicate that BSA when present in the outer phase contributes.effectively
30
r
0
CUIWE 1 CURVE2
CURVE3 CURVE4
10 2 0 30
4 0 5 0 6 0 70 8 0
90 100
DIAMETER Cm)
Fig. 49. Droplet size distribution of four emulsions prepared with(1) Span 80 + Tween 80 (9:l) in the inner phase and 0 . 2 wt% BSA in the outer aqueous phase; (2) with Span 80 in the inner phase and without BSA in the outer phase;(3) Span 80 in the inner phase and without BSA in the outer phase;(4) Span 80 in the inner phase and without BSA in the outer phase.
Aserin 490
and
Garti
to the stability of the double emulsions. Double emulsions prepared with BSA (in the outerinterface) consisted of significantly smaller droplets than any other emulsions prepared without the BSA. The improved dropletsize distribution was found for any BSA emulsion prepared with any level of monomeric emulsifiers. The photomicrographs and the Coulter counter measurementsof the droplets size distribution after 6 weeks of aging showpractically no change in droplets size distribution. The release rates as a function of the BSA concentration in the outer phase (emulsifier 11) show a minimum at 0.2 wt% BSA and a slight increase in the release rates at higher BSA concentration (Fig. 50). The Higuchi model for therelease of drug from solid polymeric matrix [321] as well as other models such Garti's as modification [318,319] for multiple emulsions (based on Fick's diffusion) have been used for testing phase. The B paramethe double emulsions prepared with BSA in the inner ter (as described previously) was plotted against time (t) in order to determine the effective diffusion coefficient, De. Figures 51 and 52 are typical plots of the parameter B versus l/Coof entrapped NaCl and B versus the time of aging of the emulsion. The De values for each BSA concentration calculated from plot B against l/Co and against t"(n=l) arelinear and quite similar, indicating that
5.
20 I(0)
t ( l week)
U
5.
-J
B
8
t(6 weeks)
10
0 0
25
50
75
100
125
DIAMETER (pm)
Fig. 50. Percentage release with time of NaCl from double emulsion prepared with 10 W% Span 80 in the inner phase and 0.1 wt% BSA in the outer phase.
491
Time (hrs)
Fig. 51. Factor B (see text) plotted against time of release for aouble emulsion stabilized with 10wt% Span 80 + 0.2 wt% BSA in the inner phase and5 wt% of Span 80-Tiveen 80(9:l) in the outer phase.
50
100
150
200
/co (wt %l of NaCl

Fig. 52. Same as Fig. 19 except plotting factor B against l/C,of the NaCl concentration in the inner aqueous phase.
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Gatti and Aserin
the Higuchi model dominates the release mechanism. However, better correlation coefficients (R,=0.998-1.0) will be obtained if the B parameter is plotted against t, where n (termed arbitrarily as the diffusion order) varies from 0.5 to 3.0 as a function of the BSA concentration (Table 11). Plots of parameter B versus to for double emulsions stabilized with BSA in the outerphase showed similar trends of linearity. Best correlation coefficients for linearity of the curves were obtained fort in which n was in the range of 0.5-1.0 for 0-0.5 wt% BSA in W, (see Table 11). The differences between the functionality and performance of the BSA present in the inner(W,) or the outer (W,) interface can be seen from the plot of the time exponent n (the diffusion order) versus the BSA concentration (Fig. 53). The effective diffusion coefficients (De) were calculated from the corrected Higuchi equation and plotted versus the BSA concentrations in the inner and outer phases (Fig. 54). Significant differences in the performance of BSA have been found. The outer BSA has only a limited retarding effect on the electrolyte transport, limited to concentration at 0-0.1 wt%, whereas the inner BSA (at 0.02 wt% levels) has a strong slowing effect on the releaseof NaC1. It has
Table 11. n Values (Diffusion Order) Obtained from Plots of B Factor Corresponding to the Percentage of Release of NaCl versus t from Double Emulsions Prepared with BSA in the Inner Phase(W,) and in the Outer Phase (W,) (see text)
BSA concentrationsin W, or W, (wt%)
0 0.05 0.1 0.2 0.3 0.5
%la
~Wzb
0.5 1.25 2.0 2.0 2.0 3.0

~
0.5 0.5 0.5 0.5 1.0 1.0
anwlis calculated from the best fit for linearity of the plot of B against t for double emulsions stabilized with BSA added to the inner water phase
(W,) bnwZ is calculated from the best fit for linearity of the plotof factor B against t of double emulsions stabilizedwith BSA added to the outer water phase (W,).
493
n(BSA in Wl) n(BSA in W2)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
CONCENTRATION of BSA (wt%)

Fig. 53. Plot of exponent n of time (t") againstBSA concentration in both inner
(0) and outer (
) interfaces.
-16.0 'p
6)
M
0
log(De)(BSA
in W 4
%4
Lag(De)(BSA ih WZ)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
CONCENTIWTION ofBSA ( @ h ]
Fig. 54. Plot of log De (effective diffusion coefficient) of NaCl vs BSA concentration in the inner and the outer interfaces.
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Garti and Aserin
Fig. 55. Schematic illustration of the two interfaces of the double emulsion stabilized by a combinationof monomeric and polymeric (BSA) emulsifiers. been assumed that the parameter nis a reflection of the nature of the film that is formed on the interface. When n s l , the film is rather thin and no significant viscoelastic gelfilm (complex between the Span and BSA) is formed. When n z l , the film is viscous owing to theformation of a strong Span-BSA complex. From Fig. 53 it can be concluded that the internal W/O film (W, interface) is more pronounced and stronger than the O N (W, interface) film. The W, film develops as the BSA concentration increases and is well defined at 0.1-0.2 wt% BSA. On the other hand, the BSA in the outer phase (W,) does not contribute to the formation of a viscoelastic network with the hydrophilic combination of Span-Tween and has only a limited effect on thediffusion coefficient. organization of the Fig. 55 is a schematic illustration of the monomeric and polymeric emulsifiers onto the inner (W,) and the outer (W,) interfaces. Note thatthe BSA is coadsorbedtogether with the monomeric emulsifier at the inner interfaceand serves only as protective colloid at the outer interface. From a careful evaluation of the release and stability results, it is possible to formulate an optimal double emulsion consisting of Span 80BSA in the outer interface and Span 80-BSA in the inner interface. XIII. DOUBLE EMULSIONAPPLICATIONS Double emulsions are excellent and exciting potential systems for slow or controlled release of active entrapped compounds. The fact that the inner W/O emulsion serves as large confined reservoir of water is very attractive
oemulsions Emulsions and
495
property for dissolving init significant amounts of water-soluble drugs. The oil membrane seems to serve as good transport barrier for the confined ionized and/or nonionized water-soluble drugs. The two amphiphilic interfaces are yet an additional barrier. The possibility to manipulate transport and release characteristics of the formulations seems to be feasible. However in spite of20 years of research with exiting in vitro release results [334-3661, no pharmaceuticalpreparation using the double-emulsiontechnology (neither for intravenous nor intramuscular administration, nor for oral or topical applications) exist inthe marketplace. It seems that the main reasons are the droplets instability (shelf life) and theuncontrolled release. We therefore present a short review summary of the studies mentioning the use of various drugs for different applications and the potential advantages that such formulation might have. Multiple emulsions have shown significantpromise in many technologies, particularly in pharmacology and separation science. Their potential biopharmaceutical applications, as a consequence of the dispersal of one phase inside droplets of another, include uses such as adjuvant vaccines [334,335], prolonged drug-delivery systems [336-3411, sorbent reservoirs of drug overdosage treatments[342,315], taste masking [313,314], and immobilization of enzymes [340,341]. In some disciplines, certain multiple emulsions have been termed liquid membranes, as the liquid film which separates one liquid phase from the other liquid phases acts as a thin semipermeable film through which solute must diffise as moves it from one phase to another. The use of multiple emulsions in the separation field has included, for example, sepathe removal of toxic materials from waste water ration of hydrocarbons and [342,345]. The following systemshave beenrecently studied and considered for pharmaceutical andmedical uses:
1. Hemoglobin multiple emulsion as a red blood cell substitute [330,346]. 2. The use of thickening agents and emulsifying agents to obtain slow release (retention to pharmaceutical dosage) of mebeverine from double emulsions [347]. 3. Prolonged release of pentazocine from O/W/Odouble emulsions [348]. 4. Sustained release of lidocaine from double emulsions stabilized [113,3491. An interesting compariwith microcrystalline cellulose son among O/W, W/O, and W / O N systems for the targeting of labeled 5-fluorouracil to lymph nodes after intratesticular administration is shown in Fig. 56. The profound response achieved
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Garti and Aserin
0.5
I 6
I 12
I
24
Time alter administration (hr)
Fig. 56. Uptake of 5-fluorouracil into regional lymph nodes after intratesticular injection into rats. W, aqueous solution; O N , oil-in-water emulsion; W/O, waterin-oil emulsion; WlOnV, multipleemulsion; IV, intravenousinjection(control). (After ref. 349.)
5.
6.
7.
8.
with the multiple emulsion is striking. Unfortunately, the reason for such an effect has not been elucidated as yet [350]. Sustained release of fluorouracil for intramuscular administration [350,351]. The use of albumin and monomeric nonionic emulsifier to control the transport of methotrexate from the internal phase of phase [328]. multiple emulsion to the external aqueous Effect of encapsulated insulin in double emulsion on the blood glucose efficiencyof experimental diabetic mice by oral administration [352]. Use of double emulsions for controlled release of topical drugs
[353].
497
9. Bioavailability and adsorption of pentazocine entrapped in double emulsion (oral intake). Its slow release effect on kidney, liver, and lung metabolism [354,355]. 10. Release of terbutaline sulfate from double emulsions prepared with paraffin oils and peanut oils, effect of pH, temperature, agitation, typeof oil on the release rates [355]. 11. Effect of acacia, gelatin, and polyvinylpyrrolidone on the chloroquine phosphase transport from W/O/W double emulsion [356]. Formation of interfacial complex films of the polymers and the surfactants and its effect on the transport rates (see Fig. 46). Reduced rate of release of chloroquine was observed. 12. Adsorption and lymphatic uptake of 5-fluorouracil in rats following oral administration of W/O/W double emulsions [350]. 13. The release of ephedrine hydrochloride from W/O/W emulsions. Efficiency of formation and rates of release as a function composition, mixing time, emulsifier concentration, and the HLB of oil and the emulsifiers. It was found that there is a linear relationship between the amount of ephedrine hydrochloride released and the square rootof the dialysis time of the double emulsions [357]. 14. In vivo releaseof 2.5% Xylocaine (lidocaine) hydrochloride from simple and multiple emulsion systems was compared with that from aqueous and micellar solution and anesthetic effects such as duration of action and tolerability were compared. The double emulsions showed a longer duration of action, less eye irritation, and improved efficacy compared with aqueous solutions [358]. 15. The use of double emulsions as useful vehicles for the administration of vaccines that gave rise to an improvement in antibody titer [359-3641. 16. In vitro release and pharmacokinetic and tissue distribution studied of doxorubicin hydrochloride (Adriamycin HCl) encapsulated in lipidolized W/O emulsions and W/O/W multiple emulsions. The results indicate that the release was sustained for both emulsions when HCO-60 (Hydrogenated CastorOil ethoxylated with 60 EO units) was used as emulsifier.The clearance of some W/O/W emulsions also decreased with the increase of the emulsifier concentration (Figs. 57 and 58) [365]. 17. Study of the release of cytarabine and fluorouracil (5-FU) from double emulsion. It was demonstrated that the release of cytarabine (Fig. 59) was very prolonged and affected by the increase of theinternal phase volume and theECO-10(Castor Oil
498
Conc.of
Garti and Aserin

Conc.of
1( r g m ~ )
Formulation A
a
o
1 [rotmlj
Formulallon E
m Tormulation F
2.010m
2.0Formulation 0 Formulotlon D
o FormulatlonC
l.0.
+
I
Formulation G
Formulation U
0.1-
025
0.5
0.75
* 1.0 2
1 0
x) 24
0.25
05
0.75
(a)
Tame [h]
(b)
1 0 2 1 0 Time [h]
20
24
Fig. 57. Serum concentrationof doxorubicin asa function of time after administration of into caroid arteryof SD rats, mean k SD, (n=6). (a) W/O/w emulsion; (b) in W/O emulsion. ethoxylated with 10EO units) (the emulsifier) concentration. The release of the 5-FU was faster and strongly depended on the pH (best at pH 10) [366]. 18. Prolonged release of bleomycin from parenteralgelatin spheresin-oil-in-water multiple emulsions. 19. Phenylephrine hydrochloride was formulated in different emulsion systems with and without viscosity builders (methylcellulose). Results indicate that the diffusion coefficient decreased with increasing viscolizer concentration. The mydriatic and intraocular pressure (IOP) were followed. Areaunderthecurve (AUC), maximum response (CMR), the time of maximum response (TMR), and the duration of drug action (DA) were found to improve in comparisonwith aqueous solution (Figs. 60 and 61). 20. Multiple W/OW emulsions containing pentazocine were prepared and tested in vitro and in vivo. The in vitro results indicated a well-controlled and higher drug release from the W/OW emulsions than the W/O emulsion. The invivo data showed prolonged tissue levels of pentazocine after oral administration of W/OW emulsions to mice in comparison with aqueous drug solution and W/O emulsion (Fig. 62).

COnC.Ol
499
1 [PQlQ]
Formulotlon B FormulotlonC FormulatlonD
0 Formulotlon A
CON. 01 1 [pg~rni]
8-1 m Formulotlon E
61
Formulotlon F
FormuiotiooG
Q FormulotionH
(b)
Lung
Liver
Heart
Spleen Kidney
Fig. 58. Tissuedistributionofdoxorubicinat 24 h afteradministration of via carotid artery of SD rats. (a) W/O emulsion; (b) WIOiW emulsion.
TlN, h
500
Garti and Aserin

a
50 7
MC
GO
120
180
240
300
360
Time (minutes)
Fig. 61. Change in pupil diameter of rabbits eye postinstallation of 2.5% phenylephrine HCl ophthalmic emulsions containing different concentrations o f (a) methylcellulose and (b) tylose.
XIV.
MICROEMULSIONS
A. DefinitionsandCharacterization
The existence of a macroscopically stable homogeneous fluid, optically transparent, and isotropic has attracted much attention in the last 20 years [46,48,50-581. The original microemulsion was first defined Hoar by and Schulman in 1959 [56] and consisted of water, benzene, hexanol, and potassium oleate. Most of Schulmans work dealt with four-component systems: hydrocarbons, ionic surfactant, cosurfactant, four- to eight-carbon chain aliphatic alcohol, and an aqueousphase. The microemulsion wasformed only when the surfactant-cosurfactant blend formed a mixed filmof the oil-water interface, resulting in interfacial pressure exceedingthe initial positive interfacial
502
Garti and Aserin
tension (so-called negative interfacial tension). The emulsion was, therefore, produced spontaneously. Rosano etal. [367,368] measured the change in the water-oil interfacial tension while alcohol was injected into one of the phases. It was found that the interfacial tension may be temporarily lowered to zero while the alcohol diffused through the interface and redistributed itself between the water and oil phases. It would, therefore, be possible for a dispersion to occur spontaneously (while yi=O).Rosano et al. [367,368] have stressed that the spontaneous formation of these dispersed swollen micelles is not dependent on simple thermodynamic stability but rather, at least in part, on theoccurrence of kinetic conditions favorable to thedispersion of the dispersed phase into the O/W system. Microemulsions are not necessarily four-component systems. It is well documented that ternary water-oil nonionic surfactant can form microemulsions [369-3721.
503
The classic structural model for microemulsion is that of a monodispersed population of dynamic microglobules in a continuous medium. The optical transparency microemulsions indicates that the diameter microglobules cannot be larger than 0.1 pm or so. Since hydrocarbon or water is the continuous phase, the microglobules will be called direct or inverse microglobules, respectively. Such organizations do not necessarily exist in any microemulsion. Inmany compositions, one finds comparable amounts of water andoil, which can bedescribed as local organizations of each of the phasesin a dynamic bicontinuous structure (as suggested by Scriven [373,374] and others[375-3811). of three-component systems can, at fixed pressure The phase behavior and temperature, best be represented using a ternary diagram [51,382-3841. is diffiThese diagramsprovide a simple perspective of phase behavior that cult to capture in any other way. Phase boundaries of systems containing more thanthree components can also be represented in a phase diagram. A ternary diagram depicting a two-phase region and a single phase region is shown in Fig. 63 [385,386]. Any system whose overall composition lies within the two-phase region will exist as two phases whose composition is represented by the endsof the tie lines. In accordance with the phase rule, the surfactant concentration in the phaselabeled M can bevaried independently over a restricted range. Also showninFig. 64 is a plait point, or critical point, which is designated as P. The ties existing in the two-phase
AMPHlPHlLE
WATER
MISCIBILITY GAP
0 1 1
Fig. 63. Ternary diagram representation of two-phase region. The sloping lines are tie lines connecting conjugate phases.
504
AMPHIPHILE
Garti and Aserin
Fig. 64. Ternary diagram showing a three-phase invariant triangular region.
region surrounding this point are quite short, indicating that the two continuous phases have nearly the same composition. If three phases coexist, the system isinvariant. Then thereis a region of the ternary diagram where systems whose overall composition fall within it and divide into three phases, having compositions which are invariant and represented by the corners of the tie triangle (Fig. 65). Any M point will represent three phases with compositions represented by corresponding to thevertices A, B, and C of the triangle. Different points within the triangle will all have compositions of A, B, and C but will differ in the proportions of the quantities of each of the phases. Fig. 66 represents a real commercial surfactant system, hexane and water. The shaded triangle in the tie-triangle with three phases, each of them with a composition represented by corresponding vertices. Thus, the composition within the tie-triangle is composed of a micellar solution in equilibrium with excess oil and water phases. The micellar phase is often called the surfactant phase or the middle phase (since it appears, in the test tube, sandwiched between the upper excess oil and the lower excess water). The tie-triangle is bounded by three two-phase regions. The twophase regions are bounded by a single-phase isotropic micellar solution designated L. The micellar solution represented by the surfactant-rich vertex of the tie-triangle is intermediate between S, and S,.
505
,A
I
C SURFACTANT
Single phase
A WATER
,
B OIL
Three phases
Fig. 65. Three-phase region for ternarysystemhaving three binarymiscibility

gaps.
9.2
Fig. 66. A phase diagramof cyclohexane, nonylphenol ethoxylated NP9, and water at 62C.
506
Garti and Aserin
AMPHIPHILE
......
OIL ..... .....
MICELLAR SOLUTION
PLAIT .AIT POINT WATER OIL
Fig. 67. A typical S1 system, also known as Winsor I type system.
At high amphiphilic concentration, two additional one-phase regions can bedepicted. The phases are also knownas theliquid crystalline phases. The D phase is lamellar and the Y phase, also known as the M, phase, is hexagonal. A typical S, system is shown in Fig. 67. The P point is a region rich with oil, and therefore the two-phase region will have a two-phase one, which is practically just oil, and a micellar solution, in which the water phase will contain practically all the surfactant. Such a system is called Winsor type I. Fig. 68 describes a Winsor type I1 system, in which the water is separated from it and an oil phase, rich with surfactant, is orgaThe transformation from S, compo'nized ona micellar structure [383,384]. sitions to anS, and vice versa can take place progressively by changing the temperature. Transformation from type I to type I1 systems can occur through an intermediate sequence of three-phase systems. These can be designated as type I11 (Fig. 69). The microstructure of a type I system may be visualized as swollen micelles surrounded by water. The hydrocarbon present in such type I system is dissolved in the interior of the micelle, forcing the micelle, owing to packing constraint, to become spherical in shape. S, or type I systems may, therefore, be imagined to be oil droplets surrounded by a sheath of amphiphiles, which separates the oil core from the continuous aqueous phase. The oil droplets are small enough so that the system is transparent

AMPHIPHILE MICELLAR SOLUTION WATER
507
WATER
OIL
Fig. 68. A typical Winsor I1 type system.
and considered to be single phased even though substantial regions of oil do exist. Determination of the structures of the different phases was, and in many cases still is, a difficult task. Methods such as light scattering [3873891, small-angle x-ray diffractions (SAXS), small angle neutron scattering (SANS) [390,394], quasi-electric light scattering (QELS) [395-3991, sedimentation [400,401], and dielectric measurements have been exercised. In spite of the recent great progress in developing the proper models (QELS and SANS) for determining the size and shapeof droplets within S, and S,, D or M areas, many investigators are not yet clear as towhat exactly is the structure of borderline composition in the phase diagrams, mainly the ones that are rich both in water and oil. When substantial oil or water quantities are added, the droplets are no longer spherical and the structure seems to be bicontinuous; that is, both the aqueous region and the oil regions are continuous and the interface separating them has essentially a constant mean curvature [373-3801. The first mesophase to appear from a swollen rodlike micelle is the middle phase, known also as the hexagonal I phase or theM, phase. If the amphiphile is dispersed in an organic phase, the middle phase will be termed consists reverse hexagonal I1 and will be defined as the M, phase. This phase of infinitely long rods in hexagonal array, as depicted in Fig. 6. X-ray diffracidentifition in the low-angle region is one of the most important methods for cation of lyotropic mesophases (Luzatti [402]). The hexagonal system is periodic in two dimensions and consists of cylindrical aggregates of which
508
Garfi and Aserin
CRITICAL END POINT
A
A
A
THREE PHASES TIE TRIANGLE
\ / i fh L C E SOLUTION M l
\
W
THREE-PHASE SYSTEM
TIE
Fig. 69. A sequence of ternary systems which are continuously transformed from type I to type 1 1 1 to type 11. This is denoted as a 1-111-11 transformation. The existence of two critical tie lines is most notable. there are two types. In hexagonal I (also called the middle phase), the lipophilic chains of the amphiphile occupy the linear coreof the cylinders, and the polar groups are arranged on the outer side in contact with the continuous waterphase. In hexagonal I1phase, the inner core of the cylinders consists of water surrounded by a shell of polar groups of the amphiphile and the hydrocarbon chains of the amphiphile make up the continuous outer phase between the cylinders. At polarized light under the microscope, the appearance of the hexagonal I and the lamellar phases is easily identified. Distinction between hexagonal I and I1 is not possible by this method. The dilution with water will reveal the differences. Type I can be diluted, since spherical micelles are formed. Dilution with water is not possible for typeI1 because of the lipophilic nature of the amphiphile.
509
At higher amphiphile concentrations, the Neat phase with lamellar structure may appear [404]. This structure is designated as the G phase, and its structure is depicted in Fig. 6. Between the M, and G phases other mesophases sometimes occur with unidentified structures. At very high amphiphile concentrations and within the M, and G phases, a more viscous phase sometimes forms. This phase is termed theV-form or theviscous-phase. Recently, the designation cubic phase was adopted. The structure of the cubic phase has beenmuch discussed and is still debatable [402,404-4061 (Fig. 6). Liquid-crystalline structures are mesophases between crystals and isotropic solutions. Some investigators treat such systems from this perspective (Table 12). The micellar or surfactant phases are of great importance, since they have practical applications. It is the goal of the food technologists to find the
Table 12. Designation of Some of the Common Lyotropic Phases
Basic
Phase
or spherical, less swollen, S1 (optically isotropic) micelles containing solubilized, organic liquids. 2. Middle phase, normal Indefinitely long, mutually parallel M, (anisotropic) rods in hexagonal array. The rods consist of more or less radically arranged amphiphiles. The hydrophiles are in contact with the surrounding continuous aqueous phase. 3. Neat phase (anisotropic) Coherent double layers of G amphiphilic molecules with the with interfaces the in hydrophiles intervening layersof water. Indefinitely long mutually parallel M, 4. Middle phase, reversed rods in hexagonal array. The (anisotropic) lipophiles are arranged so that the surrounding continuous organic phase is in contact with the lipophile. 5 . Inverted micellar solutions More or less spherical inverted S23 L2 (optically isotropic) micelles containing solubilized water.
1. Micellar solutions More
Source: From Ref. 403.
Aserin 570
and
Garti
proper oils and the right amphiphilesthat will give one-phase regions in the phase diagram, with maximumarea. Large isotropic areas mean in practice a thermodynamically stable surfactant phase thatis rich inboth oil and water. If, for example, one is interested in solubilizing substantial amounts of water (about 30 wt%), it wouldbe important tofind the surfactant or a combina,areas (Fig. 70). Larger areas in tion of surfactants that will exhibit large L the phase diagram mean more solubilized water (Fig. 71). The three-phase tie-triangle is also very important, since it was found both experimentally and by theoretical calculations that any macroemulsion prepared with the composition represented by the corners of the tie-triangle will have better thermodynamic stability than any other emulsion prepared with a composition of any two-phase regions in the diagram. Qpical phase diagrams of soybean oil-water and various amphiphilesare shown in Fig. 72, demonstrating the differences in the areas of the isotropic phases as a function of the type of the amphiphile. For further reading, the book by Bourrel and Schechter [386]on microemulsions andrelated systems is highly recommended. One must also note that the microemulsion regions are in equilibrium with the lamellar liquid crystallineregions. This is one of the reasons that in
HEXADECANE
WATER
AOT-TS/ARlACEL 20
Flg. 70. Pseudoternary phase diagram at 24C for the water/hexadecane/Arlacel 2O/AOT-75 system. The AOT-75: Arlacel20 weight ratio is 40 : 60 (A A), 30 : 70 (0 O),20 : 80 (m . ) , 10 : 90 (-), and 100% (----).

HEXADECANE
51 1
20
Fig. 71. Pseudoternaryphasediagramat25Cforthe waterkexadecanelAOT7YArlacel20 system. The ratio of AOT-75 to Arlacel20 are as follows: (- - -) for 57/ 43 wt ratio, whereas the solid line (-) is for the 54/46 AOT-75/Arlacel 20 ratio. Note the two-phase region within the single-phase region for the 54/46 AOT-75/ Arlacel20 system.
practice we add cosurfactant, with medium-chain length (C, alcohol) to the lamellar liquid crystalline structure in order to destabilize the mesophase and transform it into microemulsion. The C,-OH will upset the lamellar packing and yill facilitate formation of very small spherical panicles with high curvature. The change in curvature (from flat or almost flat to highly curved) leads to a pronounced change in the freeenergy, (e.g., the bending component of the surface free energy) and leads to thermodynamic stability (Fig. 73). XV. MICROEMULSIONS APPLICATIONS
As previously explained, microemulsions are dynamic systems in whichthe
amphiphiles, water and oil, exchange very fast within each other. Drugs incorporated in microemulsions will, therefore, separatebetween the aqueous andoil phases depending on their lipophilicity. The mass transfer constants of several drugs through the surfactant phase (hydrophilic membrane) 4 were studied [407]. It was clearly demonstrated that a linear relationship
512
Soybean 0 1 1
Garti and Aserin
25C
Emulsifier
Fig. 72. Complete phase diagram of soybean oil-water-emulsifier: (--.----.) polyglycerollinoleate, (-W--) polyglycerololeate, (-) monoglyceride stearate, and (--;-) R e e n 80.
Water
A
0
Oil
&?h
Water
Fig. 73. The curvature in an emulsion droplets (A) is extremely small and the bending componentof the surface energy is not significant.A change in curvature (B), on does not lead toa change in the free energy. In the microemulsion droplet the other hand, a change in free energy in curvature to leads a pronounced change in free energy; e.g., the bending component of the surface energy is pronounced.
513
between the release rate of the drug and itsoil-water partition coefficient exists. Several workers have reported studies in whichthe lipophilicity of the drug has been increased to enhance its solubility in the dispersed oil droplets. In this way, a reservoir of the drug is produced anda sustained release effect is achieved as the drug continuously transfers from the oil droplets to the continuous phaseto replace drug release from the microemulsion. An excellent example of such studies is theattemptmade by Gallarate [408] to add timolol into a microemulsion consisting of egg lecithin, butanol, isopropyl myristate, and octanoic acid solution buffered to pH 7.4. The drug was ion paired with octanoic acid, so that its lipophilicity wassignificantly increased. In addition, it wasshown that the in vitro permeability constants of timolol through a lipophilic membrane atpH 7.4 were about seven times higher when the timolol was present as anion pair, suggesting that an improved corneal absorption of this drug might be achieved by topical administration in this microemulsion vehicle. Similarly, the lipophilicity of propranolol has also been enhancedby the formation of lipophilic ion pairs with octanoic acid in O/W microemulsions containing isopropyl myristate, polysorbate 60, butanol, and buffer pH 6.5 [409]. The partitioning of the propranolol into the dispersed oil phase was increased by the addition of octanoic acid. As a consequence, its release rate through a hydrophilic membrane was decreased owing to its decrease in concentration in the continuous phase, producing a prolongation of drug release. Gallarate has also tried [410] to increase the solubility of the azelaic acid (dicarboxylic acid) in the dispersed oil phase of liveen 20 (ethoxylated sorbitan monolaurate)/butanol/decanolin water by lowering the pHof the aqueous phase and by adding propylene glycol to further reduce its dissociation. The partition into thelipophilic phase was improved asthe propylene glycol concentration was increased. The azelaic acid dissolved better and could be incorporated into a cream as well as be released from the microemulsion core in amore controlled or prolonged manner (within hours). A 10-fold increase in the amount of drug released was demonstrated. A similar retention effect could have been achieved if an interaction between the drug and the amphiphile was obtained. The release rates of doxorubicin [411,412] from O/W microemulsions prepared with Sodium diis0 octyl sulfosuccinate (Aerosol-OT or AOT) or polysorbate 80 and W/O microemulsions prepared with lecithin were greatly reduced owing to the formation of lipophilic complexes between this water-soluble drug and each of the surfactants. A similar modification of drug release was later noted when l-demethoxy daunorubicin was formulated in the same W/O lecithin microemulsion [413]. cosurfactant exerts on the In view of the appreciable influence that the
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properties of the microemulsion system, it is not unexpected thatit should also significantlyaffect drug release from the microemulsion. The influence of the cosurfactant concentration on the rate of release of six steroids with a range of lipophilicities from a series of O N microemulsions obtained by adding increasing volumes of butanol to fixed amounts of isopropyl myristate, water, and AOT was reported by Trotta etal. [414]. For all the drugs, the apparent partition coefficient of drug into the dispersed phase increased A with butanol addition, producing a decrease in the release rate of the drug. similar correlation between this partition coefficient and drug release was noted whenthese steroids were incorporated into microemulsionsof fixed composition preparedwith a range of alcohols as cosurfactants. The above conceptions have been exercised with slight modifications by several investigators for a variety of microemulsions indifferent areas of application. Some of the examples are grouped togetheraccording to their mode of delivery.
A. Oral Administration A cyclosporine preparation was administered as a coarse dispersion, formed by mixing a vehicle of olive oil, alcohol, and polyoxyethylated oleic glycerowing ides with water. The preparation proved to be erratic and incomplete to the pair dispersibility of the drug in the microemulsion vehicle [415]. A cyclosporine [416], preparation using a W/O microemulsion containing a sorbitan ester-polyoxyethylene glycol mono ether mixture of surfactant, a low molecular weight alcohol, fatty ester, and water as the vehicle for the drug was administered. A gel-like consistency was obtained through the addition of pyrogenic silica (Cab-o-Sil) and the microemulsions were administered in hard gel capsules. The better bioavailability of the drug [417,418] in comparison with anyother preparation (including an intravenous one) was attributed to the microemulsion droplets (Table 13). Ritschel [419] investigated the gastrointestinal absorption of three peptides using a series of O N microemulsion formulations. The peptides used were dissolved in the aqueous phaseat a suitable pH if water-soluble (insulin, vassopressin) or otherwise added to the microemulsion (cyclosporine) and dispersed by sonication. Three forms of microemulsion were used: a fluid microemulsion for in situ studies on the isolated segment rat model, a microemulsion gel formed by the addition of silicium dioxide (for rectal studies), and an encapsulated microemulsion gel for peroralabsorption studies). Improvements in the bioavailability of these peptides when administered orally were again not solely dependent on droplet size. The author Ritschel [419] concluded that the systemic peptide uptake from micro-

Table 13. Absolute (F) and Relative (EBA) Bioavailability in Rats of Cyclosporin A (AverageS D )After PerOs Solution and Two Peroral Microemulsion Formations p.0. p.0. B C
118
&
515
on p.0. oemulsion Solution Parameters Absolute bioavailability F (%) Relative bioavailability EBA(%) Rate of bioavailability C,, (pg/mL) L a x (h)
41.4 2.8 447.1
15.0 18.1 287.68 147.2
3.3 26.5
100 (standard)
-+
&
1.95 4.35
4.36 0.03 0.59
9.00
f 4.253.47
1.6Y
3.72
f
f
1.35 0.50
8Significantlydifferentfromp.0.solutions. P .05 bMicroemulsion A: W/O microemulsion containing a long chain fatty acid as lipid as surfactants, a low molecular weight alcohol as phase, Arlacel and Brij cosurfactant, and distilled water. CMicroemulsion B: as A except that branched alkyl fatty esters were used as lipid phase. Source: From ref. 388. emulsion in the gastrointestinal tract was dependent additionally on the following formulation factors: type of lipid phase of the microemulsion, digestibility of the lipid used, and types of surfactant in the microemulsion. A detailed hypothesized mechanism of peptide absorption from microemulsions given perorally was proposed.
B. Topical Drug Delivery
The effect of microemulsions on a drug vehicle was tested in various percutaneous absorptions through topical administration. The microemulsion consisted of AOT-octanol-water [420]. It was demonstrated that the tranderual flux increased sixfold as the water content of the microemulsion increased from 15 to 68%. The high water concentration served as transport vehicle to the addenda, leading to higher flux. Octanol and AOT had a synergistic effect as a penetration enhancer. Transport of glucose across human cadaver skin was demonstrated 68% water. A 30-fold enhance[421] using microemulsionscontaining up to ment of the glucose transport was achieved. FBvrier et al. [422] have reported in vitro experiments designed to simulate the percutaneous penetration of tyrosine when administered using an O W microemulsion composed of a betaine derivative (as surfactant),
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benzyl alcohol, hexadecane, and water. The release of radiolabeled tyrosine from this vehicle was compared with that from a liquid-crystal system andan emulsion using a diffusion cellequipped with rat skin. Both the microemulsion and the liquid-crystal formulation enhanced the penetration of tyrosine through the epidermis when compared with the emulsion. However, cutaneousirritation studies showed a strongly irritant effect from the liquid-crystal formulation but none from the microemulsion. In a similar study by Ziegenmeyer and Fuhrer [423], tetracycline hydrochloride was reported to show enhanced percutaneous absorption from a microemulsion compared with conventional systems. Complete diffusion from the microemulsion occurred within 5-6 h comparedwith cornplete diffusion after 12 h from a gel and after more than24 h froma cream. Martini et al. [424] showed in an in vivo study that the percutaneous absorption of D-c~-(5-methyl-3H) tocopherol in rats was more rapid from a emulsion or white petroleumjelly an O/W microemulsion than fromW/O (Vaseline). Hence, the microemulsion not only enhances the rate of penetration butalso appears toinfluence the distribution and elimination of the drug. of the difficulty Gasco et al. [425] have recently addressed the problem in applying these formulations to the skin in the development of a microemulsion for the topical administration of azelaic acid, which has reported therapeutic effects on some pigmentarydisorders and on acnevulgaris. In an earlier study [380] discussed above, these workers demonstrated the formulation conditions required to reduce the dissociation of this acidic drug. Its consequent enhanced partition into the dispersed phase of an O W microemulsion produced a reservoir of dissolved drug and a high rate of release. The viscosity of the O/W microemulsions used in this study was increased to make themsuitable for topical administration by the inclusion of Carbopol 934. A comparison of the in vitro release profiles through hairless skin membrane from this viscosized microemulsion with those from a previously reported gel containing similar components (propylene glycol, Carbopol934) is shown in Fig. 74. Clearly,a pronounced enhancement of penetration is achieved; after 8 h about 35% of the azelaic acid present in the microemulsion had been transported compared with only 1.8% from the gel. The release could be further increased by the addition of a penetration enhancer, dimethyl sulfoxide, to the microemulsion. Trotta et al. [426] havereported a similar enhancement of skin permeation of diazepam from viscosized O/W microemulsion prepared using egg lecithin, polysorbate 20, benzyl alcohol, isopropyl myristate, andwaterpropylene glycol mixtures. The potential application of microemulsions for the ocular administration of timolol was investigated [427] usingO/W lecithin microemulsionsin
51 7
time (h)
Fig. 74. Permeation profiles of azelaic acid from a "viscosized" microemulsion (A) and from a gel ( A ) . (From ref. 425.) which this drug was present as an ion pair with octanoate. The microemulsion, a solution of the ion pair, and a solution of timolol alone were instilled in the conjunctival sac of rabbits and the bioavailability of timolol under the curve fortimolol in from eachwas compared (Fig. 75). The areas aqueous humorafter administration of the microemulsion and the ion pair solution were 3.5 and 4.2 times higher, respectively, than that observed after administration of timolol alone. A prolonged absorption was achieved using the microemulsion with detectable amounts of the drug still present in the aqueous humor 120 min after administration. The enhanced corneal absorption of timolol as an ion pair might suggest the possibility of lowering the doses instilled in topical therapy, leading to a possible reduction of the side effects of this drug.
C. Perfluoro Microemulsions
Fluorinated surfactants have also been used to prepare fluorocarbon microemulsions (i.e., spontaneously formed, thermodynamicallydisperstable sions) in view of solving the long-term shelf-stability issue. F-Alkylated as surfactants amine oxides, including XMO-10 and alcohols, were proposed and cosurfactants for this purpose [428], but no toxicity data were provided. Likewise, spontaneously formed dispersions of fluorcarbons were achieved with mixtures of two F-alkylated polyoxyethylenederivatives [429]. Unfortunately, these preparations turned outto be toxic. Delpuech et al. obtained microemulsions with one single, appropriately chosen monodisperse F-
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Garti and Aserin
A
v
40
80
120
time (mln)
Flg. 75. Aqueous humor concentration-time profiles following multiple installation in rabbits eyes.+ ,timolol alone;0,timolol as an ion pairin solution; andA, timolol as an ion pair in microemulsion. (From ref. 427.) alkylated polyoxyethylenesurfactant, but biocompatibility was not achieved there either [430]. One difficulty in the microemulsion approach lies in the large proportion of surfactant required, which makes the biocompatibility issue all the morearduous.
XVI. SUMMARY
It is a very difficult task to summarize in one chapter, 100 years of extensive work done in the area of dispersed liquids in pharmaceutical and drug designs. Micelles (direct and reverse) have always been attractive vehicles for drug confinement (entrapment) andrelease, but owing to its dynamic nature and the fast exchange betweenthe continuous and the internal reservoirs, it seems to be animpractical method for encapsulation of liquid or dissolved drugs. The liposomes havesignificant better capacities and seem to be better vehicles for similar formulations. Emulsions, in spite of their thermodynamic instability and endless number of restrictions (on size of droplets, nature of the oil, and the surfactants), are excellent liquid preparations for many medical and pharmaceutical needs that require controlled and slow release. Intravenous emulsionssuffer from many limitations, but topical and oral preparation have gained a great deal of interest and areused in many such applications.
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Double emulsions are excellent potential systems for controlled and sustained releaseof drugs, but they stillsuffer from a lack of stability and a lack of the means to control the transport of matter across the two interfaces. It is our belief that futureliquid formulations will be based mainly on such formulations. Intravenous intake seems to be very difficultbecause of size limitations, but topical and oral preparations show much promise, mainly in view of the use of macromolecular amphiphiles such as modified proteins, polysaccharides, glycolipids, and other naturally occurring molecules that form viscoelastic films at the interfaces and control (slow) the release of the drugs. The progress made in the last 15 years in understanding the mechanisms of stabilization (steric) and transport of matter across the oil membrane (reverse micelles) and across the interfaces opennew possibilities in the field of liquid dispersed formulations for drugs and pharmaceuticals. REFERENCES
of emulsions, in Encyclopedia of Emulsion Technology, 1 . P. Walstra, Formation Vol. 1(P. Becher, ed.), Marcel Dekker, New York,1985, p. 57. 2. M. W. Lvnch and W. C. Griffin, Food emulsions, in Emulsions and Emulsion Technoldgy (K. J. Lissant, ed.), Marcel Dekker, New York, 1974, pp. 249290. . L. Lindner, Agricultural emulsions, in Emulsions and Emulsion Technol3. P ogy, Part I (K. J. Lissant, ed.), Marcel Dekker, New York, 1974, pp. 179-236. .Becher, Applications in agriculture, in Encyclopedia of Emulsion Tech4. D. Z nology, Vol. 2 (P. Becher, ed.), Marcel Dekker, New York,1985, p. 239. 5. P. J. Mulqueen, Surfactants for agrochemical formulation, in Industrial Applications of SurfactantsI1 (D. R. Karsa, ed.), Royal Society of Chemistry, 1990, p. 279. 6. K.Larsson and S. E. Friberg (eds.), Food Emulsions, Marcel Dekker, New York, 1990. 7. E. DickinsonandG.Stainsby(eds.),AdvancesinFoodEmulsionsand Foams, Elsevier Applied Science, London, 1988. 8. E.Dickinson andG . Stainsby (eds.), Colloids in Food, Applied Science Publishers, London, 1982. 9. E. Dickinson (ed.), Food Emulsions and Foams, Royal Society of Chemistry London, 1987. 10. N. J. b o g , T. H. Riisom, and K. Larsson, Applications inthe food industry: I, in Encyclopedia of Emulsion Technology, Vol. 2 (P. Becher, ed.), Marcel Dekker, New York, 1985, p. 321. 11, in Encyclopediaof Emul1 1 . E.N. Jaynes, Applications in the food industry: sion Technology, Vol. 2 (P. Becher, e d . ) , Marcel Dekker, New York, 1985,
520
Garti and Aserin
12. H.D.Graham,FoodColloids,AviPublishingCompany,Inc.,Westport, Connecticut,USA 1977. 13. S. S. Davis, J. Hadgraft, and J. K. Palin, Medical and pharmaceutical applications of emulsions, in Encyclopedia of Emulsion Technology, vol. 2 (P. Becher, ed.), Marcel Dekker, New York, 1983, p. 159. 14. B. A. Mulley, Medical emulsions, in Emulsions and Emulsion Technology, Part I (K. J. Lissant, ed.), Marcel Dekker, New York, 1974, p. 291. 15. J. Kreuter, Nanoparticles, in Colloidal Drug Delivery Systems (J. Kreuter, ed.), Marcel Dekker,New York, 1994, p. 219. 16. M. M. Breuer, Cosmetic emulsions, in Encyclopedia of Emulsion Technology, vol. 2 (P. Becher, ed.), Marcel Dekker, New York, 1983, p. 159. 17. M.M.Rieger (ed.), Surfactants in Cosmetics, Vol. 16, Surfactant Science Series, Marcel Dekker, New York, 1984. 18. C. Fox, in Cosmetic Science, Vol. 2 (M. M. Breuer, ed.), Academic Press, London, 1980. of 19. H.A.BampfieldandJ.Cooper,EmulsionsexplosivesinEncyclopedia Emulsion Technology, vol. 3 (P. Becher, ed.), Marcel Dekker, New York, 1988, p. 281. 20. B. W. Davis, Applications in petroleum industry, in Encyclopedia of Emulsion Technology, vol. 3 (P. Becher,ed.), Marcel, Dekker,New York, 1988, p. 307. 21. B. El-Jazairi, Surfactants widely used in the concrete industry, in Industrial Applications of Surfactants (D. R. Karsa, ed.), Royal Society of Chemistry, 1987, p. 33. 22. A. D. James and D. Stewart, Cationic surfactants in road construction and repair, in Industrial Applications of Surfactants I1 (D. R. Karsa, ed.), Royal Society of Chemistry, 1990, p. 338. 23. K. R. F. Cockett, Surfactants in textile processing, in Industrial Applications of Surfactants (D. R. Karsa, ed.), Royal Society of Chemistry, 1987, p. 195. 24. D. Klamann, Lubricants and Related Products, Verlag Chemie, Weinheim, Germany, 1984. 25. Datyner, A. (ed.), Surfactantsin Textile Processing, Vol. 14, Surfactant Science Series, Marcel Dekker, New York, 1983. 26. R. A. Morland and N. Morgan, Surfactants in the paper and board industry, of Surfactants I1(D. R. Karsa, ed.), Royal Society in Industrial Applications of Chemistry, 1990, p. 356. 27. F. J. Kenny, Use of surfactantsin mineral flotation, Surfactants in the paper and board industry, in Industrial Applications of Surfactants I1 (D. R. Karsa, ed.), Royal Societyo f Chemistry, 1990, p. 366. 28. D. Myers, Surfactant Science and Technology, VCH Publishers, New York, 1988, p. 209. NewYork, 29. D. Myers, Surfaces, Interfaces and Colloids, VCH Publishers, 1991. 30. M. J. Rosen, Surfactants and Interfacial Phenomena, Wiley, New York, 1989 p. 304. 31. Th. F. Tadros and B. Vincent, Emulsion stability, in Encyclopedia of Emul-
52 l
32. 33. 34. 35. 36. 37. 38. 39.

40.
41. 42.
44.
45. 46. 47. 48.
43.
49.
50.
51. 52. 53. 54.
sion Technology, Vol. 1 (P. Becher, ed.), Marcel Dekker, New York, 1983, p. 129. P. Becher and M. N. Yudenfreund (eds.), Emulsions, Lattices, and Dispersions, Marcel Dekker, New York, 1978. S. E. Friberg, Emulsion stability, in Emulsions-A Fundamental and Practied.), NATO AS1 Series, Kluwer Academic, cal Approach (J. Sjoblom, Dordrecht, The Netherlands, 1989, p.1. A. T. Florence and D. Whitehill, J. Colloid InterfaceSci., 79:243 (1981). S. Matsumoto and W W . . Kang, J. Dispersion Sci. Technol., 10:455 (1989). S. Matsumoto, Y. Kita, and D. Yonezawa, J. Colloid Interface Sci., 5 7 3 3 (1976). A. T. Florence and D. Whitehill, Int.J. Pharm., 11:277 (1982). Garti, N., Frenkel, M., and Shwartz, R., J. Dispersion Sci. Technol., 4:237 (1983). S. Matsumoto, J. Texture Studies, 17:141 (1986). S. S. Davis, Liquid membranes and multiple emulsions, Chem. Ind., 19583 (1981). S. Matsumoto, WIOIW-type multiple emulsions, in Nonionic Surfactants, V 23 (Schick, M.J., ed.), Marcel Dekker, New York, 1987, p. 549. N. Garti, Double emulsions for food applications, Food Microstructure Dec., 14, (1995) (in press). & Cie, Pans, 1936. G. S. Hartley, Paraffin-Chain Salts, Hermann G. S. Hartley, Rep. Prog. Appl. Chem., 45x33 (1948). J. W. McBain, Colloidal Science, D. C. Heath, Lexington, MA, 1950. of liquid crystalline phases P. Ekwall, Composition, properties and structures in systems of amphiphilic compounds, in Advances in Liquid Crystals, Vol.1 (Brown, G . H., ed.), Academic Press, New York, 1975, p. 1. G. H. Brown and J. J. Wolken, Liquid Crystals and Biological Structures, Academic Press, London, 1979. K. Larsson, Emulsions of reversed micellar phases and aqueous dispersions of cubic phases of lipids: Some food aspects, in Microemulsions and Emulsions in Foods (M. El-Nokaly and D. Cornell, eds.), American Chemistry Society, Washington, D.C., 1991, p. 44. K. Larsson and P. Dejmek, Crystal and liquid crystal structures of lipids, in Food Emulsions (K.Larsson and S. E. Friberg, eds.), Marcel Dekker, New York, 1990, p. 97. R. G. Laughlin, Phase equilibria and mesophases in surfactant systems, in Surfactants (Th.F. Tadros, ed.), Academic Press, London, 1984, p. 53. M. Bourrel and R. S. Schechter, Microemulsions and Related Systems, vol. . 30, Marcel Dekker, New York, 1988, p. 102. I. D. Robb (ed.), Microemulsions, Plenum Press, New York, 1982. S. E. Friberg and B. Lindman (eds.), Organized Solutions, Surfactant Science Series, Vol. 44, Marcel Dekker, New York, 1992. S. E. Friberg and R. L. Venable, Microemulsions, in Encyclopedia of Em Technology, Vol. I. (P. Becher, ed.), Marcel Dekker, New York, 1983, p. 287.
522
55. R.Zana(ed.),SurfactantSolutions:
Garti and Aserin
NewMethodsofInvestigation,Surfactant Science Series, Vol. 22, Marcel Dekker, New York, 1987. 56.T.P. Hoar, and J. H. Schulman, Nature, 152:102 (1943). 56a. F. B. Rosevear, Liquid Crystals: The mesomorphic phases of surfactant cornpositions. J. Soc. Cosmet. Chem. 19581 (1968). 57. J. T. Davies and E. K. Rideal, Interfacial Phenomena, Academic Press, New York, 1961, p. 16. 58. Th. F. TadrosandB.Vincent,Liquidniquidinterfaces,inEncyclopediaof EmulsionTechnology,Vol. 1 (P.Becher, ed.), Marcel Dekker, New York, 1983, p. 1. 59.N.K.Adam,ThePhysicsandChemistryofSurfaces,3rd ed., University Press, Oxford, UK, 1941, p.5 . 60. J. S. Rowlinson, Chem. Soc. Rev., 7:329 (1978). 61. B. Widom and J. S. Rowlinson, J. Chem. Phys., 52:1670 (1970). 62. J. E. Kirkwood and F. P. Buff, J. Chem. Phys., 17:338 (1949). 63. F. C. Goodrich, in Surface and Colloid Science, Vol. 3, (E. Matijevic, ed.), Wiley-Interscience, New York, p.1. 64. S. Middleman, The Flow of High Polymers, Interscience, New York, 1968. 65. R. Zana and L. G. Leal, in Proceedings of the International Colloquium on Drops and Bubbles (D. J. Collins, S. M. Plesset, andM. M. Saffren, eds.), Jet Propulsion Laboratory, Passadena, CA, 1974, p. 428. 66. R.W. Flumerfelt, Ind. Eng. Chem. Fund., 11:312 (1972). 67. R. Shinnar and J. M. Church, Ind. Eng. Chem., 52:253 (1960). 68. Y. Mlynek and W. Resnick, Am. Inst. Chem. Eng. J., 18:122 (1972). 69.J.W.Van Heuven, Ph.D. Thesis, Technical University of Delft, 1969. S. Noro, S. Ando, and M. Koishi, Chem. Pharm. Bull., 70. A. Takamura, 252617 (1977). 71. E. Lange, Z. Elektrochem., 5576 (1951). 72. R. Parsons, Modem Aspects of Electrochemistry (J. 0. M. Bockris and B. E. Conway, eds.), Butterworths, London, 1954. 73. P. Becher, Emulsions: Theory and Practice, Krieger, New York, 1977, p. 267. 74. J. Lucassen-Reynders and Lucassen, Adv. Colloid Interface Sci., 2:347 (1969). 75. E. S. R.Gopal,inEmulsionScience(P.Sherman,ed.),AcademicPress, London and New York, 1968, pp. 1-75. ed.),Pergamon, 76. E. S. Rajagopal, inRheologyofEmulsions(P.Sherman, London, 1963, pp. 15-25. 77. B. Bergenstahl, Topics in Food Emulsions, Ph.D. Thesis, University of Lund, Sweden, 1994. 78. H. L. Greenwald, J. Soc. Cosmetic Chem., 6:164 (1955). 79.M.Smoluchovski,Phys. Z . , 17593 (1916). 80. L. A. Spielman, J. Colloid Interface Sci., 33562 (1970). 81. M. Smoluchowski, Z. Phys. Chem., 92:129 (1917). 82.P. F. Saffman and J. S. 'Tbmer, J. Fluid Mech., 1:16 (1956). 83. D. H. Melik and H. S. Fogler, J. Colloid Interface Sci., 101:72 (1984).

84.
523
85.
86. 87.
88.
89. 90. 91.
92.
93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110.
111.
112. 113. 114. 115. 116. 117. 118.
D. H. Melik and H. S. Fogler, in Encyclopedia of Emulsion Technology, Vol. 3 (P. Becher, ed.), Marcel Dekker, New York, 1988, p. 3. M. Van den Temple, Rec. Trav. Chim., 72:433 (1953). Th. F. Tadros, Colloids and Surfaces 1:3 (1980). W. C. Griffin, J. Soc. Cosmetic Chemist 1:311(1949). W.C. Griffin, J. Soc. Cosmetic Chemist 5249 (1954). C. D. Moore andM. Bell, Soap, Perfum. Cosmet. 29:893 (1956). H. L. Greenwald, G. L. Brown, and M. N. Fineman, Anal. Chem. 28:1693 (1956). J. T. Davies andE. K. Rideal, Interfacial Phenomena, Academic Press, New York, 1963, pp. 371-383. J. T. Davies,Proc. Int. Congr.SurfaceActivity, 2nd ed., Vol. 1, Butterworths, London, 1959, p. 426. I. R a n and E. Orban,J. Colloid Sci., 20:99,1965. P. Becher andR. L. Birkmeier, J. Am. Oil Chem. Soc. 41:169 (1964). P. Becher, Am. Perfumer., 76:33 (1961). K. Shinoda and H. Kunieda, Phase properties of emulsions: PIT and HLB, in Encyclopedia of EmulsionTechnology,Vol. 1 (P.Becher, ed.), Marcel Dekker, New York, 1983, p. 337. S. Friberg, Emulsions and Solubilization, Wiley-InterK. Shinoda and science, New York, 1986, p.55. S. Ross, J. Soc. Cosmetic Chemists 6:184 (1955). S. U.Pickering, J. Chem. Soc. (Lond.) 91:2002 (1907). K. Shinoda and H. Saito, J. Colloid InterfaceSci., 30:2,258 (1969). K. Shinoda, J. Colloid Interface Sci., 25:396 (1969). K. Shinoda, Proceedings of the International Congress Surface Activity, 5th ed., Vol. 2, Butterworths, London, 1968, p. 275. K. Shinoda, H.Saito, and H. Arai, J. Colloid Interface Sci., 35:624 (1971). K. Shinoda and H. Takeda,J. Colloid Interface Sci., 32642 (1970). K. Shinoda, J. Colloid Interface Sci., 24:4 (1967). J. Colloid Interface Sci., 64:68 (1978). K. Shinoda and H. Sagitani, K. Shinoda, H. Kunieda, N. Obi, andS. E. Friberg,J. Colloid InterfaceSci., 80:304 (1981). J. Colloid Interface Sci., 75601 (1980). H. Kunieda and K. Shinoda, K. Shinoda andT. Ogawa, J. Colloid Interface Sci., 2456 (1967). K. Shinoda and H. Sagitani, J. Phys. Chem., 87:2018 (1983). D. H. Napper, J. Colloid Interface Sci., 58:390 (1977). K. P. Oza and S. G. Frank, J. Dispersion Sci. Technol., 7543 (1986). K. P. Oza and S. G. Frank,J. Dispersion Sci. Technol., 10:187 (1989). Y. Sela, S. Magdassi andN. Garti, Colloids and Surfaces, A., 83:143 (1994). J. A. Davidson and E. A. Collins, J. Colloid Interface Sei., 47:459 (1974). C. Holt, A. M. Kimber, B. Brooker and J. H. Prentice, J. Colloid Interface Sci., 65:555 (1978). P. Walstra and H.Oortwijn, J. Colloid InterfaceSci., 29:424 (1969). W. P. Hendrix and C. O r r ,in Particle Sue Analysis (M. J. Groves and J. L.
524
Garti and Aserin
Wyatt-Sargent, eds.), Society for Analytical Chemistry, London, 1972, pp.

133-144. 119. 120. 121. 122.
P. Sherman, J. Colloid Interface Sci., 24:97 (1967). P. Sherman, J. Phys. Chem., 67:2531(1963). P. Sherman, J. Colloid Interface Sci., 27:282 (1968). K. Yokoyama, 19th Conference on Industrial Pharmaceutical Research, Mitsugane-San, Aichi, Japan,1982. 123. J. J. Windheuser, M. L. Best, and J. H. Penin, Bull. Parenter. Drug Assoc.,
24:286 (1970). 124. A. H. Goldberg, W. I. Higuchi, N. F. H. Ho, and G. Zografi, J. Pharm. Sci., 56:1432 (1967). 125. K. Yakayama,K.Yamanouchi,M.Watanabe, et al.,Fed.Proc., 34:1478 (1975). 126. A. Fricker, W . Griem, and K. Lang, Klin. Wschr., 47:735 (1967). 127. R. P. Geyer,Uptakeandretention of fattyacidsbytissueculturecells, Wistar. Inst. Symp. Monogr. 6:33 (1967). 128. C. J. van Oss, C. F. Gillman, and A. W . Neumann, Phagocytic Engulfment 129. 130. 131. 132. 133. 134.
and Cell Adhesiveness as Cellular Surface Phenomena, Marcel Dekker, New York, 1975. R. Jeppsson and S. Rossner, Acta Pharmacol. Toxicol., 37:134 (1975). H. Joyeux and B. Astruc, Traite de nutrition artificialle del'adulte, I, SSTNA, Montpellier, France, 1980. 0 . Schuberth and A. Wretlind, Acta Chir. Scand. Suppl., 278 (1961). A. R. Vela, 0. L. Hartwig, M. Atik, et al., Am. J. Clin. Nutr., 16:80 (1965). C. D. Black and N. G. Popovich, Drug Intell. Clin. Pharm., E 1 8 4 (1981). W. R.Burnham, P. K. Hansrani,C.Knott,J.etal.,Int. J. Pharm., 13:9
(1983). 135. G. Hardy, R. Cotter, and R. Dawe, in Advances in Clinical Nutrition (I. D. A. Johnson, ed.), MTP, Lancaster, UK,1983, p. 241. 136. C. Kawilarang, K. Georghiou, and M. J. Groves, J. Clin. Hosp. Pharm., 551 (1980). 137. R. I. Lawrence, W . K. Flukes, V. J. Rust, and P. A. Braithwaite, Aust. J. Hosp. Pharm., 11540 (1981). 138. C. Solassol and H. Joyeux, Ann. Anaesthsiol. Fr. Spec., 2:75 (1974). 139. C. Solassol, H. Joyeux, R. Serrou, et al., J. Chir., 105:15 (1973). 140. I. D. A. Johnson(ed.), Advances in Clinical Nutrition, MTP, Lancaster, UK, 1983. 141. H. C. .Meng and D. W. Wilmore (eds.), Fat Emulsions in Parenteral Nutrition, American Medical Association, Chicago, 1976. 142. S. S. Davis, in Advances in Clinical Nutrition (I. D. A. Johnson,ed.), M T P , Lancaster, UK, 1983, p. 213. 143. A. Wretlind, in Fat Emulsions in Parenteral Nutrition (H. C. Meng and D. W . Wilmore, eds.), American Medical Association, Chicago, 1976, p. 109. 144. J.Molnar, S. McLain,C.Allen, et al.,Biochim.Biophys.Acta, 493:37 (1977).
525
145. C. S. J. Riggi, Exp. Mol. Pharm., T. Ashworth, N. R. DiLuzio, and l(Suppl.):83 (1963). 146. H. Tonaki, T. M. Sata, L. W. Mayron, and E. Kaplan, Exp. Mol. Pharm., l(Suppl.):83 (1963). 147. A. L. Kunz, R. E. Lewis, and G. J. Sperandio, Bull. Parenter. Drug ASSOC., 19:13 (1965). 148. S. Muchtar, M. Y. Levy, S. Sarig, and S. Benita, S.T.P. Pharm. Sci., 1:130 (1991). 149. C. Washington, Int. J. Pharm., 66:l (1990). 150. C.Washington,A.Athersuch,andD.J.Kynoch,Int.J.Pharm.,64:217 (1990). EP 220,152,1987. 151. A. Wretlind and B. Ajaxon, Eur. Patent Application 152. J. D. Sherwood, Anal. Proc., 24:277 (1987). 153. J. D. Sherwood, Anal. Proc., 24:279 (1987). 154. C. Washington andS. S. Davis, Int. J. Pharm., 44:169 (1988). 155. S. Muehlebach, R. B. Graf,andK.Sommermeyer,Pharm.ActaHelv., 62:130 (1987). 156. J.P.Reynier, J. Danho, P. Faurenfies, and A. Bovis, Pharm. Acta Helv., 62:93 (1987). 157. V.A. Parry, K. R. Harrie, and N. L. McIntosh-Lowe, Am. J. Hosp. Pharm., 43:3017 (1986). 12:116 (1988). 158. U. Julius andW. Leonhardt, J. Parenter. Enteral Nutr., 159. Y. B. Bannon, J. Corish, 0. I. Corrigan, et al., Eur. Patent Application EP 289,342 (1988). 160. R. Jeppsson, Acta Pharm. Suec., 9:81 (1972). 161. R. Jeppsson and S. Ljunberg, Acta Pharm. Suec., 10:129 (1973). 162. R. Jeppsson, Acta Pharm. Suec. 13(Suppl.):43 (1976). 163. R. Jeppsson and S. Ljunberg, Acta Pharm. Toxicol., 36:312 (1975). 164. R. Jeppsson, Acta Pharm. Toxicol., 36:299 (1975). 165. G. I. Schoefl and J. E. French, Proc. R. Soc., B169:153 (1968). 166. G. J. Jesmok, G. M. Walsh, W. Ditzler, and E. F. Woods, Proc. Soc. Exp. Biol., 162:458 (1979). 167. R. Jeppsson, J. Clin. Pharm., 1:181 (1976). 168. 0. von Dardel, C. Mebius, and T. Mossberg, Opusc. Med., 20:255 (1975). M. Ajaxon, U.S. Patent 169. K. A. Wretlind, S. Ljunberg, I. Hakansson, and B. 4,073,943 (1978). 170. R. Jeppsson, Acta Pharm. Toxicol.,3756 (1975). 171. F. J. Laska and M. R. Fennessy, Clin. Exp. Pharm. Ther.,3587 (1976). 172. H. 0. J. Collier, D. L. Francis, and C . Schneider, Nature 237:220 (1972). 173. C. L. Fortner, W. R. Grove, D. Bowie, and M. D. Walker, Am. J. Hosp. Pharm., 32582 (1975). 174. C. L.Litterst, E. G.Mimnaugh,A.C.Cowles,J.Pharm.Sci.,63:1718 (1974). 175. A.J. Repta, in Topics in Pharmaceutical Sciences (D. D. Breimer and P. Speiser, eds.), Elsevier, Amsterdam, 1981, p. 181.
526
Garti and Aserin
176. A. A. A. El-Sayed and A. J. Repta, Int. J. Pharm., 13:303 (1983). 177. B. Meyerson, Horm. Behav., 3 : l ( 1 9 7 2 ) . . Pharm. Pharmacol., 34:49 178. Y. Mizushima, T. Hamano, and K. Yokoyama,J (1982). 179. D. M. Weir, Immunology, 4th ed., Churchill, London, 1977. 180. P. Jolles and A. Paraf, Chemical and Biological Bases of Adjuvants, Chapman & Hall, London, 1973. on Adjuvantsof 181. P. MuggletonandM.Hilton,InternationalSymposium on Immunobiology, Standard 6,Utrecht, 1966, Immunity, Symposium Series Karger, Basel, 1966,p. 29. 182. W. J. Herbert, Immunology, 14:301 (1968). 51:238 (1973). 183. P. Steinberg and P. S. Norman, J. Allergy Clin. Immunol., 184. W. H. 0. Scientific Group, Who Tech. Rep. Ser. 595,World Health Organization, Geneva, 1976. 185. J. Freund and K. McDermott, Proc. Soc. Exp. Biol. Med., 49548 (1942). 186. W.J. Herbert, in Handbook of Experimental Immunology, Vol. 3 (D. M. Weir, ed.), Blackwell Scientific, London,1978,p. A31. 187. M. R. Hilleman, Prog. Med. Virol., 8:131 (1966). Modeh Trends in Immunology, Vol.2 (R. Cruickshank and 188. E. G. White, in D. M. Weir, eds.), Butterworth, London,1967,p. 28. 189. J. Lazarus and L. Lachmann, Bull. Parenter. Drug Assoc.,21:184 (1967). 190. J. A. Reynolds, Infect. Immun., 28(B):937 (1980). 191. C.A.McLaughlin, E. E. Ribi,M.B. Goren,andR.Tbubiana,Cancer Immunol. Immunother., 4:109 (1978). 192. K. Kakemi, H. Sezaki, K. Okumura, and S. Ashida, Chem. Pharm. Bull., 17:1332 (1969). Hashida, Y. Takahashi, S. Muranishi, and H. H. Sezaki, J. Phar193. M. macokinet. Bipharm., 5:225 (1977). 194. M.Hashida, S. Muranishi,and H. Sezaki,Chem.Pharm.Bull., 25:2410 (1977). 195. Y.Nakamoto, M. Fujiwara, T. Noguchi, et al., Chem. Pharm. Bull., 23:2232 (1975). 196. Y. Nakamoto,M.Hashida, S. Muranishi,andH.Sezaki,Chem.Pharm. Bull., 23:3125 (1975). 197. M. Hashida, Y. Takahashi, S. Muranishi, and H. H. Sezaki, J. Pharmacokinet. Bipharm., 5241 (1977). 198. M. Hashida, S. Muranishi, and H. Sezaki, et al., IntJ. Pharm., 2:245 (1979). 199. T. Takahashi, S. Ueda, K. Kono, and S. Majimi, Cancer, 38:1507 (1976). 200. J. N. Hunt and M.T. Knox, J. Physiol. (Lond.), 194:327 (1968). 54:1277 (1976). 201. A. Cooperman and S. A. Cook, Surg. Clin. North Am., 202. A. F . Hofman and D. M. Small, Annu. Rev. Med., 18:333 (1967). 203. C. R. Treadwell and G. V.Vahouny, in Handbook of Physiology, Sect. 6, Vol. 3 (C. F. Code, ed.), American Physiological Society, Washington, D.C., 1968,p. 1407-1438. 204. S. Muranishi, N. Muranishi, and H. Sezaki, Int. J. Pharm., 2:107 (1979).
527
205. N.Muranishi,M.Kinugawa, Y. Nakajima, et al.,Int. J.Pharm.,4:271 (1980). 206. S. M. Sieber, V. H. Cohn, andW. T. Wyn, Xenobiotica, 4:265 (1974). 207. J. E. F. Reynolds (ed.), Martindale, The Extra Pharmacopoeia, 28th ed., Pharmaceutical Press, London, 1982. 208. J. F. Hoover (ed.), Remingtons Pharmaceutical Sciences, 15th ed., Mack, Easton, PA, 1975. 209. T. Furia (ed.), Handbook of Food Additives, Chemical Rubber Company, Cleveland, 1968, p. 626. F. Delorenzo, R. Engelberg, et al.,Antibiot.Med., 210. S. E. Svenson, W. 11:148 (1956). 211. C. W. Daeschner, W. R. Bell, P. C. Stivrins,et al., Am. Med. Assoc. J. Dis. Child., 93:370 (1957). 212. P.J. Carrigan, Ph.D. Thesis, University of Connecticut, Storrs, 1974. 213. P. J. Carrigan and T. R. Bates,J. Pharm. Sci., 62:1476 (1973). Sci., 64:793 (1975). 214. T. R. Bates and J. Sequira, J. Pharm. 215. G. Gagnon and A. M. Dawson, Proc. Soc. Exp. Biol. Med., 127:99 (1968). 216. L. Diamond, Arch. Int. Pharmacodyn., 185246 (1970). 217. H. Ogata and H. Leung-Fung, Int. J. Rharm., 5:335 (1980). J. Physiol., 239:G210 (1980). 218. D. Hollander, Am. 219. J. M. Lewis, S. Q. Colhar, and A. Messina, Pediatrics,5425 (1950). 220. W. J. Warwick, L. G. Hansen, and H. Sharp, Clin. Pediatr., E 8 0 7 (1976). 221. T. De Marco and R. Levine, J. Pharmacol. Exp. Ther., 169:142 (1969). Soc. Exp.Biol.Med.,129:772 222. R. H. Engel and M. J. Fahrenbach, Proc. (1968). 223. R. Hori, K. Okumura, K. Inui,et al., Chem. Pharm. Bull., =:l974 (1977). . L. Hayton, J. Pharm. Sci., 65:328 (1976). 224. D. C. Bloedow and W 225. F . H. Mattson, R. J. Jandacek, and M. R. Webb, J. Nutr., 102:1177 (1976). 226. R. A. Volpenhein, D. R. Webb, and R. J. Jandacek, J. Toxicol. Environ. Health, 6:679 (1980). 227. K. Kakemi, H. Sezaki, S. Muranishi et al., Pharm. Bull., 20:708 (1972). 228. K.Kakemi,H.Sezaki, S. Muranishi et al.,Chem.Pharm.Bull.,20:715 (1972). 229. K. Kakemi,H.Sezaki, H. Ogata,andC.Nagai,Chem.Pharm.Bull., 20:1053 (1972). 230. T. Noguchi, Y. Tokunaga, M. Ichikawa, et al., Chem. Pharm. Bull., 25:223 (1977). Sci., 63566 231. G. M.Lin, F. Sadik, W. F. Gilmore, and J. H. Fincher, J. Pharm. (1974). Schaefer, A. Zesch, and G. Stuttgen, in Handbuch der Haut und 232. H. Geschlechtskrankheiten Erganzungswerk, Vol. 1/4B, Springer-Verlag, Berlin, 1981. 233. M. Katz and B. J. Poulsen, in Handbook of Experimental Pharmacology, Vol. 28/1 (B. B. Brodie and J. Gillette, eds.), Springer-Verlag, Berlin, 1971, pp. 103-174.
528
Garti and Aserin
234. N. J.VanAbbe, R. I. C. Spearman, and A. Jarrett, Pharmaceutical and Cosmetic Products for Topycal Administration, Heinemann, London, 1969. 235. M. Katz and B. J. Poulsen, J. Soc. Cosmetic Chemists, 23565 (1972). 236. M. Parr, J. Pharm. Sci., 51:395 (1962). for Topical Admin237. N. J. VanAbbe, in Pharmaceutical and Cosmetic Products istration, Heinemann, London, 1969, p. 98. 238. W. G. Waggoner and J. H. Fincher, J. Pharm. Sci., 60:1830 (1971). 239. F. R. Bettley, Br. J. Dermatol., 73:448 (1961). 240. I. H. Blank, E. Gould,andA.Theobald,J.Invest.Dermatol.,37:485 (1961). 241. J. Scala, D. E. McOsker,andH. H. Reller,J.InvestDermatol.,50:371 (1968). 242. L. H. McDonald and K. Himelick, J. Pharm. Assoc. Sci. Ed., 37:368 (1948). 243. P. H. Dugand and R. J. Scheuplein, J. Invest. Dermatol., 60:263 (1973). 244. B.W. Barry and G. M. Saunders, J. Colloid Interface Sci., 41:331 (1972). 245. B. W. Barry and G. M. Eccleston, J. Pharm. Pharmacol., 25244 (1973). 246. B. W. Barry, Adv. Colloid Interface Sci., 5:37 (1975). 247. G . M. Eccleston, J. Colloid Interface Sci., 57:66 (1976). 248. I. Sarkany and J. W. Hadgraft, Br. J. Dermatol., 81:98 (1969). . Barry and R. Woodford, Br. J. Dermatol., 91:323 (1974). 249. B. W 250. Z.T. Chowhan and R. Pritchard, J. Pharm. Sci., 64:754 (1975). 251. N.F. Billups and N. K. Patel, Am. J. Pharm. Ed., 34:190 (1970). 252. T. Koizumi and W. I. Higuchi, J. Pharm. Sci., 57237 (1968). 253. D. V. Barker, H. G. DeKay, and J. E. Christian, J. Am. Pharm. ASSOC., 45527 (1956). 254. L. D. Lockie andT. B. Sprowis, J. Am. Pharm. ASSOC., 38:222 (1949). 255. D. V. Barker, J. E. Christian, and H. G. DeKay, J. Am. Pharm. ASSOC., 45501 (1956). 256. J. A. Wood and L. W. Rising, J. Am. Pharm. ASSOC., 42:481(1952). 257. A. H. Fenton andM. Warren, Phann. J., 1885 (1962). 258. W . S. Singleton and M. L. Brown, J. Am. Oil Chem. Soc., 42:312 (1965). 259. J. Greiner, J. G. Riess, and P. Vierling, in Organofluorine Compounds in Medicinal Chemistry and Biomedical Applications(R. Filler, ed.), Elsevier, The Netherlands, 1993, p. 339. 260. J. G. Riessand M. Postel,Proceedingsof theInternationalSymposium BloodSubstitute,Montreal,1991,Biomat. Art.Cells,Immob.Biotech. 20:819 (1992). 261. R. P. Geyer, in Drug Design, Vol. 7 (A. J. Ariens, ed.), Academic Press, London, 1976, p. 1. 262. S. S. Davis, H.P. Round, T. S. Buscall, et al., in Recent Advances in Colloid and Interface Science, Vol. 2 M. Kerker, ed.), Academic Press,New York, 1976, p. 265. 263. S. S. Davis, H. P. Round, andT. S. Purewal,J. Colloid Interface Sci., 82:307 (1981). 264. T . M. S. Changand R. P. Geyer, eds., in Blood Substitutes, p, 411,M. Dekker, Inc., New York (1989).
529
265. J. G. Reiss, J. L. Dalfors, G. K. Hanna, et al., Biomat., Art. Cells, Immob. Biotech., 20:839 (1992). . Greiner, S. Abouhilale, and A. Milius, Prog. Colloid Polym. 266. J. G. Reiss, J Sci., 88:123 (1993). . Greiner, andJ .G. Reiss, New J, Chem., 16:771 (1992). 267. A. Milius, J 268. A. Milius, J. Greiner, andJ. G. Reiss, Colloids Surfaces, 63:281 (1992). . Greiner, A. Milius, and J .G. Reiss, Proceedings of the 3rd International 269. J Surfactants Congress, London, Sect. C, 1992, p. 31. . Greiner, and J .G . Reiss,J. Am. Oil Chem. Soc., 68:1(1991). 270. S. Abouhilale,J J. G. Reiss, Eur. J. Med. Chem., 26:953 (1991). 271. J. B. Nivet, M. LeBlanc, and .G. Riess, New J. Chem., 16:399 (1992). 272. Santaella, P. Vierling, and J E. A.O'Rear, J. ColloidInterface Sci., 273. M. Gangoda,B.M.Fung,and 116:230 (1987). J. G.Riess,andL.Zarif,Makromol.Chem., 274. A.A.Pavia,B.Pucci, 193:2505 (1992). J. FluorineChem., 58:194 275. J. Greiner,E.Myrtil,E.Dere,etal.,Pavia, (1992). 276. S. Gaentzler andP. Vierling, New J. Chem., 17:337 (1993). 277. L. Zarif, A. Manfredi, C. Varescon, et al., J. Am. Oil Chem. Soc., 66:1515 (1989). J .. Rolland, P. Vierling, 'andJ. G. Riess, New J. Chem., 278. P.M.P. Krafft, P 14:869 (1990). 279. A. M. Mahe, J. Manoux,A.Valla, et al.,Biomat.,Art.Cells,Immob. Biotech., 20:1025 (1992). 280. L. Zarif, J. Greiner, S. Pace, andJ. G. Riess, J. Med. Chem., 33:1262 (1990). . Sumaya, andK. Yokoyama, Eur. Surg. Res., 3:436 (1971). 281. T. Fujita, T .Parenter. Sci. Technol., 36:242 (1982). 282. L. Illum and S. S. Davis, J . Appl. Physiol., 283. H. A. Sloviter, M. Petkovic, S. Ogoshi, and H. Yamada, J 27566 (1969). 284. K.Yokoyama, K. Yamanouchi, M. Watanabe, et al., Fed. Proc., Fed. Am. Soc. Exp. Biol., 34:1478 (1975). 285. H. Ohyanagi, K. Toshina, M. Sekita, et al., Clin. Ther., 2:306 (1979). 286. K. Honda, S. Hoshino, M. Shafi, et al., N. Engl. J. Med., 303:391 (1980). 287. K. K. Tremper, R. Lapin, E. Levine, et al., Crit. Care Med., 8:738 (1980). 288. T. Fujita, T. Sumaya, andK. Yokoyama, Eur. Surg. Res., 3:436 (1971). 289. M. Bury and C. Boymond,S. T. P. Pharm., 6474 (1990). 290. S. S. Davis, C. Washington, P. West, et al., Lipid Emulsions as Drug Delivery Systems, Ann. NY Acad. Sci., 507:75-88 (1987). 291. S. S. Davis, in Phospholipids (I. Hanin and G. Pepeu, eds.), Plenum Press, New York, pp. 69-82 (1990). 292. J. B. Boyett and C. W. Davis, in Pharmaceutical dosage forms: Disperse Systems, Vol. 2 (H. A. Lieberman, M. M. Rieger, and G . S. Banker, eds.), Marcel Dekker, New York, 1989, pp. 379-416. 293. B. Idson, in Pharmaceutical dosage forms: Disperse Systems,Vol. 1 (H. A. Lieberman, M. M. Rieger, and G. S. Banker, eds.), Marcel Dekker, New York, 1989, pp. 199-243.
,
530
Garti and Aserin
294. S. S. Davis, Optimization of Drug Delivery (H. Bundgard, A. Bagger, Hansen and H. Kofod, eds.), Alfred Benzon Symposium 17, Copenhagen, 1982, pp. 333-347. 295.M.Y. Levy and S. Benita, Int. J. Pharm., 54:103 (1989). 296. S. Zheng, Y. Zheng, R. L. Beissinger, D. T. Wasan and D. L. McCormick, Biochim. Biophys. Acta, 115865 (1993). 297.L. C.Collins-Gold,R. T. Lyons,andL.C.Bartholow,Adv.DrugDeliv. Rev., 5:189 (1990). 298. 0 . von Dardel, C. Mebius, and T. Mossberg, Acta Anaesth. Scand., 20:221 (1976). 299. S. Benita, D. Friedman,andM.Weinstock, Int. J. Pharm.,30:47(1986). 300. R. J. Prankerd and V. J. Stella, J. Parent. Sci. Technol., 44:139 (1990). 301. M. Singh and L. J. Ravin, J. Parent. Sci. Technol., 4 0 9 (1986). 302.P.Becher,EmulsionTheoryandPractice, 2nd ed., Reinhold, NewYork, (1965), p. 2. 303. S. S. Davis, J. Hadgraft, and K. J. Palin, in Encyclopediaof Emulsion Technology, Vol. 2, (P. Becher, ed.), Marcel Dekker, New York, 1985, p. 159. 304. A.T. Florence and D. Whitehill, Int.J. Pharm., 11,277 (1982). 305. P. Docic and P. Sherman, Coll. Polym. Sci., 258:1159 (1980). 306. W . Seifriz, J. Phys. Chem. ,29, 738 (1980). 307. T. J. Lin, H. Kurihara, and H. Ohta, J. Soc. Cosmet. Chem., 26121 (1975). 308. S. Matsumoto, WlOiW-type multiple emulsions, in Nonionic SurfactantsPhysical Chemistry, Surfactants Science Series, Vol. 23, (M. J. Schick, ed.), Marcel Dekker, New York, 1985, p. 549. 309. A. T. Florence and D. Whitehill, J. Colloid InterfaceSci., 79:243 (1981). 310. S. Matsumoto and W W . . Kang, J. Dispersion Sci. Technol., 10455 (1989). 311. S. Matsumoto, Y. Kida, and D. Yonezawa, J. Colloid Interface Sci., 57:353 (1976). 312. A. T. Florence and D. Whitehill, J. Colloid Interface Sci., 79:243 (1981). 313. M. Frenkel,R.Shwartz,and N. Garti, J. ColloidInterface Sci., 94:174 (1983). 314. N. Garti, M.Frenkel, and R. Schwartz, J. Dispersion Sci. Technol.,4:237 (1983), 315. A. F. Brodin and S. G. Frank, Acta Pharm. Suec., 15111 (1978). 316.C.Chiang,G.C.Fuller, J. W. Frankenfeld, and C. T. Rhodes, J. Pharm. Sci., 67:63 (1978). 317. S. Magdassi,M. Frenkel,and N. Garti, J. DispersionSci.Technol.,5:49 (1984). 318. S. Magdassi,M. Frenkel, and N. Garti, Drug Ind. Pharm., 11:791 (1985). 319. S. Magdassi and N. Garti, J. Controll. Rel., 3:273 (1986). 320. Y. Kita, S. Matsumoto, and D. Yonezawa, J. Colloid Interface S c i . , 62237 (1977). 321. T. Higuchi, J. Pharm. Sci., 52:1145(1963). 322. T. K. Law, A. T. Florence, and T. L. Whateley,J. Pharm. Pharmacol., 3650 (1984).
53 1
323. T. K. Law, T. L. Whateley, andA. T. Florence, Int. J. Pharm., 21:277 (1984). 324. T. K. Law, T. L.Whateley,and A. T. Florence, J. Controll.Rel., 3:279 (1986). 325. A. T. Florence, T. K. Law,andT.L.Whateley,J.Colloid Interface Sci., 107584 (1985). 326. J. A. Omotosho, T. K. Law, T. L. Whateley, and A. T. Florence, Colloid and Surfaces, 20:133 (1986). 327. J. A. Omotosho, T. K. Law, T. L. Whateley, and A. T. Florence, J. Pharm. Pharmacol., 38:865 (1986). 328. J.A.Omotosho,T. K. Law, T. L. Whateley,andA. T. Florence, J. Microencaps., 6:183 (1989). 329. R. L. Beissinger, D. T. Wasan, L. R. Sehgal, and A. L. Rosen, UK Patent Application GB2,221,912 (1990). 330. S. Zheng, R. L. Beissinger, and D. T. Wasan, J. Colloid Interface Sci., 144:72 (1991). 331. K. P. Oza and S. G. Frank, J. Dispersion Sci. Technol., 7543 (1986). 332. K. P. Oza and S. G. Frank, J. Dispersion Sci. Technol., 10:187 (1989). 333. N. Garti, A. Aserin, andY. Cohen, J. Controll. Rel., 29:41 (1994). 334. W. J. Herbert, Lancet,2:771(1965). 335. R. H. Engel, S. J. Riggi, and M. J. Fahrenbach, Nature, 219:856 (1968). 336. J. A. Omotosho, Int. J. Pharm., 62:81(1990). 337. Y. Morimoto, K. Sugibayashi, Y. Yamaguchi, and Y. Kato, Chem. Pharm. Bull., 27:3188 (1979). 338. P. J. Taylor, C. L. Miller, T. M. Pollock,et al., Hygiene, 67:485 (1969). V. Mitchley, A. J.Collings,andR.Schneider,Eur. J. 339. L.A.Elson,B.C. Clin. Biochem. Res., 1587 (1970). 340. A. J. Collings, U.K. Patent 1,235,667,1971. 341. C. H.Benoy, L. A. Elson, and R. Schneider, Rev. Eur. Etudes Clin. Biol., 45:135 (1972). 342. A. F. Brodin and S. G. Frank, Acta Pharm. Suec., 1 5 1 (1978). 343. N.N. Li, U.S. Patent 3,410,794,1968. 344. N.N. Li, Amer. Inst. Chem. Eng. J. (A.I.Ch.E.J.), 17:459 (1971). L. Shrier, Recent Developments in Separation Science, 1 , 345. N. N. Li and A. Chem. Rubber Co., Cleveland, 1972, p. 163. 346. S. Zheng, Diss. Abstr. Int. B, 51:2485(1980). 347. M. S. Mohamed, F. S. Ghazy, M. A. Mahdy, and M. A. Gad, J. Pharm. Sci., 5 5 6 (1989). 348. K. P. Oza and S. G. Frank, J. Dispersion Sci. Technol., 10:163 (1989). 349. T. Takahashi, M. Mizuno, Y. Fujita, et al., Gann, 64:345 (1973). 350. J. A. Omotosho, A. T. Florence, and T. L. Whateley, Int. J. Pharm., 6151 (1990). 351. J.A.Omotosho, T. L.Whateley,andA.T. Florence,Biopharm.Drug Dispos., 10:257 (1989). 352. Q. Wu, Q. Ping, and G. Liu, Zongguo Yoyao Gongye Zashi,21:445 (1990). 353. S. C. Kundu, Diss. Abstr. Int. B 51:1763 (1990).
532
Garti and Aserin
354. 355. 356. 357. 358. 359. 360. 361. 362. 363. 364. 365. 366. 367. 368. 369. 370. 371. 372. 373. 374. 375. 376. 377. 378. 379. 380. 381. 382. 383.
B. Mishra and J. K. Pandit, J. Controll. Rel., 1453 (1990). M. Mathew andC. S. Thampi, East Pharm., 33:385 (1990). J. A. Omotosho, Int. J. Pharm., 62:81 (1990). I. Eros, J. Balazs, I. Peter, and M. Tacsi, Pharmazie, 45419 (1990). S. Ismail, A. A. Mohamed, and S. A. Hasan, Bull. Pharm. Sci. Assuit Univ., 12:68 (1989). W.J. Herbert, Lancet,2:771(1965). W . J. Herbert,Int. Symp. on Adjuvants of Immunity,Symp.Seri.Immunobiol. Standard 6, Utrecht, 1966, Karger, Basel, 1966, p. 89. W.J. Herbert, U.K. Patent 1,080,994 (1967). L. E. Elson, B. C. V. Mitchley, A. J. Collings, and R. Schneider, Eur. J. Clin. Biochem. Res., E 8 7 (1970). C.J.Benoy, L. A. Elson, andR.Schneider,Br.J.Pharmacol.,45:135P (1972). P. A. Gresham, M. Barrett, S. V. Smith, and R. Schneider, Nature, 234:149 (1971). S. Y. Lin, W. H. Wu, and W.Y. Lui, Pharmazie 46:439 (1992). S. Fukushima, K. Juni,and M. Nakanu,Chem.Pharm.Bull.,31:4048 (1983). H.L. Rosano, R. C. Peiser, and E. A. Eydt,Rev. Franc. Corps Gras, 16:249 (1969). H. L. Rosano, J. Soc. Cosmet. Chem., 25:609 (1974). S. E. Friberg, in Microemulsions: Theory and Practice (L. M. Prince, ed.), Academic Press, New York, 1977, pp. 133-146. .Shah, V. K. Bansal,K.Chan,and W . C.Hsieh,inImprovedOil D. 0 RecoverybySurfactantandPolymerFlooding (D. 0. ShahandR. S. Schechter, eds.), Academic Press, New York, 1977, pp. 293-337. L. MittalandB.Lindman, Th. F. Tadros,inSurfactantsinSolution(K. eds.), Vol. 111, Plenum Press, New York, 1984, pp. 1501-1532. A. M. Cazabat and D. Langevin, J. Chem. Phys., 74:314 (1981). L. E. Scriven, Nature, 263:123, (1976). L. E. Scriven, in Micellization, Solubilization and Microemulsions, Vol. 2 New York, 1977, p. 877. (K. L.Mittal, ed.), Plenum Press, Y. Talmon and S. Prager, Nature, 267:333 (1977). Y. Talmon and S. Prager, J. Chem. Phys., 69:2984 (1978). Y. Talmon and S. Prager, J. Chem. Phys., 76:1535 (1982). E. W. Kaler and S. Prager, J. Colloid InterfaceSci., 86:359 (1982). J. Jouffroy, P. Levinson, and P. G. De Gennes, J. Physiol. (Paris), 43:1241 (1982). E. W . Kaler, K. E. Bennet, H.T. Davis, and L. E. Scriven, J. Chem. Phys., 795673 (1983). P. A. Winsor, Trans. Faraday Soc., 44:376 (1984). L. S. Polatnik and A. I. Landau, Phase Equilibria in MulticomponentSystems, Holt, Rinehart and Winston, New York, 1964, p. 247. P. A. Winsor, Trans. Faraday Soc., 44,376,1984.
oemulsions Emulsions and
533
of Amphiphilic Compounds, Butterworth. 384. P. A. Winsor, Solvent Properties London, 1954. Clause, (eds.),MicroemulsionSystems,Vol.24, 385. H. L.RosanoandM. Surfactant Science Series, Marcel Dekker, New York, 1987. 386. M. Bourrel and R.S. Schechter, Microemulsions and Related Systems, Formulation, Solvency and Physical Properties, Vol. 30, Surfactant Science Series, Marcel Dekker, New York, 1988, p. 127. 387. A. Graciaa, J. Lachaise, A. Martinez, et al., C. R. Acad. Sci., B282547 (1976). 388. A. Graciaa, J.Lachaise,A.Martinez, et al., C. R. Acad. Sci., B285295 (1977). 389. A. A. Galje,W. G. M. Agterof, and A. Vrij, in Micellization, Solubilization and Microemulsions, Vol. 1 (K. L. Mittal. ed.), Plenum Press, NewYork, 1977. 390. C. Cabos andP. Delord, J. Appl. Crystallogr., 12502 (1979). 391. C. Cabos andP. Delord, J. Phys. Lett., 41:L455 (1980). 392. C. Taupin, J. P. Cotton, and R. Ober, J. Appl. Crystallogr., 11:613 (1978). 393. M. Dvolaitzky, M. Guyot, M. Lagues, J. P. Le Pesant, R. Ober, C. Sautery and C. Taupin, J. Phys. Chem., 69:3279 (1978). S. Huang, J. Phys.Chem.,86:3273 394. M.Kotiarchyk, S. H. Chen,andJ. (1982). 395. S. Qutubuddin, C. A. Miller, G. C.* Berry, T. Fort, Jr. and A. Hussam, in Surfactants in Solution, Vol. (K. 3 L. Mittal and B. Lindman, eds.), Plenum Press, New York, 1984. pp. 1693-1708. 396. A. Vrij, E. A. Nieuwenhuis, N. M. Fijnant, andW. G. H. Agterof, Faraday Discuss. Chem. Soc., 65:101(1978). 397. A. M. Cazabat, D. Langevin, and A. Pouchelon, J. Colloid Interface Sci., 73:1(1980). 398. C. Hermansky and R. A. Mckay, J. Colloid Interface Sci., 73:324 (1980). 399. E. Gulari, B. Bedwell, and S. Alkhafaji, J. Colloid Interface Sci., 77:202 (1980). 4 0 0 . M. B. Mathews and E. Hirschhorn, J. Colloid Interface Sci., 8:86 (1953). 401. R. N. Hwan, Ph.D. Thesis, A Mechanism for Ultralow Interfacial Tension in Systems Containing Microemulsions: Theoretical Considerations and Experi ments with Ultracentrifuge, Carnegie-Mellon University, Pittsburgh, 1978. 402. V. Luzzatti andF. Husson, J. Cell. Biol., 12:207, 1962. 403. Ekwall,P.,Composition,propertiesandstructuresofliquidcrystalline of amphiphilic compounds, in Advances in Liquid Crystals phases in systems 1. Vol. 1 (G. H. Brown, ed.), Academic Press, New York, 1975, p. 404. K.Larsson and P. Dejmek, Crystal and liquid crystal structures of lipids, in S. E. Friberg, eds.), Marcel Dekker, New Food Emulsions, (K. Larsson and York, 1990, p. 97. 405. K. Larsson, J. Phys. Chem., 73:7304,1989. 406. K. Larsson, J. Colloid Interface Sci., 113:299, 1986. 407. M. Trotta, M. R. Gasco, and S. Morel, J. Controll. Rel., 10:237 (1989).
Aserin 534
and
Garti
408. M. R. Gallarate, M. R. Gasco, and M. Trotta, Acta Pharm. Technol., 34:102 (1988). 4 0 9 . M.R.Gasco,M. E. Carlotti, and M. Trotta, Int. J.Cosmet. Sci., 10:263 (1988). 410. M. R. Gallarate, M. R. Gasco, and G. Rua, Acta Pharm. Jugoslav. 40533 (1990). 411. M. R. Gasco, F. Pattarino, and I. Voltani, I1 Farmaco Ed. Pr., 43:3 (1988). 412. M. R. Gasco, S. Morel, and R. Mazoni, I1 Farmaco Ed. Pr., 43:373 (1988). 413. F. Pattarino, M. R. Gasco, and M. Trotta, Il Farmaco Ed. Pr., 44:339 (1989). 414. M. Trotta, M. R. Gasco, andF. Pattarino, Acta Pharm. Tech. 36:226 (1990). 415. B. D. Kahan M. Ried, and J. Newburger, Transplant Proc., 15446 (1983). 416. B. Tam and S. H. Yalkowsky, Pharm. Res., 6:40 (1989). 417. W . A. Ritschel, S. Adolph, G . B. Ritschel and T. Schroeder, Meth. Find. Exp. Clin. Pharmacol., 12:127 (1990). 418. W. A. Ritschel, G. B. Ritschel, A. Sabouni, et al., Meth. Find. Exp. Clin. Pharmacol., 11:281(1989). 419. W. A. Ritschel, Meth. Find. Exp. Clin. Pharmacol., 13:205 (1991). 420. D. W. Osborne, A. J. I. Ward, and K. J. ONeill, Drug Dev. Ind. Pharm., f 14:1203 (1988). 421. D. W. Osborne, A. J. I. Ward, and K. J. ONeill, J. Pharm. Pharmacol., 43:451(1991). 422. F. FCvrier, M. F. Bobin, C. Lafforgue, and M. C. Martini, S.T.P. Pharm. Sci., 1:60 (1991). 423. J. Zeigenmeyer and C. Fuhrer, Acta Pharm. Technol., 26:273 (1980). 424. M. C. Martini, M.F. Bobin, H. Flandin, et al., Pharm. Belg.,39:348 (1984). 425. M. R. Gasco, M. Gallarate, and F. Pattarino, Int. J. Pharm., 69:193 (1991). 426. M. Trotta, M. R. Gasco, andF. Pattarino, Proceedings of 10th Pharmaceutical Technology Conf., Bologna, Italy,1991, p. 388. 427. M. R. Gasco, M. Gallarate, M. Trotta, et al., J. Pharm. Biomed. Anal., 7:433 (1989). 428. H. L. Rosano and W. E. Gerbacia,U.S. Patent, 3,778,381,1973. 429. P. Chabert, L. Foulletier, and A. Lantz, Ger. Offen., 2,452,513 (1975). 430. J.J.Delpuech,G.Mathis,J.C.Ravey,etal.,Bull.Soc.Chim.Fr., 578 (1985).
16
The Use of Drug-Loaded Nanoparticles in Cancer Chemotherapy
Jean-Christophe Leroux,Eric Doelker, and Robert Gurny
School ofPharmacy, University of Geneva, Geneva, Switzerland
I.
Introduction
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11. In Vitro Uptake of Nanoparticles by Tumoral Cells A. Interaction of Uncoated Nanoparticles with Cultured Cells B. Interaction of Antibody-Coated Nanoparticles with Cultured Cells 1 1 1 . Distribution and Pharmacokinetics of Anticancer Drugs Coupled to Nanoparticles A. Distribution of Anticancer Drugs Coupled to Nonmagnetic Nanoparticles B. Distribution of Anticancer Drugs Coupled to Magnetic Nanoparticles
IV. V. In Vivo Activity and Toxicity of Anticancer Drugs Coupled to Nanoparticles Concluding Remarks
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INTRODUCTION
Currently, the limiting factor in cancer chemotherapy is the lack of selectivity of anticancer drugs toward neoplastic cells. Generally, rapidly proliferating cells such asthose of the bone marrow or the gastrointestinal tract are affected by the cytotoxic action of these drugs. This results in a narrow therapeutic index of most anticancer compounds. Furthermore, the emergence of resistant cell sublines during the chemotherapeutic treatmentmay require the use of higher doses of anticancer drugs or the elaboration of dosing protocols combining different anticancer drugs. This, in return, may to the toxicity and enhance thetoxicity of the treatment. In order decrease to enhance the selectivity of existing drugs, many drug-delivery systems have been developed ,in recent years [1,2]. These systems include soluble drug-polymer conjugates [3], nanoparticles [4], liposomes [ 5 ] , and microparticles [6]. Nanoparticles have received a growing interest for drug targeting, because they can beeasily prepared with well-definedbiodegradable polymers [7]. The reason of targeting tumors with nanoparticles is because certain neoplastic cells have been found to exhibit an enhanced endocytotic activity [ 8 ] . In addition, since some particular tumors are blood supplied by capillaries having an increased vascular permeability, one can anticipate that nanoparticles will gain access to extravascular tumoral cells
[9,101.
This chapter covers the developments and progress made in the deliv-
ery of anticancer drugs coupled to nanoparticles and theinteractions of the

latter with neoplastic cells and tissues. Throughout the chapter, the term nunoparticles will generally refer to submicronic carriers ( 5 1 pm) consisting of either nanospheres (matrix systems) or nanocapsules (vesicular systems) [H]. Because many submicronic carriers havebeen in the past named microparticles, it is possible that some studies dealing with the present topic but using this term have been unintentionally omitted.
II. IN VITRO UPTAKE OF NANOPARTICLES BY TUMORAL CELLS
The interaction of nanoparticles with cultured cells may be either passive or mediated via determined moieties such as monoclonalantibodies which can bindspecifically to targeted cells. A. Interaction of Uncoated Nanoparticles with Cultured Cells
In vitro studies involving nanoparticles were initially performed to determine if malignant cells were able to internalize nanoparticles to a signifi-
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in Cancer Chemotherapy
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cant extent (Table 1). However, targeting drug carriers to homogeneous cell cultures is of limited value, because in this case, the carriers are directly addedtothetarget cells. Intheseconditions, no anatomical barriers prevent the nanoparticles from acceding to target cells, and no extensive clearance of the drug is encountered [10,12]. Furthermore, it still remains unclear if an increase of drug efficiency in vivoresulting from the binding of the drug to nanoparticles is related to a greater uptake of the drug by tumoral cells in vitro [13-151. So far, in vitro experiments have led to inconsistent results regarding any increase of efficacy of nanoparticle-bound anticancer drugs. In fact, the free drug remained often more or as effective as its bound counterpart [16-191. These results may be explained by the limited access of the bound drug to the internal cell compartments. Indeed, Astier et al.[20] have reported that doxorubicin-loaded poly(alky1 methacrylate) (DXR PAMA) nanospheres were more effective in inhibiting human U-937 leukemia cell growth than free DXR. However, U-937 cells belong toa monocytelike cell line with marked phagocytic properties. On the other hand, in vitro studies are useful to help to understand the mechanisms of action of bound anticancer drugs. By coupling covalently DXR to polyglutaraldehyde (PGL) nanospheres, TokCs et al. [l61 and Rogers et al. [l81 have demonstrated that DNA intercalationof DXR was not essential for its pharmacological action, and that the cell membrane was a major site of action of these DXR nanospheres. Moreover, in vitro experimentshave revealed the roleof the nanoparticlepolymer in the cytotoxic action of drug-loaded nanoparticles. In another study where it was observed thatthe binding of DXRto poly(alky1 cyanoacrylate) (PACA) nanospheres could increase its efficiency [21], it was also found that the polymers used contributed to thecytotoxicity. Although, as stated by the investigators, the polymer itself could act as a second chemotherapeutic agent, it has to be pointed out thatcolloidal carriers have a propensity to concentrate in the reticuloendothelialsystem (RES) [7,10] and thus could possibly damage the latter, especially if they are loaded with highly cytotoxic agents [lo]. Selection of appropriate polymers remains a key issue, because it was shown that even well-established biodegradable polymers such as poly(1actic acid) (PLA) cannot be considered as inert vehicles [22,23]. Regarding PACA nanospheres, it was demonstrated that themost cytotoxic polymers for tumoral cells were those with shorter side chains [15], confirmingprevious results obtained with normal cells [24]. However, the evaluation of the polymer toxicity from in vitro tests must take into account the fact that the amount of nanospheres incubated with tumoral cells is often excessively high'and sometimes corresponds to what isadministered to onemouse [15].
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One of the most interesting properties of anticancer drug-loaded nanoparticles is their capacity for overcoming pleiotropic drug resistance (see Table 1). In vitro studies evaluating the efficacy of nanoparticles on multidrug-resistant cells have been so farperformed onlywith DXR [16,18,19,21,29,34,36,37]. Generally, the sensitivity of the resistant cell sublines was completely restored when DXR-loaded nanospheres were used. The mechanisms involved in pleiotropic drug resistance are complex. Among them, the overexpression of a membrane glycoprotein, namely glycoprotein P, which pumps out the anticancer drug from theinside of the tumor cells, has been extensively investigated [2,42-441. By coupling DXR to nanospheres, DXR efflux from tumoral U-937 cells was considerably reduced (Fig. 1) [20,34]. Accordingly, increased drug retention, possibly DXR because of the inability of glycoprotein P to reject nanosphere-bound outside the cell, may partly explain how drug resistance can be counteracted. Recently, Colin de Verdibre et al. [38] demonstratedthat poly(isobutyl cyanoacrylate) (PIBCA) nanospheres were not endocytosed by P388 leukemic murine cells, and that the accumulation of doxorubicin in the resistant cells could not be explained, in this case, by a reduced efflux.
10
20
30
40
50
Incubation time after wash (h)
Fig. 1 Efflux of DXR from U-937 cells. Exponentiallygrowing cells were exposed for 4 h at 37C to 0.5 pg/mL of free DXR (0)or nanosphere (NS) bound DXR (0)(4 x lo4 NSlcell). After they had been washed in phosphate-buffered saline (PBS), the cells were resuspended in drug-free medium for 48 h and harvested at various times. Each point represents the mean of two separate experiments performed in quadruplicate (bars, SD). (From ref. 20.)
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Indeed, the increase uptakemay be attributed to the formationof an ion pair between doxorubicin and soluble oligomers of PIBCA [45]. This complex would be able to betterdiffuse through the cell membrane. Another mechanism also seems to be involved in the increase efficiency of DXR nanospheres against DRX-resistant cells. DXR bound to nanospheres can efficiently interact with the membrane of resistant cells, inducing at thefinal stage a perforation of the cytoplasmic membrane [181. This mechanism is dependent on the nanospheredensity per cell, because for thesame drug concentration, theactivity of DXR increaseswith higher nanosphere densities[20]. This issue ispotentially important for the elaboration of improved in vivo drug-administration protocols. Indeed, the drug activity may be dependent not only on its dose but also on the amountof nanoparticles administered. More recently, immunomodulators that activatemacrophages to render them cytotoxic for tumor cells have attracted increasing interest for theirincorporationinto colloidal carriers.Immunomodulators such as muramyl dipeptide (MDP)have a very short half-life whichprevents accumulation of the drug into macrophages [22]. Therefore, alipid derivative of MDP, namely muramyl dipeptide-L-alanyl-cholesterol(MTP-Chol), was incorporated intonanocapsules. These nanocapsules are made from an oily core surrounded by a polymeric wall and can be loaded with lipophilic drugs [41,46-481. MTP-Chol-loaded nanocapsules were able to activate rat alveolarmacrophages for a cytostatic effect on tumoralcells [33].However, the nanocapsule formulation was not superior to the nanoemulsion, micellar solution, and liposome formulations [33]. The possible mechanisms of action of MTP-Chol nanocapsules are depicted in Fig. 2. At low immunomodulator concentrations, MTF"Cho1 nanocapsules are generally more effective than free MDP, probably because of an improved intracellular delivery by phagocytosis [40,41]. Nanocapsules containing MTPChol were found to be slightly toxic for rat alveolar macrophages [22,33, 351. This toxicity couldbe explained by an increased sensitivity of activated macrophages to nitric oxide. Nitric oxide is produced by the immunomodulator during theactivation of the macrophages. It is a highly reactive molecule playing an important role in cytotoxic rat macrophages, but itis able to reduce the mitochondrial electron transport activity of macrophages. Since MTP-Chol nanocapsules are internalized by phagocytosis, which isan energy-dependentprocess, it is possible that the internalization of the immunomodulator by this mechanism could sensitize the macrophages to nitric oxide [22]. In recent years, there has been a growing interest for the biological therapies of cancer [49]. For instance, the insertion of new or foreigngenes into a tumoralcell may represent an alternative to conventional drug ther-
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apy. Liposomes and nanoparticles have been proposed as alternative carriers toviral capsids for gene transfer [50,51]. Bertling et al. [51] have shown that it was possible to incorporate efficiently DNA into PIBCA nanospheres and accordingly to protectDNA from degradation. However, the DNA was not any longer transcriptionally active. Other studies are needed toassess the potentiality of different nanoparticle types to increase the bioavailability of DNA to the cell nucleus, but at present time, liposomes appear to be better candidates. Promising results were recently obtained with antisense oligonucleotides adsorbed onpoly(isohexy1 cyanoacrylate) (PIHCA) nanowhen spheres [52]. It was demonstrated that the adsorbed oligonucleotides, administered locally, were able to inhibit the neoplasticgrowth of tumoral cells in nude mice. However, these preliminary results need to be further confirmed.
Drug-Loaded Nanoparticles in Cancer Chemotherapy B. Interaction of Antibody-Coated Nanoparticles with Cultured Cells
545
To achieve tumor-specific targeting, nanoparticle formulations should be

able to recognize specific cell determinants belonging only to target cells. The development of monoclonal antibody technologyby Kohlerand Milstein [53] has allowed the production of specific antibodies able to interact preferentially with tumor cell surface antigens [54,55]. From a purely pharmaceutical standpoint, the production of a drug-antibody complex is limited by the number of functional groups available per antibody molecule which can besuccessfully usedwithout significant loss of antibody activity [56]. Accordingly, a colloidal system in the form of nanoparticles with a large carrier capacity, coated with monoclonal antibodies, has been proposed as an alternative [57-591. Monoclonal antibodies (MAbs) can be fixed on nanoparticles either by nonspecific adsorption [57,58,60,61], specific adsorption [56,61-631, or covalent linkage [56,59,64-661. Nonspecific adsorption of MAbs on nanoparticles is realized by incubating the MAbs with the nanoparticles. Using this technique, it has been estimated that, theoretically, a maximum of 2000 MAbs can be adsorbed on 170 nm of poly(hexy1 cyanoacrylate) (PHCA) fixed nanospheres [57]. In vitro, the noncovalent link seems stable and the MAbs can recognize specifically tumor cells with a ratio of nonspecific binding to specific binding reaching 3:l-4:l [57,58]. These findings suggest that some MAbs are fixed to nanospheres by their Fc part with their antigen-binding site Fab protruding into the medium and thus being accessible to the antigens (Fig. 3). This type of binding can be achieved if the surface of the nanosphere is relatively hydrophobic [56].Although MAbs fixed by adsorption appear tightly bound to the nanoparticles, competitive displacement by plasma proteins can occur and may represent a drawback following intravenous administration of MAb-coated nanoparticles [57,67]. Kubiak et al. [60] showed that radiolabeling of MAbs with iodine modified the physicochemical interactions between the MAbs and the nanospheres, resulting in adecreased affinity ofthe antibody for the carrier. Therefore, it is of prime iinportance to assess the stability of the binding in biological fluids before conducting any in vivo experiments with labeled MAbs coupled to nanospheres. Apart from their possible use in drug targeting and radioimaging, Manil et al. [61] have suggested that nanospheres with adsorbed MAbs may be used as a solid phase for immunoradiometric assay for the analytical determination of tumor markers such as Q-fetoprotein. The specific adsorption of MAbs is achieved byfirst coupling to nanoparticles a ligand which can specifically bind antibodies, Then,
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Fa b
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Fig. 3 Schematic diagram of the attachment of monoclonal antibody to a nanoparticle. (From ref. 57.)
the MAbs are incubated with the precoated nanoparticles and bind to the spacer molecule via noncovalent links. Some studies report the use of staphylococcal protein A as a spacer molecule which can bind the Fc portion of most subclasses of immunoglobulin G (IgG) [56,58,62,63,68]. Although MAbs linked to protein A-precoated nanoparticles retain their specificity, it has been found in one study [61]that the presence of the protein A did not enhance the immunoreactivity of the adsorbed MAbs. Covalently bound MAbs cannot be displaced from the nanoparticle surface by plasma proteins and thus appear to be better candidates for efficient drug targeting. However, few studies have been performed in vitro with MAbs covalently linked to nanoparticle [56,64,66], and noneof them used tumoral cells. Suchexperiments have been carried out in vivo and will be discussed inSection II1.A along with the potential implications of the in vivo use of MAb-coated nanoparticles.
111. DISTRIBUTIONANDPHARMACOKINETICS OF ANTICANCER DRUGS COUPLED TO NANOPARTICLES
The localization of nanoparticles in specific tissues may be dependent only on theintrinsic characteristics of the carrier (e.g., size, surface) or bepartly governed by an external mechanism (e.g., magnetic field). Therefore, this section makes a distinction between the natural distribution of a nonmagnetized carrier and thatof magnetically directed carrier.
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A. Distribution of Anticancer Drugs Coupled to Nonmagnetic Nanoparticles The tissue distribution and pharmacokinetics of an anticancer drug can be altered by its incorporation into nanoparticles (Table 2). Generally, following intravenous administration, nanoparticles are rapidly and extensively taken up by the RES [7]. Accordingly, as soon as a few minutes after the injection of nanoparticles, the anticancer drug mainly accumulates in the liver and spleen [69-741. Thereafter,the RES drug level gradually decreases over several days depending on the biodegradability of the polymer and on the drug-release kinetics. Following intravenous injection of 14C-DXR PIHCA nanospheres, Verdun et al. [74] found that the liver concentration of radioactivity remained high during the first day and decreased rapidly during days 2 and 3 as a consequence of fecal excretion. After 8 days, only about 30% of the labeled drug could be detected in the liver. Indeed, PACA nanoparticles are biodegradable and accordingly are rapidly eliminated from the organism [4,75]. This is not the case of other nanoparticle polymers such as PAMA. PAMA nanoparticlesare well tolerated invivo but are veryslowly biodegradable [7,76]. Considering that some tumoral diseases may alter the RES, the long-term consequences of the accumulation of PAMA nanoparticles following multiple dosings of anticancer drugs should be seriously considered [ n ] . Rolland [76]suggested that the clinical use of DXR-loaded PAMA nanospheres would still be compatible with a monthly injection protocol. However, in our opinion, and because of the importance of the RES in the host defense [78,79], it seems questionable to justify the use of such polymers when other well-characterized biodegradable polymers are available. of liver DXR concentration Couvreur et al. [80] reported no increase following intravenousadministration of 3H-DXR-loaded poly(methy1 cyanoacrylate) (PMCA) nanospheres. In fact, an increase of DXR concentration in the gut and lungswas noticed. Although this unusual distribution pattern may be due to the higher hydrophilicityof PACA made of short side chains [81], these results have not been further exploited,probably because of the higher toxicity of PMCA nanospheres [24]. More recently, Bapat et al. [82] reported that DXR concentrations in the liver and spleen following intravenous injection of DXR-loaded poly(buty1 cyanoacrylate) (PBCA) nanospheres was lower than the in control and did not increase over time. They attributed these low concentrations to the slow breakdown of the polymer in the body. Nevertheless, they did not specify if their extraction procedure was able to separate the free drug from its nanosphere-bound counterpart. According to Kattanet al. [83], most of conventional methods
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of drug determination do not discriminate between nanoparticle-associated drug and free drug. Because nanoparticles are mainly taken up by the RES, nonphagocytic cells such as heart and musclecells could not theoretically accumulate nanoparticles. The incorporation of anticancer drugs into nanoparticles would thus appearsuitable for avoiding the deposition of highly toxic compounds into nontarget organs. DXR is a potent antitumor agent that is active against a wide spectrum of malignancies, but it is associated with acute and delayed cardiotoxicity [42]. When administered in the nanoparticulate form, the concentration of DXR in the mouse heart was generally lower than in the control [74,82,89]. Surprisingly, when using rabbit as the experimental animal model, Rolland [74] did not find any significant difference in tissue concentration between free and nanosphere-bound DXR in the spleen, lungs, and heart. He suggested that the bound drug could still produce a subcellular compartments decrease of cardiac toxicity byits binding to other not involved in toxicity and by a slow release of thedrugfrom the nanospheres. In the same study, no differences were found between the pharmacokinetic parametersof the bound andfree DXR[73]. These findings are in disagreement with other studies, where the administration of DXR-loaded nanospheresresulted in a reduction of DXR blood clearance during the first fewminutes after intravenous injection [74,87]. The first clinical trial using anticancer drug-loaded nanospheres was carried out by Rolland in 1989 [76].He compared the pharmacokinetics of 20-30 mg PAMA nanosphere-bound DXR intravenously administered to four patients with hepatoma against 20 mg of free DXR administered to one patientwith hepatoma. This resulted in a 15-48% increase in the dosenormalized area under the drug concentration-time curve for the patients receiving the nanosphere-bound DXR [1,76]. More recently, Kattan et al. [S31 conducted a clinical trial using DXR-loaded PIHCA nanospheres. The pharmacokinetic profiles of DXR were examined on three patients with refractory solid tumors receiving 60-75 mg/mz DXR-loaded nanospheres. These profiles were comparedwith those obtained with the administration of free DXR at the same doseto thesame patients. Contrary to the previous study, three controls with free DXR were evaluated in this trial, and it was found thatthe variability of kinetic parameters of free and boundDXR was such that no convincing conclusions could be drawn from the results. Any other comparison of these two studies is difficult, because the plasma concentrations were monitored between0.5 and 24 h in the first study and .between 5 and 90 min inthe otherone. $Althoughit has been shown that some phagocytic cells may have a higher endocytotic activity than normal cells [8,30], it still remains unclear if nanoparticles can concentrate in tumors. According to Grislain et al.
Drug-Loaded Nanoparticles Cancer Chemotherapy in
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[M], 4 h after intravenous administration of PBCA nanospheres to mice bearing a subcutaneous Lewis lung carcinoma, accumulation of the carrier in the tumoral tissue was higher than in the underlying tissue and the concentration of the nanospheres in the lungs was as much as five times higher than that of the liver. The investigators concluded that the deposition of the carrier in the lungs was possibly the consequence of nanosphere uptake by lung metastases of the primary tumor. However, as underlined by Douglas et al. [81], no histological examinations were performed in this study, and since the Lewis lung carcinoma is an hemorragic tumor that possesses poorly formed sinusoidal channels in which an endothelial lining is lacking [95],it is possible that the conditions encountered in this case were optimal for the uptake of nanospheres by tumoral cells. Other results on thedistribution of nanosphere-bound anticancer drugsto tumors are not as conclusive [70,86,88]. Even when high concentrations of bound drug were found in the tumor in comparison with the underlying tissue, the amount of nanospheres detected in the targeted area was still relatively low [88]. Chiannilkuchai et al. [l31 have administered DXR-loaded PIHCA nanospheres to mice bearing reticulosarcoma M5076 metastases. They found thatthe drug concentrated dramatically in the healthy hepatic tissue, which in turn could act as a reservoir for the anticancer drug (Fig. 4). This
10
20
30
40
Time (h)
Fig. 4 DXR concentration vs time curves in healthy hepatic(circles) and tumoral (squares) tissues after intravenous administration of DXR (10 mgkg corresponding to 133 mg/kg PIHCA) in its free (solid symbols) or nanosphere-bound form (open
symbols). (From ref. 13.)
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probably enabled a higher exposure of liver metastases to thedrug. These pharmacokinetic findings were consistent with histological examinations where a considerable number of nanospheres weredetected in the Kupffer cells, whereas nanospherescould not be foundin neoplastic cells [13]. Distribution of MAb-coated nanospheres havenot led to the results anticipated with previous in vitro experiments [59,67]. Apart from nanosphere clearance by the RES, poor localization of the nanospheres in the tumors could be explained by several factors, including competitive displacement of adsorbed MAbs, a secondary coating process where the MAb-coated nanospheres receive an opsonic layer, and most probablyan insufficient access of the nanospheres to tumor cells [10,67]. Maybe this latter issue has been so far underestimated. Initial enthusiasm for therapies using MAbs, drug-MAb complex, and MAb-macromolecular systems has been dampenedby the fact that macromolecules cannot reach all regions of a tumor in adequate quantity [3,55,96]. One method of increasing the transport of antibodies to tumoralcells could be, for instance, to use lower molecular weight agents; for example, antibody fragmentsF(ab), and Fab [96]. Nanoparticles are confronted to a greater problem in that they are much bigger than antibody molecules. Accordingly, it can be anticipated may still have potential applicathat the use of MAb-coupled nanospheres tions, but in a limited number of cases, such as nonsolid tumors, intratumoral therapy and for the treatment of solid tumors which are blood supplied by discontinuous capillaries or sinusoidal blood channels[lo]. Finally, the extensive clearance of nanoparticles and MAb-coated nanoparticles has to be lowered to achieve a better deposition of nanoparticles in tumors. Recent studies have demonstrated the efficacy of coating polystyrene nanospheres withblock copolymers of poloxamer and poloxamine and polyethylene glycol (PEG) for reducing nanosphere uptake by macrophages [97-1011 and allowing a longer in vivo circulation time of the nanospheres or their accumulation in thebone marrow [102,103]. Studies performed with Stealth@ liposomes either stabilized with PEG organglioside GM1 haveshown a greater accumulationof the stabilized carrier over the control liposomesin tumors [104,105]. Such investigations need to be performed to a greater extent with biodegradable nanoparticles [106,107]. A study carried out by our group demonstrated that coating PLA nanospheres with PEG 20,000 could produce a substantial increase of the blood circulation time of the photoactivable compound ZnPcF,,, (hexadecafluorinated zinc phthalocyanine) ascompared with plain spheres (see Table 2) [94]. After 24 and 168 h, the cumulated uptake of the compound in the liver and spleen represented 61 and 4 4 % for plain nanospheres versus 50 and 29% for PEG-coated nanospheres,respectively. resultThe reduction of the uptakeof the nanospheresby the RES and the
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ing longer blood circulation time wereassociated with a three-fold increase of the compound concentration in the tumor after 24 h.
B. Distribution of Anticancer Drugs Coupled to Magnetic Nanoparticles
In order to minimize colloidal camer uptake by the RES and to enhance their extravasation from the capillaries, magnetic nanospheres were developed in the late 1970s and have been reviewed in detail by Gupta and Hung [108]. Only considerations related to their use in cancer chemotherapy will be discussed in this section. These nanospheres (200-1000 nm) contain a magnetically active component (e.g., Fe,O,) and can be guided by an externally placed electromagnet. An electromagnetic field ranging from 1000 to 8000 G is generally applied at the target site for 5-60 min after dosing [109-1171. Very high anticancer drug concentrations in the target area (generally a section of the tail) can be obtained following the administration of magnetic 60 min nanospheres. Forinstance, Senyei et al. [l101 showed that as late as postinjection, 0.05 mg/kg DXR administered intra-arterially via magnetic nanospheres resulted in almost twice the local tissue concentration than was achieved by a 100-fold higher intravenous dose solution. The use of magnetic nanospheres can increase the extravasation and partly overcome the limited access of conventional nanoparticles to extravascular tumors. Widder et al. 30 min after infusion of albumin nanospheres to rats [l111 demonstrated that bearing subcutaneous Yoshida sarcoma, 1000-nm nanospheres were found in the extravascular compartment adjacent to tumor cells and occasionally in tumor cells. By 24 and 72 h, nanospheres were still detectable within tumor cells. Furthermore, it was shown elsewhere [l121 that DXR-loaded nanospheres could traverse the vascular endothelium asearly as 2 h after dosing and that the pharmacodynamic characteristics of the drug were not altered by its entrapment into nanospheres. In order to evaluate the efficiencyof drug delivery via magnetic nanospheres, Galloet al. [l131 introduced two indices; namely, the relative exposure (re) and the targeting efficiency (t,):
where AUC, is the dose-normalized area under the drug concentrationtime curve (AUC) for the ith tissue and the superscripts NS and S refer to
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the nanosphereand the solution dosage forms, respectively; (AUC),,,,, and (AUC)nontargct are the AUC for the target site and nontarget site, respectively. These two indices provide more valuable information on the drugdelivery than simple drug concentrations at thetarget site at different time points and should also be used for nonmagnetic nanospheres. Using these parameters, it wasfound thatDXR delivery to a selected target area can be greatly improved by its incorporation into magnetic nanospheres [113,114]. Magnetic nanospheres can also decrease the nontarget tissue exposure to as DXR. For instance, the administration of 2 mgkg DXR to ratsa solution or via magnetic albumin nanospheres provided revalues inferior to 0.6 for the heart and kidney a and reof 1.63 at the target site [1131.The efficiency of drug targeting is highly dependent of the strength of the magnetic field [114]. Table 3 suggests that the 1000-G magnetic field was only partly able to control the distribution of DXR, because the liver uptake of the drug remained high. considering a possible human application of magnetic nanospheres, this observation emphasizes the importance of defining the minimal magnetic field strength to apply to ensurean acceptable drug targeting. Moreover, it was shown thatthepharmacokinetics of magnetic nanospheres could be altered by the dose administered [116,117]. Indeed, a reduction of the carrier dose has proved to increase the targeting efficiency. The reduction of the dose decreased moreparticularly the amount of DXR delivered to the liver and heart. As underlined by Gupta et al. [116], such a dose-dependence of kinetic parameters is important since the carrier dose may often need to be altered, depending on factorslike the weight of the patient and the volume of the target tissue.
Table 3. Drug Targeting Efficiency (tJ of Magnetic Albumin Nanoparticles in

the Delivery of 0.4 mgkgDoxorubicin Administered Intra-arterially (Tail) to Wistar Rats inthe Presence or Absence of a 1000- or 8000-G Magnetic Field Applied at Target Site for 30 Min
.
L.
Tissue
~~ ~~
1000
Control 0.62 1.00 1.00

0.43
G
1.38 1.00 0.88
0.99
8000 G 15.54 6.12 6.73
Tail upstream from target site target site downstream from target site Liver Source: Adapted from ref. 114.
0.51
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Finally, .it has to be pointed out that, in most cases, the target area was easily accessibleand thatsimple intratumoral injection of drug-loaded nanospheres could provide similar results. The magnetic carriers were also often administered via the arterial route. Althoughthis mode of administration allows the carrier to avoid the hepatic first pass effect, it suffers from a lack of clinical convenience. Furthermore, in the case of disseminated tumoral diseases or nonsuperficial tumors, it can be anticipated that such highly specifictissue distribution of anticancer drugs obtainedwith animals bearing subcutaneous tumors may not be as easily achievable. In the future, target areas other than themouse or rat tail should be more investigated. Ibrahim et al. [l181 showed that the kidney levels of DXR could be enhanced by its incorporation into 220 nm-PIBCA magnetic nanospheres injected intravenously. However, in this study, the data were collected only 10 min after dosing, providing no indication on the possible retention of the carrier at the target site.
IV. IN VIVO ACTIVITY AND TOXICITY OF ANTICANCER DRUGS COUPLEDTO NANOPARTICLES
Preliminary studies regarding the in vivo activity of PACA nanospheres against sarcoma implanted subcutaneously demonstratedthe efficiency of carried anticancer drugs in reducing the tumor size [24,86,119] (Table 4). The increased efficiency of the nanoparticle dosage form was first attributed to an improved delivery of the bound drug to malignant cells. [86,119]. Although this hypothesis may be relevant for some specific tumors [lo], the modification of the drugs pharmacokinetic parameters, as discussed earlier, may be more important. Generally, an increase of life span was noted for the animals treated with nanoparticle-bound anticancer drugs. This prolonged survival was observed for animals bearing solid tumors as Interestingly, Cuvier et al. well as leukemia [24,34,84,120,122,127-1291. [34] demonstrated that it was possible using DXR-loaded nanospheres to prolong significantly the survival time of mice which were inoculated with 5 ) . This bypass of tumor cell resistance DXR-resistant leukemia cells (Table confirmed the previous in vitro observations. Recently, Allkmann et al. [l361 showed that the photodynamic activity of ZnPcF,, against EMT-6 mouse mammary tumor could be greatly improved after incorporation of the compound in PEG-coated nanospheres. One week after treatment, 63% of the mice had no macroscopic sign of tumor regrowth andat 3 weeks complete healing was observed for these mice. In contrast, with an O N emulsion formulation only 14% tumor regression was observed. In vitro investigations have revealed the importance of the binding of the drug to thecarrier on the activity of the nanoparticle suspension. A loss
Q\
W
W
2
4
vl
m m
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Table 5. Effect of Free DXR or DXR-Loaded PIHCA Nanosphereson the Survival of DBA2 Mice Bearing P388-DXR-Resistant Ascitis(Intraperitoneal Injection of lo6 P388-DXR-Resistant Cells at Day0)
50% Survival time (days)
Groups of 10 mice Nontreated (C) DXR (TI DXR-loaded NS (T)

1 2 11 14 11 11 11.7 21 21 17 18
20.2
3
4
Mean T/C P (T vs C)
13.5
115%
Not significant
164% 0.003
NS, nanospheres. See text for definitions of other abbreviations. DXR or DXR-loadedNS was given by intraperitoneal route at days 1-4 at a dose of 2 mg/kg body weighttday; the activity of unloaded NS was not significant.P is the probability that T is statistically different from C, using Students t-test. Source: From ref. 34.
of efficiency may occur if a mixture of unloaded nanoparticles and free drug is administered instead of the drug-loaded nanoparticle formulation [127,129]. This indicates that in vivo,the nanoparticle polymer is lesslikely to exerta cytostatic effect, as was noted in vitro (see Table 1). On the other hand, anticancer drugs bound to nanoparticles have often been associated with a higher acute toxicity;namely, premature deaths [86,107,119]. The increase of toxicity could be attributed to the accumulation of the carrier in the organs of the RES [86,119] or to the injection of agglomerated nanospheres that may have provoked embolisms [107]. This confirms the importance of developing drug carriers that can avoid the RES (when it is not the target site) and of making sure that the nanoparticle suspension is always fully dispersed before the injection. An increase of the bone marrow [l341 and renal toxicity [l351 has also been reported for doxorubicin-loaded nanospheres. Conversely, a reduction of the general toxicity of anticancer drugs boundto nanoparticles has beendemonstrated in a number of cases [17,24,123]. From these findings, one can infer that even if the incorporation of a cytostatic agent into nanoparticles is not always followed by an increase of drug efficiency, a reduction of the toxicity may produce an increase of the therapeuticindex [17]. One of the most promising applications of anticancer drug-loaded nanoparticles may be their usein the treatment of hepatic metastases
Drug-Loaded Nanopatticlesin Cancer Chemotherapy

200
563
150
50
No treatment
7.5
10.0
Dose (mrng)
Fig. 5 Number of hepatic metastases for C57/BL6 mice after treatmentat days 1 1 and 13 with intravenous free DXR (shaded columns) or nanosphere-bound DXR (closed columns) compared with control (open column). The mice were innoculated intravenously at day0 with 7 X Id tumoral cells (reticulum cell sarcoma M 5076). The study was performed with groups of 8-10 mice (mean 2 SD). **, significantly different from control (P C.001, Students t-test). (Adapted from ref. 129).
[128,129]. Administration of DXR-loaded PIHCA nanospheres to mice intravenously inoculated with tumoral cells resulted in a marked reduction of the number of hepatic metastases (Fig. 5 ) and in an increase of survival time (Fig. 6). These interesting results may be partly explained by the fact that in this study, the tumoral cells inoculated were macrophagic in origin [129]. Thus, these cells should have a propensity to take upcolloidal drug carriers. The activity of the immunomodulator MTP-Chol coupled to nanocapsules against metastases has also been evaluated. Because nanoparticles can incorporate peptides efficiently [l13 and have a propensity to accumulate in macrophages, MW-Chol-loaded nanocapsules could provide a stable and active pharmaceutical formulation. Indeed, MTP-Chol coupled to nanocapsules was partially able to inhibit the metastatic proliferation that normally occurs following the intravenous injection of tumoral cells. However, this formulation was efficient only when given as a prophylactic treatment [131,132,137]. According to Yu et al. [132], this would correspond in the clinic to patients undergoing surgery for a primary tumor and who could develop metastases. Furthermore, the efficacy of MTP-Chol-loaded nanocapsules is limited, and this formulation will certainly need to be combined to other chemotherapeutic drugsto ensure optimal results. It is well established that opsonization of nanoparticles
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10
15
20
25 30 Time (d)
35
40
45
Fig. 6 Survivaltime of mice(bearinghepaticmetastases)untreated
(A) or
treated with 10 mglkg intravenously of free DXR (0) or PIHCA nanosphere-bound DXR (0). Single administrationon day 7 . (From ref. 129.)
by some specific plasma proteins enhances the uptake of the carrier by macrophages [101,138].Accordingly, one possible approach to increase the efficiency of encapsulated immunomodulators could be, for instance, to use nanoparticles precoated with opsonic proteins. Encouraging results have already beenobtained in vitro and in vivo with protein-coated poly(L-lactic-co-glycolic acid) microspheres leading toan increase of macrophage activity toward tumoral cells [139]. Peroral administration of M-Chol-loaded nanocapsules has led to inconsistent results, and further studiesare needed to evaluate the potentialities of this route of administration [131]. A phase I study recently performed with DRX-loaded PIHCA nanospheres has shown that the nanosphere formulation seems to berelatively well tolerated [83]. Allergic reactions occurring at the beginning of the study were attenuated by increasing the infusion time from 10 to 60 minutes. Moreover, dextran 70, an adjuvant contained in the drug mixture, was suspected of being the causative agent of the remaining allergic episodes. Although unexpected side effects such as fever or bone pain were recorded, therewas no cardiac toxicity among the 18 patients evaluated. It was not possible to affirm that DXR-loaded nanospheres can completely avoid cardiotoxicity, because the number of patients was small and the maximal cumulative dose waslow(180mg/mz).Normally, the range of cumulative dose required to produce cardiotoxicity is 50-700 mg/m [42].
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As for free DXR, the dose-limiting factor of the DXR-loaded nanospheres was neutropenia. Interesting results have been obtained with magnetic nanospheres [121,124,125]. The administration of anticancerdrug-loaded magnetic nanospheres to rats bearing subcutaneous Yoshida sarcoma (tail) was assowidespread metasciated with a high rate of tumor remission. No deaths or tases occurred in the nanosphere-treated groups. As discussed in Section III.B, such results could be explained partly by the facilitated accessibility of the tumor to the magnetic field and by the intra-arterial injection of the magnetic nanospheres. Amongthestudies involving nanoparticles coupled toanticancer drugs, some important issues have sometimes been neglected. In many cases,thetreatment was initiated atthesamemomentor few hours following the tumor cells inoculation [84,86,107,120,122]. According to Poste [140], the implantation of tumor cells into host tissue disrupts the local microvasculature and enhances the vascular permeability at the injection site. Vascular repair is generally achieved within 2-3 days. Premature injection of nanoparticles may result in unusually high amounts of cytostaticagent in the tumor. Accordingly, positive resultsobtained with nanoparticles injected much later after the inoculation of tumoral cells providemorereliable information (see Table 4). Furthermore,transplanted tumors growing subcutaneously fail to provide a sufficiently demanding model for evaiuating the effectiveness of agents in,treating metastases [1401. In some studies (see Table 4), failures in demonstrating any difference between the activity of a nanoparticle drug formulation over the solution dosage form may be caused by the presence of a too high amount of unbound anticancerdrug in the nanoparticlesuspension. In vivo, administration of a nanoparticle suspension containing some unbound drug may be of little consequence when the entrapment efficiency of the preparation procedure is high. But, when the proportion of the unbound drug in the preparation reaches, for instance,85-92% of the total administered dose [107], it seems unlikely to anticipate important variations of the activity of the nanoparticlesuspension from the solution formulation.
V.
CONCLUDING REMARKS
Over the last 20 years, considerable progress has been made in the preparation of well-characterized nanoparticle formulations loadedwith a variety of anticancer agents [7]. Despite this considerable amount of work, still little is known about the type of neoplasia which may respond beneficially to a nanoparticulate dosage form. More in vitro and in vivo experiments
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are needed to determine the best conditions requiring the administration of anticancer drug-loaded nanoparticles. It seems possible that nanoparticles will have interesting applications only in limited tumors such as those of the mononuclear phagocyte system (e.g., monocytic leukemia, hairy cell leukemia) or for activating macrophages tumoricidal properties [141]. Increased efficiency of nanoparticle formulations against some drug-resistant leukemia and strong activity against hepatic metastases are examples which illustrate thepossible applications of nanoparticles in the future. Nanoparticles may also be used for sustained-release delivery of cytostatic agents in some specific circumstances. Magnetic targeting isan efficient but expensive and specialized technical approach. The possible accumulation of the Fe,O, in the body will probably restrict its use to the treatmentof severe superficial malignancies that are refractory to other treatments [142]. The extensive uptake of the nanoparticles by the RES and their limited access to extravascular tumors are major obstacles to an efficient drug targeting. Surface modifications of drug-loaded biodegradable nanoparticles mayincrease the half-life of the carrier and/or allow its accumulation in a wider variety of target areas. This latter approachmay open new attractive perspectives in the chemotherapy of cancer. REFERENCES
1. P. K. Gupta, Drug targeting in cancer chemotherapy: A clinical perspective, J.
Pharm. Sci., 79:949 (1990). 2. R. K. Y. &e-Cheng and C. C. Cheng, Delivery of anticancer drugs, Methods Find. Exp. Clin. Pharmacol., 11:439 (1989). 3. R. Duncan, Drug-polymer conjugates: potential for improved chemotherapy, Anti-Cancer Drugs, 3:175 (1992). 4. P. Couvreur,Polyalkylcyanoacrylatesascolloidaldrugcarriers,Crit.Rev. Ther. Drug Carrier Syst., 5:l (1988). 5. P. Couvreur, Liposomes en clinique humaine: le point sur les rksultats thkrapeutiques, in Les Liposomes ( J .Delattre, P. Couvreur, F. Puisieux, et al., eds.), Inserm, Paris, 1993, p. 214. 6. P.M.J.Flandroy,C.Grandfils,and R. J. JkrBme,Clinicalapplications of microspheres in embolization and chemoembolization: A comprehensive review and perspectives, in Pharmaceutical Particulate Carriers. Therapeutic Applications (A. Rolland, ed.), Marcel Dekker, New York, 1993, p. 321. 7. E. Allkmann, R. Gurny, and E. Doelker, Drug loaded nanoparticlesPreparation methods and drug targeting issues, Eur. J. Pharm. Biopharm., 39:173 (1993). 8. J. Kreuter, Evaluation of nanoparticles as drug-delivery systems 1 1 1 : Materials, stability, toxicity, possibilities of targeting, and use, Pharm. Acta Helv., 58:242 (1983).
567
9. R. K. Jain, Transport of molecules across tumor vasculature, Cancer Metastasis Rev., 6559 (1987). of 10. G. Poste, Drug targeting in cancer therapy, Receptor-mediated Targeting Drugs (G. Gregoriadis, G. Poste, J. Senior, and A. Trouet, eds.), Plenum Press, New York, 1984, p. 427. 11. P. Couvreur and F. Puisieux, Nano- and microparticles for the delivery of polypeptides and proteins, Adv. Drug Deliv. Syst., 10:141 (1993). 12. W.T. Bellamy, Prediction of response to drug therapy. A reviewof in vitro assays, Drugs, 44:690 (1992). 13. N. Chiannilkulchai, N. Ammoury, B. Caillou, et al., Hepatic tissue distribution ofdoxorubicin-loadednanoparticlesafteri.v.administrationinreticulomaM5076metastasis-bearingmice,CancerChemother.Pharmacol., 26: 122 (1990). 14. S. Maassen, E. Fattal, R. H. Muller, and P. Couvreur, Cell cultures for the assessmentoftoxicityanduptakeofpolymericparticulatedrugcarriers, S.T.P. Pharma Sci., 3:11 (1993). P. Speiser,Theeffects of 15. E. M. Gipps, P. Groscurth, J. Kreuter,and P. polyalkylcyanoacrylate nanoparticles on human normal and malignant mesenchymal cells in vitro, Int. J. Pharm., 40:23 (1987). 16. Z. A. Tokts, K. E. Rogers,andA.Rembaum,Synthesisofadriamycincoupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity, Proc. Natl. Acad. Sci.USA, 79:2026 (1982). 17. R. C. Oppenheim,E. M. Gipps, J. F. Forbes, and R. H. Whitehead, Development and testing of proteinaceous nanoparticles containing cytotoxics, in Microspheres and Drug Therapy (S. S. Davis, L. Illum, J. G. McVie, and E. Tomlinson, eds.), Elsevier, Amsterdam, 1984, p. 117. 18. K. E. Rogers,B. I. Carr, andZ. A. TokBs, Cell surface-mediated cytotoxicity of polymer-bound adriamycin against drug-resistant hepatocytes, Cancer Res., 43:2741 (1983). 19. L. Treupel, M. F. Poupon, P . Couvreur, and F. Puisieux, Vectorisation de la doxorubicine dans des nanosphtres et rkversion de la rksistance plCiotropiqu Sci. Paris, 313:171(1991). des cellules tumorales, C.R. Acad. 20, A. Astier, B. Doat, M.J. Ferrer, et al., Enhancement of adriamycin antitumor activity by its binding with an intracellular sustained-release form, pol methacrylate nanospheres, in U-937 cells, Cancer Res., 48:1835 (1988). 21, C. Kubiak, P. Couvreur, L. Manil, and B. Clausse, Increased cytotoxicity of nanoparticle-camed adriamycin in vitro and potentiation by verapamil and amiodarone, Biomaterials, 10553 (1989). 22. C. Morin, G. Barratt, H. Fessi, et al., Nitric oxide synthesis in rat alveolar macrophages is stimulated by an encapsulated immunomodulator, Proc. 6th Int. Conf. Pharm. Technol., 550 (1992). 23. A. Smith and I. M. Hunneyball, Evaluation of poly(1actic acid) as a biodegradable drug delivery system for parenteral administration, Int. J. Pharm., 30:215 (1986). 24.P. Couvreur, L. Grislain, V. Lenaerts, et al., Biodegradable polymeric nano-
568
Leroux et al.
25.
26. 27. 28. 29.
30.
31. 32. 33. 34. 35. 36.
37.
particles as drug camer for antitumor agents, in Polymeric Nanoparticles and Microspheres (P. Guiot and P. Couvreur, eds), CRC Press, Boca Raton, 1986, p. 27. P. A. Kramer and T. Burnstein, Phagocytosis of microspheres containing an anticancer agent by tumor cells in vitro, Life Sci., 19515 (1976). R. C. Oppenheim and N. F. Stewart, The manufacture and tumour cell uptake of nanoparticleslabelledwithfluoresceinisothiocyanate,Drug.Dev.Ind. Pharm., 5563 (1979). Z . A. Tokb, K. E. Rogers,A.M.Daniels,and J. R. Daniels, Increased cytotoxic effects by polymer-bound adriamycin are mediated through the cell surface, Proc.Am. Assoc. Cancer Res., 24:255 (1983). P. Guiotand P. Couvreur,Quantitativestudy of theinteractionbetween polybutylcyanoacrylate nanoparticles and mouse peritoneal macrophages in culture, J. Pharm. Belg., 38:130 (1983). Z .A. Tokts, K. L. Ross, andK. E. Rogers, Use of microspheres to direct the cytotoxic actionof adriamycin to the cell surface, in Microspheres and Drug Therapy (S. S. Davis,L.Illum, J. G.McVie,and E. Tomlinson,eds.), Elsevier, Amsterdam, 1984, p. 139. J. M. BtguC, A. Rolland, G. Merdrignac, et al., Etude de la ptnttration de nanoparticules de polymtthacrylate dans des cultures primaires dhbpatocy de rat et dhomme et des lignCes cellulaires dhtpatomes: application A la vectorisation dagents cytostatiques utilisCs en chimiothtrapie anticanckreuse, Arch. Int. Physiol. Biochim. 96:A413 (1988). A. Astier, G. Benoit, B. Doat, et al., Activitt antitumorale de ladriamycine J. lite B des nanoparticules sur 2 ligntes cellulaires canctreuses humaines, Pharm. Clin., 7:43 (1988). A.Rolland, J. M. Btgut, R. Le Verge,andA.Guillouzo,Increaseof doxorubicin penetration in cultured rat hepatocytes by its binding to polymethacrylic nanoparticles, Int. J. Pharm., 53:67 (1989). G. M. Barrat, W. P. Yu, H. Fessi, et al., Delivery of MDP-L-alanylcholesterol to macrophages: Comparison of liposomes and nanocapsales, Cancer J., 2:439 (1989). C. Cuvier, L. Roblot-Treupel,J. M. Millot, et al., Doxorubicin-loaded nanospheres bypass tumor cell multidrug resistance, Biochem. Pharmacol., 44509 (1992). C. Morin,G . Barratt, H. Fessi, et al., Biodegradable nanocapsules containing a lipophilic immunomodulator: drug retention and tolerance towards macrophages in vitro, J. Drug Targeting, 1:157 (1993). F. Nemati, A. C. De Verditre, C. Dubernet, et al., Cytotoxicity and uptake of free doxorubicin and doxorubicin-loaded nanoparticles in sensitive and multidrug-resistantleukaemicmurinecells,Proc.6thInt.Conf.Pharm. Technol., 3:93 (1992). F. NCmati, C. Dubernet, A. C. De Verditre, et al., Some parameters influencing cytotoxicity of free doxorubicin and doxorubicin-loaded nanoparticles in sensitive and multidrug resistant leucemic murine cells: incubation time, nu ber of nanoparticles per cell, Int. J. Pharm., 10255 (1994).
Drug-Loaded Nanoparticles in Cancer Chemotherapy
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38. A. C. De VerdiBre, C. Dubernet, F. Nemati, et al., Uptake of doxorubicin from loaded nanoparticles in multidrug-resistant leukemic murine cells, Cancer Chemother. Pharmacol. ,33504 (1994). 39. S. Bennis, C. Chapey, P. Couvreur, and J. Robert, Enhanced cytotoxicity of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres. against multidrug-resistant tumour cells in culture, Eur. J. Cancer, 30A:89 (1994). 40. C. Morin, G. Barratt, H. Fessi, et al., Improved intracellular delivery ofa J. Immunopharmuramyl dipeptide analog by means of nanocapsules, Int. macol., 16:451 (1994). 41. I. Seyler, C. Morin, G. Barratt, et al., Induction of macrophage NO-synthase by an immunomodulator entrapped within polymeric nanocapsules, Eur. J. Pharm. Biopharm., 41:49 (1995). 42. P. A. J. Speth, Q. G.C.M.VanHoesel,andC.Haanen,Clinicalpharmacokinetics of doxorubicin, Clin. Pharmacokinet., 1515 (1988). 43. V. Ling, Multidrug resistance and P-glycoprotein expression, Ann. N.Y. Acad. Sci., 507:7 (1987). 44. T. Ban, Pleiotropic, multidrug-resistant phenotype and P-glycoprotein: A review, Chemotherapy, 38:191 (1992). F. Nemati,C. De VerdiBre, et al.,Mechanism of actionof 45.C.Dubernet, polyalkylcyanoacrylate doxorubicin-loaded nanoparticles in the reversion of resistance of P388/ADR, Proc. 1st World Meeting APGI/APV, 1:473 (1995). 46. N.A1 Khouri Fallouh, L. Roblot-Treupel, H. Fessi, et al., Development of a new process for the manufacture ofpolyisobutylcyanoacrylatenanocapsules, Int. J. Pharm., 28:125 (1986). 47. S. Bonduelle, C. Foucher, J. C. Leroux, et al., Association of cyclosporin to isohexylcyanoacrylate nanospheres and subsequent release in human plasma in vitro, J. Microencaps., 9:173 (1992). 48. A. Labib, V. Lenaerts, F. Chouinard, et al., Biodegradable nanospheres containing phtalocyanines and naphthalocyanines for targeted photodynamic tumor therapy, Pharm. Res., 8:1027 (1991). 49. B.A.Chabner,Biologicalbasisforcancertreatment,Ann.Intern.Med., 118:633 (1993). 50. D. Lasic, Liposomes, Am. Sci., 80:20 (1992). 51. W. M. Bertling,M.Gareis, V. Paspaleeva, et al.,Useofliposomes,viral capsids,andnanoparticlesasDNAcarriers,Biotechnol.Appl.Biochem., 13:390 (1991). 52. G. Schwab,C.Chavany, I. Duroux, et al.,Antisenseoligonucleotidesadsorbed to polyalkylcyanoacrylatenanoparticlesspecificallyinhibitmutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice, Proc. Natl. Acad. Sci. USA, 91:10460 (1994). 53. G. Kohler and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256:495 (1975). vie, Immunoconjugates:applicationsintargeteddrug 54. B. P. Ram and P. delivery for cancer therapy, Pharm. Res., 4:181 (1987). 55. C. Kosmas, H. Linardou, and A. Epenetos, Review: advances in monoclonal antibody tumour targeting,J. Drug Targeting,1:81 (1993).
56. L. Illum and P. D. E. Jones, Attachment of monoclonal antibodies to microspheres, Methods Enzymol., 112:67 (1985). 57. L. Illum, P. D. E. Jones, J. Kreuter, et al., Adsorption of monoclonal antibodies to polyhexylcyanoacrylatenanoparticlesandsubsequentimmunospecific binding to tumour cells in vitro, Int. J. Pharm., 17:65 (1983). 58. P. Couvreur and J. Aubry, Monoclonal antibodies for the targeting of drugs: application to nanoparticles,inTopics in PharmaceuticalSciences(D.D. Breimer and P. Speiser, eds.), Elsevier, Amsterdam, 1983, p. 305. 59. Y. Akasaka, H. Ueda, K. Takayama, et al., Preparation and evaluation of bovine serum albumin nanospheres coated with monoclonal antibodies, Drug Design Deliv., 3:85 (1988). 60. C. Kubiak, L. Manil, and P. Couvreur, Sorptive properties of antibodies onto cyanoacrylic nanoparticles, Int. J. Pharm., 41:181 (1988). 61. L. Manil, L. Roblot-Treupel, and P. Couvreur, Isobutyl cyanoacrylate nanoparticlesasasolidphaseforanefficientimmunoradiometricassay,Biomaterials, 7:212 (1986). 62. K. J. Widder, A. E. Senyei, H. Ovadia, and P. Y. Paterson, Magnetic proteinAmicrospheres:arapidmethodforcellseparation,Clin.Immunol. Immunopathol., 14:395 (1979). 63. K.J.Widder, A. E. Senyei, H. Ovadia,and P. Y. Paterson,Specificcell binding using staphylococcal protein A magnetic microspheres, J. Pharm. Sc 70:387 (1981). 6 4 . M. R. Kaplan, E. Calef, T. Bercovici, and C. Gitler, The selective detection of cell surface determinants by means of antibodies and acetylated avidin attached to highlyfluorescentpolymermicrospheres,Biochim.Biophys. Acta, 728:112 (1983). Le Verge, andB. Genetet, Anew immunoreagent 65. D. Bourel, A. Rolland, R. for cell labeling-CD3 monoclonal antibody covalently coupled to fluorescent polymethacrylic nanoparticles, J. Immunol. Methods, 106:161 (1988). Le Verge, Monoclonal antibodies 66. A. Rolland, D. Bourel, B. Genetet, and R. covalently coupled to polymethacrylic nanoparticles: in vitro specific targetin to humanT lymphocytes, Int. J. Pharm., 39:173 (1987). 67. L. Illum, P. D. E. Jones, R. W. Baldwin, and S. S. Davis, Tissue distribution of poly(hexy1 2-cyanoacrylate) nanoparticles coated with monoclonal antibodies in mice bearing human tumor xenografts, J. Pharmacol. Exp. Ther., 230:733 (1984). X. 68. T. Laakso, J. Anderson, P. Artursson, et al., Acrylic microspheres in vivo. Elimination of circulating cells by active targeting using specific monoclonal antibodies boundto microparticles, Life Sci., 38:183 (1986). Y .Kato, Drug-camer property of 69. K. Sugibayashi,Y. Morimoto, T. Nadai, and albumin microspheres in chemotherapy. I. Tissue distribution of microsphereentrapped 5-fluorouracil in mice, Chem. Pharm. Bull., 25:3433 (1977). Y. Morimoto, T. Nadai, et al., Drug-carrier property of albumin 70. K. Sugibayashi, microspheres in chemotherapy. 11. Preparation and tissue distribution in mice o Chem. Pharm. Bull., 27:204 (1979). microsDhere-entraDDed 5-fluorouracil.
571
71. T. Yoshioka, M. Hashida, S. Muranishi, and H. Sezaki, Specific delivery of
mitomycin Cto the liver, spleen and lung: nano- and microspherical of carrier gelatin, Int. J. Pharm., 81:131 (1981). 72. B. Kante, P. Couvreur, V. Lenaerts, et al., Tissue distribution of [3H]actinomycin D adsorbed on polybutylcyanoacrylate nanoparticles, Int. J. Pharm.,
7:45 (1980). 73. A. Rolland, Pharmacokinetics and tissue distributionof doxorubicin-loaded J. Pharm., 42:145 (1988). polymethacrylic nanoparticles in rabbits, Int. of doxorubicin 74. C. Verdun, F. Brasseur, H. Vranckx, et al., Tissue distribution 75. 76. 77. 78. 79.
80.
81.
69:199 (1980). S. J. Douglas, S. S. Davis, and L. Illum, Nanoparticles in drug delivery, Crit. Rev. Ther. Drug Carrier Syst., 3:233 (1987). of colloidal carriers on the disposition and 82. N. Bapat and M. Boroujerdi, Effect 83. 84.
associated with polyisohexylcyanoacrylate nanoparticles, Cancer Chemother. Pharmacol., 26:13 (1990). P. Couvreur and C. Vauthier, Polyalkylcyanoacrylate nanoparticles as drug J. Control. Rel., 17:187 (1991). carrier: present state and perspectives, A. Rolland, Clinical pharmacokinetics of doxorubicin in hepatoma patients after a singleintravenousinjection of free or nanoparticle-bound anthracycline, Int. J. Pharm., 54;113 (1989). J. E. O'Mullane, P. Artursson, and E. Tomlinson, Biopharmaceutics of microparticulate drug carriers, Ann. N.Y. Acad. Sci., 507:120 (1987). K. J. Widder, A. E. Senyei, and D. F. Ranney, Magnetically responsive microspheresandothercarriersforthebiophysicaltargeting of antitumor agents, Adv. Pharmacol. Chemother., 16:213 (1979). I. J. Fidler, Macrophages and metastases. A biological approach to cancer 45:4714 (1985). therapy, Cancer Res., P. Couvreur, B. Kante, V. Lenaerts, et al., ,Tissue distributionof antitumor J. Pharm. Sci., drugs associated with polyalkylcyanoacrylate nanoparticles,
85.
86.
tissue uptake of doxorubicin: 11. Conjugation with isobutylcyanoacrylate nano' particles, Drug. Dev. Ind. Pharm., 19:2667 (1993). J. Kattan, J. P. Droz, P. Couvreur, et al., Phase I clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles, Invest. New Drugs, 10:191 (1992). Y. Morimoto, M. Akimoto, K. Sugibayashi, et al., Drug-carrier property of albumin microspheres in chemotherapy. IV. Antitumor effect of single-shot or on Ehrmultiple-shot administration of microsphere-entrapped 5-fluorouracil lich ascites or solid tumor in mice, Chem. Pharm. Bull., 28:3087 (1980). L.Grislain, P. Couvreur, V. Lenaerts, et al., Pharmacokinetics and distribution of a biodegradable drug-carrier, Int.J. Pharm., 15:335 (1983). J. Kreuter and H. R. Hartmann, Comparative study on the cytostatic effects andthetissuedistributionof5-fluorouracilinafreeformandbound to polybutylcyanoacrylate nanoparticles in sarcoma 180-bearing mice, Oncolog
40:363 (1983).
87.
L. Grislain,. P. Couvreur, and M. Roland, Modification
de la pharma-
572
Leroux et a/.
cocinttique de moltcules associbes aux nanoparticles, S.T.P. Pharma, 1:1038 (1985). J. Kreuter, et al.,Distribution ofpolyhexyl 88. E. M.Gipps,R.Arshady, cyanoacrylate nanoparticles in nude mice bearing human osteosarcoma, J. Pharm. Sci., 75256 (1986). 89. C. Verdun, P. Couvreur, H. Vranckx, et al., Development of a nanoparticle controlled-release formulation for human use, J. Control. 3:205 Rel., (1986). 90. M. Simeonova, T. Ivanova, E. Raikova, et al., Tissue distribution of polybutylcyanoacrylate nanoparticles carrying spin-labelled nitrosourea, Int. J. Pharm., 43:267 (1988). 91. G. Mukherji, R.S. R. Murthy, and B. D. Miglani, Preparation and evaluation ofpolyglutaraldehydenanoparticlescontaining5-fluorouracil, Int. J. Pharm., 50:15 (1989). 92. G. Mukherji, R. S. R. Murthy, and B. D. Miglani, Preparation and evaluation of cellulose nanospheres containing 5-fluorouracil, Int. J. Pharm., 65:l (1990). 93. V. Lenaerts, A. Labib, F. Chouinard, et al., Nanocapsules with a reduced liveruptake:targeting of phthalocyanines to EMT-6 mouse mammary tumour in vivo, Eur. J. Pharm. Biopharm., 41:38 (1995). 94. E. AllBmann, N.Brasseur, 0. Benrezzak, et al., PEG-coated poly(1actic acid) nanoparticles for the delivery of hexadecafluoro zinc phthalocyanine to EMT6 mouse mammary tumours,J. Pharm. Pharmacol., 47:382-387 (1995). 95. S. E. James and A. J. Salsbury, Effect of (~)-1,2-bis(3,5-dioxopiperazin-ly1)propane on tumor blood vessels and its relationship to the antimetastatic 342339 (1974). effect in the Lewis lung carcinoma, Cancer Res., 96. R. K. Jain, Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors, Cancer Res., 50:814s (1990). 97. S. E. Dum, A. Brindley, M. C. Davies, et al., Studies on in-vitro uptake by Kupffer cells and the in-vivo biodistribution of a range of novel polymeric colloids, J. Pharm. Pharmacol., 44(Suppl.):1082(1992). 98. S. Rudt and R. H. Muller, In vitro phagocytosis assay of nano- and microparticles by chemiluminescence. 11. Effect of surface modificationcoatby ing of particles with poloxameron the phagocytic uptake, J. Control. Rel., 2551 (1993). 99. S. Rudt, H. Wesemeyer, and R. H. Muller, In vitro phagocytosis assay of IV.Effect of surfacemodifinano- and microparticles by chemiluminescence. cation by the coating particles with poloxamine and Antarox CO on the phagocytic uptake, J. Control. Rel., 25:123 (1993). 100. J. C. Leroux, F. De Jaeghere, B. M. Anner, et al., An investigation on the role of plasma and serum opsonins on the internalization of biodegradable poly(D,L-lactic acid) nanoparticles by human monocytes, Life Sci., 57;695 (1995). 101. J. C. Leroux, P. Gravel, L. Balant, et al., Internalizationof poly(D,L-lactic acid) nanoparticles by isolated human leukocytes and analysis of plasma 28:471 (1994). proteins adsorbed onto the particles, J Biomed. Mater. Res.,
573
85
102. S. D. Troster and J. Kreuter,Influenceofthesurfacepropertiesoflow contact angle surfactants on the body distribution of 14C-poly(methyl methacrylate) nanoparticles,J. Microencaps., 9:19 (1992). to the bone marrow, 103. L. Illum andS. S.Davis, Targetingof colloidal particles Life Sci., 40:1553 (1987). .M. Allen, A. Gabizon, et al., Sterically stabilized 104. D. Papahadjopoulos, T liposomes: improvement in pharmacokinetics and antitumor therapeutic efficacy, Proc. Natl. Acad. Sci. USA, 88:11460 (1991). 105. S. Unezaki, K. Maruyama, 0. Ishida, et al., Enhanced tumor targeting of J. Drug doxorubicin by ganglioside GM1-bearing long-circulating liposomes, Targeting, 1:287 (1993). 106. S. J. Douglas, S. S. Davis, and L. Illum, Biodistribution of poly(buty1 2J. Pharm., 34:145 (1986). cyanoacrylate) nanoparticles in rabbits, Int. 107. P. Beck, J. Kreuter,R.Reszka,and I. Fichtner,Influenceofpolybutylcyanoacrylate nanoparticles and liposomes on the efficacy and toxicity of the J. Microencaps., anticancer drug mitoxantrone in murine tumour models, 1O:lOl (1993). 108. P. K. Gupta and C. T. Hung, Magnetically controlled targeted micro-camer systems, Life Sci., 44:175 (1989). 109. K. J. Widder, A. E. Senyei, and D. G. Scarpelli, Magnetic microspheres: a model system for site specific drug delivery in vivo, Proceed Soc. Exp. Biol. Med., 58:141 (1978). 110. A. E. Senyei, S. D. Reich, C. Gonczy, and K. J. Widder, In vivo kinetics of magnetically targeted low-dose doxorubicin, J. Pharm. Sci., 70:389 (1981). 111. K. J. Widder, P. A. Marino, R. M. Morris,et al., Selective targeting of magnetic albumin microspheresto the Yoshida sarcoma: Ultrastructural evaluation of microsphere disposition, Eur. J. Cancer Clin. Oncol., 19:141 (1983). 112. P. K. Gupta,C. T. Hungand N. S. Rao,Ultrastructuraldisposition of adriamycin-associated magnetic albumin microspheres in rats, J. Pharm. Sci. 78:290 (1989). 113. J. M. Gallo, P. K. Gupta, C. T. Hung, and D. G. Perrier, Evaluation of drug delivery following the administration of magnetic albumin microspheres containing adriamycin to the rat, J. Pharm. Sci., 78:190 (1989). 114. P. K. Gupta and C. T. Hung, Albumin microspheres. V. Evaluation of parameters controlling the efficacy of magnetic microspheres in the targeted delivery of adriamycin in rats, Int. J. Pharm., 5957 (1990). 115. J. M. Gallo,C. T. Hung, P. K. GuptaandD. G. Perrier,Physiological pharmacokinetic model of adriamycin delivered via magnetic albumin microspheres in therat, J. Pharmacokinet. Biopharm., 17:305 (1989). 116. P. K. Gupta and C. T. Hung, Effect of carrier dose on the multiple tissue disposition of doxorubicin hydrochloride administered via magnetic albumin microspheres in rats,J. Pharm. Sci., 78:745 (1989). 117. P. K. Gupta and C. T. Hung, Targeted delivery of low dose doxorubicin hydrochloride administered via magnetic albumin microspheres in rats, J. Microencaps., f
574
Leroux et al.
118. A. Ibrahim, P. Couvreur, M. Roland, and P. Speiser, New magnetic drug camer, J. Pharm. Pharmacol., 3559 (1983). 119. F . Brasseur, P. Couvreur, B. Kante, et al.,ActinomycinDadsorbed on polymethylcyanoacrylate nanoparticles: Increased efficiency against an experimental tumor, Eur. J. Cancer, 16:1441 (1980). 120. K. Sugibayashi, M. Akimoto,Y. Morimoto, et al., Drug-carrier property of albumin microspheres in ckemotherapy. 111. Effect of microsphere-entrapped 5-fluorouracilon Ehrlich ascites carcinoma in mice, J. Pharm. Dyn., 2:350 (1979). 121. K.J. Widder, R. M. Moms, G. Poore, et al., 'Ihmor remission in Yoshida sarcoma-bearing rats by selective targeting of magnetic albumin microspheres containing doxorubicin, Proc. Natl. Acad. Sci. USA, 78579 (1981). 122. Y. Morimoto, K. Sugibayashi, and Y. Kato, Drug-camer property of albuV . Antitumoreffectofmicrosphereminmicrospheresinchemotherapy. entrapped adriamycin on liver metastasis of AH 7974 cells in rats, Chem. Pharm. Bull., 29:1433 (1981). 123. P. Couvreur, B. Kante, L. Grislain, et al., Toxicity of polyalkylcyanoacrylate nanoparticles 11: doxorubicin-loaded nanoparticles, J. Pharm.Sci.,71:790 (1982). 124. K. J. Widder, R. M. Moms,G.A.Poore, et al.,Selectivetargeting of magnetic albumin microspheres containing low-dose doxorubicin: Total remission in Yoshida sarcoma-bearingrats, Eur.J. Cancer Clin. Oncol., 19:135 (1983). 125. R. M. Morris, G. A. Poore, D. P. Howard, and J .A. Sefranka, Selective targeting of magnetic albumin microspheres containing vindesine sulfate: total remission in Yoshida sarcoma-bearing rats, in Microspheres and Drug Therapy (S.S. Davis, L. Illum, J . G . McVie,and E. Tomlinson,eds.), Elsevier, Amsterdam, 1984, p. 439. 126. J. Kreuter, Factors influencing the body distribution of polyacrylic nanoparticles, in Drug Targeting, (P. Buri and A. Gumma, eds.), Elsevier, Amsterdam, 1985, p. 51. : Brasseur, C. Verdun, P. Couvreur, et al., Evaluation experimentale de 127. F I'efficacitC thkrapeutique de la doxorubicine associee aux nanoparticules de polyalkylcyanoacrylate, Proc. 4th Int. Conf. Pharm. Technol., 5:177 (1986). 128. N. Chiannilkulchai, Z. Driouich, J. P. Benoit, et al.,Nanoparticules de doxorubicine: vecteurs colloidaux dans le traitement des mttastases hepatiques chez I'animal, Bull. Cancer, 76:845 (1989). . P. Benoit, et al.,Doxorubicin-loaded 129. N. Chiannilkulchai, Z.Driouich, J nanoparticles: increased efficiency in murine hepatic metastases, Sel. Cancer Ther., 5:l (1989). 130. P. K. Gupta, C. T. Hung, and F. C. Lam, Applicationof regression analysis in the evaluationof tumor response following the administration of adriamycin either as a solution or via magnetic microspheres to the rat, J. Pharm. Sci., 79:634 (1990). 131. W. P. Yu, G . M. Barrat, J .Ph. Devissaguet, andF. Puisieux, Anti-metastatic
575
132. 133. 134. 135. 136. 137. 138.
139. 140. 141. 142.
activityinvivoof MDP-L-alanyl-cholesterol (MTP-CHOL)entrappedin nanocapsules, Int.J. Immunopharmacol., 13:167 (1991). W.P.Yu, C. Foucher, G. Barratt,et al., Antimetastatic activity of muramyl dipeptide-L-alanyl-cholesterol incorporated into various typesof nanocapsules, Proc. 6th Int. Conf. Pharm. Techno]., 3:83 (1992). P. Blagoeva, R. M. Balansky, T. J. Mircheva, and M. I. Simeonova, Diminished genotoxicity of mitomycin C and farmorubicin included in polybutylcyanoacrylate nanoparticles, Mutat. Res., 268:77 (1992). S. Gibaud, J. P. Andreux, C. Weingarten, et al., Increased bone marrow toxicityofdoxorubicinboundtonanoparticles,Eur. J. Cancer,30A:820 (1994). L. Manil, P. Couvreur, and P. Mahieu, Acute renal toxicity of doxorubicin (adriamycin)-loaded cyanoacrylate nanoparticles, Pharm. Res., 12:85 (1995). E. AIlCmann, S. V. Kudrevich, K. Lewis, et al., In vivo photodynamic activity of hexadecafluoro zinc phthalocyanin loaded in PEG-coated nanoparticles, Proc. 1st World Meeting APGIIAPV, 1:487 (1995). G . Barratt, F . Puisieux, W. P. Yu, et al., Antimetastatic activity of MDP-Lalanyl-cholesterol incorporated into various types of nanocapsules, Int. J. Immunopharmacol., 16:457 (1994). T. Blunk, D. F. Hochstrasser, J. C. Sanchez, et al., Colloidal camers for on surface intravenous drug targeting: plasma protein adsorption patterns modifiedlatexparticlesevaluatedbytwo-dimensionalpolyacrylamidegel electrophoresis, Electrophoresis, 14:1382 (1993). Y. Tabata andY. Ikada, Protein precoating of polylactide microspheres containingalipophilicimmunopotentiatorforenhancementofmacrophage phagocytosis and activation, Pharm. Res., 6:296 (1989). G. Poste and R. Kirsh, Site-specific (targeted) drug delivery in cancer chem therapy, Biotechnology, 1:869 (1983). R. Kirsh, P. J. Bugelski, and G. Poste, Drug delivery to macrophages forthe therapy of cancer and infectious diseases, Ann. N.Y. Acad. Sci., 507:141 (1987). D. F. Ranney and H. H. Huffaker, Magnetic microspheres for the targeted controlled release of drugsanddiagnosticagents,Ann.N.Y.Acad.Sci., 507:104 (1987).
17
Cosmetic Applications of Liposomes
Gdrard Redziniak and Pierre Perrier
Parfums Christian Dior, Saint Jean de Braye, France
I. Introduction
11. Definition: A Phospholipid Vesicle
1 1 1 . Manufacturing-From A. Methods B. Composition
IV. V. In Vivo Applications the Gram to the Ton
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Liposomes and Cutaneous Cell Interactions
1.
INTRODUCTION
Liposomes were first described by Alec Bangham [l] 30 years ago. At the end of the 1960s, liposomes were regarded as efficient and specific drug carriers capable of carrying the drugs they encapsulated directly to a targeted cellular site. However, in spite of a considerable amount of enthusiasm in the early literature, therewas still no product containing liposomes on the market by the end of 1985. At that time, the majority of investigators were rather pessimistic: the initialexcitement has given way to intense scientific activity in a num577
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ber of laboratories all over the world, but a marketable realistic product appears to be elusive [2]. Bangham [3] was more optimistic when he wrotegazing into a crista1 ball I think I can see a bright future [for liposomes]. In 1986, the first commercial productincorporating liposomes identical to those described by Bangham appeared in the market (Capture@). At the same time, a synthetic one made by nonionic surfactants [4] also was launched (Niosomes@), but these first products were not drugs; they were cosmetics.
II. DEFINITION: A PHOSPHOLIPID VESICLE
Liposomes are artificial spherical microcapsules made up of phospholipids that are able to encapsulate active ingredients in their structure (Fig. 1). The same phospholipids are the main constituent of cell membrane(s) where they act as a selective barrier and as a holder of the membrane proteins [6]. Phospholipids are amphiphilic molecules of the glycerophospholipids class made of an hydrophobic tail (fatty acids) and a polar hydrophilic head (e.g., choline, serine, inositol). When phospholipids are dispersed in water, hydrophobic tails aggregate together as far as possible from water molecules, whereas the hydrophilic heads come in contact with
Hydrophilic molecule
holipids
Amphiphilic molecule Cholesterol
Fig. l Schematic representation of a multilamellar liposome.
579
water. Thus, a layer is created in which the fattyacids tails are directed to the inside of the membrane and the polar head is the outside. When phospholipids swell and hydrate in water, they form vesicles made of one orseveral concentric bilayers which are either surrounded or divided by one orseveral aqueous compartments. These vesicles were first used by biophysicians as biological membrane models in order to study their physiological properties, especially their permeability. In 1965, Bangham called these vesicles liposomes and demonstrated that they are closed system capable of developing almost spontaneously from natural or synthetic phospholipids when they are in presence of an aqueous medium.
111.
MANUFACTURING-FROM THE GRAM TO THE TON
A. Methods
The first liposome preparation method was naturally proposed by Bangham using a rotary evaporator. This method is still today considered as the reference technique. Other techniques have been described: the socalled ethanol injection technique[7];the reverse phase evaporation technique [8]; the dialysis process [9]; the freezing-thawing method [lo]; the spray-drying technique [ll]. In this latter process, natural or synthetic phospholipids and other liposome membrane constituents (e.g., sterols, lipophilic ccactive7 ingredient, antioxidants) are dissolvedin an organic solvent (chloroform, dichloromethane, methanol). This solution is then pulverized in a fluidized gas stream to yield a preliposomesfine powder (Fig. 2). Then this powder is rehydrated in an aqueous solution containing hydrophilic activeingredient(s)(e.g.,peptides, vitamins, coenzymes, enzyme activators or inhibitors; anti-free radicals) and a dispersion of large liposomes is obtained by simple stirring. This dispersion is then refined by extrusion under high pressure [12]. Before homogenization, liposomes are multilamellar, and their size depends on the phospholipidic mixture, but varies between 0.5 and a few micronmeters. After homogenization, the average size of a liposome is about 0.1 pm. These liposomes are mainly small unilamellar vesicles (SMV) small unilamellar vesicles (SUV) (Fig. 3). Encapsulation rates varies from 1 to 30% for hydrophilic molecules. It can reach 100% for hydrophobic molecules entrapped in the phospholipidic bilayer. Used at industrial scale, this method allows the productionof large quantities of liposomes ( > l ton by batch).
580 Perrier
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Fig. 2 Preliposomes powder observed by scanning electronic microscopy (x5000). A mixture of hydrogenated phosphatidylcholin (Lucas Meyer, Germany), p. Sitosterol (Kaukas, Finland), Asiaticoside (Indena, Italy), antioxidants are dissolved in an proportion of 80/10/9/1 (w/w)insolvents (dichloromethan-methanol, U l ) . Then this solution is spray-dried.
B. Composition
The chemical composition, and more particularly the choice of the phospholipids used, is an essential step in the development of liposomes. Natural (yolk or soya) or synthetic and hemisynthetic phosphatidylcholins are mostly used. Apart from this phospholipid, it can be useful to add a sterol (cholesterol, beta-sitosterol) to modulate thetransition temperature and the lipidic bilayer microviscosity. Electrocharged componentssuchas dicetyl-phosphate or stearylamine can also be added to provide the liposomes with a negative or positive charge. These charges generate repulsions among sheets and among vesicles and improveencapsulation capacity and stability.
IV. LIPOSOMES AND CUTANEOUS CELL INTERACTIONS
Because of their biochemical nature and their structural identity with cellular membranes, liposomesare able to interact with cutaneous cells. Differ-
58 l
Fig. 3 Liposome suspension observed by transmission electronic microscopy after freeze-fracture (~45000).A preliposomes powderis dispersed in an aqueous solution of peptides and stirred during1 hour. Then the dispersion is homogenized in the high-pressure extruder.
ent interaction types can be foreseen depending on theliposome composition and the type of cells encountered [13]: endocytosis, transfer or exchange of phospholipids, fusion. Some publicationshave allowed us to support or to contradict these various possibilities; studies performed in our laboratories enable us to bring new information. It is well known that in various cellular types (e.g., neurons,lymphocytes), the membranes of the old cells are more rigid than those of the [14]. Invesyoung cells, as they contain more cholesterol sphingomyelin and tigators such as Shinitzky [l51 state that the decrease, with age, of membrane fluidity isconsistent with cellular metabolism slowing down. In vitro techniqueshave shown the influence of different effectorson the fluidity of the membranes of keratinocytes [16].In these techniques, the membrane fluidity is monitored by fluorescence polarization analysis of (DPH). the hydrophobic probe l-6-diphenyl-l-3-5-hexatriene
Redziniak and Perrier
This test has demonstrated that the addition of cholesterol in the ester culture medium reduces the membrane fluidity and blocks the endocytosis. To the contrary, a specific composition of phospholipids in the form of a calibrated liposomal dispersion (0.1 pm) reverses the effect of the cholesterol ester by increasing the membrane fluidity and reinstoring the endocytosis of the keratinocytes. As an essential element for the rigidity or fluidity of the membraneis the quantitative relationship between cholesterol and phospholipids, one can easily conceive that the unsaturated phospholipids of the liposomes, fluid as they are above their transition temperature,fuse or exchange with the cell membrane. Other in vitro investigations on cell culture have shown an increase of the efficacy of vitamin A esters and particularly the vitamin A propionate when it is encapsulated in liposomes compared with the same substance,atthesameconcentration, and in afree form [17]. These results allow us to assume that, at least in vitro, liposomes interact with the cell membrane of skin cells and transfer their contents into the cell. This interaction can be increased by a specific targeting using a lectinsugar binding strategy [18-191. of the By the discovery of particular endogenous lectins at the surface basal layer keratinocytes and human melanocytes [20], we have built new targeted liposomes for cosmetics application that also can be useful in therapeutical skin treatment.
V. IN VIVO APPLICATIONS
Even if about 40 pharmaceutical products containing liposomes are at different development levels, one can wonder why the cosmetic industry first marketed products containing liposomes. The final aimof the liposomes isthe same: they are carriershaving to convey an active principle to the target cells. For this purpose, the liposomes have to go through different obstacles in maintaining their structure, without releasing their encapsulated molecule(s). These liposomes should be able to recognize the target cells and to adhere to them. The main difficulty encountered in the preparation of liposomes for therapeutical application is their purification; that is, the completeelimination of nonencapsulated active principles, especially if they are toxic. This purification requires sophisticated processes such as gel filtration chromatography, ultracentrifugation, dialysis, and ultrafiltration [21]. In the case of cosmetic products and considering the lack of toxicity of the active ingredients, it is possible to avoid this process by over-
Applications Cosmetic
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concentrating the hydrophilic active principle, allowing its presence both inside and outside of the liposome. Another problem can be faced in therapeutics: stability and targeting or specificity after oral or parenteraladministration. It seems that we do not have to face such problems with cutaneous application: There is no need toavoid bile salts or lytic enzymes or to evade the reticuloendothelial system. The target tissue, the skin, is very visible. In cosmetics, at least for thefirst generation of active principles used, the main concern is to reach cutaneous cells while limiting the passage in blood circulation as much aspossible. In this field, the reference works are those of Mezei [22], who has studied the cutaneous penetration of labeled triamcinolone incorporated into various vehicles (ointment, lotion, and gel withor without liposomes). Compared with other vehicles, utilization of the liposomal form allows an increase five times the molecular concentration in the epidermis and three times in the dermis. On the other hand, there is an extremely low quantity of the molecule in the blood circulation. This first study on the liposome penetration in the skin was followed by numerous works confirmingthe Mezei data [23-281. We can note that the liposomes increase the active ingredients concentration around the targeted cells and then, at the same time, they inof these active ingredients on the cells. It seems logical to crease the action expect an increase of the activity of products containing liposomes. Incorporated in the lipidicphase of the vesicle, vitamin A acid has been shown to be 10 times more efficient than when presented in free form[29]. Besides, we have demonstrated that marketed products containing liposomes and tested by dermatologists revealed a whole series of positive effects;forexample, improvement of cutaneoushydration, visibleimprovement of skin texture, increase in skin glow, decrease in the depth of wrinkles, decrease in eye puffiness, and decrease in the number aging spots. When looking at the results, we understand the extreme interest of this tool in cosmetics in different claims [30]; however, the difficulties and pitfalls must not be neglected, which makes theutilization of the liposome very ticklish. In every new formula containing liposomes, it is essential to check the following: Safety of the preparation,as it can potentiate theeffect of ingredients applied on the skin Physical and chemical stability, since a marketed product must be stored for a long time without modification of its properties
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To be efficient, such controls need sophisticated equipment, for instance electronic microscopy, and checking of size of the liposomes by laser light scattering. It is now taken for granted that liposomes representa major step in cosmetics formulation. However, this requires research studies and severe controls. The choice of the type of liposome andof its formulation must be according to theactive ingredient to be encapsulated and also according to the required target. The final product must be formulated and tested regarding its stability as well as its safety and efficacy.
REFERENCES
1. A. D. Bangham, M. M. Standish, and J. C. Watkins, J. Mol. Biol., 13:238
(1965). 2. M. J. Groves, S.T.P. Pharma Sci., 3:664 (1987). 3. A. D. Bangham, in Lipids and Membranes: Past, Present and Future (J.A.F. Op den Kamp, B. Roelofen, andK. Witz, eds.), Elsevier, Amsterdam, 1986, p. 106. 4. R. M. Handjani-Vila, A. Ribier, B. Rondot, and G. Vanlerberghe, Int. J. Cosmet. Sci., 1:303 (1979). 5. J. F. Danielli andH. Dawson, J. Cell Comp. Physiol., 5:495 (1935). 6. S. J. Singer and G. L. Nicolson, Sciences, 175:720 (1972). 7. J. Batzri and E. D. Korn, J. Cell. Biol., 66:621 (1975). 8. F. Szoka and D. Papahadjopoulos, P.N.A.S., 75:4194 (1978). 9. 0 .Zumbuehl and H. G. Weder, Biochem. Biophys. Acta, 640:252 (1981). 10. G. Strauss in liposome Technology, Vol. 1 (G. Gregoriadis, ed.), CRC Press, Boca Raton, FL, 1984, p. 197. 11. G.RedziniakandA.Meybeck, U.S.Patent 4,508,703,ParfumsChristian Dior Assignee, 1985. 12. G. RedziniakandA.Meybeck, U.S. Patent 4,621,023,ParfumsChristian Dior Assignee, 1986. 13. G. Redziniak, Seminaire INSERM, 214:129 (1991). 14. G. Rouser and A. Yamamoto, Lipids, 3:284 (1968). 15. M. Shinitzky, in Physiologyof Membrane Fluidity, Vol.1(M. Shinitzky, ed.), CRC Press, Boca Raton,FL, 1984, p. 1. 16. T. M.Callaghan, P. Metezeau,H.Gachelin, et al., J. Invest.Dermatol., 92:410 (1989). 17. J. Franchi, M.C. Coutadeur, J. C. Archambault, et al., Nouv. Dermatol., 12:443 (1993). 18. D.Cerdan, C. Grillon, M. Monsigny, et al., Biol. Cell., 73:35 (1991). 19. D. Cerdan, G. Redziniak, C. A. Bourgeois, et al., Exp. Cell Res., 203:164 (1992). 20. A. Denis, C. Kieda, P.Monsigny,andG.Redziniak, Eur. Patent 464077, Parfums Christian Dior Assignee, 1989.
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21. 22. 23. 24. 25. 26.
F. J. Martin, Drug Pharm. Sci., 41:267 (1990).
M. Mezei andV. Gulasekharam, LifeSci., 26:1473 (1980).

J. Lash and W. Wohlrab, Biomed. Biochem. Acta, 45:1295 (1986). W. Wohlrab and J. Lash, Dermatologica, 174:18 (1987). A. Kato, I. Yasuo, and M. J. Yasuo, Pharm. Pharmacol., 39:399 (1987). R. Natmki, S. Tomomichi, R. Matsuo, et al., Pharmacobiol. Dyn., 9:S-12
(1986). 27. N. Weiner, N. Williams, G. Birch, et al., Microb. Agents Chemother., 34:107 (1990). D. Kittayanond, and N. Weiner, Antimicrob. 28. K.Egbaria, C. Ramachandran, Agents Chemother., 34:107 (1990). 29. A. Meybeck, R. Michelon, C. Montastier, and G. Redziniak, French Patent 2591105, Parfums Christian Dior Assignee, 1985. in Liposome Dermatics,GriesbachConference (0. Braun30. A. Meybeck H. I. Maibach,eds.),Springer-Verlag,Berlin, Falco,M.C.Korting,and 1992, p. 341.
18
Cosmetic Applications of Vesicular Delivery Systems
Simon Benita
The Hebrew University ofJerusalem, Jerusalem, Israel
Marie-Claude Martini
Institut des Sciences Phunnaceutiques et Bwlogiques, Lyon, France
Monique Seiller
Universitk de Caen, Caen, France
I. Introduction
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11. %es of Vesicular Delivery Systems A. Liposomes B. Nanoparticulate Systems C. Microemulsions D. Multiple Emulsions E. Liquid Crystals
111. Composition A. Liposomes B. Nanoparticulate Systems C. Microemulsions D. Multiple Emulsions E. Liquid Crystals
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W. Production
A. B. C. D.
Liposomes Nanoparticulate Systems Microemulsions Multiple Emulsions E. Liquid Crystals
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V. Characterization A. Microscopic Analyses B. Rheological Analysis C. Encapsulation or Incorporation Efficiency of the Active Substance
VI. Stability
A. B. C. D. Liposomes Nanoparticles Microemulsions Multiple Emulsions E. Liquid Crystals
A. B. C. D.
VII. Cosmetic Uses

Liposomes Nanoparticles Microemulsions Multiple Emulsions E. Liquid Crystals
1.
INTRODUCTION
Cosmetic technology is constantly evolving in term of raw materials, excipients, and formulations of active ingredients. The new surfactant molecules, the search for original active substances and efficient combinations, and the design of novel vehicles or carriers led to the implementation of new cosmetic systems incontrast to the classic forms such ascreams or gels. The achievements of the extensive research conducted over the last 15 years have resulted in the development of well-controlled innovative delivery systems. Some of these systems have been extensively investigated for their therapeutic potential at the same time that they were being examined quitesuccessfully for theirpossible cosmetic uses. The main objective of this chapter is to concentrate on and fully describe the preparation, characterization,and fate of the various delivery systems following topical application. The recent sophisticated cosmetic preparations based on theinnovative carriers are more attractive than the regular cosmetic preparations, because the sensitive and active cosmetic substances are more fully protected when incorporated in the innovative carrier systems. Furthermore, the carrierscan promote and enhance the cutaneous permeation of specific substances that normally exhibit low skin permeability. This can occur because the stratum corneum, the outermost skin layer, consists of dense,
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overlapping laminates of dead cells with each cell packed with keratin filaments in an amorphousmatrix of proteins with lipids and water-soluble substances, The novel carrier therefore favors the penetrationof the active substance through the stratum corneum, which is recognized as the ratelimiting barrier to the ingress of materials. This is an important feature, since the novel camer can retain oreven sustain the releaseof these active substances in the epidermis leading to skin targeting. Among the various delivery systems or camers, the liposomes have been the most widely studied and successfully marketed by the cosmetic industry. Furthermore,other vesicularsystems(such as nanocapsules, nanospheres, and multiple emulsions) also have been developed and are currently under investigation. If their claimed efficiency and potential is proven, there is no doubt that these vesicular systemswill be rapidly developed and will be present in the cosmetic market in the next decade. It is believed that effective and pioneering cosmetic applications of these vesicular systems stillremain to be discovered. When discovered and developed, such systemswill probably open up a new era of effective and sophisticated cosmetic preparations.
II. TYPES OF VESICULAR DELIVERY SYSTEMS
A. Liposomes
Liposomes were first studied around 1965 as models of biological membranes [l-61. By 1970, their structureand physical-chemicalcharacteristics had led researchers in a number of fields to investigate the potential of liposomes as camers of therapeutical active ingredients. Research on the use of liposomes in the cosmetics sector started more recently. This research has included attempts to achieve an optimal modulation in the release of active substances introduced within this type of vesicle by means of phospholipids. Although research on liposomesin the cosmetics field started fairly late, the well-established ability of liposomes to protect encapsulated active substances and above all the similarity between most of their components and cutaneouslipids turned liposomes quite quickly into the prime camers of dermatological ingredients. Furthermore, they are compatible withmany active, biodegradable substances of limited toxicity. Thus, for a period of someyears now, the use of liposomes in cosmetology has continued to grow. Liposomes are spherical vesicles with anaqueous cavity at their center (Fig. 1). The encasing envelope is made up of a varying number of bimolecular sheets (lamellae)composed of phospholipids. They are either unilamellar, and thus have a single bilayer, or oligolamellar, with multiple
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Fig. 1 Schematic descriptions of unilamellar liposomes.

bilayers, or multilamellar, with a large number of bilayers. Liposomes are either homogeneous with a narrow distribution, or heterogeneous with a broad distribution. Their size varies from approximately 15 to 3500 nm. The type of liposomes-small unilamellar vesicles (SUV),large multilamellar vesicles (MLV), and so forth-depends mainly on: Nature of the amphiphilic lipids selected Composition of the iso-osmotic dispersing solution Method of preparation (ultradispersion) Since their appearance in 1971 as carriers of active substances, a whole series of names other than liposomes can be found in the literature, and have been given to commercial products depending on the nature of the components which form their envelope. Thus, if the envelope is formed of sphingolipids, the perfectly correct nameof sphingosome is given; if some stabilizing agent or active substance is introduced into thevesicles, a more or less imaginative trade name is given; for example, Dermosome, Glycosome, and Brookosome. Thetrade name Ufasome, forexample, was coined to describe vesicles made of a long chain of unsaturated fattyacids. Vesicles whose envelopes are made up of nonionic surfactants are called Niosomes. Their discovery resulted from the physicochemical problems exhibited by liposomes. In fact, the large extent of leakage andthe mediocre stability of liposomes led a number of researchers to design new systems containing amphiphilic lipids different from those used for liposomes; thus, synthetic nonionic lipids were created, allowing the formation of
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vesicles whichpresent the same properties exhibited by liposomes without the disadvantages previously mentioned. These systems have also found their place in all sorts of cosmetic preparations. These syntheticnonionic lipid systems varyin size, shape, and structure but remain in the same ranges as liposomes. They appeared as commercial products in the market shortly after liposomes. The first publication concerning original amphiphilic lipids capable of forming such vesicles, and the first patents dealing with these lipids, came out in1972 and 1975, respectively. llvo other systems may be likened to liposomes, particularly as they are composed primarily of phospholipids. 1. Supramolecular biocarriers (Fig. 2) are very small vesicles (20nm) formed by a gelified polysaccharide hydrophilic core capable of capturing the active substances in the links of a network. This central core is surrounded by a crown of fatty acids attached to the coreby covalent bonds. The whole iscovered by an external sheet of phospholipids attached to the lipid crown by hydrophobic interactions, with their polar heads facing the periphery. The hydrophilic active substances are attached with more orless stability to theheart of the core, and the active lipophilic substances penetrate through the double-lipid membrane. These supramolecular carriers can be either of nautral or synthetic origin. (Fig. 3). 2. A second liposome-like system is totally lipid in nature and acts as a carrier forlipophilic substances (Lipomicrons). The phospholipid molecules, fatty acids, and cholesterol are arranged so as to form spherical
I I
stabilizdby Coulombian interactions
<
20 nm
>
Fig. 2 Schematic description of supramolecularbiocamers (biovectors).
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Fig. 3 Comparative description between pramolecular biocarriers.
(A) natural and
(B) synthetic su-
globules of 400-500 nm. Their periphery is hydrophilic, but their interior, completely lipophilic, may be loaded with liposoluble vitamins or sunscreen agents.
B. Nanoparticulate Systems
The entities known as particulate systems are becoming increasingly popular, just as the delivery systems discussed above, both in the fields of cosmetology and pharmacy. Although in principle particulate systems are generally well known, their proliferation and sometimes their definitions cause some confusion as to their capacity as carriers, as well as their biofate in the stratum corneum. The size of particulate systems, which ranges from a few nanometers to a millimeter, allows the formation of two groups which offer completely different carrier possibilities, as theboundary is around 1pm. For particulate sizes smaller than 1pm (nanoparticles), the insertion of vesicles between corneal cells is possible. Thus, theseare true carriers of active agents, the release kinetics of which are a function of the stability and structureof the system. For sizes larger than 1 pm, the fate of the particulate forms remains superficial, with the possible release of agents or surface activity likely due to the components of the matrix itself. These latter systems are not addressed in this chapter because of their lack of uniqueness as compared with that of nanoparticles, and also because of the tremendous lack of
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Fig. 4 Schematic description of a nanosphere (A) and a nanocapsule (B).
precision in their names anddefinitions. Microspheres, microcapsules, millispheres, thallaspheres, microbeads, or pearls are included in this category. All these different systems share a common spherical form, which is either a matrix or reservoir type. They vary in size ranging from 5 pm (microsphere) to some millimeters (microbeads), and in the materials from which they are composed (e.g. , polyamides, collagens, polysaccharides). Among nanoparticulate systems [7], nanospheres are generally distinguished from nanocapsules. Nanospheres are matrix systems of polymers composed of a solid core with a porous structure and a discontinuous envelope (Fig. 4A). Substances are adsorbed uponthe polymeric materials and generally dissolve inthe polymerization environmental medium,which is most often oriented toward hydrophilic ingredients. Nanocapsulesare reservoir systems made up of a continuous polymerized envelope surrounding a liquid or gelified core (Fig. 4B). The substances are most often of a lipophilic nature, andthey may be composedof a dispersion or oily mixture. Certain nanoparticles are hybrid nanocapsules-nanospheres insofar as theactive substances are intimately integrated into thegelified core of a nanocapsule.
C. Microemulsions
The study of microemulsions [8] is often coupled with the study of micellar solutions because of their structural similarity. The distinctions between the two systems remain to be defined (Fig. 5 ) . Microemulsionsare stable dispersions of a liquid inthe form of spherical droplets whose diameter is lessthan 100 nm. Theyare composed of oil, water, and one ormore surface-active agents. The dispersion is micellar in nature, formed by the aggregation of amphiphilic molecules around either an aqueous core (normal micelles) or around a lipid core (inverse micelles),
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A$
5nm
7nm
20nm
Solution
Micelle
Solubilization Microemulsion
Fig. 5 Formation and schematic description of a microemulsion.

resulting in oil-in-water ( O N ) or water-in-oil (W/O) microemulsions, respectively. They form spontaneously without the use of energy subject to the appropriate choice of surface-active agents that must sufficiently lower the interfacial tension to make it either negligible or a negative value. The main characteristics of microemulsions are low viscosity associated with a Newtonian-type flow, a transparent or translucid appearance associated with the isotropic character of the system, and thermodynamic stability within a specific temperature setting. Certain microemulsions may thus be obtained without heating simply by mixing the components as long as they are in a liquid state.
D. Multiple Emulsions
Multiple emulsions are emulsions of emulsions [9,10]. They can comprise, in each of theirthree constituent phases active hydrosoluble and/or liposoluble substances. Although interesting as cosmetological forms, multiple emulsions are not widely usedin cosmetic preparations. Nevertheless, it is anticipated that these limitations will be removed in the very near future, and that multiple emulsions will be very much in demand in the coming years, particularly if their extended-release abilities are confirmed. The use of multiple emulsions could be almost as broad as thatof the simple emulsions from which they are derived and at least equal to other vesicular systems to which they are similar. One of the considerations in their favor is that they are capable of exhibiting the same properties as those of simple emulsions, and, like these, are applicable directly to the skin, since they can be constituted in the form of white, oily creams with a consistency well suited to proper spreading. Multiple emulsions are emulsions in which the dispersion phase contains another dispersion phase. Thus, a water-in-oil-in water (WlOnV) emulsion is a system in which the globules of water are dispersed in globules of oil, and the oil globules are
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themselves dispersed in an aqueous environment. A parallel arrangement exists in oil-in-water-in oil (O/W/O) type of multiple emulsions in which an internal oily phase is dispersed in aqueous globules, which are themselves dispersed within an external oily phase (Fig. 6). These emulsions are composed of at least two nonmiscible liquids. Emulsifiers, most often selected from amongsyntheticsurface-active agents, are thus required in order to formulate them. In orienting themselves along the two interfaces, these synthetic surface-active agentscalled, respectively, primary surface-active agent (SAA 1) and secondary or surface-active agent ( S A A 2)-each forms a film which, during a longer shorter period of time, gives the system a certain degree of stability. In the case of W/O/W-type emulsions, the only ones which will be described in this chapter, the S A A 1 molecules, which tend to be lipophilic, are arranged on the internal W/O interface, and the S A A 2 molecules, which have a hydrophilic tendency, are arrangedalong the externalO/W surface. Thus, if the natureof the surface-active agents is welladapted to that of the oily phase, two monomolecular films will be formed: the apolar part of each emulsifier is localized in the oil, whereas the polar part is situated in the internal orexternal aqueous phase. It follows that the emulsifiers are organized into abimolecular layer along the interfaces,forming with the oil the envelopeof the vesicle itself.The idea that these systems could serve as useful vehicles for the transport of active ingredients has crystallized only during the 1990s despite thefact that multiple emulsions have been known for a numberof decades. Certain productsnow on the market are called triphase emulsions or triple-phase emulsions. Some of these are not truemultiple emulsions but rather complex preparations composed, for example, of three main components, a simple gelified emulsion, a simple emulsion containing microparticles in suspension, or a simple emulsion in which three active substances are incorporated and present three different activities. One must remember that true multiple emulsions are also sometimes called triple emulsions. Although in theory quadruple and even quintuple emulsions exist, a more precise name would be double emulsion, since in these systems two emulsions co-exist: one emulsion with a continuous oily phase and one emulsion with a continuous aqueous phase.
E. Liquid Crystals
Discovered a centuryago, liquid crystals have been used in many different fields for a number of decades, but their value in cosmetology has been confirmed only in the last few years [11,12]. Liquid crystals are intermediate anisotropic fluids which are between the conventional solid and liquid
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et
Water
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w/o/w
emulsion
Oil
@/W/O emulsion
Water
Oil
+. hydrophilic ADS
0-
hydrophobic ADS
Fig. 6 Schematic representation of O/W/O and WIONV multiple emulsions.
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Fig. 7 Schematic representationof lamellar phases.
isotropic phases. This structure is attributable to a very specific morphology of molecules which must be elongated and present an irregular distribution of electrical charges. These molecules must be arranged in a specific order (Fig. 7).
111.
COMPOSITION
A. Liposomes
1. Phospholipids Phospholipids and specific additives are considered the most important primarymaterials for vesicular delivery systems[13-181. Some phospholipids, like phosphatidylcholine, are of natural origin, and some, like dimyristoyl or phosphatidylcholine dipalmitoyle, are of semisynthetic or synthetic origin. The phospholipids that are most commonly used are the glycerophospholipids. In these substances, glycerol, whichmay be considered the skeleton of the molecule, is esterified in and 2 by long-chain fatty acids and in position 3 by phosphoric positions 1 acid. A number of different hydrophilic molecules can be attachedto this acid, for example, choline and ethanolamine. Although the length of the chain of fatty acids and/or the degree of saturation may vary from C14 to C 1 8 and from1to 2 double bonds,the lipophilic nature of the alkyl chain is relatively constant compared with the hydrophilic part of the chain. In fact, this hydrophilic part of the chain presents different properties and has considerable influence on thecharacteristics of liposomes. A few other phospholipids shouldbementioned in addition to glycerophospholipids. These are less commonly used, but because of their
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great similarity to the lipidsin the skin, they are good candidates for forming liposomes for dermatological use. Sphingolipids are a good example of these: They have a sphingosine skeleton upon which a number of derivatives are grafted. All these phospholipids have the fundamental property of forming flat lamellar sheets in the presence of water within which aqueous compartments alternate with lipid sheets. Under certain temperature and agitation conditions, these flat bilamellar structures may fragment, fold in on themselves, and ultimately become fastened at their ends. They thus become the external wall of water-trapping vesicles, which are themselves in suspension in the water. By choosing certain specific phospholipid fractions, even dry mixtures can be achieved. In thepresence of water, these allow the immediate formation of unilamellar liposomes of the LUV type (proliposomes).
2. Additives I In addition to the phospholipids which constitute the main envelope, two types of additives may be used (see Table 1).The first isa sterol (including phytosterol, dihydrocholesterol, and cholesterol). By localizing themselves in the phospholipid bilayer, these sterols permit phospholipids to modulate the physical and chemical characteristicsof the envelope, which becomes more rigid. As a result of the modified compactness, the permeability will change according to the proportion and the location of these substances. The second type of additive, in addition to, for example, buffers, electrolytes, pH modifiers, and preservatives, comprises ionic substances. Theseareanionic derivatives (phosphatidic acid, dicetylphosphate) or cationic derivatives (stearylamine). The function of these additives is to confer a negative or positive charge on vesicles, thus giving them a greater stability with regard to their aggregation and fusion. They may also cause an increase in the interlamellar space resulting in a greater capacity for the encapsulation of certain active substances. Thus, it is said that, except for liposomes, the essential components of the nonionic surfactant agent vesicle envelopes are, in this case, not phospholipids but rather nonionic synthetic surfactants. They are essentially surface-active agents containing ester or ether bonds. Their hydrophilic part consists of condensation products of polyoxyethylene, polyoses, and most of all polyglycerols (the most effective). The lipophilic part is usually composed of one ortwo hydrocarbon chains, between C12 and C18, either saturated or unsaturated. The additives used are the sameas for liposomes and have the same function. B. Nanoparticulate Systems
The primary constituents of nanospheres and nanocapsules are identical acrylic derivafor both of these types of systems [19]. In essence, they are
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tives, most frequently of the polyalkylcyanoacrylatetype (PACA) or derivatives of copolymers of styrene, lactic and glycolic acids, cross-linked polysiloxames, or biological macromolecules such as albumin, gelatine, or dextran. In order to ensure and to control theparticle sue of nanoparticles of the SAA type, dodecylsulfate or sodium oleate and polysorbates are added. Sometimes, cross-linking agents (glutaraldehyde) or stabilizing substances (polyvinyl alcohol, cellulose derivatives), salt buffers, and protective colloids are incorporated in order to facilitate the formation of the individual nanoparticles in either an aqueous oroily environment. The active substances thus incorporated may be hydrophilic in the case of nanospheres or lipophilic in the case of nanocapsules (Table 2). It should be added that both types of active ingredients may be incorporated invariably into these two types of nanoparticles but that depends on the methods of fixation or incorporationused as depicted in Fig. 8.
C. Microemulsions
Microemulsions are generally systems with four components: water, oil, surfactant, and co-surfactant [20-261. The aqueous phase may contain hydrophilic active ingredients and preservatives, and the fattyphase may be composed of mineral oil,silicone oil, vegetable oil, or esters of fatty acids, all of which are classic ingredients
Table 1. TypicalFormula of a Liposomal Suspension 200 mg Soya lecithin 25 mg Cholesterol Phosphatidic acid 30 mg Hyaluronicic acid 10 mg 5-10 mg Preservative Water to l o g Table 2. Typical Formula of a
Nanocapsule Suspension acid Polylactic Mink oil Poloxamer 188 (Puronic F 68) Phosphatidylcholine Purified water to
125 mg
0.5 m 1
75 mg 75 mg 20 m 1
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D
Fig. 8 Description of the different incorporation patterns of active ingredients in nanospheres and nanocapsules. Active substance association: (A) dissolved in the nanosphere matrix, (B) dissolved in the liquid phase nanocapsule, (C) adsorbed at the nanosphere surface, (D) adsorbed at the nanocapsule surface.
for cosmetic products. The lipid phase may also contain lipophilic active ingredients. The surfactants chosen are generally among the nonionic group because of their good cntaneous tolerance. Certain derivatives of sugar are currently being studied (Table 3). To a lesser degree, and only for specific cases, amphoters are being investigated. The cosurfactants were originally short-chain fatty alcohols (pentanol,hexanol, benzyl alcohol).Theseare most often polyols, esters of polyol, derivatives of glycerol, and organic acids. Theirpurpose is to make the interfacial filmfluidbywedging themselves between the surfactant molecules. Thus, they create a bicontinuous structure allowing a continuous phase inversion; the undetectable transition from a W/O microemulsion into a O W microemulsion when the water to oil proportions are altered.
The nature of the componentshas no significant effect either on the multiple character or on the stability of multiple emulsions [27-301 provided that a specific ratio is maintained between the SAA 1 and SAA 2 rates, the HLB of their mixture, and an optimal concentration of additives. The primary materials capable of producing this sort of system are quite numerous and practically identical to those generally used for simple
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emulsions. The oily phases most frequently utilized in order to form multiple W/O/W emulsions are hydrocarbons, esters, and triglycerides. The emulsifiers are usually nonionic surfactant agents. Someexamples are: for SAA 1 emulsions, the long hydrocarbon chain ester sorbitan, perfluorocarbons derivatives,and most importantly polymeric surfactants; for SAA 2 emulsions, esters of polyoxyethylene sorbitan, copolymers of ethylene propylene oxide, strongly ethoxylated fatty alcohols, and condensation products of polyglycerol (Table4). As in vesicular systems, additives are often introduced in order to control and increase the stability of the system. Along with electrolytes, sugars or glycols are used, most frequently hydrophilic polymers (xantham gum, cellulose derivatives, and carboxyvinylic compounds) introduced in one of the aqueous phases, most commonly in the external phase. Lipophilic substances (waxes, acids or fatty alcohols, silicone derivatives) are introduced into theoily phase.
D. Liquid Crystals
There are two types of liquid crystals: thermotropics and lyotropics [31]. Thermotropic liquid crystals are made up of identical molecules or of a Table 3 . Example of a
Formulation of O N Microemulsion Sucroester Glycerol Jojoba oil Elastin Preservative Purified water q.s.p.
30 5 2 2 0.1
1 0 0
Table 4. Example of a Formulation of

OIWIO Multiple Emulsion
Almond oil Urea Sodium Sorbitan oleate (Span 80) ulfate Magnesium Polyoxyethylated sorbitan stearate (Tween 60) Preservative water Distilled q.s.p.
25 2 3 8 0.7 1
1 0 0
0.1
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Table 5. Example of a Gel Formulation Containing Lyotropic Liquid Crystals
Oxyethylaled (2 OE) oleyl alcohol Oxyethylated (10 OE) oleyl alcohol lose Hydroxyethyl Preservative ater Distilled
2 0.1 100
combination of molecules of the same geometrical form. They are cholesteric liquid crystals-so named because of their helical structure even though they are not always derived from cholesterol. In fact, liquid crystals dispersed in cosmetic preparations are generally basedon derivatives of methyl butyl Rhenol (Licritherm) or equivalent molecules. There are also combinations of cholesteric and synthetic chiral nematic esterson themarket. Liquid crystals constitute more or less thick mesomorphic structures in which the molecules are arranged in a certain order, leading to characteristics somewhere between those of a liquid state and a crystalline state. The kind of liquid crystals known as lyotropics are systems with two or three constituents: water, oil, and surfactant (Table 5). The soluble molecules are amphiphilic. One example is compounds of glycerol stearate and polyethylene glycol, which provide a lamellar structure beyonda given concentration, generally higher than 40%. They may be formed from concentrated micellar solutions or during the production of microemulsions wherein the proportions of the various components lead to a gelified phase. These lamellar structures may also form at the interface of an emulsion.
IV. PRODUCTION A. Liposomes
There area number of processes available for the productionof liposomes [32-401, and appropriate selection would depend on the type of liposome desired: multilamellar or unilamellar and large or small. Only twoof these are described briefly below. The reference method (schematically presented in Fig. 9) was initially developed by Bangham, allowing the obtention of multilamellar vesicles. The process is carried out in four main stages:
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Dissolution of phospholipids and other constituents of the wall coating in a volatile solvent
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U
Evaporation in a rotorevaporator
Solution of wall constituents
Addition of an aqueous solution
p J
... . .*.
Suspension of liposomes
Fig. 9 Description of a typicalprocess for thepreparation of multilamellar liposomes.
1. Dissolution of the components of the envelope in a volatile solvent 2. Evaporation of the solvent under reduced pressure in a rotating evaporator in order to form a lamellar layer of phospholipids along the wall of the evaporator 3. Addition of a generally buffered aqueous solution at a temperature higher than the phase-transition temperature of the phospholipid 4. Stirring the suspension until it has cooled completely
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The active substance tobe encapsulated is added to the organic solvent, if it is lipophilic, or in the aqueous solution,if it is hydrophilic. This method generally produces multilamellar liposomes and is carried out under nitrogen atmosphere. This is one of the oldest methods in use; for purposes of comparison, one of the most recent methods is mentioned below. The stages areas follows: Preparation of a first liquid phase made up primarily of a solution of phospholipids in a single solvent or acombination of solvents Preparation of a second liquid phase, miscible in the solvent or solvents described above, and insoluble for phospholipids, and made up primarily of water Addition, with moderate stirring,of the first phase to the second followed by partial or total elimination of the pure solvent in a way which willpermit the production a colloidal suspension of liposomes adequately loaded
Just as with the preceding procedure, the active ingredients are added to the solvent most suited to themaccording to their solubility properties. This procedure yields multilamellar and especially oligolamellar liposomes of a very uniform size. Small unilamellar liposomes form when multilamellar liposomes obtained through some of the methods described above, for example, are subjected to high-frequency ultrasound waves during a fairly long period of time or to a high-pressure homogenization process with a two-stage homogenizing valve assembly. It is also possible to obtain small unilamellar liposomes by means of other methodswhich circumvent the need for ultrasound; like the method which involves injection of an ethanolic phospholipid solution into the aqueous phase or the method known as detergent removal, which consists of preparing mixed micellesof phospholipid detergent and then eliminating the latter. Just as unilamellar liposomes may be obtained from multilamellar liposomes, it is also possible to obtain large unilamellar liposomes from small unilamellar liposomes. Small vesicles fuse and provoke the formation of large lamellar structures by the addition of calcium ions which function as complexing agents promoting the formationof large vesicles. The above method, which requires thepresence of phosphatidic acid, is less popular than two methods which make use of the injection of ether and evaporation in the inverse phase. The first of these two methods consists of dissolving the phospholipids in ether and then injecting this solution slowly into the aqueoussolution of the active hydrophilic substance to be encapsulated at a specified temperature. The evaporation of the ether causes the spontaneous formation of vesicles inthe aqueous phase.
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The second method makes use of the production of an emulsion with a continuous oily phase after dissolving phospholipids in a volatile lipophilic solvent and the active hydrophilic ingredient to be encapsulated in the aqueous phase. Large liposomes form spontaneously after evaporation of the externalphase of the emulsion. All of the methods cited involve both advantages and disadvantages which will not be detailed in this chapter, since they have been described thoroughly in the literature. Each process is characterized by its possible induced degradation, the extent of its encapsulation efficiency which can be attained, its simplicity, its reproducibility, and its control of the particle size distribution of the liposomes obtained. As for this last point, when the liposome populations are tooheterogeneous, but include the type and size of liposomes desired, common separation methods such as gel filtration, ultrafiltration, ultracentrifugation, dialysis, or membrane diffusion under pressure are employed. used for liposomes is satisfactory for theproducNone of the methods tion of large quantities of monionic surfactant vesicles. Indeed, large quantities of solvents are necessary, and.moreover, ultimately the encapsulation rate is low, since the dispersions are not rich enough in vesicles. A number of patents have been issued on specific methods that do not present any of the disadvantages mentioned above. The most common (and simplest) of these makes use of the formation of a lamellar phase. In summary, it involves the following stages: fusion of the amphiphilic lipid phase at a very high temperature; formation of a lamellar phase by mixing of the amphiphilic lipid phase with the aqueous phase containing the hydrophilic substances; gradual introduction of an iso-osmotic aqueous solution to the aqueous phase previously described; and homogenization, with the help of an efficient homogenizer, during a predetermined period of time, during which the preparation is allowed to reach the ambient temperature and the vesicles are formed.
B. Nanoparticulate Systems
As for liposomes, a large number of processes exist for obtaining nanotype of particulate systems [41,42], and theprocess selected depends on the nanoparticles desired (either nanospheres or nanocapsules), their particle size, and the materials used to form the nanoparticles. Nanospheres are most commonly obtained using polymerization reactions, purified natural macromolecules, or preformed polymers. Nanocapsules are most commonlyobtained through the use of interfacial polymerization reactions preformed polymers. A typical method for the preparation of nanospheres is described in Fig. 10.
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Anionic Polymerization
Fig. 10 Schematic representationof the nanosphere preparationtechnique using the anionic polymerization approach. Most of the methods used for obtaining nanospheres are based on the polymerization of monomers introduced in the dispersed phase of an emulsion or W/O microemulsion or dissolved in a nonsolvent polymer environment. 73vo separate phases are distinguished, a nucleation phase and a growing phase.
1. Methods of Producing Nanospheres There are four main methods for obtaining nanospheres.
a. Preparation in emulsion. Thecontinuous phase is composed of a mixture of monomer S A A water. The monomer + S A A combination produces micelles from 1to 10 nm. Polymerization occurs in the interiorof the micelle, and the particles expand owing to the continuous insertionof monomer molecules into the interior of the micelle until they reach asize of 200 nm. The monomer is soluble in the continuous phase. It is in this phase which promote the initiation of polymerthat free radicals (FR) are formed, ization with the formation of an insoluble polymer. This process involves three stages: (1)the monomeris in the micelle; (2) a gradual incorporation
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of monomer molecules into the micelle is produced, giving rise to a polymerization initiated by FRS, and subsequently a reorganization of oligomers surroundedby SAA, which causes further growth of the micelle; and (3) The fusion of two micelles leads to a certain increase in size.
b. Dispersion-polymerization. The monomer is dissolved in the continuous phase, and nucleation is directly induced in the monomer solution without any diffusion. The oligomers which have formed then turn into aggregates stabilized by molecules of S A A . The polymer is obtained by growth of the aggregates.
c. Polymerization in inverse microemulsion. Dilated nonaqueous micelles are stabilized by a layer of SAA and dispersed in the oil.The process of polymerization is continuous. The number of polymer particles which have formed increases over time, but theirsize remains constant. The formation of polymer particles results from the fusion of many micelles by collision. Thus, each polymer particle contains relatively few chains, which is in contrast to particles formed through other processes. d. Evaporation of the solvent. An organic polymer solution is emulsified in anaqueous phase and followed by evaporation of the solvent.If the solvent and nonsolvent are not chosen carefully, the organic solvent in which the polymer is dissolved can diffuse rapidly into the aqueous solution resulting in the precipitation of the polymer. Therefore, the choice of solvent and nonsolvent is a very delicate matter. They must be conducive to the formation of nanoparticles. Once the nanospheres are formed, the polymer solvent is evaporated under reduced pressure. 2. Methods of Producing Nanocapsules There aretwo principal methods of obtaining nanocapsules. Emuls@cation. Fora lipophilic substanceto be encapsulated, two phases, A and B, are mixed: monomer + alcohol + lipophilic substance represents the dispersed phase, whereas water + S A A represents the continuous phase. The monomer, which isinsoluble in water, is polymerized at the interfaceof the O M emulsion. For a hydrophilic substance, two phases, A and B, are both mixed. The hydrophilic substance is dissolved in water which is emulsified in the lipophilic phase containing the monomerusing anappropriate S A A resulting in a W/O emulsion. The monomer can be methyl, ethyl, or butyl cyanoacrylate. The monomer, which isinsoluble in water, is polymerized at the interfaceof the W/O emulsion. In case of an inverse microemulsion (W/O), the individual size is small. Rinsing is necessary for the solvent to be eliminated.
a.
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b. Evaporation of the solvent. WO different environments areused: S1 = polymer in solution in an organic solvent, and S2 = polymer nonsolventsolution. Oil to becapsulated must be miscible withS1and nonmiscible inS1+ S2. Oil must be minutely dispersed in S1 S2. The polymer capsulates the oil. inwhich lipophilic substances are dissolved. The encapsulation of lipophilic substances can thus be performed easily, whereas the encapsulation of hydrophilic substances is difficult. Whatever method is used, the critical point is the thickness of the membrane, which ranges between 3 and 10 nm, for a total size reaching between 150 and 800 nm.
C. Microemulsions
Owing to the stable thermodynamic character of microemulsions, it is straightforward and easy to obtain them [43-481. A stable W/O emulsion obtained with a lipophilic SAA may be used as a base for preparing an O/W microemulsion. To this emulsion, a hydrophilic aqueous SAA solution is added followed by stirring. A gelified phase appears because of the cubic structure of the product. If hydrophilic SAA is again added, an O/W microemulsion is obtained. In order to prepare a W/O microemulsion, anO/W emulsion should be used stabilized either by an ionic or a nonionic SAA. By means of titration, a cosurfactant (COS) is added. As in the preceding example, a gelified phase appears which becomes fluid and results in the formation of a W/O microemulsion. However, these methods are empirical and relatively crude. Likewise, a microemulsion is almost always created by the establishment of a pseudoternary diagram for which a ratio of SAA/CoS is fixed, representing a sole constituent. The establishment of a ternary diagram is generally accomplished for the purpose of locating the microemulsion or the microemulsion zonesby titration. Using a specific ratio of SAA/CoS, various combinations of oil and SAA/CoSare produced. The water is added drop by drop. After the addition of each drop, the mixture is stirred and examined through a crossed polarized filter. The appearance (transparence, opalescence, isotropy) is recorded, along with the number of phases. In this way, an approximate delineation of the boundaries can be obtained in whichit is possible to refine through the production of compositions point by point beginning with the four basic components.
The operational technique plays an even more important role in the production of multiple emulsions than in the production of simple emulsions
[49-541.
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Aqueous Phase
Oil + lipophilic surfactant
W10 Emulsion
(4
WKb Emulsion
(B)
Hydrophilic surfactant in water
WIOW Emulsion
Fig. 11 Preparation of a multiple emulsion by a two-step procedure. (A) Step 1: formulation of W/O emulsion. (B) Step 2: formulation of W/Oi?V emulsion. Four main types of protocols are recommended:
1. The two-stage procedure as depicted inFig. 11 (the most frequently utilized method). The name is not precisely indicative, since other methodsinvolve two stages. 2. Dispersion of a lamellar phase. This method is still rarely used. It is similar to the procedure described above for obtaining nonionic surfactant vesicles. 3. Dispersion of an isotropic oil solution in water. This procedure is recommended more for the formation of W/O/W-type microemulsions. 4. Phase inversion. This procedure isbecoming more commonly used. Here again, the name is inaccurate, since it does not actually involvea phase inversion.
Each of the fourprotocols is executed in almost identical fashion: First, either a continuous oily phase simple emulsion,alamellar phase, or an isotropic oil solution is produced at 70-80C ? 1. Whichever system is used, the water, the oil, and the emulsifiers (the proportionsof the three components vary depending on which
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type of system is desired) are combined with the help of a classic turbine mixer during a period of approximately 30 min. IA e r , for the protocol designated double-stage, the continuous oil phase emulsion is poured slowly into the aqueous phase. For the other three procedures, it is the water which is gradually introduced either into the lamellar phase or the isotropic oil emulsion or the continuous oily phase emulsion. The second dispersion is likewise produced by means of a turbine stirrer, most commonly at room temperature, during a period of approximately 30 min.
Of course, each procedure offers both advantages and disadvantages. The two-stage procedure has the major advantage of having very well-regulated steps; in theory, it is possible to fix the amount of internal water. Its disadvantage is that it is not very reproducible, although this is likely because of the second emulsification, which is a critical stage in the procedure. In fact, theprimary W/O emulsion is quite viscous and is difficult to disperse. This is the stage when a shearingshould be executed, and certain globules of oil which have just formed are atrisk of breaking down. If this happens, someof the internal water may become mixed withsome of the externalwater. The process which involvesdispersion in the waterof a lamellar phase has the advantage of necessitating only a single emulsification stage. The initial phase is an anisotropic phase; it is thermodynamically stable, and easy to achieve. One of the limitations of this procedure stems from the fact that not all of the surfacantsform a lamellar phase. Where such a phase does exist,the HLB (hydrophile lipophile balance) is often elevated,which is undesirable for the stability of a multiple emulsion. Furthermore, only a small quantity of oil (rarely exceeding 10%) is incorporated in the lamellar phase. This procedure is best adapted to obtaining nonionic surfactant vesicles, since, in this kind of vesicle, the amount of apolar substancesis smaller. The procedure thatinvolves the dispersion in water of an isotropic oil solution presents more or less the same advantage as the procedure detailed in the preceding paragraph, in that only one emulsification process is involved. The initial phase is a pure phase, stable, and easy to obtain. The main disadvantage is the incorporationof a low concentration of water-soluble compounds in inverse micelles (seldomly exceeding 10%). The disadvantages which are common to these two procedureshave to do with the necessarily elevatedquantities of surface-active agents needed. Furthermore,because of the dispersion in water of the lamellaror isotropic oil phases, in which the initial water is not truly emulsified in the form of globules, it is difficult to know whether the water transported by
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this dispersion still remains soluble, and if so, then to determine the total water quantityin the internal aqueous phase. The advantages of thephase inversion procedure arethat itis easy to perform and that it provides a precisely known rate of internal aqueous phase similar to thedouble-stage procedure.Moreover, unlike the doublestage procedure,it is reproducible. However, it entails the disadvantage of having two emulsification stages, andof being a very delicate procedure to perform. Indeed, even a very slight excess of water might be sufficient to transform the multiple emulsion into an aqueous simple emulsion type.
E. Liquid Crystals
Special laboratory skills are necessary in order toobtain thermotropicliquid crystals [55,56]. These crystals are, like any chemical molecule, added at the same time as the other constituents of the vehicle in whichthey must, following a change in temperature, present a certain appearance. Obtaining lyotropic liquid crystals is quite easy, and any formulator can produce them. The process consists of mixing, usually by means of a simple turbine mixer, the solvent and the ingredients (e.g., fatty ethoxyl alcohol, phospholipid stearate of glycerol or PEG) which will produce the desired structureabove a certain concentration level. Thus, a lamellar structure could easily be obtained. Most often this structure is not preparedby itself but rather is obtained during the preparation of the cosmetic formulations. Thus, acream or gel may be formed on the spot with ingredients which will produce liquid crystals equally well.
V . CHARACTERIZATION
Since all the particulate systems described above are basically dispersions, the approach used for their characterization is almost identical [57-731. The following features pertain to all of them: particle size distribution, morphology analysis, electrical charge nature, creaming or sedimentation rate, and so forth; rheological behavior; and rate.and extent of the encapsulated active substances. WO approaches are used for the characterization of these systems. The first is simple and of a systematic nature. Its objective is to determine rapidly and without difficulty the type of system obtained.
A. Microscopic Analyses
Microscopic examination is the first test performedin order toidentify the type of particulate systems obtained [57-651. Moreover, it constitutes an,
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Fig. 12 TEM photomicrograph of multilamellar liposomes following negative staining with phosphotungstic acid.
excellent means of following up on the physical stability of these systems as a functionof prolonged storage times. The most studied characteristic of liposomes is their dimensions. If the vesicles are medium sized (on the orderof 1pm), they can be examined under any ordinary optical microscope, a polarized light microscope, or a fluorescent microscope using a markerlike carboxyfhoresceine. However, examination under an electron microscope is necessary for nanoparticles and liposomes smaller than 1 pm, as depicted in Fig. 12. In any event, in order to measure the size, and in order to ascertain the particle distribution of these two types of systems, other methods which are faster and more accurate than microscopic techniques must be used. As for any other dispersed system, the technique used involves the electronic counting of the particles for vesicles that have (for the most part) a diameter greater than 600 nm. Photon-correlation spectroscopy is used for size determination of vesicles of smaller size. For multiple emulsions, optical microscopy isalso used as a common method of analysis, as shown in Fig. 13, to keep track of all these systems and
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Fig. 13 Photograph of a WIOIW multiple emulsion obtained with a normal light microscope (at a magnificationof 1 O O O ) .
to determine the particle size distribution. This method allows the direct measurement of the size of multiple globules with a diameter greater than 0.5 pm, as well as a rough estimate of the percentage of multiple globules compared with simple globules. Furthermore, thesize of the internal aqueous globules (usually on the orderof a micrometer) can be determined by this method. Prior to observation, and in order to measure the actual size of the globules, the multiple emulsions must be well diluted with a solution whose osmotic pressure should be similar to that of the internal aqueous phase. Indeed, dilution with a solution of lower molar concentration would, after the ingress of external water into the internal water, cause swelling and then rupture of the internal aqueous globules. Inversely, dilution with a more highly concentrated solution would precipitate the escape of the internal water and hence a shrinking of the internal aqueous globules. Furthermore, examination under polarized light could sometimesdetect a texture corresponding to a lamellar phase indicating the directional orientation which the two emulsifiers take in the oily phase. As for the vesicular systems described above, microscopy following cryofracture of the multiple emulsion is used in order to enablea precise analysis of the internal aqueous globules. The objective here is to generate accurate information regarding their exact morphology.
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Fig. 14 Photograph of lyotropic liquid crystals forms with polarizing microscope. Some researchers instead of obtaining a direct estimate following observation prefer to use photographs for measurement, and in this way, they follow up on the size and stability of the emulsions. Finally, examination under a polarizing microscopeis also a valid tool for detecting andobserving liquid crystals, as shown in Fig. 14.
B. Rheological Analysis
In order to gain a deeper knowledge of particulate systems, we often turn to rheology [66-701. Because of the diversity offered by this technique, itis possible to characterize the structure of such systems and to follow their evolution over time. In termsof rheological analyses, it means:
l . ViscoelasticOscillator Analyses. The oscillatory experiment consists of applying a sinusoidal stress and recording theconsecutive strain defined as follows:
, C O S W t T(f) = T E(t) = ,COS(Wt
+ S)
where T, and eo are maximal amplitudes of stress and strain, W = 27rN, with N the frequency of strain and S the phase angle of stress/strain. The basic viscoelastic parameters which describe the rheological behavior are:
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G* = TJE,, is the rigidity modulus, and S the phase angle of the stress with respect to the strain (for a viscoelastic material, it is included 0" and for a between 0 and 90"; for apurely elastic material, it equals is the critical stress at perfect newtonian liquid, it equals 90"); and (T")~ which the sample becomes more viscous than elastic.The comparison and G*/27r values gives information about the form and the of (T")~ homogeneity of the droplets.
2. Analyses of the Permanent Flow System.

During the process of sweeping, the system is sheared in a cycle of increasing constraint, then constant constraint, and then diminishing constraint. Thiskind of test provides information about the functionof shearing in the destructuration of the dispersion. It also provides information about themore or less reversible character of the dispersion. More simply stated, in this kind of analysis, the Newtonian character of these systems, taking microemulsions for example,can be exposed.
C. Encapsulation or Incorporation Efficiency of the Active Substances

The assay of the active substance incorporated into one of the dispersion phases provides useful information on the encapsulation ability of the systems [71-731. The location of the active substance in the dispersion phase depends on its affinity for the various constituents of the formulation. Hydrophilic substances are dissolved in the aqueous phases, whereas lipophilic substances are dissolved in the oily phases. The rateof encapsulation depends on a number of factors, like the concentration of the active substance, the nature of the constituents, the type and size of the vesicles. Prior to the determination of the amountof active substance encapsulated, it is nevertheless preferable, once the dispersed systems have been formed, to eliminate the nonencapsulated active substance. Separation is accomplished by means of various procedures, for example filtration on gel, ultracentrifugation, ordialysis.
VI.
STABILITY Liposomes
A.
To date, the production of stable liposomes isstill delicate and chancy

[74-791. It should nevertheless be kept in mind that, as for all dispersions, these vesicles have a tendency toward flocculation and fusion and later sedimentation.
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Likewise, instability is also associated with the increase in permeability of the envelope and the resulting leakage of the encapsulated active substance, as, for example, it is likely to occur when the rate of cholesterol in the envelope is insufficient. Degradation of phospholipids is largelyrelated to autoxidation, which can be markedly decreasedif antioxidants are added. Furthermore,oxidation of liposomes can be avoided by using hydrogenated phospholipids. Another cause of degradation is hydrolysis of the constituents of the membrane which results in the release of fatty acids, an increase in the an in membrane permeability. All of fluidity of the membrane, and increase these phenomena can lead to fusion of the vesicles and either partial or total leakage of the encapsulated active ingredient. Protection against hydrolysis and autoxidation as well can be ensured by coating the liposomal vesicles with a membrane comprising biological macromolecules,atelocollagen, and glycosaminoglycanes. The second coating ensures a diminution in the liposomal membrane fluidity as a result of interaction between the phospholipids and the macromolecules. This leads to a reduction in the permeability of the membranes. Various physical procedures, such as irradiation with gamma rays or cryodessication, promise to provide answers to increasing the stability of these systemsin the future. Generally speaking, the stability of liposome in gelified aqueous or hydroalcoholic environments ranges between 2 and 3 years at temperature between 4" and 2 5 C . However, liposomes remain stable for only a few months if they are dispersed (and this is quite frequently the case in cosmetic products) in a lipid-rich environment or in a solution containing surfactants in which the phospholipid envelop dissolves or gradually becomes soluble.
B.
Nanoparticles
As long as organic solvents, residual monomers, and polymerization inducers are eliminated, then problems in the stability of nanoparticles are practically nonexistent. Nevertheless, in order toavoid anyeventual degradation of the polymeric materials in aqueous suspension or hydrolysis of the active substance after release, cryodessication can be performed without causing any alteration in the size.
C. Microemulsions
Since microemulsions are stable thermodynamic systemswithin a defined temperature range, they present no problems for storage under normal conditions.

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Multiple emulsions are unstable thermodynamic systems; over time they progress inevitably toward their rupture [77-791. In addition to the traditional factors of instability inherent in simple emulsions (separation, creaming, coalescence, phase inversion), specificunstable conditions occur within multiple emulsions such as the diffusion of internal water into the external aqueous continuous phase. These instabilities are all irreversible. They may appear separately orconcurrently. Today, through the use of very effective polymeric surface-active agents and thickeners that increase the viscosity of the various phases (particularly cross linking of the intermediateoily phase through the use of chemical or physical processes like gamma radiation), we are able significantly to slow down the destabilization process of these systems. In fact, stablemultiple emulsions can be prepared for a periodof 2 years without any alteration of their characteristics or release of the encapsulated substance. E. Liquid Crystals Liquid crystals do not exhibit any problem of stability provided they are stored within specific temperature ranges, since above such temperatures they lose theirproperties (including even lyotropic liquid crystals). It should be noted that the presence of lamellar structures at the emulsion interface greatly increases their stability.
VII. COSMETIC USES
A. Liposomes
Liposomes are theprincipal vehicles for the transport of active ingredients, which, depending on their molecular size and solubility properties, are localized at different sitesof the liposomes [80-991. If the active ingredients are small hydrophilic molecules such as sugars, aminoacids, peptides, ornormal moisturizing factors (NMF), they are localized at the center of the vesicle or between the bilayers. If they are lipophilic molecules such as liposoluble vitamins and their esters, they are located in the lipid bilayer. The transformation of these lipid vesicular carriers when they come is still the subjectof a strong controinto contactwith the stratum corneum versy. The presence of globular structures in the first layers of corneal cells was firstassumed and later confirmed through various transmission electron microscopic (TEM) techniques. Furthermore, the presence of multilamellar structures in the deep layers of the stratum corneum has also been observed through cryofracture. Given the fact that intercellular distances are either of
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6.4 or 13.4 nm, itis surprising to find entirely intact vesicular structures with of bilayers with such a diameter of between 50 nm and 0.5 pm in the interior anarrow thickness. It is now known thatthe molecules constituting liposomes become dispersed during thecourse of their intercellularmigration, and their reactions with the cutaneous lipids are more or less a function of their chemical structure. An accumulation of phospholipids develops at certain lipid sites, and this leads to the formation of new vesicular structures at these sites. It has been demonstrated that phosphatidylcholine, having a relatively modest hydrophilic moiety, is more likely to react than phosphatidyl inositol choline, since the latter has a heavier hydrophilic moiety group. It is thus a dynamic structure whose initial globular form is indispensable in penetrating the cutaneous barrier. Extensive and thorough knowledge regarding interactions between liposomes and living cells is currently available. These interactions studied with routes of administration other than topical may call into play four different phenomena:
1. Absorption, facilitated by the charge on the vesicles due to the ionization of the primary or secondary component. 2. Lipidic transfer, through a mediation of surface proteins (here an analogy between lipids, liposomes, and membrane lipidscan be seen). It is important to emphasize the role played by molecules such as lipoproteins in the constitutionof the membrane. 3. Endocytosis, also influenced by the charge on the particles. This permits thedigestion of liposomes by lysosomes inthe interiorof the cell, and this promotes the releaseof the active ingredient at the same location. 4. Fusion, which results from the insertion of constituents of the liposome membrane into thecell membrane.
This simple and attractive hypothesis has, however, not been proven to date. Because of their biomimetism with the cellular membranes, all of these lipid systems are designed as vehicles for carrying active ingredients, particularly hydrophilic ones, sometimes lipophilic, andensuringprolonged release of such ingredients. Although the natureof interactions between liposomes and the skin remains unexplained to date, these systems have been used continuously f a "liposomed" cosmetic product manufactured since the initial creationo by Dior more than a decade ago and called Capture. The first cosmetic applications of liposomes were in the field of hydration of the skin. It was demonstrated that products containingliposomes, into which propylene glycol was added, possessed excellent moisturizing properties. This hydrating capacity was prolonged and extended with a
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product containing liposomes constituted from glucosylceramides, an ester of glucose of fatty acid, and an ester of sucrose or trehalose of fatty acid. The following products have been tested: Creams or lotions made up of lysophospholipids, an aqueous phase containing monovalent and polyvalent alcohols, an oily phase able to containhydrocarbons, esters, triglycerides, fatty acids, and fatty alcohol as well as silicone. Dispersions constituted from a mixture of an emulsion containing elastin, collagen, casein, and fibroin, and a liposomal suspension containing vitamins E, F, and A. Liposomes made of cutaneous lipids dispersed in gel.
1
Currently, a large number of active substances used in cosmetology have been encapsulated, presented, and dispersed in hydroalcoholic liquids, aqueous gels, or creams with a continuous aqueous phase. Most cosmetic compounds, whether intended foruse on the face, the hands, the body, or thehair, are appreciatedby those who use them. Because of their bilamellar structure, nonionic surfactant vesicles have the same properties as liposomes. However, since the nature of lipids found in the envelope is different, their properties are not entirely identical. Of course,thesame controversies existwith regardtononionic surfactant vesicles as with regard to liposomes insofar as their passage through the skin is concerned. Even if we accept that only vesicles on the penetrate the corneal layer intact, it is still order of 20 nm in size can undeniable that vesicles with nonionic surface-active agents have some effect after topical application. The first results were reported by Handjani-Vila and colleagues [88,91,96,97], whoshowed that sodium carboxylate pyrolidone has the greatest hydrating power when it is encapsulated in nonionic surfactant vesicles and not when it is encapsulated in emulsions or even in liposomes. Recently, nonionic surfactant vesicles without any active ingredients, composed of fatty ethoxyl alcohols and cholesterol, were tested on human skin. Microscopic examination following cryofracture of a cross section of skin reveals that the vesicles would be located in intercellular lipid sites in a depth of a few micrometers. This confirms the stratum corneum to results whichshow,with the aid of images obtained by Electron Paramagnetic Resonance (EPR) and plates obtained through microscopy following cryofracture of biopsy a of human skin, that intercellular restructuring of the epidermis takes place after contact withsuch systems. Like liposomes, nonionic surfactant vesicles will then restore to a delipidified corneal layer the lamellar structures normally contained therein. It was shown that incorporation of estradiol in nonionic surfactant vesicles (par-
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ticularly multilamellar structures) substantially enhances penetration of this ingredient into the stratum corneum of a piece of skin. Although themechanism by which these carriers operate remains to be explained, theynevertheless constitute very useful systems for cosmetic applications. Without any active substance, like liposomes, the nonionic surfactant vesicles enhance the supply of lipids and water of the stratum corneum. Even when they do not penetrate the stratum corneum intact, they facilitate theaccumulation of hydro- or lipophilic active substances in the upper layers of the epidermis. Finally, like liposomes, nonionic surfactant vesicles exhibit a high cutaneous tolerance. Preparations containing nonionic surfactant vesicles were first placed on the market at approximately the same time as the liposomes described in the preceding sections. They too achieve satisfactory results for people who use them knowledgeably. For these systems as well, the first cosmetic applications were intended as hydrating agents, andthey were followed by self-tanning ingredients of the skin. Prevention of dry skin has been studied with empty vesicles. With their component ingredients, thesesystems work against water loss by forming an occlusive film on the surface of the epidermis. As mentioned earlier, their hydrating capacity has been studied by encapsulating sodium carboxylate pyrolidone (PCNa), a componentof the skins natural hydrating system (NMF). The hygroscopic substances which make up the NMF are often used as hydrating agents in cosmetic compositions. However, these composites, which are soluble in water, develop a weak affinity for the corneal layer once they are incorporated into aqueous solutions or in oily emulsions within water. They penetrate the stratum corneum with onlymoderate success, and are, aside from that, easily eliminated by washing with water. If such substances are administeredin a lipid environment, their diffusion and the resistance to washing are increased. The application of nonionic surfactant vesicles containing 10% PCNa will, even afterrinsing, markedly improve the hydration of the skin by 40-90% as compared with the initial moisturizing level before treatment. One of the first tests in the study of coloration of the skin was peras controls, formed with vesiclescontaining 0.6% of tartaric aldehyde and, aqueous solutionsof various concentrations of the same component. Four hours after application, both before and after washing, the intensity of coloration produced by these vesicles was as high as the intensity produced by the aqueous solutioncontaining 10 times the quantityof tartaric aldehyde. A second, parallel test was conducted with vesicles containing 1.5% of tartaric aldehyde and 3% of dihydroxyacetone and an O/W emulsion. An intense tan was obtained with these vesicles, which was resistant to
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washing with soap and water, whereas with the O/W emulsion, the tan, which was already weak before washing, disappeared almost completely after washing. These nonionic surfactant vesicles have been in continuous use since their first commercial exploitation as Niosomes by L'Oreal. They are more and more commonly used, and a number of active substances have been encapsulated. Evenif, as for liposomes, the precise mechanism by which they operate on application to the skin remains yet to be determined, their biomimetic approach, like that of liposomes, has inspired a growing number of researchers, as their vesicular carriers constitute an undeniably valuable asset to cosmetology.
B. Nanoparticles
Although cosmetic applications of nanoparticles proliferate (numerous patents have been granted), publications, studies, or reports on their biofate in the skin following topical application have been rare. The incorporation of active cosmetic hydrophilic substances (e.g., amino acids, vegetable extracts, organic and mineral elements) in the nanospheres attempts to modulate the release of the substances in the skin. Where nanocapsules are concerned, theactive substances (AS) are usually of lipophilic nature, and they can be composed of an oily compound or a dispersion. Here again the objective is to control the release of the AS, since the molecule is protected, during a shorter longer or period, from biodegradation in the organism. The release profile of the AS (by, e.g., erosion, diffusion, elution) depends on the nature of the constituents. Recently, Lancome launched a cosmetic product containing nanocapsules of vitamin E (Primordiale). As for the vesicular systems described in the preceding sections, except for very small sizes which can be detected in the pilosebaceous apparatus, it is unlikely that they penetrate thestratum corneum intact, since their size varies from 100 to 800 nm.
C. Microemulsions
Over the last 10years, many studies have dealt with the percutaneous absorption of various active ingredients carried by microemulsions both from a pharmacological point of view and a cosmetological perspective [100-107]. Overall, hydrosoluble active ingredients have been the most sought after and (rathercuriously) the ingredients most often added to the external phase of O/W microemulsions. Various investigators have shown that microemulsions acted as absorptionenhancers of both liposoluble and hydrosoluble active substances. This action was not just attributed to the high proportion of surface-active agents. Still, when the hydrosoluble active element is in the internal phase of a W/O microemulsion, its physicochemical characteristics are altered. Be-
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cause of this, and owing to the components of the fatty phase, it can accumulate in the cutaneous lipophilic structures or in the cutaneous fats (orotic acid).Liposoluble active ingredients have been introduced both in the internaland in the externalmicroemulsion phases compared with W/O or O/W emulsions or with petroleum jelly (Vaseline). Nevertheless, they are more frequently incorporated in the internal phase, where they are solubilized. This is the case with alpha-tocopherol, azelaic acid, and octyl dimethyl para-aminobutyric acid, for which the absorption rate is significantly increased, and which no longer behave like lipophilic compounds but like hydrophilic ones. When estradiol or prednisone is introduced in the internalO/W microemulsion phase, it is stored in the intercellularlipids of the stratum corneum. Estradiol placed in the external phase is better retained. Thus, it can be observed that depending on the formulation and the type of the microemulsion, the absorption of an active ingredient can be modulated on request. There are numerous cosmetic products in the form of microemulsions; these products range from body care to facial and hair treatments. They include bath oils, body thinning products, fixatives for hair, hardeners fornails, hydrating products, antiwrinkle products, productsto prevent seborrhea, and antiaging serums marketed principally in France, Italy, Belgium, and the United States.
The behavior of the multiple emulsions on the skin following topical application has not yet been addressed [108-1101. Do small-sized globule vesicles in the range of 20 nm penetrate intact? In what manner does the rupture of larger-sized globule vesicles take place? Are they reformed on contact with the intercellular lipids? For multiple emulsions, these questions do not present a problem. Although the releaseof the encapsulated active substance is complicated, since a number of different mechanisms exist, themultiple emulsions behavior after application to theskin appears to be relatively simple, since it is quite similar to the behavior observed with regard to simple emulsions. If evaporation of the water after application to the skin leading to other structuresis not taken into consideration, two principal hypotheses may be proposed. In the first hypothesis, the encapsulated active substance has a near zero diffusion rate through the oily membrane. The active substance is thus released in the internal phase only by virtue of the rupture of the multiple oily globules. This rupture takes place either by shearing of the preparation or after swelling of the internal aqueous globules. Shearing may be induced by massaging or rubbing performed when the creamis applied to theskin.
623
Swelling of the internal aqueous globules may be causedby dilution of the multiple emulsion in water. It is possiblethat theinstructions for use could require ,mixing immediately prior to application of a given quantity of the multiple emulsion with a specific volume of water. The strong osmotic gradient thus created causes the external water to enter the internal phase, causing swelling of the internal water globules. When theseglobules reach their maximum size, they burst. In both of these cases, rupture by shearing or rupture after swelling, the active substance, having reached the external aqueous phase, becomes immediately available. The multiple emulsion behaveslike a simple emulsion with a continuous aqueous phase. In this case, multiple emulsions serve the purpose of protecting (by encapsulation) an active substance or of permitting the incorporation into the same preparation of incompatible active substances which will not come intocontact until the cream is used. In the second hypothesis, the encapsulated active substance diffuses through the oily membrane, and the multiple emulsion keeps its multiple globules intact when applied to theskin. The release profile of the active substance from a multiple emulsion would, in this case, not be similar to that froma simple emulsion. It would be slower and gradual, and it would depend on the following factors: the 1ocalization.andphase partition distribution of the active substance and its permeability or diffusion rate through the oily membrane; the rigidity, the viscosity, and the thickness of the interfacial film as well as thepresence or absence of liquid crystals at this level; the particle size and distribution of the dispersion, and otherfactors. Since they do not concentrate or accumulate the active substance at the vesicular systems described above do, level of the stratum corneum as the they at least extend the release rate of the active substance. Their fate after application to the skin has been thesubject of few publications to date; only the works of Kundu [l101 and Ferreira [l081 on thesubject are well known. Multiple emulsions used as cosmetic preparations generally come in the form of lotions or creams of varying density. As soon as they are constituted, they can be used directly; unlike the vesicular systems described above, it is not necessary to disperse them in a gel environment or in a creamin order toobtain an acceptable cosmetic product. Cosmetic applications of multiple emulsions have been protected in the patents issued for theircomposition. One example of an application is perfume encapsulated in the internal phase: very small amounts of it are released over a long period of time. However, it could be released instantaneouslyby intentionally breaking the primary emulsion; for example, by rubbing during application to the skin. The patents show that multiple emulsions are highly recommended for all kinds of cosmetic applications:
'
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for example, sunscreens, make-up removers, cleansers, nutritive, hydrating, and refreshing and cooling products. The mixture of a continuous oily phaseemulsion with an aqueous solution containing, in addition to a hydrophilic surface-active agent, saccharides (such as xylose, lactose, or sorbitol), polysaccharides (such as xantham gum, pectin, carraghenate, or dextran), or synthetic polymers(such as acrylic derivatives) allows the obtention of multiple emulsions presenting cosmetic qualities of good spreading and unctuousness, which can be modified on request. In these systems, it is possible to incorporate a number of active substances both in the internal and external aqueous phases and even in the intermediateoily phase without significantly altering their stability. We cite as an examplea formula which produces a fairly viscous cream that remains stable for a 5 C . period of 36 months at2 All other things being equal, W/O/W multiple emulsions are more effective than simple emulsionswith a continuous aqueous phase and are more pleasant to use than simple emulsions with a continuous oily phase because of a less greasy feel. Multiple emulsions will certainly enjoy a future in cosmetics at least as bright as that of the vesicular systems described above. Simple emulsions are the building blocks of cosmetology. Multiple emulsions, like their simpler form, give skin both water and oil; they can comprise a large number of ingredients, both hydrophilic and lipophilic; they are easy to administer, since they can be applied directly to the skin; and theypresent good cosmetic qualities. Like any vesicular system, multiple emulsions constitute a new form which could prove extremely fruitful even if only for the possibilities it presents for the protection of encapsulated substances and perhaps for prolonged release of substances. The firstcommercial use of a W/O/W-type multiple emulsion is Unique Moisturizing by Lancaster, which was introduced to the marketin 1991.
E. Liquid Crystals
For reasons of cosmetic appeal due to thecolored appearance theygive to preparations into which they are introduced, and for reasons of active substance solubilization, or simply because they increase the stability of dispersed systems, liquid crystals are enjoying a growing popularity [ l l l ] . Until now, liquid crystalshave beenselected only rarely as absorption enhancers, because they contain such a high proportion of surface-active agents. In fact, poor cutaneoustolerance was observed whenliquid crystals were usedas the principal vehicle. After dispersion in a gel, their tolerance is no longer a problem.
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Thermotropic liquid crystals were introduced in a cosmetic product for the first time by the Estee Lauder Company, with the launching of Eyxone, a translucid gel.Today, liquid crystals incorporated in microcapsules made of gelatin which rupture ontopical application are available (Merck). Lyotropicliquid crystals are incorporatedin special dermatological formulations thatexhibit hydrating properties (Liphaderm). Most of all, liquid crystals are used as excipients to protect sensitive, active substances (vitamins, antioxidants, oils). Liquid crystals may enhance the stability of emulsions while creating arheological barrier resulting inan increase in the viscosity and a decreasein coalescence by modification of Van der Waals forces. It is also claimed that they enhance cutaneous penetration. REFERENCES
1 . G. Gregoriadis,P. D. Leathwood, and B. E. Ryman, Enzyme entrapment in liposomes, FEBS Lett., 14:95-99 (1971). 2. C. G . Knight, Liposomes, in Physical Structure to Therapeutic Applications, Elsevier, Amsterdam,1981. 1983. 3. M. J. Ostro, Liposomes, Marcel Dekker, New York, 4. F. Puisiew and J. Delattre,LesLiposomes:applicationstherapeutiques, Lavoisier Tecet Doc, Paris,1985. 5. G. Gregoriadis, J. Wiley, and S. Chichester, Liposomes as Drug Carriers, Wiley, Chichester, UK,1988. 6. J. Delattre, P. Couvreur, F. Puisiew, et a1 al., Les liposomes: Aspects 7.
8.
9. 10. 1 1 . 12. 13. 14.
technologiques, biologiques, et pharmacotechniques, Lavoisier Tec et Doc, Paris, 1993. B. Magenheim andS. Benita, Nanoparticles characterisation. A Comprehensive physicochemical approach, STP Pharma Sci., 4:221-241 (1991). F. Fevrier, Etude de microemulsions non ioniques comme agentde penetration cutanee, Doctoral thesis, de 1Universitie de Technologie de Compiegne, France, 1991. W. J. Herbert, Multiple emulsion, a new form of mineral oil antigen adjuvant, Lancet, 2:771-773 (1965). M. DeLuca,C.Vaution,A.Rabaron,andM.Seiller,Classification et obtention des emulsions multiples, STP Pharma Sci., 4:679-687 (1988). J. P. Caquet andT. Bemoud, Les cristaux liquides dans les cosmetiques, Parf. Cosmet. Aromes, 91:77-84 (1990). G. Cioca and L. Calvo, Liquid crystals and cosmetic applications, Cosmet. Toiletries, 10557-62 (1990). A. D. Bangham, M. W. Hill, and N. G. A. Miller, Preparation and use of liposomes as a model of biological membranes, in Methods in Membrane Biology (E. D. Kom, ed.), Plenum Press, New York, 1974, pp. 1-68. J. M. Gebicki and M. Hicks, Preparation and propertiesof vesicles enclosed 16:142-160 (1976). by. fatty acid membranes, Chem. Phys. Lipids,
626
Benita et a/.
15. C. Kirby, J. Clarke, and G. Gregoriadis, Effect of cholesterol content of small unilamellarliposomes on their stability in vivo and in vitro, Biochem.J., 186~591-598 (1980). 16. A. W. T. Konings, Lipid peroxidation in liposomes, in Liposome Technology FL, 1984,pp. 139-161. (G. Gregoriadis, ed.), CRC Press, Boca Raton, 17. S. Stainmesse, H. Fessi,J. Devissaguet, andP. Puisieux, Procede de preparation de systemes colloidaux dispersibles de lipides amphiphilessous forme de liposomes submicroniques, Brevet No.88.08.874,France, June,1988. 18. D. Marsh, Handbook of Lipid Bilayers, CRC Press, Boca Raton, FL, 1990. on the 19. D. J. A. Crommelin, Influence of lipid composition and ionic strength 73:1559-1263 (1990). physical stability of liposomes, J. Pharm. Sci., 20. L. M. Prince, Microemulsions: Theory and Practice, Academic Press, New York, 1977,p. 177. 21. L.M.Prince,Micellization,solubilizationandmicroemulsions,inMicellization,SolubilizationandMicroemulsions, (K. L. Mittal,ed.),Plenum Press, New York, 1977,pp. 45-54. 22. S. E. Friberg and L. G a m o , Microemulsions with esters, J. Soc. Cosmet. Chem., 34:73-81 (1983). 23. I. Rico, Les microemulsions: definition et applications pratiques, Vol. VII, Les entretiens du Carla, (P. Fabre, ed.), Castres,1986,pp. 22-26. 24. A. Ceglie, K. P. Das,andB.Lindman,Microemulsionstructureinfourcomponent system for different surfactants, Colloid and Surfaces, 28:29-40 (1987). on the microscopic 25. A. Ceglie, K. P. Das,andB.Lindman,Effectofoil structure in four component cosurfactant microemulsions, J. Colloid and Interface Sci. B, 115:115-120 (1987). 26. C.H.ChewandL. M. Gan,Monohexyletherofethyleneglycolanddiethylene glycol as microemulsion cosurfactants, J. Dispersion sci. Technol., 11~49-68 (1990). 27. A. T. Florence and D. Whitehill, The formulation and stability of multiple emulsions, Int.J. Pharm, 11:277-308 (1982). N. Garti, Correlation between nature of emulsi28. S. Magdassi, M.Frenkel, and fiersandmultipleemulsionstability,DrugDev.Ind.Pharm., 11:791-798 (1985). 29. C. Fox, An introduction to multiple emulsions, Cosmet. Toiletries, 62:lOl112 (1986). 30. C. Prybilski, M. de Luca, J. L. Grossiord, et al., w/o/w multiple emulsions 106:97manufacturing and formulation considerations, Cosmet. Toiletries, 100 (1990). 31. T. Suzuki, M. Nakamura, H. Sumida, and I. Shigeta, Liquid crystal make-up remover:conditionsofformationanditscleansingmechanisms,J.Soc. Cosmet. Chem., 43:21-36 (1992). 32. L. Saunders, J. Pemn, and D. B. Gammack, Aqueous dispersion of phospholipids by ultrasonic radiations, J. Pharm. Pharmacol., 14567-572 (1962). 33. A. D. Bangham, M. M. Standish, and J. C. Watkins, Diffusion of univalent
627
34. 35. 36. 37. 38. 39. 40. 41. 42. 43.
4 4 .
45.
46.
47.
4 8 .
49.
50.
ions across the lamellae of swollen phospholipids, J. Mol. Bioi., 13:238-252 (1965). C. H. Huang, Studies on phosphatidylcholine vesicles. Formation and physical characteristics, Biochemistry, 8:344-352 (1969). S. Batzri and E. D. Kom, Single bilayer liposomes prepared without sonication, Biochem. Biophys. Acta, 298:1015-1019 (1973). D. W. Deamer and A. D. Bangham, Large volume liposomes by an ether evaporation method, Biochem. Biophys. Acta, 443:629-634 (1976). J. M. H. Kremer, M. W, J. Esker, N. C. Pathmamanohara, and P . H. Wiersema, Vesicles of variable diameter prepared by a modified injection method, Biochemistry, 16:3932-3935 (1977). F. C. Szoka and D. Papahadjopoulos, Procedure for preparation of liposome with large internal aqueous surfacehigh and captureby reverse phase evaporation, Proc. Natl. Acad. Sci. USA, 75:4194-4198 (1978). Y .Barenholz, S. Amselem, and D. Lichtenberg, A new method for preparation of phospholipid vesicles (liposomes), FEBS Lett., 99:210-215 (1979). T .Oshawa, H. Miura, and K. Harada, A novel method for preparing liposomes with a high capacity to encapsulate protein and drugs, Chem. Pharm. Bull., 32:2442-2445 (1984). C. Vauthier-Holzcherer, S. Benabbou, G. Spenlehauer, et al., Methodology for the preparation of ultradispersedpolymersystems, STP PharmaSci., 1:109-116 (1991). M. Bornschein, P. Melegari, C. Bismarck, andS. Keipert, Mikro- und Nanopartikeln alsarzneistofftragersysteme unter besonderer Berucksichtigung der herstellungsmethoden, Pharmazie, 44585-593 (1989). N. Kamenka, G. Haouche, B. Brun, and B. Lindman, Microemulsions in zwitterionic surfactant systems, J. Colloid Interface Sci., 86:359-369 (1982). J. Franz (S. A. Sandoz), Compositions pharmaceutiques topiques sous forme dune microemulsion, Fr. Patent Application 2-502-951 (30/03/1982). B. W . Muller and P. Kleinebudde, Untersuchengen an sogenannten mikroemulsions systemen1Teil: Unter suchungen an arzneistoffhaltigen systemen, Pharm. Ind., 50:370-378 (1988). B.W. Muller and P. Kleinebudde, Untersuchengen an sogenannten Mikroemulsions systemen 2 Teil: Unter suchungen an arzneistoffhaltign systemen, Pharm. Ind., 50:1301-1306 (1988). F. Fevrier, Formulation de microemulsions cosmetiques, Nouv. Dermatol., 10:84-87 (1991). M. F. Bobin, N. Dejour, A. Revillon, and M.C. Martini, Formulation of ultrafine emulsions with surcroesters for Pharmaceutic and Cosmetic applic tions, Congres Mondial de lemulsion, Paris, France, 1993. S. Matsumoto, Y. Kita, and D. Yonesawa, An attemptat preparing water in oil in water multiple phase emulsions, J. Colloid Interface Sci., 57:353-361 (1976). D. R. Kavaliunas and S. G. Franck, Liquid crystal stabilization of multiple emulsions, J. Colloid InterfaceSci., 66586-588 (1978).
628
51.
et
Benita
al.
S. Matsumoto andP. Sherman, A preliminary study of wlolw emulsions with a J. Texture Studies,12:243-257 (1981). view to possible food applications, 52. S. Matsumoto, Development of wlolw type dispersion during phase inversion of 94:362-368 (1983). concentratedwlo emulsions,J. Colloid and Interface Sci., 53. S. Magdassi,M. Frenkel, N. Garti, On thefactorsaffectingtheyield of
5~49-59(1984). 54. M. de Luca,Les emulsions multiplesW L J H . Obtention, validation,et libera191, 1991. tion, These de 1Universite Paris XI, No. 55. C.Colinet,ContributionaIetudedesystemesmesomorphesapartir de surfactifs non ioniques, These de pharmacie, Lyon I, 1991. 56. E. Coupier, Contribution a letude de la stabilite des systemes mesomorphes. 57.
preparation and stability of multiple emulsions, J. Dispersion Sci. Technol.,
58.
59.
60.
61. 62.
63.
Influence de la temperature et des divers adjuvants, These de pharmacie, Lyon I, 1991. P. Schurtenberger and H. Hauser, Characterization of the size distribution of unilamellar vesicles by gel filtration quasi-elastic light scattering and electron microscopy, Biochim. Biophys. Acta,778:470-480 (1984). H. Ruf, Y. Georgalis, and E. Grell, Dynamic laser light scatteringto determine size distributions of vesicles, Methods Enzymol., 172:364-372(1989). Z .Kojro, S. Q. Lin, E. Grell, and H. Ruf, Determination of internal volume and volume distribution of lipid vesicles from dynamic light scattering data, Biochim. Biophys. Acta,9851-8 (1989). R. R. C.New,Characterization of liposomes,inLiposomes,APractical Approach (R. R.C. New, ed.), IRL Press, McLean, Virginia, 1990. D. Atwood and G . Ktistis, A light scattering study on olw microemulsion,Int. J. Pharm, 52:165-171 (1989). R. C. Baker,A. T. Florence, R. H. Ottewill, andT. H. Tadros, Investigations into the formation and characterization of microemulsions. 11. Light scattering conductivity and viscosity studies of microemulsions,J .Colloid Interface Sci., 100:332-349 (1984). S. Keipert, I. Siebenbrodt, F. Luders, and M. Bornschein, Mikroemulsionen 44:433-444 undihrepotentiellepharmazentischenutzung,DiePharmazie,
(1989).
6 4 . J. C. Selser, Y.Yeh,and R. J. Baskin, A light scattering measurement
of membrane vesicle permeability, Biophysics, 16:135-139 (1976). 65. S. S. Davis, A.S. Burbage, Electron micrography of wlolw emulsions, J. Coll. Interface, 62(2):361-362 (1977). 66. Y. Kita, S. Matsumoto, and D. Yonezawa, Viscosimetric method for estimating the stability of wlolw type multiphase emulsions, J. Colloid Interface Sci.,
67.
T. Ota, Anattempttoestimate J. Colstability of the oil layer in wlolw emulsions by means of viscosimetry, loid Interface Sci., 77564-564 (1980). 68. J. I. Zatz and G . H. Cueman, Assessment of stability in water in oil in water multiple emulsions,J. Soc. Cosmet. Chem., 39:211-222 (1988).
62:87-94 (1977). S. Matsumoto, T. Inoue, M.Koda,and
629
69. A. A. Elbary, S. A. Nour, and I. Ibrahim, Physical stability and rheological propertiesofw/o/wemulsionsasafunctionofelectrolytes,Pharm. Ind., 52~357-363 (1990). 70. J. L. Grossiord, M. Seiller, andF. Puisieux, Apport des analyses rheologiques dans l'etude des emulsions multiples I", Rheologica Acta, 32:168-180 (1993). 71. E. H. Ralston,L. M. Jelmeland, R. D. Klausner, et al., Carboxyfluoresceine as a probefor liposome cell interactions. Effect of impurities and purification of the dye, Biochim. Biophys. Acta, 649:133-137 (1981). 72. S. S. Davis and I. Walker, Measurement of the yield of multiple emulsion droplets by a fluorescent technique, Int. J. Pharm., 17:203-213 (1983). T. K. Law, and A. T. Florence, The nature 73. J. A. Omotosho, T. L. Whateley, of the oil phase and release of solutes from multiple wlolw emulsions, J. Pharm. Pharmacol., 58:865-870 (1986). de contraste iode. Influence de la formula 74. P. A. Poly, Liposomes d'un produit tion sur l'encapsulation Etude de la conservation par lyophillisation. Docto thesis No. 65,3 " '' cycle, UniversitC Paris-Sud, 1983. 75. S. Henry Micheland, P. A. Poly, F. Puisieux, et al., Etude du comportement desliposomeslorsd'experiencesde congelatioddecongelation et de lyophillisation. Influence des cryoprotecteurs, 3rd International Conference on Pharmaceutical Technology APGI, Paris, 1983, pp. 223-133. et al., Prevention of fusion and 76. L. M. Crowe, C. Womersley, J. H. Crowe, leakage infreeze-driedliposomes,Biochim.Biophys.Acta,861:131-140 (1986). 77. T. K. Law, T. L. Whateley, A. T. Florence, Stabilization of wlolw multiple emulsions by interfacial complexation of macromolecules and non-ionic surfactants, J. Controlled Rel., 3:279-280 (1986). 78. A. 02, P. Kamles, P. Franck, and G. Sylvan, Multiple emulsions stabilized by colloidal microcrystalline cellulose, J. Dispersion Sci. Technique, 10:163-185 (1989). 79. M. de Luca, J. L. Grossiord, J. M. Medard, et al., A stable w/o/w multiple emulsion, Cosmet. Toiletries, 10565-69 (1990). 80. M. Mezei andV. Gulasekharam, Liposomes. A selective drug delivery system for the topical route of administration, Life Sci., 26:1473-1477 (1980). 81. A. Rougier, D. Dupuis, C. Lotte, et al., In vivo correlation between stratum corneumreservoirfunctionandpercutaneousabsorption.J.Invest.Dermatol., 81:275-278 (1983). 82. J. Lasch, S. Zellmer, W. Pfeil, and R. Schubert, Interaction of liposomes wit the human skin lipid bamer: hSCLLD as model system-DSc of intact corneum and in situ CLSM of human skin, J. Liposome Res., 5:99-108 (1995). P. Pemer, New vehicleto enhance the biologi83. G. Redziniak, A. Meybeck, and cal performances of active cosmetic ingredient, Vol.11, XIVth International Federation Societies of Cosmetic Chemists Congress, Barcelona, 1986, pp. 299-307. ;&l 8 4 . Skincosmeticscontainingoilinwaterdispersionscontainingnaturalsur-
630
Benira er al.
factantsandliposomewaterdispersions,PolaChemicalIndustries,Inc., French Patent 261 4787 (10 1 11988). 85. Skin treatment products containing lysophospholipids, Eur. Patent 25 59 37 (17 02 1988). 86. Topical compositions containing liposomes and acrylic gels, Estee Lauder 06 1989). Inc., Eur. Patent 3 196 38 (14 87. A. Meybeck, F. Bonte, G. Redziniak, Improvement of comedolytic activity andtoleranceofvitaminAacidintopicalliposomeformulation,Vth Congres Int. Tech. Pharm., Pans, 1989. 88. R. M. Hanjani-Vila andJ. Guesnet, Les liposomes. Un avenir prometteur en dermatologie, Ann. Dermatol. Venerol., 116:423-430 (1989). 89. W. Abraham and D. T. Downing, Interaction between corneocytes and strain vitro, Biochim. Biophys. Acta, 1021:119-125 tum corneum lipid liposomes (1990). 90. H. Lautenschlager, Liposomes in dermatological preparations. Part I, Cosmet. Toiletries, 105:89-96 (1990). 91. E. Clar and A. Ribier, Lipid vesicles in cosmetology, Cosmet. Toiletries Mf., 2~138-147 (1991). 92. M. Ghyczy, J. Roding, and E. Hoff, Control of skin humidity by liposomes, Cosmet. Toiletries Mfg., 2:148-152 (1991). 93. H. E. J. Hofland, J. A. Bouwstra, F. Spies, et al., Interactions betweennonionic surfactant vesicles and human stratum corneum in vitro, J. Liposome Res., 5241-263 (1995). 94. F. Bonte, J. M. Chevalier, and A. Meybeck, Determination of retinoic acid liposomalassociationlevelinatopicalformulation,DrugDev.Indus. Pharm., 20:25-34 (1994). 95. R. M. Handjani-Vila and G. Vanlerberghe, Les Niosomes, in Formes Pharmaceutiques nouvelles. Aspects technologique, biopharmaceutique, et medical, (P. Bun, F. Puisieux, and J. B. Benoit eds.), Lavoisier Tec et Doc, Pans, 1985. Les Niosomes des structures 96. G.VanlerbercheandR.M.Handjani-Vila, restructurantes pour les soins de laNouv. peau, Dermatol., 5259-262 (1986). 97. G . Vanlerberghe, R. M. Handjani-Vila, and A. Ribier, Les niosomes. Une nouvelle famille de vesicules a base damphiphiles non ioniques Colloques Nationaux du CNRS No. 938, Masson,Pans, 1989, pp. 303-311. . Gabnjedeie, M. Sentjure, and J. Kristl, Evaluation of liposomes as drug 98. V J. Pharm., carriers into the skin by onedimensionalEPRimaging,Int. 62~75-79 (1990). 99. H. Schreier, J. Bouwstra, Liposomes and niosomes as topical drug carriers: dermal and transdermal drug delivery, J. Controlled Rel., 3O:l-15 (1994). . Cotte, Absorption and fate of 100. M. C. Martini, M. F. Bobin, H. Flandin, J orotic acid after topical application in different vehicles, J. Appl. Cosmetol., 2~19-26 (1984). 101. M. C. Martini, M. F. Bobin, H. Flandin, and F. Caillaud, Role des microemulsions dans labsomtion Dercutanee de lalpha-tocopherol,J. Pharm. Belg., 39:348-354 (1984).
631
102. M. Gallarate, M. R. Gasco, and G. Rua, In vitro release of azelaic acid from oil in water microemulsions, Acta Pharm. Jugosl., 40533438 (1990). 103. E. E.Linn, R. C. Pohlan, and T. K. Byrd, Microemulsion for intradermal delivery of cetyl alcohol and octyl dimethyl PABA, Drug Dev. Indus. Pharm., 16:899-920 (1990). 104. F. Fevrier, M. F. Bobin,C.Lafforgue,andM.C.Martini,Advancesin STP Pharma microemulsions and transepidermal penetration of tyrosine, Sci., 1:60-63 (1991). 105. V. Burigana, Mise au point de dosagede la N-acetyl hydroxyproline.Etude I, de l'absorption cutanee de cette molecule, These de pharmacie, Lyon 1993. In vitro cutaneous penetration of 106. F. Fevrier, M. F. Bobin, and M. C. Martini, proline with microemulsion formulation compared to emulsion, Symposium International Federation Societies of Cosmetic Chemists, Barcelona, 1993. 107. F. Fevrier, Microemulsions in cosmetology, LCR Intetnational Federation Societies of Cosmetic Chemists Congress, pp. 105-118, Barcelona, 16-19/9/ 1993. 108. L. A. M. Ferreira, Emulsions multiples WWH et simples LEI et WL. Etude comparative pur l'approche de la voie topique, These Universite de ParisSud, No. 370,1994. 109. M. Seiller, A. M. Orecchioni, and C. Vautions, Vesicular Systems in H. I. Maibach, eds.), cosmetology, in Cosmetic Dermatology (R. Baran and London, Martin Dunitz, 1994, pp. 27-35. 110. S. C. Kundu, Preparation and evaluation of multiple emulsions as controlled release topical drug delivery systems, Avail Univ. Microfilms, Dep. Abstra. Int. Order no. DA 9224664 from Dirs Abstr. Int. B 1990 51 (1990) No. 4 S1763-4. 112. G. Cioca and L. Calvo, Liquid crystals and cosmetic application. Cosmet. Toiletries, 10557-62 (1990).
Index
Adsorbents, 359 Aedes aegvpti, 404 Aggregation, 307 Albumin, 166, 172,599 Alginate, 366 Alkylcyanoacrylates,364 Alum, 401 Amphotericin B, 394 Anesthetics local, 389 Anomalous diffision, 174 Anopheles srephensi, 404 Anthracyclines, 320 Antibiotics, 394 Antibody, 351,362 IgG, 401 monoclonal, 545-546,554,368 Antifly case study, 22,23,24 Arabic gum, 51 Arginase, 360 Atomization, 50,61 tower, 53,57,61 Atomizer, 55,57 method, 52 AUC, 404 Bangham, 602 Benzalkonium chloride, 363 Biocarrier, 591 Biocompatibility, 369
Biodegradable polymers, 36,385 Biovector, 591 Blood cells, 359 Box and Wilson test, 104 Brij96,33 1 Bromocriptine, 53 Bupivacaine, 385,389 Busereline, 53 Caking, 22 Carbon dioxide, 59,61 Carrageenan, 389 Cells, 35 1 artificial, 360 microencapsulated, 367 Centrifugation, 160, 161 filtration, 161 Centrisart@,161 Characterization, 611 Chemotherapy for cancer, 535-537 Chloroform, 356 Chlorpromazine, 163 Cholesteryl hexadecyl ether, 407 Chymotrypsin, 360 Coacervation, 22,23,27,36-37,41 Coated particles, 168 Colloidal drug carrier, 214-216 lipid-based, 215-218
633
634
[Colloidal drug carrier] polymeric, 214-215 Composition, 597 liposomes, 597 liquid crystals, 601 microemulsions, 599 multiple emulsions, 600 nanoparticulate systems, 598 Copolymer of ethylenepropylene oxide, 601 of styrene, 599 Cosmetics, 299 Cosmetic uses liposomes, 617-621 liquid crystals, 624 microemulsions, 621 multiple emulsions, 622-624 nanoparticles, 621 Co-surfactant, 599 Counting of particles electronic, 612 Critical point, 57 Crystallinity of colloidal glyceride crystals, 239 of stored tripalmitate nanoparticles,
242
Index
Drug delivery of, 299,362 leakage of, 216-252 release of, 156-181 resistance, 542-543,557 EGF, 265-266,288 Embryonic microparticles, 50 Emulsifiers, 413,424,432 in baked goods, 448 fany acids, 464 fatty alcohols, 464 HLB of, 435 lecithin, 450,457 lysolecithins, 474 in milk, 427,448 monodiglycerides, 45 1,465,5 12 monomeric, 464 polymeric, 419,440,466 spans, 465,480,484,487 Sodium stearoyl lacrylate (SSL), 464 stabilization by ,419,440,466,484 Tweens, 465,480,486,487 Emulsion, 24,26,3 1 , 159, 163 double vs. multiple, 475,494 adjuvants, 494 anticancer, 497 diffusion controlled, 479 droplet size distribution, 473,375 flux, 483 formation, 475 intravenous, 495 oil-in-water-in-oil (OIWIO), 475,
494
Crystallization in the dispersed state, 232 Cyclohexane, 356 Cytotoxicity, 406 Deconvolution, 162 DEET, 403 Denaturation heat, 25 surface, 26 Dermal uptake,403 Dexamethasone, 379 Dextran, 599 Dialysis, 159, 164 reverse, 160 tubing, 386 Diazepam, 160,387 Differential scanning calorimetry, 236 of melt-emulsified glyceride dispersions, 236-243 Differential thermal analysis (DTA),
135
oral intake, 496 osmotic pressure, 480 release of drugs, 494 release of foods, 494 release mechanism, 477,482 by reverse micelles, 480 stability, 477,482 transdennal, 495 transport through, 477,482 water-in-oil-in-water (W/OIW), 475,
494
multiple, 47,49, 170, 172,

608,616,622 non aqueous,47 oil-in-oil, 47 oil-in-water, 45
594,
599,
Diffision, 156-181 Diffisional exponent, 174
l-B-Diphenyl-l-3-5-hexatriene, 581
DLVO, 31 2 Doxorubicin, 321,537,542-543,547,552553,555-557,562-565
DPH (see 1-6-Diphenyl-l-3-5-hexatriene)
oil-in-water-in-oil, 594 stabilization, 440,444 aggregation, 427,4267 bridging flocculation, 427 coalescence, 419
Index
[Emulsion] colloidal, 445 creaming, 427 depletion, 428, DLVO theory, 430 electrostatic, 429 flocculation, 427 HLB, 435 by hydrocolloids, 440,460 mechanical, 444 by monomeric emulsifiers, 419 by polymeric emulsifiers, 440,466,
484
635
y-irradiation, 380 Gelati, 172 Gelatin, 25,27,31,599 Gel formation of lipid suspensions, 227-229,230 prevention by coemulsifying agents,
227
by proteins, 440,484 required HLB, 435 sedimentation steric, 440 water-in-oil, 351 water-in-oil-in-water, 47,594 Encapsulation, 22,23,453,518 or incorporation efficiency of the active substances, 615 Endocytosis, 581 Enzymes, 61,351,359 kinetics, 361 Epitaxical effect of emulsifier in lipid-in-water dispersions, 246 of phospholipids in melt-emulsified glyceride dispersions, 239 Ester sorbitan, 601 Estradiol, 336 Ethoxylated fatty alcohols, 601 Etoposide, 388 Experimental design, 97 principle application, 97 of the Z3type, 104 of the 33type, 105 methodology, 100 Exponential function, 165-168, 175 Fibrinogen, 360,361 Film, 27 Filtration, 161 Flow system permanent, analyses of, 615
of phospholipid stabilized solid lipid nanoparticles, 232-233 in phospholipid-stabilizedtripalmitate suspensions, 242-249 model of, 247-248 prevention of, 243-245 studied by transmission electron .. microscopy, 245-249 Genes as therapy, 543,544 Glycolic acid, 36,53,599 Grinding technique, 62 Growth factors, 265-266,276,288 Hadamard matrix, 100 Hard fat nanoparticles isothermal recrystallization of, 241 recrystallization of, 236 supercooling of, 240-241 synchroton radiation x-ray diffraction Of, 234-236 Hepatocytes, 365 Hexamethylenediamine, 352,353 1,6-Hexanediamine, 352,,353 High pressure homogenization, 225,229 Higuchi law, 165, 169 Hisitidase, 360 Histidinemia, 360 HLB, 357 Hyaluronic acid, 172, 173 Hydrocolloids, 460,486 emulsifiers, 484 galactomannans, 460 gelatin, 486 gum arabic, 486 stabilizers, 484 xanthan gum,460 Hydrolysis, 356 Hydrophile-lipophile-balance(HLB), 357 Immune system, 299 Immunoadsorbents,359 Immunoliposomes, 272,275-277 . Immunomodulator muramyl dipeptide, 543 muramyl dipeptide-L-alanyl-cholesterol,
543,563,564
Foods,421,440,448
colloids, 448 emulsifiers, 424,427,448 emulsions, 427,448 margarine, 421,427 Formaldehyde, 365 Freeze-drying, 164 Fusion, 581
Indomethacin, 169
636
Insect repellent, 403 Interfacial addition polymerization, 349 complexation, 349,366 condensation polymerization, 349 polyelectrolyte complexation, 350,366 polymerization, 349,352 reactions, 605 transport, 163,166-168 Intralipid, 156 Ion exchange, 166 Islets of Langerhans, 368 Isocarbacyclin, 163,164 Kelvin equation, 240 Lack of fit test, 107 Lactic acid, 36 ,53,599 Lamellar structure, 602,611 Lanreotide, 43 Lattice, imperfections, 236,239 Lecithin, 164 Leuprolide, 47 LHRH analogue of, 41,47,62 Linked polysiloxames, 599 Lipid A, 402 Lipid emulsion, 216 Lipid nanopellets, 218-219 Lipid suspensions (see also Solid lipid nanoparticles) as alternative delivery system for poorly water-soluble drugs, 216-218 incorporation of drugs into, 249-25 1 of melt-emulsified glycerides, 225-231 crystallinity of, 236-243,250 crystallinity index of, 239,242 degree of crystallinity, 236,243 gel formation of, 243-249 recrystallization process of, 236-243 time course of polymorphic transitions, 236 prepared by melt-emulsification, 225231 physical state o f , 233-236 physicochemical characterization of, 232-249 synchrotron radiation x-ray diffraction of, 234-236,240 prepared by precipitation in emulsified organic solvents, 223-225 polarized microscopy of, 246-247 Lipomicron, 591 Lipoproteins, 215-216 low-density (LDL),215-216
Index
Liposomes, 164, 168, 173,215,259-295, 297,329,333,401,578,589,597, 602,615,617 (see also Vesicles, lipid) bilayer mechanics, 305 bioadhesive, 276 cutaneous cell interactions, 580 drug biological activities. 287-290 drug encapsulation, 261,262,267,278279,281-283 drug release from, 261,267,279,281, 283-286 as pharmaceutical products, 259-295 stability, 260-261,267,276-281,300 biological, 309 chemical, 306 colloidal, 307 physical, 301 sterically stabilized, 300,311 sterility, 277-278 surface modification, 274-277 thermodynamics, 303 types and sizes, 260-264 Lipospheres, 377-410 antibiotics, 394 blank, 393 characterization, 380 composition, 396 drug. dlstribution, 386 loading, 385 release from, 386,396 elimination of, 399 insect repellent, 403 for intramuscularor subcutaneous injection, 222 local anesthetics, 389 morphology, 380 nabolipospheres, 406 particle size, 384 preparation, 379 prepared from microemulsions, 219-221 stearic acid, 220 sterilization, 382,384 for sustained drug release, 221-223 timolol-containing, 221 toxicity of, 393 vaccines, 399 Liquid crystals, 595,601,611,617,624 cubic, 417,509 hexagonal I, 417,506 lamellar, 415,506 reverse hexagonal, 417,506 stabilization, 415 L-lysine, 353,354
Index
L W (see Vesicles, large unilamellar) Lyotropics, 601
Magnetic, 362 Maltodextrin, 51 Marangoni effect, 197,205 Marcaine, 387 Matricial systems, 52 Melt-homogenization, 225 Melting point depression of melt-emulsified lipid suspensions, 240 of dispersed glycerides, 239 Membrane fluidity, 582 rigidity, 582 Methotrexate, 172 Micelles, 329,334 Micellizations aggregation number, 427 critical micellar concentration, 413 formation, 413 rod-like micelles, 417,507 size, 4 17 solubilization, 413 spherical, 417,506,508 surfactants exchange, 427 swollen micelles, 416,418,502 Microcapsules, 36-37,52,61,349,350 definition, 1 shell materials of alginate, 15 enteric polymers, 13 ethyl cellulose, 6 fats, 13 gelatin, 3, 10 gum arabic, 3, 10 hot-melt, 15, 17 maltodextrins, 11 melamine-fomaldehyde, 9 modified starch, 10 nylon, 7 polyamide, 7 polyurea, 7 polyurethane, 7 urea-formaldehyde, 9 Microemulsions, 593,599,608,616,621 Microencapsulationprocesses, 36,46,50 centrifugal extrusion in, 15 classification of, 2,3 complex coacervation i n ,3 fluidized bed coating in, 13 in situ polymerization in, 9 interfacial polymerization in, 7 polymer/polymer incompatibility in, 5
637
[Microencapsulation processes] rotating submerged orifice in, 10 rotational suspension separation in, 16 spray drying in, 10,25,27,50,56 static submerged nozzle in, 10 Microparticle, 156-18 1 Microscope analyses, 611 fluorescent, 612 ordinary optical, 612 polarized light, 612 Microsomes, 367 Microspheres, 36-38,41-44,47,50-53,55, aging of, 138 cyanoacrylate, 163 lipid, 218 magnetic, 161, 173 morphology cisplatin, 151 hydrocortisone, 144 lomustine, 145 poly(d,l lactide), 134-135 progesterone, 135 wax, 222 Mifepristone, 53 Milling methods, 62 Mitomycin C , 159 MLV (see Vesicles, multilamellar large) Mosquitoes, 403 Motor blockade, 391 Nanocapsules, 191-197,204-206,207,593 definition, 94 Nanolipospheres, 406 Nanoparticles, 183-208,616,621 cholesteryl acetate, 223-225 morphology of, 224 transmission electron microscopy of, definition, 94 fatty acid, 219-221 formation mechanism, 109, 124 hard fat, 234 magnetic cancer chemotherapy, 565-566 distribution, 555-557 polymeric, 2 14-2 15 systems, 592,599,605 tripalmitate, 224,234 Nanospheres, 184-191, 197-204,207,593 albumin, 204 definition, 94 gelatin, 203 polyacrylamide, 202
223-224 61,350,448,458,482
638
[Nanospheres] polyalkylcyanoacrylate,202 poly-c-caprolactone, polylactide-coglycolic, 202 polymethacrylate, 202 polystyrene, 202 Nebulization, 50, 52,55 laboratory, 53 Niosomes, 590 N,N-diethyl-m-tolumide, 403 Nonionic surfactant vesicles, 329,330 drug entrapment, 329,333 interaction with skin, 336 penetration enhancement of, 377 transport, 336 physical state, 329,333 preparation, 329,331 ether injection method, 332 handshaking method, 332 reversed phase evaporation, 332 sonication method, 331 Nylon 6-10,353 Oil silicone, 37-40 Opsonization, 564 Oxytetracycline, 394 Particle, 156-181 size, 384 distribution, 156, 163 Partition coefficient, 159,355 PEG, 407 Perfluorocarbon derivative, 601 Permeability, 363,368 Pesticides, 1 formulation q.c., 15, 16, 17 introduction to formulations of, 1-5 pH-activity curve, 361 Pharmacokinetics, 393,546-557 Phase inversion, 609 separation, 10, 13, 14,23 PHCA, 36-37,40-41,49,62-63 Phenylketonuria, 359 pH-Activity curve, 361 pH measurement, 163 Phospholipids, 379,589,597 Piperazine, 352,353 PLA, 36,40-41,46,55-57,61-62,95,98,109 PLAGA, 36-38,40-41,53,55,57,61 Plasmodiumfalciparum, 400 Polarizing microscopy of lipid-in-water dispersions, 232,233, 246-247
Index
Polarography, 163 Poloxamer, 228 as steric stabilizer of lipid suspensions, 228 Polyacrylate, 367 Poly(a-hydroxy-carboxylicacids), 35,47 Polyalkylcyanoacrylate,599 Poly(alkyl-2-cyanoacrylates), 364 Polyamide, 35 1 Polyamine, 353 Polyanhydrides, 171 Polycaprolactone, 402 Poly(d,l-lactic acid-coglycolic acid), 37 Poly(d-l lactide), 169, 171 Polyester, 95, 353 Polyethylene glycol, 300,407 Polyglycerol, 601 Poly(iminocarbonates), 173 Poly(1actide-co-glycollide), 169, 171, 173 Polylysine, 366 Polymer albumin, 555-556 brush, 312 desolvatation, 202 membrane, 35 1 phase-separation 36,43 poly(alky1 cyanoacrylate), 537,547,557 poly(alky1 methacrylate), 537,547,552 poly(buty1 cyanoacrylate), 547,553 polyglutaraldehyde,537 poly(hexy1cyanoacrylate), 545 poly(isobuty1cyanoacrylate), 542-543, S57 ". poly(isohexy1cyanoacrylate), 544,547, 552-553.563-564 poly(1actic acid), 537,554 Polymeric barrier, 50 core, 385 surfactant, 601 Polymerization, 183-208 anionic, 195 dispersion, 198 emulsion, 185, 191, 195, 198, 199 interfacial 6,9, 191, 192, 193, 197, 198, 204,349 interfacial condensation, 349 Polymorphic transitions of glycerides, 233 in melt-emulsifiedglyceride dispersions, 236-243 time course of, 241 Poly(ortho-esters), 173 Polyoxyethylene alkyl ether surfactants, 331
lndex
Polyoxyethylene sorbitan, 601 Polyurea, 353,363 Polyurethane, 353 Preferential adsorption of surfactants to glyceride nanocrystals, 248-249 Production liposomes, 602 liquid crystals, 61 1 microemulsions, 608 multiple emulsions, 608 nanoparticulate systems, 605 Products thermally sensitive, 55 Progesterone, 46 Proliposomes, 598 Proteins, 61,351,440,484 absorption on latex, 440 absorption on liquid interfaces, 5 18 Bovine serum albumin (BSA), 440,443, 487,490 denaturation, 203-204 as emulsifiers, 440 as food colloids, 440,484 lysosyme, 440,484 modified chemically, 519 modified enzymatically, 519 soy proteins, 440,484 Pulsed release, 173 Randall-Selitto instrument, 389 Recrystallization from dispersed lipid melt, 232,240-241 of melt-emulsified glyceride nanoparticles, 236-243 of melt-emulsified trilaurate, 229 Release measurement, 158-163 model, 157,165-176 rate, 156-181 Response surface methodology, 99 Rheological analysis, 614 Rose bengal, 162 Sebacoyl chloride, 352,353,354 Sensory block, 391 Skin absorption, 403 SMV (see Vesicles, small multilamellar) Sodium dodecyl sulfate, 27,31 Sodium glycocholate, 228 Solidification of lipid suspensions, 230 Solid tipid nanoparticles (see also Lipid suspensions) drug release from, 253
639
[Solid lipidnanoparticles] incorporation of drugs into, 249-251 in vitro cytotoxicity of, 252 of melt-emulsified glycerides, 225-23 1 crystallinity of, 236-243,250 crystallinity index of, 239,242 degree of crystallinity, 236,243 recrystallization process of, 236243 time course of polymorphic transitions, 236 prepared by melt-emulsification,22523 1 f , 227-229,230 gel formation o lyophilization of, 230-231 mean particle size of, 227 spray-drying of, 230 storage stability of, 225,227-228 prepared by sonication, 227 Solvent evaporation, 43,45 extraction residual, 43,56,61-62 Somatostatin f , 62 analogue o Spectroscopy photon correlation, 612 Spheronization, 62-63 Spray congealing, 62 Stability, 615 liposomes, 615 liquid crystals, 617 microemulsions, 616 multiple emulsions, 617 nanoparticles, 616 window, 37-40 Steric stabilization, 228 Sterol, 598 Storage stability of solid lipid nanoparticles, 225,227228,252 Sucrose ester Wasag-7,331 Supercooling degree of, 232 of hard fat and tripalmitate dispersions, 240-241 of melt-emulsified trilaurate, 229 Supercritical conditions, 57-60 fluid, 58,60-61 phases, 58,61 Supramolecularbiocarriers, 591 Surface activity, 25,26 Surface modification of colloidal drug carriers, 231
640
[Surface modification] by modifying agents, 554-557(see also Antibody, monoclonal) to reduce uptake by reticuloendothelial system, 231 of tripalmitate nanoparticles, 231 Surfactants, 357,413,424,432,599 anionic, 464,467 cationic, 464 HLB, 435,465,477,497 hydrophilicity, 438 micellization, 413,487 non-ionic, 438,450,460,464,600, non-ionic vesicles, 590,610 surface tension, 472,502 wetting, 473 zwitterionic, 471 SUV (see Vesicles, small unilamellar) Targeting, 259-277 Taxol, 406 Technologies solvent-free, 62 Terephthaloyl chloride, 352,353,354 Ternary diagrams, 37 Therapy photodynamic, 554455,557 Thermotropics, 601 Tissue distribution, 407 Toxicity, 537,543,562 cardiotoxicity, 552,564 embolism, 562 neutropenia, 565 Transferosomes, 329,334 Transmission electron microscopy of cholesteryl acetate nanoparticles, 223-224 of melt-emulsified tripalmitate suspensions, 245-249 Triglycerides polymorphic behavior of, 233 Tripalmitate nanoparticles crystallinity of stored, 242 crystal shape of, 245 DSC heating scans of, 239 gel formation of, 243-249 gel structure of, 246 prepared by precipitation in emulsified solvent, 224 stabilized by phospholipids only, 239 DSC of, 239 f , 240-241 supercooling o surface-modificationof, 23 1 synchrotron radiation x-ray diffraction Of, 234-236
lndex
pripalmitate nanoparticles] transmission electron microscopy of, 245-246 ubidecarenone-loaded, 250 zeta potential of, 23 1 Triptoreline, 43 Tumor EMT-6 mouse mammary tumor,557 hepatoma, 552 leukemia, 537,557,566 Lewis lung carcinoma, 553 metastases, 553454,562463,565466 models, 322 sarcoma, 553,555,557,565 Turbine method, 52 Two-stage procedure, 609 Tyloxapol, 228 as steric stabilizer of lipid suspensions, 228 Ultracentrifugation, 164 Ultrafiltration, 161 Ultraviolet spectroscopy, 161-163 Uptake cellular, 536-546 Urea, 359 Urease, 359 Vaccines, 399 Vesicles large unilamellar, 330 lipid, 330 multilamellar large, 330,590 phospholipid, 578 skin interaction with, 329,335 small multilamellar, 579 small unilamellar, 330,579,590 in colloidal tripalmitate suspensions, 246,247-248 transdermal application of, 329,334 Viscoelastic oscillator analyses, 614 Vitamin A acid, 583 Witepsol W35,233,249 X-ray diffraction, 234 synchrotron radiation, 234 of melt-homogenized glyceride dispersions, 234-236 time-resolved during temperature scans, 236 Zeta potential of surface-modifiedtripalmitate nanoparticles, 23 1

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Methods and Industrial Abblicalions

edited by Simon Benita

Marcel Dekker, Inc. New York.

Basel Hong Kong

96- 1569 CIP

This book is printed on acid-free paper.

Part I: Methods of Encapsulation and Advances in Production Technology

2. Microencapsulation of Oil-in-Water Emulsions by Proteins Shlomo Magdassi and Yelena Vinetsky

Patrick Couvreur, Guy Couarraze, Jean-Philippe Devissaguet, and Francis Puisieux

Kirsten Westesen and Britta Siekmann

Rimona Margalit and Noga Yerushalmi

Part IIk Applications of Particulate Delivery Systems

Nissim Garti and Abraham Aserin

Jean-Christophe Leroux, Eric Doelker, and Robert Gurny

17. CosmeticApplications of Liposomes G6rard Redziniak and Pierre Perrier

This Page Intentionally Left Blank

PROCESSES FOR PREPARING MICROCAPSULES

Microencapsulation of A Processes Survey

Table 1 List of Types A and B Encapsulation Processes

Form Emulsion of Core In Solulion Gelalln (40-60C)

Core Malerial (Llquicl)

7 Crosslink with Glutaraldehyde

A Survey of Microencapsulation Processes

Core Material (Small Particles)

Dispersion of Core Material in Hot, Phase-Separated Polymer Solution

Wash and Harvest Capsules

A Survey of Microencapsulation Processes

Core Material ["oil")

icroencapsulation of A Processes Survey

A Survey of Microencapsulation Processes

Shell Material (e.g.. Warm Gelatin Solution)

Warm Carrier Phase (e.g.,Vegetable Oil)

Gel Beads that Core can be harvested Material and dried

Fig. 5 Schematicdiagram of a submerged two-fluid nozzle used microcapsules. (Courtesyof C. Thies.)

A Survey of Microencapsulation Processes

Core Material Being Coated Formulation Gas Flow

A Survey of Microencapsulation Processes

3. Centrifugal Extrusion Fig. 7 is a schematic diagram of a centrifugal extrusion process [20].

Shell gelled b y immersion in gelling bath

Fig. 7 Schematicdiagram of centrifugaltwo-fluidnozzleused crocapsules. (Courtesy of C. Thies.)

A Survey of Microencapsulation Processes

Free Coating Material

Fig. 8 Schematic diagram

of rotational suspension separation encapsulation

A Survey of Microencapsulation Processes

11. Methods of Encapsulation A. Phase Separation B. Spray Drying C. Heat Denaturation

Microencapsulation of Oil-inEmulsions Water

Magdassi and Vinetsky

Hardening of coacervate-capsule wall by chemical or physical means

Microencapsulation of Oil-inEmulsions Water

Microencapsulation Emulsions Water of Oil-in-

Magdassi and Vinetsky

Microencapsulation of Oil-in- Water Emulsions

Magdassi and Vinetsky

Aqueous solution of SDS, pH=6.1

Removal of water (lyophilization, evaporation)

Microencapsulation Emulsions Water Oil-in- of

12. 13. 14. 15. 16. 17. 18.

Microencapsulation Emulsions Water Oil-in- of

Biodegradable Microspheres: Advancesin Production Techno

Hew6 Rolland and Vincent Vande Velde

Benoit et al. INTRODUCTION

bad polymer dissolution

k PLA ACE DCM PLAACE PLADCM ACE*DCM PLA~