SOLID LIPID NANOPARTICLES AS COLLOIDAL DRUG CARRIER SYSTEMS

Antnio J. Almeida
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmcia Universidade de Lisboa Portugal aalmeida@ff.ul.pt

A.J. Almeida, 2007

Universidad de Sevilla, Facultad de Farmacia, November 2007

MICROPARTICLES AND NANOPARTICLES: THE BASIC CONCEPTS

A.J. Almeida, 2007

INOVATIVE DRUGS

The recent tremendous advances in health science technology and inexorable progress in related scientific innovations, coupled with changes in population demographics and the colouring of political agendas with the economics of disease, are all acting in concert to take the pharmaceutical industry into exciting and challenging new dimensions. G. Gregoriadis (2002)

A.J. Almeida, 2007

INOVATIVE DRUGS

New drug delivery systems Genomics Biotechnology Neurosciences Gene Therapy Bioinformatics Proteomics Metalolomics

Inovative Drugs

AIDS Cancer Ebola Tuberculosis Alzheimer SARS

Economics Demographic changes Globalization

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DIFFERENT APPROACHES

PHYSICAL APPROACH Macroscopic systems activated by physical processes (capsules, granules, etc)

CHEMICAL APPROACH Chemical modification of drugs (derivative preparation, salts, pro-drugs, association to polymers, etc.) PHARMACEUTICAL APPROACH (pharmaceutical formulation)

BIOCHEMICAL / BIOTECHNOLOGICAL APPROACH Naturally occurring macromolecules Cells Colloidal polymeric or lipid systems

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GENERAL CONCEPTS IN MODIFIED DRUG RELEASE in vitro

C

Time

in vivo
Drug concentration in serum
0

12

18

Time (hour)
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GENERAL CONCEPTS IN MODIFIED DRUG RELEASE

Convencional Release Modified Release Delayed Release Prolonged Release Controlled Release Drug Targeting

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Chien (1992). Novel Drug Delivery Systems

NEW DRUG DELIVERY SYSTEMS

SKF
Spansule

ALZA

Progestasert
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Oros

Ocusert

FORMULATION OF NEW DRUG MOLECULES PROTEIN DRUGS Physicochemical problems

Molecular weight and size Complex struture Conformational stability Solubility Sensitivity (light, temperature, pH) Crystallization Molecularar interactions Adsorption Aggregation Presence of protein impurities

Biological problems
Biodegradation by digestive enzymes Short in vivo half-life Imunogenicity Complex metabolism Dificulty in crossing mucosal barriers No access to some compartments

Pharmacokinetic Problems Analytical Problems

A.J. Almeida, 2007

PROTEIN DRUGS

9 Investigation of alternative administration routes (nasal, rectal, pulmonary, etc.); 9 Investigation of alternative prolonged or pulsed release drug delivery systems; 9 Investigation of new dosage forms that can provide protection against premature
degradation;

9 Invstigation of new site specific drug delivery systems.

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PARTICULATE SYSTEMS

liposomes cochleates nanocapsules iscoms Particulate cubosomes Systems microemulsions niosomes microspheres transfersomes nanoparticles micelles nanosuspensions virosomes

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COLLOIDAL SYSTEMS

Definition IUPAC

COLLOIDAL

The term refers to a state of subdivision, implying that the molecules or polymolecular particles dispersed in a medium have at least in one direction a dimension roughly between 1 nm and 1 m, or that in a system discontinuities are found at distances of that order.

A.J. Almeida, 2007

COLLOIDAL SYSTEMS
Microparticles Solid particles made of polymeric or solid lipid materials Podem conter o frmaco disperso, encapsulado ou adsorvido superfcie. Microspheres (1 m) Microcapsules (reservoir systems) Microparticles (matrix systems)

Nanospheres (<1 m) Nanocapsules (reservoir systems) Nanoparticles (matrix systems)

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POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS MAIN POLYMERS

Biocompatibility versus Biodegradation Biodegradation mechanisms: Type I, II e III

9 9 9 9 9 9 9 9 9 9 9
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poly(lactide-co-glycolide) (PLGA) poly--caprolactone (PCL) poly(hydroxibutiric acid) (PHB) polyorthoesters polyanhydrides polyphosfazenes polyalkylcianoacrylates (PIBCA ou PHCA) proteins (albumine, gelatine, gliadines) starch chitosan alginate

POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS

Biodegradation Mechanisms Type I

Type II

Type III

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POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS

BIODEGRADABLE IMPLANTS Poly(lactide-co-glycolide) (PLGA)

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POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS

9 Drug release by polymer enzymatic degradation. 9 Drug release rate is controlled by the rate of the enzymatic reaction. 9 Predominat use in controlled release from biodegradable polymers often used in
particulate carriers (e.g. albumine, gelatine, polyalkylcianoacrylates).

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POLYMERIC MICRO- AND NANOPARTICULATE SYSTEMS

Main Technologic and Therapeutic Uses

Diagnostic systems Controlled drug delivery systems

Implants Aerosols Solid dosage forms Vaccines Site-specific drug delivery

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MAIN MICRO / NANOENCAPSULATION TECHNIQUES

Colloidal Systems
1. Spray coating; Pan-coating, Spray-drying 2. Droplet extrusion 3. Coacervation / Phase separation 4. Emulsion solidification 4.1. Solvent evaporation (simple or multiple emulsion) 4.2. Solvent extraction (simple or multiple emulsion) 4.3. Melting and solidification (simple or multiple emulsion) 5. Polymerization methods 5.1. Interfacial polymerization 5.2. Emulsion polymerization

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MAIN MICRO / NANOENCAPSULATION TECHNIQUES Solvent Evaporation/ Extraction

simple emulsion
A Polymer in organic solvent Drug Aqueous phase + surfactant Drug suspended in polymer solution

multiple emulsion
Polymer in organic solvent Drug + water + emulsifying agent w/o emulsion B Aqueous phase + surfactant

simple [A] or multiple [B] emulsion Evaporation or Extraction of organic solvent

Particle isolation by filtration or centrifugation

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MAIN MICRO / NANOENCAPSULATION TECHNIQUES Melting and Solidification (1)

simple emulsion
A Melted lipid Melted lipid Drug Aqueous phase heated at same temperature Drug suspended in melted lipid

multiple emulsion
Drug+ Water + Emulsifying agent w/o emulsion B Aqueous phase heated at same temperature

Simple [A] or multiple [B] emulsion Cooling to solidify disperse phase

Particle isolation by filtration or centrifugation

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MAIN MICRO / NANOENCAPSULATION TECHNIQUES Melting and Solidification (2)

Oil Protein Solution (albumin or gelatine) Emulsion 1 Drug

Oil at 180C + surfactant

Emulsion 2

Cooling

Particle isolation by filtration or centrifugation

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MAIN MICRO / NANOENCAPSULATION TECHNIQUES Emulsion Polymerization

9 A single liquid (or disolved) reacting monomer 9 Drug disolved or suspended 9 Polymerisation started by chemical agent or radiation 9 Polymer MW and particle size dependent on: 9monomer concentration 9Initiator concentration 9Temperature 9 Emulsion stabilizer needed 9 Particles >20 nm

Ex.: PIBCA; polyacrylamide

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MAIN MICRO / NANOENCAPSULATION TECHNIQUES Droplet Extrusion

Gelatine + alginate + drug

Washing CaCl2

Fluid-bed drying

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Ferreira Almeida and Almeida (2004). J Control Release.

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MICRO- AND NANOPARTICULATE SYSTEMS CONTROL OF FORMULATIONS

Particle size 9Electron microscopy 9Electric sensing zone 9Laser diffraction 9Photon correlation spetroscopy (PCS) 9Optical sensing zone Encapsulation efficiency Drug loading Surface charge (zeta potential) Hydrofobicity 9Contact angle 9Partition coeficient 9Hydrophobic interaction chromatography Release profile

Sterilization

Stability

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MICRO- AND NANOPARTICULATE SYSTEMS DRUG RELEASE

Dependent on: 9 Drug location inside the particle 9 Type and amount of polymer 9 Particle size and density 9 Cross-linking 9 Physicochemical properties of drug and polymer 9 Drug MW and concentration 9 Presence of excipients 9 Release medium

General drug release kinetics

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PARTICULATE CARRIERS IN THE PHARMACEUTICAL MARKET

LIPOSOMES 9physical and chemical stability problems 9no cheap liposome available 9marketed products but behind expectations

POLYMERIC NANOPARTICLES 9input: 30 years of research 9no output: no products on the market

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SITE-SPECIFIC DRUG DELIVERY

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SITE-SPECIFIC DRUG DELIVERY

The magic bullet concept

The term used to describe a specific cure for syphilis, which would attack the syphilis spirochaete while having no effect whatsoever on human tissue.

Paul Ehrlich (1854-1915)

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RATIONALE FOR SITE-SPECIFIC DRUG DELIVERY

Specificity

Translocation across biological barriers

Protection against inactivation

Target site Other sites

Carrier Drug

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Puisieux and Roblot-Treupel (1989) STP Pharma

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RATIONALE FOR SITE-SPECIFIC DRUG DELIVERY

Exclusive delivery to specific compartments (and/or diseases) Access to previously inaccessible sites (e.g. intracellular infections) Protection of drug and body from unwanted deposition, which could lead to unwanted reactions and metabolism, etc. Controlled rate and modality of delivery to pharmacological receptor Reduction in the amount of drug employed

Drug safety Drug efficacy Patient compliance

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SITE-SPECIFIC DRUG DELIVERY: DRUG TARGETING

PASSIVE TARGETING
Uptake by MPS and tropism for the lysosomes macrophages Kupffer cells

Translocation to the tissues hepatocytes bone marrow splenocites tumors (?)

Capillar embolism lung targeting (iv) specific regions (ia)

0,01

10

100

ACTIVE TARGETING Magnetic fields Lynphotropic adjuvants MPS blocking Receptor mediated processes Immunotherapy 0,1 Diametre (m)
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DRUG TARGETING

Utilizao de lipossomas como transportadores de frmacos

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Crommelin et al. (http://www.drugdeliverypartnerships.com/lip.html)

MAIN DRUG TARGETING SYSTEMS

Soluble carriers (monoclonal antibodies, dextrans, soluble synthetic polymers) Particulate carriers (liposomes, microspheres, nanoparticles, etc.) Target-specific recognition moieties (monoclonal antibodies, carbohydrates) Antibody-directed enzyme/prodrug therapy Virus-directed enzyme/prodrug therapy

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SOLID LIPID NANOPARTICLES

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LIPID NANOPARTICLES

9 Lipophilic colloidal delivery system (R.H. Mller, Berlin; M.R. Gasco, Turin); 9 Efficient and non-toxic drug carrier specially for lipophilic drug molecules; 9 Composed of physiological / well tolerated excipients e.g. GRAS (= similar to emulsions and liposomes); 9 Possess solid matrix (= similar to polymeric nanoparticles); 9 Protective properties 9 Controlled release properties 9 Colloidal dimensions and controlled release behaviour enable drug protection and administration by parenteral and non-parenteral routes.

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LIPID NANOPARTICLES
9 Versatility 9Parenteral administration (Fundar et al, 2000) 9Brain delivery (Fundar et al, 2000) 9Ocular delivery (Cavalli et al, 2002) 9Rectal delivery (Sznitowska et al, 2001) 9Oral delivery (Garca-Fuentes et al, 2002) 9Topical delivery (Souto et al, 2004) 9 Potential vaccine delivery systems (Almeida et al, 1997) 9 Large scale production possible using lines available in pharmaceutical plants (i.e. lines for parenteral emulsions (Mller et al, 2001)

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SLN PREPARATION

High Pressure Homogenization (R.H. Mller et al.)

-SkyePharma PLC, Berlin, Germany -PharmaSol GmbH, Berlin, Germany

Microemulsion Technology (M.R. Gasco)

- Vectorpharma/Eurand Spa, Turin, Italy

Solvent Evaporation

Phase Inversion Type

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SLN PREPARATION BY HIGH PRESSUE HOMOGENIZATION (HPH)

HOT HOMOGENIZATION PROCESS A
Drug Aqueous phase at same temperature Drug solution in melted lipid Emulsion w/o Aqueous phase at same temperature Melted Lipid Melted Lipid Drug + Water + surfactant

Emulsification using Ultra-turrax single [A] or double [B]

High-Pressure Homogenisation

Cooling for solidification and particle hardening

SLN suspension

A.J. Almeida, 2007

HIGH PRESSURE HOMOGENIZATION (HPH)

APV Gaulin LAB40 discontinuous (40 g batch)

A.J. Almeida, 2007 E. Souto (2005)

PhD Thesis

Dept. Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin

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SLN PREPARATION BY HPH

HOT HOMOGENIZATION PROCESS

Basic principles: 9equipment can be qualified and validated 9accepted by regulatory authorities in production lines used for parenterals 9existing industrial production lines for i.v. parenteral emulsions can be used 9all production lines developed for SLN are usable

A.J. Almeida, 2007

SLN PREPARATION BY HPH

APV Gaulin LAB40 continuous (0.5-2 Kg batch)

APV Gaulin LAB60 (2-10 Kg batch)

APV Gaulin 5.5 (150 Kg/h)

A.J. Almeida, 2007

Dept. Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin

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SLN PREPARATION BY HPH

COLD HOMOGENIZATION PROCESS
Melted Lipid

Drug Drug suspension in melted lipid

Liquid nitrogen

Micronization

Suspension in water using Ultra-turrax

High-Pressure Homogenisation

SLN suspension

A.J. Almeida, 2007

Dept. Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin

DRUG ENTRAPMENT INTO SLN

m.p. drug m.p. lipid

homogeneous matrix

m.p. drug < m.p. lipid

lipid-enriched core

m.p. drug > m.p. lipid

drug-enriched core

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Mller et al (2000). Eur J Pharm Biopharm

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DRUG ENTRAPMENT INTO SLN

PROBLEMS ASSOCIATED WITH SLN Formation of perfect crystalline structure during storage ( modification) drug expulsion

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Souto (2007)

SLN PROCESSING DISADVANTAGES

9 Physical stability of aqueous solution 9 Gel formation 9 Particle aggregation 9 High water content of dispersions (70 - 95%) 9 Need to remove too much water in tablet / pellet production 9 Dosing problems (e.g. dispersion for soft gelatin capsules, lipid particle content max. 30%)

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Souto (2007)

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SLN PROCESSING DISADVANTAGES

SLN Solid Lipid Nanoparticles

9 Produced from solid lipids 9 Tend to form perfect crystals drug expulsion

NLC Nanostructured Lipid Carriers

9 Produced from blend of solid and liquid lipids 9 Particles are in solid state at body temperature 9 Inhibit crystallization process by mixing spatially very different molecules imperfections in lattice

not just mixing solid lipids but: controlled nanostructuring of lipid matrix
9 to accommodate drug 9 to control release 9 to trigger release
A.J. Almeida, 2007

Souto (2007)

LIPID NANOPARTICLES

NLC a multiple carrier

SLN: one phase (= solid lipid) NLC: multiple O/F particle (oil nanodroplets in solid fat nanoparticles)

Advantages:
higher drug load, firm incorporation (e.g. 1% Retinol in SLN, 6% in NLC)

Solid lipid particle

Oil-loaded particle

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Souto et al (2007) Pharm Tech Europe (in press)

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LN AS A POTENTIAL PROTEIN / ANTIGEN DELIVERY SYSTEM

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PROTEIN NANOENCAPSULATION IN SLN

Lipid + poloxamer 188 Aqueous Lysozyme

melting

Solidification in liquid nitrogen

Micronisation in a powder-mill

Solubilisation using a rotary evaporator

Pre-mix using a highspeed homogeniser Lipid composition Hard fats (Softisan 142 and Witepsol E85) Cetyl alcohol Propyleneglycol palmitostearate (Monosteol) PEG 300 mono-, di-stearate (Superpolystate) Surfactant Poloxamer 188 Tween 80

HPH at room temperature

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Almeida et al (1997). Int J Pharm

25

STABILITY OF LYSOZYME THROUGHOUT FORMULATION

Influence of temperature (50)

MW rt 0,5h 1h 2h 3h 4h 5h
Lysozyme activity (Units/ml) 500 400 300 200 100 0 Distilled water 3% Tween 80 3% Na Cholate 3% Poloxamer 188

Day 1 Day 10

Influence of HPH treatment (N cycles/Pressure)

MW Ct 3 4 5 6 7 8 9 10

500 bar/1 cy/ 25C

1000 bar/3 cy/ 50C

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Almeida et al (1997). Int J Pharm

NANOENCAPSULATION OF LYSOZYME IN LN

5000 Lysozyme activity (Unit/g of lipid) 4000 3000 2000 1000 0

Softisan 142/Cetyl alcohol Witepsol E85/Cetyl alcohol Monosteol Superpolystate

MW

Water

S/CA

W/CA

Mono

Sup

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Almeida et al (1997). Int J Pharm.

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NANOENCAPSULATION OF LYSOZYME IN LN
140 Lysozyme activity (%) 120 100 80 60 40 20 0

MW 97.0 KDa 66.2 KDa 45.0 KDa 31.0 KDa 21.5 KDa 14.4 KDa

Ct

Initial

Sup Soft/CA

LN

Initial

Sup Wit/CA

LN

Lipid matrix

Lys Content (%) 0.22 0.30

Particle Size Laser diffraction (m sd) D50 0.600.00 D90 1.860.02 D50 0.580.00 D90 0.930.01

Particle Size PCS (nm sd) 64412.9 5497.0

Zeta Potential (mV sd) -11.00.2 -9.80.7

S 142/CA W E85/CA

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Almeida et al (1997). Int J Pharm

DEVELOPMENT OF OVA-CONTAINING LN

Lipid melting heating heating Aq.phase emulsification

Lipid composition
gliceryl behenate (Compritol 888 ATO) gliceryl palmitosterarate (Precirol ATO 5) cetyl Alcohol

high-speed homogenization 8 000 - 10 000 rpm

Surfactant
Tween 80

Speed rate
8 000 - 10 000 rpm

solidification

Particle size (PCS) 100-400 nm PI - 0,250

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Videira and Almeida (1998). Proceed III SPLC-CRS Conf

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NANOPARTICLE CHARACTERISATION: PHYSICAL STABILITY

SLN (nm) Lipid (%) 6 Pr Cp W/AC Pr Cp W/Ac Pr Cp W/Ac dmt=3d 103 171 284 196 240 252 230 373 307 dmt=30d 115 168 280 205 253 265 200 378 289

OVA SLN (nm) dmt=3d 128 196 314 dmt=30d 153 205 320

214 206 280 305 290 312 300 390 400 nd nd nd

10

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Videira et al. (2002). Proceed V SPLC-CRS Conf

ENTRAPMENT EFFICIENCY

Tween 80

Lipid
Pr
3 30 3 30 3 30 3 30 3 30 3 30 3 30 3 30 3 30

2%

6%

Cp W/CA Pr

8%

Cp W/CA Pr

10%

Cp W/CA

60%

100%

OVA-6SLN 92% EE with 2% surfactant

A.J. Almeida, 2007

Videira et al (2002). Proceed V SPLC-CRS Conf

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FORMULATION STUDIES OF OVA OF LYSOZYME IN LN

80 70 OVA released (%) 60 50 40 30 20 10 0 45.0 KDa 0 5 10 15 20 25 30 35 Time (day) 31.0 KDa 97.0 KDa 66.2 KDa

Protein Integrity
MW LNsup 1W 2W 3W

Conc.OVA (%)

40 30 20 10 0 0 50 100 150 200 250 300 350 400 time (min)

21.5 KDa 14.4 KDa

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Videira et al (2002). Proceed V SPLC-CRS Conf

ADSORPTION STUDIES

OVA/lipid (%) 4 A.E (%) 25C 15C 90 80

7 80 70

10 82 79

Desorption profiles
Conc. OVA (%) 100 80 60 40 20 0 0 250 500 750 time (min) 1000 1250 1500 25C 15C

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Videira et al (2002). Proceed V SPLC-CRS Conf

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LN AS A POTENTIAL PROTEIN / ANTIGEN DELIVERY SYSTEM

9 LN are potential protein carriers with high protein binding 9 Formulations are stable 9 Partial adsorption of protein may not be precluded 9 Production conditions seem not to damage protein integrity 9 Following a burst effect, a sustained release profile is obtained 9 Loading capacity is suitable to the usual antigen doses

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Videira et al (2002). Proceed V SPLC-CRS Conf

PULMONARY DELIVERY OF SLN

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PULMONARY ADMINISTRATION

The Lung

Unique features that can facilitate systemic delivery via pulmonary administration of drugs:

Large surface rea (75 m2). Good vascularisation. Large capacity for solute exchange. Ultra-thineness of the alveolar epithelium (0.1-0.5 mm). First-pass metabolism is avoided.

A.J. Almeida, 2007

PULMONARY DELIVERY USING PARTICULATE CARRIERS

Advantages

100 80
Lung

60

An alternative non-invasive means for both local and

Tobramycin (%)

40 20 0 80 60 40 20 0 80 60 40 20 0 6 Time (hour)
Free Liposomal (0,7 m) PLGA Microencapsulated (0,7 m) Kidney Blood

systemic drug delivery using particulate carriers.

Avoids instability in blood stream and uptake by the MPS. Allows high concentrations of drug in the lungs

minimizing side toxic effects.

A potential route for drug therapy and immunisation.

24

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Poyner et al (1995). J Controlled Release

31

LYMPHATIC TARGETING AND LUNG CANCER THERAPY

A leading cause of death around the world, accounting for 13% of newly diagnosed cancers and 30% of all cancer deaths. Very low survival rate (14% in 5 years; mean survival time 14 months). Metastatic disease starts at very early stages, particularly in SCLC, the majority of patients with lung cancer having metastatic disease at the time of diagnosis. Metastasis spread mainly via the lymphatics to the lymph nodes, liver, brain, bones and adrenal glands. Early diagnosis is crucial to increase survival rate and staging is critically dependent upon the involvement of the lymph nodes that drain the region containing the tumour. Chemotherapy plays an important role alongside surgery and radiation therapy, but lung cancer is usually diagnosed at an incurable stage.

A.J. Almeida, 2007

IMAGING USING PARTICULATE CARRIERS

Lung perfusion imaging is based on the trapping of i.v. injected radiolabelled albumin microspheres (10-50m) in the capillary bed of the lung: Technetium [99m Tc] Microspheres Injection (Ph Eur).

I.v. injected radiolabelled PLGA microspheres have also been proposed for lung imaging (Delgado et al. 2000).

Several

particulate

carriers

have

been

used

for

lymphoscintigraphy, such as liposomes, sulfur colloid, PMMA and PHCA nanoparticles.

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PULMONARY ADMINISTRATION OF LIPID NANOPARTICLES

Lipid Nanoparticles Administration have been studied by parenteral and non-parenteral routes.

To investigate LN as a potential drug carrier to the lungs and, through alveolar airways, to the lymphatic system, thus optimising concentration at the tumour site or at distant metastasis sites.

To evaluate LN in vivo fate after pulmonary absorption upon nebulisation and delivery to laboratory animals.

A.J. Almeida, 2007

NANOPARTICLE PRODUCTION

Lipid melting heating heating Aq.phase emulsification

high-speed homogenization 8 000 - 10 000 rpm

Lipid composition
gliceryl behenate (Compritol 888 ATO) gliceryl palmitosterarate (Precirol ATO 5) cetyl Alcohol solidification

Surfactant
Tween 80

Speed rate
8 000 - 10 000 rpm

A.J. Almeida, 2007

Videira et al (2002). J Drug Targeting

33

LABELLING AND ADMINISTRATION

Me

Me

99mTcO 4

NaCl

Me

NH

NH

Me

Me

N OH

N OH

Me

D,L-hexamethylpropyleneamine oxime (HMPAO)

Lipid Nanoparticles

Radiolabelled LN

- Scintigraphy
Labelling efficiency

Ultrasonic nebulization
anaesthetised male Wistar rats

Particle size

A.J. Almeida, 2007

Videira et al (2002). J Drug Targeting

CHARACTERISATION OF 99mTc-HMPAO-LN

ASPECT

PARTICLE SIZE

Before aerosolisation D 50 (nm ) PI 197.5 194.8 196.1 0.231 0.291 0.261

After aerosolisation D 50 (nm ) PI 218.3 220.3 219.3 0.378 0.379 0.379

A.J. Almeida, 2007

Videira et al (2002). J Drug Targeting

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CHARACTERISATION OF 99mTc-HMPAO-LN
Radiochemical Purity (TLC) Labelling efficiency: 972%
99mTc-HMPAO

reduced-hydrolysed 99mTc Na 99mTcO4 Other hydrophilic species

99mTc-HMPAO

99mTc-HMPAO-LN

t0

t0

Sys I t30

Sys II t30

Sys I

Sys II

butanone
A.J. Almeida, 2007

0.9% aq. NaCl

butanone

0.9% aq. NaCl

BIODISTRIBUTION

99mTc-HMPAO

99mTc-HMPAO-LN

A.J. Almeida, 2007

Videira et al (2002). J Drug Targeting

35

BIODISTRIBUTION

3 min

8 min

60 min

99mTc-HMPAO

99mTc-HMPAO-LN

A.J. Almeida, 2007

Videira et al (2002). J Drug Targeting

LYMPHATIC DISTRIBUTION OF 99mTc-HMPAO-LN

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Videira et al (2002). J Drug Targeting

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LYMPHATIC DISTRIBUTION
Lungs
T1/2 10 min

Axillary nodes

Periaortic nodes

Inguinal nodes

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Videira et al (2002). J Drug Targeting

BIODISTRIBUTION IN RATS 4h AFTER INHALATION

35 30 % activity/g of tissue 25 20 15 10 5 0
Se ru ly m m Ax ph illa no ry de ly In m s gu ph in no al de ly m s ph no de s Li ve r Sp le en Th yr oi d Ki dn ey s rt Br Sm ai al n li nt e La st in rg e e in te st in e Te st ic le s s Lu ng He a Ur in e

Pe r

ia o

r ti c

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Videira et al (2002). J Drug Targeting

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BIODISTRIBUTION OF 99mTc-HMPAO IN RATS AFTER i.v. INJECTION

Tissue 2 min Brain Blood Lungs Kidneys Intestines Liver Urine 2.10.1 11.00.4 3.70.8 6.51.1 15.41.7 13.91.9 0.40.4

% Dose in Organ 10 min 2.10.2 10.51.1 3.10.2 6.30.2 15.80.2 10.20.4 3.20.3 30 min 1.80.2 9.40.2 3.10.1 6.60.4 19.80.3 10.40.4 9.50.1 60 min 2.00.2 8.60.3 2.80.3 6.41.2 21.51.5 8.61.0 14.60.3

A.J. Almeida, 2007

Sharp et al (1986). J Nucl Med

RADIOACTIVITY IN RAT LUNGS AFTER ENDOTRACHEAL ADMINISTRATION

120 100
% Activity/g % activity/g

80 60 40 20 0 3 8 15 Time (min)
(n=4; meansd)

30

60

A.J. Almeida, 2007

Videira et al (2006).J Microencapsulation

38

LYMPHATIC DISTRIBUTION IN RATS AFTER ENDOTRACHEAL ADMINISTRATION

99mTc-HMPAO-LN

90 80 70

% activity/g

60 50 40 30 20 10 0 3 8 15 Time (min) 30 60

Stomach Liver Aortic lymph nodes Axillary lymph nodes Inguinal lymph nodes

(n=4; meansd)

A.J. Almeida, 2007

Videira et al (2006).J Microencapsulation

LYMPHATIC DISTRIBUTION IN RATS AFTER ENDOTRACHEAL ADMINISTRATION

99mTc-HMPAO

90 80 70
% activity/g

60 50 40 30 20 10 0 3 8 15 Time (min) 30

Stomach Liver Axillary lymph nodes Inguinal lymph nodes

60

(n=4; meansd)

A.J. Almeida, 2007

Videira et al (2006).J Microencapsulation

39

ALVEOLAR CLEARANCE
Uptake of 1.1 m fluorescent polystyrene particles administered i.n. to mice
Eyles et al (2001). Vaccine

15 min

24 h

Alveolar clearance of 400 nm fluorescent polystyrene particles administered i.n. to mice

Byersdorfer et al (1995). J Immunol

Dendritic cells

Peribronchial LN

6h
A.J. Almeida, 2007

24 h

PARTICLE UPTAKE AT THE LUNGS

LN
B MA L T

Lymphatics

Possible Mechanisms

Dissolution Macrophage phagocytosis Direct passage Uptake by vascular system Movement into the lymphatic system Axonal translocation to CNS

Alveolus Blood circulation

Interstitium

Pleura

Elimination Mucociliary escalator Exhaled air

A.J. Almeida, 2007

Valentine and Kennedy (2001) In: Principles and Methods of Toxicology

40

TREATMENT OF BREAST CANCER LUNG METASTASIS

PRELIMINARY STUDY

% mice metastised per group

non-treated LN-paclitaxel aero (last 15 days) Taxol i.v. (last 15 days) LN aero control LN-paclitaxel aero (first 15 days) Taxol i.v. (first 15 days) LN iv control
25 100 100 75 100 100 100

A.J. Almeida, 2007

Videira (2007). PhD Thesis

LIPID NANOPARTICLES AS TOPICAL DELIVERY SYSTEMS

A.J. Almeida, 2007

41

LIPID NANOPARTICLES AS TOPICAL DELIVERY SYSTEMS

BACKGROUND
Topical application of LN have been used with promising results either for therapeutic or

cosmetic purposes (Jenning et al. 2000; Mller et al. 2002).

SLN have shown some protective activity on skin surface, such as a UV-blocking

potential (Wissing et al. 2003).

SLN may be formulated in creams, gels,sprays. SLN allow modulated drug release.

A.J. Almeida, 2007

MECHANISMS OF UV PROTECTION Particulate sunscreen Molecular sunscreen heath, light

UV

Background:
Topical application of SLN have been used for therapeutic or cosmetic
purposes.

SLN act as an efficient particulate UV blocker (scattering effect) SLN and

molecular sunscreens have synergistic effect.

Reduced release of sunscreens from SLN when compared to emulsions Reduced side effects SLN may be formulated in creams, gels, sprays, allowing modulated drug
release.

Is it so ?

A.J. Almeida, 2007

Jenning et al. (2000); Mller et al. (2002); Wissing et al. (2003)

42

NANOPARTICLE PRODUCTION
Lipid melting melting heating heating Aq.phase Emulsification

Lipid composition gliceryl behenate (Compritol 888 ATO) gliceryl palmitosterarate (Precirol ATO 5) Surfactant Tween 80 Speed rate 8 000 - 10 000 rpm Model Drugs Octylmethoxycinamate (OMC)

Homogenization solidification

A.J. Almeida, 2007

NANOPARTICLE CHARACTERISATION: PHYSICAL STABILITY

Formulation

C (empty)

C-OMC

P (empty)

P-OMC

Time (months) 1 12 16 1 12 16 1 6 16 1 6 16

D50 147,0 80,4 102,2 16,5 33,9 14,9 221,6 269,4 289,1 144,7 59,3 87,2

Particle size (nm) D90 174,3 320,4 201,8 83,6 41,4 49,5 247,3 269,4 289,1 203,3 139,8 202,4

PI D95 206,6 320,4 201,8 105,3 41,4 49,5 247,3 269,4 289,1 241,0 186 268 0,516 0,546 0,529 0,541 0,443 0,418 0,541 0,176 0,614 0,204 0,589 0,586

A.J. Almeida, 2007

Toscano et al. (2004) Eur J Pharm Sci, 23 (Suppl. 1)

43

OMC RELEASE STUDIES

350 300

NL PR/OMC

NL CP/OMC

2 Amount released ( g/cm)

250 200 150 100 50 0 0,0

0,5

1,0

1,5

2,0

2,5

3,0

SQRT time (h)

Membrane: Silicone Receiving medium: 2% albumin in phosphate buffer (pH=7.4)

A.J. Almeida, 2007

Toscano et al. (2004) Eur Conf Drug Delivery Pharm Technol

IN VITRO PERCUTANEOUS ABSORPTION OF OMC

OMC amount in receptor phase (ug/cm 2 )

15 12 9 6 3 0 0,0

NL CP/OMC

NL PR/OMC

3,0

6,0 Time (h)

9,0

12,0

Membrane: Human skin Receiving medium: 2% albumin in phosphate buffer (pH=7.4)

A.J. Almeida, 2007

Toscano et al. (2004) Eur Conf Drug Delivery Pharm Technol

44

IN VIVO STUDIES
16 female volunteers (24 to 54 years old; phototypes II and III 10-day application trial on the antero-external surface of both legs 3 different areas studied corresponding: LN-OMC formulation LN blank formulation non-treated area (1 negative control per leg). Experimental challenge: controlled exposure of volunteers to a UV radiation source (Sunny HB-406 Solarium, Phillips) Parameter measured: skin colorimetry (erythema and melanisation indexes), TEWL and elasticity.

50 cm

Fonte de Radiao

A.J. Almeida, 2007

IN VIVO RESULTS

Erythema
10 9 8 7 6 5 4 3 2 1 0 0 3 5 Time (days) 7 10
70 68
Melanisation (AU)
LN Neg Cont LN-OMC Neg Cont

Melanisation

Erythema (AU)

66 64 62 60 58 56 0 3 5
Time (days)

LN Neg cont LN-OMC Neg Cont

10

A.J. Almeida, 2007

Toscano et al. (2004) Eur J Pharm Sci

45

MARKETED PRODUCTS

Nanobase Yamanouchi/Poland

Cutanova Dr. Rimpler GmbH/Germany

A.J. Almeida, 2007

PRODUCTION OF LIPID MICRO- OR NANOPARTICLES USING SUPERCRITICAL FLUIDS

A.J. Almeida, 2007

46

LIPID MICROPARTICLES USING SUPERCRITICAL FLUIDS

AIM Production of drug-lipid micro- and nanoparticles by a PGSS (particles from gas saturated solutions) process, as an alternative method.

MAIN ADVANTAGES
9 9 9

Avoid the use of solvents Particles are obtained as a dry powders, instead of suspensions Mild pressure and temperature conditions

A.J. Almeida, 2007

LIPID MICROPARTICLES USING SUPERCRITICAL FLUIDS

PGSS System
(A) (B) (C) (D) (E) CO2 supply cylinder Gas compressor CO2 storage cylinder Mixing cell Collecting chamber

Lipid composition Tripalmitin Temperature < 60C

Pressure 120-180 Bar Model drug theophiline

A.J. Almeida, 2007

Rodrigues et al (2004). J Supercrit Fluid

47

LIPID MICROPARTICLES USING SUPERCRITICAL FLUIDS

14 10 8 6 4 2 0 0,10 0,46 2,2

Dia m ete
Percentage of particles

12

180 160 10
r( m )

140 46 120
su Pres

ar) re (b

100 80

% Released

60 40
T F L c o m m erc ia l

20 0 0 6 12

160 bar 180 bar

18

24

Time (hours)

A.J. Almeida, 2007

Rodrigues et al (2004). J Supercrit Fluid

LYSOZYME-CONTAINING LN PREPARED USING SUPERCRITICAL CO2

Lysozyme Biological Activity Lysozyme particles produced from solutions with an ethanol mole fraction of C = 0.28 showed no significant loss of activity.

A.J. Almeida, 2007

Rodrigues et al (2007). submitted

48

CONCLUSIONS (I)
LN show suitable properties to become one of the most versatile nanoparticulate carriers for drug and protein delivery. Several drug incorporation techniques are applicable, including protein adsorption. Inhalation may be an effective route to deliver LN, representing an alternative to the intraperitonial route for targeting colloidal carriers to the lymphatics. LN present lymphatic tropism, thus providing the possibility of using radiolabelled LN as a lymphoscintigraphic agent, and allowing the direct delivery of cytotoxic drugs to lung cancer in limited or extensive stage. The limited amount of particle retention in lungs may allow the use of LN as a sustained release formulation in chronic lung therapy. LN show an effective control release of lipophilic drugs, whici is suitable for percutaneous absorption, with a minimum permeation thus making LN a safe vehicle for sunscreen agents.

A.J. Almeida, 2007

CONCLUSIONS (II)
In vivo results do not confirm early reports, i.e. LN on their own no not improve significantly TEWL and elasticity (results not shown).

Melanisation index was the only parameter that showed a trend, demonstrating that LN present some protective capacity against UV radiation in vivo.

SCF techniques can be successfully applied to the formulation of solid lipid microand nanoparticles with controlled-release characteristics.

Using a modified SCF technique, it is possible to produce LN containing intact and biologically active protein molecules.

A.J. Almeida, 2007

49

ACKNOWLEDGEMENTS
Faculdade de Farmcia, Lisboa Ana Cerdeira Ana Filipa Azevedo Helena Florindo Jorge Galhardas Jorge Moita Jos A. G. Morais Lus F. Gouveia Lus M. Rodrigues Mafalda Videira Paulo Ferreira Almeida Freie Universtt, Berlin Rainer H. Mller Stephan Runge IBILI, Coimbra Ana Cristina Santos Filomena Botelho J.J. Pedroso de Lima Institut de Recerca Oncolgica, Barcelona Angels Fabra
A.J. Almeida, 2007

UNFAB, INETI, Lisboa Eugnia Cruz Lusa Corvo Manuela Gaspar IBET, Oeiras Manuel Carrondo Antnio Cunha Ldia Gonalves IST, Lisboa Edmundo Gomes de Azevedo Henrique Matos Jun Li Miguel ngelo Rodrigues LEF, Lisboa Ascenso Farinha Cristina Toscano

Instituto Tecnolgico Nuclear, Lisboa Maria dos Anjos Neves Lurdes Gano Laboratrio Sorolgico, SA Patrcia Cruz London School of Pharmacy (CDDR) H.Oya Alpar S. Pandit

Fundao para a Cincia e a Tecnologia Tecnimede, SA Laboratrio Sorolgico, SA SINDEPEDIP- IMUNOPOR Fundao C. Gulbenkian DAAD, Germany INVOTAN

OLD LISBON

A.J. Almeida, 2007

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MODERN LISBON

A.J. Almeida, 2007

Thank you for your attention !

A.J. Almeida, 2007

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