Journal of Colloid and Interface Science 342 (2010) 283292

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Journal of Colloid and Interface Science

www.elsevier.com/locate/jcis

Formation of organic nanoparticles from volatile microemulsions

Katrin Margulis-Goshen a, Hadas Donio Netivi a, Dan T. Major b, Michael Gradzielski c, Uri Raviv a, Shlomo Magdassi a,*
a

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Chemistry Department and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University, Ramat-Gan, Israel c Technische Universitt Berlin, Stranski-Laboratorium fr Physikalische und Theoretische Chemie, Institut fr Chemie, Berlin, Germany
b

a r t i c l e

i n f o

a b s t r a c t
A method for preparation of nanoparticles of poorly water-soluble organic materials is presented. By this method, an oil-in-water microemulsion containing a volatile solvent with dissolved model material, propylparaben, undergoes solvent evaporation and conversion into nanoparticles by spray drying. The resulting powder can be easily dispersed in water to give a clear, stable dispersion of nanoparticles with a high loading of propylparaben. By ltration of this dispersion it was found that more than 95 wt.% of the dispersed propylparaben is in particles of less than 450 nm. X-ray diffraction revealed that propylparaben is present as nanocrystals of 4070 nm. After dispersion of the powder in water, formation of large crystals rapidly occurs. Addition of polyvinylpyrrolidone (PVP) prevented crystal growth during dispersion of the powder in water. The inhibition of propylparaben crystal growth by PVP was studied by molecular dynamic simulations that addressed the binding of PVP to the propylparaben crystal. A comparison was made between PVP and polyvinylalcohol, which did not display crystal inhibition properties. 2009 Elsevier Inc. All rights reserved.

Article history: Received 18 August 2009 Accepted 13 October 2009 Available online 17 October 2009 Keywords: Organic nanoparticles Microemulsion Evaporation Poorly-soluble drug Crystallization prevention Crystallization inhibitor PVP

1. Introduction Nanoparticles have unique physical, mechanical, chemical, electrical, optical, magnetic, electro-optical, and magneto-optical properties [14]. Therefore, their processes of formation have been studied extensively in recent years. In pharmaceutics nanoparticulate dosage forms enhance bioavailability of poorly water-soluble drugs. Reducing the size of class 2 and class 4 drugs to nanoscale leads to a great improvement in their solubility and dissolution rates [5,6]. A well-known method for the preparation of organic micro and nanoparticles is organic solvent evaporation from an oil-in-water emulsion [710]. In this method, nanoparticles are prepared by dissolving the organic compound in a volatile water-immiscible solvent followed by emulsifying this solution in water. Solvent evaporation from the resulting emulsion yields formation of particles in a size range comparable to that of the emulsion droplets. During the emulsication process, high energy consuming equipment is applied in order to reach the required size of the nal emulsion droplets. High pressure homogenization, colloid milling

* Corresponding author. Address: Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Fax: +972 2 658 4350. E-mail addresses: katymargulis@yahoo.com (K. Margulis-Goshen), hnetivi@ yahoo.com (H.D. Netivi), majort@mail.biu.ac.il (D.T. Major), michael.gradzielski@ tu-berlin.de (M. Gradzielski), raviv@chem.ch.huji.ac.il (U. Raviv), magdassi@cc. huji.ac.il (S. Magdassi). 0021-9797/$ - see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.10.024

with rotorstator apparatus, and ultrasonic devices are required to reduce the droplet size to submicron range. Microemulsions are spontaneously formed systems, with no high shear force investment. Since their formation is easy and inexpensive, microemulsions can become very attractive conned structures for the preparation of nanoparticles. The synthesis of inorganic particles in microemulsions is already widespread [11 15]. However, there are only a few reports on the formation of organic nanoparticles from microemulsions [12,1624]. Cholesterol, Rhodiarome, Rhovanil, nimesulide, and retinol nanoparticles were prepared by direct precipitation of those active substances in aqueous cores of water-in-oil microemulsions [1618]. Nanoparticles of griseofulvin, an antifungal drug, were prepared from water-dilutable microemulsion by the solvent diffusion technique [20]. This technique involves solubilization of the drug in oil-inwater microemulsion followed by dilution of this microemulsion with a large quantity of water. The displacement of solvent with an excess of water from the internal phase of the microemulsion into the external phase results in formation of drug nanoprecipitates dispersed in water. In this report we present a method for obtaining highly wettable organic nanometric particles with minimal energy investment. The proposed method was recently successfully applied for several hydrophobic materials [22]. Obtaining nanoparticles of waterinsoluble dye by ink-jet printing of oil-in-water microemulsion was previously developed by our research group [23]. Additionally, preparation of water-dispersible akes containing nanoparticles of

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a poorly water-soluble drug, simvastatin, by lyophilization of an oil-in-water microemulsion was recently described [24]. The method is based on the following steps: A. The water insoluble organic material is dissolved in a hydrophobic solvent, which has an evaporation rate greater than water; B. The isotropic, thermodynamically stable liquid, called microemulsion, is spontaneously formed by the addition of proper surfactants, co-surfactants, and water to this organic solution; C. Solvent evaporation is performed either at reduced air pressure to remove organic solvent and yield an aqueous suspension of the nanoparticles, or by spray drying to remove all the solvents (water and organic) and yield a dry nanometric powder. In case of a dry powder, the resultant particles have nanometric dimensions and are highly water wettable which makes them easily dispersible in water. This method has several advantages over commonly used systems: (A) The microemulsion formation is spontaneous and simple and does not require use of any high energy equipment. (B) Nanoparticles of less than 100 nm are usually achieved since the size of the microemulsion droplets is less than a few tens of nanometers. (C) The nal form of a solid, dry, free-owing nanometric powder facilitates its potential use in different elds (for instance, enabling easy transportation and prolonged storage of the nal product). (D) The process can be easily scaled-up. In the present report we describe the preparation and characterization of water-dispersible nanoparticles of a model poorly water-soluble material, propylparaben (Fig. 1) Propylparaben is a propyl ester of 4-hydroxybenzoic acid having saturation water solubility of 0.05 wt.% at 25 C [25]. It is widely used as antimicrobial preservative agent in food and cosmetics and as antifungal pharmaceutical aid. The hydrophobicity of propylparaben makes its use in water-based formulations challenging. Formation of highly wettable and freely water-dispersible nanoparticles of this material will potentially facilitate its use in water-based formulations and will enable reduction of the concentration required for its antimicrobial activity.

In all experiments water was deionized and ltered through a 0.1 lm lter (Millex-VV-PVDF lter produced by Millipore, Carrigtwohill, Ireland). 2.2. Preparation of microemulsions Microemulsions were prepared at room temperature by mixing n-butyl acetate with iso-propanol and adding propylparaben (in propylparaben-loaded microemulsions) to the resulting solution to create an oil phase. The surfactant (SDS) was dissolved in water to create an aqueous phase. Whenever water soluble crystal inhibitor was introduced to the microemulsion, it was dissolved in the aqueous phase. Afterwards, the oil and aqueous phases were united and the mixture was magnetically stirred at room temperature until an optically transparent system was formed. A phase diagram was prepared for empty microemulsions (not loaded with propylparaben). Only systems that remained transparent and homogeneous were attributed to the monophase area in the phase diagram. 2.3. Electrical conductivity measurements Electrical conductivity measurements of microemulsions were performed at room temperature using an Oyster conductivity meter (Extech Instruments, Waltham, MA, USA). In order to increase the electric conductivity of the aqueous phase, water was replaced with 0.0025 M NaCl aqueous solution. For the reference, electrical conductivities of various microemulsion components were measured. 2.4. Viscosity measurements The viscosity measurements of the microemulsions were performed at 25 C using a DV2 type viscometer (Brookeld, Middleboro, MA, USA). The viscosity was measured at various shear rates (13.2132 s1). 2.5. Small angle neutron scattering (SANS) measurements The droplet size in the microemulsion was estimated by SANS measurements, which were done on the instrument LOQ of ISIS (Rutherford Appleton Laboratory, Oxford, UK) at 25 C. In these experiments a q-range of 0.010.2 1 was covered, q being the magnitude of the scattering vector as given by:

q 4p=k sinh

2. Experimental 2.1. Materials Sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP) average MW 40,000, polyvinylalcohol (PVA) average MW 50,000, n-butyl acetate 99.5 wt.% and iso-propanol 99.8 wt.% were purchased from SigmaAldrich (Rehovot, Israel). Propylparaben was obtained from Sharon Laboratories (Ashdod, Israel). Ethanol anhydrous was purchased from Gadot (Netanya, Israel).

Fig. 1. Chemical structure of propylparaben.

where k is the neutron wavelength and h the half scattering angle. The sample was contained in 1 mm thick Hellma QS quartz cells. The data were radially averaged and corrected for transmission and detector efciency (accounted for by comparison with the isotropic scatterer (H2O)). They were then converted into absolute scattering intensities by comparison to the scattering intensity of a 1 mm thick H2O sample. For a quantitative analysis of the scattering data the scattering length densities SLD (and therefore the mass densities q) of the various microemulsion components have to be known. The employed values (at 25 C) are listed in Table 1 (Supporting material). The collected data could be tted with a model of interacting spherical aggregates, where for the interaction potential we assumed a screened Coulomb potential, and for the particle form factor P(q), that of homogeneous polydispersed spheres. The screened Coulomb potential then leads to a structure factor S(q) that accounts for the interparticle interactions, for which we chose an RPA approximation [26] as frequently employed, for instance for the case of ionic microemulsion droplets [27]. Accordingly, the experimental scattering intensity is given as:

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285

Iq1 N

Z
0

dr f r Pq; r Sq

where 1N is the number density of particles, f (r) their size distribution function, P(q, r) the particle form factor, and S(q) the structure factor. For f (r) we employed a Schulz distribution as given by [28]:

f r

   t1 t1 rt t1 r exp Rm Rm Ct 1

with t + 1 = 1/p2, which is directly related to the polydispersity index pp2 hR2 i=hR2 i. The form factor of homogeneous spheres can be written as:

2. All solvents were removed by spray drying using a Mini-laboratory Spray Dryer B-290 equipped with inert loop dehumidier B296 (Buchi, Flawil, Switzerland). Spray drying is a widely applied, technical method to dry aqueous or organic solutions, emulsions etc. in pharmaceutics, industrial chemistry, and the food industry. Process conditions applied for drying the microemulsions: air inlet temperature 110 C, drying chamber temperature (outlet temperature) 60 C liquid introduction rate (peristaltic pump rate) 5 ml/ min, spray ow rate 414 normliter/h, aspirator rate 35 m3/h, nitrogen pressure six atmospheres. This method of evaporation leads to a dry, free-owing powder. Residual solvent in the product by both evaporation methods was evaluated by gas chromatography (GC) (GC-5890A equipped with Rtx-530 0.25 0.50 column, Hewlett Packard, USA), after extraction with ether. Detection limit for this instrument is 0.0025 wt.% for both n-butyl acetate and iso-propanol. 2.8. Powder dispersion in water Powders obtained at the end of the spray drying process were dispersed at 5 wt.% in distilled water. The samples were magnetically stirred at room temperature for 20 min. 2.9. Propylparaben concentration in nanoparticles The concentration of propylparaben in nanoparticles following dispersion of the powder in water was determined after ltration of the dispersions (using a 0.45 lm lter Millex VV-PVDF lter produced by Millipore, Ireland). The ltrate was diluted 800 times with 90 wt.% ethanol, and propylparaben concentration was determined by UV spectrophotometer (UVvisible spectrophotometer Cary 100, Varian, Palo Alto, CA, USA). It was found that absorbance of propylparaben in ethanol 90 wt.% solution at 258 nm wavelength is linearly dependent on the concentration of propylparaben in the concentration range 5 1051.2 103 wt.%. Due to technical difculties, no correction was made for quantity of propylparaben possibly adsorbed onto the lter during the ltration process. In any case, this quantity was considered to be the large particles, so the actual propylparaben concentration in nanoparticles might be even greater than measured. 2.10. Visualization of crystals Large crystals were observed using a trinocular phase contrast light microscope Model ME-643 (Lieder, Ludwigsburg, Germany). 2.11. Zeta (f) potential measurement f potential measurements were performed at 25 C using a Zetamaster (Malvern, UK). The voltage in the measurement cell was kept at 150 V. f potential was evaluated after powder re-dispersion in 10 mM NaCl. The measurements were performed in triplicate. 2.12. X-ray diffraction (XRD) X-ray powder diffraction measurements were performed using the D8 Advance diffractometer (Bruker AXS, Karlsruhe, Germany) with a goniometer radius of 217.5 mm, Gbel Mirror parallel-beam optics, 2 Sollers slits, and 0.2 mm receiving slit. Standard sample holders were carefully lled with the samples. The specimen weight was approximately 0.5 g. XRD patterns within the range of 535 2h were recorded at room temperature using CuKa radiation (k = 1.5418 ) with the following measurement conditions: tube voltage of 40 kV, tube current of 40 mA, step-scan mode

( Pq 16 p DSLD
2 2

)2 sinq R q R cosq R q R3

where DSLD is the difference in the scattering length densities (SLD) of aggregate and solvent, and R the sphere radius. Essentially, Eq. (2) yields the absolute scattering intensity and shape of the curve where the location of the correlation peak determines the mean particle spacing and thereby also the droplet size. With Eq. (2), and taking into account the experimental resolution, the mean droplet radius (R) and the number of charges per aggregate (z) were derived from the scattering curve. 2.6. Small angle X-ray scattering (SAXS) measurements The periodicity measurements in water suspensions were performed at room temperature using SAXS. Scattering experiments were performed using CuKa radiation (k = 0.154 nm) from a Rigaku RA-MicroMax 007 HF X-ray generator operated at a power rating up to 1.2 kW generating a 70 70 lm2 spot size and focus. The direct beam then goes through a vacuum Osmic CMF12-100CU8 focus unit and, dened by a set of two scatterless slits [29], the beam size at the sample position is 0.7 0.7 mm2. The scattered beam goes through a ight path lled with He and reaches a Mar345 Image Plate detector. The sample was inserted into 1.5 mm quartz capillaries that were then ame-sealed. Each sample was checked before and after the experiment to verify that no uid had been lost during the time of exposure, approximately 3 h. The temperature was maintained at 23 1 C. The sample to detector distance was calibrated using silver behenate and was 1840.5 mm. Background correction was veried by measuring the scattering of a capillary lled with distilled water and correcting for sample absorption. Integration of scattering density was performed using FIT2D software. Scattered intensity was plotted as a function of the scattering vector q = (4p/k) sinh, where k is X-ray wavelength and h is half the angle between the incident and scattered wavevectors. 2.7. Converting microemulsion to nanoparticles Two methods of converting microemulsion into nanoparticles were used: 1. Removal of organic solvent using a rotovapor apparatus (Rotovapor R-114, Buchi, Flawil, Switzerland). This apparatus is operating at reduced air pressure and enables solvent evaporation, providing the solvent exhibits a sufciently high vapor pressure [8]. The microemulsion sample was placed in a 100 ml ask and a vacuum was applied (1 mm Hg) at 50 C for 20 min. The vacuum was released several times during the evaporation and the ask was weighed. The loss in sample weight was restored with water. This method of evaporation leads to an aqueous suspension.

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with a step size of 0.02 2h, and counting time of 1 s/step. The average crystal size has been calculated using TOPAS V3.0 (Bruker AXS, Karlsruhe, Germany) software from the full-width at half-maximum of the XRD peaks using the DebyeScherrer equation. 2.13. Control experiments

After heating (25 ps) and brief equilibration (25 ps) in the constant particle-volumetemperature (NVT) ensemble, the systems were equilibrated for 250 ps in the NPT ensemble before data collection. The time step was 1 fs in all MD simulations.

3. Results and discussion Several control experiments were performed to prove the necessity of the microemulsion system route for propylparaben nanoparticles preparation. In all those experiments the concentration of dissolved/solubilized propylparaben was detected by ltering the dispersion using a 0.45 lm lter (Millex-VV-PVDF lter produced by Millipore, Carrigtwohill, Ireland) and determining the concentration in a ltrate by UV spectrophotometer (UVvisible spectrophotometer Cary 100, Varian, Palo Alto, CA, USA) as detailed in Section 2.8. In the rst control experiment, propylparaben at concentrations of 15 wt.% was added to 4 wt.% iso-propanol solution in water and stirred for 48 h at room temperature. This experiment was conducted to eliminate the possibility of simply dissolving the active material in the initial composition without the formation of the microemulsion. In the second control experiment, 3 wt.% propylparaben was added to 8 wt.% SDS solution in water and the resultant suspension was stirred for 48 h at room temperature. This experiment evaluates possible solubilization of propylparaben in a micellar solution of SDS in the initial microemulsion composition. A third control experiment was conducted to evaluate the possible solubilization enhancement effect by crystal inhibitor, PVP, when a nal product is dispersed in water. In this experiment, the following components were dispersed in water (at concentrations similar to those in 5 wt.% water dispersion of a dry powder containing 17 wt.% propylparaben, 39 wt.% PVP, and 44 wt.% SDS): 0.85 wt.% Propylparaben, 1.95 wt.% PVP, 2.2 wt.% SDS. The dispersion was sonicated for 20 min and stirred for 80 h at room temperature. 2.14. Molecular dynamics (MD) simulations The simulation studies were done as follows: (1) validation of forceeld for the propylparaben crystal; (2) determination of propylparaben crystal morphology; (3) propylparaben and water MD simulations; (4) propylparaben, water, and PVP/PVA MD simulations; (5) docking studies of PVP/PVA to the propylparaben crystal by MD simulations. All simulations employed the Material Studio 4.0 (MS4) program (Accelrys Software Inc., USA). In stage 1, the experimental crystal structure of propylparaben determined at 173 K was employed as the starting point for the simulations. Initially the suitability of the COMPASS force eld for the crystal system was investigated [30]. The propylparaben crystal was simulated by constant particle-pressuretemperature (NPT) [31] MD in a 27 28 31 3 triclinic cell for 0.6 ns, with lattice angles a = 90, b = 11, and c = 90. Subsequently, in stage 2 the propylparaben crystal morphology was determined employing the Growth Morphology algorithm implemented in the Morphology module in MS4 [32]. In Table 2 (Supporting material), the three most stable propylparaben crystal surfaces are enumerated. From the morphology calculations, it can be seen that the most stable surface in propylparaben is (1 0 0) surface. Thus, this surface cut was employed throughout the simulations. For stages 3 and 4 MD simulations, a water layer interacting with the propylparaben crystal was generated using MS4 Amorphous Cell. A water slab of thickness $30 and the same lattice vectors as the propylparaben crystal was equilibrated. In simulations involving PVP/PVA, the polymers were modeled as monomers to reduce the computational complexity and the uncertainty related to the polymer conformation in solution, allowing more rapid diffusion through the aqueous medium. 3.1. Microemulsion system The microemulsion system chosen for nanoparticle preparation was based on solvents with high evaporation rates and contained n-butyl acetate, iso-propanol, SDS, and water. A pseudoternary phase diagram of this microemulsion is shown in Fig. 2. The grey area indicates the formation of one-phase (microemulsion) systems. The broad one-phase region of this system may be attributed to the co-surfactant role of the short-chain alcohol, iso-propanol, which increases the mobility of SDS interfacial monolayer and enables an additional reduction of the interfacial tension [33]. Electrical conductivity measurements of microemulsions located on dilution line 2:8 of the phase diagram (2:8 w/w ratio SDS to water-shown in Fig. 2) were performed at room temperature. All the microemulsions located on this line were visually clear and appeared dark under cross-polarized light microscopy (no birefringence). Viscosity measurements of the same microemulsions were performed at room temperature and all samples exhibited Newtonian ow behavior, as expected from the microemulsions [34]. The dilution line representing descending concentration of oil was chosen to characterize this system since during the solvent evaporation process, the concentration of oil phase will be gradually reduced (both n-butyl acetate and iso-propanol have evaporation rates greater than that of water). The dependence of electrical conductivity of the microemulsions on the oil phase concentration can be seen in Fig. 3. At oil phase concentration of 10 wt.%, the microemulsion conductivity is very high, similar to that of the aqueous phase with the surfactant only (18 mS/cm for 20 wt.% solution of SDS in water), indicating an oil-in-water microemulsion structure. With the increase in oil phase concentration, there is a gradual decrease in the electrical conductivity, but it still remains signicantly higher than the conductivity of the n-butyl acetate = 2 lS/cm and

Fig. 2. Phase diagram describing the formation of microemulsions (grey area) at room temperature. Concentrations are given in weight fractions. Electric conductivity measurements were performed on the microemulsion compositions located on dilution line 2:8. Microemulsion composition chosen for propylparaben incorporation is labeled with an asterisk.

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20.00 18.00 16.00

1.8

1.6

Conductivity mS*cm -1

14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00

I(q) / cm

-1

1.4

1.2

1.0

0.8 0.01

0.1

0.2

q/
0 10 20 30 40 50 60 70 80 90 100

-1

Oil phase percentage wt%

Fig. 3. Electrical conductivity measurements of microemulsions located on the dilution line 2:8 of the phase diagram (Fig. 1). These microemulsions have a constant concentration ratio between the surfactant (SDS) and water (20/ 80 wt.%).

Fig. 4. SANS intensity as a function of the magnitude of the scattering vector for microemulsion composed of 8 wt.% SDS/3.5 wt.% butyl acetate/3.5 wt.% iso-propanol/3 wt.% propylparaben/H2O 82 wt.% (at 25 C). Fitted curve (Eq. (2)) is given as solid line.

These results indicate that the microemulsion contains small droplets of oil containing dissolved propylparaben, in a continuous aqueous medium. 3.3. Formation of nanoparticles Solvent evaporation was performed either by removal of organic solvents under reduced pressure in rotavapor, leading to particles dispersed in water, or by immediate removal of all liquids by spray drying, resulting in formation of a dry powder. While the solvent removal was performed under reduced pressure, large (micron size range) crystals were formed immediately after the evaporation and they could be observed by light microscopy (Fig. 5). After the spray drying process, a dry, free-owing powder was obtained. This powder was composed of 27 wt.% propylparaben and 73 wt.% SDS. No solvent remained in the powder as veried by GC (total quantity of solvent was less than 0.005 wt.%, the detection limit of the instrument). XRD measurements performed on this powder indicated that the propylparaben is fully crystalline (Fig. 6).

iso-propyl alcohol = 5 lS/cm. At oil phase concentration of 50 60 wt.%, there is a change in conductivity slope. This may indicate the transition of oil-in-water microemulsion structure to a bicontinuous region.

3.2. Addition of propylparaben The following microemulsion composition was chosen for obtaining nanoparticles of propylparaben (labeled by an asterisk in Fig. 2): 8 wt.% SDS, 3.5 wt.% n-butyl acetate, 3.5 wt.% iso-propanol, 3 wt.% propylparaben, 82 wt.% water. This system has a low organic solvent content (which is benecial for the evaporation process) and it allows reaching a low surfactant:active material ratio in the nal powder. The conductivity measured for this microemulsion was 12.4 0.7 mS/cm and the viscosity was 4.8 0.04 mPa*s. Droplet size of the microemulsion was estimated from SANS measurements. A broad correlation peak was observed at q = 0.0765 1, which indicates a mean spacing of 8.21 nm for the contained aggregates (Fig. 4). The scattering length density for the microemulsion (under the assumption that the iso-propanol is dissolved in the aqueous phase and all the rest is in the oil phase) is 10.4 109 cm2. We used H2O as solvent in our experiments which reduces the contrast largely, but guarantees to study exactly the microemulsions system in question and one does not have to worry about potential effects of the isotopic substitution by D2O. The scattering of this microemulsions is rather well-described by a model of spherical aggregates that interact via a screened electrostatic repulsion. In this t we neglected the low q-range due to the large error bar of the scattering intensity in this region (Fig. 4). The mean droplet radius (R) and the mean number of charges per aggregate (z) were calculated using Eq. (2). The microemulsion droplets have an average radius of 2.25 nm (average diameter of 4.5 nm). About 14 effective charges per aggregate are obtained, which would correspond to about 11% of the theoretical charge, a value typically observed for micelles and small microemulsion droplets [35,36].

Fig. 5. Crystals of propylparaben observed with light microscope after dispersion of powder in water (in the absence of crystal growth inhibitor-PVP).

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1000 900 800 700

Intensity (a.u.)

600 500 400 300 200 100 0 2 10 20 30 4

2-Theta - Scale
Fig. 6. XRD pattern of powder received from microemulsion without PVP. Bars indicate peaks of crystalline propylparaben.

All propylparaben crystals in the dry powder were 4070 nm (approximate crystal size of propylparaben from XRD peaks using the DebyeScherrer equation). When the solid powder was dispersed in water (5 wt.%), the dispersion was initially clear but rapidly became turbid due to crystal growth. Formation of the crystals in the micron size range was observed visually and under the microscope. It should be noted that the f-potential of the particles was 46 3 mV, which is high enough to provide electrostatic stabilization and prevent particle aggregation. The high negative value of zeta potential is attributed to the negatively charged SDS molecules which are adsorbed on the particle surface. It can be expected that the formation of large crystals is due to Oswald ripening of the dispersed nanocrystals in water. During the solvent evaporation process, the concentration of propylparaben in each microemulsion droplet increases signicantly. At some point the labile supersaturation zone is reached, where spontaneous and rapid crystal nucleation occurs. The nucleation process is simultaneously accompanied by crystal growth. When the process continues, the polydispersity of the system increases, since previously formed crystals become larger than the newer ones. As a result, in the dry powder there could be signicant differences in the crystal size (as indicated by the XRD experiment). When the powder of these nanocrystals is dispersed in water, the differences in crystal sizes may lead to Oswald ripening, since the saturation solubility of the smaller crystals is greater than that of the larger ones, causing dissolution of the former and gradual growth of the latter (providing bulk water solubility is not negligible). Since the initial propylparaben dispersion is probably polydispersed, and since propylparaben has low but nite water solubility (28 mM), it is susceptible to ripening. 3.4. Crystallization inhibition Since crystal growth was observed when the nanocrystals were dispersed in water, the next step was an attempt to prevent or retard the crystallization process. Recently, Lindfors et al. [37] reported on retardation of the crystal growth of bicalutamide in

aqueous medium by addition of polyvinylpyrrolidone (PVP). They were able to separate the two steps of the crystallization process nucleation and crystal growth. They found that the crystal growth rate of bicalutamide is signicantly retarded by PVP (MW 360,000) due to the strong adsorption of the polymer to crystals exceeding the critical radius of nucleation. The nucleation rate of this drug was not signicantly altered since PVP binding to the monomer/subcritical crystal was weak. This suggests that the small critical radius of bicalutamide prevents interference of PVP in nucleation step. Other reports also mention alteration of the crystallization process of various drugs in aqueous medium by PVP. Thus, PVP was reported to retard the nucleation rate of hydrocortisone acetate [38] and felodipine [39], and to retard the crystal growth rate of sulphathiazide [40], acetaminophen [41], and nifedipine [42]. Incorporation of PVP of various molecular weights in microemulsions was previously reported by Koetz et al. [43] Microemulsions containing SDS showed noticeable change in the spontaneous curvature of the surfactant, probably due to adsorption of the polymer at the head groups of SDS. Based on these ndings, it was decided to incorporate PVP in the microemulsion loaded with propylparaben in order to retard crystallization during solvent evaporation and upon dispersion of the powder in water. PVP of various molecular weights was successfully introduced into the above microemulsion. It was possible to incorporate as much as 10 wt.% PVP (MW 10,000360,000) in the microemulsion without causing phase separation. The experiments indicated that PVP with an average MW of 40,000 had the most profound effect on crystallization of propylparaben. Microemulsions containing 010 wt.% PVP were spray dried to yield a ne, free-owing powder. The microemulsion compositions were: 010 wt.% PVP, 8 wt.% SDS, 3.5 wt.% n-butyl acetate, 3.5 wt.% iso-propanol, 3 wt.% propylparaben, 8272 wt.% water. In preliminary experiments, the resulting powders were dispersed (5 wt.%) in water; when the PVP concentration was P7 wt.%, the dispersion remained clear and transparent, and no

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crystal formation was visually observed. At lower concentrations of PVP, the crystallization process was kinetically retarded, but eventually the dispersion became turbid and crystallization was observed. It is well known that SDS is capable of forming complexes with PVP [4447]. As found by Minnati et al. [45] these complexes are likely to be formed due to the attraction between the partially negative ether oxygen on PVP and the positive sodium ion of SDS, thus allowing the dodecyl sulfate to attach to the PVP. In excess of SDS, the previously neutral PVP becomes a highly charged polyanion. Zeta potential of the particles which were dispersed in the presence of PVP was 29 3 mV. The decrease in zeta potential can be explained by elevated ionic strength of the dispersion due to increased population of dissociated ions [45]. However, the measured zeta potential is still high enough to provide electrostatic stabilization of the nanoparticles. Moreover, it should be expected that the attachment of the polymeric complex to the particle surface would provide additional stabilization by a steric mechanism, and thus further contribute to the prevention of particle aggregation. Furthermore, both the adsorbed PVP and PVPSDS complex may interfere with the crystallization process, hence leading to obtaining stable nanoparticles in dispersion. For a microemulsion containing 7 wt.% PVP, the composition of the powder after drying is: 17 wt.% propylparaben, 39 wt.% PVP, 44 wt.% SDS. XRD performed on this powder did not reveal any peaks of crystalline propylparaben (Fig. 7). This result implies that PVP interfered in the nucleation step of the crystallization process and led to obtaining a fully amorphous product. This result can probably be explained by a strong interaction between the polymer and individual propylparaben molecules or subcritical crystals. Possible interactions with the growing crystals were modeled by MD simulations and will be discussed later. When the powder is stored at room temperature, propylparaben remains amorphous for months. In order to obtain quantitative information about the effect of PVP concentration in the fraction of propylparaben that is present

as nanoparticles, the dispersion was ltered through a 0.45 lm lter, followed by measurement of the concentration of propylparaben in the ltrate. It was found that when the initial microemulsion contained P7 wt.% PVP, more than 95 wt.% of the propylparaben was present as particles having a diameter of less than 0.45 lm for at least 1 week after the dispersion was performed (Fig. 8). The dispersion remained visually stable for at least 2 months at room temperature without any turbidity. When the initial microemulsion contained less than 7 wt.% PVP, the initial fraction of propylparaben that is present as nanoparticles was smaller and turbidity was observed. SAXS measurement was performed on 5 wt.% dispersion in water of the powder that was prepared from microemulsion containing 7 wt.% PVP. The scattering pattern revealed a maximum at q = 0.55 nm1 (Fig. 9). Another SAXS measurement was performed on powder dispersed 0.5 wt.% in water (10 times diluted dispersion). No change in scattering maximum location was observed in the diluted sample. This result implies that the contribution of the structure factor to the scattering pattern is relatively low in this q-range, suggesting that the data are perhaps a form factor alone. After a coarse t to a hard cylinder model, an approximate particle diameter of 16 nm could be deduced. Hard cylinder model was chosen for the evaluation since it provided the best t for the collected data. At present we do not know the explanation for the suitability of that model, and this issue will be investigated in future studies. It can be seen from the scattering measurements that the average particle size is larger than the average microemulsion droplet size. Growth of particles formed from water-in-oil microemulsion droplets was previously explained using crystallization models [18]. A possible explanation for the larger size of the amorphous particles in our system is the coalescence of the microemulsion droplets during the evaporation process. Initially, the components of the dispersed phase of the microemulsion were selected with much higher evaporation rate than the aqueous phase, in order to achieve rapid transformation of the droplets into solid particles.

700

600

500

Intensity (a.u.)

400

300

200

100

0 2 10 20 30 4

2-Theta - Scale
Fig. 7. XRD pattern of powder obtained from microemulsion with 7% PVP. Bars indicate 2h angles for peaks of crystalline propylparaben.

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Fraction of propylparaben in particles < 450nm (wt%)

100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 0 wt% PVP 2 wt% PVP 3 wt% PVP 4 wt% PVP 5 wt% PVP 6 wt% PVP 7 wt% PVP

Days in dispersion
Fig. 8. Fraction of propylparaben dissolved/found in nanoparticles smaller than 450 nm as a function of time after dispersion in water.

500 450 400

Intensity (a.u)

350 300 250 200 150 100 50 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

For comparison, the same experiment was conducted with another hydrophilic polymer, polyvinylalcohol (PVA, average MW 50,000). It was found that large crystals were formed shortly after dispersing the powder, indicating that PVA do not retard crystal growth, emphasizing the special role of PVP. 3.5. Control experiments Bulk propylparaben was mixed with iso-propyl alcohol/water solution and it was found that the solubility of propylparaben in this solution at room temperature is 0.12 wt.%. In the second control experiment (solubilization of propylparaben in a micellar solution of SDS), only 5.6 wt.% of the propylparaben was solubilized in SDS micelles. In the third control experiment (solubilization of propylparaben in the micellar solution of SDS in presence of PVP), 19.9 wt.% of the propylparaben was solubilized. 3.6. MD simulations of crystal growth inhibition The inhibition of propylparaben crystal growth by PVP was studied by MD simulations. The purpose was to address the binding of PVP to the propylparaben growing crystal. A comparison was made between PVP and PVA, which did not display crystal growth inhibition properties. It is evident from analysis of the MD trajectory of pure propylparaben that the crystal remains stable throughout the simulations: the stacking interactions between different layers of the propylparaben crystal remain intact (Fig. 1, Supporting material) and the hydrogen bonds within different layers are stable (Fig. 2, Supporting material). Nonetheless, considerable thermal motion causing local disorder is observed throughout the simulations. The mean square displacement is 2.5 2 and remains stable for the last 0.5 ns of the control simulation. Thus, the COMPASS force eld is deemed suitable for the current study involving propylparaben crystal. In the combined propylparabenwater-PVP/PVA simulations, the PVP and PVA monomeric units were added at the center of water layer. Both monomers diffused through the water to the propylparabenwater interface within the rst few hundred ps of the simulations (Fig. 3, Supporting material). The simulations of PVA indicate that this monomeric unit has rather nonspecic

Q [nm^-1]
Fig. 9. Radially integrated SAXS data of aqueous dispersion of powder obtained from microemulsion with 7% PVP.

However it is possible that the actual differences in the evaporation rates in the nal system are not that signicant, as it is well known that in microemulsions and emulsions the evaporation rates of the dispersed droplets might be retarded [8,48]. Therefore, we can not rule out the possible collapse of the microemulsion during the evaporation process which results in droplets coalescence, and eventually leads to particles larger than the original microemulsion droplets. From the results obtained so far, it can be concluded that PVP inhibits crystal nucleation during spray-drying. In order to evaluate the potential ability of PVP to also retard the growth of already formed propylparaben crystals, the following experiment was performed: powder prepared from microemulsion without PVP (which is composed of crystalline nanoparticles, as described above) was dispersed (5 wt.%) in 3.2 wt.% PVP aqueous solution (this concentration was selected in order to have the same proportions between the components as in the powders prepared with 7 wt.% PVP). It was found that the obtained dispersion was clear and stable, and the fraction of propylparaben present in particles having a diameter less than 0.45 lm was 98 1 wt.%. This result indicates that PVP is effective in retardation of crystal growth as well as in inhibition of crystal nucleation.

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interactions with the surface and no steric t with the surface is evident. Indeed, the PVA monomeric unit diffuses back into the aqueous medium to maximize hydrogen bonds. However, in the case of PVP, the monomeric unit attaches to hydrophobic clefts in the crystal surface of propylparaben. This cleft is sterically and electronically complementary with the hydrophobic part of the pyrrolidone side chain (Fig. 10). No specic hydrogen bonding interactions seem to be involved in PVP recognition at the propylparaben surface. PVP interactions with the propylparaben crystal are seemingly hydrophobic in nature and the pyrrolidone ring is involved in hydrophobic interactions with both the propylparaben propyl tail and the aromatic ring. These ndings are in agreement with the results obtained by Molyneux and Cornarakis-Lentzos, who studied binding interactions between paraben esters and PVP and evaluated the contribution of hydrophobic interactions between methylene groups in the paraben alkyl tail and PVP to the binding strength [49]. In some cases, the PVP monomeric unit exchanged with propylparaben molecules, intercalating between propylparaben layers (between the p-stacked propylparaben aromatic rings). In the later stages of the simulations (approaching 1 ns) the propylparaben crystal lost its ordered, layered structure near the surface due to the presence of the PVP monomeric unit. Such an effect was not observed in the case of PVA. Similar conclusions are drawn from the pure crystal PVP/PVA simulations. During the course of the simulations, the PVP monomeric unit did not detach from the surface, once attached. Moreover, the PVA monomeric unit was considerably more mobile during the simulations and binding to the surface would involve a greater entropy loss than in the case of PVP. Thus, the thermodynamic driving force, which is to minimize the total free energy, is likely obtained by competing forces: (1) Minimize enthalpy thereby maximizing adsorbate density at the surface. In the case of PVP, there is a greater enthalpy gain due to favorable hydrophobic interactions with the propylparaben surface than for PVA, while unfavorable interactions with the water is avoided. Adsorption of PVA to the surface would entail a loss in H-bonding interactions with water, and is therefore disfavored. (2) Maximize entropy the adsorption of PVP which is larger, bulkier, and more rigid than PVA is favored due to less translational and conformational entropy loss on binding. It should be noted that more complicated interactions of subcritical crystals with PVPSDS complexes might have also occurred

in this system. But since SDS alone does not inhibit crystal growth, most probably such complexes would still interact with propylparaben growing crystal via the segments of the PVP molecules which are the source of a specic interaction. However, verifying this assumption by performing a detailed simulation with added SDS would be much more complicated and therefore is beyond the scope of the current study. The results of the MD simulations are in good agreement with the results published by other research groups. Simonelli et al. [40] proved strong and irreversible binding of the PVP molecule to sulfathiazole growing crystal surface, which enabled increasing inhibition even if the crystal was removed from PVP solution and introduced to supersaturated solution not containing PVP. Wen et al. [41] tested PVA and PVP as acetaminophen crystal growth inhibitors. They revealed that PVA 15 K was unable to suppress crystal growth due to the high mobility of its functional groups involved in hydrogen bonding interactions with the crystal surface, while PVP 40,000 was an excellent crystal growth inhibitor due to both strong interactions with crystal surface and low mobility of its functional groups involved in the specic van der Waals interactions.

4. Conclusions A process for obtaining powders of nanoparticles of a poorly water-soluble organic material, propylparaben, by solvent evaporation from volatile microemulsions was described. The process has several advantages over commonly used methods, such as low cost, simplicity of preparation, and no need for any special equipment to prepare the nanometric emulsion droplets. Since the resultant nanoparticles are in the form of dry, ne powder, high chemical and microbial stability is expected, thus prolonging the shelf life of the preparation. The nanoparticle powder can be easily dispersed in water in the presence of a crystal growth inhibitor, such as PVP. It was found that at a PVP:propylparaben weight ratio of 7:3 in the microemulsion, more than 95 wt.% propylparaben was present as nanoparticles after dispersion in water, and the resultant dispersions were optically clear and stable for months. The results indicate that PVP in the microemulsion prevents the nucleation of propylparaben during the spray-drying and inhibits crystal growth in aqueous medium. MD simulations of propylparaben crystal growth in the presence of PVP (successful inhibitory agent) and PVA (unsuccessful inhibitory agent) monomeric units were carried out with and without water. The simulations suggest that the determining factor in crystal growth inhibition is molecular recognition of the monomer units on the propylparaben

Fig. 10. Propylparaben crystal (down) with PVP (monomer) (left up) and PVA (monomer) (right up) during molecular dynamics simulations.

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surface. PVP binds to hydrophobic parts on the growing crystal surface that is stereo-electronically complimentary to the pyrrolidone ring. PVA, which lacks such a hydrophobic unit, does not possess the same stereo-electronic t with the propylparaben surface and is therefore very loosely bound at the surface. Subsequently, after initial adsorption on the propylparaben particle surface, PVP intercalates in between the p-stacked propylparaben aromatic rings. This is seemingly the initial stage of destroying the ordered structure at the face of the growing crystal, which inhibits further crystal growth. During the course of the simulations, the PVP monomeric unit did not detach from the surface, once attached. The PVA monomer was considerably more mobile during the simulations and its binding to the surface likely involves a greater entropy loss than in the case of PVP. The results of the MD simulations are in agreement with the results on crystal growth inhibition by PVP for various drugs. We expect that the described process of direct conversion of microemulsion droplets into nanoparticles can be utilized for a variety of functional organic molecules, mainly in pharmaceutics. Acknowledgments We would like to thank Sarah Rogers (ISIS) and Sylvan Prvost (TUB) for the SANS measurements and Carmen Tamburu (HUJI) for performing the SAXS measurements. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2009.10.024. References
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