Formation of Organic Nano Particles

8/3/2019 Formation of Organic Nano Particles

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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, Israelb Chemistry Department and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University, Ramat-Gan, Israelc Technische Universitt Berlin, Stranski-Laboratorium fr Physikalische und Theoretische Chemie, Institut fr Chemie, Berlin, Germany

a r t i c l e i n f o

Article history:

Received 18 August 2009Accepted 13 October 2009Available online 17 October 2009

Keywords:

Organic nanoparticlesMicroemulsionEvaporationPoorly-soluble drugCrystallization preventionCrystallization inhibitorPVP

a b s t r a c t

A method for preparation of nanoparticles of poorly water-soluble organic materials is presented. By thismethod, an oil-in-water microemulsion containing a volatile solvent with dissolved model material, pro-pylparaben, undergoes solvent evaporation and conversion into nanoparticles by spray drying. Theresulting powder can be easily dispersed in water to give a clear, stable dispersion of nanoparticles witha high loading of propylparaben. By filtration of this dispersion it was found that more than 95 wt.% of thedispersed propylparaben is in particles of less than 450 nm. X-ray diffraction revealed that propylparabenis present as nanocrystals of 4070 nm. After dispersion of the powder in water, formation of large crys-tals rapidly occurs. Addition of polyvinylpyrrolidone (PVP) prevented crystal growth during dispersion ofthe powder in water. The inhibition of propylparaben crystal growth by PVP was studied by moleculardynamic simulations that addressed the binding of PVP to the propylparaben crystal. A comparisonwas made between PVP and polyvinylalcohol, which did not display crystal inhibition properties.

2009 Elsevier Inc. All rights reserved.

1. Introduction

Nanoparticles have unique physical, mechanical, chemical, elec-trical, optical, magnetic, electro-optical, and magneto-optical prop-erties [14]. Therefore, their processes of formation have beenstudied extensively in recent years. In pharmaceutics nanoparticu-late dosage forms enhance bioavailability of poorly water-solubledrugs. Reducing the size of class 2 and class 4 drugs to nanoscaleleads to a great improvement in their solubility and dissolutionrates [5,6].

A well-known method for the preparation of organic micro andnanoparticles is organic solvent evaporation from an oil-in-wateremulsion [710]. In this method, nanoparticles are prepared bydissolving the organic compound in a volatile water-immisciblesolvent followed by emulsifying this solution in water. Solventevaporation from the resulting emulsion yields formation of parti-cles in a size range comparable to that of the emulsion droplets.During the emulsification process, high energy consuming equip-ment is applied in order to reach the required size of the finalemulsion droplets. High pressure homogenization, colloid milling

with rotorstator apparatus, and ultrasonic devices are requiredto reduce the droplet size to submicron range.

Microemulsions are spontaneously formed systems, with nohigh shear force investment. Since their formation is easy and inex-pensive, microemulsions can become very attractive confinedstructures for the preparation of nanoparticles. The synthesis ofinorganic particles in microemulsions is already widespread [1115]. However, there are only a few reports on the formation of or-ganic nanoparticles from microemulsions [12,1624]. Cholesterol,Rhodiarome, Rhovanil, nimesulide, and retinol nanoparticleswere prepared by direct precipitation of those active substancesin aqueous cores of water-in-oil microemulsions [1618]. Nano-particles of griseofulvin, an antifungal drug, were prepared fromwater-dilutable microemulsion by the solvent diffusion technique[20]. This technique involves solubilization of the drug in oil-in-water microemulsion followed by dilution of this microemulsionwith a large quantity of water. The displacement of solvent withan excess of water from the internal phase of the microemulsioninto the external phase results in formation of drug nanoprecipi-tates dispersed in water.

In this report we present a method for obtaining highly wetta-ble organic nanometric particles with minimal energy investment.The proposed method was recently successfully applied for severalhydrophobic materials [22]. Obtaining nanoparticles of water-insoluble dye by ink-jet printing of oil-in-water microemulsionwas previously developed by our research group[23]. Additionally,preparation of water-dispersible flakes containing nanoparticles of

0021-9797/$ - see front matter 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2009.10.024

* Corresponding author. Address: Casali Institute of Applied Chemistry, TheHebrew University of Jerusalem, Jerusalem 91904, Israel. Fax: +972 2 658 4350.

E-mail addresses: [email protected] (K. Margulis-Goshen), [email protected] (H.D. Netivi), [email protected] (D.T. Major), [email protected] (M. Gradzielski), [email protected] (U. Raviv), [email protected] (S. Magdassi).

Journal of Colloid and Interface Science 342 (2010) 283292

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

www.elsevier .com/locate / jc is
http://dx.doi.org/10.1016/j.jcis.2009.10.024mailto:[email protected]:hnetivi@%20yahoo.commailto:hnetivi@%20yahoo.commailto:[email protected]:michael.gradzielski@%20tu-berlin.demailto:michael.gradzielski@%20tu-berlin.demailto:[email protected]:magdassi@cc.%20huji.ac.ilmailto:magdassi@cc.%20huji.ac.ilhttp://www.sciencedirect.com/science/journal/00219797http://www.elsevier.com/locate/jcishttp://www.elsevier.com/locate/jcishttp://www.sciencedirect.com/science/journal/00219797mailto:magdassi@cc.%20huji.ac.ilmailto:magdassi@cc.%20huji.ac.ilmailto:[email protected]:michael.gradzielski@%20tu-berlin.demailto:michael.gradzielski@%20tu-berlin.demailto:[email protected]:hnetivi@%20yahoo.commailto:hnetivi@%20yahoo.commailto:[email protected]://dx.doi.org/10.1016/j.jcis.2009.10.024


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a poorly water-soluble drug, simvastatin, by lyophilization of anoil-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 hydro-phobic solvent, which has an evaporation rate greater thanwater;

B. The isotropic, thermodynamically stable liquid, calledmicroemulsion, is spontaneously formed by the addition ofproper surfactants, co-surfactants, and water to this organicsolution;

C. Solvent evaporation is performed either at reduced air pres-sure to remove organic solvent and yield an aqueous suspen-sion of the nanoparticles, or by spray drying to remove allthe solvents (water and organic) and yield a dry nanometricpowder. In case of a dry powder, the resultant particles havenanometric dimensions and are highly water wettablewhich makes them easily dispersible in water.

This method has several advantages over commonly usedsystems:

(A) The microemulsion formation is spontaneous and simpleand does not require use of any high energy equipment.

(B) Nanoparticles of less than 100 nm are usually achieved sincethe size of the microemulsion droplets is less than a few tensof nanometers.

(C) The final form of a solid, dry, free-flowing nanometric pow-der facilitates its potential use in different fields (forinstance, enabling easy transportation and prolonged stor-age of the final product).

(D) The process can be easily scaled-up.

In the present report we describe the preparation and charac-terization of water-dispersible nanoparticles of a model poorlywater-soluble material, propylparaben (Fig. 1)

Propylparaben is a propyl ester of 4-hydroxybenzoic acid hav-ing saturation water solubility of 0.05 wt.% at 25 C [25]. It iswidely used as antimicrobial preservative agent in food and cos-metics and as antifungal pharmaceutical aid. The hydrophobicityof propylparaben makes its use in water-based formulations chal-lenging. Formation of highly wettable and freely water-dispersiblenanoparticles of this material will potentially facilitate its use inwater-based formulations and will enable reduction of the concen-tration required for its antimicrobial activity.

2. Experimental

2.1. Materials

Sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP) aver-age MW 40,000, polyvinylalcohol (PVA) average MW 50,000, n-bu-tyl acetate 99.5 wt.% and iso-propanol 99.8 wt.% were purchasedfrom SigmaAldrich (Rehovot, Israel). Propylparaben was obtainedfrom Sharon Laboratories (Ashdod, Israel). Ethanol anhydrous waspurchased from Gadot (Netanya, Israel).

In all experiments water was deionized and filtered through a0.1lm filter (Millex-VV-PVDF filter 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 (inpropylparaben-loaded microemulsions) to the resulting solutionto create an oil phase. The surfactant (SDS) was dissolved in waterto create an aqueous phase. Whenever water soluble crystal inhibi-tor was introduced to the microemulsion, it was dissolved in theaqueous phase. Afterwards, the oil and aqueous phases were unitedandthe mixturewasmagnetically stirred at roomtemperature untilan optically transparent system was formed. A phase diagram wasprepared for empty microemulsions (not loaded with propylpara-ben). Only systems that remained transparent and homogeneouswere attributed to the monophase area in the phase diagram.

2.3. Electrical conductivity measurements

Electrical conductivity measurements of microemulsions wereperformed at room temperature using an Oyster conductivitymeter (Extech Instruments, Waltham, MA, USA). In order to in-crease the electric conductivity of the aqueous phase, water wasreplaced with 0.0025 M NaCl aqueous solution. For the reference,electrical conductivities of various microemulsion componentswere measured.

2.4. Viscosity measurements

The viscosity measurements of the microemulsions were per-formed at 25 C using a DV2 type viscometer (Brookfield, Middle-boro, MA, USA). The viscosity was measured at various shearrates (13.2132 s1).

2.5. Small angle neutron scattering (SANS) measurements

The droplet size in the microemulsion was estimated by SANSmeasurements, which were done on the instrument LOQ of ISIS(Rutherford Appleton Laboratory, Oxford, UK) at 25C. In theseexperiments a q-range of 0.010.2 1 was covered, q being themagnitude of the scattering vector as given by:

q 4p=k sinh 1

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 transmis-sion and detector efficiency (accounted for by comparison with

the isotropic scatterer (H2O)). They were then converted into abso-lute scattering intensities by comparison to the scattering intensityof a 1 mm thick H2O sample.

For a quantitative analysis of the scattering data the scatteringlength densities SLD (and therefore the mass densities q) of thevarious microemulsion components have to be known. The em-ployed values (at 25 C) are listed in Table 1 (Supporting material).

The collected data could be fitted with a model of interactingspherical aggregates, where for the interaction potential we as-sumed a screened Coulomb potential, and for the particle form fac-tor P(q), that of homogeneous polydispersed spheres. The screenedCoulomb potential then leads to a structure factor S(q) that ac-counts for the interparticle interactions, for which we chose anRPA approximation [26] as frequently employed, for instance for

the case of ionic microemulsion droplets [27]. Accordingly, theexperimental scattering intensity is given as:Fig. 1. Chemical structure of propylparaben.

284 K. Margulis-Goshen et al./ Journal of Colloid and Interface Science 342 (2010) 283292
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Iq1N

Z10

drfr Pq; r Sq 2

where 1Nis the number density of particles, f(r) their size distribu-tion function, P(q, r) the particle form factor, and S(q) the structurefactor.

For f(r) we employed a Schulz distribution as given by [28]:

fr t 1

Rm

t1 rt

Ct 1exp

t 1

Rmr

3

with t+ 1 = 1 /p2, which is directly related to the polydispersity in-dex pp2 hR2i=hR2i.

The form factor of homogeneous spheres can be written as:

Pq 16 p2 DSLD2

R3 sinq R q R cosq R

q R3

( )24

whereDSLD 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 andshape of the curve where the location of the correlation peak deter-

mines the mean particle spacing and thereby also the droplet size.With Eq. (2), and taking into account the experimental resolu-

tion, the mean droplet radius (R) and the number of charges peraggregate (z) were derived from the scattering curve.

2.6. Small angle X-ray scattering (SAXS) measurements

The periodicity measurements in water suspensions were per-formed at room temperature using SAXS. Scattering experimentswere performed using CuKa radiation (k = 0.154 nm) froma RigakuRA-MicroMax 007 HF X-ray generator operated at a power ratingup to 1.2 kW generating a 70 70lm2 spot size and focus. The di-rect beam then goes through a vacuum Osmic CMF12-100CU8 fo-cus unit and, defined by a set of two scatterless slits [29], the beam

size at the sample position is 0.7 0.7 mm2. The scattered beamgoes through a flight path filled with He and reaches a Mar345 Im-age Plate detector. The sample was inserted into 1.5 mm quartzcapillaries that were then flame-sealed. Each sample was checkedbefore and after the experiment to verify that no fluid had beenlost during the time of exposure, approximately 3 h. The tempera-ture was maintained at 23 1 C. The sample to detector distancewas calibrated using silver behenate and was 1840.5 mm. Back-ground correction was verified by measuring the scattering of acapillary filled with distilled water and correcting for sampleabsorption. Integration of scattering density was performed usingFIT2D software. Scattered intensity was plotted as a function ofthe scattering vector q = (4p/k) sinh, where k is X-ray wavelengthand h is half the angle between the incident and scattered

wavevectors.

2.7. Converting microemulsion to nanoparticles

Two methods of converting microemulsion into nanoparticleswere used:

1. Removal of organic solvent using a rotovapor apparatus (Roto-vapor R-114, Buchi, Flawil, Switzerland). This apparatus is operat-ing at reduced air pressure and enables solvent evaporation,providing the solvent exhibits a sufficiently high vapor pressure[8]. The microemulsion sample was placed in a 100 ml flask anda vacuum was applied (1 mm Hg) at 50 C for 20 min. The vacuumwas released several times during the evaporation and the flask

was weighed. The loss in sample weight was restored with water.This method of evaporation leads to an aqueous suspension.

2. All solvents were removed by spray drying using a Mini-labora-tory Spray Dryer B-290 equipped with inert loop dehumidifier B-296 (Buchi, Flawil, Switzerland). Spray drying is a widely applied,technical method to dry aqueous or organic solutions, emulsionsetc. in pharmaceutics, industrial chemistry, and the food industry.Process conditions applied for drying the microemulsions: air inlettemperature 110 C, drying chamber temperature (outlet tempera-

ture) 60 C liquid introduction rate (peristaltic pump rate) 5 ml/min, spray flow rate 414 normliter/h, aspirator rate 35 m3/h, nitro-gen pressure six atmospheres. This method of evaporation leads toa dry, free-flowing powder.

Residual solvent in the product by both evaporation methodswas evaluated by gas chromatography (GC) (GC-5890A equippedwith Rtx-530 0.25 0.50 column, Hewlett Packard, USA), afterextraction with ether. Detection limit for this instrument is0.0025 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 weredispersed at 5 wt.% in distilled water. The samples were magneti-cally stirred at room temperature for 20 min.

2.9. Propylparaben concentration in nanoparticles

The concentration of propylparaben in nanoparticles followingdispersion of the powder in water was determined after filtrationof the dispersions (using a 0.45lm filter Millex VV-PVDF filterproduced by Millipore, Ireland). The filtrate was diluted 800 timeswith 90 wt.% ethanol, and propylparaben concentration was deter-mined by UV spectrophotometer (UVvisible spectrophotometerCary 100, Varian, Palo Alto, CA, USA). It was found that absorbanceof propylparaben in ethanol 90 wt.% solution at 258 nm wave-length is linearly dependent on the concentration of propylparabenin the concentration range 5 1051.2 103 wt.%. Due to tech-

nical difficulties, no correction was made for quantity of propylpar-aben possibly adsorbed onto the filter during the filtration process.In any case, this quantity was considered to be the large particles,so the actual propylparaben concentration in nanoparticles mightbe even greater than measured.

2.10. Visualization of crystals

Large crystals were observed using a trinocular phase contrastlight 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 cellwas kept at 150 V. f potential was evaluated after powder re-dis-persion in 10 mM NaCl. The measurements were performed intriplicate.

2.12. X-ray diffraction (XRD)

X-ray powder diffraction measurements were performed usingthe D8 Advance diffractometer (Bruker AXS, Karlsruhe, Germany)with a goniometer radius of 217.5 mm, Gbel Mirror parallel-beamoptics, 2 Sollers slits, and 0.2 mm receiving slit. Standard sampleholders were carefully filled with the samples. The specimenweight was approximately 0.5 g. XRD patterns within the rangeof 535 2h were recorded at room temperature using CuKa

radiation (k = 1.5418 ) with the following measurement condi-tions: tube voltage of 40 kV, tube current of 40 mA, step-scanmode

K. Margulis-Goshen et al./ Journal of Colloid and Interface Science 342 (2010) 283292 285


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

2.13. Control experiments

Several control experiments were performed to prove thenecessity of the microemulsion system route for propylparabennanoparticles preparation. In all those experiments the concentra-tion of dissolved/solubilized propylparaben was detected by filter-ing the dispersion using a 0.45 lm filter (Millex-VV-PVDF filterproduced by Millipore, Carrigtwohill, Ireland) and determiningthe concentration in a filtrate by UV spectrophotometer (UVvisi-ble spectrophotometer Cary 100, Varian, Palo Alto, CA, USA) as de-tailed in Section 2.8. In the first control experiment, propylparabenat concentrations of 15 wt.% was added to 4 wt.% iso-propanolsolution in water and stirred for 48 h at room temperature. Thisexperiment was conducted to eliminate the possibility of simplydissolving the active material in the initial composition withoutthe formation of the microemulsion. In the second control experi-ment, 3 wt.% propylparaben was added to 8 wt.% SDS solution inwater and the resultant suspension was stirred for 48 h at roomtemperature. This experiment evaluates possible solubilization ofpropylparaben in a micellar solution of SDS in the initial micro-emulsion composition. A third control experiment was conductedto evaluate the possible solubilization enhancement effect by crys-tal inhibitor, PVP, when a final product is dispersed in water. In thisexperiment, the following components were dispersed in water (atconcentrations similar to those in 5 wt.% water dispersion of a drypowder containing 17 wt.% propylparaben, 39 wt.% PVP, and44 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 hat room temperature.

2.14. Molecular dynamics (MD) simulations

The simulation studies were done as follows: (1) validation offorcefield for the propylparaben crystal; (2) determination of pro-pylparaben crystal morphology; (3) propylparaben and water MDsimulations; (4) propylparaben, water, and PVP/PVA MD simula-tions; (5) docking studies of PVP/PVA to the propylparaben crystalby MD simulations. All simulations employed the Material Studio4.0 (MS4) program (Accelrys Software Inc., USA).

In stage 1, the experimental crystal structure of propylparabendetermined at 173 K was employed as the starting point for thesimulations. Initially the suitability of the COMPASS force fieldfor the crystal system was investigated [30]. The propylparabencrystal was simulated by constant particle-pressuretemperature(NPT) [31] MDina27 28 31 3 triclinic cell for 0.6 ns, with lat-

tice angles a = 90, b = 11, and c = 90. Subsequently, in stage 2 thepropylparaben crystal morphology was determined employingthe Growth Morphology algorithm implemented in the Morphol-ogy module in MS4 [32]. In Table 2 (Supporting material), the threemost stable propylparaben crystal surfaces are enumerated. Fromthe morphology calculations, it can be seen that the most stablesurface in propylparaben is (1 0 0) surface. Thus, this surface cutwas employed throughout the simulations.

For stages 3 and 4 MD simulations, a water layer interactingwith the propylparaben crystal was generated using MS4 Amor-phous Cell. A water slab of thickness $30 and the same latticevectors as the propylparaben crystal was equilibrated. In simula-tions involving PVP/PVA, the polymers were modeled as monomersto reduce the computational complexity and the uncertainty re-

lated to the polymer conformation in solution, allowing more rapiddiffusion through the aqueous medium.

After heating (25 ps) and brief equilibration (25 ps) in the con-stant particle-volumetemperature (NVT) ensemble, the systemswere equilibrated for 250 ps in the NPT ensemble before data col-lection. The time step was 1 fs in all MD simulations.

3. Results and discussion

3.1. Microemulsion system

The microemulsion system chosen for nanoparticle preparationwas based on solvents with high evaporation rates and containedn-butyl acetate, iso-propanol, SDS, and water. A pseudoternaryphase diagram of this microemulsion is shown in Fig. 2.

The grey area indicates the formation of one-phase (microemul-sion) systems. The broad one-phase region of this system may beattributed to the co-surfactant role of the short-chain alcohol,iso-propanol, which increases the mobility of SDS interfacialmonolayer and enables an additional reduction of the interfacialtension [33].

Electrical conductivity measurements of microemulsions lo-cated on dilution line 2:8 of the phase diagram (2:8 w/w ratioSDS to water-shown in Fig. 2) were performed at room tempera-ture. All the microemulsions located on this line were visually clearand appeared dark under cross-polarized light microscopy (nobirefringence). Viscosity measurements of the same microemul-sions were performed at room temperature and all samplesexhibited Newtonian flow behavior, as expected from the micro-emulsions [34]. The dilution line representing descending concen-tration of oil was chosen to characterize this system since duringthe solvent evaporation process, the concentration of oil phase willbe gradually reduced (both n-butyl acetate and iso-propanol haveevaporation rates greater than that of water). The dependence ofelectrical conductivity of the microemulsions on the oil phase con-centration can be seen in Fig. 3.

At oil phase concentration of 10 wt.%, the microemulsion con-

ductivity is very high, similar to that of the aqueous phase withthe surfactant only (18 mS/cm for 20 wt.% solution of SDS inwater), indicating an oil-in-water microemulsion structure. Withthe increase in oil phase concentration, there is a gradual decreasein the electrical conductivity, but it still remains significantly high-er than the conductivity of the n-butyl acetate = 2lS/cm and

Fig. 2. Phase diagram describing the formation of microemulsions (grey area) atroom temperature. Concentrations are given in weight fractions. Electric conduc-tivity measurements were performed on the microemulsion compositions located

on dilution line 2:8. Microemulsion composition chosen for propylparaben incor-poration is labeled with an asterisk.

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iso-propyl alcohol = 5lS/cm. At oil phase concentration of 5060 wt.%, there is a change in conductivity slope. This may indicatethe transition of oil-in-water microemulsion structure to a bicon-tinuous region.

3.2. Addition of propylparaben

The following microemulsion composition was chosen for

obtaining nanoparticles of propylparaben (labeled by an asteriskin Fig. 2): 8 wt.% SDS, 3.5 wt.% n-butyl acetate, 3.5 wt.% iso-propa-nol, 3 wt.% propylparaben, 82 wt.% water.

This system has a low organic solvent content (which is benefi-cial for the evaporation process) and it allows reaching a low sur-factant:active material ratio in the final powder. The conductivitymeasured for this microemulsion was 12.4 0.7 mS/cm and theviscosity was 4.8 0.04 mPa*s.

Droplet size of the microemulsion was estimated from SANSmeasurements. A broad correlation peak was observed atq = 0.0765 1, which indicates a mean spacing of 8.21 nm forthe contained aggregates (Fig. 4).

The scattering length density for the microemulsion (under theassumption that the iso-propanol is dissolved in the aqueous phase

and all the rest is inthe oil phase) is 10.4 109

cm2

. We used H2Oas solvent in our experiments which reduces the contrast largely,but guarantees to study exactly the microemulsions system inquestion and one does not have to worry about potential effectsof the isotopic substitution by D2O.

The scattering of this microemulsions is rather well-describedby a model of spherical aggregates that interact via a screenedelectrostatic repulsion. In this fit we neglected the low q-rangedue to the large error bar of the scattering intensity in this region(Fig. 4). The mean droplet radius (R) and the mean number ofcharges per aggregate (z) were calculated using Eq. (2). The micro-emulsion droplets have an average radius of 2.25 nm (averagediameter of 4.5 nm). About 14 effective charges per aggregate areobtained, which would correspond to about 11% of the theoretical

charge, a value typically observed for micelles and small micro-emulsion droplets [35,36].

These results indicate that the microemulsion contains smalldroplets of oil containing dissolved propylparaben, in a continuousaqueous medium.

3.3. Formation of nanoparticles

Solvent evaporation was performed either by removal of organ-ic solvents under reduced pressure in rotavapor, leading to parti-cles dispersed in water, or by immediate removal of all liquids byspray drying, resulting in formation of a dry powder.

While the solvent removal was performed under reduced pres-sure, large (micron size range) crystals were formed immediately

after the evaporation and they could be observed by light micros-copy (Fig. 5).

After the spray drying process, a dry, free-flowing powder wasobtained. This powder was composed of 27 wt.% propylparabenand 73 wt.% SDS. No solvent remained in the powder as verifiedby GC (total quantity of solvent was less than 0.005 wt.%, thedetection limit of the instrument).

XRD measurements performed on this powder indicated thatthe propylparaben is fully crystalline (Fig. 6).

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

0 10 20 30 40 50 60 70 80 90 100

Oil phase percentage wt%

ConductivitymS

*cm-1

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

0.10.010.8

1.0

1.2

1.4

1.6

1.8

I(q)/cm

-1

q / -1

0.2

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

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



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All propylparaben crystals in the dry powder were 4070 nm(approximate crystal size of propylparaben from XRD peaks usingthe DebyeScherrer equation). When the solid powder was dis-persed in water (5 wt.%), the dispersion was initially clear but rap-idly became turbid due to crystal growth. Formation of the crystalsin the micron size range was observed visually and under themicroscope. It should be noted that the f-potential of the particles

was 46 3 mV, which is high enough to provide electrostatic sta-bilization and prevent particle aggregation. The high negative va-lue of zeta potential is attributed to the negatively charged SDSmolecules which are adsorbed on the particle surface.

It can be expected that the formation of large crystals is due toOswald ripening of the dispersed nanocrystals in water. During thesolvent evaporation process, the concentration of propylparaben ineach microemulsion droplet increases significantly. At some pointthe labile supersaturation zone is reached, where spontaneous andrapid crystal nucleation occurs. The nucleation process is simulta-neously accompanied by crystal growth. When the process contin-ues, the polydispersity of the system increases, since previouslyformed crystals become larger than the newer ones. As a result,in the dry powder there could be significant differences in the crys-

tal size (as indicated by the XRD experiment). When the powder ofthese nanocrystals is dispersed in water, the differences in crystalsizes may lead to Oswaldripening, since the saturation solubility ofthe smaller crystals is greater than that of the larger ones, causingdissolution of the former and gradual growth of the latter (provid-ing bulk water solubility is not negligible). Since the initialpropylparaben dispersion is probably polydispersed, and sincepropylparaben has low but finite water solubility (28 mM), it issusceptible to ripening.

3.4. Crystallization inhibition

Since crystal growth was observed when the nanocrystals weredispersed in water, the next step was an attempt to prevent or re-

tard the crystallization process. Recently, Lindfors et al. [37] re-ported on retardation of the crystal growth of bicalutamide in

aqueous medium by addition of polyvinylpyrrolidone (PVP). Theywere able to separate the two steps of the crystallization process nucleation and crystal growth. They found that the crystalgrowth rate of bicalutamide is significantly retarded by PVP (MW360,000) due to the strong adsorption of the polymer to crystalsexceeding the critical radius of nucleation. The nucleation rate ofthis drug was not significantly altered since PVP binding to the

monomer/subcritical crystal was weak. This suggests that thesmall critical radius of bicalutamide prevents interference of PVPin nucleation step.

Other reports also mention alteration of the crystallization pro-cess of various drugs in aqueous medium by PVP. Thus, PVP was re-ported to retard the nucleation rate of hydrocortisone acetate [38]and felodipine [39], and to retard the crystal growth rate of sulpha-thiazide [40], acetaminophen [41], and nifedipine [42].

Incorporation of PVP of various molecular weights in micro-emulsions was previously reported by Koetz et al. [43] Microemul-sions containing SDS showed noticeable change in the spontaneouscurvature of the surfactant, probably due to adsorption of the poly-mer at the head groups of SDS.

Based on these findings, it was decided to incorporate PVP in

the microemulsion loaded with propylparaben in order to retardcrystallization during solvent evaporation and upon dispersion ofthe powder in water. PVP of various molecular weights was suc-cessfully introduced into the above microemulsion. It was possibleto incorporate as much as 10 wt.% PVP (MW 10,000360,000) inthe microemulsion without causing phase separation.

The experiments indicated that PVP with an average MW of40,000 had the most profound effect on crystallization of propyl-paraben.

Microemulsions containing 010 wt.% PVP were spray dried toyield a fine, free-flowing powder. The microemulsion compositionswere: 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 dis-

persed (5 wt.%) in water; when the PVP concentration wasP7 wt.%, the dispersion remained clear and transparent, and no

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



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crystal formation was visually observed. At lower concentrations ofPVP, the crystallization process was kinetically retarded, but even-tually the dispersion became turbid and crystallization was ob-served. It is well known that SDS is capable of formingcomplexes with PVP [4447]. As found by Minnati et al. [45] thesecomplexes are likely to be formed due to the attraction betweenthe partially negative ether oxygen on PVP and the positive sodiumion of SDS, thus allowing the dodecyl sulfate to attach to the PVP.In excess of SDS, the previously neutral PVP becomes a highlycharged polyanion.

Zeta potential of the particles which were dispersed in the pres-ence of PVP was 29 3 mV. The decrease in zeta potential can beexplained by elevated ionic strength of the dispersion due to in-creased population of dissociated ions [45]. However, the mea-sured zeta potential is still high enough to provide electrostaticstabilization of the nanoparticles. Moreover, it should be expectedthat the attachment of the polymeric complex to the particle sur-face would provide additional stabilization by a steric mechanism,and thus further contribute to the prevention of particle aggrega-tion. Furthermore, both the adsorbed PVP and PVPSDS complexmay interfere with the crystallization process, hence leading toobtaining stable nanoparticles in dispersion.

For a microemulsion containing 7 wt.% PVP, the composition ofthe powder after drying is: 17 wt.% propylparaben, 39 wt.% PVP,44 wt.% SDS. XRD performed on this powder did not reveal anypeaks of crystalline propylparaben (Fig. 7).

This result implies that PVP interfered in the nucleation step ofthe crystallization process and led to obtaining a fully amorphousproduct. This result can probably be explained by a strong interac-tion between the polymer and individual propylparaben moleculesor subcritical crystals. Possible interactions with the growing crys-tals were modeled by MD simulations and will be discussed later.When the powder is stored at room temperature, propylparabenremains amorphous for months.

In order to obtain quantitative information about the effect ofPVP concentration in the fraction of propylparaben that is present

as nanoparticles, the dispersion was filtered through a 0.45 lmfilter, followed by measurement of the concentration of propylpar-aben in the filtrate. It was found that when the initial micro-emulsion contained P7 wt.% PVP, more than 95wt.% of thepropylparaben was present as particles having a diameter of lessthan 0.45 lm for at least 1 week after the dispersion was per-formed (Fig. 8). The dispersion remained visually stable for at least2 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 nanoparti-cles was smaller and turbidity was observed.

SAXS measurement was performed on 5 wt.% dispersion inwater of the powder that was prepared from microemulsion con-taining 7 wt.% PVP. The scattering pattern revealed a maximumat q = 0.55 nm1 (Fig. 9).

Another SAXS measurement was performed on powder dis-persed 0.5 wt.% in water (10 times diluted dispersion). No changein scattering maximum location was observed in the diluted sam-ple. This result implies that the contribution of the structure factorto the scattering pattern is relatively low in this q-range, suggest-ing that the data are perhaps a form factor alone. After a coarse fitto a hard cylinder model, an approximate particle diameter of16 nm could be deduced. Hard cylinder model was chosen forthe evaluation since it provided the best fit for the collected data.At present we do not know the explanation for the suitability ofthat model, and this issue will be investigated in future studies.

It can be seen from the scattering measurements that the aver-age particle size is larger than the average microemulsion dropletsize. Growth of particles formed from water-in-oil microemulsiondroplets was previously explained using crystallization models[18]. A possible explanation for the larger size of the amorphousparticles in our system is the coalescence of the microemulsiondroplets during the evaporation process. Initially, the componentsof the dispersed phase of the microemulsion were selected withmuch higher evaporation rate than the aqueous phase, in orderto achieve rapid transformation of the droplets into solid particles.

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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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However it is possible that the actual differences in the evapora-tion rates in the final system are not that significant, as it is wellknown that in microemulsions and emulsions the evaporationrates of the dispersed droplets might be retarded [8,48]. Therefore,we can not rule out the possible collapse of the microemulsionduring the evaporation process which results in droplets coales-

cence, and eventually leads to particles larger than the originalmicroemulsion droplets.

From the results obtained so far, it can be concluded that PVPinhibits crystal nucleation during spray-drying. In order to evaluatethe potential ability of PVP to also retard the growth of alreadyformed propylparaben crystals, the following experiment was per-formed: powder preparedfrommicroemulsion withoutPVP(whichis composed of crystalline nanoparticles, as described above) wasdispersed(5 wt.%) in 3.2 wt.%PVP aqueous solution(this concentra-tionwas selectedin order to have thesame proportions betweenthecomponents 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 adiameter less than 0.45 lm was 98 1 wt.%. This result indicates

that PVP is effective in retardation of crystal growth as well as ininhibition of crystal nucleation.

For comparison, the same experiment was conducted with an-other hydrophilic polymer, polyvinylalcohol (PVA, average MW50,000). It was found that large crystals were formed shortly afterdispersing the powder, indicating that PVA do not retard crystalgrowth, emphasizing the special role of PVP.

3.5. Control experiments

Bulk propylparaben was mixed with iso-propyl alcohol/watersolution and it was found that the solubility of propylparaben inthis solution at room temperature is 0.12 wt.%. In the second con-

trol experiment (solubilization of propylparaben in a micellar solu-tion of SDS), only 5.6 wt.% of the propylparaben was solubilized inSDS micelles. In the third control experiment (solubilization of pro-pylparaben 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 wasstudied by MD simulations. The purpose was to address the bind-ing of PVP to the propylparaben growing crystal. A comparison wasmade between PVP and PVA, which did not display crystal growthinhibition properties.

It is evident from analysis of the MD trajectory of pure propyl-

paraben that the crystal remains stable throughout the simula-tions: the stacking interactions between different layers of thepropylparaben crystal remain intact (Fig. 1, Supporting material)and the hydrogen bonds within different layers are stable (Fig. 2,Supporting material). Nonetheless, considerable thermal motioncausing local disorder is observed throughout the simulations.The mean square displacement is 2.5 2 and remains stable forthe last 0.5 ns of the control simulation. Thus, the COMPASS forcefield is deemed suitable for the current study involving propylpar-aben crystal.

In the combined propylparabenwater-PVP/PVA simulations,the PVP and PVA monomeric units were added at the center ofwater layer. Both monomers diffused through the water to thepropylparabenwater interface within the first few hundred ps of

the simulations (Fig. 3, Supporting material). The simulations ofPVA indicate that this monomeric unit has rather nonspecific

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Formation of Organic Nano Particles