Journal of Controlled Release 97 (2004) 291 300 www.elsevier.

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Preparation and characterization of propranolol hydrochloride nanoparticles: a comparative study

Nathalie Ubrich a, Philippe Bouillot b, Christina Pellerin c, Maurice Hoffman a, Philippe Maincent a,*
nique, EA 3452, Faculte de Pharmacie, 5, rue Albert Lebrun, B.P. 403, 54001 Nancy cedex, France Laboratoire de Pharmacie Gale b Pfizer, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK c matologie et de Physiologie, EA 3452, Faculte de Pharmacie, 5, rue Albert Lebrun, B.P. 403, 54001 Nancy cedex, France Laboratoire dHe Received 6 January 2004; accepted 24 March 2004 Available online 30 April 2004
a

Abstract The water-in-oil-in-water (w/o/w) emulsification process is the method of choice for the encapsulation inside polymeric particles of hydrophilic drugs such as proteins and peptides which are high molecular weight macromolecules. Our objective was to apply this technique in order to formulate nanoparticles loaded with both a hydrophilic and a low molecular weight drug such as propranolol-HCl. Nanoparticles were prepared using a pressure homogenization device with various polymers (poly-qcaprolactone, poly(lactide-co-glycolide), ethylcellulose) and different amounts of drug and were compared in terms of particle size, encapsulation efficiency and drug release. Higher encapsulation efficiencies were obtained with both PCL (77.3%) and PLGA (83.3%) compared to ethylcellulose (66.8%). The in vitro drug release was characterized by an initial burst and an incomplete dissolution of the drug. When decreasing the polymer/drug ratio, the release appeared more controlled and prolonged up to 8 h. It can be concluded that nanoparticles prepared by w/o/w emulsification followed by solvent evaporation might be potential drug carriers for low molecular weight and hydrophilic drugs. D 2004 Elsevier B.V. All rights reserved.
Keywords: Nanoparticle; w/o/w emulsion; Hydrophilic drug; Low molecular weight drug; Propranolol hydrochloride

1. Introduction Nanoparticles are colloidal polymeric drug carriers that hold promise for peroral drug delivery which represents by far the most common and convenient route of administration. In addition, their potential uptake as well as their stability in the gastrointestinal
* Corresponding author. Tel.: +33-3-83-68-2296; fax: +33-383-68-2301. E-mail address: maincent@pharma.uhp-nancy.fr (P. Maincent). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.03.023

tract indicate that nanoparticles are expected to be promising carriers for the transport of drugs or proteins. Moreover, since the drug targeting at the cellular or tissular level is size-dependent, smaller particles are taken up to a higher degree than larger ones. However, the difficulty in designing water-soluble drug-loaded nanoparticle formulations combining both a high drug entrapment and an effective drug release with the adapted method of preparation represents a real challenge. Biodegradable nanospheres including polyalkylcyanoacrylate [1] and poly(lactide-co-glyco-

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lide) [2] nanospheres have already been studied. Gautier et al. [3] demonstrated likewise the efficacy of biodegradable poly(isohexyl cyanoacrylate) nanoparticles with a sustained delivery of growth hormone releasing factor (GRF). Unexpected results have been et al. [4] with insulin-loaded also obtained by Damge poly(isobutyl cyanoacrylate) nanocapsules prepared by interfacial polymerization, reducing glycemia by 50 60% in diabetic rats. Niwa et al. [5] developed a novel spontaneous emulsification solvent diffusion method in an aqueous phase for the preparation of PLGA nanospheres with an LH RH analogue. However, a poor entrapment of the drug was observed. In order to improve the entrapment efficiency by preventing the leakage of the peptide into the continuous phase, the same authors employed a nontoxic oil as the outer phase [6]. Novel emulsion solvent diffusion methods in water or oil were also developed to prepare tyrotropin releasing hormone and elcatonin-loaded PLGA nanospheres via the coprecipitation of drug and polymer in the emulsion droplets induced by the diffusion of the solvent [7]. Nevertheless, the high toxicity of methanol used in the organic phase could be a considerable disadvantage. The nanoprecipitation method [8] was also widely used by several research groups to prepare nanoparticles [9,10]. However, this technique suffers the drawback of a poor incorporation efficiency of watersoluble drugs owing to the rapid migration and therefore loss of drug into the aqueous phase. Govender et al. [11] have recently improved the incorporation of a water-soluble drug into poly(DLlactide-co-glycolide) nanoparticles prepared by this technique by either increasing the aqueous phase pH and thereby decreasing the solubility of the drug, or including various excipients into the formulations. Another well-known technique (initially developed for microparticles and not nanoparticles), based on the water-in-oil-in-water (w/o/w) emulsification and solvent evaporation, was also employed for the encapsulation of high molecular weight molecules such as peptides or proteins inside polyester nanoparticles [12]. Moreover, it was demonstrated to be superior to other incorporation methods, mainly in terms of stability for drugs [13]. Macromolecules such as heparins were also successfully entrapped into nanoparticles [14,15] and showed promising future prospects as an oral delivery system [16].

To the best of our knowledge, no study has been carried out on the encapsulation of low molecular weight hydrophilic drugs inside polymeric nanoparticles prepared by the w/o/w emulsion. Taking into account this information, the primary goal of this study has been to formulate nanoparticles loaded with propranolol hydrochloride, chosen as a model drug, by w/o/w emulsification. Therefore, the research was based on the encapsulation and drug release of propranolol hydrochloride (propranolol-HCl) both with different polymers (ethylcellulose, poly-q-caprolactone and poly(lactide-co-glycolide)) and various amounts of drug.

2. Materials and methods 2.1. Materials The two biodegradable polymers, poly(D,L-lacticco-glycolic acid) (PLGA 50:50; MW 40,000 Da) and poly(q-caprolactone) (PCL; MW 42,000 Da), were purchased from Medisorb Technologies International L.P. (Welmington, DE, USA) and Aldrich Chemicals (Steinheim, Germany), respectively. The nonbiodegradable polymer, ethylcellulose (EC; MW 146,000 Da), was supplied by Sigma (St. Louis, MO, USA). Polyvinylalcohol (PVA; MW 30,000 Da, 88% hydrolyzed) was purchased from Sigma and chosen as a surface active agent for the second emulsion. The highly water-soluble drug, propranolol hydrochloride (MW 135 Da, water solubility >150 mg/ml), was purchased from Sigma and used as the model drug. All other chemical reagents were obtained from Sigma and Prolabo (Strasbourg, France) and were of analytical grade. 2.2. Experimental 2.2.1. Nanoparticles preparation The preparation of nanoparticles (NP) was achieved by conveniently adjusting the multiple emulsion (w/o/w) technique, previously applied to the preparation of both micro- and nanoparticles [17]. The adjustment was based on the use of an homogenizer for the second emulsification step, thus reducing considerably the size of the dispersed droplets. Briefly, the drug previously dissolved in 1

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ml of distilled water was emulsified in 10 ml of methylene chloride containing the polymer (PLGA 50:50, PCL or EC, 250 mg) under magnetic stirring at 1500 rpm. In order to decrease the aqueous solubility of propranolol-HCl (pKa 9.5), this first emulsion was thereafter poured into 200 ml of 0.25% PVA aqueous solution at pH 9.7 and homog rin, enized with the homogenizer (AML 2, Gue Mauze, France) in an ice bath for 3 min. After solvent evaporation under reduced pressure, the NP were isolated by centrifugation at 70,000 g for 30 min (Beckmann Model J2-21) and washed three times with Milli-Q water before freeze-drying. The effect of the amount of propranolol hydrochloride (20, 30, 40 and 60 mg) on both the entrapment efficiency and the drug release was evaluated. 2.2.2. Encapsulation efficiency The amount of propranolol hydrochloride entrapped within NP was determined by measuring by UV spectrophotometry at 289 nm the amount of nonentrapped drug recovered in the supernatant after ultracentrifugation of the NP at 70,000 g for 30 min. 2.2.3. Particle size The nanoparticles were analyzed for their size distribution using a Zetasizer IIR (Malvern Instruments, UK). The results were normalized with respect to a polystyrene standard solution (Malvern Instruments, UK). Each sample was measured in triplicate and an average particle size was expressed as the mean diameter. 2.2.4. In vitro release experiments One hundred milligrams of freeze-dried nanoparticles prepared with each polymer and 20 or 60 mg of propranolol hydrochloride were resuspended in a 100 ml of phosphate buffer (pH 7.4) flask and incubated into a bath at 37 jC under gentle magnetic stirring (150 rpm). At appropriate intervals, 2 ml samples were removed and centrifuged for 30 min at 70,000 g . The supernatant was removed and assayed for drug release. Fresh PBS (2 ml) was then added to the nanoparticles suspension at each sampling point and agitation was continued. The amount of propranolol-HCl in the release medium was determined by UV spectrophotometry at 289 nm. All release experiments were performed in triplicate.

2.2.5. Surface tension of propranolol-HCl in water The surface tension of propranolol-HCl was determined in water according to Faivre et al. [18]. Briefly, various amounts of propranolol-HCl (from 0 to 60 mg/ml) were dissolved in tridistilled water and the aqueous solutions were transferred into measurement cells. The surface was cleaned by suction and the solutions were left for 24 h at room temperature. The surface tension measurements were performed using a digital tensiometer (Kru ss K10T, Germany) at 22 jC. The results are expressed as the mean of three independent measurements. The accuracy was estimated to 0.2 mN/m. 2.2.6. Water content The water content within nanoparticles was analyzed via Karl Fisher titration. Assays were performed on an Aquatest 10 Karl Fischer Coulorimetric Titrator (Seradyn, Indianapolis, USA) calibrated with anhydrous methylene chloride. Typically, 1 ml of anhydrous methylene chloride was added in 2 ml vials containing 50 mg of freeze-dried propranolol hydrochloride-loaded nanoparticles. The vials were then subjected to brief sonication and mild shaking to facilitate the moisture extraction process. The samples (200 500 Al) were then injected into the reaction vessel. Each sample was monitored in triplicate and an average of the three determinations was reported and expressed on a percent total weight basis (%, w/w). 2.2.7. Residual solvent The amount of residual solvent was determined by gas chromatography equipped with flame ionization detector (GC-14A, Shimadzu). The temperatures were selected as follows: oven temperature 100 jC, injector temperature 150 jC and detector temperature 220 jC. Each nanoparticle batch was analyzed in triplicate. 2.2.8. Differential scanning calorimetry A scanning calorimeter (DSC 92 Setaram) was used and calibrated using indium as the standard. Samples weighing 2 5 mg were heated in sealed aluminium pans. The resulting thermograms covering a range of 100 to 200 jC were recorded at heating rate of 10 jC/min. Analysis were conducted firstly with samples of pure polymers and propranolol-HCl pure drug, then the resulting nanoparticles were analyzed and compared for possible drug polymer interactions.

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3. Results and discussion 3.1. Size and encapsulation efficiency The choice of a particular method of encapsulation is usually determined by the solubility characteristics of the drug. In our approach, the double emulsion solvent evaporation process was adopted for the encapsulation of propranolol-HCl, a water-soluble drug. It involves two major steps, the formation of droplets of the first emulsion and the subsequent removal of solvent from the droplets of the second emulsion. The size of the resulting particles depends mainly on the agitation velocity of the preparation process. The more vigorous the agitation, the smaller the particles. Nanoparticles have thus been previously prepared by the water-in-oil-in-water technique by using sonication [12]. However, the polydispersity index still remains relatively high. In our study, the choice of the nanoparticle preparation consisted in a high pressure homogenization process which allowed to reduce considerably the mean particle size and simultaneously to narrow the width of the size distribution, i.e. reduce the polydispersity index [19]. As reported in Fig. 1, the size of nanoparticles prepared with each polymer fluctuated between 220 and 270 nm, with a relatively monodispersed distribution. The larger nanoparticles were obtained with ethylcellulose, the smaller with PLGA. This might be due to a slight

Fig. 1. Comparison of the size of unloaded and loaded nanoparticles prepared by w/o/w emulsification with EC (n), PCL (E) PLGA ( w ) and various amounts of propranolol-HCl (20, 30, 40 and 60 mg). Data are shown as mean F S.D. (n = 3).

swelling of EC NP in water. Indeed, after freezedrying, the water content was higher for EC nanoparticles than that observed with both PLGA and PCL nanoparticles (Fig. 2). Furthermore, the nanoparticle size was affected by the presence of the drug, since significant differences were observed for both loaded and unloaded EC, PLGA and PCL nanoparticles. Moreover, when the polymer/drug ratio was decreased by increasing the amount of propranolol-HCl in the inner aqueous phase, a decrease in nanoparticle size was observed. These results are correlated with those observed by Verrachia et al. [20]. Indeed, they stated that the nanoparticle size decreased with increasing drug concentration in the internal aqueous phase. Since the particle size is related to a great extent to the stability of the first emulsion, they claimed that the drug can act as a surfactant by stabilizing the first emulsion and consequently hampering the fast coalescence of the droplets. The chemical structure of propranolol-HCl, an hydrophilic molecule with an hydrophobic aliphatic chain, may confer a relative amphiphilicity to the drug which could behave as a surfactant. In order to check this hypothesis, the surface tension of different concentrations of propranolol-HCl in water was measured (Fig. 3). It is obvious that propranolol hydrochloride decreases the surface tension of all concentrations tested, which explains the decrease in size of drug loaded NP compared to unloaded NP. Furthermore, from 20 to 60 mg/ml of propranolol-HCl (corresponding to the concentrations used for the different nanoparticulate formulations), the surface tension did not change significantly. Consequently, the decrease in size does not seem to be due only to the tensioactive properties of the drug. Other parameters such as molecular weight and viscosity of each polymer as well as the internal aqueous phase viscosity may also affect the NP size. Indeed, we have already demonstrated that a 5% PLGA organic solution (intrinsic viscosity = 0.3 dl/g) gives smaller NP (140 nm) than a PCL organic solution at the same concentration (intrinsic viscosity = 0.55 dl/g, average diameter = 270 nm) [21]. The entrapment efficiency within NP was significantly affected by the nature of the polymers as well as the ratio polymer/drug (Fig. 4). The highest encapsulation efficiency was obtained with PLGA, the lowest with ethylcellulose. Despite the high hydrosolubility of propranolol hydrochloride, favouring the leakage of

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Fig. 4. Comparison of the encapsulation efficiency of propranolol hydrochloride-loaded nanoparticles prepared by w/o/w emulsion with EC (n), PCL (E), PLGA ( w ) and various amounts of propranolol-HCl (20, 30, 40 and 60 mg). Data are shown as mean F S.D. (n = 3). Fig. 2. Comparison of the water content within freeze-dried nanoparticles prepared by w/o/w emulsion with propranolol-HCl (60 mg) and EC (black), PCL (grey), and PLGA (white). Data are shown as mean F S.D. (n = 3).

the drug into the external aqueous phase, entrapment efficiencies were rather high. It is assumed that propranolol hydrochloride is localised at the interfaces (either internal water in oil or external oil in water). Therefore a significant amount of drug is supposed to be adsorbed at the outer surface for the 20 mg/ml drug concentration. With increasing propranolol hydrochloride concentration, there is a saturation of the outer surface. At that time, the drug would dissolve more in the aqueous core and the encapsulation efficiency will also consequently increase. This hypothesis is

Fig. 3. Surface tension of various concentrations of propranolol-HCl in water. Data are shown as mean F S.D. (n = 3).

strengthened by the measurement of the pH of the inner aqueous solution of propranolol hydrochloride going from 5.51 to 4.8 for drug solutions of 20 and 60 mg/ml, respectively. Therefore, the solubility of propranolol hydrochloride would be enhanced at pH 4.8 which would be in favour of a larger amount of drug dissolved in the inner core and, consequently, in the nanoparticles prepared with 60 mg/ml of propranolol hydrochloride. In addition, the degree of ionization of the drug and the pH of the continuous phase are critical parameters for the encapsulation of ionizable drugs such as propranolol-HCl. Increasing the pH of the aqueous PVA solution up to 9.7 (i.e. above the pKa of the drug) resulted in a decrease of its solubility and consequently an increase of its encapsulation [22,23]. Indeed, the saturation solubility in the PVA solution (pH 9.7) was 135 mg/ml whereas the saturation solubility in purified water is more than 150 mg/ml. Therefore it has been confirmed that working at a pH higher than the drugs pKa allows a better encapsulation efficiency than in pure water. In addition, the removal of the organic solvent under reduced pressure favours its fast evaporation followed by the polymer precipitation, thus reducing the migration of the drug to the external phase. Indeed, the faster the solvent evaporation, the higher the encapsulation efficiency. Nevertheless, it has been noted that the pH of the external phase and the solvent evaporation duration were the same for the four concentrations of propranolol-HCl. One possible explanation could concern the

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et al. [6] who prepared nafarelin acetate PLGA submicron nanospheres (200 300 nm) by a spontaneous emulsification solvent diffusion method. Compared with microparticles [24], the amount of residual solvent is much lower. It can be postulated that smaller particles characterized by a larger surface area may involve a more complete evaporation of methylene chloride. In addition, during lyophilization, the diffusion of methylene chloride from nanoparticles will be favoured, leading to low residual amounts of solvent. Moreover, the affinity of the solvent to the polymers must also be taken into account, as well as the physicochemical properties of each polymer. A higher residual solvent content of PCL nanoparticles could be explained by the semi-crystalline nature of the polymer which confers a slower diffusion of the solvent from the particles.
Fig. 5. Comparison of the residual methylene chloride within nanoparticles prepared with propranolol-HCl (60 mg) and EC (black), PCL (grey) and PLGA (white) by w/o/w emulsion. The measurements were carried out using a gas chromatography system with flame ionization detector. Data are shown as mean F S.D. (n = 3).

3.3. Thermoanalytical analysis As shown in Table 1, bulk propranolol-HCl powder exhibited a melting peak at 167 jC. The two amorphous polymers PLGA and EC showed a Tg around 38 and 123 jC, respectively. The semicrystalline polymer PCL was characterized by a melting peak at 62.9 jC and a Tg around 62 jC. All these findings are well-known and were expected. In case of propranolol-HCl-loaded nanoparticles, a shift to much lower values was observed for the melting temperature of the drug. At the same time, a shift in the Tg was also observed for each polymer for NP prepared with 60 mg of propranolol hydrochloride (NP 60) whereas no significant shift occurred for unloaded and NP formulated with 20 mg (NP 20). The rise of Tg for EC and PLGA drug-

increase of the internal phase viscosity due to the different propranolol hydrochloride concentrations studied which could reduce the leakage of the drug towards the external aqueous phase. 3.2. Residual organic solvent The residual methylene chloride content into propranolol-HCl-loaded nanoparticles prepared with the three polymers was below 15 ppm (Fig. 5). The results are correlated with those observed by Niwa

Table 1 Thermal characteristics of propranolol-HCl, ethylcellulose, poly(q-caprolactone) and poly(lactide-co-glycolide) (50:50) as well as unloaded and loaded nanoparticles prepared by the double emulsion and evaporation method with each polymer and 20 (NP20) or 60 (NP60) mg of propranolol-HCl Polymers Ethylcellulose Poly(q-caprolactone) Poly(lactide-co-glycolide) (50:50) PropranololFormulations Polymer Unloaded Loaded Loaded Polymer Unloaded Loaded Loaded Polymer Unloaded Loaded Loaded HCl NP NP20 NP60 NP NP20 NP60 NP NP20 NP60 123.5 125.1 130.2 84.4 152.5 57.1 61.9 62.9 62.3 61.8 63.8 70.7 38.7 60.7 59.9 84.9 56.9 39.9 41.5 86.2 49.7 57.3 167.0

Tg (jC) Tf polymer (jC) Tf drug (jC)

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loaded NP may be attributed to significant interactions occurring between the drug and the polymers during nanoparticles preparation. These results are correlated with those previously observed by Miyajima et al. [25] who stated that incorporation of various drugs raised the Tg of PLGA rods, presumably due to interactions between the polymer and the drug. Bodmeier and Chen [26] reported likewise the existence of interactions between the amine groups of basic drugs such as quinidine or propranolol hydrochloride and the acid functions of lactic and glycolic acid copolymers. On the other hand, the increase of Tg noted for EC and PLGA NP 60 loading may also be a classical effect of fillers as it is well-known in polymer chemistry. Usually, when there are strong interactions between the drug and the polymer, the Tg is significantly decreased. This phenomenom observed with PCL NP 60 loading means that the drug has a plasticizer effect on the PCL matrix. In addition, for each propranolol-HClloaded nanoparticles, the drop of the melting temperature of the drug could be also induced by interactions between propranolol hydrochloride and polymers. Since PCL is still characterized by a Tg and a melting temperature for drug-loaded NP, it can be concluded that its semi-crystalline structure was preserved. In addition, although the melting temperature of the drug decreased significantly after encapsulation, its crystalline state was maintained, probably as very small and unstable crystals. It has to be noted that in other studies, the melting temperature of the encapsulated drug was totally disappearing from the DCS runs, meaning a transformation to an amorphous state [27]. 3.4. Drug release Fig. 6 illustrates the in vitro release profile of each formulation, in pH 7.4 phosphate buffer, by representing the percentage of propranolol-HCl release with respect to the amount of propranolol-HCl encapsulated. The saturation concentration of propranolol hydrochloride in pH 7.4 phosphate buffer is 73 mg/ml which definitely demonstrates that the sink conditions (defined as 30% of the concentration saturation) were maintained during the whole dissolution experiment. Therefore the non-complete release cannot be explained by non-sink conditions.

For all formulations, a first initial burst release occurred. It was demonstrated that it was not due to the adsorption of propranolol-HCl onto the outer surface of the nanoparticles since no significant adsorption ( < 1.5%) was detected when blank nanoparticles were suspended in a propranolol-HCl solution. However, when particles are prepared by the w/o/w method, water-soluble drugs exhibit a tendency to migrate to the aqueous medium, thereby concentrating at the surface of the particles and involving the burst effect [28]. Moreover, the burst release could also be explained by the imperfect encapsulation of the drug inside nanoparticles, resulting from the unstable nature of the emulsion droplets during the solvent removal step. This potential instability may cause a part of the loaded drug to relocate at the nanoparticle surface, thereby rapidly released [29]. When the excipients, i.e. polymers, are not soluble in water, it has been also suggested that drug molecules dissolved in water might be very close to the outer NP surface, forming a layer of molecules, susceptible to be easily and rapidly released [30]. The incomplete release of the drug from each formulation could result from the rather short time of the release study (8 h), but still allows to see interesting differences between 20 and 60 propanolol-HCl NP. For propranolol hydrochloride-loaded NP20, the burst was higher than for NP60. This initial release was followed by a rather stable plateau between 2 and 8 h. On the other hand, for NP60, there is a constant release of the drug up to 8 h after the burst. The interactions between propranolol hydrochloride and the polymers may slow down the drug release or hamper its complete dissolution. Our results are consistent with Okada et al. [31] who reported an increase in Tg of PLGA microspheres (as observed with EC and PLGA NP) with increasing basic drug content, implying an incomplete and slower drug release. The lower drug release was observed from EC NP. Although the slightly higher water content in EC NP could involve an increase in molecular mobility by acting as a plasticizer [30], the increase in Tg correlated with a decrease in chain mobility was predominant. Indeed, the significantly large number of polymer/drug interactions due to increased drug content are likely reflected in the rise of Tg as these interactions made the matrix more rigid. Fig. 6 also

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Fig. 6. Release profiles of propranolol-HCl from nanoparticles prepared with (A) 20 mg and (B) 60 mg of drug and EC (n), PCL (E), PLGA ( w ). Experiments were performed in phosphate buffer at 37 jC and pH 7.4. Data are shown as mean F S.D. (n = 3).

shows that the drug release is faster from polyesters, probably displaying the action of the drug on the polymers. Indeed, the continuous drug release observed from NP60 may be attributed to the higher amine concentration, whereas in NP20, the amine amount is not able to display any action in the short time of the experiment.

4. Conclusion Interestingly, propranolol-HCl-loaded nanoparticles were prepared successfully by water-in-oil-inwater emulsion followed by solvent evaporation. Our results also suggest that drug-loaded nanoparticles displayed significant encapsulation efficiency when

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prepared with either polyester or nonpolyester polymers. The amorphous and/or semi-crystalline properties of each polymer did not dramatically change during the NP preparation. In case of propranololHCl, there was a major change in the melting temperature although its crystalline state was also preserved. Therefore, the double emulsion technique can also be applied to the preparation of NP loaded with both low molecular weight and hydrophilic drugs opening a new field of investigation for this technique. Acknowledgements ronique Rosilio The authors are grateful to Dr. Ve (UMR CNRS 8612, School of Pharmacy, Paris XI, France) for her participation in the research by performing surface tension measurements. References
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