The Design of Nanoparticles Obtained by Solvent Evaporation: A

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The Design of Nanoparticles Obtained by Solvent Evaporation: A Comprehensive Study Ste´phanie Desgouilles,†,‡ Christine Vauthier,*,† Didier Bazile,‡ Joe¨l Vacus,‡ Jean-Louis Grossiord,† Michel Veillard,‡ and Patrick Couvreur† UMR CNRS 8612, Universite´ de Paris-Sud, Chatenay-Malabry, France, AVENTIS, Vitry-sur-Seine, France Received June 6, 2003. In Final Form: August 28, 2003 The preparation of nanoparticles by emulsion solvent evaporation is a very popular method. The purpose of the present study was to clarify the mechanism by which nanoparticles of ethylcellulose (EC) and poly(lactic acid) (PLA) are formed during the emulsion solvent evaporation procedure. This study was mainly based on the measure of the variation of the emulsion and nanoparticle surface charge and size during the solvent evaporation process. From the data obtained and depending on the polymer used (EC or PLA), two different models are proposed to explain the nanoparticle formation. In the EC model, after shrinkage of the emulsion droplets as the direct consequence of solvent evaporation, coalescence occurred before stable and solvent-free nanoparticles were formed. On the contrary, in the PLA model, no or limited coalescence was found to occur so that the picture is that one PLA nanoparticle originated from one (or only a few) PLA emulsion droplet after its shrinkage.

Introduction Following systemic administration of a drug molecule either by intravenous injection or orally, the drug substance distributes throughout the body as a function of its physicochemical properties and molecular structure. The final amount of drug reaching the biological target is thus only a small fraction of the administered dose, which leads to poor efficacy and adverse reactions (side effects). One way to circumvent this problem by controlling body distribution of drugs at the tissular and cellular level is to entrap the drug into submicroscopic carriers. In this view, polymer-based nanoparticles have been extensively investigated since the early 1980s, and numerous medical applications have been proposed.1-3 Among the different processes for nanoparticle preparation, the solvent evaporation method is well established.4-8 It is a very popular method because it is easy and it mainly allows efficient encapsulation of numerous compounds of lipophilic nature.8-13 It is based on the emulsification of an organic solution of a polymer in an * Corresponding author. Mailing address: UMR CNRS 8612, Universite´ de Paris Sud, 5 Rue J. B. Cle´ment, 92296 CHATENAYMALABRY Cedex, France. E-mail: christine.vauthier@ cep.u-psud.fr. Tel: + 33 1 46 83 53 86. Fax: +33 1 46 61 93 34. † Universite ´ de Paris-Sud. ‡ AVENTIS. (1) Couvreur, P.; Kante´, B.; Lenaerts, V.; Scailteur, V.; Roland, M.; Speiser, P. J. Pharm. Sci. 1980, 69, 199-202. (2) Kreuter, J.; Hartmann, H. R. Oncology 1983, 40, 363-366. (3) Damge´, C.; Michel, C.; Aprahamian, M.; Couvreur, P. Diabetes 1988, 37, 246-251. (4) Gurny, R.; Peppas, N. A.; Harrington, D. D.; Banker, G. S. Drug. Dev. Ind. Pharm. 1981, 7, 1-25. (5) Bodmeier, R.; Chen, H. J. Controlled Release 1990, 12, 223-233. (6) Sanchez, A.; Vila-Jato, J. L.; Alonso, M. J. Int. J. Pharm. 1993, 99, 263-273. (7) Plard, J. P.; Bazile, D. Colloids Surf., B 1999, 16, 173-183. (8) De Jaeghere, F.; Doelker, E.; Gurny, R. In The Encyclopedia of Controlled Drug Delivery; Mathiowitz, E., Ed.; Wiley and Son, Inc.: New York, 1999; pp 641-664. (9) Song, C. X.; Labhasetwar, V.; Murphy, H.; Qu, X.; Humphrey, W. R.; Shebuski, R. J.; Levy, R. J. J. Controlled Release 1997, 43, 197-212. (10) Lemos-Senna, E.; Wouessidjewe, D.; Lesieur, S.; Duchene, D. Int. J. Pharm. 1998, 170, 119-128. (11) Vinogradov, S.; Batrakova, E.; Kabanov, A. Colloids Surf., B 1999, 16, 291-304.

aqueous phase followed by the evaporation of the organic solvent.14-17 This leads to the precipitation of the polymer as nanoparticles of a few hundred nanometers in diameter. Although this method is certainly the more widely used,4-13 the mechanism of nanoparticle formation has never been investigated from a fundamental physicochemical point of view. It is expected that the number of the nanoparticles that are formed after the evaporation of the solvent depended on the mechanism that governed the transition from the emulsion droplet state to the nanoparticle state. This mechanism is actually unknown, but two hypotheses could be proposed: either one emulsion droplet gives one nanoparticle, or one nanoparticle arises from several emulsion droplets. According to the first hypothesis, the emulsion droplets, which are stabilized by surfactants, are perfectly stable during the solvent evaporation stage; thus, the formation of a nanoparticle resulted from a single volume shrinkage of the initial emulsion droplet. On the contrary, the second hypothesis is based on the assumption that the emulsion droplets could fuse together during the solvent evaporation stage so that one nanoparticle descended from several emulsion droplets. The determination of an aggregation ration (A) should distinguish these phenomena. It is not questionable whether a better knowledge of how nanoparticles are formed will allow one to control nanoparticle characteristics also improving the industrialization processes for nanoparticle preparation. Thus, this paper investigates in detail how solvent evaporation allows an emulsion of a polymer solution to form nanoparticles. (12) Legrand, P.; Barratt, G.; Mosqueira, V.; Fessi, H.; Devissaguet, J. P. Polymeric nancapsules as drug delivery systems. A review. S.T.P. Pharma Sci. 1999, 9, 411-418. (13) Barratt, G.; Couarraze, G.; Couvreur, P.; Dubernet, C.; Fattal, E.; Gref, R.; Labarre, D.; Legrand, P.; Ponchel, G.; Vauthier, C. In Polymeric Biomaterials, second ed.; Dumitriu, S., Ed.; Marcel Dekker Inc.: New York, 2001, pp 753-782. (14) Kwon, S. S.; Nam, Y. S.; Lee, J. S.; Ku, B. S.; Han, S. H.; Lee, J. Y.; Chang, I. S. Colloids Surf., A 2002, 210, 95-104. (15) Verrecchia, T.; Huve, P.; Bazile, D.; Veillard, M.; Spenlehauer, G.; Couvreur, P. J. Biomed. Mater. Res. 1993, 27, 1019-1028. (16) Julienne, M. C.; Alonso, M. J.; Gomez Amoza, J. L.; Benoit, J. P. Drug. Dev. Ind. Pharm. 1992, 18, 1063-1077. (17) Coffin, M. D.; McGinity, J. W. Pharm. Res. 1992, 9, 200-205.

10.1021/la034999q CCC: $25.00 © 2003 American Chemical Society Published on Web 10/03/2003

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Table 1. Polymer Concentration, c, in Ethyl Acetate for Three Isoviscosities of the Polymer Solutions and Apparent Density (Ggrad) of EC7 and EC22 Nanoparticles as Determined by Isopycnic Centrifugationa EC7 Fo ) 1.139 g/mL viscosity c Fgrad (Pa‚s) (% w/v) (g/mL) 0.02 0.06 0.08 a

5.8 8.3 9.4

σ

1.136 0.001 1.137 0.005 1.138 0.001

EC22 Fo ) 1.123 g/mL c Fgrad (% w/v) (g/mL) 2.5 4.0 4.4

σ

1.128 0.001 1.129 0.001 1.130 0.003

PLA c (% w/v) 8.8 b 15.0

σ is the standard deviation. b Not determined.

Materials and Methods Materials. Ethyl acetate (Normapur) was purchased from Prolabo (Fontenay-sous-Bois, France). Ethylcellulose EC7 (MW 55 600), ethylcellulose EC10 (MW 69 800), and ethylcellulose EC22 (MW 98 900) were from Aqualon Hercules (Antony, France), and polylactic acid (PLA) (MW 108 700) was from Boerhinger Ingelheim (Paris, France). Cellulose acetate (CA, MW 117 600) and cellulose acetobutyrate (CAB, MW 130 800) were purchased from Eastman Chemical (Hamburg, Germany). Sodium dodecyl sulfate (Rectapur) was obtained from Prolabo (Fontenay-sousBois, France). All chemicals were of analytical grade. Viscosity Measurements of Polymer Solutions. The experimental determination of the intrinsic viscosity of EC7, EC10, EC22, CA, CAB, and PLA solutions in ethyl acetate was performed with an Ubbelohde-type “suspended level” viscosimeter, which was mounted vertically in a thermostated bath (AVS 400, Schott Gera¨te, Hofsheim, Germany). The measurements were carried out with a high precision (timing of the flow to within 0.01 s, temperature control to within 0.05 °C), which allowed detection of the very small differences in viscosity between the pure solvent and the very dilute solutions of the polymers. The viscosity of the polymer solutions was also measured with a controlled stress rheometer (CSL100, Carri-Med, RHEO, Chaylan, France) by using a cone and plate geometry. The temperature was controlled to within 0.1 °C with a Peltier device, which was incorporated into the plate. The surroundings were saturated with ethyl acetate. Nanoparticles Preparation. Nanoparticles were prepared by the emulsion solvent evaporation method.15 Practically, ethylcellulose EC7 (MW 55 600), ethylcellulose EC10 (MW 69 800), ethylcellulose EC22 (MW 98 900), cellulose acetate (CA, MW 117 600), cellulose acetobutyrate (CAB, MW 130 800), or poly(lactic acid) (PLA, MW 108 700) were dissolved in ethyl acetate (10 mL) at different concentrations given in Table 1 to obtain polymer solutions with viscosity of 0.02, 0.06, and 0.08 Pa‚s. These polymer solutions were preemulsified using an ultraturrax (T25, Janke and Kunkel IKA TM Labotechnik) with 40 mL of an aqueous solution of sodium dodecyl sulfate (2 g/L) saturated with ethyl acetate. The preemulsions obtained were then further homogenized using a microfluidizer (Microfluidics 110 T, Sodexim, Reims, France) by recycling for 3 min at 20 °C. Ethyl acetate was then evaporated under controlled and mild vacuum (170 mmHg, rotating evaporator) for 30 min at 30 °C. During the evaporation, the emulsions were swept by a continuous nitrogen flow with a constant flow rate (70 L/h). Samples were collected at different time intervals during the course of the evaporation (120 min) by means of a catheter introduced into the emulsion to avoid the interruption of the solvent evaporation process. The final volume of the suspension was 40 mL. It was checked that no aggregate was formed during the preparation of the nanoparticles by filtration of the suspensions through a 1.2 µm cellulose acetate filter (ministar TM NML, Sartorius, Palaiseau, France). Size and ζ-Potential Measurements. The mean diameter of the emulsion droplets and nanoparticles was measured by quasi-elastic light scattering (QELS) using a BI 90 photon correlation spectrometer (Brookhaven Instruments Corporation, New York) working at an angle of 90°. The analysis was performed right after the preparation of the emulsions or of the nanoparticle dispersions. ζ-potentials of emulsions and nanoparticles were determined from their electrophoretic mobility using a BI-

ZETAPLUS (Brookhaven Instruments, New York). Both of these measurements were also performed on samples collected during the course of the evaporation of the solvent. Before analysis, the samples were diluted with their dispersion medium (i.e., an aqueous solution of SDS 2 g/L saturated with ethyl acetate). PLA-containing samples were diluted 50 times, whereas EC-containing samples were diluted 10 times to reach a level of light scattering signal recommended by the supplier of the light scattering apparatus. Each sample was analyzed in triplicate. Ethyl Acetate Determination. The amount of residual ethyl acetate in the emulsion has been evaluated by gas chromatography (GC) during the solvent evaporation process. GC apparatus (gaz chromatograph Fison GC 8000 series, Rodano, Italia) was equipped with an automatic injector (Fison HS 800 CE instrument Rodano, Italia, incubation time 30 min, injection volume 1 mL), a flame ionization detector, and a Megabore (60 m × 0.53 mm) column filled with a stationary phase consisting of methylated silicon with 5% phenyl units (SPB-5 SUPELCO, L’Isle D’Albeau, France). The mobile phase consisted of helium gas with a flow rate of 10 mL/min. Oven temperature conditions were isotherm at 35 °C during 10 min with a following gradient of temperature of 3 °C/min until 250 °C. Temperature of the injector was 150 °C. Before analysis, the emulsion samples were diluted in distilled water (dilution from 1/100 to 1/10000). Density Measurements. The measure of the true density of EC and PLA polymers has been performed using a helium pycnometer 1320TM (Micrometrics, Creil, France). The density of EC and PLA nanoparticles has been determined by isopycnic centrifugation on sucrose gradients.18 Practically, linear gradients of sucrose of density ranging from 1.21 to 1.29 g/mL for PLA nanoparticles and from 1.12 to 1.15 g/mL for EC nanoparticles were prepared at +4 °C within an ultracentrifuge tube. Then, a suspension of EC or PLA nanoparticles was loaded on the top of the sucrose gradient before ultracentrifugation (Combi, Sorvall TM, Rotor T865, Kendro Laboratory Product, Courtaboeuf, France) at 260 000g during 6 h. Gradients were collected at 0.1 mL/min to give 40 fractions of 1 mL each. The identification of the band corresponding to the nanoparticles was performed on the collected fractions by spectrophotometry at 405 nm (Lambda 2, Perkin-ElmerTM, Courtaboeuf, France) and by the evaluation of the density of the corresponding fractions by measuring their refractive index. Small Angle Neutron Scattering. Small angle neutron scattering determinations were performed on EC7, EC22, and PLA nanoparticles. The suspensions were diluted in deuterium oxide (D2O) to give a final polymer concentration of 1 g/L. The small angle neutron scattering experiments were performed on the spectrometer PACE at the Laboratory Leon Brillouin (Saclay, France). The samples were placed in 2 mm thick quartz cells (Helma, Mullheim, Germany), and the sample to detector distance was 1.62 or 4.62 m with incident wavelengths of 6 or 15 Å, respectively. Examinations were carried out over the Q range 5 × 10-3 to 10-1 Å -1. Cryomicroscopy Experiments. A drop of the nanoparticle suspension was deposited on a glow-discharged grid coated with a perforated carbon film. The grid was mounted on a guillotinelike frame, and the emulsion excess was blotted with a filter paper. Then the frame was released, and the grid was plunged into liquid nitrogen cooled liquid propane. The grid was transferred from liquid propane to the Gatan transfer chamber and loaded in a Gatan 626 stage. The samples were observed in a Philips CM12 electron microscope operated at 100 keV. Micrographs were recorded on Kodak image plate S0 163 developed 12 min in D19 full strength.

Results and Discussion Viscosity Measurements of Polymer Solutions. Ethyl acetate solutions of ethylcellulose with 2.45% ethoxylation (EC7, MW 55 600), of ethylcelluloses with 48%-49.5% ethoxylation (EC10, MW 69 800, and EC22, (18) Vauthier, C.; Schmidt, C.; Couvreur, P. J. Nanopart. Res. 1999, 1, 411-418.

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Figure 1. Variation of the viscosity coefficient, µ, as a function of the polymer concentration in ethyl acetate: (9) EC7; (f) EC10; (b) EC22; (2) CA; (1) CAB; ([) PLA. Table 2. Molecular Weight, MW, Intrinsic Viscosity, [µ], and Hydrodynamic Volumes, RH3, of the Different Polymers

a

polymer

MW (g/mol)

[µ] (L/g)

RH3‚ψa (L)

EC7 EC10 EC22 CA CAB PLA

55 600 69 800 98 900 117 600 130 800 108 700

0.096 0.107 0.149 0.119 0.137 0.066

5 338 7 469 14 736 13 994 17 920 7 174

ψ ) Flory constant.

MW 98 900), of cellulose acetate (CA, MW 117 600), of cellulose acetobutyrate (CAB, MW 130 800), and of poly(lactic acid) (PLA) (MW 108 700) were used for viscosity measurements. Figure 1 shows the viscosity coefficient, µ, as a function of the weight concentration of the polymer in ethyl acetate (% w/v). All of the tested solutions displayed a Newtonian-type behavior. However, the analysis of the µ values according to polymer concentration showed two types of behavior: in the case of PLA, µ values remained very low whatever the polymer concentration was, whereas with the ethylcellulose polymer family a strong correlation could be observed between the MW of the polymers and their viscosity. The determination of the intrinsic viscosity has been done using the Ubbelohde viscosimeter to approach the hydrodynamic radius and to give insights concerning the conformation of the solvated macromolecules in ethyl acetate. The intrinsic viscosity [µ] is given by the slope of the equation (µ - µο)/µο ) f(c), where c is the weight concentration of the polymer (% w/v) and µο is the viscosity of the solvent (ethyl acetate, 0.47 mPa‚s at 20 °C). The values of [µ] given in Table 2 confirmed the strong dependency that existed in the ethylcellulose polymer family between the intrinsic viscosity [µ] and the weight molecular mass of the polymer (MW), which is in agreement with the Mark-Houwink-Sakurada relationship: [µ] ) KMvR, where K and R are constants depending on the polymer, the solvent, and the temperature, and Mv is the viscosimetric molecular mass, which is close to MW.19 According to the theory of Fox and Flory,20-22 [µ] ) ψ(RH3/ MW), the individual hydrodynamic volume of the different polymers (RH3) may be calculated (Table 2). From the values of RH3, it may be concluded that the conformation of PLA molecules was much more coiled than that of EC22 for a comparable MW (MW of 98 900 and 108 900 for EC22 and PLA, respectively). Indeed, RH3 was twice (19) Walstra, P. Dechema Monogr. 1974, 77, 87-94. (20) Fowkes, F. M. In Chemistry and Physics at the interfaces; Ross, S., Ed.; American Chemical Society: Washington, DC, 1965. (21) Israelachvili, J. N. In Principles of colloids and surface chemistry; Hunter, R. J., Ed.; Royal Australian Chemical Society: Sydney, 1982. (22) Graessley, W. W. Adv. Polym. Sci. 1982, 47, 67-117.

Figure 2. Size of the emulsions prepared with solutions of (s) EC7, (‚‚‚) EC22, and (- - -) PLA50 at a viscosity of (A) 0.02 and (B) 0.08 Pa‚s.

as high for EC22 than for PLA (Table 2). Thus, the values for the molecular volumes of ethylcellulose polymers suggested that the topological organization of ethylcellulose macromolecules was swollen in ethyl acetate solvent. It is noteworthy that CBA and CA showed very similar values of RH3 compared to EC22 meaning that these polymers behave more like EC22 in ethyl acetate. If the macromolecular chains would be assimilated to dense sphere-type particles, then the hydrodynamic volume should be proportional to the MW of the polymer whatever the type of polymer is. This was not the case in this study. Because the increase in RH3 from EC7 to EC22 was more important than the ratio between the respective MWs of those polymers (i.e., 55 600-98 900), the excluded volume existing inside of the polymer coils was filled by the solvent. Thus, this expansion of ethylcellulose in ethyl acetate may be explained by favorable interactions between the solvent and these polymers. Taken together, these data show that of the cellulose derivatives EC, CA, and CAB were better solvated by ethyl acetate than PLA. Size of Emulsion Droplets. The sizes of the emulsion droplets (Figure 2) have been measured by QELS for the emulsions prepared at polymer concentrations in ethyl acetate corresponding to isoviscosities of either 0.02 or 0.08 Pa‚s for EC7, EC22, and PLA. Table 1 shows the values of those polymer concentrations as determined graphically by linear extrapolation from Figure 1. For the same polymer, the size and size distribution of the droplets were unchanged according to the viscosity. In isoviscosity conditions, sizes were comparable for EC7- and EC22based emulsions, although polymer concentration was more than twice as high for EC7 compared to EC22 emulsions (Table 1). On the contrary, whatever the value of the viscosity, PLA-based emulsions displayed always bigger emulsion droplet sizes than EC7 or EC22 emulsions (Figures 2). Thus, the diameter of the emulsion droplets was strongly dependent on the nature of the polymer (PLA or ethylcellulose) dissolved in the inner phase. This experimental observation shows an analogy with the law of Walstra and Kumar,23,24 leading to a relationship (23) Koshy, A.; Das, T. R.; Kumar, R. Chem. Eng. Sci. 1988, 43, 649654. (24) Walstra, P. In Encyclopedia of Emulsion Technology, 1 Basic Theory; Becher, P., Ed.; Marcel Dekker Inc.: New York, 1983; Vol. 1, Chapter 2, pp 57-127.

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between the diameter of the droplets (ddrop) and the viscosity µ of the organic phase:

ddrop ∝ µβ

(1)

In this relationship, β includes many factors such as the temperature, the agitation parameters (speed and geometry of the apparatus), the viscosity of the continuous phase, the dispersed to continuous volume ratio, the properties of the polymers and of its interactions with the solvent, and the surfactant concentrations. Because during the preparation of the emulsions all of these parameters were kept constant except the nature of the polymer, it was concluded that β reflected only the effect of the polymer in our experimental conditions. The value of β deduced from eq 1 was calculated for emulsions prepared with EC7, EC22, and PLA ethyl acetate solutions. The two polymers arising from the same family (EC7 and EC22) had, although very different MW, almost the same value for β (0.05 and 0.03, respectively, for EC7 and EC22). These values were very different from the one calculated for PLA (0.28). Thus, it was suggested that β should be strongly dependent on the polymer characteristics and may be affected by the interactions between the solvent and the polymer. Especially, in the model of Walstra and Kumar,23,24 the time needed for the viscoelastic relaxation of the stress to occur is a critical point because it is a condition for the rupture of the droplets to take place. In a binary solvent/polymer system, it depends not only on the viscosity of the droplet but also on the time needed for the own rearrangement of the polymer’s own molecules to occur. In our case, we have shown from the calculated values of the polymer’s hydrodynamic volume (RH3) that the conformation of PLA molecules in ethyl acetate was much more folded than that of the ethylcelluloses even at similar MW values (EC22 and PLA). In those conditions, the relaxation viscoelastic time should be more important for PLA than for ethylcellulose molecules.25 Thus, the breaking of a droplet of ethyl acetate containing PLA became less easy than that of the same droplet containing ethylcellulose during the period of time corresponding to the droplet deformation due to the shearing process. This may explain why ethylcellulose emulsion was smaller than PLA emulsion at isoviscosities condition. Size of Nanoparticles. After solvent evaporation, the size of the obtained nanoparticles have been measured by QELS. Figure 3 shows that, in conditions of isoviscosity (0.08 or 0.02 Pa‚s), the size of the nanoparticles was almost the same for all of the cellulose polymers used, whatever their MW or chemical structure (EC7, EC10, EC22, CA, and CAB). Thus, for the cellulose-derivative family, the control of the size of the emulsion droplets by the viscosity allowed also control of the size of the resulting particles after solvent evaporation. This was true whatever the concentration of the cellulose polymer in the inner phase of the emulsion. On the contrary, it was observed that for a same viscosity value, the size of the PLA nanoparticles was always bigger than the size of the cellulose-based nanoparticles (Figure 3). It is noteworthy that the same difference was already observed previously with the emulsion droplets (in isoviscosity conditions, PLA-based emulsions showed bigger droplet diameters than ethylcellulose-based emulsions). Therefore, it was suggested that there was a correlation between the size of the mother emulsion and the size of the resulting nanoparticles. (25) Ferry, J. D. Viscoelastic properties of polymers, 3rd ed.; J. Wiley and Sons: New York, 1980.

Figure 3. Size of the nanoparticles obtained after solvent evaporation from emulsions prepared with different polymers dissolved in ethyl acetate and at different isoviscosity: (0) EC7; (g) EC10; (O) EC22; (4) CA; (3) CAB; (]) PLA. Isoviscosity values were (open symbols) 0.02, (left side filled symbols) 0.04, (right side filled symbols) 0.06, (closed symbols) 0.08 Pa‚s. Solid lines indicate the same polymer; dotted lines indicate the same isoviscosity.

Figure 4. Small angle neuton scattering of the nanoparticle suspension obtained with (A) EC7 and (B) EC22. The solid line corresponds to the fit for monodispersed particles responding to the hard sphere model.

Structure Characterization of Nanoparticles. The apparent density (Fgrad) of EC22 and EC7 nanoparticles has been measured by isopycnic centrifugation on sucrose gradients. Results given in Table 1 show that Fgrad values were very close to the true density (F0) of the polymers as measured by helium picnometry. This suggested that the nanoparticles are formed either (i) by a dense polymer network without any porosity or (ii) by a matrix with open pores allowing the sucrose solution (at the same F0 as the polymer) to diffuse into the pores. To choose between these two possible models of nanoparticle and to investigate more in detail the structure of the particles, small angle neutron scattering has been used as a complementary approach. Figure 4 shows the slope of the neutron scattering spectra for the relationship I(Q) ) f(log Q) with EC7 and EC22 nanoparticles prepared from the emulsion in isoviscosity conditions (0.08 Pa‚s). These slopes followed the Porod law in Q-4, which is a characteristic of spherical, isotropic, dense, and nonporous particles with a smooth surface.26 Observations of the nanoparticles by cryo-TEM confirmed the spherical shape of the EC nanoparticles (data not shown). The contrast inside the nanoparticles appeared homogeneous suggesting that the nanoparticles (26) Cabane, B. In Surfactants solutions: new methods of investigation; Zana, R., Ed.; Surfactant sciences series; Marcel Dekker Inc.: New York, 1987; pp 57-145.

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Figure 5. Variation of the hydrodynamic diameter and of the ethyl acetate content of the emulsion/nanoparticle suspension during the course of the evaporation of ethyl acetate. Hydrodynamic diameter of the systems prepared with (A) (0) EC7 and (9) EC22 and (B, 2) PLA is shown. Panel C shows the residual ethyl acetate remaining in the emulsion/nanoparticle suspension during the course of the preparation of the nanoparticles by the emulsion solvent evaporation method. Viscosity of polymer solutions in ethyl acetate was 0.08 Pa‚s.

were nonporous spheres27 in agreement with the model proposed by the small angle neutron scattering experiments. Mechanism of Nanoparticle Formation. To investigate the mechanism by which emulsion droplets transform into nanoparticles, the size and ζ-potential of the emulsion droplets and resulting nanoparticles have been determined at different time points during the evaporation process. It is noteworthy that the samples were taken by mean of a catheter at different time intervals during the evaporation of ethyl acetate so that there was no breaking in this process. Measurements were performed immediately after sampling. The medium used to carry out the analysis consisted of water saturated with ethyl acetate containing 2 g/L of SDS to keep constant the equilibrium between the different phases. Size Measurements. In these conditions, the size of the EC emulsion droplets decreased significantly before it increased again to reach a plateau. This was observed for both EC7 and EC22 emulsions (Figure 5A). The minimum values of the diameter (dmin) of the EC emulsions were between 66 and 68 nm and were obtained after 30 and 40 min of evaporation for EC22 and EC7, respectively. The maximum diameter values (dmax) were reached after 90 min for both EC7 and EC22 emulsions (Figure 5A). (27) Dubochet, J.; Adrian, M.; Chang, J. J.; Lepault, J.; McDowall, W. In Cryoelectron microscopy of vitrified specimens, Cryotechniques in Biological Electron Microscopy, Steinbrecht, R. A., Zerold, K., Eds.; Spring-Verlag: Berlin, 1987; pp 114-131.

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Although dmax was 74 nm for EC7, it was 83 nm for EC22. This difference was attributed to the swelling of the polymer in ethyl acetate (size determined in water saturated with ethyl acetate), which was more important for EC22 than for EC7 because the EC22 concentration was lower than the concentration of EC7. Figure 5A suggested that the decrease of the size of the emulsion droplets was the consequence of the evaporation of ethyl acetate; then, after approximatly 30 min, droplet or nanoparticle nuclei fusions occurred and led to an increase of the diameter of the dispersed species (emulsion droplet or nanoparticle nuclei). In our experimental conditions (constant volumes of the emulsion, isoviscosity conditions, etc.), it may be considered that there was almost no modification in the number of emulsion droplets during the first stage of evaporation until a maximum shrinkage was reached (dmin). Afterward, aggregation and coalescence would certainly modify the number of species (emulsion droplets or nanoparticle nuclei) because it was defined that the fusion between several droplets or nanoparticle nuclei results in only one nanoparticle at the end of the preparation; this process found expression in the diameter increase of the dispersed species (Figure 5A). Concerning PLA, the curve profile of the droplet size during evaporation was very different: a size decrease was observed until a minimum value (dmin) 147 nm) was reached after 40-50 min of evaporation, but no further increase in the size was observed later on (Figure 5B). This may be explained by the fact that either no droplet or nanoparticle nuclei aggregation and coalescence occurred with PLA and that the final nanoparticles were obtained after shrinkage of the emulsion droplet volumes until a stable value of particle diameter was reached or droplet and nanoparticle nuclei fusion occurred in a restrictive manner so that it could not be highlighted by QELS measurements. It could be hypothesized that the difference of behavior between EC and PLA emulsions would be a consequence of a different kinetic in the solvent evaporation because the two types of polymers used in this study behave very differently in ethyl acetate. Indeed, their molecules could be able to retain the solvent more or less during the evaporation process. By measuring the remaining concentration of ethyl acetate in the two systems (EC and PLA) during the course of the evaporation, we found that it was actually not the case. The dosage of the residual solvent in the emulsion droplets and nanoparticles did, indeed, not show any difference between EC and PLA emulsions (Figure 5C). It is noteworthy that the period of time during which both EC and PLA emulsion droplet size decreased corresponded to the time during which the solvent evaporation was the more pronounced. ζ-Potential Measurements. ζ-potential values of the emulsions before evaporation were found to be -58 mV ((10 mV) for EC22 and -67 mV ((10 mV) for PLA. These values were very reproducible. With EC22 emulsions, the ζ-potential remained close to -58 mV during the first 30 min of the evaporation of ethyl acetate. Afterward, in the time interval 35-80 min, ζ-potential increased to values close to 0 mV, although the measures were in general quite irreproducible. This observation correlated very well with the increase of the emulsion droplet diameter as observed by QELS (Figure 5A). Then, after 80-90 min of evaporation, the values of the ζ-potential dropped again to reach a rather stable value around -60 mV, which was also the measured value for EC22 nanoparticles after complete evaporation of ethyl acetate. Similar results were obtained with EC7 emulsion/nanoparticle suspensions

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Figure 6. Hypothesis about the mechanisms of formation of the nanoparticles by emulsion solvent evaporation using solutions (A) of EC in ethyl acetate and (B) of PLA in ethyl acetate.

(data not shown). The data obtained with PLA emulsion/ nanoparticle suspensions were quite different because the ζ-potential was constant over the 120 min period of the solvent evaporation process. Thus, on the contrary to what was observed with EC emulsions, no increase of the ζ-potential was highlighted with PLA emulsions during this process. These data clearly showed that with EC emulsions there was a temporary perturbation of the ζ-potential of the emulsion droplets. Because SDS is a negatively charged surfactant, it may be reasonably considered that the increase of the ζ-potential during the period of time 3580 min was due to a temporary depletion of SDS surfactant from the interface of EC emulsion droplets. Such decrease in the SDS concentration at the droplet interface could be explained by a competition between SDS and EC which, in turn, induced droplet or nanoparticle nuclei fusion as evidenced by the increase of the diameter of the dispersed species in the EC preparation. Thus EC nanoparticles were clearly believed to form after aggregation and coalescence of EC droplets or nanoparticle nuclei. On the contrary, PLA nanoparticles were considered to form in a very different way. Indeed, ζ-potential values did not change during the evaporation, and no size increase was observed. This suggested that with PLA no or only limited coalescence occurred so that one nanoparticle was supposed to originate, after solvent evaporation, from only one or from a few emulsion droplets. Determination of the Aggregation Ratio A. Because nanoparticles of EC and PLA obtained by solvent evaporation may be considered as nonporous spheres, the polymer mass (mp) per nanoparticle (mp/np) may be calculated as follows:

(mp/np) ) Fo(π/(6dnp3))

(2)

where Fo is the true density of the polymer and dnp is the diameter of the nanoparticles. In the same way, the polymer mass per emulsion droplet (mp/drop) is

(mp/drop) ) c(π/(6ddrop3))

(3)

Table 3. Calculation of the Aggregation Ratio (A) for Polymers EC7, EC22, and PLA 50 polymer EC7 EC22 PLA EC7 EC22 PLA a

dnp (nm)

F0 (g/mL)

A

Viscosity 0.02 Pa‚s 5.8 72 55 2.5 73 56 8.8 121 79

1.139 1.123 1.25

9 20 4

Viscosity 0.08 Pa‚s 76 74 77 83 183 143

1.139 1.123 1.25

11 32 4

ca (% w/v)

9.4 4.4 15.0

ddrop (nm)

Polymer concentration in EtOAc.

where c is the concentration of the polymer in the organic phase and (ddrop) is the diameter of the emulsion droplets. The aggregation ratio (A) may be deduced from eq 2 and eq 3 as

A ) (mp/np)/(mp/drop) ) Fo/(c(dnp/ddrop)3)

(4)

The aggregation ratio for EC7, EC22, and PLA nanoparticles when prepared in isoviscosity conditions (0.02 or 0.08 Pa‚s) are shown in Table 3. For EC7 and EC22 nanoparticles, the A values were important, ranging from 9 to 32, respectively. The lower values were obtained with EC7 at the viscosity of the EC solution of 0.02 Pa‚s, whereas the highest value was 32 for EC22 used at a viscosity of 0.08 Pa‚s. Thus, coalescence was found to be two to three times more important with EC22 compared with EC7. However, because EC7 and EC22 nanoparticles were prepared from emulsions with the same viscosity of the polymer solution (0.02 or 0.08 Pa‚s), the amount of EC22 was two times lower in each droplet than the amount of EC7. Therefore, because of these contradictory effects (coalescence and amount of polymer per initial emulsion droplet), final EC7 and EC22 nanoparticles had roughly comparable polymer mass per particle, which explained that EC7 and EC22 nanoparticles had a similar size and density in isoviscosity conditions of preparation as observed previously. Thus the aggregation ratio determined here could also represent the ratio between the number

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Langmuir, Vol. 19, No. 22, 2003

of macromolecular chains contained in the final nanoparticles and in the emulsion droplets for the different systems. For PLA nanoparticles, the aggregation ratio (A) was much smaller than that for EC without any influence of viscosity conditions: A was 4 for both µ ) 0.02 and µ ) 0.08 Pa‚s. As in isoviscosity conditions, droplets of PLA were more concentrated in polymer compared to EC emulsions (Table 1), and because droplet or nanoparticle nuclei fusions were inhibited when the polymer became insoluble and solid during solvent evaporation, aggregation and coalescence should stop earlier with PLA than with EC during the solvent evaporation process. This was probably the reason coalescence leading to an increase of particle size was not observed with PLA using QELS measurements (Figure 5B). Conclusion The process named emulsion solvent evaporation is currently used for the manufacture of injectable nanoparticles as drug carrier systems. The present study was the first to investigate nanoparticle formation at the molecular and supramolecular level. It was found that the conditions in which the nanoparticles were formed were greatly influenced by the equilibrium conformation of the polymer chains in the solvent, by their dynamic evolution through mechanical shear or solvent evapora-

Desgouilles et al.

tion, and by the organic phase viscosity. Taken together, the data obtained show the importance of the polymer/ solvent pair on the final nanoparticle architecture and mechanism of formation. Hence, two different models were proposed for nanoparticle formation with two polymer types (EC and PLA). In the EC model, after shrinkage of the emulsion droplets due to solvent evaporation, EC molecules were able to compete with the surfactant SDS at the interface inducing emulsion instability, aggregation, and coalescence of the dispersed species before stable and solvent-free nanoparticles were obtained (Figure 6A). On the contrary, in the PLA model, no or only limited aggregation and coalescence occurred so that one PLA nanoparticle arose from one or only few PLA emulsion droplets (Figure 6B). It is noteworthy that this study, which has identified physicochemical parameters that govern nanoparticle formation, will help soon to improve the process leading to a subsequently benefit scale-up work for the pharmaceutical industry. Acknowledgment. The authors thank the Laboratoire Leon Brillouin, Saclay, France, for the access to the PACE spectrometer for the Small Angle Neuton Scattering Experiments. LA034999Q