Microencapsulation of Amoxicillin in Poly(l-lactic acid) by Supercritical

Nov 3, 2006 - Dependence of SAS particle morphologies on the ternary phase equilibria. R. Campardelli , E. Reverchon , I. De Marco. The Journal of Sup...
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Ind. Eng. Chem. Res. 2006, 45, 8738-8743

Microencapsulation of Amoxicillin in Poly(L-lactic acid) by Supercritical Antisolvent Precipitation Constantinos G. Kalogiannis, Chrysoula M. Michailof, and Constantinos G. Panayiotou* Department of Chemical Engineering, Aristotle UniVersity of Thessaloniki, 54124 Thessaloniki, Greece

Microparticles of poly(L-lactic acid) (PLLA), loaded with the antibiotic amoxicillin, have been produced with the SEDS (solution enhanced dispersion by supercritical fluid) process. Encapsulation of amoxicillin in PLLA was initially attempted from a suspension of amoxicillin microparticles in a solution of PLLA in dichloromethane (DCM). In addition, several mixtures of dichloromethane and dimethyl sulfoxide (DMSO), in which amoxicillin and PLLA were dissolved, were employed in an attempt to encapsulate the drug in the biodegradable matrix. The effect of process parameters such as pressure, temperature, concentration of solutes, and DCM/DMSO ratio on the encapsulation efficiency was investigated. In our study, high pressures and a coaxial nozzle for the introduction of the organic solution and the supercritical antisolvent were employed, resulting in the increased mixing of the two flows. An attempt was made to explain the different encapsulation percentages obtained at different operating conditions. Introduction Controlled drug delivery from biodegradable matrixes has been a subject of great scientific interest in the past two decades. The efficacy of drugs is often compromised by their poor bioavailability, chemical instability, and poor dissolution.1 The design of drug delivery systems such as that of the biodegradable microparticle matrixes represents one of the most innovative aspects of pharmaceutical technology. Micronization and encapsulation for industrial purposes is carried out by techniques such as recrystallization from solution, spray and freeze drying, evaporation, organic antisolvent precipitation, and comminution. However, these methods have several drawbacks such as wide size distribution, high thermal and mechanical stresses, environmental pollution, large quantities of residual organic solvent, low drug loading, multistage processes, and degradation of sensitive drugs. Supercritical antisolvent processes, on the other hand, have several advantages. Tuning the process variables may control properties of supercritical antisolvents such as density, mass and energy coefficients, and solvent power. Carbon dioxide specifically is the most widely used supercritical antisolvent due to its low critical point, its low price, and its being an environmentally benign solvent. For the above reasons, supercritical fluids have been employed as solvents in the case of the RESS (rapid expansion of supercritical solutions) process,2 and as antisolvents in the cases of the GAS (gas antisolvent) and SAS (supercritical antisolvent)3 processes. Proteins have been successfully encapsulated in polymers with the SEDS (solution enhanced dispersion by supercritical fluid) process4,5 as well. Due to the low solubility of polymers in supercritical CO2, the processes of supercritical antisolvent have been the focus of interest in past years. In these processes, a solution of the polymer and the drug to be encapsulated is introduced into a high-pressure precipitation vessel, where it comes in contact with a stream of supercritical CO2. The supercritical CO2 dissolves the organic solvents of the polymer and the drug stream, causing supersaturation. The great supersaturation gradients achieved lead to the formation * To whom correspondence should be addressed. Tel.: +30-2310996223. Fax: +30-2310-996232. E-mail: [email protected].

of microparticles of the solutes. The precipitation of the polymer and the drug takes place simultaneously, leading in most cases to the encapsulation of the drug in the polymer matrix. Different types of substances have been encapsulated in polymers, namely, proteins, enzymes, and herbicides. In this work an antibiotic, amoxicillin, is encapsulated in a biodegradable matrix, poly(L-lactic acid) (PLLA). Amoxicillin is a penicillin-like antibiotic used in treatment of certain infections caused by bacteria, such as bronchitis, pneumonia, venereal disease (VD), and ear, lung, nose, urinary tract, and skin infections. It is not, however, easily soluble in water, which leads to overdosing. Moreover, the sustained release of an antibiotic such as amoxicillin may improve the effectiveness of such a drug against infections and diseases that require longterm administration. PLLA is a semicrystalline biodegradable polymer. Its monomer unit is lactic acid, which is produced in the human organism during normal metabolism. PLLA can thus be fully absorbed by the human organism after its degradation to its monomer units. This is why PLLA along with PGA (poly(glycolic acid)) and their copolymers are approved by the American and European Food and Drug Administrations. In our previous work,6 amoxicillin microparticles were produced from solutions in dimethyl sulfoxide (DMSO), ethanol (EtOH), and methanol (MeOH). The effect of the process parameters on the mean sizes and the size distributions was investigated. In continuation of the previous work, the object of the present work is to encapsulate the drug amoxicillin in PLLA microparticles. The effect of the process parameters will be investigated, and an attempt will be made to improve the drug loading and the encapsulation efficiency of the system. Materials and Methods Chemicals and Solutions. Amoxicillin was obtained from Ranbaxy Laboratories (India) (purity 99%). Poly(L-lactic acid) was purchased from Galactic Laboratories (Brussels) (Mw ) 189 000). Instrument-grade CO2 (purity ) 99.9%) was purchased from Air Liquide Mediterrane´e (Vitrolles, France). Analytical-grade dichloromethane (DCM) and dimethyl sulfoxide (purity ) 99.9%) were purchased from Riedel-de Haen (Germany). HPLC-grade water, methanol, and acetonitrile were purchased from Merck (Germany).

10.1021/ie060529q CCC: $33.50 © 2006 American Chemical Society Published on Web 11/03/2006

Ind. Eng. Chem. Res., Vol. 45, No. 25, 2006 8739 Table 1. Conditions and Results for Production of Microparticles from 0.5% w/w PLLA in DCM with the SEDS Processa T (K)

P (bar)

mean size (µm)

SD

308.15 308.15 308.15 308.15 323.15 323.15 323.15 323.15 333.15

75 150 250 350 100 150 200 250 250

1.379 1.354 1.112 1.33 1.15 1.06 1.01 1.16 1.03

0.44 0.36 0.281 0.312 0.352 0.329 0.38 0.42 0.396

a Flow rates of polymer solution and CO are 0.2 mL/min and 5 SLPM 2 (standard liters per minute), respectively.

Figure 1. Schematic diagram of the SAS apparatus: (1) precipitation chamber, (2) Milton Roy pump of the organic solution, (3) filters, (4) coaxial nozzle, (5) Tescom back pressure regulator, (6) extraction vessel, and (7) dry test flow meter.

SEDS Experiments. (A) Apparatus. The apparatus employed for the production of microparticles with the process of supercritical antisolvent has been described elsewhere.6 For convenience, a schematic layout of the process is shown in Figure 1. (B) Experimental Procedure. At the beginning of each experiment, the precipitation chamber was pressurized with CO2. The temperature was controlled and maintained constant by placing the precipitation cell (1) in an air oven. The pressure and temperature were kept constant within (0.2 bar and (0.1 K, respectively. Once steady-state conditions were reached, the organic solution containing the pharmaceutical compound and the polymer was pumped into the precipitation chamber simultaneously with the supercritical CO2, via a coaxial nozzle (4). The internal diameter of the nozzle that sprayed the organic solution was 1 mm. During the introduction of the organic solution, the solutes precipitated and could be visually observed as a fine white cloud. The sample was collected on filters (3) in powder form. The flow of organic solution was maintained for 15 min to ensure that enough material was collected for analysis. After this, the solution feed was stopped while CO2 flow was kept constant for another 40 min and under the same conditions of pressure and temperature in order to wash the formed particles and remove any residual solvent that would dissolve them upon depressurization. After washing, the vessel was gradually depressurized to atmospheric conditions and the precipitated powder was collected for analysis. Morphological Studies. Scanning electron microscopy (SEM; JEOL, Model JSM-840A) was used for characterizing the formed particles. Prior to the analysis, the samples were coated with graphite to avoid charging under the electron beam. The morphology of the microparticles was examined and related to the conditions of the experiment. Amoxicillin Loading Determination. The amoxicillin loading was determined by high-pressure liquid chromatography (HPLC). The HPLC system used was comprised of two LC10ADVP Shimadzu HPLC pumps controlled by an SCL-10AVP pump controller, a manual Rheodyne injector with 20 µL loop (USA), a column oven at 35 °C, and a UV-DAD, Model SPDM6A by Shimadzu. For data collection and area calculations, software Class-LC10 (Shimadzu) was used. The column used was ODS-2 Spherisorb, 250 mm × 4.6 mm, 5 µm (Waters). Amoxicillin was eluted using a mixture of 80% MeCN-20%

MeOH (A) and 20 mM phosphate buffer pH 2 (B) as mobile phase at a ratio of (A) 20%-(B) 80%, with 1 mL/min flow rate, and was detected at 230 nm. The powder samples of each experiment were first washed with ethanol five times to remove any surface-bound or unentrapped amoxicillin. The samples were then dried in an oven under vacuum and weighed to determine the amount of each sample. A 400 µL volume of CH2Cl2 was added to each sample to dissolve the PLLA microparticles, followed by addition of 1 mL of 20 mM phosphate buffer pH 2. The mixture was vigorously shaken for 15 min and then centrifuged. An aliquot of the aqueous phase was retrieved and injected into the HPLC system. The loading percentage (LP, %) and loading efficiency (LE, %) are defined as follows:

LP % )

mass of encapsulated amoxicillin × 100% mass of amoxicillin + mass of PLLA (1)

LE % )

actual loading percentage × 100% theoretical loading percentage

(2)

theoretical loading percentage ) mass of amoxicillin in the organic solution total mass of amoxicillin and PLLA in the organic solution × 100% (3) Results and Discussion In the first set of experiments, solutions of PLLA in CH2Cl2 were sprayed into the high-pressure vessel. The effects of pressure and temperature on the characteristics of the produced microparticles were studied. Table 1 summarizes the conditions of the experiments and the mean sizes and standard deviations of the produced particles. The mean sizes and the size distributions were calculated by measuring 350 randomly picked particles from each sample. The increase of pressure resulted in a reduction of the mean size and the standard deviation of the formed particles. This was due to the increase of the solvent power of CO2, which enabled it to extract the organic solvent faster, leading to greater supersaturation. Greater supersaturation leads to the birth of a larger number of nuclei.7 The solute is thus spent in the initial stage of nucleation, leaving a small amount of material to be spent in the growth stage of the formed nuclei. However, at 350 bar the effect of pressure was reversed, yielding microparticles with a larger mean size. This was attributed to the greater solvent power that the CO2 + DCM mixture has at high pressures. The micronization process is characterized by two antagonistic phenomena. On one hand, greater pressures result in greater solvent powers of the carbon dioxide, causing greater supersaturations and therefore leading to smaller particles. On

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Figure 2. SEM image of PLLA microparticles produced at 75 bar and 308.15 K from a 0.5% w/w solution in DCM.

Figure 4. SEM image of PLLA microparticles produced at 250 bar and 323.15 K from a 0.5% w/w solution in DCM.

Figure 3. SEM image of PLLA microparticles produced at 100 bar and 323.15 K from a 0.5% w/w solution in DCM.

Figure 5. SEM image of PLLA microparticles produced at 250 bar and 333.15 K from a 0.5% w/w solution in DCM.

the other hand, an increase in pressure leads to the increase of solubility of PLLA in the mixed solvent of carbon dioxide and dichloromethane, which leads to the decrease of the achieved supersaturation. Above a certain pressure the decrease in supersaturation due to the greater solubility of PLLA may lead to particles with larger mean sizes and broader distributions. Moreover, an increase in the pressure of CO2 may cause a great drop in the glass transition temperature,8 leading to coalescence of the formed nuclei and particles.9,10 The same effect was observed here with an increase in temperature. At higher temperatures the particles tend to coalesce more. Figures 2-5 present the effect of increasing pressure and temperature on the PLLA microparticles. It is clear that an increase in pressure and temperature causes the produced particles to plasticize, an effect that can be very advantageous in the case of the encapsulation of a drug. Amorphous polymers have been reported to encapsulate pharmaceuticals in greater percentages.11,12 Amorphous polymers, however, are difficult to precipitate in discrete well-defined microparticles because of the tendency of supercritical CO2 to dissolve in them. A semicrystalline polymer such as PLLA may therefore be the ideal biodegradable matrix for the encapsulation process, especially if it is precipitated in a way that facilitates the incorporation of drugs in its amorphous regions. To further understand the effect of the antisolvent process on the characteristics of the produced microparticles, PLLA was precipitated

from a 50/50 v/v DCM/DMSO mixture of the two solvents. Liquid CO2 could not fully extract DMSO, resulting in highly aggregated particles. Supercritical CO2 could extract both solvents, but the formed particles had a tendency to coalesce. A similar observation was made by Elvassore et al.13 Figures 6 and 7 present SEM images of particles formed with subcritical and supercritical CO2. Suspensions of Amoxicillin Microparticles in PLLA Solutions in DCM. Based on preliminary experiments for PLLA and amoxicillin,6 the encapsulation of amoxicillin in PLLA was attempted. To ascertain the ability of PLLA to encapsulate amoxicillin, a suspension of amoxicillin microparticles in a 1 w/v % solution of PLLA in DCM was created. The amoxicillin microparticles had been precipitated with the aid of supercritical CO2 and had a mean size of 0.5 µm. Amoxicillin microparticles were added to the above solution, yielding a suspension with an amoxicillin/PLLA ratio ) 1/5. Previous experiments with PLLA and amoxicillin have shown that their mean sizes are between 0.7 and 2 µm and 0.3 and 0.8 µm, respectively.14 PLLA microparticles can therefore incorporate amoxicillin microparticles from a dimensional point of view since PLLA particles have larger diameters. Table 2 summarizes the conditions of the experiments and the loading percentages and loading efficiencies of the produced particles. Encapsulation was successful over a range of pressures and temperatures. The suspended particles of amoxicillin assisted

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Figure 6. SEM image of particles produced at 100 bar and 302.15 K from a 0.25 w/w % solution of PLLA in a 50/50 v/v DCM/DMSO mixture.

Figure 7. SEM image of particles produced at 100 bar and 308.15 K from a 0.25 w/w % solution of PLLA in a 50/50 v/v DCM/DMSO mixture. Table 2. Conditions and Results for Amoxicillin Encapsulation with the SEDS Process from a Suspension of Amoxicillin Microparticles (Amoxicillin/PLLA Ratio ) 1/5) in 1 w/v % PLLA in DCM Solutiona T (K)

P (bar)

LP (%)

LE (%)

302.15 302.15 313.15 313.15 323.15

100 250 100 250 250

5.4 6.4 4.1 4 4.4

32.5 38.5 24.7 24.1 26.5

a Flow rates of organic solution and CO are 0.1 mL/min and 5 SLPM 2 (standard liters per minute), respectively.

the formation of PLLA nuclei, resulting in high supersaturation. The nuclei of PLLA were formed on the suspended particles, encapsulating them during their growth. Higher encapsulation percentages were observed when liquid CO2 (302.15 K, 250 bar) was employed, due to the greater supersaturation achieved. Solutions of Amoxicillin and PLLA in Mixtures of DCM/ DMSO. Having ascertained that PLLA can incorporate amoxicillin, solutions of PLLA and amoxicillin in mixtures of DCM and DMSO were studied. Table 3 summarizes the solutions prepared, the conditions employed, and the loading percentages and efficiencies of the produced particles. The first set of experiments (runs 1-5) was characterized by a mixture rich in DCM and PLLA. In Figure 8 an SEM image of the produced microparticles is shown. The PLLA

particles produced were much like those produced from DCM solutions of PLLA. The DCM/DMSO ratio is high enough (80/ 20, v/v) that liquid CO2 extracts both solvents successfully. However, the loading percentages were low, especially at low pressures and high temperatures. It seems that, at these conditions, amoxicillin is either poorly extracted or precipitated as separate particles. Because the mixture of solvents was rich in DCM, the precipitation process was controlled mostly by the precipitation of the PLLA particles, resulting in low loading percentages and efficiencies. It should be noted at this point that the ideal solvent mixture and conditions of precipitation would be such that it would result in the simultaneous birth of amoxicillin nuclei and growth of PLLA nuclei. In this case, the amoxicillin nucleation could occur on previously formed PLLA nuclei, enhancing its incorporation in the polymer matrix. A different ratio of solvents was thus considered, one that shifted the control of the microparticle precipitation toward amoxicillin. Runs 6-10 employed a 50/ 50 v/v ratio of DCM/DMSO. It is easily noted that increasing the ratio of DMSO in the mixture of organic solvents had a dramatic effect. First, the loading percentage at low temperatures (302.15 K) dropped along with the loading efficiency. At high temperatures, however (323.15 K), the loading percentage increased. This was due to the inability of liquid CO2 to fully extract DMSO and, therefore, to fully precipitate the dissolved amoxicillin from the solution. DMSO is a much more difficult solvent to be extracted compared to DCM. Elvassore et al.13 have reported that, in the case of a mixture of two liquid solvents, the thermodynamic properties of the mixture tend to be closer to those of the low expandable liquid, in this case DMSO. DMSO has a high boiling point (462.15 K) in contrast to DCM, which has a low boiling point (313.15 K). An increase of temperature has an effect on the diffusion of both DCM and DMSO from the organic solution to the supercritical CO2 rich environment. Higher temperatures and pressures result in a better extraction especially of DMSO, leading to higher supersaturation, which in turn leads to the formation of a multitude of small nuclei of amoxicillin, increasing its incorporation in PLLA. Runs 11-19 were characterized by a DCM/DMSO v/v ratio equal to 20/80. The bulk of the liquid mixture of organic solvents was DMSO and its expansion was similar to that of pure DMSO. The first set of experiments, featuring runs 11-13, employed amoxicillin and polymer concentrations of 2 g/L DCM/DMSO mixture. To incorporate amoxicillin in PLLA, a high supersaturation must be achieved for PLLA. A high supersaturation ratio ensures that PLLA precipitates first. During the stage of the birth of PLLA nuclei, the precipitation of amoxicillin must be held off so that it coincides with the growth of the aforementioned nuclei. Both these requirements are met with the increase of the DMSO ratio in the liquid mixture. DMSO does not dissolve PLLA and, thus, a mixture of 20/80 v/v DCM/ DMSO has a low solvent power for PLLA. This leads to a fast precipitation of PLLA. It is clear from Table 3 that the increase of DMSO has a beneficial effect, leading to the increase of the loading percentages and efficiencies. Figure 9 presents an SEM image of the produced microparticles. It is evident that the particles are strongly coalesced, as was the case in the production of PLLA microparticles presented in Figure 7. The coalescence of the particles can be attributed to two main factors. First, the increase of pressure causes a drop in the glass transition temperature (Tg) of the polymer. It has been reported that CO2 may cause a drop in the Tg of polymers up to 230 K.8 Thus, CO2 plasticizes the polymer matrix, assisting the incor-

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Table 3. Summary of Encapsulation Experiments with the SEDS Processa run

amoxicillin concn (g/L)

polym concn (g/L)

1 2 3 4 5

1 1 1 1 1

8 8 8 8 8

DCM/DMSO (80/20, v/v) 302.15 302.15 313.15 323.15 323.15

6 7 8 9 10

1 1 1 1 1

5 5 5 5 5

11 12 13 14 15 16 17 18 19

2 2 2 4 4 4 1 1 1

2 2 2 2 2 2 1 1 1

a

T (K)

P (bar)

LP (%)

LE (%)

SD for LP (%)

100 200 200 100 200

4.3 2.52 0.22 0.1 0.41

38.7 22.7 1.98 0.9 3.7

0.1 0.04 0.01 0 0.01

DCM/DMSO (50/50, v/v) 302.15 302.15 313.15 323.15 323.15

100 200 100 200 100

3.18 1.55 3.15 1.62 5.92

11.1 5.4 11 5.7 20.6

0.1 0.1 0.1 0.1 0.1

DCM/DMSO (20/80, v/v) 313.15 313.15 323.15 313.15 313.15 323.15 313.15 313.15 323.15

100 200 200 100 200 200 100 200 200

8.95 12.88 17.51 7.07 15.42 20.38 10.3 16.21 21.46

17.9 25.76 35 10.6 23.1 30.6 20.6 32.4 42.9

0.5 0.82 1.2 0.51 1.2 1.83 0.84 1.47 1.9

Flow rates of the organic solution and the CO2 were 0.1 mL/min and 5 SLPM, respectively.

Figure 8. SEM image of PLLA particles incorporating amoxicillin that precipitated from a 80/20 v/v DCM/DMSO ratio, run 1 of Table 3.

Figure 9. SEM image of PLLA particles incorporating amoxicillin that precipitated from a 20/80 v/v DCM/DMSO ratio, run 13 of Table 3.

poration of amoxicillin. The Tg of PLLA is between 333.15 and 337.15 K.15,16 Thus, an increase in temperature will affect the PLLA microparticles formed. Therefore, precipitation at high

pressures and temperatures leads to coalescing particles, as was pointed out through Figures 2-5. It is noticed that a liquid mixture rich in DMSO favors greater loading percentages. To further increase the loading percentages and efficiencies, runs 14-16 were conducted. In these runs, the amoxicillin concentration was doubled to 4 g/L, while the polymer concentration was maintained constant at 2 g/L. The loading percentages were increased, indeed, in these runs. However, the loading efficiencies decreased, revealing that a greater amoxicillin concentration in the liquid mixture may not necessarily be the best choice. The increase of amoxicillin causes an increase in the mean size of the produced particles15 since more material is spent during the growth stage of precipitation. This results in more amoxicillin precipitating outside or on the surface of the polymer matrix, leading to lower loading efficiencies. Moreover, an increase in amoxicillin concentration shifts the formation of amoxicillin nuclei closer in time to the formation of PLLA nuclei. In this manner, instead of simultaneous nucleation of amoxicillin and growth of PLLA nuclei, simultaneous nucleation of both solutes takes place, resulting in the increase of amoxicillin particles that precipitate outside the polymer matrix. To ascertain the validity of the above hypothesis, runs 1719 were conducted. In these runs, both, the polymer and the amoxicillin concentrations were 1 g/L. Thus, the amoxicillin/ PLLA ratio was equal to that of runs 11-13, while the absolute concentrations of the solutes were decreased by 50% compared to those of runs 11-13. Both loading percentages and efficiencies were increased in these runs. Decreasing amoxicillin concentration causes the precipitated material to be spent mainly in the stage of nucleation, which leads to the production of many and small in size nuclei. These nuclei may be more easily incorporated in the polymer matrix. This results in a noticeable increase in loading percentages and efficiencies. At 200 bar and 323.15 K the loading percentages and efficiencies were 21.46% and 42.9%, respectively. It should be mentioned that encapsulation from the 20/80 v/v DCM/DMSO mixture was attempted at 100 bar and 323.15 K. However, DMSO could not be fully extracted at these conditions, leading to the formation of droplets at the bottom of the high-pressure cell. Figures 10 and 11 present the loading percentages and efficiencies, respectively, of runs

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addressed.6 Encapsulation of amoxicillin in PLLA was then attempted first from a suspension of amoxicillin microparticles in a solution of PLLA in DCM to ascertain the ability of PLLA to incorporate amoxicillin. Coprecipitation of the drug and polymer took place at various pressures and temperatures and from various solutions. The effects of pressure, temperature, and solvent ratio were investigated, and the increase of the loading percentages and efficiencies are reported. This study could contribute to properly appreciating the potential of the SEDS process for the encapsulation of active substances in polymer matrixes. Acknowledgment Financial support for this work has been provided by Greek GSRT/PENED 2001. Literature Cited

Figure 10. Loading percentages of amoxicillin in PLLA, precipitated from solutions of 20/80 v/v DCM/DMSO ratio.

Figure 11. Loading efficiencies of amoxicillin in PLLA, precipitated from solutions of 20/80 v/v DCM/DMSO ratio.

11-19. It should be noted that good reproducibility was noted for the majority of the experiments. All encapsulation experiments were done twice. Conclusions In this work, the encapsulation of amoxicillin in PLLA microparticles via an SEDS process was studied. A preliminary study of the precipitation of PLLA particles at various conditions and from DCM and DCM/DMSO solvents took place in order to clarify the effect of the external parameters on the efficiency of the SEDS process. In addition, the effect of the SEDS process parameters on the precipitation of amoxicillin particles had been

(1) Otsuka, M.; Kaneniwa, N. Effect of grinding on the crystallinity and chemical stability in the solid state of cephalothin sodium. Int. J. Pharm. 1990, 62, 65. (2) Matson, D. W.; Fulton, J. L.; Petersen, R. C.; Smith, R. D. Rapid expansion of supercritical fluid solution: solute formation of powders, thin films and fibers. Ind. Eng. Chem. Res. 1987, 26, 2298. (3) Taki, S.; Badens, E.; Charbit, G. Controlled release system formed by supercritical anti-solvent coprecipitation of a herbicide and a biodegradable polymer. J. Supercrit. Fluids 2001, 21, 61. (4) Yeo, S. D.; Lim, G. B.; Debenedetti, P. G.; Bernstein, H. Formation of microparticulate protein powders using a supercritical fluid antisolvent. Biotechnol. Bioeng. 1993, 41, 341. (5) Winters, M. A.; Knuston, B. L.; Debenedetti, P. G.; Sparks, H. G.; Przybycien, T. M.; Stevenson, C. L.; Prestrelski, S. J. Precipitation of protein in supercritical carbon dioxide. J. Pharm. Sci. 1996, 85, 586. (6) Kalogiannis, C. G.; Pavlidou, E.; Panayiotou, C. G. Production of Amoxicillin Microparticles by Supercritical Antisolvent Precipitation. Ind. Eng. Chem. Res. 2005, 44, 9339. (7) Shekunov, B. Yu.; Hanna, M.; York, P. Crystallization process in turbulent supercrtial flows. J. Crystal Growth 1999, 198/199, 1345. (8) Condo, P.; Paul, D.; Johnston, K. Glass Transitions of Polymers with Compressed Fluid Diluents: Type II and III Behavior. Macromolecules 1994, 27, 365. (9) Bodmeier, K.; Wang H.; Dixon, D. J.; Mawson, S.; Johnston, K. P. Polymeric Microspheres Prepared by Spraying into Compressed Carbon Dioxide. Pharm. Res. 1995, 12, 1211. (10) Dixon, D. J.; Johnston, K. P. Polymeric Materials Formed by Precipitation with a Compressed Fluid Antisolvent. AlChE J. 1993, 39, 127. (11) Engwicht, A.; Girreser, U.; Muller, B. W. Critical properties of lactide-co-glycolide for the use in microparticle preparation by the aerosol solvent extraction system. Int. J. Pharm. 1999, 185, 61. (12) Ghaderi, R.; Artursson, P.; Carlfors, J. A new method for preparing biodegradable microparticles and entrapment of hydrocortisone in DL-PLG microparticles using supercritical fluids. Eur. J. Pharmacol. Sci. 2000, 10, 1. (13) Elvassore, N.; Bertucco, A. Cacileti P. Production of protein-loaded polymeric microcapsules by compressed CO2 in a mixed solvent. Ind. Eng. Chem. Res. 2001, 40, 795. (14) Kalogiannis, K. Use of supercritical fluids for the treatment of biodegradable polymers and pharmaceutical substances. Ph.D. Thesis. Thessaloniki, Greece, 2006. (15) Ray, S. S.; Yamada, K.; Okamoto M.; Ueda, K. New polylactidelayered silicate nanocomposites. 2. Concurrent improvements of material properties, biodegradability and melt rheology. Polymer 2003, 44, 857. (16) Sato, Y.; Inohara, K.; Takishima, S.; Masuoka, H.; Imaizumi, M.; Yamamoto, H.; Takasugi, M. Pressure-volume-temperature behavior of polylactide, poly(butylenes succinate), and poly(butylenes succinate-coadipate). Polymer 2000, 40, 2602.

ReceiVed for reView April 26, 2006 ReVised manuscript receiVed September 9, 2006 Accepted September 20, 2006 IE060529Q