Nanosized Paclitaxel Particles from Supercritical Carbon Dioxide

Jan 23, 2007 - predominantly micron-sized particles.15-17 Recently, Sun and co-workers made a ... Temple University School of Medicine. (1) (a) Rowins...
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Nanosized Paclitaxel Particles from Supercritical Carbon Dioxide Processing and Their Biological Evaluation Pankaj Pathak,† Gaddamanugu L. Prasad,*,‡ Mohammed J. Meziani,† Attalla A. Joudeh,† and Ya-Ping Sun*,† Department of Chemistry and Laboratory for Emerging Materials and Technology, Hunter Hall, Clemson UniVersity, Clemson, South Carolina 29634, and Department of Physiology, Temple UniVersity School of Medicine, Philadelphia, PennsylVania 19140 ReceiVed September 19, 2006. In Final Form: December 6, 2006 The rapid expansion of a supercritical solution into a liquid solVent (RESOLV) technique with benign supercritical carbon dioxide was applied to obtain aqueous suspended nanoparticles of the highly potent anticancer drug paclitaxel. The paclitaxel nanoparticles were protected from agglomeration by using a known nontoxic stabilization agent. The aqueous suspended paclitaxel nanoparticles of different average particle sizes were evaluated in vitro against human breast cancer cells. The results suggest that the nanosized paclitaxel particles are effective, with an antineoplastic activity comparable to that of the commercial paclitaxel formulation. The technique should be generally applicable to the processing of nanoparticles from other important drugs with aqueous solubility problems.

Introduction Paclitaxel is one of the best antineoplastic drugs, with excellent therapeutic efficacy against a wide spectrum of cancers, especially ovarian, breast, lung, colon, head, and neck cancers.1 However, paclitaxel is practically insoluble in water, which represents a significant limitation of the drug.2,3 In current clinical administration, paclitaxel is delivered in a vehicle composed of Cremophor EL (polyethoxylated castor oil) and dehydrated alcohol.2-4 These reagents cause serious side effects (hypersensitivity reaction, nephrotoxicity, neurotoxicity, cardiotoxicity, etc.).2,4 Alternative paclitaxel formulations without the use of Cremophor EL have been pursued, especially those based on colloidal nanoparticle carriers.5-10 Among more promising recently developed formulations are the paclitaxel albumin-bound * Corresponding author. E-mail: [email protected] (Y.-P.S.); [email protected] (G.L.P.). † Clemson University. ‡ Temple University School of Medicine. (1) (a) Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. J. Natl. Cancer Inst. 1990, 82, 1247. (b) Lopes, N. M.; Adams, E. G.; Pitts, T. W.; Bhuyan, B. K. Cancer Chemother. Pharmacol. 1993, 32, 235. (c) Thigpen, J. P. Semin. Oncol. 2001, 27, 11. (2) Weiss, R. B.; Donehower, R. C.; Wiernik, P. H.; Ohnuma, T.; Gralla, R. J.; Trumph, D. L.; Baker, J. R.; Vanecho, D. A.; Vonhoff, D. D.; Leylandjones, B. J. Clin. Oncol. 1990, 8, 1263. (3) Panchagnula, R. Int. J. Pharm. 1998, 172, 1. (4) Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Eur. J. Cancer 2001, 37, 1590. (5) (a) Tarr, B. D.; Sambandan, T. G.; Yalkowsky, S. H. Pharm. Res. 1987, 4, 162. (b) Sharma, A.; Straubinger, R. M. Pharm. Res. 1994, 11, 889. (c) Sharma, U.; Balasubramanian, S. V.; Straubinger, R. M. J. Pharm. Sci. 1995, 10, 1223. (d) Kan, P.; Chen, Z. B.; Lee, C. J.; Chu, I. M. J. Controlled Release 1999, 58, 271. (6) (a) Feng, S. S.; Huang, G. F. J. Controlled Release 2001, 71, 53. (b) Liggins, R. T.; Burt, H. M. AdV. Drug DeliVery ReV. 2002, 54, 191. (c) Koziara, J. M.; Lockman, P. R.; Allen, D. D.; Mumpher, R. J. J. Controlled Release 2004, 99, 259. (7) (a) Singla, K.; Garg, A.; Aggarwal, D. Int. J. Pharm. 2002, 235, 179. (b) Zamboni, W. C. Clin. Cancer Res. 2005, 11, 8230. (c) Hennenfent, K. L.; Goindan, R. Ann. Oncol. 2006, 17, 735. (8) Ibrahim, N. K.; Desai, N.; Legha, S.; Soon-Shiong, P.; Theriault, R. L.; Rivera, E.; Esmaeli, B.; Ring, S. E.; Bedikian, A.; Hortobagyi, G. N.; Ellerhorst, J. A. Clin. Cancer Res. 2002, 8, 1038. (9) Takimoto, C. H.; Schwartz, G.; Romero, O. Patnaik, A.; Tolcher, A.; Garrison, M.; Oldham, F.; Bernareggi, A.; Rowinsky, E. Proc. Am. Soc. Clin. Oncol. 2005, 23, 145. (10) Dordunoo, S. K.; Vineck, W.; Hoover, R.; Dang, W. Proc. Am. Assoc. Cancer Res. 2005, 46, 985.

nanoparticle suspension ABI-007 (Abraxane),8 poly-(L)-glutamic acid-paclitaxel conjugates (Xyotax),9 and paclimer (microsphere formulation of paclitaxel).10 The nanosizing of drug particles has been identified as a highly promising approach in the formulation of water-insoluble drugs.11,12 Various traditional and emerging techniques,11,13 including those based on supercritical fluid processing technology,14 have been developed or investigated for such a purpose. However, the classical supercritical fluid particle formation methods, such as supercritical anti-solvent (SAS) and rapid expansion of supercritical solutions (RESS), generally produce predominantly micron-sized particles.15-17 Recently, Sun and co-workers made a simple but significant modification to the traditional RESS by using a liquid at the receiving end of the supercritical fluid expansion, or rapid expansion of a supercritical solution into a liquid solVent (RESOLV).18,19 The RESOLV method has been used to generate exclusively nanoscale particles from a variety of materials, including inorganic, organic, and polymeric compounds.19-21 The specific experimental configuration of RESOLV with rapid expansion into a liquid-receiving (11) (a) Muller, R. H.; Jacobs, C.; Kayser, O. AdV. Drug DeliVery ReV. 2001, 47, 3.(b) Muller, R. H.; Cornella, M. K. J. Biotechnol. 2004, 113, 151. (12) Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R. Eur. J. Pharm. Sci. 2003, 18, 113. (13) (a) Stella, V.; Rajewski, R. Pharm. Res. 1997, 14, 556. (b) Nakano, M. AdV. Drug. DeliVery ReV. 2000, 45, 1. (c) Leuner, C.; Dressman, J. Eur. J. Pharm. Biopharm. 2000, 50, 47. (14) (a) Tom, J. W.; Debenedetti, P. G. J. Aerosol. Sci. 1991, 22, 555. (b) York, P. Pharm. Sci. Tech. Today 1999, 2, 430. (c) Reverchon, E.; Della-Porta, G. Pure Appl. Chem. 2001, 73, 1293. (d) Jung, J.; Perrut, M. J. Supercrit. Fluids 2001, 20, 179. (15) Helfgen, B.; Turk, M.; Schaber, K. Powder Technol. 2000, 110, 22. (16) Ginosar, D. M.; Swank, W. D.; Mcmurtery, R. D.; Carmack, W. J. Flowfield studies of the RESS process. Proceedings of 5th International Symposium on Supercritical Fluids, April 8-12, Atlanta, 2000. (17) Weber, M.; Thies, M. C. In Supercritical Fluid Technology in Materials Science and Engineering: Synthesis, Properties, and Applications; Sun, Y.-P., Ed.; Marcel Dekker: New York, 2002; p 387. (18) Sun, Y.-P.; Rollins, H. W. Chem. Phys. Lett. 1998, 288, 585. (19) Sun, Y.-P.; Rollins, H. W.; Bandara, J.; Meziani, M. J.; Bunker, C. E. In Supercritical Fluid Technology in Materials Science and Engineering: Synthesis, Properties, and Applications; Sun, Y.-P., Ed.; Marcel Dekker: New York, 2002; p 491. (20) Meziani, M. J.; Pathak, P.; Hurezeanu, R.; Thies, M. C.; Enick, R. M.; Sun, Y.-P. Angew. Chem., Int. Ed. 2004, 43, 704. (21) Sun, Y.-P.; Meziani, M. J.; Pathak, P.; Qu, L. Chem.sEur. J. 2005, 11, 1366.

10.1021/la062739d CCC: $37.00 © 2007 American Chemical Society Published on Web 01/23/2007

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Figure 1. Experimental RESOLV setup. Shown in the photo is an aqueous suspension of stabilized paclitaxel nanoparticles.

solution is particularly amenable to the preparation of nanosized drug particles suspended in an aqueous solution,22,23 which is compatible with commonly employed drug administration systems. In order to demonstrate the potential of RESOLV as a nanosizing method with a real and important drug and the fact that the biological consequences of the processing are mostly beneficial, here we report the nanosizing of paclitaxel via RESOLV with benign supercritical solvent carbon dioxide to obtain exclusively aqueous suspended drug nanoparticles. The paclitaxel nanoparticles were protected from agglomeration by using a stabilization agent. The aqueous suspended paclitaxel nanoparticles of different average particle sizes were evaluated in vitro against human breast cancer cells. The results suggest that the nanosized paclitaxel particles are effective, with an antineoplastic activity comparable to that of the commercial paclitaxel formulation. Experimental Section Materials. Paclitaxel and poly(N-vinyl-2-pyrrolidone) (PVP; Mw of 40 000 and 360 000) were purchased from Sigma. Carbon dioxide (high-purity SCF grade) was obtained from Air Products. All other chemicals and reagents of analytical grades were used as supplied. Water was deionized and purified by being passed through a Labconco WaterPros water purification system. Human breast adenocarcinoma cells (MDA-MB-231) were procured from American Type Culture Collection (ATCC, Rockville, MD). The cell culture media were purchased from Invitrogen Corp. The solution of paclitaxel in ethanol for bioassay was prepared in 1 mg/mL concentration and stored at 4 °C. RESOLV and Particle Characterization. The RESOLV setup for nanosizing paclitaxel particles was similar to what was reported previously (Figure 1).22 In a typical experiment, paclitaxel (2 mg) was added to the syringe pump, followed by the filling of CO2 to form a solution. The solution was pushed through the heating unit (a cylindrical solid copper block tightly wrapped with a stainless steel tubing coil) to reach the desired supercritical temperature of 40 °C before the expansion nozzle. The nozzle was a laser-drilled orifice (50 µm in inner diameter and an aspect ratio of 5), one end of which was attached to the pre-expansion section of the tubing, and the other end was inserted into the collection chamber containing the ambient receiving solution. The rapid expansion was carried out (22) Pathak, P.; Meziani, M. J.; Desai, T.; Sun, Y.-P. J. Am. Chem. Soc. 2004, 126, 10842. (23) Pathak, P.; Meziani, M. J.; Sun, Y.-P. Expert Opin. Drug DeliVery 2005, 2, 747.

at a pre-expansion pressure of 310 bar. The receiving solution was either neat water or an aqueous solution containing a stabilization agent, depending on the targeted nanoparticle properties. Differential scanning calorimetry (DSC) was performed on a Mettler-Toledo DSC820 system. Typically, a carefully weighed sample (10 mg) under nitrogen atmosphere was scanned over a temperature range of 25-300 °C at a heating rate of 5 °C/min. The instrumental temperature and heat flow were calibrated with standard indium samples. Powder X-ray diffraction (XRD) measurements were carried out on a Scintag XDS-2000 powder diffraction machine with a Cu KR source. The scan of diffraction angle 2θ was over the range of 5-90° at a rate of 0.04° per second. Scanning electron microscopy (SEM) images were obtained on a Hitachi S4800 FieldEmission SEM system. The specimen for the SEM imaging was prepared by depositing a few drops of a dilute aqueous suspension of the nanoparticles onto a carbon tape, followed by drying under ambient conditions. The specimen was coated with platinum to minimize charging effects. The particle sizes were obtained from the SEM images by manually identifying and measuring the particle spots. A collection (typically about 200 particles) of the thus determined particle sizes was used for the statistical analysis to yield the average particle size and size distribution results. In Vitro Cytotoxicity Assay. Actively growing MDA-MB-231 cells (10 000-20 000) were plated in 12-well cluster dishes in regular growth media and allowed to adhere overnight. For paclitaxel nanoparticles of 38 or 530 nm in average size, the particle suspension was sonicated in a water bath, diluted directly into cell growth medium, and added to the cells at a desired paclitaxel concentration. The same procedures were used with a paclitaxel solution in ethanol at an equimolar concentration. In assays for all three samples, cells were harvested at various time points from 4 h of plating to 72 h in culture. Cells treated without any additions were used as controls. At the time of harvest, the growth medium was replaced and rinsed with phosphate-buffered saline (PBS). Cells were fixed and stained in 0.5% crystal violet in 50% ethanol at room temperature for 15 min, washed to remove excess dye, and allowed to dry. The dye was extracted with 50% ethanol for 15 min, and the absorbance was recorded at 540 nm. Confocal Microscopy and Flow Cytometry. MDA-MB-231cells were plated in Nunc chamber slides and allowed to attach overnight. After the treatment with paclitaxel nanoparticles (38 or 530 nm in average size) or the paclitaxel ethanol solution (10 µM paclitaxel concentration for all three samples) for 6-24 h, or no treatment as control, the cells were fixed with paraformaldehyde and stained for microfilaments with an anti-actin antibody followed by a rhodamineconjugated antibody, and then reacted with DAPI to stain nuclei (DNA). Samples were mounted using a Prolong Antifade kit

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Figure 2. XRD patterns of paclitaxel samples before (top) and after (bottom) the RESOLV processing. (Molecular Probes) for imaging on a Zeiss LSM 510 confocal microscope.24 For flow cytometric studies, the drug-treated (for 3-24 h) cells were trypsinized and fixed in 70% ethanol for at least 24 h. Cell pellets were resuspended in PBS (100-150 mL), stained with propidium iodide, and briefly treated with RNAse for analyses on a BD FACS Star Plus flow cytometer. Gated populations of cells in the G0-G1, S, and G2-M phases of the cell cycle as well as those in the subG1 and hypertetraploidy states (>4N DNA) were calculated.24

Results and Discussion Paclitaxel is practically insoluble in water, but has some solubility in supercritical CO2. According to results available in the literature,25 the estimated paclitaxel solubility in CO2 is about 0.03-0.04 mg/mL under our experimental conditions of 40 °C and 310 bar. The solubility was adequate for the nanosizing of paclitaxel particles via RESOLV. Nanosizing Paclitaxel Particles. In a RESOLV experiment, a solution of paclitaxel in CO2 was prepared in the syringe pump (Figure 1), and the solution was pushed through the heating unit to reach the desired supercritical temperature (40 °C) before the expansion nozzle. The rapid expansion (at a pressure of 310 bar) was into a solution instead of ambient air, as found in traditional RESS experiments. Here, the aqueous receiving solution plays two major functions. One is to interfere with the particle agglomeration process in the expansion jet. Experimental and computational results available in the literature suggest that, in traditional RESS, there are both nanoscale and micron-sized particles in the expansion jet, with the latter being a result of efficient condensation and coagulation and becoming dominant in the end product.16,17 Thus, the expansion into a solution instead of air is critical to keeping the drug particles exclusively at the nanoscale.22,23 The other function of the aqueous receiving solution is to provide a desirable medium for the suspension of the nanosized drug particles as they form. The RESOLV process produces exclusively nanoscale drug particles, but the resulting suspension of drug nanoparticles is subject to the kind of aggregation phenomenon found in many other colloidal particle suspensions. Stabilization agents are often (24) Bharadwaj, S.; Hitchcock-DeGregori, S.; Thorburn, A.; Prasad, G. L. J. Biol. Chem. 2004, 279, 14039. (25) (a) Vandana, V.; Teja, A. S. Fluid Phase Equilib. 1997, 135, 83. (b) Nalesnik, C. A.; Hansen, B. N.; Hsu, J. T. Fluid Phase Equilib. 1998, 146, 315. (c) Suleiman, D.; Estevez, L. A.; Pulido, J. C.; Garcia, J. E.; Mojica, C. J. Chem. Eng. Data 2005, 50, 1234.

Figure 3. An SEM image of nanosized paclitaxel particles (average size 530 nm) from RESOLV with PVP40K protection.

used to protect the colloidal suspensions. For the RESOLVprocessed paclitaxel nanoparticles suspended in water, there was typical aggregation of the nanoparticles, although the aggregates still exhibited different properties from those of the bulk paclitaxel. For example, according to DSC analyses of the aggregated paclitaxel nanoparticle sample, there was a 10 °C decrease in the melting point from that of bulk paclitaxel. The lower melting point is probably an indication of reduced crystallinity in the nanosized drug particles (despite the aggregation). Shown in Figure 2 is a comparison for the XRD patterns of paclitaxel samples before and after the RESOLV processing. The weaker and broader peaks for the processed sample are consistent with reduced crystallinity. A well-known polymeric stabilization agent, PVP, with molecular weight Mw ∼ 40 000 (PVP40K) was used to protect the aqueous suspended paclitaxel nanoparticles from aggregation. The agent was added to the receiving solution in RESOLV, that is, the supercritical solution rapid expansion was into an aqueous PVP40K solution. (0.33 mg/mL). It should be pointed out that PVP is benign in use with drugs26 and is classified as nontoxic according to pharmaceutical guidelines.27 There have also been (26) (a) Torchilin, V. P.; Shtilman, M. I.; Trubetskoy, V. S.; Whiteman, K.; Milstein, A. M. Biochim. Biophys. Acta 1994, 1195, 181. (b) Kamada, H.; Tsutsumi, Y.; Yamamoto, Y.; Kihira, T.; Kaneda, Y.; Mu, Y.; Kodaira, H.; Tsunoda, S.-I.; Nakagawa, S.; Mayumi, T. Cancer Res. 2000, 60, 6416. (c) D’souza, A. J. M.; Schowen, R. L.; Topp, E. M. J. Controlled Release 2003, 94, 91. (d) Le Garrec, D.; Gori, S.; Luo, L.; Lessard, D.; Smith, D. C.; Yessine, M. A.; Ranger, M.; Leroux, J. C. J. Controlled Release 2004, 99, 83. (e) Sharma, D.; Chelvi, T. P.; Kaur, J.; Chakravorty, K.; De, T. K.; Maltra, A.; Ralhan, R. Oncol. Res. 1996, 8, 281. (27) (a) Kibbe, A. H. Handbook of Pharmaceutical Excipients, 3rd ed.; AphA and PhP: Washington, DC, 2000. (b) Osol, A., Hoover, J. E., et al., Eds.; Remingtons Pharmaceutical Sciences, Mack Publishing Co.: Easton, PA, 1975.

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Figure 5. Cytotoxicity assays of the paclitaxel nanoparticles (smaller 38 nm and larger 530 nm average sizes) and the paclitaxel ethanol solution at equimolar concentration (0.5 µM) against MDA-MB231 cells (normalized to the cell growth of the control after a 72 h culture), with significant inhibition in comparison with the controls (p values < 0.05). Error bars indicate standard deviation, and the data points are offset slightly on purpose for easier viewing.

Figure 4. SEM images of nanosized paclitaxel particles from RESOLV with the protection of higher-molecular-weight PVP360K (top, average size 200 nm) and higher-concentration PVP40K (bottom, average size 38 nm).

many examples for the use of PVP to enhance drug delivery and performance.26 The RESOLV into the aqueous PVP40K solution yielded a stable suspension of paclitaxel nanoparticles that appeared homogeneous and solution-like (see the picture in Figure 1) for an extended period of time (at least months). According to SEM imaging of the sample (Figure 3), the paclitaxel nanoparticles thus prepared are 530 nm in average diameter (about 85 nm in size distribution standard deviation). Varying Particle Sizes. In RESOLV processing of drugs, the resulting particle sizes could be altered by varying experimental parameters.23 In this work, the stabilization agent was present in the receiving solution during the rapid expansion, so its effect was beyond the protection of the final drug nanoparticles from aggregation. Rather, its presence should also affect the nanoparticle formation in RESOLV, contributing to the likely complicated overall process that determines particle sizes in the end product. With PVP as the stabilization agent, the parameters including the PVP molecular weight and its concentration in the receiving solution were exploited to obtain paclitaxel nanoparticles of different average particle sizes. The use of a higher PVP40K concentration in the receiving solution resulted in smaller paclitaxel nanoparticles. For example, at the PVP40K concentration of 1 mg/mL while other experimental parameters were kept the same, the paclitaxel nanoparticles thus prepared were 38 nm in average diameter (Figure 4), with a size distribution standard deviation of about 9 nm. The resulting aqueous nanoparticle suspension was equally stable. These

particles appear less spherical, for which a mechanistic explanation is not available. One possibility might be that the particle shape is affected by competition between the particle growth and the hindrance associated with the higher PVP concentration. Higher molecular weight PVP in the receiving solution of the same concentration also resulted in smaller paclitaxel nanoparticles. For PVP with molecular weight Mw ∼ 360 000 (PVP360K) at 0.33 mg/mL in the receiving solution, the RESOLV yielded paclitaxel nanoparticles of 200 nm in average diameter (Figure 4), with a size distribution standard deviation of about 30 nm. In Vitro Evaluation. The evaluation was based on the antiproliferation effects on the human malignant breast cell line MDA-MB-231.28 Treatment of cells with small (38 nm) and larger (530 nm) paclitaxel nanoparticles resulted in marked inhibition of cell proliferation. The estimated IC50 values (based on the results after 48 h exposure) are 100 nM (paclitaxel equivalent concentration in the nanoparticle samples), comparable with what is reported in the literature for paclitaxel dissolved in DMSO against the same cancer cell line.28 The use of paclitaxel ethanol solution under the same experimental conditions yielded similar results (Figure 5). However, the control experiments on PVP only (0.33 and 1.0 mg/mL) showed no toxicity to the cells, consistent with the known conclusion that the polymer is generally nontoxic.26,27 A known function of paclitaxel is to stabilize microtubules, thus interfering with the formation of mitotic spindles, inhibiting cell division, and inducing apoptosis,30,31 as observed in the cytotoxicity assay above. For MDA-MB-231 cells treated with the paclitaxel nanoparticles of small or larger sizes (10 µM drug (28) (a) Patel, N. M.; Nozaki, S.; Shortle, N. H.; Nakshatri, P. B.; Newton, T. R.; Rice, S.; Gelfanov, V.; Boswell, S. H.; Goulet, R. J.; Sledge, G. W.; Nakshatri, H. Oncogene 2000, 19, 4169. (b) Shenoy, D.; Little, S.; Langer, R.; Amiji, M. Mol. Pharmacol. 2005, 2, 257. (29) Liaoa, C.-H.; Pana, S.-L.; Guhb, J.-H.; Teng, C.-M. Biochem. Pharmacol. 2004, 67, 2031. (30) (a) Horowitz, S. B. Trends Pharmacol. Sci. 1992, 13, 134. (b) Woods, C. M.; Zhu, J.; McQueney, P. A.; Bollag, D.; Lazarides, E. Mol. Med. 1995, 1, 506. (31) Judson, P. L.; Watson, J. M.; Gehrig, P. A.; Fowler, W. C., Jr.; Haskill, J. S. Cancer Res. 1999, 59, 2425.

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Figure 6. The cellular organization of microtubule network in MDA-MB-231 cells with (10 µM drug concentration for all) and without paclitaxel treatment. All scale bars represent 10 µm.

concentration) for 24 h, there was a marked disruption of microtubule architecture and nuclear morphology according to confocal microscopy images (Figure 6).29,31 The disruption of microtubule architecture and the accumulation of R-tubulin around the nucleus were evident in drug-treated cells. Furthermore, nuclear examination revealed the presence of fragmented, diffusely stained, and multinucleated nuclei, which are consistent with apoptosis (Figure 6). Colocalization of microtubules with fragmented DNA was also detected. The interference of paclitaxel nanoparticles with the cell cycle progression was probed in terms of flow cytometry. For MDAMB-231 cells treated 24 h with the nanoparticles of a small (38 nm) or larger (530 nm) average size, there was a marked increase (about 6-fold) in the G2-M phase and a concomitant decrease in G0-G1 populations. This is consistent with the blockade of mitosis. In a comparison with paclitaxel dissolved in ethanol, the nanoparticles appeared to be more effective, with a higher population of cells accumulated in the G2-M phase (about 70% vs about 50% for the paclitaxel ethanol solution).

The RESOLV with supercritical CO2 is apparently a clean technique to nanosize water-insoluble paclitaxel particles. The nanosizing causes no degradation effect on the drug, as confirmed by the NMR results of paclitaxel before and after the processing. The stable aqueous suspension of paclitaxel nanoparticles, with PVP as a stabilization agent, serves as an ideal platform for the reported in vitro assays and other future bioevaluations (including investigation on the mechanism of drug accumulation in cells by using fluorescently labeled paclitaxel nanoparticles) and potential new formulation applications. The use of solid drug particles associated with such a platform may also be advantageous by leveraging the aqueous insolubility and enabling high drug loading and delivery via a number of administration routes. Many other water-soluble oligomeric and polymeric stabilization agents, including natural proteins such as bovine serum albumin, can be used with the RESOLV process, providing opportunities to alter or manipulate the physicochemical and pharmaceutical properties of their protected drug nanoparticles.23 The effectiveness of the nanosizing technique is validated by the in vitro

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evaluation results of the nanosized paclitaxel (comparable to a nonaqueous paclitaxel solution against specific cancer cells), although more investigations on other cell lines (MCF-7, for example, and those known to be more drug resistant) are necessary. Broader applications of RESOLV to the processing of nanoparticles from other important drugs, such as the antiparasitic drug Amphotericin B and conjugates of Doxorubicin with antioxidants, may also be pursued.

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Acknowledgment. Y.-P.S. acknowledges financial support from the NSF and the Center for Advanced Engineering Fibers and Films (an NSF-ERC at Clemson University). A.A.J. was a participant of the Summer Undergraduate Research Program sponsored jointly by the NSF and Clemson University. LA062739D