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Recrystallization and Micronization of Taxol Using the Supercritical Antisolvent (SAS) Process Xiuhua Zhao,†,‡ Xiaoqiang Chen,†,‡ Yuangang Zu,*,†,‡ Ru Jiang,†,‡ and Dongmei Zhao†,‡ †

State Engineering Laboratory of Bio-Resources Eco-Utilization, Northeast Forestry University, Harbin, Heilongjiang 150040, China Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin, Heilongjiang 150040, China

‡

ABSTRACT: Recrystallization and micronization of taxol solubilized in EtOH has been performed using the supercritical antisolvent precipitation (SAS). An orthogonal array design (OAD), OAD16 (45), was employed to optimize parameters of the SAS process. We obtained microparticles and nanoparticles of taxol, the minimum mean particle size (MPS) of which was about 150.5 nm under the selected conditions, 2.5 mg/mL of the concentration of the drug solution, 57 °C of process temperature, 20 MPa of process pressure, 150 μm of nozzle internal diameter (ID), and 6.6 mL/min of drug solution flow rate. The micronized product has also been characterized using scanning electron microscopy (SEM), high performance liquid chromatography-mass spectrometry (LC-MS), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy (FTIR), to verify the influence of the micronization process on the final product properties. Results showed that SAS process had not induced degradation of taxol and that micronized taxol particles had lower crystallinity. These results suggest that micronized powder of taxol has a great potential to be a drug delivery system in cancer therapy.

1. INTRODUCTION Taxol (Figure 1) is a diterpenoid extracted from the bark of a rare, slowly growing Pacific yew or Western yew tree (Taxus

However, because taxol is a hydrophobic drug with poor aqueous solubility, practical application is limited by its low solubility in water due to low therapeutic index.6,7 Presently, the only dosage-form of taxol for clinical application is Taxol, which is formulated with alcohol and Cremophor ELP as acceptable solubilizing solvents pharmaceutically.8,9 Development of new drug delivery system (DDS) for taxol is still one of the most important goals in cancer chemotherapy today. A great deal of effort has been focused on the development of various taxol delivery systems such as liposomes, emulsions, micelles, microspheres, polymeric nanoparticles,6,10,11 and rapid expansion of supercritical solutions (RESS).12 However, the limit of many delivery systems is that water-insoluble ingredients were introduced into the systems, which have the potential risks to health. Among these, emulsion may be a promising way because it provides good biocompatibility, good solubilization of poorly water-soluble drugs, and high concentration of lipophilic drugs in aqueous media. Micronized taxol has many specific properties different from unprocessed taxol, such as solubility, surface area, and so on. It has been reported that Nuray Yildiz et al.12 have done taxol micronization through rapid expansion of supercritical solutions (RESS); ethanol was used as cosolvent to increase the solvating power of CO2. Moreover, Kang, Y. et al.13 prepared taxolloaded poly(L-lactic acid) nanoparticles using SEDS process. However, very few reported that nanobased taxol with supercritical antisolvent (SAS). SAS precipitation, an alternative to the liquid antisolvent precipitation, can potentially overcome the limitations of liquid antisolvent processing, with the complete elimination of the solvents.14 SAS is a steady system;

Figure 1. Chemical structure of taxol (C47H51NO14, M = 853 g mol−1).

brevifolia). It is a novel antimicrotubule agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network that is essential for vital interphase and mitotic cellular functions. Taxol is approved in many countries for its use as second line treatment of ovarian cancer, breast cancer, and lung cancer. It is also used in the treatment of Kaposi’s sarcoma.1 It is recommended in National Institute for Health and Clinical Excellence (NICE) guidance of June 2001 that it should be used for non-small-cell lung cancer in patients’ unsuitable for curative treatment, and in first-line and second-line treatment of ovarian cancer. It was approved by the United States Food and Drug Administration (FDA) for ovarian cancer in 1992, for advanced breast cancer in 1994, and for early stage breast cancer in October 1999.2−5 © 2012 American Chemical Society

Received: Revised: Accepted: Published: 9591

May 6, 2012 June 15, 2012 June 20, 2012 June 20, 2012 dx.doi.org/10.1021/ie3011726 | Ind. Eng. Chem. Res. 2012, 51, 9591−9597

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separated from the organic solvent by a decompression (pressure < 5 MPa). A stainless steel frit vessel was placed at the bottom of the precipitation chamber to collect the micronized particles. Once injected the fixed quantity of taxol/EtOH solution, the liquid pump was stopped. After that, CO2 was continuously delivered to remove the residual solvent in the precipitator. This drying stage lasted for about 2 h. The precipitation vessel was then depressurized gradually to atmospheric pressure. Finally, the samples of micronized particles were taken from the precipitator for further characterization analysis. Further information and a schematic representation of the apparatus have been reported by others.15,16 An orthogonal OA16 (4)5 test design was used to investigate the optimal micronization condition of taxol. The range of each factor level was based on the results of preliminary experiments. As shown in Table 1, the SAS experiment was fulfilled with 5

there are not so many effect factors such as pre-expansion temperature or pressure of RESS, and it is more easy to control the particle size of taxol. The aims of this work are to study the feasibility of recrystallization and micronization of taxol from EtOH by SAS process, to optimize a SAS process, and to evaluate the factors that influence the mean particle size of micronized taxol. Moreover, characterization of micronized taxol was analyzed by scanning electron microscopy (SEM), liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared (FTIR), and X-ray diffraction (XRD), with the purpose of developing a suitable drug delivery system of cancer chemotherapy; meanwhile, micronized taxol could improve the drug-loading rate and decrease irritant properties.

2. MATERIALS AND METHODS 2.1. Materials. Taxol, with a purity of mass fraction of more than 98.5%, was provided kindly by Zhejiang Hisun Pharmaceutical Co. Ltd. (Zhejiang, P.R. China). CO2 (purity 99%) was supplied by Liming Gas Company of Harbin (Heilongjiang, PR China). EtOH (purity 99.5%) was purchased from Sigma-Aldrich. 2.2. Apparatus and Procedures. The schematic representation of SAS process apparatus is shown in Figure 2. The

Table 1. Factors and Levels of the Orthogonal Array Design

factor levels 1 2 3 4

A

B

C

D

E

concn. of taxol soln (mg/mL)

drug soln flow rate (mL/min)

precip. temp. (°C)

precip. pressure (MPa)

nozzle ID (μm)

2.5 5.0 7.5 10.0

3.3 6.6 9.9 13.2

35 46 57 68

10 15 20 25

150 200 300 1000

factors and 4 levels, namely, concentration of taxol solution (2.5, 5.0, 7.5, 10.0 mg/mL), drug solution flow rate (3.3, 6.6, 9.9, 13.2 mL/min), precipitation temperature (35, 46, 57, 68 °C), precipitation pressure (10, 15, 20, 25 MPa) and nozzle inner diameter (nozzle ID) (150, 200, 300, 1000 μm). The mean particle size (MPS) of micronized taxol (nm) was the dependent variable changing with the every factor. 2.3. Characterization Analysis. 2.3.1. Surface Morphology Analysis. The morphology of unprocessed taxol and micronized taxol particle were examined by using SEM (Quanta 200, FEI). 2.3.2. Dynamic Light Scattering. MPS and particle size distribution of micronized taxol were determined by dynamic light scattering (DLS) using an electrophoretic light scattering spectrophotometer. The samples were prepared by dilution of the micronized taxol powder in pure water and special care was taken to eliminate dust and to avoid the aggregation of particles. To avoid dissolution of the micronized particles, the water must be presaturated with taxol. The scattered light was measured at 90°. The temperature was set to 25° ± 0.1 °C. The suspensions were analyzed in DLS data (ZetaPALS, Brookhaven Instruments,). Every measurement was repeated three times. MPS and standard deviations (SD) obtained were used to fit the particle size distribution (PSD) to a log-normal distribution. 2.3.3. FTIR Analysis. Unprocessed and processed taxol (1 mg) was mixed with 100 mg KBr and pressed to obtain selfsupporting disks, separately. Samples were scanned at 4 cm−1 resolution with 100 scans in the spectral range of 4000−400 cm−1 (mid-IR region) at room temperature. Analysis of the spectral data was performed by using Grams 32 (Galactic Industries, U.S.A.) software.

Figure 2. Schematic diagram of the SAS apparatus. 1: CO2 cylinder. 2, 14, 15, 19, 21, 23, 24, 26, and 27: valves. 3: check valve. 4: CO2 cooler. 5: liquid solution supply. 6: liquid pump. 7 and 9: flow meter. 8: CO2 pump. 10, 13, and 20: heat exchangers. 11, 12, and 25: filters. 16: nozzle. 17: stainless steel frit vessel of 200 nm. 18: precipitation chamber. 22: gas−liquid separation chamber.

SAS apparatus mainly consists of three sections for carbon dioxide supply, precipitation, and depressurization (gas−liquid separation chamber). High-pressure carbon dioxide was fed into the system from the gas cylinder through a high-pressure pump. A precipitator consisted of a tainless steel nozzle, reducing union, and stainless steel frits vessel with pore sizes of 100 nm. The volume of the precipitator was 1000 mL. In the depressurization section, a metering valve was used to control the gas flow rate, a cold trap was used for solvent recovery, and a liquid flow meter was used to record the flow rate of CO2. A SAS process begins by delivering supercritical CO2 to the precipitation chamber until reaching the desired pressure. As a steady state was achieved, pure EtOH was pumped, preheated, and fed to the 1000 mL precipitation chamber through the nozzle, with the aim of obtaining steady state composition conditions during the taxol precipitation. At this point, the flow of the pure EtOH was stopped and the taxol/EtOH liquid solution was delivered through the nozzle at a given flow rate. The flow rate of the mixture that left the precipitator was controlled by a valve located between the precipitation chamber and the gas−liquid separation chamber. Here, the CO2 was 9592

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Table 2. Analysis of OA16 (45) Test Results run

concn. (mg/mL)

drug soln feed rate (mL/min)

temp. (°C)

pressure (MPa)

nozzle ID (μm)

MPS ± SD, n = 3 (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K1a K2 K3 K4 Rb optimal level

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 807.2 ± 56.9 811.7 ± 33.8 857.2 ± 51.6 1285.9 ± 64.9 478.8 A1

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1130.7 ± 58.8 428.9 ± 26.8 1126.1 ± 59.2 1076.3 ± 63.0 701.8 B2

1 2 3 4 2 1 4 3 3 4 1 2 4 3 2 1 952.9 ± 59.1 1379.6 ± 66.1 283.9 ± 15.2 1145.6 ± 66.8 1095.7 C3

1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3 908.8 ± 54.1 1071.3 ± 68.3 845.6 ± 35.8 936.4 ± 49.0 225.8 D3

1 2 3 4 4 3 2 1 2 1 4 3 3 4 1 2 890.3 ± 42.4 951.3 ± 52.5 900.4 ± 54.7 1020.0 ± 57.7 129.7 E1

927.8 ± 64.1 876.3 ± 63.3 201.1 ± 14.6 1223.4 ± 85.6 1425.6 ± 45.0 268.3 ± 16.1 1181.5 ± 59.1 371.5 ± 14.8 397.5 ± 19.9 405.6 ± 16.2 1265.6 ± 88.6 1360.2 ± 81.6 1771.9 ± 106.3 165.5 ± 11.6 1856.3 ± 74.3 1350.0 ± 67.5

Ki A = Σ(mean particle size at Ai) /4, the mean values of mean particle size for a certain factor at each level with standard deviation. bRi A = max{Ki A } − min{Ki A }. a

Figure 3. Effect of each parameter on the MPS of micronized taxol. (A) Level of concentration of taxol solution. (B) Level of taxol solution flow rate. (C) Level of precipitation temperature. (D) Level of precipitation pressure. (E) Level of nozzle ID.

sample is run at a scanning rate of 5 °C/min under nitrogen atmosphere. The temperature for the scan ranged from 20 to 300 °C.

2.3.4. LC-MS Analysis. The unprocessed and processed taxol were dissolved in methanol, separately. LC-MS information was obtained by analyst 1.4 of AB API 3000 (U.S.A.). The mass spectrometer was operated in positive ion mode. 2.3.5. XRD Analysis. The unprocessed and processed taxol powder XRD patterns were obtained using Ni-filtered Cu Kα radiation with the X-ray powder diffractometer equipped with a rotating anode (Philips, Xpert-Pro, Netherlands). The samples were filled to the same depth inside the sample holder by leveling with spatula, and the scanning rate (4°/min.) was same for all XRD analysis. 2.3.6. DSC Analysis. A differential scanning calorimeter (DSC), TA Instruments, model DSC 204, was used. Each

3. RESULTS AND DISCUSSION 3.1. Effect of Operating Conditions on the MPS of Micronized Taxol. The particle size is dominated by two possible mechanisms, evaporation of the solvent into the antisolvent phase and diffusion of the antisolvent into the droplets.17 The density of supercritical CO2, which is influenced by pressure and temperature, plays an important 9593

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role for mass transfer between organic solvents and CO2 during particle formation.18 In the SAS process, the concentration of drug solution, precipitation temperature, precipitation pressure, drug solution flow rate, and nozzle ID are generally considered to be the most important factors. In the present study, all selected factors were examined using an orthogonal OA16 (45) test design. The total evaluation index was used to analysis by statistical method. The analysis results of orthogonal test, performed by Design Expert 7.0 software. The assignment of the experiment and the collected data for MPS of micronized taxol is shown in Table 2. Although the minimum MPS of micronization taxol was 165.5 ± 11.6 nm, we cannot choose the corresponding conditions as the best technology. In view of orthogonal analysis, we adopt statistical software to calculate the values of K and R. The factors influencing the MPS of micronization taxol were listed in a decreasing order as follows: C > B > A > D > E according to the R value. So, the minimum MPS of micronization taxol was obtained when precipitation temperature, flow rate, concentration, precipitation pressure, nozzle ID of taxol solution were C3 > B2 > A1 > D3 > E1 (57 °C, 6.6 mL/min, 2.5 mg/mL, 20 MPa, 150 μm), respectively. Through confirmatory test, the smaller micronization taxol was obtained, with a MPS of 150.5 ± 10.3 nm, which greatly improved the bioavailability and taxol value. 3.2. Univariate Effect on the MPS of Micronized Taxol. The relationships between the MPS of micronized taxol and variables factors are shown in Figure 3. The MPS revealed increased, decreased, and then increased when the precipitation temperature increased from 35 to 68 °C, precipitation pressure increased from 10 to 25 MPa, and nozzle ID increased from 150 to 1000 μm, respectively. However, the concentration, ranging from 2.5 to 5.0 mg/mL, has no significant influence on the MPS of micronized taxol, and when it ranges from 5.0 to 10.0 mg/mL, the MPS increased continually. The MPS of micronized taxol significantly decreased and then increased when feed rate increased from 3.3 to 9.9 mL/min, and the MPS reached the largest point, then it showed a slightly decrease, with the increase of feed rate. Generally speaking, there is no obviously sequence effect from the Figure 3 about the precipitation temperature, precipitation pressure, flow rate, nozzle ID, except the concentration of taxol solution. 3.3. Morphology of Micronized Taxol. Figure 4 shows SEM micrographs of unprocessed taxol particles, which are irregular lamelliform crystals, ranging in length from 1 to 80 μm. Orthogonal tests showed that micronized taxol have different morphologies and MPS under different operating conditions. Figure5 gives the SEM images of micronized taxol precipitated from EtOH under optimum conditions. Micronized taxol particles are spherical like other conditions. It can be noted a tendency to form aggregates because of smaller particle size and surface activity of the high reunion. It should be mentioned that these aggregates are completely separated when the powder is suspended in water. This phenomenon can be explained by the good dispersibility of these aggregates in water and that the polydispersity of these aggregates was just 0.005, which was determined by DLS using electrophoretic light scattering spectrophotometer when suspended in water. The morphology of the micronized powder can be a function of the position of the operating point with respect to the mixture critical point (MCP) of the ternary system. EtOH is a versatile and powerful solvent in SAS process due to no tendency to produce strong interactions with solutes. There-

Figure 4. SEM micrographs of unprocessed taxol particles.

Figure 5. SEM micrographs of processed taxol particles.

fore, SAS micronization process of Taxol was analyzed under the corresponding binary system (EtOH−CO2). Catinca Secuianu et al.19 and Seung Nam Joung et al.20 determined experimental phase diagram data on CO2-EtOH at 303.15 ± 0.1 K, 313.40 K, 322.50 K, 333.40 K, and 344.75 K. From the critical pressure 6.52 MPa at 303.15 ± 0.1 K, 8.16 MPa at 313.40 K, 9.21 MPa at 322.50 K, 10.64 MPa at 333.40 K, and 10.97 MPa at 344.75 K (Figure 6), it is evident that all the points of the orthogonal array except trial no. 7, 14, and the optimum condition, were made in the completely developed supercritical phase region. Spherical microparticles were successfully obtained under these conditions. Irregular spheres were obtained in trial no. 14 because the operation condition is near supercritical phase region. Meanwhile, trial no. 7 was performed at subcritical conditions inside the two-phase region, and needle-like microparticles were obtained. 3.4. Characterization of Micronized Taxol. 3.4.1. FTIR Analysis. FTIR analysis was performed to study the chemical composition of the microparticles prepared by the SAS process 9594

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Figure 6. Phase diagram of CO2-EtOH (303.15 ± 0.1 K, 313.40 K, 322.50 K, 333.40 K, and 344.75 K).

were no varieties in the chemical structure of taxol before and after the SAS process. For this reason, the SAS process has not caused degradation of taxol. 3.4.3. XRD Analysis. X-ray diffraction imaging is a novel modality that synthesizes two important characteristics of Xrays: namely, their ability to form images (radiography) and their capacity to analyze material via the technique of X-ray diffraction (XRD).22 The XRD results obtained in our investigation are presented in Figure 9. The presence of several distinct peaks in the XRD of unprocessed taxol at the diffraction peaks 2θ = 5.48°, 12.44°, 8.9°, 17.08°, 5.12°, and 16.76° reveals that the drug is present as a crystalline form. However, the micronized taxol presents a diffraction pattern with peaks at 12.22°, 5.52°, 5.22° of 2θ. This result suggests that micronized taxol particles are less crystalline after SAS processing. Less crystalline and smaller drug particles are higher in the dissolution rate or bioavailability than crystals, and the therapeutic action is obtained in shorter times.23 Therefore, micronized taxol should have higher dissolution rate or bioavailability than unprocessed taxol. 3.4.4. DSC Analysis. DSC is a useful technique for measuring the energy necessary to establish a nearly zero temperature difference between a substance and an inert reference material, as the two specimens are subjected to identical temperature regimes in an environment heated or cooled at a controlled rate, and whether solutes have been dispersed or dissolved in polymeric matrices.24,25 Figure 10 shows the DSC curves of unprocessed taxol and micronized taxol prepared by the SAS process. Pure crystalline taxol has a characteristic endothermic melting peak at approximately 223.0 °C.26 In our study, unprocessed taxol is observed as a crystalline form with a melting temperature (Tm) of about 221.9 °C. After the SAS process, the Tm is shifted to 212.7 °C, implying that the crystallinity had slightly decreased through the SAS process, and this result is consistent with the XRD results.

and to evaluate the presence of EtOH residues in micronized taxol. FTIR spectra of unprocessed and micronized taxol are reported in Figure 7. The specta of unprocessed taxol show

Figure 7. FTIR traces of unprocessed and SAS processed taxol.

strong absorption bands in the ranges 1750−1600 cm−1, 1300− 1180 cm−1, and 770−630 cm−1 as the report.21 These characteristic bands all exist in the spectra of micronized taxol with negligible rightward shifts, the main absorption bands of micronized taxol are as follows: 1734 cm−1 (CO stretching vibration), 1244 cm−1 and 1072 cm−1 (C−O stretching vibration), and 710 cm−1 (C−H out-of-plane bending vibration). From their comparison, we concluded that no variations in composition have been induced by SAS processing and that no solvent residue is detectable within the range of sensitivity of this analysis. 3.4.2. LC-MS Analysis. Figure 8 shows LC-MS spectra of unprocessed and micronized taxol. It can be seen that there were no modifications that occurred in molecular weight between unprocessed and micronized taxol, the molecular weight of unprocessed and micronized particles were 854.70 g/ mol, from mass spectrum. This result could explain why there

4. CONCLUSIONS In the present study, recrystallization and micronization of taxol was carried out by the SAS process. An orthogonal array design OA16 (45) was used to determine the optimum conditions. We 9595

dx.doi.org/10.1021/ie3011726 | Ind. Eng. Chem. Res. 2012, 51, 9591−9597

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Figure 8. LC-MS spectra of unprocessed and processed taxol.

Figure 9. X-ray diffraction patterns of taxol before/after SAS process. Figure 10. DSC curves of unprocessed and processed taxol.

obtained microparticles and nanoparticles of taxol, whose mean particle size was about 150.5 nm under the optimum conditions: 2.5 mg/mL concentration of the liquid solution, 57 °C process temperature, 20 MPa process pressure, 150 μm nozzle internal diameter, and 6.6 mL/min of drug solution flow rate. Moreover, the SEM, LC-MS, XRD, DSC, and FTIR were used to characterize micronized taxol. The results showed that the SAS process has not induced degradation of taxol and that micronized taxol particles have lower crystallinity. Micronization improves the solubility of drug substances with a particle size of less than 1 μm. This is supported by the Ostwald−Freundlich equation, which demonstrates that solubility increases exponentially as a function of particle size.

This approach could be used for pharmaceutical products with a high drug-loading.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 451 8219 1517. Fax: +86 451 8210 2082. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9596

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Article

A. M. F. Supercritical antisolvent micronization of minocycline hydrochloride. J. Supercrit. Fluids 2008, 44 (2), 238−244. (17) Mamata, M.; V, D. S. Mass and heat transfer analysis of SAS: effects of thermodynamic states and flow rates on droplet size. J. Supercrit. Fluids 2004, 30 (3), 333−348. (18) Min-Soo, K. I. M.; Sibeum, L. E. E.; Jeong-Sook, P.; Ong-Soo, W. O. O.; Sung-Joo, H. Micronization of cilostazol using supercritical antisolvent (SAS) process: Effect of process parameters. Powder Technol. 2007, 177 (2), 64−70. (19) Secuianu, C.; Feroiu, V.; Geana, D. Phase behavior for carbon dioxide plus ethanol system: Experimental measurements and modeling with a cubic equation of state. J. Supercrit. Fluids 2008, 47 (2), 109−116. (20) Seung Nam, J.; Chang Woo, Y. O. O.; Hun Yong, S.; Sun Young, K. I. M.; Ki-Pung, Y. O. O.; Chul Soo, L. E. E.; Wan Soo, H. U. H.; F, E. L. Y. J. Measurements and correlation of high-pressure VLE of binary CO2-alcohol systems (methanol, ethanol, 2-methoxyethanol and 2-ethoxyethanol). Fluid Phase Equilibria 2001, 185 (1−2), 219− 230. (21) Yu, L. I. U.; Guo-Song, C.; Yong, C.; Dong-Xu, C. A. O.; ZhiQiang, G. E.; Ying-Jin, Y. Inclusion complexes of paclitaxel and oligo(ethylenediamino) bridged bis(-cyclodextrin)s: solubilization and antitumor activity. Bioorg. Med. Chem. 2004, 12 (22), 5767−5775. (22) Harding, G. X-ray diffraction imaging: A multi-generational perspective. Appl. Radiat. Isot. 2009, 67 (2), 287−295. (23) Viktor, M.; Gérard, C.; Elisabeth, B.; Géza, H.; Laszlo, S.; Nathalie, B.; Eric, T. Bioavailability enhancement of an active substance by supercritical antisolvent precipitation. J. Supercrit. Fluids 2007, 40 (1), 101−110. (24) Dubernet, C.; Ford, J. L. Thermoanalysis of microspheres. Thermochim. Acta 1995, 248, 259−269. (25) Corrigan, O. I.; Ford, J. L. Thermal analysis of spray dried products. Thermochim. Acta 1995, 248, 245−258. (26) Liggins, R. T.; Hunter, W. L.; Burt, H. M. Solid-state characterization of paclitaxel. J. Pharm. Sci. 1997, 86 (12), 1458−1463.

ACKNOWLEDGMENTS The authors are grateful for the precious comments and careful corrections made by anonymous reviewers. The authors would also like to acknowledge the financial support from the Special Fund for Forestry Scientific Research in the Public Interest (201204601) and the Forestry Science and Technology Promotion Project ([2011]08). The authors are also grateful to Dr. Lin Zhang for performing the LC-MS analysis.

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REFERENCES

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