http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.950272
RESEARCH ARTICLE
Preparation, physical characterization and pharmacokinetic study of paclitaxel nanocrystals
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Lisha Wei1, Yanxia Ji2, Wei Gong1, Zhenqiao Kang2, Meng Meng3, Aiping Zheng1, Xiaoyan Zhang1, and Jianxu Sun1 1
Department of Pharmaceutics, Beijing Institute of Pharmacology and Toxicology, Beijing, P.R. China, 2Handan Central Hospital, Hebei Province, P.R. China, and 3Department of Pharmaceutics, School of Pharmacy, Hebei Medical University, Hebei, P.R. China
Abstract
Keywords
Paclitaxel (PTX) is a natural broad-spectrum anticancer drug with poor aqueous solubility. PTX nanocrystals were formulated to improve the water solubility, and PTX nanosuspensions were prepared using anti-solvent precipitation, and then organic solvent and surfactants were removed by filtering through a vacuum system. The physical characterization of PTX nanocrystals were measured by transmission electron microscope, X-ray diffraction and differential scanning calorimetry. In addition, saturation solubility, in vitro release, stability and pharmacokinetic characteristics were examined. The average particle size of PTX nanocrystals was 200 nm, and they had a stable potential and a uniform distribution. Paclitaxel nanocrystals can effectively improve drug solubility and in vitro release. PTX pharmacokinetic and tissue distribution studies were compared after intravenous administration of nanocrystals versus a commercial injection formulation. PTX nanocrystals were rapidly distributed with a longer elimination phase. Moreover, tissue distribution indicated that PTX nanocrystals are mainly absorbed by the liver and spleen and may offer reduced renal and cardiovascular toxicity which may reduce side effects.
Nanocrystals, paclitaxel, pharmacokinetics, tissue distribution, X-ray diffraction
Introduction Paclitaxel (PTX) is a complex secondary metabolite originally developed by Wani et al.1 derived in 1971 from the bark of Taxus chinensis (Taxus brevifolia). PTX promotes microtubule assembly in cells which interfere with various cellular functions, especially cessation of mitotic cell division, blocking normal cell division2,3. According to phase II and III clinical studies, PTX is chiefly indicated for ovarian, breast and non-small cell lung cancer, and may be useful for esophageal, head and neck cancers4. PTX has low aqueous solubility and is chiefly administered by injection after dissolving PTX in polyoxyethylene castor oil (Cremophore EL) and ethanol (1:1). However, many studies suggest that Cremophore EL in this PTX injection can cause gastrointestinal and allergic reactions, hypotension, renal and cardiac toxicity, and bone marrow suppression, among other toxic reactions, greatly limiting this application5,6. At this time, pretreatment for allergic reactions include oral dexamethasone, intramuscular or oral diphenhydramine, or intravenous cimetidine or ranitidine, and these also cause adverse reactions6. Specifically, adverse reactions occur in 30% of patients7. Therefore, the design of safe, effective and convenient PTX formulation would be of significant clinical value. A PTX albumin suspension injection (Abraxane) lacking Cremophor EL
Address for correspondence: Aiping Zheng, Department of Pharmaceutics, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing 100850, P.R. China. Tel: +86 10 66931694. Mob: +86 13520467936. E-mail: [email protected]
History Received 12 May 2014 Revised 25 July 2014 Accepted 27 July 2014 Published online 26 August 2014
was made available in 20058 and this low-toxicity formulation did not require allergy pretreatment9. However, studies suggest that Abraxane to treat breast cancer in Phase III clinical trials caused more sensory neuropathy compared with the group not treated with the experimental drug10. A PTX liposome for injection (L-PTX) was approved by the CFDA (China Food and Drug Administration) and was marketed in 200311. A PTX liposome drug carrier system is presently in clinical trials worldwide12–14. However, the encapsulation rate of PTX liposome is low along with PTX leak15. Recent studies indicate that a larger liposomal particle radius of liposomes and an abundance of cholesterol in the liposome lipid bilayer can activate the complement system to cause a pseudo-allergy (CARPA)16, capable of damaging the heart and lung, thereby limiting clinical PTX liposome applications17,18. In the late 1990s, a nanoparticle drug delivery system was developed19. Nanocrystals need no carrier material and consist of a colloidal dispersion system of submicron particles formed from pure drug with no constrained of encapsulation efficiency. The adjustable dose is broad, and the stability of nanocrystals relies on potential and dimensional protective agents19. As a new formulation technique, nanocrystals can be used for enhancing oral bioavailability of poorly soluble drugs20, and nanocrystals can be suspended in water (nanosuspensions) for IV or pulmonary drug delivery21,22. Two approaches are used to prepare nanocrystals: top–down and bottom–up technologies23. Top–down technologies mainly consist of bead/pearl milling and high-pressure homogenization. No organic solvent is needed, and it is especially suitable
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for drugs that are insoluble both in aqueous and organic phases. The bottom–up – or precipitation – process involves the dissolution of the drug in a solvent to create a homogeneous precipitation by mixing with a non-solvent. Nanocrystal technology is promising for poorly soluble drugs and the preparation is universal and simple, with applicability to most drugs. Industrial production is also highly feasible. Great interest has been generated for formulating poorly soluble drugs into nanocrystals, specifically anticancer agents24. We developed a precipitation process for preparing PTX nanocrystals and we characterized nanocrystal particle size, morphology and crystalline structure as well as documented pharmacokinetics.
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Physical characterization Particle morphology The particle dimension and morphology was observed with transmission electron microscope (TEM) (H-7650; Hitachi, Tokyo, Japan), with an accelerating voltage of 80 kV. Freshly prepared PTX nanocrystals (5 ml) were dropped onto a 300-mesh carbon-coated copper grid and incubated (5 min) at room temperature. Grids were then negatively stained with 5 ml 3% uranyl acetate for 5 min, and then air-dried. The crystallographic morphology of PTX nanocrystals was observed using TEM under which images were obtained. X-ray diffraction
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Materials PTX (purity 498%) material and Commercial PTX injection was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. (Zhejiang, China). Docetaxel was provided by Guilin Huiang Biochemical Pharmaceutical Co. Ltd. (Guilin, China). Ethanol and glacial acetic acid (analysis grade) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Poloxamer 407 was provided by BASF (Florham Park, NJ). Docusate sodium (DOSS) and lauryl sodium sulfate (SDS) were purchased from Beijing Fengli Jingqiu Commercial and Trade Co. Ltd. (Beijing, China). Methanol and acetonitrile (HPLC grade) were from Fisher Scientific (Pittsburgh, PA). Methyl tertiary butyl ether (MBTE, analysis grade) was provided by Beijing Modern Oriental Fine Chemicals Co. Ltd. (Beijing, China). Preparation of PTX nanocrystals Preparation method PTX nanocrystals were prepared by anti-solvent precipitation. Firstly, 50 mg PTX was dissolved in 1 ml of ethanol (50 mg/ml) to form the organic phase. Two surfactants (poloxamer 407 and DOSS) were dissolved in 10 ml water to form the aqueous phase. Then the aqueous phase was rapidly stirred at 1200 rpm for 5 min at room temperature, after which 1 ml of the organic phase was injected into the aqueous phase under rapid stirring (1200 rpm) for 5 min. The mixed phases were sonicated (KQ3200DE sonicator from Kun Shan Sonicator Company, Kun Shan, China) at 80 W for several min. The final product was filtered by a vacuum system through a 0.22-mm filter membrane and nanocrystals were washed in 10 ml water 3 times to remove ethanol and most surfactant. Nanocrystals were re-dispersed in 10 ml water and sonicated to create stable PTX nanosuspensions (5 mg PTX/ml H2O). PTX nanosuspensions were lyophilized into dry powder (LGJ18C freeze-dryer, Si Huan Instrument Factory, Beijing, China). In brief, nanosuspensions were dispensed into a vial, and protectant (5%, w/v, g/ml) was added. Samples were frozen at 70 C. Then, the vial was transferred to a freeze-dryer and dried at 45 C for 40 h with a 30-Pa vacuum. The freeze-dried products were used for X-ray diffraction and differential scanning calorimetry (DSC) experiments or re-dispersed in 0.9% saline for IV administration. The re-dispersion was sonicated at 80 W for 1–5 min before use. Particle size and zeta potential The particle size and the polydispersity index (PDI) were measured by dynamic light scattering (DLS; Malvern Zeta Sizer, Nano-ZS90, Worcestershire, UK), and the ZP (Zeta potential) values were measured. Before DLS measurements, each sample was diluted and sonicated at 80 W for 10 s25.
The X-ray diffraction patterns for freeze-dried nanocrystals products were obtained through an X-ray power diffractometer (D/MAX 2000; Rigaku, Japan). The X-ray source was Cu filtered flat graphite monochromator, with a wavelength of 1.5406 A. The sample amount was 50 mg. The 2 range scanned was 2.0–40 at a rate of 4 /min. The step size26was 0.015. DSC analysis The DSC analysis for freeze-dried nanocrystals products was by differential scanning calorimeter (Q2000; New Castle, DE). The temperature scope was from 40 C to 250 C, heating at 5 C/min with a N2 flow rate of 20 ml/min (sample 50 mg). Saturation solubility of PTX nanocrystals To measure the saturation solubility of PTX, 0.5 ml suspensions of PTX nanocrystals (5 mg PTX/ml H2O) were placed in a 25-ml bottle to which 2 ml water was added. In addition to this, 0.5% lauryl sodium sulfate (SDS), 1.0% SDS and PBS (pH 7.4) was used as solvent (final PTX ¼ 1 mg/ml). After shaking, the mixed solution was ultrasonicated to achieve supersaturated state. The supersaturated solutions were shield from light and airtight before being placed in the shaker. Afterwards, solutions were shaken at 200 rpm at 37 C under constant shaking. After 24 h, 1 ml of the solution was centrifuged at 12 000 rpm for 5 min. The supernatant was filtered through a 0.45-mm filter membrane, and the filtrate was analyzed with HPLC analysis to measure PTX. For comparison, the PTX material was also measured the same way. Final PTX was 1 mg/ml and all experiments were performed in triplicate. In vitro release of PTX nanocrystals To measure in vitro release, a ZRS-8G dissolution tester (Tianjin Xinzhou Technology, Tianjin, China) was used. Then, 1 ml of the 5 mg/ml PTX nanocrystal suspension was pulled into a 2-ml needle and then injected into 200 ml 0.5% SDS at 37 C under constant revolution (50 rpm). At a predetermined time, 2 ml of the dissolution medium was centrifuged at 12 000 rpm for 5 min, and 2 ml of fresh medium was added subsequently. Supernatant was filtered through a 0.45-mm filter membrane, and the PTX in the filtrate was measured using HPLC. Also, 5 mg of PTX material dispersed in 1 ml water was injected into 200 ml 0.5% SDS, and PTX was quantified. All experiments were performed in triplicate. Stability study Dilution stability The distribution of particle size in PTX nanocrystals was measured by dynamic light scattering (DLS; Malvern Zeta Sizer, Nano-ZS90, Worcestershire, UK). To study the effect of
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DOI: 10.3109/03639045.2014.950272
dilution on size stability, 1 ml of original nanosuspension sample (5 mg PTX/ml H2O), a 10-fold diluted sample, a 50-fold diluted sample and a 100-fold diluted sample were measured. Temperature stability To study the effect of temperature on stability of PTX nanocrystals, 4 C, room temperature, 30 C and 37 C were selected as a temporary placement condition. Particle size changes were measured at 0, 24 and 72 h. Storage stability
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PTX nanocrystal suspensions were stored at 4 C or room temperature. Periodically, samples were removed and sample particle sizes were measured at 0, 0.25, 0.5, 1, 1.5, 2, 3, 6 and 9 months. Pharmacokinetics and tissue distribution study Kun Ming female mice (16–20 g) were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China). PTX nanocrystals were lyophilized into dry powder and suspended in 0.9% saline (PTX ¼ 1 mg/ml). Commercial PTX injection was diluted in 0.9% saline to 1 mg/ml. PTX nanocrystals and the commercial injection were administered to the mice (N ¼ 6) via the tail vein (IV, 10 mg/kg). At predetermined time points (2, 5, 10 and 30 min, and 1, 2, 4, 8 and 24 h), blood was collected into heparinized test tubes and centrifuged immediately at 12 000 rpm for 5 min to obtain plasma. Mice were euthanized at predetermined time points (5 and 30 min, and then 2, 8 and 24 h) and the heart, liver, spleen, lung and kidneys were excised, washed, dried and weighed. Plasma and tissue samples were stored at 70 C for further analysis. Analysis of PTX HPLC analysis of PTX PTX concentrations in solution were detected with HPLC, comprised of a pump (Spectra System P2000, Thermo Separation Products, Fremont, CA) and an ultraviolet detector (Spectra System Uv1000, Thermo Separation Products). Using a column (5 mm, 250 mm 4.6 mm, reverse-phase C-18 Phenomenex). The mobile phase consisted of methanol, water and triethylamine (80:20:0.1, v/v), and the pH was adjusted to 6.3 with glacial acetic acid. The mobile phase was filtered through a 0.45-mm filter membrane before analysis. The column was eluted at a flow rate of 1.0 ml/min at room temperature. The PTX concentrations were measured with the UV detector at 228 nm (20 ml sample volume). LC-MS analysis of PTX To measure the pharmacokinetics and tissue distribution of PTX, LC-MS was used. The LC-MS consists of an Agilent 1260 Infinity (G1322A Vacuum Degassers, G1312C quaternary pump, G1367E an automatic sampler and a G1316A column temperature box, Palo Alto, CA) and MS (G6460A triple level four pole tandem mass spectrometry, Palo Alto, CA). An MP C18 column (3 mm, 2.1 mm 50 mm, Venusil) was used. The mobile phase was a gradient elution, which included A (0.1% acetic acid:acetonitrile ¼ 70:30) and B (0.1% acetic acid:acetonitrile ¼ 10:90). The elution procedure was as follows: 0–0.5 min, 25% B; 0.5–1.5 min, 100% B; 1.5–5 min, 25% B. The flow rate was 0.4 ml/min and the column temperature was 25 C (5 ml sample volume).
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For plasma samples, 100 ml of plasma was obtained and combined with 10 ml of 500 ng/ml docetaxel. Each sample was vortexed for 3 min (Vortex geniu-2, Scientific Industries, Bohemia, NY) after adding 1 ml MBTE, and then centrifuged (centrifuge 1–14, Sigma, Munich, Germany) at 14 000 rpm for 5 min. Organic supernatant was removed to clean test tubes and evaporated at 37 C under a stream of nitrogen with a speed vacuum concentrator (Labconco, Kansas City, MO). For tissue samples, 5 times the organ weight of 0.1% acetic acid was added and homogenated. Then, 100 ml of tissue homogenate was added to 10 ml 500 ng/ml docetaxel. Tissue samples were treated in the same way. Before sample introduction, residues were dissolved in 100 ml of the mobile phase and vortexed for 1 min, and then centrifuged at 14 000 rpm for 5 min. Next, 5 ml of supernatant was injected into the LC-MS for analysis. Statistical analysis Graphpad-Prism curves were generated for each result. In vitro release profile was processed using one-site binding for curve fitting. A two-tailed T test was used to determine significance among groups. A p value of 50.05 was considered to be a significant difference.
Results Nanocrystal preparation To prepare PTX nanocrystals by anti-solvent precipitation, surfactants were needed to maintain the stability of PTX nanosuspensions. Dimensional surfactant was poloxamer 407 (P407), and DOSS was selected as the potential surfactant. As seen in Figure 1(A), particle size varied with different proportions of surfactant. The smallest particle size ratio was 1:5. Using this 1:5 proportion of PTX/P407, different proportions of DOSS did not change ZP values (jZPj 20 mV). Figure 1(B) depicts particle sizes with each ratio of potential surfactant. Screening results showed that the particle size was the smallest at a ratio of 1:2. From these data, the final combination was poloxamer 407 and DOSS at a proportion of 1:5 and 1:2, respectively. Process optimization is depicted in Table 1 and included removal of organic reagents using two methods. First rotaryevaporation with a 40 C water bath under reduced pressure for 30 min was used and nanocrystals were re-dispersed in 10 ml water and sonicated for stabilization. Next, vacuum-filtration separated nanocrystals from surfactants. The final product was filtered through a vacuum system (0.22-mm filter membrane) and nanocrystals were washed in 10 ml water 3 times to remove ethanol and residual surfactants. Then, nanocrystals were re-dispersed in 10 ml water and sonicated to stabilize and obtain PTX nanosuspensions. Data show that after rotary evaporation, PTX re-suspensions were larger with respect to particles. The 40 C water bath may have caused nanocrystal aggregation. After vacuum-filtration to remove residual ethanol and re-dispersion, the PTX nanosuspension was more stable with smaller particles (jZPj425 mV). Ultimately, the optimal formulation conditions were determined, and PTX nanocrystals were prepared using a preparation method described in the section titled ‘‘Preparation method’’. PTX nanosuspensions were lyophilized to a dry powder and a uniform particle size (Table 2) with a sable ZP. Physical characterization Transmission electron microscope images of PTX nanocrystals after processing are depicted in Figure 2, revealing a smooth rodlike morphology (200–500 nm). PTX crystalline structures were obtained by X-ray diffraction (see Figure 3 for diffraction peaks
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for PTX material, pure surfactant dry powder and PTX nanocrystals plus dry powder). PTX alone had crystalline diffraction peaks and the product was chiefly crystalline with some amorphous crystalline structures. Comparing with pure surfactants, the peak profiles of PTX nanocrystals were highly
similar, but the crystal formations of the two samples had some difference. The phase transition temperature of PTX nanocrystals was obtained from DSC. Figure 4 endothermic peaks for PTX material, pure surfactant dry powder and PTX nanocrystals plus dry powder. The main PTX nanocrystal endothermic peak moved slightly to the right compared to pure surfactants and the peak was 209.86 C. After nanocrystal preparation, the main endothermic peak was reduced sharply to 165.77 C. DSC measurements indicted that the phase transition temperature of PTX nanocrystals decreased.
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Saturation solubility of PTX nanocrystals The saturation solubility of two PTX formulations was measured in four solvents. As shown in Table 3, nanocrystal saturation solubility was greater than PTX material in four solvents and this difference was statistically significant (p50.001) between nanocrystals and PTX material in water
Table 1. Particle properties of PTX nanosuspensions with process optimization (mean ± SD; n ¼ 3).
Parameter
Before removing organic reagents
After revolvingevaporation
355.7 ± 25.3
725.05 ± 157.6
0.218 ± 0.03 19.2 ± 2.7
0.389 ± 0.30 20.35 ± 4.9
Particle size (nm) PDI ZP (mV)
After vacuumingfilter 240.9 ± 33.1 0.145 ± 0.01 29.25 ± 2.8
Table 2. Particle properties of PTX nanocrystals with freeze-drying (mean ± SD; n ¼ 3).
Figure 1. The influence of different proportion of surfactants on particle size. (A) Different proportion of P407 and (B) different proportion of DOSS (n ¼ 3).
Parameter
Before freeze-drying
After freeze-drying
Particle size (nm) PDI ZP (mV)
226.9 ± 32.0 0.149 ± 0.02 28.7 ± 1.9
232.7 ± 11.6 0.167 ± 0.05 28.9 ± 2.3
Figure 2. Transmission electron microscopy of PTX nanocrystals (40 000). Scale bar: 500 nm.
DOI: 10.3109/03639045.2014.950272
Paclitaxel nanocrystals
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Figure 3. X-ray diffraction peaks of (A) PTX material, (B) pure surfactants dry powder and (C) PTX nanocrystals plus dry powder.
(which was 150 times more soluble) and PBS (pH 7.4) in which the solubility was 50 times greater. However, with the addition of the surfactant SDS (0.5%), the difference was not statistically significant between nanocrystals and PTX material. SDS had solubilizing effects that were similar for both PTX formulations (high solubility). Next, we tested this solvent for in vitro release of nanocrystals versus PTX material.
In vitro release of PTX nanocrystals Release data for PTX nanocrystals and PTX material are depicted in Figure 5. PTX nanocrystals released rapidly compared with the PTX material. Specifically, within 5 min, nanocrystals released 490% of the drug and this was maintained for 8 h. PTX material released 50% of the drug in the same time period.
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(A) 0.0
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Figure 4. DSC endothermic peaks of (A) PTX material, (B) pure surfactants dry powder and (C) PTX nanocrystals plus dry powder.
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Then, PTX material released 80% of the drug after 30 min, and 490% of the drug was released after 2 h, before reaching equilibrium.
The effect of dilution on size stability was depicted in Figure 6(A). Data show that particle size of every dilution multiple was 250 nm. All samples did not undergo particle size changes, indicating that dilution did not cause aggregation. Figure 6(B) shows that for four temperatures, PTX particle sizes changed at 30 C and 37 C (4300 nm). At 4 C and room temperature, particle size fluctuated narrowly. Data show that PTX nanosuspensions were sensitive to temperature changes. Long-term stability was then tested. PTX nanocrystals suspensions were stored at 4 C and at room temperature. Particle size changed as depicted in Figure 6(C), with size increasing with increased storage time. At the lower temperature (4 C), particle size changed little. At room temperature, particle size increased by 60% over 9 months. Thus, long-term storage should be at 4 C. Pharmacokinetics and tissue distribution study Pharmacokinetic analysis was carried out using DAS 2.2.1 pharmacokinetic software. Model discrimination was based on Akaike information criteria (AIC). Statistical significances for pharmacokinetics among the treatment groups were analyzed using a two-tailed T test. Analytical methods for plasma PTX measurements are established27–29. Figure 7 depicts plasma concentration–time data for PTX and a two-compartment model
Table 3. The saturation solubility of two forms of PTX (mean ± SD; n ¼ 3).
Solvent
Material solubility (mg/ml)
Nanosolubility (mg/ml)
Nano/material
Water pH 7.4 PBS 0.5% SDS 1.0% SDS
0.081 ± 0.041 0.023 ± 0.039 196.562 ± 5.405 385.331 ± 31.174
12.416 ± 0.861*** 1.181 ± 0.021*** 200.138 ± 20.245 429.957 ± 14.019
153.284 51.348 1.018 1.116
***p50.001, statistical significance compared with material.
describes these data (Table 4). Figure 7 shows that after IV administration of PTX nanocrystal or injection, plasma PTX rapidly declined for both. After administration (2 or 5 min), no significant differences were observed between the two formulations. However, PTX plasma concentrations of nanocrystals were significantly lower than the PTX injection group (p50.001) from 10 min to 8 h. Data show that both t1/2,a and t1/2,b indicated a relatively rapid distribution phase and a longer elimination phase for PTX nanocrystals compared to commercial PTX injection. The AUC(0–1) for PTX injection was 2-fold greater than PTX nanocrystals, but the MRT for PTX nanocrystals were longer than for PTX injection. Moreover, the Vz for PTX nanocrystals was 5-fold larger than the PTX injection formulation, and PTX nanocrystals cleared faster suggesting that PTX nanocrystals can be distributed more quickly to the surrounding organs. After 24 h, the plasma concentrations of both formulations were equivalent. Distribution of PTX in the blood and major organs was examined after the IV PTX nanocrystal injection and commercial PTX injection (10 mg/kg). As shown in Figure 8, IV nanocrystals distributed as follows (greatest to lowest concentration): liver4spleen4lung4kidney4heart. After IV commercial PTX administration, drug concentrated (high to low) as follows: liver4kidney4lung4spleen4heart prior to 30 min and liver4spleen4kidney4lung4heart after 2 h. Therefore, the highest drug concentration of two formulations was the liver, and the lowest drug concentration was in the heart. The concentration ratio of two forms of PTX was showed in Table 5. After PTX injection (5 min), except for the heart and kidney, PTX nanocrystals accumulated more than the PTX commercial formulation. In the spleen, PTX nanocrystal accumulation was significantly greater (p50.001), specifically 45-fold greater than commercial PTX. At 30 min and 2 h, PTX nanocrystals accumulated more than the commercial injection in the spleen and liver, but nanocrystals were lower in the heart, kidney, lung and blood. PTX in the liver and spleen was continuously higher after nanocrystal administration until 24 h compared to commercial injection. Nanocrystals accumulated in the liver and spleen at 24 h and this was 4 times greater than the commercial formulation. So, IV administration of PTX nanocrystals offered greater liver and spleen absorption compared to commercial PTX injection. Thus, PTX nanocrystals may target these two organs. A lower accumulation of nanocrystals in the heart and kidney (until 8 h) may reduce toxicity in these areas. Both formulations accumulated similarly in the lung.
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Figure 5. In vitro release of PTX nanocrystals and material in 0.5% SDS (n ¼ 3).
100 Amount dissolved (%)
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Stability study
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Figure 6. Size stability of PTX nanocrystals. (A) Dilution stability, (B) temperature stability and (C) storage stability.
Figure 7. Mean plasma drug concentration curves versus time in mice after IV PTX nanocrystals and commercial PTX injection (n ¼ 6).
25000 nanocrystals Concentration of paclitaxel (ng/ml)
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Discussion PTX nanocrystals were prepared by anti-solvent precipitation via the addition of poloxamer 407 and DOSS. Ethanol was the chosen solvent due to low toxicity, water miscibility and appropriate PTX solubility. Addition of ethanol to a non-solvent with rapidly stirring, dissolved PTX rapidly formed small crystals due to supersaturation. Nanocrystal particle uniformity and size depended on stirring, rate of oil injection, and ultrasonic power and time. Ethanol slowed nanocrystal dissolution which can decrease jZPj value and increase system instability.
Ethanol removal and separation of the nanocrystals from surfactants reduced polymer toxicity. Combined with X-ray diffraction and DSC data, the reduction of phase transition of PTX nanocrystals depicted a decreased melting point, due to less energy of the amorphous crystalline structure. PTX nanocrystals have greater solubility in water and in a neutral environment. This phenomenon can be explained by the relationship between the saturation solubility of a drug and its particle size, which is described by the equation: log (Cs/Ca) ¼ 2sV/2.303RTr30. Here, Cs is the saturation solubility, Ca is the solubility of the solid consisting of large particles
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Table 4. Pharmacokinetic parameters in mice after intravenous injection of PTX nanocrystals and PTX injection (mean ± SD; n ¼ 6).
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Table 5. The concentration ratio of two forms of PTX (n ¼ 6). Nanocrystals/injection
Parameters
Unit
PTX nanocrystals
A B Vz t1/2,a t1/2,b AUC(0–1) CL MRT(0–1)
min1 min1 l kg1 Min min mg min l1 l min1 kg1 min
0.24 ± 0.012 0.010 ± 0.002 10.48 ± 0.41 2.91 ± 0.067 69.41 ± 0.73 276 700 ± 960 0.036 ± 0.011 86.3 ± 3.1
PTX injection 0.19 ± 0.013 0.013 ± 0.002 2.41 ± 0.11 3.70 ± 0.063 53.94 ± 0.62 464 160 ± 710 0.022 ± 0.010 80.0 ± 2.0
Time 5 min 30 min 2h 8h 24 h
Liver
Spleen
Heart
Kidney
Lung
Blood
1.27 1.13 1.47** 1.97*** 4.19**
5.20*** 1.78*** 1.45 2.00** 6.56**
0.41*** 0.63*** 0.64*** 0.95 0.58
0.68* 0.76*** 0.70** 1.05 1.34
1.05 0.87 0.95 1.71* 0.94
0.86 0.35*** 0.24*** 0.58*** 1.29
*p50.05, **p50.01, ***p50.001, statistical significance compared with injection.
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dc/dt ¼ (DA) (Cs Cx)/h30; here A is the surface area, Cs is the saturation solubility and Cx is the bulk concentration. The surface area of a particle increases drastically with size reduction, so nanocrystals rapidly dissolved. In addition, the large proportion of amorphous crystalline structure in nanocrystals has higher solubility and dissolution rate, which can also explain this phenomenon. Storage stability study showed that increased PDI may be attributed to widening of particle size distribution. Due to the Ostwald ripening phenomenon31, small particles are dissolved and molecules are re-deposited into larger particles and this phenomenon is correlated with temperature, which increases the speed of thermal motion, accelerating this phenomenon. Therefore, storage temperature affected nanocrystal stability and storage at 4 C was optimal, or prepared as a lyophilized powder to store. Pharmacokinetics and tissue distribution study showed that nanocrystals may target the liver and spleen. As abundant reticular endothelial system (RES) exist in the liver and spleen, which can phagocytize the particle in 0.2 3 mm. Thus, a majority of PTX nanocrystals were accumulated in the two organs though phagocytosis because of their nanoparticle size. As such, a lower accumulation of nanocrystals in the heart and kidney may reduce toxicity in these areas. Meanwhile, as some research proved that particles 47 microns are usually intercepted by pulmonary capillary bed, then into the lung tissue or lung bubble32. That both formulations accumulated similarly in the lung may be explained by the relatively small and uniform particles of PTX nanocrystals.
Conclusions
Figure 8. Tissue distribution in mice after 5 and 30 min, 2, 8 and 24 h of PTX nanocrystals and commercial PTX injection. Each bar represents the mean ± SE (n ¼ 6).
In conclusion, this study selected PTX as a model drug for a new nanocrystal drug delivery system. PTX nanocrystals were prepared via anti-solvent precipitation, with a rod-like morphology and a higher potential. Poloxamer 407 and DOSS were utilized as stereo and potential protectant, respectively, to obtain the stable nanosuspensions. During the formulation process, the drug molecule was transformed into an amorphous crystalline structure which had a decreased phase transition temperature. PTX nanocrystals can provide a variety of benefits including nanoparticle size, high solubility and quick release rate. PTX nanocrystals were stable in low temperature. Pharmacokinetic and tissue distribution assays indicated that nanocrystals were rapidly distributed with a longer elimination phase, chiefly accumulated in the liver and spleen because of their nanoparticle size, and effectively reducing cardiovascular and renal toxicity. Paclitaxel nanocrystals may represent a promising formulation for reducing side effects in cancer treatment.
Declaration of interest and r is the radius of particle. A decrease in particle size (r) results in an increase in saturation solubility (Cs) of the drug. Meanwhile, data were obtained with the equation
This work was supported by the National Key Technologies Research and Development Program for New Drugs of China (No 2012ZX09301003001-009 and No 2013ZX09J13104-02B).
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References 1. Wani MC, Taylor HL, Wall ME, et al. Plant antitumor agents. VI. The isolation and structure of Taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 1971;93: 2325–7. 2. Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by Taxol. Nature 1979;277:665–7. 3. Schiff PB, Horwitz SB. Taxol stabilizes microtubes in mouse fibroblast cells. Proc Natl Acad Sci 1980;77:1561–5. 4. Spencer CM, Faulds D. Paclitaxel. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer. Drugs 1994;48:794–847. 5. Dorr RT. Pharmacology and toxicology of Cremophor EL diluent. Ann Pharmacother 1994;28:S11–14. 6. Weiss RB, Donehower RC, Wiernik PH, et al. Hypersensitivity reactions from taxol. J Clin Oncol 1990;8:1263–8. 7. Rowinsky EK, Eisenhauer EA, Chaudhry V, et al. Clinical toxicities encountered with paclitaxel (Taxol). Semin Oncol 1993;20:S1–5. 8. Stinchcombe TE. Nanoparticle albumin-bound paclitaxel: a novel Cremphor-EL-free formulation of paclitaxel. Nanomedicine (Lond) 2007;2:415–23. 9. Nyman DW, Campbell KJ, Hersh E, et al. Phase I and pharmacokinetics trial of ABI-007, a novel nanoparticle formulation of paclitaxel in patients with advanced nonhematologic malignancies. J Clin Oncol 2005;23:7785–93. 10. Vishnu P, Roy V. Safety and efficacy of nab-paclitaxel in the treatment of patients with breast cancer. Breast Cancer (Auckl) 2011;5:53–65. 11. Xu X, Wang L, Xu HQ, et al. Clinical comparison between paclitaxel liposome (LipusuÕ ) and paclitaxel for treatment of patients with metastatic gastric cancer. Asian Pac J Cancer Prev 2013;14:2591–4. 12. Lim WT, Tan EH, Toh CK, et al. Phase I pharmacokinetic study of a weekly liposomal paclitaxel formulation (genexol-PM) in patients with solid tumors. Ann Oncol 2010;21:382–8. 13. Wang X, Zhou J, Wang Y, et al. A Phase I clinical and pharmacokinetic study of paclitaxel liposome infused in non-small cell lung cancer patients with malignant pleural effusions. Eur J Cancer 2010;46:1474–80. 14. Sahu PK, Mishra DK, Jain N, et al. Mannosylated solid lipid nanoparticles for lung-targeted delivery of Paclitaxel. Drug Dev Ind Pharm 2014. [Epub ahead of print]. 15. Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm 2002;235:179–92. 16. Moghimi SM, Hamad I, Andresen TL, et al. Methylation of the phosphate oxygen moiety of phospholipid-methoxy (polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production. FASEB J 2006;20: 2591–3.
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17. Szebeni J. Complement activation-related pseudoallergy: a new class of drug-induced acute immune toxicity. Toxicology 2005;216: 106–21. 18. Szebeni J, Muggia F, Gabizon A, Barenholz Y. Activation of complement by therapeutic liposomes and other lipid excipientbased therapeutic products: prediction and prevention. Adv Drug Deliv Rev 2011;63:1020–30. 19. Junghanns JU, Mu¨ller RH. Nanocrystals technology, drug delivery and clinical applications. Int J Nanomedicine 2008;3:295–309. 20. Gao L, Liu G, Ma J, et al. Application of drug nanocrystals technologies on oral drug delivery of poorly soluble drugs. Pharm Res 2013;30:307–24. 21. Gao L, Zhang D, Chen M. Drug nanocrystals for the formulation of poorly soluble drugs and its application as a potential drug delivery system. J Nanopart Res 2008;10:845–62. 22. Ganta S, Paxton JW, Baguley BC, Garg S. Formulation and pharmacokinetic evaluation of an asulacrine nanocrystalline suspension for intravenous delivery. Int J Pharm 2009;367:179–86. 23. Shegokar R, Mu¨ller RH. Nanocrystals: industrially feasible multifunctional formulation technology for poorly soluble actives. Int J Pharm 2010;399:129–39. 24. Liu F, Park JY, Zhang Y, et al. Targeted cancer therapy with novel high drug-loading nanocrystals. J Pharm Sci 2010;99:3542–51. 25. Wang Y, Jiang G, Qiu T, Ding F. Preparation and evaluation of paclitaxel-loaded nanoparticle incorporated with galactose-carrying polymer for hepatocyte targeted delivery. Drug Dev Ind Pharm 2012;38:1039–46. 26. Hirun N, Sangfai T, Tantishaiyakul V. Characterization of freezedried gallic acid/xyloglucan. Drug Dev Ind Pharm 2013. [Epub ahead of print]. 27. Wang Y, Li X, Wang L, et al. Formulation and pharmacokinetic evaluation of a paclitaxel nanosuspension for intravenous delivery. Int J Nanomedicine 2011;6:1497–507. 28. Zhu Z, Li Y, Li X, et al. Paclitaxel-loaded poly (N-vinylpyrrolidone)-b-poly(epsilon-caprolactone) nanoparticles: preparation and antitumor activity in vivo. J Control Release 2010;142:438–46. 29. Belete A, Metz H, Mueller T, Maeder K. Benchtop MRI for pharmacokinetic evaluation of two aqueous-based nano-scaled formulations of oleic acid stabilized magnetite nanocrystals. Drug Dev Ind Pharm 2013. [Epub ahead of print]. 30. Nagarwal RC, Kumar R, Dhanawat M, et al. Nanocrystal technology in the delivery of poorly solubility drugs: an overview. Curr Drug Deliv 2011;8:398–406. 31. Mu¨ller RH, Jacobs C. Buparvaquone mucoadhesive nanosuspension: preparation, optimisation and long-term stability. Int J Pharm 2002; 237:151–61. 32. Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery: the need for precision in reporting particle size parameters. Eur J Pharm Biopharm 2008;69:1–9.
RESEARCH ARTICLE
Preparation, physical characterization and pharmacokinetic study of paclitaxel nanocrystals
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Lisha Wei1, Yanxia Ji2, Wei Gong1, Zhenqiao Kang2, Meng Meng3, Aiping Zheng1, Xiaoyan Zhang1, and Jianxu Sun1 1
Department of Pharmaceutics, Beijing Institute of Pharmacology and Toxicology, Beijing, P.R. China, 2Handan Central Hospital, Hebei Province, P.R. China, and 3Department of Pharmaceutics, School of Pharmacy, Hebei Medical University, Hebei, P.R. China
Abstract
Keywords
Paclitaxel (PTX) is a natural broad-spectrum anticancer drug with poor aqueous solubility. PTX nanocrystals were formulated to improve the water solubility, and PTX nanosuspensions were prepared using anti-solvent precipitation, and then organic solvent and surfactants were removed by filtering through a vacuum system. The physical characterization of PTX nanocrystals were measured by transmission electron microscope, X-ray diffraction and differential scanning calorimetry. In addition, saturation solubility, in vitro release, stability and pharmacokinetic characteristics were examined. The average particle size of PTX nanocrystals was 200 nm, and they had a stable potential and a uniform distribution. Paclitaxel nanocrystals can effectively improve drug solubility and in vitro release. PTX pharmacokinetic and tissue distribution studies were compared after intravenous administration of nanocrystals versus a commercial injection formulation. PTX nanocrystals were rapidly distributed with a longer elimination phase. Moreover, tissue distribution indicated that PTX nanocrystals are mainly absorbed by the liver and spleen and may offer reduced renal and cardiovascular toxicity which may reduce side effects.
Nanocrystals, paclitaxel, pharmacokinetics, tissue distribution, X-ray diffraction
Introduction Paclitaxel (PTX) is a complex secondary metabolite originally developed by Wani et al.1 derived in 1971 from the bark of Taxus chinensis (Taxus brevifolia). PTX promotes microtubule assembly in cells which interfere with various cellular functions, especially cessation of mitotic cell division, blocking normal cell division2,3. According to phase II and III clinical studies, PTX is chiefly indicated for ovarian, breast and non-small cell lung cancer, and may be useful for esophageal, head and neck cancers4. PTX has low aqueous solubility and is chiefly administered by injection after dissolving PTX in polyoxyethylene castor oil (Cremophore EL) and ethanol (1:1). However, many studies suggest that Cremophore EL in this PTX injection can cause gastrointestinal and allergic reactions, hypotension, renal and cardiac toxicity, and bone marrow suppression, among other toxic reactions, greatly limiting this application5,6. At this time, pretreatment for allergic reactions include oral dexamethasone, intramuscular or oral diphenhydramine, or intravenous cimetidine or ranitidine, and these also cause adverse reactions6. Specifically, adverse reactions occur in 30% of patients7. Therefore, the design of safe, effective and convenient PTX formulation would be of significant clinical value. A PTX albumin suspension injection (Abraxane) lacking Cremophor EL
Address for correspondence: Aiping Zheng, Department of Pharmaceutics, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing 100850, P.R. China. Tel: +86 10 66931694. Mob: +86 13520467936. E-mail: [email protected]
History Received 12 May 2014 Revised 25 July 2014 Accepted 27 July 2014 Published online 26 August 2014
was made available in 20058 and this low-toxicity formulation did not require allergy pretreatment9. However, studies suggest that Abraxane to treat breast cancer in Phase III clinical trials caused more sensory neuropathy compared with the group not treated with the experimental drug10. A PTX liposome for injection (L-PTX) was approved by the CFDA (China Food and Drug Administration) and was marketed in 200311. A PTX liposome drug carrier system is presently in clinical trials worldwide12–14. However, the encapsulation rate of PTX liposome is low along with PTX leak15. Recent studies indicate that a larger liposomal particle radius of liposomes and an abundance of cholesterol in the liposome lipid bilayer can activate the complement system to cause a pseudo-allergy (CARPA)16, capable of damaging the heart and lung, thereby limiting clinical PTX liposome applications17,18. In the late 1990s, a nanoparticle drug delivery system was developed19. Nanocrystals need no carrier material and consist of a colloidal dispersion system of submicron particles formed from pure drug with no constrained of encapsulation efficiency. The adjustable dose is broad, and the stability of nanocrystals relies on potential and dimensional protective agents19. As a new formulation technique, nanocrystals can be used for enhancing oral bioavailability of poorly soluble drugs20, and nanocrystals can be suspended in water (nanosuspensions) for IV or pulmonary drug delivery21,22. Two approaches are used to prepare nanocrystals: top–down and bottom–up technologies23. Top–down technologies mainly consist of bead/pearl milling and high-pressure homogenization. No organic solvent is needed, and it is especially suitable
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for drugs that are insoluble both in aqueous and organic phases. The bottom–up – or precipitation – process involves the dissolution of the drug in a solvent to create a homogeneous precipitation by mixing with a non-solvent. Nanocrystal technology is promising for poorly soluble drugs and the preparation is universal and simple, with applicability to most drugs. Industrial production is also highly feasible. Great interest has been generated for formulating poorly soluble drugs into nanocrystals, specifically anticancer agents24. We developed a precipitation process for preparing PTX nanocrystals and we characterized nanocrystal particle size, morphology and crystalline structure as well as documented pharmacokinetics.
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Physical characterization Particle morphology The particle dimension and morphology was observed with transmission electron microscope (TEM) (H-7650; Hitachi, Tokyo, Japan), with an accelerating voltage of 80 kV. Freshly prepared PTX nanocrystals (5 ml) were dropped onto a 300-mesh carbon-coated copper grid and incubated (5 min) at room temperature. Grids were then negatively stained with 5 ml 3% uranyl acetate for 5 min, and then air-dried. The crystallographic morphology of PTX nanocrystals was observed using TEM under which images were obtained. X-ray diffraction
Methods Drug Dev Ind Pharm Downloaded from informahealthcare.com by University of Maastricht on 10/27/14 For personal use only.
Materials PTX (purity 498%) material and Commercial PTX injection was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. (Zhejiang, China). Docetaxel was provided by Guilin Huiang Biochemical Pharmaceutical Co. Ltd. (Guilin, China). Ethanol and glacial acetic acid (analysis grade) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Poloxamer 407 was provided by BASF (Florham Park, NJ). Docusate sodium (DOSS) and lauryl sodium sulfate (SDS) were purchased from Beijing Fengli Jingqiu Commercial and Trade Co. Ltd. (Beijing, China). Methanol and acetonitrile (HPLC grade) were from Fisher Scientific (Pittsburgh, PA). Methyl tertiary butyl ether (MBTE, analysis grade) was provided by Beijing Modern Oriental Fine Chemicals Co. Ltd. (Beijing, China). Preparation of PTX nanocrystals Preparation method PTX nanocrystals were prepared by anti-solvent precipitation. Firstly, 50 mg PTX was dissolved in 1 ml of ethanol (50 mg/ml) to form the organic phase. Two surfactants (poloxamer 407 and DOSS) were dissolved in 10 ml water to form the aqueous phase. Then the aqueous phase was rapidly stirred at 1200 rpm for 5 min at room temperature, after which 1 ml of the organic phase was injected into the aqueous phase under rapid stirring (1200 rpm) for 5 min. The mixed phases were sonicated (KQ3200DE sonicator from Kun Shan Sonicator Company, Kun Shan, China) at 80 W for several min. The final product was filtered by a vacuum system through a 0.22-mm filter membrane and nanocrystals were washed in 10 ml water 3 times to remove ethanol and most surfactant. Nanocrystals were re-dispersed in 10 ml water and sonicated to create stable PTX nanosuspensions (5 mg PTX/ml H2O). PTX nanosuspensions were lyophilized into dry powder (LGJ18C freeze-dryer, Si Huan Instrument Factory, Beijing, China). In brief, nanosuspensions were dispensed into a vial, and protectant (5%, w/v, g/ml) was added. Samples were frozen at 70 C. Then, the vial was transferred to a freeze-dryer and dried at 45 C for 40 h with a 30-Pa vacuum. The freeze-dried products were used for X-ray diffraction and differential scanning calorimetry (DSC) experiments or re-dispersed in 0.9% saline for IV administration. The re-dispersion was sonicated at 80 W for 1–5 min before use. Particle size and zeta potential The particle size and the polydispersity index (PDI) were measured by dynamic light scattering (DLS; Malvern Zeta Sizer, Nano-ZS90, Worcestershire, UK), and the ZP (Zeta potential) values were measured. Before DLS measurements, each sample was diluted and sonicated at 80 W for 10 s25.
The X-ray diffraction patterns for freeze-dried nanocrystals products were obtained through an X-ray power diffractometer (D/MAX 2000; Rigaku, Japan). The X-ray source was Cu filtered flat graphite monochromator, with a wavelength of 1.5406 A. The sample amount was 50 mg. The 2 range scanned was 2.0–40 at a rate of 4 /min. The step size26was 0.015. DSC analysis The DSC analysis for freeze-dried nanocrystals products was by differential scanning calorimeter (Q2000; New Castle, DE). The temperature scope was from 40 C to 250 C, heating at 5 C/min with a N2 flow rate of 20 ml/min (sample 50 mg). Saturation solubility of PTX nanocrystals To measure the saturation solubility of PTX, 0.5 ml suspensions of PTX nanocrystals (5 mg PTX/ml H2O) were placed in a 25-ml bottle to which 2 ml water was added. In addition to this, 0.5% lauryl sodium sulfate (SDS), 1.0% SDS and PBS (pH 7.4) was used as solvent (final PTX ¼ 1 mg/ml). After shaking, the mixed solution was ultrasonicated to achieve supersaturated state. The supersaturated solutions were shield from light and airtight before being placed in the shaker. Afterwards, solutions were shaken at 200 rpm at 37 C under constant shaking. After 24 h, 1 ml of the solution was centrifuged at 12 000 rpm for 5 min. The supernatant was filtered through a 0.45-mm filter membrane, and the filtrate was analyzed with HPLC analysis to measure PTX. For comparison, the PTX material was also measured the same way. Final PTX was 1 mg/ml and all experiments were performed in triplicate. In vitro release of PTX nanocrystals To measure in vitro release, a ZRS-8G dissolution tester (Tianjin Xinzhou Technology, Tianjin, China) was used. Then, 1 ml of the 5 mg/ml PTX nanocrystal suspension was pulled into a 2-ml needle and then injected into 200 ml 0.5% SDS at 37 C under constant revolution (50 rpm). At a predetermined time, 2 ml of the dissolution medium was centrifuged at 12 000 rpm for 5 min, and 2 ml of fresh medium was added subsequently. Supernatant was filtered through a 0.45-mm filter membrane, and the PTX in the filtrate was measured using HPLC. Also, 5 mg of PTX material dispersed in 1 ml water was injected into 200 ml 0.5% SDS, and PTX was quantified. All experiments were performed in triplicate. Stability study Dilution stability The distribution of particle size in PTX nanocrystals was measured by dynamic light scattering (DLS; Malvern Zeta Sizer, Nano-ZS90, Worcestershire, UK). To study the effect of
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dilution on size stability, 1 ml of original nanosuspension sample (5 mg PTX/ml H2O), a 10-fold diluted sample, a 50-fold diluted sample and a 100-fold diluted sample were measured. Temperature stability To study the effect of temperature on stability of PTX nanocrystals, 4 C, room temperature, 30 C and 37 C were selected as a temporary placement condition. Particle size changes were measured at 0, 24 and 72 h. Storage stability
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PTX nanocrystal suspensions were stored at 4 C or room temperature. Periodically, samples were removed and sample particle sizes were measured at 0, 0.25, 0.5, 1, 1.5, 2, 3, 6 and 9 months. Pharmacokinetics and tissue distribution study Kun Ming female mice (16–20 g) were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China). PTX nanocrystals were lyophilized into dry powder and suspended in 0.9% saline (PTX ¼ 1 mg/ml). Commercial PTX injection was diluted in 0.9% saline to 1 mg/ml. PTX nanocrystals and the commercial injection were administered to the mice (N ¼ 6) via the tail vein (IV, 10 mg/kg). At predetermined time points (2, 5, 10 and 30 min, and 1, 2, 4, 8 and 24 h), blood was collected into heparinized test tubes and centrifuged immediately at 12 000 rpm for 5 min to obtain plasma. Mice were euthanized at predetermined time points (5 and 30 min, and then 2, 8 and 24 h) and the heart, liver, spleen, lung and kidneys were excised, washed, dried and weighed. Plasma and tissue samples were stored at 70 C for further analysis. Analysis of PTX HPLC analysis of PTX PTX concentrations in solution were detected with HPLC, comprised of a pump (Spectra System P2000, Thermo Separation Products, Fremont, CA) and an ultraviolet detector (Spectra System Uv1000, Thermo Separation Products). Using a column (5 mm, 250 mm 4.6 mm, reverse-phase C-18 Phenomenex). The mobile phase consisted of methanol, water and triethylamine (80:20:0.1, v/v), and the pH was adjusted to 6.3 with glacial acetic acid. The mobile phase was filtered through a 0.45-mm filter membrane before analysis. The column was eluted at a flow rate of 1.0 ml/min at room temperature. The PTX concentrations were measured with the UV detector at 228 nm (20 ml sample volume). LC-MS analysis of PTX To measure the pharmacokinetics and tissue distribution of PTX, LC-MS was used. The LC-MS consists of an Agilent 1260 Infinity (G1322A Vacuum Degassers, G1312C quaternary pump, G1367E an automatic sampler and a G1316A column temperature box, Palo Alto, CA) and MS (G6460A triple level four pole tandem mass spectrometry, Palo Alto, CA). An MP C18 column (3 mm, 2.1 mm 50 mm, Venusil) was used. The mobile phase was a gradient elution, which included A (0.1% acetic acid:acetonitrile ¼ 70:30) and B (0.1% acetic acid:acetonitrile ¼ 10:90). The elution procedure was as follows: 0–0.5 min, 25% B; 0.5–1.5 min, 100% B; 1.5–5 min, 25% B. The flow rate was 0.4 ml/min and the column temperature was 25 C (5 ml sample volume).
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For plasma samples, 100 ml of plasma was obtained and combined with 10 ml of 500 ng/ml docetaxel. Each sample was vortexed for 3 min (Vortex geniu-2, Scientific Industries, Bohemia, NY) after adding 1 ml MBTE, and then centrifuged (centrifuge 1–14, Sigma, Munich, Germany) at 14 000 rpm for 5 min. Organic supernatant was removed to clean test tubes and evaporated at 37 C under a stream of nitrogen with a speed vacuum concentrator (Labconco, Kansas City, MO). For tissue samples, 5 times the organ weight of 0.1% acetic acid was added and homogenated. Then, 100 ml of tissue homogenate was added to 10 ml 500 ng/ml docetaxel. Tissue samples were treated in the same way. Before sample introduction, residues were dissolved in 100 ml of the mobile phase and vortexed for 1 min, and then centrifuged at 14 000 rpm for 5 min. Next, 5 ml of supernatant was injected into the LC-MS for analysis. Statistical analysis Graphpad-Prism curves were generated for each result. In vitro release profile was processed using one-site binding for curve fitting. A two-tailed T test was used to determine significance among groups. A p value of 50.05 was considered to be a significant difference.
Results Nanocrystal preparation To prepare PTX nanocrystals by anti-solvent precipitation, surfactants were needed to maintain the stability of PTX nanosuspensions. Dimensional surfactant was poloxamer 407 (P407), and DOSS was selected as the potential surfactant. As seen in Figure 1(A), particle size varied with different proportions of surfactant. The smallest particle size ratio was 1:5. Using this 1:5 proportion of PTX/P407, different proportions of DOSS did not change ZP values (jZPj 20 mV). Figure 1(B) depicts particle sizes with each ratio of potential surfactant. Screening results showed that the particle size was the smallest at a ratio of 1:2. From these data, the final combination was poloxamer 407 and DOSS at a proportion of 1:5 and 1:2, respectively. Process optimization is depicted in Table 1 and included removal of organic reagents using two methods. First rotaryevaporation with a 40 C water bath under reduced pressure for 30 min was used and nanocrystals were re-dispersed in 10 ml water and sonicated for stabilization. Next, vacuum-filtration separated nanocrystals from surfactants. The final product was filtered through a vacuum system (0.22-mm filter membrane) and nanocrystals were washed in 10 ml water 3 times to remove ethanol and residual surfactants. Then, nanocrystals were re-dispersed in 10 ml water and sonicated to stabilize and obtain PTX nanosuspensions. Data show that after rotary evaporation, PTX re-suspensions were larger with respect to particles. The 40 C water bath may have caused nanocrystal aggregation. After vacuum-filtration to remove residual ethanol and re-dispersion, the PTX nanosuspension was more stable with smaller particles (jZPj425 mV). Ultimately, the optimal formulation conditions were determined, and PTX nanocrystals were prepared using a preparation method described in the section titled ‘‘Preparation method’’. PTX nanosuspensions were lyophilized to a dry powder and a uniform particle size (Table 2) with a sable ZP. Physical characterization Transmission electron microscope images of PTX nanocrystals after processing are depicted in Figure 2, revealing a smooth rodlike morphology (200–500 nm). PTX crystalline structures were obtained by X-ray diffraction (see Figure 3 for diffraction peaks
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for PTX material, pure surfactant dry powder and PTX nanocrystals plus dry powder). PTX alone had crystalline diffraction peaks and the product was chiefly crystalline with some amorphous crystalline structures. Comparing with pure surfactants, the peak profiles of PTX nanocrystals were highly
similar, but the crystal formations of the two samples had some difference. The phase transition temperature of PTX nanocrystals was obtained from DSC. Figure 4 endothermic peaks for PTX material, pure surfactant dry powder and PTX nanocrystals plus dry powder. The main PTX nanocrystal endothermic peak moved slightly to the right compared to pure surfactants and the peak was 209.86 C. After nanocrystal preparation, the main endothermic peak was reduced sharply to 165.77 C. DSC measurements indicted that the phase transition temperature of PTX nanocrystals decreased.
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Saturation solubility of PTX nanocrystals The saturation solubility of two PTX formulations was measured in four solvents. As shown in Table 3, nanocrystal saturation solubility was greater than PTX material in four solvents and this difference was statistically significant (p50.001) between nanocrystals and PTX material in water
Table 1. Particle properties of PTX nanosuspensions with process optimization (mean ± SD; n ¼ 3).
Parameter
Before removing organic reagents
After revolvingevaporation
355.7 ± 25.3
725.05 ± 157.6
0.218 ± 0.03 19.2 ± 2.7
0.389 ± 0.30 20.35 ± 4.9
Particle size (nm) PDI ZP (mV)
After vacuumingfilter 240.9 ± 33.1 0.145 ± 0.01 29.25 ± 2.8
Table 2. Particle properties of PTX nanocrystals with freeze-drying (mean ± SD; n ¼ 3).
Figure 1. The influence of different proportion of surfactants on particle size. (A) Different proportion of P407 and (B) different proportion of DOSS (n ¼ 3).
Parameter
Before freeze-drying
After freeze-drying
Particle size (nm) PDI ZP (mV)
226.9 ± 32.0 0.149 ± 0.02 28.7 ± 1.9
232.7 ± 11.6 0.167 ± 0.05 28.9 ± 2.3
Figure 2. Transmission electron microscopy of PTX nanocrystals (40 000). Scale bar: 500 nm.
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Figure 3. X-ray diffraction peaks of (A) PTX material, (B) pure surfactants dry powder and (C) PTX nanocrystals plus dry powder.
(which was 150 times more soluble) and PBS (pH 7.4) in which the solubility was 50 times greater. However, with the addition of the surfactant SDS (0.5%), the difference was not statistically significant between nanocrystals and PTX material. SDS had solubilizing effects that were similar for both PTX formulations (high solubility). Next, we tested this solvent for in vitro release of nanocrystals versus PTX material.
In vitro release of PTX nanocrystals Release data for PTX nanocrystals and PTX material are depicted in Figure 5. PTX nanocrystals released rapidly compared with the PTX material. Specifically, within 5 min, nanocrystals released 490% of the drug and this was maintained for 8 h. PTX material released 50% of the drug in the same time period.
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(A) 0.0
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Figure 4. DSC endothermic peaks of (A) PTX material, (B) pure surfactants dry powder and (C) PTX nanocrystals plus dry powder.
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Then, PTX material released 80% of the drug after 30 min, and 490% of the drug was released after 2 h, before reaching equilibrium.
The effect of dilution on size stability was depicted in Figure 6(A). Data show that particle size of every dilution multiple was 250 nm. All samples did not undergo particle size changes, indicating that dilution did not cause aggregation. Figure 6(B) shows that for four temperatures, PTX particle sizes changed at 30 C and 37 C (4300 nm). At 4 C and room temperature, particle size fluctuated narrowly. Data show that PTX nanosuspensions were sensitive to temperature changes. Long-term stability was then tested. PTX nanocrystals suspensions were stored at 4 C and at room temperature. Particle size changed as depicted in Figure 6(C), with size increasing with increased storage time. At the lower temperature (4 C), particle size changed little. At room temperature, particle size increased by 60% over 9 months. Thus, long-term storage should be at 4 C. Pharmacokinetics and tissue distribution study Pharmacokinetic analysis was carried out using DAS 2.2.1 pharmacokinetic software. Model discrimination was based on Akaike information criteria (AIC). Statistical significances for pharmacokinetics among the treatment groups were analyzed using a two-tailed T test. Analytical methods for plasma PTX measurements are established27–29. Figure 7 depicts plasma concentration–time data for PTX and a two-compartment model
Table 3. The saturation solubility of two forms of PTX (mean ± SD; n ¼ 3).
Solvent
Material solubility (mg/ml)
Nanosolubility (mg/ml)
Nano/material
Water pH 7.4 PBS 0.5% SDS 1.0% SDS
0.081 ± 0.041 0.023 ± 0.039 196.562 ± 5.405 385.331 ± 31.174
12.416 ± 0.861*** 1.181 ± 0.021*** 200.138 ± 20.245 429.957 ± 14.019
153.284 51.348 1.018 1.116
***p50.001, statistical significance compared with material.
describes these data (Table 4). Figure 7 shows that after IV administration of PTX nanocrystal or injection, plasma PTX rapidly declined for both. After administration (2 or 5 min), no significant differences were observed between the two formulations. However, PTX plasma concentrations of nanocrystals were significantly lower than the PTX injection group (p50.001) from 10 min to 8 h. Data show that both t1/2,a and t1/2,b indicated a relatively rapid distribution phase and a longer elimination phase for PTX nanocrystals compared to commercial PTX injection. The AUC(0–1) for PTX injection was 2-fold greater than PTX nanocrystals, but the MRT for PTX nanocrystals were longer than for PTX injection. Moreover, the Vz for PTX nanocrystals was 5-fold larger than the PTX injection formulation, and PTX nanocrystals cleared faster suggesting that PTX nanocrystals can be distributed more quickly to the surrounding organs. After 24 h, the plasma concentrations of both formulations were equivalent. Distribution of PTX in the blood and major organs was examined after the IV PTX nanocrystal injection and commercial PTX injection (10 mg/kg). As shown in Figure 8, IV nanocrystals distributed as follows (greatest to lowest concentration): liver4spleen4lung4kidney4heart. After IV commercial PTX administration, drug concentrated (high to low) as follows: liver4kidney4lung4spleen4heart prior to 30 min and liver4spleen4kidney4lung4heart after 2 h. Therefore, the highest drug concentration of two formulations was the liver, and the lowest drug concentration was in the heart. The concentration ratio of two forms of PTX was showed in Table 5. After PTX injection (5 min), except for the heart and kidney, PTX nanocrystals accumulated more than the PTX commercial formulation. In the spleen, PTX nanocrystal accumulation was significantly greater (p50.001), specifically 45-fold greater than commercial PTX. At 30 min and 2 h, PTX nanocrystals accumulated more than the commercial injection in the spleen and liver, but nanocrystals were lower in the heart, kidney, lung and blood. PTX in the liver and spleen was continuously higher after nanocrystal administration until 24 h compared to commercial injection. Nanocrystals accumulated in the liver and spleen at 24 h and this was 4 times greater than the commercial formulation. So, IV administration of PTX nanocrystals offered greater liver and spleen absorption compared to commercial PTX injection. Thus, PTX nanocrystals may target these two organs. A lower accumulation of nanocrystals in the heart and kidney (until 8 h) may reduce toxicity in these areas. Both formulations accumulated similarly in the lung.
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Figure 5. In vitro release of PTX nanocrystals and material in 0.5% SDS (n ¼ 3).
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Figure 6. Size stability of PTX nanocrystals. (A) Dilution stability, (B) temperature stability and (C) storage stability.
Figure 7. Mean plasma drug concentration curves versus time in mice after IV PTX nanocrystals and commercial PTX injection (n ¼ 6).
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Discussion PTX nanocrystals were prepared by anti-solvent precipitation via the addition of poloxamer 407 and DOSS. Ethanol was the chosen solvent due to low toxicity, water miscibility and appropriate PTX solubility. Addition of ethanol to a non-solvent with rapidly stirring, dissolved PTX rapidly formed small crystals due to supersaturation. Nanocrystal particle uniformity and size depended on stirring, rate of oil injection, and ultrasonic power and time. Ethanol slowed nanocrystal dissolution which can decrease jZPj value and increase system instability.
Ethanol removal and separation of the nanocrystals from surfactants reduced polymer toxicity. Combined with X-ray diffraction and DSC data, the reduction of phase transition of PTX nanocrystals depicted a decreased melting point, due to less energy of the amorphous crystalline structure. PTX nanocrystals have greater solubility in water and in a neutral environment. This phenomenon can be explained by the relationship between the saturation solubility of a drug and its particle size, which is described by the equation: log (Cs/Ca) ¼ 2sV/2.303RTr30. Here, Cs is the saturation solubility, Ca is the solubility of the solid consisting of large particles
Paclitaxel nanocrystals
DOI: 10.3109/03639045.2014.950272
Table 4. Pharmacokinetic parameters in mice after intravenous injection of PTX nanocrystals and PTX injection (mean ± SD; n ¼ 6).
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Table 5. The concentration ratio of two forms of PTX (n ¼ 6). Nanocrystals/injection
Parameters
Unit
PTX nanocrystals
A B Vz t1/2,a t1/2,b AUC(0–1) CL MRT(0–1)
min1 min1 l kg1 Min min mg min l1 l min1 kg1 min
0.24 ± 0.012 0.010 ± 0.002 10.48 ± 0.41 2.91 ± 0.067 69.41 ± 0.73 276 700 ± 960 0.036 ± 0.011 86.3 ± 3.1
PTX injection 0.19 ± 0.013 0.013 ± 0.002 2.41 ± 0.11 3.70 ± 0.063 53.94 ± 0.62 464 160 ± 710 0.022 ± 0.010 80.0 ± 2.0
Time 5 min 30 min 2h 8h 24 h
Liver
Spleen
Heart
Kidney
Lung
Blood
1.27 1.13 1.47** 1.97*** 4.19**
5.20*** 1.78*** 1.45 2.00** 6.56**
0.41*** 0.63*** 0.64*** 0.95 0.58
0.68* 0.76*** 0.70** 1.05 1.34
1.05 0.87 0.95 1.71* 0.94
0.86 0.35*** 0.24*** 0.58*** 1.29
*p50.05, **p50.01, ***p50.001, statistical significance compared with injection.
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dc/dt ¼ (DA) (Cs Cx)/h30; here A is the surface area, Cs is the saturation solubility and Cx is the bulk concentration. The surface area of a particle increases drastically with size reduction, so nanocrystals rapidly dissolved. In addition, the large proportion of amorphous crystalline structure in nanocrystals has higher solubility and dissolution rate, which can also explain this phenomenon. Storage stability study showed that increased PDI may be attributed to widening of particle size distribution. Due to the Ostwald ripening phenomenon31, small particles are dissolved and molecules are re-deposited into larger particles and this phenomenon is correlated with temperature, which increases the speed of thermal motion, accelerating this phenomenon. Therefore, storage temperature affected nanocrystal stability and storage at 4 C was optimal, or prepared as a lyophilized powder to store. Pharmacokinetics and tissue distribution study showed that nanocrystals may target the liver and spleen. As abundant reticular endothelial system (RES) exist in the liver and spleen, which can phagocytize the particle in 0.2 3 mm. Thus, a majority of PTX nanocrystals were accumulated in the two organs though phagocytosis because of their nanoparticle size. As such, a lower accumulation of nanocrystals in the heart and kidney may reduce toxicity in these areas. Meanwhile, as some research proved that particles 47 microns are usually intercepted by pulmonary capillary bed, then into the lung tissue or lung bubble32. That both formulations accumulated similarly in the lung may be explained by the relatively small and uniform particles of PTX nanocrystals.
Conclusions
Figure 8. Tissue distribution in mice after 5 and 30 min, 2, 8 and 24 h of PTX nanocrystals and commercial PTX injection. Each bar represents the mean ± SE (n ¼ 6).
In conclusion, this study selected PTX as a model drug for a new nanocrystal drug delivery system. PTX nanocrystals were prepared via anti-solvent precipitation, with a rod-like morphology and a higher potential. Poloxamer 407 and DOSS were utilized as stereo and potential protectant, respectively, to obtain the stable nanosuspensions. During the formulation process, the drug molecule was transformed into an amorphous crystalline structure which had a decreased phase transition temperature. PTX nanocrystals can provide a variety of benefits including nanoparticle size, high solubility and quick release rate. PTX nanocrystals were stable in low temperature. Pharmacokinetic and tissue distribution assays indicated that nanocrystals were rapidly distributed with a longer elimination phase, chiefly accumulated in the liver and spleen because of their nanoparticle size, and effectively reducing cardiovascular and renal toxicity. Paclitaxel nanocrystals may represent a promising formulation for reducing side effects in cancer treatment.
Declaration of interest and r is the radius of particle. A decrease in particle size (r) results in an increase in saturation solubility (Cs) of the drug. Meanwhile, data were obtained with the equation
This work was supported by the National Key Technologies Research and Development Program for New Drugs of China (No 2012ZX09301003001-009 and No 2013ZX09J13104-02B).
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