Phospholipid was provided by Taiwei Pharmaceutical Factory (Shanghai, China, M n = 760.08). Polysorbate 80 was purchased from Shanghai Shenyu Pharmaceutical & Chemical Co. Ltd (Shanghai, China, M n = 1309.65). DTX (99.4%) was obtained from Shanghai Junjie Bio-Engineering Co. Ltd. (Shanghai, China, M n = 807.88). Heparinsodium injection, 10,000 IU/mL was purchased from Shanghai No. 1 Biochemical Pharmaceutical Co. Ltd. (Shanghai, China). Glucose solution 5% was obtained from Shanghai Baite Medical Product Co. Ltd. (Shanghai, China). Spectra-grade reagents were used as the mobile phase in high-performance liquid chromatography (HPLC) analysis, and all other reagents were analytical grade and used without further purification. Distilled and deionized water was used in all experiments.
Male Sprague-Dawley (SD, 8 weeks old, 200 ± 20 g) rats and female Kunming strain mice (8 weeks old, 20 ± 2 g) were obtained from Second Military Medical University of Chinese PLA. All the pathogen-free animals were acclimatized at a temperature of 25 ± 2°C and a relative humidity of 70 ± 5% under natural light/dark conditions for at least 24 h before dosing. The experiments were carried out in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Preparation of DTX-loaded Polysorbate 80/Phospholipid mixed micelles
DTX-loaded Polysorbate 80/Phospholipid mixed micelles were prepared by means of the self-assembly method as we described previously . Briefly, the response surface methodology was used to optimize the preparation of the mixed micelles: DTX (5 mg), Polysorbate 80 (125 μL), and Phospholipid (30 mg) were dissolved in 0.3 mL of dehydrated ethanol with the help of stirring at room temperature, then the homogeneous phase was injected rapidly into the 5% glucose solution in order to obtain a clear mixed micelle solution with a final volume of 10 mL, then the mixed micelles solution was filtrated through a 0.22-μm pore-sized membrane for further investigation. The final concentration of DTX is 0.62 mM, Polysorbate 80 is 10.16 mM, and Phospholipid is 3.95 mM.
The mean diameter and particle size distribution of the DTX-loaded Polysorbate 80/Phospholipid mixed micelles were determined by a particle-size analyzer (Mastersizer 2000, Malvern Instruments, Ltd., Malvern, U.K.), based on the laser dynamic light scattering technique. Sample solutions filtered through a 0.22-μm filter membrane were transferred into the light scattering cells. The intensity autocorrelation was measured at a scattering angle of 90° at room temperature. The morphological examination of micelles was performed using a JEOL JEM-2010 transmission electron microscope (TEM) at an acceleration voltage of 120 kV. In practice, one drop of solution with the sample was placed on a 400-meshes copper grid precoated with a carbon film and allowed to dry further for 30 min, then examined with the electron microscope.
An aliquot of DTX-loaded Polysorbate 80/Phospholipid mixed micelles was treated with four times volume of dehydrated ethanol to disrupt the micelle structure. Level of encapsulated DTX was measured using a reverse phase HPLC method. Stock solutions of DTX (0.2 mg/mL) were prepared by dissolving 10 mg of DTX in 10 mL dehydrated ethanol, followed by addition of 40 mL distilled water, and standard curve was set up with satisfactory linearity. The HPLC system consisted of a HP HPLC (3D) series equipped with G1322A online degasser and G1311A quaternary pump (Agilent Technologies, Palo Alto, CA, USA) was used. Chromatographic separations were achieved using a Diamonsil C18 column (5 μm, 250 × 4.6 mm, Dikma Technologies Inc, Lake Forest, CA, USA) at 25C. The mobile phase consisted of deionized water and HPLC grade acetonitrile [45:55 (V/V)]. The samples were delivered at a flow rate of 1 mL/min and detected at 230 nm using G1314A VWD detector (Agilent Technologies, Palo Alto, CA, USA).
Sprague-Dawley rats were used to examine the pharmacokinetics of DTX encapsulated in Polysorbate 80/Phospholipid mixed micelles. Rats were randomly divided into following two groups (n = 6, half male and half female): (1) DTX encapsulated in Polysorbate 80 micelles (75 mg/m2); (2) DTX encapsulated in Polysorbate 80/Phospholipid mixed micelles (75 mg/m2). Drugs were intravenously administrated trough the tail vein. The blood samples (0.5 mL) were collected into heparinized tubes via the femoral vein at 5, 15, 30 min, 1, 2, 4, 6, 8, and 12 h. The plasma was obtained by centrifugations at 900 × g for 10 min. Plasma samples were frozen and maintained at -20°C before analysis.
Tissue biodistribution study
To assess the effect of Polysorbate 80/Phospholipid mixed micelle formulation of DTX on tissue distribution, 72 female Kunming strain mice were randomly divided into two groups. The administration protocol of tissue distribution study was as same as that used in the pharmacokinetics. At 5, 15, 30 min, 1, 2, 3, 4, 6, and 8 h after drug injection, each animal (n = 4 for each time point) was euthanized and heart, spleen lung, liver, kidney, uterus and ovaries, brain as well as blood samples were collected. Tissue samples were blotted with paper towel, rinsed in ice-cold saline, blotted to remove excess fluid, weighed, and stored at -50°C until required for analysis.
Aliquots of 0.1 g tissue samples were minced into small pieces (1 mm3 on average), homogenized in the mixed solution of acetonitrile and water (50:50, V/V) with a ultrasonic Cell Disrupter System (JY92-, Ningbo Scientz Biotechnology Co., Ltd, Ningbao, China), and vortexed for 1 min. After centrifugation at 15000 × g for 3 min, the 100-μL clear supernatant was removed and extracted by 600-μL tert-butyl methyl ether, the organic phase was separated and evaporated under a gentle stream of nitrogen. Then, the residue was dissolved in 40 μL of acetonitrile, centrifuged at 1400 × g for 5 min, and aliquots of 20 μL were injected into the HPLC system. The concentrations of DTX in tissue samples were determined by the HPLC method as same as the analysis of the mice plasma samples.
Serum sample analysis
DTX levels in plasma and tissue were measured by reverse-phase HPLC method. Briefly, 200 μL of plasma was extracted twice with 200-μL tert-butyl methyl ether. The total clear organic layer was separated by centrifugation at 15000 × g for 3 min, and evaporated under a gentle stream of nitrogen. The residue was then dissolved by 40 μL acetonitrile, centrifuged at 1400 × g for 5 min, and aliquots of 20 μL were injected into the HPLC system. The rat plasma samples employed the mobile phase consisted of HPLC grade acetonitrile, deionized water, tetrahydrofuran, ammonium hydroxide solution (25%), and acetic acid solution (36%) [55:45:3:0.03:0.06 (V/V)]; the mice plasma samples employed the mobile phase consisted of HPLC grade acetonitrile, deionized water, and tetrahydrofuran [55:45:4 (V/V)]. All the analyses with a flow rate of 1.0 mL/min for the mobile phase, the retention time of DTX in rat plasma samples and mice plasma samples were approximately 10.7 and 11.2 min, respectively.
Rat blood was used to test the hemolysis effect of DTX-loaded Polysorbate 80/Phospholipid mixed micelles. Briefly, the fibrinogen was removed from 10 mL of rat blood by stirring the blood with glass rod. Ten milliliters of 5% glucose injection solution was added into defibrinogen blood sample, and supernatant was removed after centrifugation at 900 × g for 10 min. The erythrocyte pellets at the bottom of centrifuge tube were washed for four times (centrifugation followed by re-dispersion) with 5% glucose injection solution. Finally, after repeated washing and centrifugation, an adequate amount of 5% glucose injection was added to the erythrocyte pellets to give a 2% erythrocyte standard dispersion and stored at 4°C for further use. The DTX-loaded Polysorbate 80 micelles (Taxotere®), and DTX-loaded Polysorbate 80/Phospholipid mixed micelles were dispersed in 5% glucose injection solution with DTX concentration of 0.5 mg/mL and Polysorbate 80 of 1.25%, respectively. The different amounts of micelle solution with volume of 0.1, 0.2, 0.3, 0.4, and 0.5 mL (NO. 1, 2, 3, 4, and 5) were added into six tubes with 2.5 mL of 2% erythrocyte dispersion in each. Then adequate amounts of 5% glucose injection solution were added in every tube to obtain a final volume of 5 mL. Negative control (NO. 6) was 5% glucose injection, and positive control (NO. 7) was prepared by adding 2.5 mL of ultra- pure water into 2.5 mL of 2% erythrocyte dispersion instead of 5% glucose injection and micelle solution. After vortexing, the tubes were incubated at 37°C and observed microscopically from 15 min to 1 h. Then, the tubes were centrifuged at 900 × g for 10 min. At last, the optical density (OD) was obtained from a fast wavelength scanning between 200 and 1100 nm by a UV-spectrophotometer (11000, Beijing analysis Instrument Co., Ltd, Beijing, China) at 418 nm. The hemolysis ratio (HR) was calculated according to the equation: HR = [(ODt - ODn)/(ODp - ODn)] × 100%. Here, the ODt means the OD value of tested group, the ODn and ODp were OD value of negative and positive control, respectively.
Plasma protein binding test
Equilibrium retrodialysis was used to evaluate the plasma protein binding ability of DTX captured in Polysorbate 80 micelles and DTX captured in Polysorbate 80/Phospholipid mixed micelles, respectively. Firstly, 4-mL rat plasma was full mixed with 1.2 mL micelles solution for 0.5 h in 10 mL sealed glass cells with 120 rpm magnetic stirring at 36.5 ± 0.5°C, then precise ultra- pure water was added into the mixed solution for making up the loss of weight; secondly, dialysis bags (cellulosic membranes with molecular weight cut-offs of 10000 Da; Millipore, USA) filled with 0.5 mL 5% glucose injection were put into the sealed cells and the retrodialysis was carried out for 6 h at 36.5 ± 0.5°C with 120 rpm magnetic stirring in temperature controlled water bath. After cooling to the ambient temperature and making up the loss of weight with ultra-pure water, aliquots of 200 μL plasma and resulting dialysate were promptly recovered from the glass cells and analyzed by HPLC method mentioned above, respectively. The percentage of plasma protein binding rate of DTX (B%) was calculated as:
. The C
o and C
i were the drug concentrations in the plasma outside the dialysis bags and the dialysate inside the dialysis bags (ng/mL). 0.5 and 5.2 means the volume of the solution inside and outside the dialysis bags respectively (mL).
The compartment of model was simulated by 3p87 program (Practical Pharmacokinetic Program, 1987, China) and the parameters of pharmacokinetics were obtained. The calculation of AUC was based on statistical moment theory. The pharmacokinetic parameters were analyzed for statistical significance by unpaired Student's t- test. For this purpose, the level of significance was set at α < 0.05. In the tissue distribution studies, the AUC could not be determined in individual mice because of the destructive study design.