| In order to explore a new dosage form for the traditional antitumour drug,vinblastine nanoparticles were prepared with poly(buty1 cyanoacrylate) nanoparticles as a carrier. BT325 and C6 were served as models to investigate its antitumour activities, and quality nature and pharmacokinetic in rabbit were systematically studied.Part 1: The preparation of vinblastine nanoparticles.1. Analytical method establishment of vinblastine nanoparticles content determination. The article established analytical method of vinblastine nanoparticles content determination, and the method was systematically validated. The results showed that the good line range was emerged from 10 to 320μg·mL-1, and RSD of mean recovery, mean retention time, intra-day precision, inter-day precision respectively was 0.34%, 0.93% , 2.27%, and 3.33%. The HPLC analytical method for content determination of vinblastine nanoparticles was stable, reliable, and specificity, repetitiveness were good. The method would provide a platform for preparation of vinblastine nanoparticles.2. The preparation and quality evaluation of vinblastine nanoparticles. Vinblastine nanoparticles were prepared by emulsion polymerization process, and quality was evaluated by shape, size, drug entrapment efficiency, drug loading efficiency and stability. The results showed optimal preparation method as follow: pH=2, 1%(W/V)dextran70 , 0.5%(W/V)vinblastine, 1%(V/V)BCA, 600 r·min-1 stirring speed and stirring time 8 h. According to the optimal preparation method, mean diameter of vinblastine nanoparticles was 74.4 nm, and drug entrapment efficiency, loading efficiency was 78.39% and 39.20% respectively. Distribution of particle diameter is narrow. Nanoparticles'shape were globular, smooth and glossy without conglutination. Vinblastine nanoparticles were stable in room temperature when they were freeze-dried.3. The safety evaluation on vinblastine nanoparticles. Vinblastine nanoparticles'safety were systematically evaluated by haemolyticus test, cytotoxicity test and acute toxicity experiment in vitro and in vivo. The results showed vinblastine nanoparticles didn't emerge haemolysis and conglomeration. Proliferation inhibition rate of rat astrocyte in 5000 ng·mL-1 vinblastine nanoparticles group was (42.27±3.75)%, and degraded 9.56% compared with vinblastin physiologic saline. The difference was significant between vinblastine nanoparticles and vinblastin physiologic saline (P<0.05). Relative growth rate of L929 cells in 5000 ng·mL-1 vinblastine nanoparticles group was (30.07±1.44)%, and increased 9.62% compared with vinblastin physiologic saline. The difference was very significant between vinblastine nanoparticles and vinblastin physiologic saline (P<0.01). The median lethal dose of vinblastine nanoparticles was 26.38 mg·kg-1, and had a 16.21% enhancement compared with vinblastine physiologic saline. The results suggest that vinblastine nanoparticles degrade toxicity to organism.Part 2: Antitumour activities of vinblastine nanoparticles.1. The effects of vinblastine nanoparticles on the growth and apoptosis of tumour cell lines BT-325 and C6. Antiproliferation effects of vinblastine nanopraticles on tumour cell lines BT-325 and C6 were investigated by MTT assays, evaluation of growth curve, clone formation. The whole appearance of tumour cell lines BT325 and C6 were evaluated by inverted microscope and HE staining. Surface structure and ultramicrostructure changes were observed by transmission electron microscope and scanning electron microscope. The effects of vinblastine nanoparticles on tumour cell lines BT325 and C6 nucleolus morphous were observed by Hoechst33342 fluorescein stain. Microtubule morphous of tumour cell line C6 were observed by indirect immunofluorescence FITC stain. The results showed that IC50 of tumour cells lines BT325 and C6 in vinblastine nanoparticles group were 33.88 and 97.72 ng·mL-1, and degraded respectively 4.62 and 1.14 times compared with vinblastine physiologic saline group. Amount of tumour cells BT325 and C6 degraded significantly from the second day to the seventh day (P<0.05). Clone formation rate of tumour cell lines BT325 and C6 in vinblastine nanopaticles groups were 22.27% and 18.8%, and degraded respectively 9.31% and 5.68% compared with vinblastine physiologic saline group. The results of tumour cells morphology showed that amount of cells was distinctly degraded, and the space between two cells was greaten compared with vinblastine physiologic saline group. At the same time, a great deal of cells floated in culture fluid, and the shape of cells with shrinkage, round, nuclear dyeing deepen, and processes disappearances were multiple in vinblastine nanoparticles group. The results by electron microscope showed that cell's microvillus disappeared, cellular membrane perforated, chromosome adjoined verge, and vacuole emerged. Cells missed structure integrality, and presented typical apoptosis state. Apoptosis degree was more apparente than vinblastine physiologic saline group. Microtubule protein of tumour cell line C6 depolymerized and green fluorescence dispersed in the whole cell. Some tumour cells emerged shrinkage and processes disappearance. The results suggest that vinblastine nanoparticles strengthen antiproliferation effects and induction of apoptosis on tumour cell lines BT325 and C6.2. Inhibitory action research of vinblastine nanoparticles on tumour cell line BT325's nude mice transplantation tumor. Antitumour activity in vivo of vinblastine nanoparticles were studieded by measurement of transplantation tumor's volume, tumour's inhibition rate and observation of tumour's pathological section. Myelosuppression toxicity of vinblastine nanoparticles in nude mice were evaluated by measurement of peripheral blood leucocyte amount. The results showed that tumor weight in vinblastine nanoparticles group was (0.87±0.15) g, and tumour's growth inhibition rate increased 18.42% compared with vinblastine physiologic saline group. The difference is significant between vinblastine nanoparticles and vinblastin physiologic saline (P<0.05). Amount of typical cells with nuclear dyeing deepen was multiple, and there was apparent necrosis area in tumour's core compared with vinblastine physiologic saline group. Amount of peripheral blood leucocyte in vinblastine nanoparticles was (1.30±0.23)×109/L. It was 1.71 times to vinblastin physiologic saline, and the difference was very significant (P<0.01). The results suggest that vinblastine nanoparticles strengthen inhibition effects on tumour cell line BT325's nude mice transplantation tumor, and degrade myelosuppression toxicity in nude mice which vinblastine caused.3. Inhibitory action research of vinblastine nanoparticles on cell line C6 rats'transplantation tumor. Antitumour activity in vivo of vinblastine nanoparticles were further studied by measurement rats'mean survival time and observation of tumour pathological section. Myelosuppression toxicity of vinblastine nanoparticles in rats were evaluated by measurement of peripheral blood leucocyte amount. The results showed that mean survival time of tumor-bearing rats of vinblastine nanoparticles and tween80 coated vinblastine nanoparticles were (72.16±4.04) and (89.54±4.51) d, extended 35.31% and 67.90% respectively compared with vinblastine physiologic saline group. The difference was very significant (P<0.01). Results of tumour pathological section indicated that amount of blood vessel and tumour cells invasion brain tissue grew downwards after vinblastine nanopartiles treatment than vinblastine physiologic saline. Amount of peripheral blood leucocyte was (15.93±2.89)×109/L. It was 2.52 times to vinblastin physiologic saline, and the difference was significant (P<0.05). The results suggest that vinblastine nanoparticles strengthen inhibition effects on tumour cell line C6 rats'transplantation tumor, and degrade myelosuppression toxicity in rats which vinblastine caused. Part 3: The pharmacokinetics of vinblastine nanoparticles in rabbits.HPLC analytical method was firstly established in rabbits plasma in order to understand dynamic state in blood. The kinetic equation was fitted by method of residual, and then parameter of dynamics were calculated. Eventually, the differences were studied between vinblastine nanoparticles and physiologic saline. The results showed the HPLC analytical method for content determination of vinblastine nanoparticles in rabbits plasma was stable, reliable, and repetitiveness, specificity is good. The good line range was emerged from 10 to 320μg·mL-1, and RSD of mean recovery, mean retention time, intra-day precision, inter-day precision respectively was 1.54%, 1.25% , 3.23% and 2.65%. Result of pharmacokinetic studies showed that vinblastine nanopartcles and vinblastine physiologic saline were fit for the two room open model in rattits. Elimination half life of vinblastine nanoparticles was 9.071 h, and it was 2.39 times to vinblastine physiologic saline. Plasma clearance rate of vinblastine nanoparticles was 1/3.17 to vinblastine physiologic saline. AUC of vinblastine nanoparticles was 3.16 times to vinblastine physiologic saline.Preparation technology of vinblastine nanoparticles is feasible, and performance is stable. Vinblastine nanoparticles can significantly increase antitumour activity, and degrade toxicity and side effect of vinblastine. The research provides science foundation for clinic application of vinblastine repeatedly and long term. |
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