Study On Long-circulation Thermosensitive Liposomes Loaded Vincristine

Background In 1962, Beeret al. extracted and separated the vinblastine and vincristine from catharanthus. After this, about more than 70 diferent alkaloids have be found from catharanthus. Vinblastine, vincristine, vinorelbine and vindesine are commonly used in clinical. In these alkoids, the anti-tumor activity of vincristine was strongest, while aslo being able to inhibit RNA polymerase activity and interfere the tubulin synthesis. Vincristine used for treatment of lymphoma and leukemia, but also for gastric cancer, liver cancer, ovarian cancer, breast cancer, soft tissue sarcoma, bronchogenic carcinoma and neuroblastoma, malignant tumor, in addition, vincristine also as an immunosuppressant with in platelet therapy for thrombocytopenic purpura. The ADR of vincristine is very common in clinical use due to its non-selectivity. Clinical often use combination therapy to reduce the dose of vincristine, which limits its effect.Liposome use phospholipid as main membrane material, like bursa bubble structure. The membrane of limposome like cell membrane, through this structure, lipid soluble and water-soluble drug could be loaded and increased the drug stability. Long circulation thermo-sensitive liposome is usually modified PEG basing on traditional liposme, containing thermosensitive lipids. Using PEG can reduce liposme bingding with plasma protein and avoid RES phagocytosis by increase steric. As the result, the liposomes in vivo circulation time was extended. In addition, by using temperature-sensitive phospholipid to prepare liposome can make it have phase transition temperature(TM). When the temperature is below TM the phospholipid bilayerperform like gel phase, as temperature approach TM the membrance change to liquid state. In this phase drug can be realeased quickly. A small amount of lysophospholipids in thermosensitive liposomes can improve the sensitivity to temperature, accelerated change of state in the phase transition temperature.Objective To solve vincristine some problems in clinical like adverse reaction ofdrugs, systemic toxicity, no targeting and improve clinical dose. By using new way to prepare long circulation thermosensitive liposome loaded vincristine, and expect to extend clinical application. Evaluatevincristine liposome and vincristine injection and compare difference. Explore liposome anti-tumor activity and toxicity.Contents By using film dispersion method and pH gradient loading method to prepare vincristine long circulating thermosensitive liposomes. To optimize prescription by particle size, determination of content, entrapment efficiency, thermal stability characteristics as the main evaluation index.Establish methods of determination of content and related substances using HPLC. The encapsulation efficiency of liposomes was determined by ultrafiltration centrifugation.By using transmission electron microscopy and laser scattering particle analyzer toobserve the liposome morphology and particle size.To track vincristine long circulating thermosensitive liposomes for more than 5 months stability using stability analyzer, and analyze relevant evaluation inde. Determination of in vitro cytotoxicity by MTT assay, to observe the fluorescence labeled liposomes of cell uptake using confocal laser scanning microscopy. Establish transplant tumor models on nude mice to evaluate anti-tumor activity and toxicity ofvincristine long circulating thermosensitive liposomes combined with heating.Results DPPC, DSPE-PEG2000, MSPC were membrance phospholipid. By optimizing hydration solution, compression thecnic, cryoprotectant, et al. determin film dispersion method was used to prepare the blank liposomes, and pH gradient method with 0.2M, pH 3.0 tartaric acid buffer.The average particle size of liposomes was about 90 nm, good appearance of liposome was observed by transmission electron microscope. The entrapment efficiency of liposomes was above 95%.The reproducibility tests show that the formulation of good reproducibility, the batch production of liposomes prepared indicators have reached expection.The liposome was stable in 5 months at -20℃. Vincristine long circulating thermosensitive liposomes with heating the activity of sw620 cell was less than 5% and in PANC cell was 20%, after 72 hours, in cell toxicity test. Heating can help cell uptake of liposome in cell experiment. In vivo experiments, the liposme shows obvious advantages of inhibiting tumor, and the tumor inhibition rate reached 70% and above.Conclusion In this study, we prepared vincristine sulfate long-circulation thermo-sensitive liposomes with good physical and chenmical properties. The preparation technique was stable and the reproducibility was good.Inhibition of tumorwas good, and expected to develop into a new drug.