【作者】 朱陵君；

【导师】 刘宝瑞；

【作者基本信息】 南京中医药大学 ， 中西医结合肿瘤内科学， 2009， 博士

【摘要】 目的:探讨小檗胺(Berbamine,BA)与常用化疗药物紫杉醇(Paclitaxel,PTX)联合给药对于人胃癌细胞株的影响;并且采用自行合成的聚己内酯—聚乙二醇(methoxypoly(ethylene-glycol)-polycaprolactone,mPEG-PCL)二亲嵌段共聚物,通过纳米沉淀法,分别制备小檗胺载药纳米微球及小檗胺与紫杉醇双药载药纳米微球;通过考察其各项理化表征、释放性质以及体外协同抗肿瘤活性来明确载药纳米微球在肿瘤治疗中的优势,为这种新型的控释纳米给药系统的进一步开发提供科学根据。方法:应用中效原理(Median-effect Principle,Chou—Talalay联合指数法)进行统计分析,绘制小檗胺与紫杉醇单药及联合用药对肿瘤细胞生长的剂量-效应曲线,确定联合用药时对肿瘤细胞的效应(Fa)与合用指数(CI)的关系,判定药物间的相互作用。通过开环聚合法,制备mPEG-PCL二亲嵌段共聚物,采用优化的o/w乳化扩散/挥发法,制备负载小檗胺的纳米粒子;优化制备工艺并考察载体的结构、分子量、纳米粒子的形态、粒径分布、体外释药特性等性质。以MTT法,评价载药纳米微球在体外对于胃癌细胞株MKN-28、BGC-823的协同抗肿瘤活性。结果:第一,在一定的浓度范围内小檗胺单药与紫杉醇单药对BGC-823与MKN-28细胞均显示出了明显的生长抑制效果。当将各个对应浓度的两药联合作用于胃癌细胞株时生长抑制效果进一步提高,低浓度的小檗胺与紫杉醇的协同杀伤效果明显强于高浓度下的两药协同效果。第二,所制备的小檗胺和紫杉醇的单药及双药载药纳米粒子呈规整的球形,粒径小于100nm,小檗胺最高载药量为15.28%,包封率为87±3.4%。体外释放实验显示,载药粒子具有缓释特征。荧光标记粒子的细胞摄取实验证实,微球可通过胞吞作用进入细胞,在细胞内释放药物,这是微球不同于裸药的抗肿瘤机制。小檗胺载药纳米微球与裸药小檗胺对于肿瘤细胞的杀伤作用相差不大,且都具有浓度以及时间依赖性。第三,小檗胺与紫杉醇双药载药微球的粒径小于100nm。双药微球中紫杉醇的载药量为16.9±2.1%,包封率为16.9±2.1%;而双药微球中小檗胺的载药量与紫杉醇接近,为14.8±1.9%,包封率达到了84.7±7.4%。体外释放曲线显示双药微球表现为一个缓慢释放的特点。由细胞生存率曲线可以见到在BGC-823细胞系上,双药载药微球所造成的细胞杀伤明显强于同样浓度的紫杉醇单药,显示了小檗胺与紫杉醇被载入高分子微球后仍然具有良好的协同生长抑制效果。结论:小檗胺在体外胃癌细胞系上显示了其具有增强紫杉醇生长抑制效果的协同作用,而以小檗胺为模型药物构建的载药纳米微球及小檗胺与紫杉醇双药载药纳米微球具有一定的缓释特征,同时在体外也显示了一定的协同生长抑制作用,具有良好的应用前景和价值。更多还原

【Abstract】 Objectives: To evaluate the effect of Berbamine (BA) and Paclitaxel (PTX) on human gastric cancer cell lines. The nanoparticles loading BA or BA together with PTX were prepared from methoxy poly(ethylene-glycol)-polycaprolactone (mPEG-PCL) by nanopreticipation method. The physiochemical properties, release properties and the in vitro synergistic antitumor effect of the nanoparticles was evaluated to elucidate the superiorty of polymeric nanoparticles in the treatment of cancer and the prospect of this nanoscale controlled release drug delivery system.Methods: Median effect analysis was employed to determine the interaction between BA and PTX by analyzing the relationship between fraction affected (Fa) and the combination index (CI) acquired from the dose-effect curve. The amphiphilic block copolymer was prepared by ring opening polymerization. The o/w emulsion method was applied to prepare BA-loaded nanoparticles. The structure and molecular weight of the copolymer as well as the shape, diameter and in vitro release properties of the nanoparticles was studied. The synergistic antitumor effect of the nanoparticles on gastric cancer cell lines MKN-28 and BGC-823 was determined using MTT assay.Results: Both BA and PTX inhibited the growth of MKN-28 and BGC-823 cells. Synergistic effect of BA and PTX was observed, which was more obvious at lower concentrations. The BA-PTX nanoparticles was spherical with the diameter less than 100nm. The maximum loading content and encapsulating efficiency of BA were 15.28% and 87±3.4%, respectively. The nanoparticles exhibited sustained release pattern as to the in vitro release test. The nanoparticles entered cells via endocytosis, which was proved in the cellular uptake studies. BA nanoparticles showed similar cytotoxicity compared to BA free drug with concentration and time dependence. The BA-PTX nanoparticles was also smaller than 100nm. The maximum loading content and encapsulating efficiency of PTX were 16.9±2.1% and 16.9±2.1%, which were 14.8±1.9% and 84.7±7.4% as to BA. The release profile of the double nnaoparticles was also sustainable. As to the cytotoxicity against BGC-823 cell lines, the double nanoparticles showed much more prominent effectiveness compared to free PTX. This confirmed that BA and PTX showed satisfactory effect after being loaded into the copolymeric nanoparticles. Conclusions: BA can reinforce the antitumor effect of PTX on gastric cancer cell lines. At the same time, the BA-loaded and BA-PTX-loaded nanoparticles showed sustained release pattern as well as synergistic antitumor effect in vitro, which implied the prospect of this drug delivery system.更多还原