叶酸-壳聚糖介导survivin shRNA基因纳米载体的制备

Preparation of folic acid/chitosan-mediated gene survivin shRNA nano-composites

背景:叶酸-壳聚糖纳米粒是一种新型的高靶向纳米载体,可进一步提高药物的靶向性,并进一步实现缓释和控释给药。目的:探讨叶酸-壳聚糖纳米粒作为survivinshRNA重组质粒载体传递系统的可行性以及对大肠癌细胞(SW480)的转染效率。设计、时间及地点:对比观察实验,于2008-06/2009-01在中南大学卫生部纳米生物技术重点实验室完成。材料:壳聚糖(脱乙酰度>90%)由上海伯奥生物科技有限公司提供,叶酸由国药集团化学试剂有限公司提供,survivinshRNA重组质粒由上海吉凯基因化学技术有限公司提供。方法:首先制备粒径均匀的叶酸偶联壳聚糖纳米粒,然后将20mg/LsurvivinshRNA重组质粒和10mg/L叶酸-壳聚糖混合制备基因纳米复合物,同时以阳离子脂质体基因复合物作为对照,将上述两者转染大肠癌细胞。主要观察指标:基因纳米复合物的理化性质及转染效率;Westernblotting检测转染后肿瘤细胞中survivin蛋白的表达。结果:成功制备叶酸-壳聚糖介导的survivinshRNA重组质粒基因纳米复合物。该基因纳米复合物转染大肠癌细胞的效果强于阳离子脂质体基因复合物。且转染后大肠癌细胞中survivin蛋白的表达显著低于阳离子脂质体基因复合物。结论:叶酸-壳聚糖纳米粒作为载体系统能将survivinshRNA重组质粒高效递送到大肠癌细胞,从而诱导人大肠癌细胞的凋亡。

BACKGROUND： Folic acid-chitosan nanoparticle is a new nano-high-targeted vector, which can further improve drug targeting as well as the achievement of sustained-release and controlled-release drug delivery. OBJECTIVE： To study the feasibility of folic acid-chitosan nanoparticles as a recombinant survivin shRNA plasmid vector delivery system, as well as the transfection efficiency to colon cancer cells （SW480）. DESIGN, TIME AND SETTING： A contrast experiment was performed at the Key Laboratory of Nanobiotechnology, Ministry of Public Health, Central South University between June 2008 and January 2009. MATERIALS： Chitosan （deacetylating degree 〉 90%） was provided by Shanghai Bo＇ao Biological Technology Co., Ltd., folio acid was provided by sinopharm Chemical Reagent Co., Ltd., and survivin shRNA recombinant plasmid was provided by Shanghai GeneChem Co., Ltd. METHODS： Folate-conjugated chitosan nanoparticles with the uniform diameter were prepared, and then survivin shRNA （20 mg/L） recombinant plasmid and folic acid-chitosan （10 mg/L） were mixed to obtain nanocomposites. Meanwhile, cationic liposome gene complexes were considered as a control. Both the above-mentioned samples were used to transfect colorectal cancer cells. MAIN OUTCOME MEASURES： Physico-chemical property and transfection efficiency of nanocomposites and survivin protein expression in tumor cells after transfection using Western blotting. RESULTS： Folic acid-chitosan-mediated survivin shRNA recombinant plasmid nanocomposites were successfully prepared. The transfection efficiency of the nanocomposite was superior to that of cationic liposome gene complex to transfect colorectal cancer cells. In addition, survivin protein expression in the transfected colerectal cancer cells was less than cationic liposeme gene complex. CONCLUSION： Folic acid-chitosan nanoparticles as a vector system can high-effectively delivery survivin shRNA recombinant plasmid into colon cancer cells so as to induce the apoptosis of human colorectal cancer cells.