mPEG-CS纳米粒介导livinshRNA基因纳米复合物的制备及转染效率

Preparation and transfection efficiency of mPEG-CS/livin shRNA complex

背景:作为非病毒基因转染载体,由可降解的高聚物形成的纳米载体目前被广泛由于基因转染,因为他们具有良好的缓释性,靶向性和生物相容性。目的:制备mPEG-CS纳米粒,探讨mPEG-CS作为Livin shRNA基因转染载体的可行性。方法:通过离子交联法制备mPEG-CS纳米粒,利用聚乙二醇对壳聚糖进行改性,通过静电吸附法制备载livin shRNA的基因纳米复合物。Zeta-size分析仪和透射电镜检测空白纳米粒和载livin shRNA的基因纳米复合物的形态、粒径和zeta电位,测定基因纳米复合物的包封率,凝胶电泳阻滞实验和DNase I酶消化实验验证纳米粒对基因的保护作用。利用最佳条件下制备的基因纳米复合物,转染大肠癌HT-29细胞,考察转染效率。结果及结论:成功制备出约60nm的mPEG-CS纳米粒,当纳米粒与基因体积比为3∶1时,得到的基因纳米复合物形较规则,粒径100nm左右;其包封率为(94.32±0.35)%。凝胶电泳阻滞实验表明纳米粒能够紧密结合DNA,对基因具有良好的基因保护作用。该基因纳米复合物转染大肠癌细胞的转染效率高,持续作用时间长。mPEG-CS纳米粒作为基因转染载体,对基因具有保护作用,能够将livin shRNA重组质粒高效转染入大肠癌细胞,能够在大肠癌细胞内长时间表达,克服了RNA干扰在基因治疗肿瘤中基因作用时间较短的缺点。

BACKGROUND： Nanoparticles formulated from biodegradable polymers such as mono methyl poly （ethylene glycol）-g-chitosan are being extensively investigated as non-viral gene delivery systems due to their sustained release characteristics, targeting and biocompatibility. OBJECTIVE： To prepare mPEG-CS nanoparticles and to explore the feasibility of mPEG-CS as livin shRNA gene carrier, and to study the gene transfection efficiency to colorectal cancer cell HT-29 mediated mPEG-CS nanoparticles. METHODS： mPEG-CS nanoparticles were prepared by ionic cross-linking method and mPEGylated -chitosan/ livin shRNA complex by Static adsorption. The pattern, size and Zeta potential of blank nanoparticles and mPEGylated-chitosan/ livin shRNA complex were detected by Zeta-size analyzer and transmission electron microscope （TEM）. The encapsulation efficiency of gene-nano complex was measured. The protection of nanoparticles for gene was validated through Gel electrophoresis block experiment and DNase I enzyme digestion experiment. The transfection efficiency of livin shRNA was compared to that of CRC cell HT-29 mediated by mPEG-CS nanoparticles or not. RESULTS AND CONCLUSION： mPEG-CS nanoparticles of the size about 60 nm were successfully prepared out, when nanoparticles /gene volume ratio was 3∶1, size of gene-nano complex was 100 nm and encapsulation efficiency was （94.32±0.35）%. The gel electrophoresis blocking test showed that nanoparticles could be effectively combined with the plasmid, and Dnase I test proved that the nanoparticles could protect the plasmid. Transfection efficiency mediated by mPEG-CS was higher than naked gene and working a longer time. As gene carrier, mPEG-CS nanoparticles can protective gene well, and it is able to transfect livin shRNA recombinant plasmid into colorectal cancer cells and express in a long time. The gene-nano complex overcomes the shortage of relatively short time of RNA interference in gene therapy for tumor.