新型纳米非病毒基因输送体系PCL-PEG-chitosan的构建与特性

Construction and characteristics of novel PCL-PEG-chitosan nano non-viral gene delivery system

目的优化新型纳米非病毒载体聚己内酯(PCL)-聚乙二醇(PEG)-壳聚糖(chitosan)(PCL-PEG-chitosan)在体外与质粒的复合条件,并进行转染活性测定。方法在体外利用凝胶电泳方法确定非病毒载体与携带绿色荧光蛋白基因的质粒(pEGFP)复合的最小氮磷比。利用动态光散射粒径测量仪确定复合物在不同氮磷比、不同缓冲溶液体系下粒径的变化情况。将pEGFP与PCL-PEG-chitosan进行复合后转染293T细胞,荧光显微镜观察转染效率。结果凝胶电泳结果显示,非病毒载体PCL-PEG-chitosan与pEGFP复合的最小氮磷比为4:1,PCL-chitosan与pEGFP复合的最小氮磷比也为4:1,chitosan与pEGFP复合的最小氮磷比为1:1。动态光散射实验结果显示:非病毒载体PCL-PEG-chitosan与pEGFP复合而成的复合物的粒径随着氮磷比的增大而减小;在醋酸盐缓冲体系与DMEM缓冲体系中,复合物的粒径基本一致,相对稳定。荧光显微镜观察发现,经过接枝改性的纳米载体PCL-PEG-chitosan的转染效率有所提高。结论 PCL修饰可减弱chitosan对pEGFP的复合能力,提高最低复合氮磷比。经PCL和PEG修饰的纳米载体PCL-PEG-chitosan的细胞转染活性有所提高,但尚需进一步改进。

Objective To optimize the complexation condition of the novel nano non-viral gene vectors of polycaprolactone （PCL）- polyethylene glycol （PEG）-ehitosan with plasmid in vitro, and evaluate the transfection activity of the complexes. Methods The minimum N/P ratio of the complexation of non-viral gene vector with plasmid carrying green fluorescent protein gene （pEGFP） was measured by gel electrophoresis in vitro. The particle sizes of the complexes under different N/P ratios and different buffering conditions were determined by dynamic light scattering method. The 293T cells were transfected with complexes of pEGFP with PCL-PEG-chitosan, and the transfection efficiency of complexes was observed under fluorescence microscope. Results The results of gel electrophoresis demonstrated that the minimum N/P ratio of complexation of PCL-PEG-chitosan with pEGFP was 4： 1, the minimum N/P ratio of complexation of PCL-chitosan with pEGFP was also 4：1, and the minimum N/P ratio of complexation of chitosan with pEGFP was 1：1. Dynamic light scattering test revealed that the particle sizes of the complexes of PCL-PEG-ehitosan with pEGFP decreased with the increase of N/P ratios, and the particle sizes in acetate buffer system and DMEM buffer system were similar and maintained stable. Fluorescence microscopic observation indicated that the transfection efficiency of PCL-PEG-chitosan improved to some extent. Conclusion PCL modification can attenuate the complexation ability of chitosan with pEGFP, and increase the minimum complexation N/P ratio. The transfection efficiency of PCL-PEG-chitosan improves after PCL and PEG modification, and needs to be further improved.