PEG化壳聚糖/DNA自组装复合物的制备、表征和体外Hela细胞转染研究

Preparation and Characterization of PEGylated Chitosan/DNA Self-assemble Complex and the Research on Transfection on Hela Cell in Vitro

通过有机合成和高分子聚合等方法将亲水性的聚乙二醇接枝到壳聚糖的氨基侧链上 ,得到了改性的壳聚糖 -聚乙二醇接枝共聚物 ,应用现代波谱等技术对中间产物和最终产物进行了表征 .采用绿色荧光蛋白基因质粒 p EGFP-N1为 DNA模型 ,在溶液中通过自动 (静电 )吸附得到 PEG化的壳聚糖 /DNA自组装复合物 .初步研究了该自组装复合物对 Hela细胞的体外转染效率 .结果表明 ,活化的聚乙二醇被成功地接枝到壳聚糖上 ,使不溶于水的壳聚糖改性为水溶性的 PEG化的壳聚糖 . PEG化壳聚糖 /DNA自组装复合物在Hela细胞体外转染率达到 81 % .因此 ,PEG化的壳聚糖有可能成为基因转染的非病毒载体 .

Chitosan, as a non-viral delivery system for gene therapy, has been increasingly proposed because of its biodegradable, cationic, non-toxic, good biocompatibility. As hydrophobic polymers are usually taken up by reticuloendothelial-system(RES) and have a short residence time in blood, hydrophilic, flexibleand non-ionic polymer, methoxypolyethyleneglycol(mPEG) was grafted to the amino group of chitosan to modify the chitosan′s property in this paper. mPEG-Chitosan is obtained through three steps of esterification reactions. The structures of the activated mPEG and mPEG-Chitosan were confirmed with 1H NMR, 13C NMR and Fourier transform infrared(FTIR) spectrum. The solubility of PEGylated chitosan in water is 53.6 mg/mL, which becomes dissolved in water while chitosan is undissolvable. The percentage of PEG grafted onto the amino group of chitosan was 16.71%(molar fraction). Plasmid pEGFP-N 1 was chosen as model DNA. The PEGylated chitosan/DNA complex was prepared by using an auto-coacervation process. The transfection efficiency of the PEGylated chitosan/DNA complex in Hela cells in vitro was 81% analyzed by flow cytometer. Thus, The PEGylated chitosan could be a candidate of effective non-viral vectors for gene delivery system.