聚乙二醇-壳聚糖共聚物作为基因传递载体的体外研究

本文通过将单甲氧基聚乙二醇(mPEG)的端羟基氧化为醛基，进而与壳聚糖(CS)链节上的氨基反应，合成了聚乙二醇-壳聚糖(mPEG-CS)共聚物。用MTT法检验不同浓度共聚物对HeLa细胞和A549细胞的毒性，结果显示5~100 μg·mL^-1聚合物的细胞毒性较低。通过考察不同PEG取代度的共聚物与质粒DNA所形成复合物的粒径、zeta电位及凝胶阻滞分析，筛选出最佳共聚物为取代度3.55%的mPEG(3.55)-CS。将mPEG(3.55)-CS作为基因传递载体，介导绿色荧光蛋白基因(pEGFP-C1)转染HeLa细胞和A549细胞，荧光显微镜下观察到荧光蛋白的表达，流式细胞仪测定HeLa细胞与A549细胞的最高转染率分别为8.1%和4.8%，证实了mPEG-CS共聚物是一种有效的非病毒类基因传递载体。

In vitro study on polyethylene glycol-chitosan copolymer as a gene delivery vector

Chitosan and its derivatives are extensively studied as non-viral gene delivery vectors nowadays. Polyethylene glycol-chitosan (mPEG-CS) copolymers were synthesized by oxidation of mPEG-OH and then combined mPEG-CHO with amino groups on chitosan chains. The in vitro cytotoxicity of copolymers was evaluated by MTT method. The results showed >70% cell viability of HeLa and A549 cells after incubation with mPEG-CS copolymer from concentration 5 to 100 μg·mL^-1. The mPEG-CS copolymers with various degrees of PEG substitution were combined with DNA and the properties of mPEG-CS/DNA complexes were investigated such as nanoparticle size, zeta potential and agarose gel analysis. The best one among all these mPEG-CS copolymers was mPEG(3.55)-CS, for its capability to condense plasmid DNA was most efficient. For this reason, mPEG(3.55)-CS was picked out to mediate plasmid enhanced green fluorescence protein (pEGFP) and transfect HeLa and A549 cells. The expression of green fluorescence protein was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. The gene expression mediated by mPEG-CS was resistant to serum, and the optimal transfection efficiency (8.1% for HeLa cells and 4.8% for A549 cells) of mPEG-CS/EGFP system was obtained under the condition of N/P 40 and 48 h transfection time. These results indicate that mPEG-CS copolymer is an efficient non-viral gene vector.