以硅烷基保护壳聚糖分子中-NH2和部分羟基,与D,L-丙交酯接枝聚合,制备壳聚糖与聚乳酸接枝共聚物(CS-g-PDLLA).利用CS—g—PDLLA作为DNA载体,复合pEGFP-N1质粒,分别采用动态激光散射及原子力显微镜(AFM)分析复合物粒子的粒径分布及形态,凝胶电泳试验考察载体与质粒DNA的结合情况,人体胃癌细胞培养进行细胞毒性评估.测试表明CS-g-PDLLA/DNA复合物平均粒径为91.59—234.56nm,AFM结果显示,在D,L-丙交酯与壳聚糖单元的摩尔比等于3,N/P等于3所形成的壳聚糖聚乳酸共聚物/pEGFP-N1复合物粒径小于100nm.提高载体材料的PDLLA比例及复合物N/P比,复合物对DNaseI显示高稳定性,并且具有较好的DNA缓释效果.细胞培养显示载体良好的生物相容性.结果表明壳聚糖与聚乳酸共聚物可作为一种新型的非病毒 型基因载体.
Trimethylsilyl chloride (TMS-C1) was used to selectively protect the - NH2 groups of chitosan molecules. By changing the molar ratios of D, L-LA and glucosamine units of chitosan in the reac- tion, CS-g-PDLLA could be prepared with different D, L-LA moisture. The particles size and distribution, particle morphology of CS-g-PDLLA / pEGFP-N1 complex were analyzed by laser scatter and atomic force microscope (AFM). Agarose gel electrophoresis analysis and MTT assay were performed to assess the DNA combination ability and cellular toxicity of CS -g- PLA vectors. The results confirmed the formation of CS-g-PLA. The results of laser light scattering (LLS) showed that CS-g-PLA / pEGFP-N1 complex was between 91 and 234 nm. AFM observation confirmed that the particles size of CS-g-PDLLA (3:1 )/pEGFP-N1 (N/P = 3 ) complex was less than 100 nm. When the molar ratios of D, L-LA/chitosan monomer and the N/P of carrier and DNA were greater than 3, the CS-g-PDLLA and DNA complex particles could be efficiently protected from the degradation of DNase. The trials in cell model showed that CS- g-PLA exhibited non-cytotoxicity and good sustained release of DNA in vitro. Taken together, our results suggest that CS-g-PDLLA would be a new effective non-virus DNA carrier.