聚-羟丙基-天冬氨酸-谷氨酸-聚乙烯亚胺共聚物作为非病毒载体的研究

Poly-aspartamide-glutamic acid grafted low molecular weight polye-thylenimine as a novel non-viral gene vector

目的:选用聚-羟丙基-天冬氨酸-谷氨酸材料作为骨架偶联低分子量聚乙烯亚胺,以构建低毒、高效的新型非病毒性基因载体。方法:用聚-羟丙基-天冬氨酸-谷氨酸(PHPAG)为基本骨架,偶联低分子量的聚乙烯亚胺(PEI1.8kDa)形成聚-羟丙基-天冬氨酸-谷氨酸-聚乙烯亚胺(PHPAG-PEI1.8kDa)的载体材料。通过核磁共振氢谱(1H-NMR)、粒径测定、凝胶体积排除色谱法(GPC)等化学物理方法,凝胶电泳阻滞实验、MTT细胞毒性实验、细胞转染等生物学实验,对聚合物的结构及性能进行研究。结果:成功合成载体材料PHPAG-PEI1.8kDa。通过1H-NMR证实材料PHPAG-PEI1.8kDa在5或6个氨基酸上能偶合1个PEI1.8kDa。GPC结果表明PHPAG、PHPAG-PEI1.8kDa2种材料的分子量约为1.2×104。粒径检测结果显示,PHPAG-PEI/pDNA复合物的平均粒径为200nm左右。凝胶电泳阻滞实验表明,PHPAG-PEI/pDNA复合物在N/P为3.5∶1时可以完全阻滞DNA。细胞毒性实验表明,在COS-7和A2932种不同的细胞中,载体材料显示出较低的毒性,与对照组PEI1.8kDa相近。在B16细胞、Hela细胞上的转染实验表明,PHPAG-PEI/pCAG-Luc3的复合物在N/P为25∶1时的转染效率最高,高于对照组PEI25kDa。结论:PHPAG-PEI聚合物载体材料是一种有潜在用途的非病毒基因药物载体。

Objective： To develop a novel gene delivery vector with poly aspartamide-glutamic acid and polyethylenimine as the backbone. Methods： α,β- poly-（N 2-hydroxypropyl）-D, L aspartamide glmamic acid （ PHPAG ） was symhesized and low molecular weight polyethylenimine （PEI 1. 8 kDa） was grafled to form PHPAG PEI 1 800. Chemical and biological characterization of the polymer was identified. Results： The polymer was confirmed by tH-NMR, and the molecular weight was about 1.2 ×10^4. The ability of DNA binding was showed by gel retardation assay at N/P ratio of 3. 5. MTT assay showed thal the polymer was non toxic in COS-7 and A293 cell lines. In vitro test demonstrated that it had high transfection efficiency in B16 and Hela cell lines. Conclusion： PHPAG-PEI 1 800 was successfully synthesized,which might he a potential vector for gene delivery.