论文部分内容阅读
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 select poly-hydroxypropyl-aspartic acid-glutamic acid as a framework-conjugated low molecular weight polyethyleneimine to construct a new non-viral gene vector with low toxicity and high efficiency. Methods: Poly - hydroxypropyl - aspartic acid - glutamic acid (PHPAG) was used as basic framework to conjugate low molecular weight polyethyleneimine (PEI 1.8 kDa) Glutamate-polyethyleneimine (PHPAG-PEI 1.8 kDa). By means of chemical and physical methods such as nuclear magnetic resonance (1H-NMR), particle size determination and gel exclusion chromatography (GPC), gel electrophoresis retardation, MTT cytotoxicity and cell transfection, Polymer structure and properties of the study. Results: The vector material PHPAG-PEI1.8kDa was successfully synthesized. It was confirmed by 1H-NMR that the material PHPAG-PEI 1.8 kDa could couple 1 PEI 1.8 kDa on 5 or 6 amino acids. GPC results show that the molecular weight of PHPAG, PHPAG-PEI1.8kDa2 material is about 1.2 × 104. The particle size analysis showed that the average particle size of PHPAG-PEI / pDNA complex was about 200nm. Gel electrophoresis retardation experiments showed that PHPAG-PEI / pDNA complexes completely blocked DNA at a N / P ratio of 3.5: 1. Cytotoxicity experiments showed that the vector material showed lower toxicity in COS-7 and A2932 different cells, similar to the control PEI 1.8 kDa. Transfection experiments on B16 cells and Hela cells showed that the transfection efficiency of PHPAG-PEI / pCAG-Luc3 complex was the highest when the N / P ratio was 25:1, which was higher than that of control group PEI25kDa. Conclusion: PHPAG-PEI polymeric carrier material is a potential non-viral gene drug carrier.