含RGD肽CP9修饰的聚乙烯亚胺复合物的理化特性及其转基因功能的研究

目的：构建含精氨酸-甘氨酸-天冬氨酸（RGD）肽CP9修饰的聚乙烯亚胺（PEI），观察其理化特性和转基因功能。方法：通过琥珀酰亚胺-3-（2-嘧啶二硫）丙酸酯（N-Succinimidyl-3-（2-pyridyldithio）]propionate，SPDP）将CP9偶联到PEI上，合成新型转基因载体CP9-PEI。用^1H-NMR和FT-IR验证CP9的偶联；用凝胶电泳阻滞实验、电镜和粒径检测，观察CP9-PEI浓缩质粒DNA的能力以及浓缩质粒DNA后形成的转染颗粒形态和粒径；通过CP9-PEI在人肝癌细胞株HepG2中的转染实验来验证CP9对PEI的转染效率的影响；通过游离CP9肽的竞争抑制实验验证CP9-PEI的整合素靶向功能。结果：CP9成功偶联到PEI上；CP9-PEI能有效浓缩质粒DNA，形成的转染颗粒形态为圆形或类圆形，在N／P比为10时，粒径约为200nm。CP9-PEI的转染效率是PEI的2倍，游离CP9肽能抑制CP9-PEI的转染效率。结论：修饰了CP9的PEI能有效增加转染效率，具有整合素的靶向能力，是一种具有应用前景的转基因载体。

Physicochemical characters and gene delivery function of polyethylenimine modified with peptide CP9 containing RGD sequence

Objective: To construct a novel gene delivery vector using polyethylenimine(PEI) as backbone modified with the peptide CP9 containing Arginine-Glycine-Aspartic acid(RGD)sequence and to verify its physicochemical characters and the gene delivery function.Methods: The chemical linker [N-Succinimidyl-3-(2-pyridyldithio)] propionate(SPDP) was employed to bind CP9 onto PEI to form a novel gene delivery vector CP9-PEI.The()~(1)H-NMR and FT-IR were used to verify the linkage of CP9.The plasmid DNA condensing ability of CP9-PEI,the shape and the particle size of the polyplexes formed with CP9-PEI-plasmid DNA were demonstrated by gel retardation assay,electron microscope observation and particle size assay,respectively.The enhanced transfection efficiency and the integrin targeting capacity were detected by the transfection experiments in HepG2 cells and free RGD peptide inhibition test.Results: CP9 was linked onto PEI successfully.The new synthesized vector CP9-PEI could efficiently condense plasmid DNA at N/P ratio of 4 and when N/P ratio was equal to 10,the shape of polyplexes formed with CP9PEI-plasmid DNA was round or round-alike with particle size of about 200 nm.The transfection efficiency of CP9-PEI was nearly 2 times of PEI in HepG2 cells and the free RGD peptide had the inhibition effect on the efficiency of CP9-PEI. Conclusion: The modification of CP9 on PEI can improve the transfection efficiency of PEI and has the integrin targeting ability.