普鲁兰多糖为骨架的新型阳离子非病毒基因载体

通过琥珀酸酐将低分子量支化聚乙烯亚胺(PEI, 分子量1000)偶联到普鲁兰多糖(Pullulan)上, 合成了新型基因载体P-PEI. 利用 ¹H NMR、 FTIR、 粒度仪、 Zeta电位仪、 透射电镜和凝胶电泳对聚阳离子载体及其与质粒pDNA 的复合物进行了表征. 凝胶阻滞实验结果证明, 载体P-PEI在体外可以通过静电相互作用稳定结合pDNA, 并能有效抑制DNA水解酶及血清成分对pDNA的降解. 噻唑蓝(MTT)细胞毒性测试、 绿色荧光蛋白表达质粒(pGFP)及荧光素酶表达质粒(pGL3)转染实验结果表明, 载体P-PEI在N/P高达12.5时对细胞MCF-7, HeLa和COS-7的毒性低于PEI; 当N/P 为6.25时能有效将pGFP和pGL3带入Hela 细胞并表达, 最佳转染效率及荧光素酶活分别为, 比Lipo 2000(49.13±0.61)%, (58.47±7.62)×10⁸ RLU/mg蛋白) 略低. 因此以Pullulan为骨架材料的P-PEI是一种新的有潜在应用价值的非病毒基因载体.

Novel Pullulan-based Non-virus Cationic Gene Delivery Vector

A novel non-virus gene delivery vector(P-PEI) was synthesized by grafting low-molecular branched PEI(molecular weight: 1000) onto the backbone of pullulan with succinic acid as a spacer.1H NMR,FTIR,particle size analysis and zeta-potential measurements,TEM and gel electrophoresis were performed to characterize P-PEI and P-PEI/pDNA complexes.Gel electrophoresis retardation assay demonstrated that P-PEI can wrap pDNA with electrostatic interaction excellently even in the presence of heparin,and protect pDNA from the degradation by DNase I and serum.Compared with PEI/pDNA complexes,the P-PEI/pDNA complexes show relative lower cytotoxicity against MCF-7,HeLa and COS-7 cells,even at high N/P(up to 12.5) demonstrated by MTT assay.Flowcytometry revealed that the highest transfection efficiency(36.67±0.25)%] for P-PEI as a delivery agent was obtained at N/P ratio of 6.25,which was comparable with that of Lipofectamine 2000(49.13±0.61)%],and consistent with the results of luciferase expression.These results suggest that the pullulan-grafted branched polyethylenimine polymer(P-PEI) might be an excellent biocompatible candidate for gene delivery systems.