摘要
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[(36.67±0.25)%和(28.73±7.19)×108RLU/mg蛋白,RLU为光强度],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.
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.
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
2012年第8期1757-1764,共8页
Chemical Journal of Chinese Universities
基金
国家自然科学基金(批准号:20876024)资助
