聚乙烯亚胺-聚乙二醇-siRNA纳米复合物的制备和理化性质的研究

目的 探索聚乙烯亚胺-聚乙二醇(PEI-PEG)-siRNA纳米复合物的制备方法及其理化性质,以提高siRNA的细胞转染率.方法 设计合成了PEI-PEG共聚物基因载体,使其与针对细胞表面受体CD44v6的siRNA形成纳米复合物.通过粒径与电位测定、凝胶阻滞电泳、扫描电镜、流式细胞仪测定等方法,观察不同N/P比的纳米复合物的复合效果、表面形态和大小、基因转染率等.结果 电镜下纳米复合物呈近球形、大小较一致、分散良好的纳米颗粒.N/P=5、10时复合物粒径分别为(174.6±1.2)nm,(267.7±1.8)nm.当N/P超过10时纳米复合物粒径减小,zeta电位为正值且增大.此时,siRNA被PEI-PEG完全复合,产生一种荧光淬灭作用.流式细胞仪结果表明纳米复合物的基因转染率随着N/P的增加而增大.当N/P比为30时,其转染率为(75.6±9.2)%.结论 PEI-PEG是一种有潜力的阳离子基因载体,它的制备为下一步体外实验及动物实验提供了条件.

Preparation of polyethyleneimine- polyethylene glycal- siRNA nanoparticle and investigation of its characteristics

Objective To investigate the preparation method and characteristics of polyethyleneimine- polyethylene glycal (PEI- PEG) siRNA nanocomplex to improve the cell transfection efficiency of siRNA. Methods PEI-PEG copolymer was synthesized as gene carrier and complexed with siRNA targeting to CD44v6 to form PEI-PEG-siRNA nanoparticle. The size and zeta potential was measured. The morphology of the complex was observed by scanning electron microscopy. The complex abilities of the nanocomplex were validated by gel retardation assay. And the transfection efficiency of nanocomplexes at different N/P ratios was measured by flow cytometry. Results The nanoparticles appeared spherical,uniform in size, and well-dispersed. When N/P ratio was over 10, the size of nanocomplex decreased as N/P value increased. Meanwhile, the zeta potential was positive and increased. At this time, siRNA was completely complexed with PEI-PEG, producing a fluorescence quenching phenomenon. The flow cytometry showed that the transfection efficiency of nanocomplexes was improved as N/P value increasied. When N/P was 30, the transfection efficiency was (75.6±9.2)%. Conclusion PEI-PEG may be a promised gene carrier and can provide evidences for related research of PEI-PEG-siRNA nanoparticles in animals and in vitro experiments.