聚乙烯亚胺修饰纳米金基因载体制备及体外实验研究  

Preparation of PEI capped nanoparticles and their in vitro physicochemical properties

作　　者：蒋家云[1] 邹孟达 陈施瀚 邓青松[1] 谭运华[1] 夏峰[1] 马宽生[1] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所,重庆400038

出　　处：《第三军医大学学报》2016年第9期921-926,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81272688);重庆市应用开发计划重点项目(CSTC2014yykf B10002)~~

摘　　要：目的制备聚乙烯亚胺修饰纳米金基因载体并研究其理化性质的表征参数和体外转染效率。方法通过化学还原法制备聚乙烯亚胺修饰的纳米金基因载体,用绿色荧光蛋白质粒(pAc GFPN1)做报告基因,纳米基因载体可通过静电吸附的方式结合质粒DNA。用紫外分光光度计检测其吸收光谱,用透射电镜观察其形态特征,激光粒度分析仪测定其粒度分布、表面电位(Zeta电位),1%琼脂糖凝胶电泳检测该基因载体与质粒DNA的结合稳定性,CCK-8实验检测聚乙烯亚胺修饰纳米金基因载体及DNA-纳米金复合物对HEK293细胞的细胞毒性作用,通过荧光显微镜观察聚乙烯亚胺纳米基因载体介导pAcGFP-N1在体外培养的HEK293细胞中的表达,并分析其转染效率。结果聚乙烯亚胺还原氯金酸可以得到带正电荷的纳米颗粒,呈单分散球形分布,其粒径为(12.3±3.3)nm。在pH=7.2时,Zeta电位为+(29.7±5.1)m V。1%琼脂糖凝胶电泳结果表明,当纳米金/质粒DNA≥0.5时,质粒DNA可完全结合到纳米金表面。体外转染实验表明,聚乙烯亚胺修饰纳米金基因载体能介导pAcGFPN1转染HEK293细胞并在细胞中表达绿色荧光蛋白,其转染效率可达25%。结论聚乙烯亚胺修饰纳米金是一种新型非病毒基因载体,具有转染效率高、对细胞毒性小等优势。Objective To prepare polyethyleneimine（ PEI） capped Au nanoparticles for gene vector and investigate their physicochemical prosperities and transfection efficiency in vitro. Methods PEI-capped Au nanoparticles acting as gene carrier were prepared by using PEI as the reductant of hydrogen tetrachloroaurate（ HAu Cl4）. pAcGFP-N1 DNA was used as reporter gene bind to Au nanoparticles by electrostatic adsorption. The appearance and morphology of nanoparticles were characterized by ultraviolet spectrophotometry and transmission electron microscopy（ TEM）,respectively. The grain distribution and zeta potentials of the nanoparticles were determined with laser grain analyzer. DNA binding capacity of PEI capped Au nanoparticles was detected by agarose gel electrophoresis. The cytotoxicity of PEI-capped Au nanoparticles to HEK293 cells was examined by CCK-8 assay. The transfection activities of PEI-capped Au nanoparticles was assessed by in vitro gene transfection. The transfection of the vector p Ac GFP-N1 to HEK293 cells was observed by fluorescence microscopy and the efficiency was determined. Results Our prepared PEI capped nanoparticles were distributed dispersely,in spherical shape of 12. 3 ± 3. 3 nm in diameter,with zeta potential of 29. 7 ± 5. 1 m V under the p H value of 7. 2,and could obtain positively charged nanoparticles. Agarose gel electrophoresis indicated that when the weight ratio of Au NP to DNA ≥ 0. 5,DNA could bind to the Au nanoparticles completely. In vitro transfection showed that the DNA-PEI nanoparticles could be transfected into HEK293 cells, and the gene could be expressed in the cells with a transfection efficiency of 25%.Conclusion PEI capped Au nanoparticles is a novel non-viral gene vector,with the advantages of high transfection efficiency and low cytotoxicity.

关 键 词：聚乙烯亚胺 纳米金 基因载体 转染 

分 类 号：R394-33[医药卫生—医学遗传学] R945[医药卫生—基础医学]