超顺磁Fe3O4/SiO2-聚乙烯亚胺复合微球的制备及基因传递应用

The preparation of superparamagnetic Fe_3O_4/SiO_2-polyethyleneimine composite particles and their applications in gene delivery

背景:在基因治疗中选择合适、低毒、对人体和环境无害的载体,使基因高效地转移至靶向部位并有效表达相关产物尤为关键。目的:制备超顺磁性Fe3O4/SiO2-聚乙酰亚胺复合微球。方法:通过乳化溶剂挥发法制备Fe3O4纳米粒子聚集体,再利用stober法合成超顺磁性Fe3O4/SiO2核壳型微球,进一步在该微球表面修饰聚乙酰亚胺,得到超顺磁性Fe3O4/SiO2-聚乙酰亚胺复合微球,并对其进行透射电镜、Zeta电位和磁性等结构性能表征。将Fe3O4/SiO2-聚乙酰亚胺复合微球与Plasmid DNA按照不同的质量比(29∶1,39∶1,49∶1,59∶1,68∶1,78∶1,88∶1)混合,通过凝胶电泳测定该复合微球与绿色荧光蛋白基因的结合能力。将Plasmid DNA分别与Fe3O4/SiO2-聚乙酰亚胺、聚乙酰亚胺混合,通过共聚焦荧光显微镜观测其在HeLa细胞中转染绿色荧光蛋白基因的情况。结果与结论:成功合成了Fe3O4/SiO2-聚乙酰亚胺复合微球,分散性良好,粒径分布均匀,约为100 nm,表面电荷为21.07 mV,饱和磁化强度为28.05 emu/g,为超顺磁性。随着复合微球与Plasmid DNA质量比的不断增加,越来越多的Plasmid DNA质粒被吸附在Fe3O4/SiO2-聚乙酰亚胺复合微球上,此时Plasmid DNA质粒过量,当质量比达到59∶1时,所有的pDNA质粒都被吸附在复合微球上;质量比大于59∶1时,复合微球过量,因此质量比为59∶1时二者均无过量,结果较好,用于HeLa细胞转染。与聚乙酰亚胺相比,Fe3O4/SiO2-聚乙酰亚胺复合微球可显著提高Plasmid DNA的转染效率。

BACKGROUND：It is vital to choose the appropriate carrier with low toxicity and high gene transfection efficiency in gene therapy, which is harmless to human body and environment. OBJECTIVE： To prepare superparamagnetic Fe3O4/SiO2-polyethyleneimine （PEI） composite particles. METHODS： Fe3O4 nanoparticles were prepared via an emulsion solvent evaporation method and superparamagnetic Fe3O4/SiO2 core shel microspheres were prepared successfuly subsequently via a modified stober method. The microspheres were further modified with PEI to obtain superparamagnetic Fe3O4/SiO2-PEI composite particles. The structures and properties of resultant composite particles microspheres were characterized by transmission electron microscopy, zeta potential and vibrating sample magnetometer. Superparamagnetic Fe3O4/SiO2-PEI composite particles were mixed with plasmid DNA at different mass ratios （29∶1, 39∶1, 49∶1, 59∶1, 68∶1, 78∶1, 88∶1）. Thein vitro gene transfection ability was evaluated by Hela＆amp;nbsp;cels with the transfection of plasmid DNA encoded with green fluorescent protein and the transfection efficiency was determined by confocal fluorescence microscopy. RESULTS AND CONCLUSION： We successfuly synthesized the Fe3O4/SiO2-PEI composite particles with good dispersibility and even size distribution （about 100 nm）. The surface charge was 21.07 mV, and the saturation magnetization was 28.05 emu/g that meant superparamagnetism. When the mass ratio was 59∶1, al the plasmid DNA was adherent to the Fe3O4/SiO2-PEI composite particles; when the mass ratio was ＆gt; 59∶1, there were excessive Fe3O4/SiO2-PEI composite particles. Therefore, the mass ratio of 59：1 could lead to a better outcome for HeLa celltransfection. These results indicate that the Fe3O4/SiO2-PEI composite particles can dramaticaly improve the transfection efficiency of plasmid DNA compared with PEI.