利用均匀磁场提高聚乙烯亚胺-Fe3O4纳米颗粒复合物的磁转染效果

摘要

The effect of uniform magnetic field(MF)on magnetofection was studied for the first time.The uniformity of the unilateral Halbach magnet and the commercialized 96 hole magnet were measured.Polyethyleneimine(PEI)modified ferroferric oxide (Fe3O4)nanoparticles (PEI-Fe3O4)were prepared by chemical coprecipitation.The morphology,composition and the DNA binding ability of PEI-Fe3O4 were charaterized by transmission electron microscope (TEM),vibrating sample magnetometer (VSM),atomic force microscopy and agarose gel electrophoresis.The green fluorescent protein(GFP)expression efficiency in human renal epithelial cells (HEK293)under different uniformity of magnetic field was detected with inverted fluorescence microscope and flow cytometry. The phagocytosis of PEI-Fe3O4 nanoparticles was observed by TEM.The results showed that the difference of uniformity of the two magnetic fields was about 100 times.The superparamagnetic PEI-Fe3O4 has a good compound ability for plasmid pDNA,and its op-timal combination ratio is N/P=0.5.Flow cytometry showed that the transfection efficiency was:PEI-Fe3O4-pDNA+ uniform MF group (77.75%±0.07%)＞PEI-Fe3O4-pDNA+ nonuniform MF group (30.65%±0.49%)＞PEI-Fe3O4-pDNA without MF group (7.90%±0.56%)(p＜0.05).PEI-Fe3O4 magnetic nanoparticles can be effectively consumed by cells and have little effect on cell morphology.It can be concluded that when the magnetic field intensity is certain,higher uniformity of magnetic field contributes to the higher the magnetic transfection efficiency.The combination of the unilateral Halbach magnet and the magnetosfection can be used as a new method to improve the transfection rate,which may potentially be employed in gene therapy.%首次研究了不同均匀度磁场对磁转染效果的影响.测定了单边 Halbach 磁体和商品化96孔磁板的磁场均匀度.以化学共沉淀法制备聚乙烯亚胺(Polyethyleneimine,PEI)修饰的四氧化三铁(Fe3O4)纳米颗粒(PEI-Fe3O4),并用透射电子显微镜(TEM)、振动样品磁强计(VSM)、原子力显微镜、琼脂糖凝胶电泳等对其形貌、组成、DNA结合能力等进行表征.用倒置荧光显微镜、流式细胞术观察不同均匀度磁场下人肾上皮细胞(HEK293)对带有绿色荧光蛋白报告基因(GFP)的质粒 pDNA(pAdTrack-OK)的表达效果,并采用TEM观察PEI-Fe3O4磁性纳米颗粒进入细胞的过程.结果显示,所选取的两种磁场均匀度相差约100倍.制备的PEI-Fe3O4纳米复合物具有超顺磁性,对质粒 pDNA(pAdTrack-OK)有较好的复合能力,其最佳结合氮磷比为0.5;流式细胞术显示转染效率为 PEI-Fe3O4-pDNA+均匀磁场组(77.75%±0.07%)＞PEI-Fe3O4-pDNA+不均匀磁场组(30.65%± 0.49%)＞PEI-Fe3O4-pDNA不加磁场组(7.90%±0.56%)(p＜0.05);PEI-Fe3O4磁性纳米颗粒能有效被细胞吞噬,且对细胞形态的影响不大.结果表明,当磁场强度一定时,磁场均匀度越高,磁转染效率越高,单边 Halbach磁体与磁转染结合可以作为一种提高转染效率的新手段,也可以进一步应用在基因治疗中.