超顺磁性聚乙烯亚胺纳米粒的制备及其骨肉瘤 细胞、组织靶向转染效果观察.

Objective To prepare the polyethylenimine-superparamagnetic iron oxide nanoparticles-nanoparticles (PEI-SPION-NPs) and to observe the feasibility of PEI-SPION-NPs as targeted gene delivery vector to transefect into osteosarcoma cells and tissues in vivo. Methods We used the polyethylenimine (PEI) modified superparamagnetic iron oxide nanoparticles (SPION) to prepare the PEI-SPION-NPs by the dehydration condensation reaction. Their molecular structure, morphology, particle size, saturation magnetization and Zeta potential were detected. PEI-SPION-NPs were mixed with pDNA in different proportions，and agarose gel electrophoresis was performed to observe the binding ability and protective effect of PEI-SPION-NPs on pDNA. MG-63 cells were selected as target cells for transfection, which were cul‐ tured and then were divided into the group 1 (PEI-SPION-NPs/pDNA＋the static magnetic field), group 2（PolyMag200/ pDNA＋the static magnetic field), group 3（PEI-NPs/pDNA ), and the control group（naked pDNA)；the cell survival rate was determined by CCK-8 assay, the transfection effect was detected by inverted fluorescence microscope, and the transfection efficiency was measured by flow cytometry. The hypodermal osteosarcoma models pared, which were then divided into the control group（subcutaneous injection of normal saline), PEI-SPION-NPs group (subcutaneous injection of PEI-SPION-NPs/pGL3-Luc＋the static magnetic field), and PEI-NPs group (subcutaneous injection of PEI/pGL3-Luc complexes＋the static magnetic field). Fluorescence intensity of injection site was detected and body weight of nude mice in each group was measured at 5,6,7,8 and 9 weeks after osteosarcoma modeling, respectively. Results The hydrodynamic diameters of PEI-SPION-NPs were shorter than those of PEI-SPION-NP/pDNA complexes, the saturation magnetization of PEI-SPION-NPs was（21. 5±1. 6）emu/g and the zeta potential was (31. 2±1. 5) mV； PEI-SPION-NPs could bind to and protect pDNA，and can be successfully transfected into MG-63 cells. The cell survival rates of the group 1 and control group were higher than those of the group 2 and group3, nd the transfection efficiency of the group 1 was higher than those of the group 3 and control group（all P<0. 05). Both PEI-SPION-NPS/pGL3-Luc and PEI-NPS/pGL3-Luc could be successfully transfected into and expressed in the osteosarcoma tissues of nude mice, however, the fluorescence of the PEI-SPION-NPS group was more concentrated than that of the PEI-NPS group, and the body weight of nude mice in the PEI-SPION-NPS group was higher than that in the PEI-NPS group at 7 weeks after osteosarcoma modeling（both P<0. 01). Conclusion PEI-SPION-NP is an effective non-viral gene vector, which can be transfected into osteosarcoma cells and tissues in vivo under the guidance of the magnetic field. [ABSTRACT FROM AUTHOR]

目的 制备超顺磁性聚乙烯亚胺纳米粒（PEI-SPION-NPs），观察其作为靶向基因输送载体转染骨肉瘤细 胞和组织的可行性。方法 通过脱水缩合反应将低分子量聚乙烯亚胺（PEI）修饰超顺磁性氧化铁纳米粒（SPION), 制备PEI-SPION-NPs，对其分子结构、形态、粒径、饱和磁化强度、Zeta电位进行检测。将PEI-SPION-NPs与pDNA分 别以不同比例混合，琼脂糖凝胶电泳观察PEI-SPION-NPs对pDNA的结合能力和保护作用。培养骨肉瘤MG-63细 胞，分为实验1组（加入PEI-SPION-NPs/pDNA＋磁场作用）、实验2组（加入PolyMag200/pDNA＋磁场作用）、实验3组 （加入PEI/pDNA）及对照组（加入pDNA），采用CCK-8法检测细胞存活率，倒置荧光显微镜观察转染情况，流式细胞仪 测算转染率。制备裸鼠皮下骨肉瘤模型，分为Control组（皮下注射生理盐水）、PEI-SPION-NPs组（皮下注射PEI-SPI⁃ ON-NPs/pGL3-Luc+磁场作用）、PEI-NPs组（皮下注射PEI/pGL3-Luc复合物+磁场作用），检测注射部位荧光强度，分 别于造模第5、 6、 7、 8、 9周测量各组裸鼠体质量。结果 PEI-SPION-NPs的粒径低于PEI-SPION-NPs/pDNA；PEI-SPI⁃ ON-NPs的饱和磁化强度为（21.5±1.6）emu/g，Zeta电位为（31.2±1.5）mV；PEI-SPION-NPs可结合并保护pDNA，并 可成功转染MG-63细胞。实验1组和对照组细胞存活率高于实验2组和实验3组，实验1组转染效率高于实验3组和 对照组（P均<0.05）。PEI-SPION-NPs/pGL3-Luc与PEI-NPs/pGL3-Luc均可在裸鼠骨肉瘤组织中成功转染，PEI-SPI⁃ ON-NPs组较PEI-NPs组荧光更为集中。造模第7周后PEI-SPION-NPs组裸鼠体质量高于PEI-NPs组（P均<0.01）。结 论 PEI-SPION-NPs是有效的非病毒基因载体，在磁场作用下可成功转染骨肉瘤细胞和组织。 [ABSTRACT FROM AUTHOR]