多功能纳米基因载体转染人脂肪间充质干细胞及体外细胞磁共振显像

Multifunctional nano-vector for gene delivery into human adipose derived mesenchymal stem cells and in vitro cellular magnetic resonance imaging

目的 观察多功能纳米基因载体聚乙二醇-聚乙烯亚胺-超顺磁性氧化铁（PEG-g-PEI-SPION）转染人脂肪间充质干细胞（hADMSCs）的可行性、效率及体外细胞磁共振显像功能.方法 根据先前报道的方法合成PEG-g-PEI-SPION.采用凝胶阻滞实验评估其复合外源性基因的能力;动态光散射法测量PEG-g-PEI-SPION/pDNA纳米复合物的粒径大小及表面电位;体外细胞毒性实验检测其对hADMSCs的细胞毒性.使用流式细胞分析仪检测PEG-g-PEI-SPION转染hADMSCs的效率.通过激光共聚焦显微镜及锥虫蓝染色实验研究hADMSCs对PEG-g-PEI-SPION/pDNA纳米复合物的摄入.PEG-g-PEI-SPION的磁共振显像功能通过体外细胞磁共振扫描验证.结果 PEG-g-PEI-SPION细胞毒性小,复合外源性基因后能够形成稳定的纳米复合物,粒径80 ～ 100 nm.在N/P=20时PEG-g-PEI-SPION转染hADMSCs最高取得了22.8％±3.6％的转染率,高于商品化的阳离子脂质体Lipofectamine最高11.2％±2.6％的转染率（P＜0.05）.同时,PEG-g-PEI-SPION中的SPION可有效标记hADMSCs,在磁共振T2加权图像上呈低信号.结论 PEG-g-PEI-SPION是一种磁共振可视的,能有效转染hADMSCs的纳米基因载体.

Objective To examine the feasibility and efficacy of using superparamagnetic iron oxide nanoparticles coated with polyethylene glycol-grafted polyethylenimine （PEG-g-PEI-SPION） as a carrier for gene delivery into human adipose derived mesenchymal stem cells （hADMSCs） and in vitro cellular magnetic resonance imaging （MRI）.Methods PEG-g-PEI-SPION was synthesized as previously reported.Gel electrophoresis was performed to assess the pDNA condensation capacity of PEG-g-PEI-SPION.The particle size and zeta potential of PEG-g-PEI-SPION/pDNA complexes were determined by dynamic light scattering.Cytotoxicity of PEG-g-PEI-SPION was evaluated by CCK-8 assay with hADMSCs.Gene transfection efficiency of PEG-g-PEI-SPION in hADMSCs was quantified by flow cytometry.The cellular internalization of PEG-g-PEI-SPION/pDNA nanocomplexes was studied by confocal laser scanning microscopy and Prussian blue staining.MRI function of PEG-g-PEI-SPION was studied by in vitro cellular MRI scanning.Results PEG-g-PEI-SPION condensed pDNA to form stable complexes of 80-100 nm in diameter and showed low cytotoxicity in hADMSCs.At the optimal N/P ratio of 20,PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of 22.8％ ± 3.6％ in hADMSCs.And it was higher than that obtained with lipofectamine 11.2％ ± 2.6％ （P ＜ 0.05）.Furthermore,hADMSCs labeled with PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2-weighted images in vitro.Conclusion PEG-g-PEI-SPION is an efficient and MRI-visible nano-vector for gene delivery into hADMSCs.