人骨髓间充质干细胞靶向纳米基因载体制备及体外细胞磁共振成像

目的 探讨靶向纳米基因载体单链神经节苷脂抗体-聚乙二醇-聚乙烯亚胺-超顺磁性氧化铁（scAbGD2-PEG-g-PEI-SPION）转染人骨髓间充质干细胞（hBMSCs）的可行性、效率及体外细胞MR显像能力。方法 合成scAbGD2-PEG-g-PEI-SPION后，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scAbGD2-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hBMSCs的细胞毒性。采用流式细胞仪检测scAbGD2-PEG-g-PEI-SPION靶向转染hBMSCs的效率，并设置PEG-g-PEI-SPION组、cAbGD2-PEG-g-PEI-SPION组、抗体竞争抑制（scAbGD2-PEG-g-PEI-SPION+free AbGD2）组和同型抗体（scAbIgG2a-PEG-g-PEI-SPION）组，通过激光共聚焦显微镜及普鲁士蓝染色观察hBMSCs对纳米复合物的摄入。通过体外细胞MR扫描验证scAbGD2-PEG-g-PEI-SPION的MR成像功能。结果 scAbGD2-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。在相同的N/P比值下，scAbGD2-PEG-g-PEI-SPION组的转染率明显高于其他组（P<0.001）。N/P=20时，靶向组具有最高转染率（59.60±4.50）%]。同时，scAbGD2-PEG-g-PEI-SPION中的SPION可有效标记hBMSCs，在MR T2/T2^*加权图像上呈低信号。结论 scAbGD2-PEG-g-PEI-SPION是一种MRI可视的、可有效转染hBMSCs的靶向纳米基因载体。

Targeted nano-vector for gene delivery into human bone marrow mesenchymal stem cells and cellular MR imaging in vitro

Objective To explore the feasibility and efficacy of an MRI-visible, targeted, nano-vector which is synthesized by attaching a targeting ligand, the GD2 single chain antibody (scAb GD2), to the distal ends of PEG-g-PEI-SPION as a carrier for gene delivery into human bone marrow mesenchymal stem cells (hBMSCs) and in vitro cellular MR imaging. Methods scAbGD2-PEG-g-PEI-SPION was synthesized as previously reported. Gel electrophoresis was performed to assess the pDNA condensation ability of scAbGD2-PEG-g-PEI-SPION. The particle size and Zeta potential of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes were observed by dynamic light scattering. Cytotoxicity of scAbGD2-PEG-g-PEI-SPION was evaluated by CCK-8 assay using hBMSCs. Gene transfection efficiency of scAbGD2-PEG-g-PEI-SPION in hBMSCs was quantified by flow cytometry, PEG-g-PEI-SPION, scAbGD2-PEG-g-PEI-SPION, scAbGD2-PEG-g-PEI-SPION+free AbGD2 and scAbIgG2a-PEG-g-PEI-SPION group was established. The cellular internalization of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes was observed by confocal laser scanning microscopy and Prussian blue staining. MRI of scAbGD2-PEG-g-PEI-SPION was performed by cellular MRI scanning in vitro. Results scAbGD2-PEG-g-PEI-SPION condensed pDNA to form stable nanocomplexes of 80-100 nm in diameter and showed low cytotoxicity to hBMSCs. At the same N/P ratio, the transfection efficiency of scAbGD2-PEG-g-PEI-SPION group was significantly higher than those of other groups (P<0.001). At the optimal N/P ratio of 20, scAbGD2-PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of (59.60±4.50)% in hBMSCs. Furthermore, hBMSCs labeled with scAbGD2-PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T₂/T₂^*-weighted images in vitro. Conclusion scAbGD2-PEG-g-PEI-SPION is an efficient MRI-visible targeted nano-vector for gene delivery into hBMSCs.