Lv Juan, Yang Jing, Hao Xuefang, Ren Xiangkui, Feng Yakai, Zhang Wencheng
School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072, China.
J Mater Chem B. 2016 Feb 7;4(5):997-1008. doi: 10.1039/c5tb02310f. Epub 2016 Jan 11.
In recent years, gene therapy has evoked an increasing interest in clinical treatments of coronary diseases because it is a potential strategy to realize rapid endothelialization of artificial vascular grafts. The balance of high transfection efficiency and low cytotoxicity of nonviral gene carriers is an important issue to be solved. In this study, we aim to establish a gene delivery system offering an elegant way to tune the transfection activity and cytotoxicity. Biodegradable complex micelles were prepared from polyethylenimine-b-poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)-b-polyethylenimine (PEI-b-PLMD-b-PEI) and methoxy-poly(ethylene glycol)-b-poly(lactide-co-3(S)-methyl-morpholine-2,5-dione) (mPEG-b-PLMD) copolymers by a co-assembly method. Then the ZNF580 gene plasmid (pDNA) was encapsulated into the complex micelles. The hydrodynamic size and zeta potential of these complex micelles and micelles/pDNA complexes indicated that they were feasible for use in cellular uptake and gene transfection. As expected, the transfection efficiency and cytotoxicity of these micelles/pDNA complexes could be conveniently tuned by changing the mass ratio of mPEG-b-PLMD to PEI-b-PLMD-b-PEI (3/1, 2/2, 1/3 and 0/4) in the mixed mPEG/PEI shell. The transfection efficiency increased as the mass ratio of mPEG-b-PLMD/PEI-b-PLMD-b-PEI decreased from 3/1 to 0/4, while the cytotoxicity showed an opposite tendency. Moreover, ZNF580 protein expression determined by Western blot analysis and the migration of transfected endothelial cells (ECs) by wound healing assay were consistent with the result of transfection efficiency. All these results indicated that the co-assembled complex micelles could act as suitable gene carriers with tunable gene transfection efficiency and cytotoxicity, which should have great potential for the transfection of vascular ECs.
近年来，基因治疗在冠心病临床治疗中引起了越来越多的关注，因为它是实现人工血管移植物快速内皮化的一种潜在策略。非病毒基因载体高转染效率和低细胞毒性之间的平衡是一个亟待解决的重要问题。在本研究中，我们旨在建立一种基因递送系统，提供一种优化的方法来调节转染活性和细胞毒性。通过共组装方法，由聚乙烯亚胺-b-聚（丙交酯-共-3(S)-甲基吗啉-2,5-二酮）-b-聚乙烯亚胺（PEI-b-PLMD-b-PEI）和甲氧基聚（乙二醇）-b-聚（丙交酯-共-3(S)-甲基吗啉-2,5-二酮）（mPEG-b-PLMD）共聚物制备了可生物降解的复合胶束。然后将ZNF580基因质粒（pDNA）包裹到复合胶束中。这些复合胶束和胶束/pDNA复合物的流体力学尺寸和zeta电位表明它们可用于细胞摄取和基因转染。正如预期的那样，通过改变混合mPEG/PEI壳层中mPEG-b-PLMD与PEI-b-PLMD-b-PEI的质量比（3/1、2/2、1/3和0/4），可以方便地调节这些胶束/pDNA复合物的转染效率和细胞毒性。随着mPEG-b-PLMD/PEI-b-PLMD-b-PEI的质量比从3/1降至0/4，转染效率增加，而细胞毒性呈现相反的趋势。此外，通过蛋白质免疫印迹分析测定的ZNF580蛋白表达以及通过伤口愈合试验检测的转染内皮细胞（ECs）的迁移与转染效率结果一致。所有这些结果表明，共组装的复合胶束可以作为具有可调基因转染效率和细胞毒性的合适基因载体，在血管内皮细胞转染方面具有巨大潜力。
