Tay Andy, Melosh Nicholas
Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305.
Department of Biomedical Engineering, National University of Singapore, Singapore 117583.
Adv Ther (Weinh). 2019 Dec;2(12). doi: 10.1002/adtp.201900133. Epub 2019 Sep 30.
Transfection is a critical step for gene editing and cell-based therapies. Nanoscale technologies have shown great promise to provide higher transfection efficiency and lower cell perturbation than conventional viral, biochemical and electroporation techniques due to their small size and localized effect. Although this has significant implications for using cells post-transfection, it has not been thoroughly studied. Here, we developed the nano-electro-injection (NEI) platform which makes use of localized electric fields to transiently open pores on cell membrane followed by electrophoretic delivery of DNA into cells. NEI provided two-folds higher net transfection efficiency than biochemicals and electroporation in Jurkat cells. Analysis of cell doubling time, intracellular calcium levels and mRNA expression changes after these gene delivery methods revealed that viruses and electroporation adversely affected cell behavior. Cell doubling times increased by more than 40% using virus and electroporation methods indicative of higher levels of cell stress, unlike NEI which only minimally affected cell division. Finally, electroporation, but not NEI, greatly altered the expression of immune-associated genes related to immune cell activation and trafficking. These results highlight that nanoscale delivery tools can have significant advantages from a cell health perspective for cell-based research and therapeutic applications.
转染是基因编辑和基于细胞的治疗的关键步骤。纳米技术已显示出巨大的潜力，因其尺寸小和局部效应，能比传统的病毒、生化和电穿孔技术提供更高的转染效率和更低的细胞扰动。尽管这对转染后细胞的使用有重大影响，但尚未得到充分研究。在此，我们开发了纳米电注射（NEI）平台，该平台利用局部电场瞬时打开细胞膜上的孔，随后通过电泳将DNA导入细胞。在Jurkat细胞中，NEI的净转染效率比生化方法和电穿孔高两倍。对这些基因递送方法后细胞倍增时间、细胞内钙水平和mRNA表达变化的分析表明，病毒和电穿孔对细胞行为有不利影响。使用病毒和电穿孔方法时，细胞倍增时间增加超过40%，这表明细胞应激水平较高，而NEI对细胞分裂的影响最小。最后，电穿孔而非NEI极大地改变了与免疫细胞激活和运输相关的免疫相关基因的表达。这些结果突出表明，从细胞健康的角度来看，纳米级递送工具在基于细胞的研究和治疗应用中具有显著优势。
