Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy
Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy
复制标题
DOI：
10.1038/s41598-020-63172-8
复制
发表时间：
2020-04-08
影响因子：
4.6
通讯作者：
Wang, Shengnian
中科院分区：
文献类型：
--
作者：
Chang, An-Yi;Liu, Xuan;Wang, Shengnian
毫秒级脉冲的标准电穿孔已被用作将药物或遗传探针递送到细胞中的有效工具，而纳秒级脉冲的不可逆电穿孔被探索用于改变脉冲诱导的细胞凋亡的细胞内活性。采用纳秒脉冲和毫秒脉冲相结合的方法，使DNA质粒快速穿过细胞质膜和核膜，促进转基因在贴壁细胞和悬浮细胞中的表达水平和动力学。持续时间为400- 800 ns的纳秒脉冲被发现有效地破坏核膜以促进质粒DNA的核递送。额外的微流体操作还有助于抑制负面影响，例如焦耳加热和来自普通纳秒脉冲处理的气泡演变，其导致高毒性和/或无效转染。在具有不同脉冲持续时间的两种类型的处理之间具有适当的顺序和小的延迟对于保证有效性是至关重要的：2倍或更高的转染效率增强和GFP质粒的快速转基因表达动力学，而不损害细胞活力。这种新的电穿孔方法的实施可能有益于许多需要高效递送外源性探针的生物学研究和临床实践。
Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400-800ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.