Faculty of Electronics, Vilnius Gediminas Technical University, Vilnius, Lithuania.
State Research Institute Centre for Innovative Medicine, Department of Immunology, Vilnius, Lithuania.
Bioelectrochemistry. 2020 Dec;136:107594. doi: 10.1016/j.bioelechem.2020.107594. Epub 2020 Jul 3.
Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300-700 ns) high frequency pulsed electric fields of 2-15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2-5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 μs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.
微秒范围内的电场脉冲已被用于促进 DNA 向细胞和组织转移数十年，而越来越多的临床试验突显了 DNA 电穿孔的强大潜力。在这项工作中，我们提出了新的亚微秒范围方案和方法，使亚微秒范围内的电转染能够成功进行。为了促进 DNA 转移，研究中应用了 3 kV/60 A 和高频（1 MHz）亚微秒范围方波发生器。作为模型，使用中国仓鼠卵巢（CHO-K1）细胞。施加了亚微秒范围（300-700 ns）的高频脉冲电场，其强度为 2-15 kV/cm。使用两个不同大小（3.5 kbp 和 4.7 kbp）的绿色荧光蛋白编码质粒评估电转染效率。结果表明，在达到一定阈值后，随着脉冲数量的增加，转染效率不能有效地提高，然而，与质粒大小无关，所提出的亚微秒范围脉冲方法（2-5 kV/cm；n = 250）在效率上相当于 1.5 kV/cm×100 μs×4 次电穿孔程序。该研究结果有助于进一步开发使用较短脉冲的有效体外和体内 DNA 电转移方法。
