論文

査読有り 本文へのリンクあり 国際誌
2020年12月4日

Mechanistic studies of gene delivery into mammalian cells by electrical short-circuiting via an aqueous droplet in dielectric oil

PLOS ONE
  • Hirofumi Kurita
  • ,
  • Hirohito Nihonyanagi
  • ,
  • Yuki Watanabe
  • ,
  • Kenta Sugano
  • ,
  • Ryuto Shinozaki
  • ,
  • Kenta Kishikawa
  • ,
  • Rika Numano
  • ,
  • Kazunori Takashima

担当区分
筆頭著者, 責任著者
15
12
開始ページ
e0243361
終了ページ
e0243361
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1371/journal.pone.0243361
出版者・発行元
Public Library of Science (PLoS)

We have developed a novel methodology for the delivery of cell-impermeable molecules, based on electrical short-circuiting via a water droplet in dielectric oil. When a cell suspension droplet is placed between a pair of electrodes with an intense DC electric field, droplet bouncing and droplet deformation, which results in an instantaneous short-circuit, can be induced, depending on the electric field strength. We have demonstrated successful transfection of various mammalian cells using the short-circuiting; however, the molecular mechanism remains to be elucidated. In this study, flow cytometric assays were performed with Jurkat cells. An aqueous droplet containing Jurkat cells and plasmids carrying fluorescent proteins was treated with droplet bouncing or short-circuiting. The short-circuiting resulted in sufficient cell viability and fluorescent protein expression after 24 hours’ incubation. In contrast, droplet bouncing did not result in successful gene transfection. Transient membrane pore formation was investigated by uptake of a cell-impermeable fluorescence dye YO-PRO-1 and the influx of calcium ions. As a result, short-circuiting increased YO-PRO-1 fluorescence intensity and intracellular calcium ion concentration, but droplet bouncing did not. We also investigated the contribution of endocytosis to the transfection. The pre-treatment of cells with endocytosis inhibitors decreased the efficiency of gene transfection in a concentration-dependent manner. Besides, the use of pH-sensitive dye conjugates indicated the formation of an acidic environment in the endosomes after the short-circuiting. Endocytosis is a possible mechanism for the intracellular delivery of exogenous DNA.

リンク情報
DOI
https://doi.org/10.1371/journal.pone.0243361 本文へのリンクあり
URL
https://dx.plos.org/10.1371/journal.pone.0243361 本文へのリンクあり

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