Hasan, Mahadi Department of Biophysical Chemistry, Kyoto Pharmaceutical University
ニシモト, アキノリ Department of Biophysical Chemistry, Kyoto Pharmaceutical University
オオギタ, タカシ Department of Biophysical Chemistry, Kyoto Pharmaceutical University
ハマ, ススム Department of Biophysical Chemistry, Kyoto Pharmaceutical University
カシダ, ヒロム Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University
アサヌマ, ヒロユキ Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University
Faint electric treatment
Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.
Journal of Controlled Release
© 2016 Elsevier B.V. All rights reserved.
© 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/
Journal of Controlled Release (2016) Vol. 228 p.20-25
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