title: Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm.
abstract: 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.
authors.affiliation: Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
authors.forename: Mahadi
authors.initials: M
authors.lastname: Hasan
authors.affiliation: Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Nishimoto
authors.affiliation: Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohgita
authors.affiliation: Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Hama
authors.affiliation: Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Kashida
authors.affiliation: Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Asanuma
authors.affiliation: Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan; Department of Pharmaceutical Health Chemistry, Institute of Biomedical Sciences, Tokushima University, Graduate School, Shomachi 1, Tokushima 770-8505, Japan. Electronic address: kogure@tokushima-u.ac.jp.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kogure
mesh_terms: D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D004560:Electricity; D057927:Hydrophobic and Hydrophilic Interactions; D007478:Iontophoresis / Q000379:methods; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008564:Membrane Potentials; D051379:Mice; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics
chemical_list: D034741:RNA, Small Interfering; D008156:Luciferases
keywords: Cellular uptake; Cytoplasmic delivery; Faint electric treatment