Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes

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• 作者：Shinji Sakuma^a ; ^{sakuma@pharm.setsunan.ac.jp} ; Masaya Suita^a ; ^b ; Takafumi Yamamoto^a ; Yoshie Masaoka^a ; Makoto Kataoka^a ; Shinji Yamashita^a ; Noriko Nakajima^b ; Norihiro Shinkai^b ; Hitoshi Yamauchi^b ; Ken-ichiro Hiwatari^c ; Akio Hashizume^d ; Hiroyuki Tachikawa^c ; Ryoji Kimura^c ; Yuki Ishimaru^e ; Atsushi Kasai^e ; Sadaaki Maeda^e ; ^{smaeda@pharm.setsunan.ac.jp}
• 关键词：Cell-penetrating peptides ; Oligoarginines ; d-Octaarginine ; Poly(N-vinylacetamide-co-acrylic acid) ; Cell-penetrating peptide-linked polymers ; Penetration enhancer
• 刊名：European Journal of Pharmaceutics and Biopharmaceutics
• 出版年：2012
• 期刊代码：16_09396411
• 类别：pha
• 出版时间：May, 2012
• 卷：81
• 期：1
• 页码：64-73
• 文件大小：615 K

摘要

We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30 min, approximately 0.1%of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2 mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120 min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone.