Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes.
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 0.2mg/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.
Department of Pharmaceutics, Setsunan University, University, 45-1 Nagaotoge-cho, Hirakata, Osaka, Japan. sakuma@pharm.setsunan.ac.jp
Shinji
S
Sakuma
Masaya
M
Suita
Takafumi
T
Yamamoto
Yoshie
Y
Masaoka
Makoto
M
Kataoka
Shinji
S
Yamashita
Noriko
N
Nakajima
Norihiro
N
Shinkai
Hitoshi
H
Yamauchi
Ken-Ichiro
K
Hiwatari
Akio
A
Hashizume
Hiroyuki
H
Tachikawa
Ryoji
R
Kimura
Yuki
Y
Ishimaru
Atsushi
A
Kasai
Sadaaki
S
Maeda
D000081:Acetamides / Q000737:chemistry*; D000179:Acrylates / Q000737:chemistry; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D057846:Cell-Penetrating Peptides / Q000737:chemistry; D005452:Fluoresceins / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000737:chemistry*; D010873:Pinocytosis / Q000187:drug effects; D011145:Polyvinyls / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D013997:Time Factors
D000081:Acetamides; D000179:Acrylates; D057846:Cell-Penetrating Peptides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011145:Polyvinyls; D012235:Rhodamines; C448619:octaarginine; C539745:poly(N-vinylacetamide); C024098:6-carboxyfluorescein; D000584:Amiloride; C036658:acrylic acid; C039614:ethylisopropylamiloride