title: Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles.
abstract: The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R9 enters GUVs of various lipid compositions.
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Sharmin
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
authors.forename: Md Zahidul
authors.initials: MZ
authors.lastname: Islam
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
authors.forename: Mohammad Abu Sayem
authors.initials: MA
authors.lastname: Karal
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
authors.forename: Sayed Ul
authors.initials: SU
authors.lastname: Alam Shibly
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Dohra
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Yamazaki
mesh_terms: D001693:Biological Transport, Active; D002232:Carbocyanines; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C569686:Alexa Fluor 647; D002232:Carbocyanines; D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D009842:Oligopeptides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D053835:Unilamellar Liposomes; C491427:nonaarginine; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine