title: Acid wash in determining cellular uptake of Fab/cell-permeating peptide conjugates.
abstract: Successful intracellular delivery of various bioactive molecules has been reported using cell-permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-(125)I-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-(125)I-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-(125)I-Fab conjugates. The kinetics of internalization of REV-(125)I-Fab conjugate was rapid, and a considerable fraction of REV-(125)I-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV-(125)I-Fab > or = TAT-(125)I-Fab > or = ANP-(125)I-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs.
authors.affiliation: Research Planning, Bipha Corporation, Chitose, Hokkaido, Japan. kameyama@oxy-genix.com
authors.forename: Shouju
authors.initials: S
authors.lastname: Kameyama
authors.forename: Mayo
authors.initials: M
authors.lastname: Horie
authors.forename: Takeo
authors.initials: T
authors.lastname: Kikuchi
authors.forename: Takao
authors.initials: T
authors.lastname: Omura
authors.forename: Akiko
authors.initials: A
authors.lastname: Tadokoro
authors.forename: Toshihide
authors.initials: T
authors.lastname: Takeuchi
authors.forename: Ikuhiko
authors.initials: I
authors.lastname: Nakase
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sugiura
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
mesh_terms: D000143:Acids; D002463:Cell Membrane Permeability / Q000187:drug effects; D006367:HeLa Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D018796:Immunoconjugates / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D010873:Pinocytosis / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects
chemical_list: D000143:Acids; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D006493:Heparin