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Acid wash in determining cellular uptake of Fab/cell‐permeating peptide conjugates
Author(s) -
Kameyama Shouju,
Horie Mayo,
Kikuchi Takeo,
Omura Takao,
Tadokoro Akiko,
Takeuchi Toshihide,
Nakase Ikuhiko,
Sugiura Yukio,
Futaki Shiroh
Publication year - 2007
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.20689
Subject(s) - chemistry , conjugate , endocytosis , internalization , cell penetrating peptide , pinocytosis , peptide , immunoglobulin fab fragments , intracellular , biochemistry , hela , cell , peptide sequence , mathematical analysis , mathematics , gene , complementarity determining region
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 (0.2 M glycine–0.15 M 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 ≥ TAT‐ 125 I‐Fab ≥ 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. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 98–107, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com