Document Detail

Acid wash in determining cellular uptake of Fab/cell-permeating peptide conjugates.
MedLine Citation:
PMID:  17252560     Owner:  NLM     Status:  MEDLINE    
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.
Shouju Kameyama; Mayo Horie; Takeo Kikuchi; Takao Omura; Akiko Tadokoro; Toshihide Takeuchi; Ikuhiko Nakase; Yukio Sugiura; Shiroh Futaki
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biopolymers     Volume:  88     ISSN:  0006-3525     ISO Abbreviation:  Biopolymers     Publication Date:  2007  
Date Detail:
Created Date:  2007-04-23     Completed Date:  2007-05-22     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0372525     Medline TA:  Biopolymers     Country:  United States    
Other Details:
Languages:  eng     Pagination:  98-107     Citation Subset:  IM    
Research Planning, Bipha Corporation, Chitose, Hokkaido, Japan.
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MeSH Terms
Cell Membrane Permeability / drug effects
Hela Cells
Heparin / pharmacology
Immunoconjugates / metabolism*
Immunoglobulin Fab Fragments / metabolism
Peptides / metabolism*
Pinocytosis / drug effects
Protein Transport / drug effects
Reg. No./Substance:
0/Acids; 0/Immunoconjugates; 0/Immunoglobulin Fab Fragments; 0/Peptides; 9005-49-6/Heparin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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