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‐125I‐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‐125I‐Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP‐125I‐Fab conjugates. The kinetics of internalization of REV‐125I‐Fab conjugate was rapid, and a considerable fraction of REV‐125I‐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‐125I‐Fab ≥ TAT‐125I‐Fab ≥ ANP‐125I‐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

Cell Penetrating Peptides: How Do They Do It?

“…However, these pathways might not be as general and could depend on the specific sequence of the CPP and the cargo attached to it. Several recent reports indicate that these peptides get inserted into the cell through endocytosis and pinocytosis [60][61][62][63][64]. We observed in our simulations that these peptides interact very strongly with the membrane, introducing lipid rearrangements and stress.…”

Cell Damage in Light Chain Amyloidosis

“…Cells surrounded by fibrils are likely to be excluded from cell-cell contact, a vital mechanism for maintaining cell viability. Cell treatment with trypsin (40,48,59), chondroitinase ABC, and heparinase II (60, 61) did not detach the external amyloid from the cell membrane (data not shown). We propose that membrane surfaces facilitate fibril attachment and act as an anchor point for cell-mediated seeding mechanism.…”

Cell-penetrating and cell-targeting peptides in drug delivery

“…In several studies, initial ionic interactions at the cell membrane 7 surface have been however shown to be key determinants for the uptake of all the cationic CPPs since the peptide entry could be strongly reduced by competition with polyanionic compounds [51][52][53][54] or by stringent cell washes with solutions at acidic pH [55].…”

“…Park et al described the equivalent cell translocation potency of a short protamine derivative rich in arginine residues which was equivalent to that of the Tat peptide combined with the absence of toxicity [85]. Cardozo et al showed that concentrations of up to 100 M of Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic [86]. In addition, it is noteworthy that peptide concentrations used in toxicity tests are in their very large majority far above the concentrations used for delivering various drugs into cells (which range from 1 to 10 M).…”