Cellular uptake mechanisms and potential therapeutic utility of peptidic cell delivery vectors: Progress 2001–2006

Abstract

Cell delivery vectors (CDVs) are short amphipathic and cationic peptides and peptide derivatives, usually containing multiple lysine and arginine residues. They possess inherent membrane activity and can be conjugated or complexed with large impermeable macromolecules and even microscopic particles to facilitate cell entry. Various mechanisms have been proposed but it is now becoming clear that the main port of entry into cells of such CDV constructs involves adsorptive-mediated endocytosis rather than direct penetration of the plasma membrane. It is still unclear, however, how and to what extent CDV constructs are capable of exiting endosomal compartments and reaching their intended cellular site of action, usually the cytosol or the nucleus. Furthermore, although many CDVs can mediate cellular uptake of their cargo and appear comparatively non-toxic to cells in tissue culture, the utility of CDVs for in vivo applications remains poorly understood. Whatever the mechanisms of cell entry and disposition, the overriding question as far as potential pharmacological application of CDV conjugates is concerned is whether or not a therapeutic margin can be achieved by their administration. Such a margin will only result if the intracellular concentration in the target tissues necessary to elicit the biological effect of the CDV cargo can be achieved at systemic CDV exposure levels that are non-toxic to both target and bystander cells. It is proposed that the focus of CDV research now be shifted from mechanistic in vitro studies with labeled but otherwise unconjugated CDVs to in vivo pharmacological and toxicological studies using CDV-derivatized and other cationized forms of inherently non-permeable macromolecules of true therapeutic interest.

Targeting antibodies to the cytoplasm

“…124 Although early results that were potentially affected by methodological artifacts suggested an energy-independent mode of uptake, it is now generally accepted that PTDs are primarily taken up by endocytosis. 121,125 The positive charge of the HIV1-TAT-PTD and other argininerich PTDs may help to concentrate the peptide on the cell surface by electrostatic interactions with negatively charged glycoproteins, 124,[126][127][128] similar to the mechanism described for transfection. Heparan sulfate proteoglycans (HSPGs) were proposed to assist in promoting attachment of HIV1-TAT protein and HIV1-TAT-PTD to the cell surface.…”

“…As Fischer suggests, mechanistic studies should focus on applications for PTDs rather than "profiling yet another CDV (cell delivery vector) in unconjugated form" and, according to him, "One of the most exciting applications of CDVs would be the delivery of antibodies against intracellular targets." 121 The appealing idea of creating "transbodies" or "transmabs" by linking a PTD to an antibody had already been promoted earlier by others. 137,138 An extensive use of this exciting approach, however, has been missing so far.…”

“…Naturally-derived peptides include the herpes simplex virus structural protein VP22 and antimicrobial or pore-forming peptides. 121 Rational design has yielded peptides such as transportan, which is a chimeric peptide composed of a neuropeptide and a wasp venom peptide toxin. 122 Despite differences in amino acid sequence, most PTDs share some common physicochemical properties, such as amphipathicity and positive charge.…”

“…122 Despite differences in amino acid sequence, most PTDs share some common physicochemical properties, such as amphipathicity and positive charge. 121 The class of arginine-rich PTDs, which includes HIV1-TAT-PTD, nona-arginine and Antennapedia-PTD, is one of the best-studied and most widely used group of PTDs. Mechanistic aspects mentioned here mainly refer to this class of PTDs.…”

“…Misinterpretation of flow cytometry data and fixation artifacts suggested an apparent endocytosisindependent uptake of high efficiency. 121,133 In 2003, Richard and colleagues showed that even mild fixation of cells caused redistribution of membrane parts, which internalized membraneassociated peptides. 184 Consequently, live cell imaging became the preferred method for examination of cells.…”