Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems

Abstract

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.

Cell-penetrating peptides as a promising tool for delivery of various molecules into the cells

“…However, regardless of the model, the translocation of large molecules requires a large destabilization of cell membrane, which is not correlated with the observed low cytotoxicity of CPPs and their conjugates. Thus, none of the presented models can fully explain the results of experiments with different CPPs, suggesting a contribution of alternative mechanisms of peptides' internalization, especially during penetration combined with molecular delivery [19].…”

“…The results of internalization of CPPs into cells even at low temperatures excluded endocytosis as the main mechanism of transport of peptides into the cell and suggested the existence of an alternative mechanism which required no energy [19]. Such transport mechanism was naturally observed for CPPs isolated from venoms: mastoparan [20] from V. lewisii wasp or crotamine [21] from rattlesnake, which interact directly with cell membrane (Table 1).…”

“…-In the inverted cell model, interactions between CPPs and cell membranes result in the formation www.fhc.viamedica.pl of concave fragments of the membrane, and in the 'toroidal' model, membrane protrusions occur. -In the 'barrel-stave' and 'toroidal' models, formation of pores within the membrane, through the homo-oligomerization of peptides introduced in the membrane, allows the prediction of well-defined structures, unlike the highly unpredictable and variable elements constituting the 'carpet' model [19]. -The driving forces of the direct translocation processes are CPP gradient for the 'toroidal' model and the inverted micelle or membrane potential in case of adaptive translocation model.…”

“…Despite the fact that results of many studies have shown the contribution of endocytosis in the CPP internalization mechanism and their cargo molecules, there are controversies surrounding the exact definition of the type of endocytosis involved in this process ( Figure 2) [19]. Hence, cell biology techniques were used to determine the type of endocytosis contribution or the transport without energy contribution in the process of CPP absorption: -incubation of cells with CPPs at low temperatures (< 4°C) or under energy deficiency; -application of trypsin to remove surface receptor proteins; -use of confocal or freeze-fracture electron microscopy [43,44]; -incubation with compounds facilitating selective transport pathway, e.g.…”

Polioviral receptor binding ligand: A novel and safe peptide drug carrier from polioviral capsid

“…It has been reported that the excess CPP could not affect the amount of uptake cargo. Trabulo et al (2010) demonstrated that there was no enhanced cellular internalization of siRNA after extensive purification of CPP/siRNA conjugates from the excess cationic peptide in the conjugation reaction. The result from cytotoxicity study showed that V peptide was safer than Tat peptide, indicated by higher cell viability.…”