Date Published: January 23, 2019
Publisher: Public Library of Science
Author(s): Claudia Almeida, Ofelia Maniti, Margherita Di Pisa, Jean-Marie Swiecicki, Jesus Ayala-Sanmartin, Ludger Johannes.
Cell penetrating peptides (CPPs) are able to transport hydrophilic molecules inside cells. To reach the cytosol, the peptide associated with a cargo must cross the plasma or the endosomal membrane. Different molecular mechanisms for peptide internalisation into cells have been proposed and it is becoming clear that the cellular internalisation mechanisms are different depending on the peptide sequence and structure and the target membrane. Herein, the penetration of three peptides into large unilamellar vesicles were studied: the homeodomain derived 16-residues penetratin, nona-arginine (R9), and a small peptide containing 6 arginine and 3 tryptophan residues (RW9). The membrane models were composed of phospholipids from natural sources containing different molecular species. We observed that among the three peptides, only the amphipathic peptide RW9 was able to cross the membrane vesicles in the liquid disordered state. The changes in the distribution of the previously characterized cholesterol-pyrene probe show that cholesterol-pyrene molecules dissociate from clusters upon membrane interaction with the three peptides and that the cholesterol environment becomes more disordered in the presence of RW9. Finally, we studied the effect of the peptides on lipid ordering on giant plasma membrane vesicles. The amphipathic peptides RW9 and its longer homologue RW16 induced lipid de-packing in plasma membrane vesicles. Overall, the data suggest that a disordered membrane favours the translocation of RW9, that the membrane cholesterol is redistributed during peptide interaction, and that the peptide amphipathic character is important to increase membrane fluidity and peptide membrane translocation.
Cell penetrating peptides (CPPs) are potential therapeutic vectors used to introduce molecules into mammalian cells, from small active molecules to siRNAs and DNA oligonucleotides. They are frequently enriched in arginine and lysine residues. For reviews see [1–3]. The molecular mechanisms involved in cellular membrane translocation of CPPs depend on peptide sequence, structure, concentration and also the nature of the cargo [2,4,5]. Several plasma membrane or endosomal membrane crossing mechanisms have been proposed which include the electroporation-like process , the neutralisation charges by guanidinium-phosphate hydrogen bonds , membrane inverted micelles formation [8,9], pore formation  and also direct translocation through the membrane .
The purpose of this work was to study the role of lipid organisation during CPP-membrane interaction. We performed experiments with membrane containing phospholipid from natural sources mimicking biological membranes which contain acyl chains of different length and degree of unsaturation. Secondly, we studied the peptides effects on lipid order in giant plasma membrane vesicles obtained from epithelial cells.
In conclusion, penetratin induced small modifications in the cholesterol-pyrene distribution and did not cross the LUVs membranes. R9 triggered a diminution in cholesterol-pyrene aggregation and a slight increase in the Lo membrane environment contribution. Therefore, R9 modifies the membranes following the cholesterol movements illustrated in Fig 8A. Similarly, RW9 reduced the cholesterol-pyrene aggregation. However, RW9 favours the Ld environment of cholesterol-pyrene. This behaviour is represented in the scheme of Fig 8B. Moreover, the propensity of RW9 to favour the Ld domains was supported by its effect on membrane lipid de-packing observed in GPMVs. Overall, our data suggest that the membrane cholesterol is redistributed during peptide-membrane interaction, that the peptide-induced disorder favours the translocation of RW9, and that the peptide amphipathicity is important to induce both, membrane fluidity and peptide translocation through the membrane. We suggest that in cells, the peptides are able to induce lipid reorganization including cholesterol movements. This lipid rearrangements result in the preferential peptide translocation through the plasma membrane in liquid disordered (non-raft) domains or at the interface of ordered-disordered domains.