Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43–58)

Abstract

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.

rBPI21 Promotes Lipopolysaccharide Aggregation and Exerts Its Antimicrobial Effects by (Hemi)fusion of PG-Containing Membranes

“…Apparently, the interaction of the peptide with neutral liposomes is weaker, when compared to the negatively charge membranes, and must be limited to the surface of the bilayer, making this interaction difficult to detect by other methods [21]. rBPI 21 interaction with pure POPC bilayers may be explained by the repulsion of the positive charged end group of the choline towards the aqueous phase, due to the high density of the positive amino acids in rBPI 21 , exposing the hydrophobic region of the lipid for the interaction with the peptide [38], [39]. Despite the lower aggregation state of the POPC∶LPS in the presence of rBPI 21 , the peptide is able to interact with negative charges of the LPS lipid A.…”

The Cell‐Penetrating Peptide TAT(48‐60) Induces a Non‐Lamellar Phase in DMPC Membranes

“…For several CPPs, binding, membrane insertion, bilayer disruption, translocation and/or fusogenicity have been characterized in such model systems. [30,32,33,[37][38][39] For example, some peptides derived from the HIV protein Tat (transacting activator of transcription) were shown to induce phospholipid mixing and vesicle fusion without detectable dye leakage, as seen by fluorescence. [32] Recently, it was also demonstrated that they can translocate through liposomes consisting of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) [33] and through giant vesicles consisting of soybean phosphatidylcholine, but not through large unilamellar ones.…”

Properties of cell penetrating peptides (CPPs)

“…The mechanism of uptake of cationic CPPs is still unclear and some existing concepts, particularly the role of endocytosis in this process has been revised (40,42,70,71). Until recently, it was believed that internalization of CPPs involves the mechanism of endocytosis similar to the one described for bacterial and plant toxins that include their own transmembrane transfer apparatus (72).…”