Abstract
The passive translocation of cell-penetrating peptides (CPPs) through the cellular membrane has been extensively studied for its importance in virus life cycles and promising drug-delivery potential. The penetration mechanism, however, remains unresolved.
To elucidate membrane structure in the presence of CPPs, we study three different peptides in experimental systems of various degrees of complexity: nonaarginine (R9), a cationic peptide known for its ability to passively penetrate cellular membranes, the equally-charged nonalysine (K9) as a non-penetrating negative control and tetraarginine (R4), addressing oligoarginine size-dependent penetration properties. Firstly, we employ Umbrella sampling molecular dynamic simulations, providing insight on an atomistic level as well as the free energy profiles of peptide-membrane interactions.
Secondly, the spectral changes of membrane incorporated fluorescent dye Laurdan show the effect of CPPs induced in membranes' lateral organization. We employ lipid-only large unilamellar vesicles (LUVs), as the simplest model of biological membranes as well as extracellular vesicles (EVs), similar in size to LUVs but biologically complex.
Lastly, we use live imaging confocal microscopy to study human bone osteosarcoma epithelial cells interacting with CPPs. Our results show similar effect of CPPs on LUVs and EVs and we monitored passive translocation of fluorescently labelled R9 into cells dissimilar to Transferrin endocytic uptake.