Intracellular Uptake and Membrane Binding of Cell-penetrating Peptides: The Influence of Arginines, Tryptophans and Proteoglycans
One of the major limitations in the realization of functional gene therapy is efficient intracellular transport of the therapeutic macromolecules. Amongst several different techniques, cell-penetrating peptides (CPPs) have emerged as promising candidates for this purpose. CPPs may cross cellular membranes by both endocytotic and non-endocytotic pathways, but the mechanisms by which uptake is mediated are not yet fully understood. It has been suggested that a major determinant in this issue is the interaction between the CPPs and the cell membrane, which is dependent on both the peptide sequence and membrane constituents.
The focus of the work presented in this thesis is CPP-membrane interactions, and how they are influenced by the amino acids arginine and tryptophan, as well as by membrane proteoglycans. For this purpose cellular uptake and membrane affinity of penetratin and two homologues enriched in or lacking arginines were investigated in both wild type cells and cells lacking proteoglycans. Furthermore, uptake in live cells as well as induced liposome leakage and binding affinity to liposomes, of six newly designed peptides containing arginine and tryptophan in different combinations, were investigated. This was followed by investigating the orientation of the tryptophans and the secondary structure of these peptides when interacting with liposomes. We show that, with higher number of arginines both intracellular uptake and membrane affinity increase while the dependence of proteoglycans for uptake decreases. Moreover, we find that the spacing of tryptophans in the peptide sequence seems to influence intracellular uptake and that the variations in peptide secondary structure, as reflected by the angles of the transition moments of the tryptophans, also suggest variations in membrane interactions.