Membrane Interactions of Arginine-Rich Peptides for the Intracellular Delivery of Gene-Targeted Drugs

Doctoral thesis, 2003

A major obstacle for the therapeutic use of e.g. oligonucleotides is their inherently poor cellular uptake. A recently discovered class of peptides, denoted cell-penetrating peptides, has been shown to traverse cell membranes, even when conjugated to oligonucleotides or even full-length proteins. This has raised the hope of improving the bioavailability of such gene-targeted drugs. The mechanism by which these peptides enter cells is poorly understood but has been suggested to involve a previously unknown, receptor-independent and non-endocytotic uptake pathway, via direct interactions with membrane lipids. The main part of this Thesis focuses on studies of the membrane interactions of three peptides: penetratin, Tat(48-60) and oligoarginine, using model systems. The latter two peptides are particularly rich in arginine residues, the role of which was investigated. All peptides were found to be able to translocate across the membranes of cell-sized vesicles. Smaller vesicles, on the other hand, are impermeable to the peptides. It remains to be elucidated whether these results are relevant for our understanding of the uptake mechanism. The membrane affinity of the peptides was quantified by a novel analysis of tryptophan emission spectra and was found to be highly dependent on the fraction of anionic lipids in the membrane. A model based on the Gouy-Chapman theory in combination with a surface partition equilibrium was found to be in excellent agreement with the experimental data. A lysine-substituted Tat peptide exhibits a weaker affinity for the vesicles than its parent peptide. The difference is, however, not large enough to account for the observed substantial differences in affinity for the cell surface, which suggests an important role for other constituents in the cell membrane. A live cell study included in this thesis points to the existence of both endocytotic and non-endocytotic uptake pathways for these peptides. The results suggest that only arginine-rich peptides are taken up via a non-endocytotic uptake pathway. Interestingly, oligoarginine and Tat were found to induce vesicle fusion at elevated peptide to lipid ratios, while penetratin merely aggregates the vesicles. The observed variations in membrane destabilizing properties of the peptides may be related to their different uptake characteristics and variations in cargo delivery efficiency.