Internalization mechanisms of cell-penetrating peptides : Structure-activity relationships, cellular distribution and membrane toxicity studies

Abstract: Transport of substances with pharmaceutical potential over the plasma membrane has been pushed a great step forward with the discovery of short cationic polypeptides, known as cell-penetrating peptides (CPP). Since then, the internalization mechanism and cargo delivery properties of CPPs has been extensively studied. Unfortunately, no consensus over the internalization mechanism has been achieved so far. Much attention has been paid to the delivery of various macromolecules into the cytoplasm and other subcellular compartments with the help of CPPs, while the structural requirements and toxicity of the peptides and their conjugates has not been studied as thoroughly.The focus of this thesis is on the characterization of internalization mechanism, toxicity and prediction of CPPs as currently most discussed issues in the field of research.The studies included in the present thesis indicate that for the majority of CPPs the uptake is dependent of endocytic processes. However, these studies do not reveal one major endocytosis pathway, responsible for the uptake of all tested CPPs. Instead it is most likely that different mechanisms are involved in the uptake of different CPPs. Peptides with apparent amphipathic natures are mainly internalized via clathrin-mediated endocytosis while the CPPs with low amphipathic moment utilize macropinocytosis.Disturbance of plasma membrane by CPPs in different cell lines was also studied. Results show that peptides with higher amphipathic moment have, in general, higher toxicity, both long- and short-term. However, the amphipathic nature of CPPs is not the only criterion for membrane perturbation. Results also demonstrated the difference in membrane perturbation in different cell lines.Taken together the studies involved in this thesis provide useful information about the internalization mechanisms and cytotoxicity of cell-penetrating peptides.