The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.