This stapling system, further developed by the Verdine and Walensky groups, has been shown to significantly increase α-helical content in short peptide sequences, binding affinity, resistance to proteolysis and importantly to promote efficient cellular uptake. One strategy to overcome this entropic penalty and induce peptide folding into a biologically active α-helix is the introduction of an all-hydrocarbon staple in the peptide sequence, through ring-closing metathesis (RCM) reaction. However, the amino acid sequence of natural peptides, even if similar to a natural protein helix, is not sufficient to allow its proper α-helix folding. Therefore, in this study we decided to further expand and explore the domain of structured cell penetrating peptide by designing and synthesizing helical peptides for cytosolic delivery. More recently, it has been demonstrated that conformational constraints of amphipathic peptides, through multicyclization or introduction of α, α-disubstituted residues in the sequence as helical promoters, can dramatically increase their cellular uptake. In most cases, the cell delivery of such peptides is improved by their folding, as it was previously observed for some CPPs that naturally adopt a helical fold when they interact with the biological membranes. CPPs are often amphipathic oligomers with positively charged residues (arginine or lysine), allowing them to enter into the cell and drive the internalization of a wide range of bioactive cargos such as nanoparticles, RNA, proteins or cytotoxic agents. In this study, we focused on the last group of cargo carriers, CPPs, which represent short peptides composed of less than 30 amino acids. Indeed, many different siRNA delivery systems have been investigated such as viral vectors, liposomes, micelles, polymeric nanoparticles, and cell penetrating peptides (CPPs). Thus, one of the main current challenges to develop siRNA therapies is to improve their delivery into the cytosol while preventing their degradation. Furthermore, naked siRNA molecules are rapidly degraded by serum RNases, preventing their direct blood injection as therapeutic treatment. Unfortunately, siRNAs are anionic molecules that are unable to cross the cell membrane by themselves because of electrostatic repulsion with the anionic phospholipids of the biological membranes. The ability of siRNAs to “switch off” specific genes makes them attractive to develop new highly specific therapies. Small interfering RNA (siRNA) represents an interesting class of molecules that specifically target and downregulate their mRNA and the corresponding expressed proteins.
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