The Construction, Screening And Internalization Mechanism Of Cell Penetrating Peptides For Direct Cytosolic Delivery In Vivo

Cell-penetrating peptides (CPPs) are described as a kind of short peptides which can penetrate into cells and trigger a range of membrane-impermeable molecules into living cells. For their low toxicity, high uptake efficiency, bio-compatibility and the ability to cross brain-blood barrier, CPPs have attracted increasingly attentions as potential vehicles for therapeutics.However, there are still some problems hindering the development of CPPs. Firstly, the internalization mechanism of CPPs have not been fully understood yet. Secondly, although D-CPPs are usually preferred to be applied in vivo to improve their bio-stability, D-CPPs are also more toxic and the effect of chirality is still unclear. Thirdly, endosomal entrapment after internalizations and/or subsequently proteolysis in lysosomes is the most critical problem for the application of CPPs in vivo, which would severely restrict the bioavailability of cargos. Until now, there have been no reports on CPPs which have been showed to be distributed diffusely in cytosolic without endosomal entrapments after administrations in vivo. The direct cytosolic delivery of CPPs in vivo presents a major challenge for applications of CPPs in vivo.Trying to resolve above problems, we constructed and synthesized a series of novel chimeras consisting of various numbers of D-and L-arginine residues, investigated their cellular uptake behaviors, summarized the disciplines of D-Arg’s effect on the property of CPPs and picked out the best CPP (rR)3R2. Utilizing this CPP, we achieved the efficient cytosolic delivery not only in cultured cells but also in living tissue cells in mice after intravenous injection. And we still tried to explain the internalization mechanism of the series of CPPs. The main contents are summarized as followed:(1) To explore the effect of chirality, a series of novel chimeras consisting of various numbers of D-and L-arginine residues were constructed, synthesized and characterized.(2) The intracellular distributions, uptake efficiency, cytotoxicity, systemic toxicity and proteolytic stability and serum stability of these CPPs were tested and analyzed in different incubation conditions. The disciplines of D-Arg’s effection on the property of CPPs were summarized. The best CPP (rR)3R2was picked out. It possessed low systemic toxicity, high uptake efficiency, and remarkably, achieved efficient cytosolic delivery not only in cultured cells but also in living tissue cells in mice after intravenous injection.(3) The arginines-rich CPPs were showed to have two independent internalization mechanisms:endocytosis and directly membrane translocation. The direct membrane translocation is driven by the membrane potential and cell energy. Based on this mechanism, CPPs penetrate into cells directly from the membrane regions, accumulate in cells rapidly and are distributed diffusely in cytosol.