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Intracellular and especially cytosolic delivery with cell penetrating peptides (CPPs) can be severely limited by endocytotic uptake, leading to unwanted vesicular entrapment. A correlation between cell-surface thiols and uptake efficiency of disulfilde-containing CPPs was discussed and it was shown that these CPPs possess much better cytosolic targeting capability [1].Our newly developed, cysteine-rich CPP, CyLoP-1 (Cytosol Localizing Peptide 1, CRWRWKCCKK) showed pronounced cytosolic delivery already in its non-oxidized form. Aim of the present study was to evaluate if a controlled oxidation of the cysteines, generating disulfide linkages in CyLoP-1, further increases its cytosolic targeting. To test this hypothesis the pro-apoptotic peptide AVPIAQK (SmacN7) was attached to CyLoP-1. SmacN7 alone can not pass cellular membranes and needs to bind to its cytosolic target to exert a pro-apoptotic action and thus can be used to prove cytosolic delivery of CPP-conjugates. CyLoP-1 (covalently bound to lysine-FITC) was subjected to oxidizing conditions. SmacN7 was covalently conjugated to the N-terminus, yielding the construct SmacN7-K(FITC)-CyLoP-1, which was also oxidized. Internalization of all 4 compounds was evaluated on 3T3 fibroblasts (2.5µM, 18 hours, for method details see [2]). Caspase-3 activity as parameter for apoptosis-induction by the reduced and oxidized SmacN7-K(FITC)-CyLoP-1 was measured in HeLa cells. Intracellular uptake (measured by FITC-fluorescence) was significantly higher for the oxidized versions of both the peptide itself and SmacN7-CyLoP-1. This was confirmed by fluorescence microscopy of the same cells, displaying higher cytosolic fluorescence for the oxidized versions of the compounds. However, cargo-attachment reduced the internalization efficacy of CyLoP-1 in its reduced as well as oxidized form. SmacN7-CyLoP-1 was capable of inducing apoptosis in HeLa cells, while SmacN7 alone had no effect, proving efficient delivery of the bioactive cargo into the cytosol. Oxidation of SmacN7-CyLoP-1 further enhanced the amount of apoptotic cells (i.e. increased Caspase-3 activity). Thereby demonstrating that oxidative modification of cysteine-residues in CyLoP-1 enhances its cytosolic targeting capability. Conclusion: Our results suggest an important role of disulfides in the uptake mechanisms of cysteine-containing CPPs and also in their capability to deliver to the cytosol. The use of disulfide-motifs in CPPs might also be applicable for other types of cargos, like imaging agents. [1]Aubry,FASEB J.23(2009)2956 [2]Mishra,Bioconjug.Chem.2009
