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INTRODUCTION: Recently, exploiting cell-surface thiols (also known as exofacial protein thiols (EPTs)) to obtain better cellular delivery when using thiolated biomolecules such as cell penetrating peptides (CPPs), has gained increasing attention as promising new mechanism. Still, plenty questions remain to be answered. For disulfide-containing CPPs a correlation between EPTs and uptake efficiency was discussed [1]. Our cysteine-rich CPP CyLoP-1 (Cytosol Localizing Peptide-1) has been shown to be more efficient in cytosolic targeting than other established CPPs [2]. Aim of the present study was to evaluate if modification of available EPTs
influences the cytosolic delivery of CyLoP-1. Furthermore, the question was asked how the uptake of the reduced and oxidized version of CyLoP-1 (CyloP-1 [RED.]/[OX.]) is influenced by this EPT-modification. METHODS: To test our hypothesis, B16.F10 cancer cells, that are known to have a high number of EPTs [3] were subjected to tris(2-carboxethyl)phosphine (TCEP) or N-ethylmaleimide (NEM)
to increase or reduce the number of available EPTs, respectively [3]. Internalization of CyLoP-1 [RED.] or [OX.] (both covalently bound to lysine-FITC) was evaluated according to [2] (both 2.5M, after 3 and 18 hours). RESULTS: Pre-treatment of B16.F10 cells with TCEP (i.e. higher number of available EPTs) increased the intracellular delivery of CyLoP-1 [RED.] and CyLoP-1 [OX.] after 3h of incubation (Fig. 1A). While no significant effect with NEM-treatment was seen for CyLoP-1 [OX.], pre-treatment with 20M NEM significantly decreased delivery of CyLoP-1 [RED.] (Fig. 1A). In contrast to [3] (who used up to 200M NEM) in our cell model, higher concentrations of NEM lead to pronounced cell death already after 15min (data not shown). Similar effects as seen after 3h of incubation with CPPs were also observed for 18h incubation with both CyLoP-1 [RED.]/[OX.] (Fig. 1B). In these experiments, even
lower NEM-concentrations had to be used, due to otherwise massive cell death. CONCLUSION: Using a cell line which is known for its high amount of EPTs in combination with EPT-modifications we were able to demonstrate, that intracellular delivery of the cysteine-rich CPP CyLoP-1 is highly dependent on the number of available EPTs (i.e. reduced surface thiols). By this, we further strengthen the
notion of an important role for EPTs when using thiolated compounds for cell delivery.
