CyLoP-1, a new tool for the cytosolic delivery of intracellular targeted probes

Cell Penetrating Peptides (CPPs) have gained attention in biomedical field because of their ability to deliver a variety of hydrophobic moieties through the membrane barrier. CPPs are generally taken up by endocytotic pathways and vesicular encapsulation limits the interaction of the attached cargo with targets present in the cytosol. Here we present the systematic development of a cysteine rich CPP (derived from polypeptide Crotamine [1]) by Structure Activity Relationship (SAR) studies. Various peptides were synthesized by Fmoc/tBu strategy and labeled with fluorescein isothiocyanate at the N-terminus. After purification by RP-HPLC, the products were lyophilized and characterized by ESI-MS. Uptake of fluorescently labeled peptides was assessed in NIH-3T3 mouse fibroblasts by fluorescence spectroscopy and microscopy. Our studies identified a novel CPP, CyLoP-1, which is efficiently delivered into the entire cytoplasm of cells besides being encapsulated in vesicles at low concentrations of 2.5 µM in a non-cytotoxic and temperature independent fashion. It proved to be most efficient in its natural L-form as SAR studies indicated a clear negative effect of chiral inversion of the residues on cellular uptake and distribution of CyLoP-1. The additional feature of cytosolic gain was further explored by the covalent attachment of different cargoes to CyLoP-1. Payload consisted of fluorophore, a Magnetic Resonance Imaging agent (Gd)-DOTA, a bio-active peptide (SmacN7), the larger peptide Penetratin, or a PNA molecule. The regular decrease in the uptake and localization pattern concomitant with the increase in the cargo size was observed. This indicates the dependence of internalization and distribution profile of CyLoP-1 constructs on the nature, charge as well as size of the cargo. Efficient intracellular delivery into the entire cytosol and a low cytotoxicity makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes like intracellular targeted probes. [1] Kerkis A. et al, FASEB J. 2004, 18, 1407