The uptake mechanism for the drug delivery vector Tat-derived peptide

Tat (Transactivating transcriptional activator) peptide, derived from HIV type-1 Tat protein, is the most commonly studied cell penetrating peptide (CPP) as a peptide or as a gene carrier. As a gene carrier (GDS), its low transfection efficiency remains a disadvantage when compared with the viral vectors. As it may be due to a loss of cell penetrating activity upon binding to DNA, improved efficiency was sought by coupling membrane active peptides e.g. LK15 to Tat peptide. While it has been shown that the resulting Tat-LK15 is orders of magnitude more efficient than Tat at mediating transfection in cells its trafficking mechanism as well as the influence of the amphipathic peptide upon binding remain unclear thus its limits refinement. Saleh et al (ref) have shown that Tat-LK15 DNA complexes enter cells by endocytosis but could not elucidate the cellular trafficking in particular the endocytosis route remained unclear and needed a more detailed approach. Based on this we hypothesised that (i) the endocytosis route may depend on the size and the concentration of the GDS and (ii) blocking of one endocytosis route may stimulate or induce another pathway to compensate the effect consequently. The initial phase consisted in assessing the concentration and of incubation times of specific inhibitors to observe a significant shift by FACS. The chosen inhibitors are chloropromazin, filipin and N-ethyl isopropyl amiloride (EIPA) which inhibit clathrin mediated, caveolae mediated endocytosis and macropinocytosis respectively. To test the effect of the inhibitors, specific marker s were used i.e. transferrin, albumin and dextran for clathrin mediated-, caveolae mediated endocytosis and macropinocytosis respectively and the effect of these inhibitors [chloropromazin, filipin and (EIPA)]. In parallel, Tat-LK15 has been labelled with TAMRA and purified to follow the fate of the peptide during cellular trafficking in later experiments.