Mechanism Of Internalization Of Cell Penetrating Peptides Into HFF Cells

BERBERIÁN, VICTORIA; DELGUI, LAURA R.; FEDERICO, FERNANDA

Simposio; II Simposio Internacional de Medicina Traslacional; 2019

Introduction: The development of tools that facilitate the cellular uptake of therapeutic molecules constitutes an active field of research. Cell penetrating peptides (CPPs) have been introduced as novel biocarriers, since they are able to translocate cell membranes by a mechanism which is still poorly understood. Experimental evidence suggests that CPPs internalization may occur by physical diffusion across cell membranes, or may involve endocytic processes, or a combination of both. Objectives: The objectives of this study were to optimize the translocation of CPPs into Human Foreskin Fibroblasts (HFF) and to elucidate the mechanism of cellular uptake.Methods: We combine confocal fluorescence microscopy and flow cytometry to analyze the effect of basic parameters such as the concentration of CPP, culture medium, time and temperature of incubation, on the internalization of the CPP: TAT. The mechanism of cellular uptake was investigated using endosomal and cytoplasmic molecular markers. Results: The results showed that the translocation kinetics of the TAT peptide depends on the CPP concentration, as well as the medium, temperature and incubation time. For example, when we increase the CPP concentration (0,5-2 μM), the time (5-60 min) and the temperature (4-37 oC) TAT increases the translocation rates and the concentration of CPP in the cytosol cellular. Moreover, experiments with endosomal and cytoplasmic markers indicated that HFF cells internalize the TAT peptide through the diffusive pathway, as TAT does not co-localize with the endosomal Dextran-Texas Red marker. Conclusion: We have demonstrated that TAT translocates the cell membrane of Human Foreskin Fibroblasts and that the translocation efficiency can be modulated by varying some basic parameters. Moreover, we have demonstrated that only the diffusive mechanism of internalization operates in these cells. These results show promising use of TAT peptide to introduce into HFF cells proteins capable of modulating processes as complex as viral replication.