MECHANISM OF INTERNALIZATION OF CELL PENETRATING PEPTIDES INTO HFF CELLS

FERNANDA FEDERICI; LAURA DELGUI; MARIA VICTORIA BERBERIAN

Mendoza

Jornada; XIII Jornadas de Investigación de la Facultad de Ciencias Médicas; 2019

FCM y FCEN

MECHANISM OF INTERNALIZATION OF CELL PENETRATING PEPTIDES INTO HFF CELLSAutores: María Fernanda Federici, Laura Ruth Delgui, María Victoria BerberiánIntroduction: 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, 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 involved.Methods: We combine 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 kinetic 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 TAT only through the diffusive pathway since TAT does not co-localize with the endosomal Dextran-Texas Red marker. Conclusion: We have demonstrated that TAT translocate the cell membrane of Human Foreskin Fibroblasts after co-incubation 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 the HFF cells, proteins capable of modulating processes as complex as viral replication.