M918: A novel cell penetrating peptide for effective delivery of HIV-1 Nef and Hsp20-Nef proteins into eukaryotic cell lines.

BACKGROUND: HIV-1 Nef protein is a possible attractive target in development of therapeutic HIV vaccines including protein-based vaccines. The most important disadvantage of protein-based vaccines is their low immunogenicity which can be improved by heat shock proteins (Hsps) as an immunomodulator, and cell penetrating peptides (CPPs) as a carrier.

METHODS: In this study, the HIV-1 Nef and Hsp20-Nef proteins were generated in E.coli expression system for delivery into the HEK-293T mammalian cell line using a novel cell penetrating peptide, M918, in a non-covalent fashion. The size, zeta potential and morphology of the peptide/protein complexes were studied by scanning electron microscopy (SEM) and Zeta sizer. The efficiency of Nef and Hsp20-Nef transfection using M918 was evaluated by western blotting in HEK-293T cell line.

RESULTS: The SEM data confirmed the formation of discrete nanoparticles with a diameter of approximately 200-250 nm and 50-80 nm for M918/Nef and M918/Hsp20-Nef, respectively. The dominant band of ~ 27 kDa and ~ 47 kDa was detected in the transfected cells with the Nef/ M918 and Hsp20-Nef/ M918 nanoparticles at molar ratio of 1:20 using anti-HIV-1 Nef monoclonal antibody. These bands were not detected in the un-transfected and transfected cells with Nef or Hsp20-Nef protein alone indicating that M918 could increase the penetration of Nef and Hsp20-Nef proteins into the cells.

CONCLUSION: These data suggest that M918 CPP can be used to enter HIV-1 Nef and Hsp20-Nef proteins inside mammalian cells efficiently as a promising approach in HIV-1 vaccine development.