Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo.

Macromolecules including DNA and proteins serve as important human therapeutics but are limited by their general inability to cross cell membranes. Supercharged proteins have been known as potent tools for delivery of macromolecules into mammalian cells. Thus, the use of these delivery systems is important to reduce the human papillomavirus (HPV)-associated malignancies through improvement of vaccine modalities. In this study, we used a supercharged green fluorescent protein (+36 GFP) for delivery of the full-length HPV16 E7 DNA and protein into mammalian cells and evaluated immune responses, and protective/therapeutic effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7 DNA/+36 GFP and also E7 protein/+36 GFP form stable nanoparticles through non-covalent binding with an average size of ∼ 200-300 nm. The efficient delivery of E7 DNA or protein by +36 GFP was detected in HEK-293T cell line for 4 h and 24 h post-transfection. Mice immunization with E7 protein/+36 GFP nanoparticles elicited a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein, E7 DNA, E7 DNA/+36 GFP and control groups (p < .05). Moreover, the E7 DNA/+36 GFP and E7 protein/+36 GFP nanoparticles similarly protected mice against TC-1 tumor challenge (∼67%) as compared to E7 DNA and E7 protein (∼33%), respectively. These data suggest that +36 GFP may provide a promising platform to improve protein and DNA delivery in vitro and in vivo.