Recombinant protein-based nanocarriers and their association with cationic liposomes: Characterization and in vitro evaluation

Abstract

A major bottleneck in the development of efficient protocols for gene therapy and DNA vaccination is the low efficiency of gene transfer by non-viral vectors. This is mainly attributed to the fact that, during the traffic to target cell nuclei, vectors must overcome a series of enzymatic, physical, and diffusional barriers. The objective of this work was the development and characterization of new multifunctional non-viral vectors, based on proteins and lipids, that are able to efficiently deliver the foreign plasmid DNA (pDNA) to the nucleus of mammalian cells. A model pDNA containing the reporter gene GFP was complexed to protamine or the recombinant modular protein T-Rp3 to form binary complexes. In addition, we studied the ability of the cationic liposome composed of EPC/DOPE/DOTAP to encapsulate the binary complexes to form pseudo-ternary complexes (pDNA/protein/liposome). Characterization of the complexes were performed by dynamic light scattering (DLS), zeta potential, transmission electron microscopy (TEM) and pDNA accessibility assay. The assays revealed that both proteins were able to condense pDNA and form positively charged complexes, that could be efficiently encapsulated, leading to the formation of pDNA/protein/liposome complexes. Transfection studies using HeLa cells indicated that pDNA/protein formed by T-Rp3 were far more efficient for pDNA delivery than protamine. The complexes formed by pDNA/T-Rp3/liposome presented the highest transfection level (25%). On the other hand, cytotoxicity assays showed a significant decrease on cell viability when using pDNA/T-Rp3/liposome, indicating that the association of T-Rp3 with liposome significantly increase the delivery efficiency whilst prompting a proportional negative impact on cytotoxicity. A better understanding of the mechanisms of cell uptake and intracellular trafficking regarding the synergic effect between proteins and lipids in these vectors may, in the near future, lead to the development of more efficient non-viral vectors able to mimic the abilities of viral nucleic acid delivery.