RESUMO
Previously, our group carried out a series of studies using branched polyethyleneimine with 25 000 g mol-1 molar mass (bPEI-25k) as a gene delivery vector and came up with the theory that free cationic chains un-complexed with plasmid DNA (pDNA) can greatly increase the gene transfection efficiency and influence the intracellular delivery process. These free chains can penetrate the membrane quickly, with some of them embedded inside the lipid bi-layers. The "stuck-out" cationic chain ends would shield the signal protein, prevent/delay the development of the later endolysosomes and enhance the efficiency of gene delivery. To mimic the effect of cationic polymers, we selected to use vesicle associated membrane protein-8 (VAMP8) and modified its N-terminus with different cationic cell penetrating peptides (CPPs). The modified fusion proteins are expressed in an Escherichia coli system and purified after extraction. These modified VAMP8 proteins are used as free chains for gene transfection, while using bPEI-25k to condense the pDNA. The results show that the gene transfection efficiency of bPEI-25k/pDNA polyplexes is obviously enhanced in the 293 T cell line. Furthermore, the gene sequences encoding these modified VAMP8 proteins are sub-cloned to pcDNA-3.1 vector and then transferred to 293 T before the treatment with bPEI-25k/pDNA polyplexes. From the result, the transfection efficiency of bPEI-25k/pDNA complexes is enhanced at a similar level to that using modified VAMP8 as free chains. Our current results prove that free cationic chains are probably embedded with the membrane and influence intracellular trafficking, pointing out a new idea to design an effective non-viral gene delivery system.
