Agmatine-grafted bioreducible poly(l-lysine) for gene delivery with low cytotoxicity and high efficiency

Bioreducible cationic polymers have gained considerable attention in gene delivery due to their low cytotoxicity and high efficiency. In the present work, we reported a cationic polymer, poly(disulfide-l-lysine)-g-agmatine (denoted as SSL-AG), and evaluated its ability to transfer pEGFP-ZNF580 plasmid (pZNF580) into human umbilical vein endothelial cells (HUVECs). This SSL-AG polymeric carrier efficiently condensed pZNF580 into positively charged particles (<200 nm) through electrostatic interaction. This carrier also exhibited excellent buffering capacity in the physiological environment, good pDNA protection against enzymatic degradation and rapid pDNA release in a highly reducing environment mainly because of the responsive cleavage of disulfide bonds in the polymer backbone. The hemolysis assay and in vitro cytotoxicity assay suggested that the SSL-AG carrier and corresponding gene complexes possessed both good hemocompatibility and great cell viability in HUVECs. The cellular uptake of the SSL-AG/Cy5-oligonucleotide group was 3.6 times that of the poly(l-lysine)/Cy5-oligonucleotide group, and its mean fluorescence intensity value was even higher than that of the PEI 25 kDa/Cy5-oligonucleotide group. Further, the intracellular trafficking results demonstrated that the SSL-AG/Cy5-oligonucleotide complexes exhibited a high nucleus co-localization rate (CLR) value (36.0 ± 2.8%, 3.4 times that of the poly (l-lysine)/Cy5-oligonucleotide group, 1.6 times that of the poly(disulfide-l-lysine)-g-butylenediamine/Cy5-oligonucleotide group) at 24 h, while the endo/lysosomal CLR value was relatively low. This suggested that SSL-AG successfully delivered plasmid into HUVECs with high cellular uptake, rapid endosomal escape and efficient nuclear accumulation owing to the structural advantages of the bioreducible and agmatine groups. In vitro transfection assay also verified the enhanced transfection efficiency in the SSL-AG/pZNF580 group. Furthermore, the results of CCK-8, cell migration and in vitro/vivo angiogenesis assays revealed that pZNF580 delivered by SSL-AG could effectively enhance the proliferation, migration and vascularization of HUVECs. In a word, the SSL-AG polymer has great potential as a safe and efficient gene carrier for gene therapy.