Multifunctional REDV-G-TAT-G-NLS-Cys peptide sequence conjugated gene carriers to enhance gene transfection efficiency in endothelial cells

Rapid endothelialization on small diameter artificial blood vessels is an effective strategy to facilitate long-term patency and inhibit thrombosis. The gene delivery can enhance the proliferation and migration of endothelial cells (ECs), which is beneficial for rapid endothelialization. REDV-G-TAT-G-NLS-Cys (abbreviated as TP-G) peptide could weakly condense pEGFP-ZNF580 (pZNF580) and transfect ECs, but its transfection efficiency was still very low because of its low positive charge, low stability and weak endosome escape ability. In order to develop more stable and efficient gene carriers with low cytotoxicity, in the present study, we conjugated different amounts of TP-G peptide onto poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-g-PEI) amphiphilic copolymers via a hetero-poly(ethylene glycol) spacer (OPSS-PEG-NHS). The TP-G peptide and PEI could cooperatively and strongly condense pZNF580. The carrier’s cytotoxicity was reduced by the introduction of poly(ethylene glycol) spacer. They condensed pZNF580 to form gene complexes (PPP-TP-G/pZNF580) with suitable size and positive zeta potential for gene delivery. The transfected ECs promoted their migration ability as demonstrated by cell migration assay. The results of cellular uptake and confocal laser scanning microscopy showed significantly high internalization efficiency, endosomal/lysosomal escape and nucleus location of pZNF580 by this multifunctional TP-G peptide sequence conjugated gene delivery system. Furthermore, several inhibitors were used to study the cellular uptake pathways of PPP-TP-G/pZNF580 complexes. The results showed that PPP-TP-G2/Cy5-oligonucleotide complexes exhibited the optimized endocytosis pathways which facilitated for cellular uptake. In conclusion, the multifunctional TP-G peptide conjugated gene carriers could promote the transfection efficiency due to the multifunction of REDV, cell-penetrating peptide and nuclear localization signal in the peptide sequence, which could be a suitable gene carrier for endothelialization.