Multifunctional Gene Carriers with Enhanced Specific Penetration and Nucleus Accumulation to Promote Neovascularization of HUVECs in Vivo

Recently, gene therapy has attracted much attention, especially for the treatment of vascular disease. However, it is still challenging to develop the gene carriers with high biocompatibility as well as highly efficient gene delivery to overcome multiple barriers. Herein, a frequently used cell-penetrating peptide PKKKRKV (TAT) was selected as a functional sequence of the gene carrier with distinctive cell-penetrating ability. REDV peptide with selectively targeting function for endothelial cells (ECs) and nuclear localization signals (NLS) were integrated with this TAT peptide to obtain a highly efficient gene delivery system with ECs specificity and nucleus accumulation capacity. Besides, the glycine sequences with different repeat numbers were inserted into the above integrated peptide. These glycine sequences acted as a flexible spacer arm to exert the targeting, cell-penetrating, and nucleus accumulation functions of each functional peptide. Three tandem peptides REDV-Gm-TAT-Gm-NLS (m = 0, 1, and 4) complexed with pZNF580 plasmid to form gene complexes. The results of hemocompatibility and cytocompatibility indicated that these peptides and gene complexes were nontoxic and biocompatible. The internalization efficiency and mechanism of these gene complexes were investigated. The internalization efficiency was improved as the introduction of targeting REDV and glycine sequence, and the REDV-G4-TAT-G4-NLS/pZNF580 (TP-G4/pZNF580) complexes showed the highest cellular uptake among the gene complexes. The TP-G4/pZNF580 complexes also presented significantly higher internalization efficiency (∼1.36 times) in human umbilical vein endothelial cells (HUVECs) than human umbilical artery smooth muscle cells. TP-G4/pZNF580 complexes substantially promoted the expression of pZNF580 by confocal live cell imaging, gene delivery efficiency, and HUVECs migration assay. The in vitro and in vivo revascularization ability of transfected HUVECs was further enhanced obviously. In conclusion, these multifunctional REDV-Gm-TAT-Gm-NLS peptides offer a promising and efficacious delivery option for neovascularization to treat vascular diseases.