Abstract
BackgroundGene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. But slow endosomal escape rate is still one of the major barriers to successful gene delivery. In order to evaluate whether introducing oligohistidine (Hn) sequence into gene carriers can promote endosomal escape and gene transfection or not, we designed and synthesized Arg-Glu-Asp-Val (REDV) peptide functionalized TAT-NLS-Hn (TAT: typical cell-penetrating peptide, NLS: nuclear localization signals, Hn: oligohistidine sequence, n: 4, 8 and 12) peptides with different Hn sequence lengths. pEGFP-ZNF580 (pZNF580) was condensed by these peptides to form gene complexes, which were used to transfect human umbilical vein endothelial cells (HUVECs).ResultsMTT assay showed that the gene complexes exhibited low cytotoxicity for HUVECs. The results of cellular uptake and co-localization ratio demonstrated that the gene complexes prepared from TAT-NLS-Hn with long Hn sequence (n = 12) benefited for high internalization efficiency of pZNF580. In addition, the results of western blot analysis and PCR assay of REDV-TAT-NLS-H12/pZNF580 complexes showed significantly enhanced gene expression at protein and mRNA level. Wound healing assay and transwell migration assay also confirmed the improved proliferation and migration ability of the transfected HUVECs by these complexes. Furthermore, the in vitro and in vivo angiogenesis assay illustrated that these complexes could promote the tube formation ability of HUVECs.ConclusionThe above results indicated that the delivery efficiency of pZNF580 and its expression could be enhanced by introducing Hn sequence into gene carriers. The Hn sequence in REDV-TAT-NLS-Hn is beneficial for high gene transfection. These REDV and Hn functionalized TAT-NLS peptides are promising gene carriers in gene therapy.
Highlights
Gene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis
Ji et al proved that the combination of rapamycin-loaded polymer base layer and REDV peptide tethered top layer could promote the competitive adhesion of human umbilical vein endothelial cells (HUVECs) over human aortic smooth muscle cells, and enhanced in situ endothelialization [12]
Because H residue was hydrophobic at physiological condition, the amphiphilic REDV-TAT-nuclear localization signal (NLS)-Hn peptides could form stable micelles with relatively compact structure compared with REDV-TAT-NLS-H0
Summary
Gene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. Small-diameter artificial blood vessels (< 6 mm) have been used in clinical treatment of cardiovascular diseases, but their long-term patency rate remains low [1, 2] To solve this problem, many surface modification strategies have been developed to enhance. Surface modification with bioactive peptides, such as Arg-Gly-Asp (RGD) [8], Cys-Ala-Gly (CAG) [9] and Arg-Glu-Asp-Val (REDV), can promote the attachment to endothelial cells (ECs). Among these peptides, REDV peptide can be specially recognized by α4β1 integrin, which is enriched in ECs but lacked in smooth muscle. Our studies demonstrated that REDV-modified gene carriers could specially recognize ECs and selectively enhance transfection efficiency by transferring DNA into ECs so as to promote their proliferation and migration as well as vascularization in vitro and in vivo [13,14,15,16]
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