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.

在小直径人造血管上快速内皮化是促进长期通畅和抑制血栓形成的有效策略。基因递送可以增强内皮细胞（EC）的增殖和迁移，这对于快速内皮化是有益的。REDV-G-TAT-G-NLS-Cys（缩写为TP-G）肽可以弱凝聚pEGFP-ZNF580（pZNF580）并转染EC，但是由于其低正电荷，低稳定性和低转染率，其转染效率仍然很低。内体逃逸能力弱。为了开发具有低细胞毒性的更稳定和有效的基因载体，在本研究中，我们将不同量的TP-G肽缀合到聚（丙交酯-共-乙交酯）-g-聚乙烯亚胺（PLGA- g）上。-PEI）两亲共聚物通过杂聚乙二醇间隔物（OPSS-PEG-NHS）。TP-G肽和PEI可以协同并强烈凝聚pZNF580。载体的细胞毒性通过引入聚乙二醇间隔物而降低。他们凝结了pZNF580，形成了基因复合物（PPP-TP-G / pZNF580），具有合适的大小和正的ζ传递基因潜力。如细胞迁移测定所证明的，转染的ECs促进了它们的迁移能力。细胞摄取和共聚焦激光扫描显微镜的结果显示，通过该多功能TP-G肽序列共轭基因递送系统，pZNF580的内在化效率，内体/溶酶体逸出和核位置显着提高。此外，使用了几种抑制剂来研究PPP-TP-G / pZNF580复合物的细胞摄取途径。结果表明，PPP-TP-G2 / Cy5-寡核苷酸复合物表现出优化的内吞途径，其促进细胞摄取。综上所述，多功能的TP-G肽共轭基因载体由于REDV的多功能性，细胞穿透肽和肽序列中的核定位信号的多功能性，可以提高转染效率，可能是适合内皮化的基因载体。