School of Chemical Engineering and Technology, Tianjin University , Yaguan Road 135, Tianjin 300350, China.
Collaborative Innovation Center of Chemical Science and Chemical Engineering , Tianjin 300350, China.
ACS Appl Mater Interfaces. 2017 Oct 18;9(41):35613-35627. doi: 10.1021/acsami.7b11615. Epub 2017 Oct 5.
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-G-TAT-G-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-G-TAT-G-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-G-TAT-G-NLS peptides offer a promising and efficacious delivery option for neovascularization to treat vascular diseases.
最近，基因治疗引起了广泛关注，特别是在治疗血管疾病方面。然而，开发具有高生物相容性和高效基因传递能力的基因载体仍然具有挑战性，需要克服多种障碍。在此，选择一种常用的细胞穿透肽 PKKKRKV（TAT）作为基因载体的功能序列，该序列具有独特的细胞穿透能力。将内皮细胞（ECs）选择性靶向功能的 REDV 肽和核定位信号（NLS）与该 TAT 肽整合，获得具有 ECs 特异性和核积累能力的高效基因传递系统。此外，在上述整合肽中插入了不同重复数目的甘氨酸序列。这些甘氨酸序列充当柔性间隔臂，发挥每个功能肽的靶向、细胞穿透和核积累功能。三个串联肽 REDV-G-TAT-G-NLS（m=0、1 和 4）与 pZNF580 质粒复合形成基因复合物。血液相容性和细胞相容性结果表明，这些肽和基因复合物均无毒性且生物相容性良好。研究了这些基因复合物的内化效率和机制。通过引入靶向 REDV 和甘氨酸序列，内化效率得到提高，并且 REDV-G-TAT-G-NLS/pZNF580（TP-G4/pZNF580）复合物在基因复合物中的细胞摄取率最高。与人脐动脉平滑肌细胞相比，TP-G4/pZNF580 复合物在人脐静脉内皮细胞中的内化效率（约 1.36 倍）也显著提高。TP-G4/pZNF580 复合物通过共聚焦活细胞成像、基因传递效率和 HUVEC 迁移实验显著提高了 pZNF580 的表达。转染 HUVECs 的体外和体内血管生成能力也明显增强。总之，这些多功能 REDV-G-TAT-G-NLS 肽为治疗血管疾病的血管新生提供了一种有前途和有效的递药选择。
