Shen Wen-Juan, Tian Duo-Mei, Fu Le, Jin Biao, Liu Yu, Xu Yun-Sheng, Ye Yong-Bin, Wang Xiao-Bo, Xu Xiao-Jun, Tang Chun, Li Fang-Ping, Wang Chun-Fei, Wu Gang, Yan Le-Ping
Department of Critical Care Medicine, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen 518107, China.
Department of Dermatovenereology, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen 518107, China.
Pharmaceutics. 2023 Feb 16;15(2):670. doi: 10.3390/pharmaceutics15020670.
Cell-penetrating peptides (CPPs) are attractive non-viral gene delivery vectors due to their high transfection capacity and safety. Previously, we have shown that cell-penetrating peptide RALA can be a promising gene delivery vector for chronic wound regeneration application. In this study, we engineered a novel peptide called RALA-E by introducing elastin-derived VGVAPG fragment into RALA, in order to target the elastin-binding protein on the cell surface and thus improve delivery efficacy of RALA. The transfection efficiency of RALA-E was evaluated by transfecting the HEK-293T and HeLa cell lines cells with RALA-E/pDNA complexes and the flow-cytometry results showed that RALA-E significantly increased the transfection efficiency by nearly 20% in both cell lines compared to RALA. Inhibition of pDNA transfection on HEK-293T cells via chlorpromazine, genistein and mβCD showed that the inhibition extent in transfection efficiency was much less for RALA-E group compared to RALA group. In addition, RALA-E/miR-146a complexes showed up to 90% uptake efficiency in macrophages, and can escape from the endosome and enter the nucleus to inhibit the expression of inflammation genes. Therefore, the developed RALA-E peptide has high potential as a safe and efficient vector for gene therapy application.
细胞穿透肽（CPPs）因其高转染能力和安全性而成为有吸引力的非病毒基因递送载体。此前，我们已经表明细胞穿透肽RALA有望成为用于慢性伤口再生的基因递送载体。在本研究中，我们通过将弹性蛋白衍生的VGVAPG片段引入RALA构建了一种名为RALA-E的新型肽，以靶向细胞表面的弹性蛋白结合蛋白，从而提高RALA的递送效率。通过用RALA-E/pDNA复合物转染HEK-293T和HeLa细胞系来评估RALA-E的转染效率，流式细胞术结果表明，与RALA相比，RALA-E在两种细胞系中均显著提高了近20%的转染效率。通过氯丙嗪、染料木黄酮和甲基-β-环糊精对HEK-293T细胞上pDNA转染的抑制作用表明，与RALA组相比，RALA-E组转染效率的抑制程度要小得多。此外，RALA-E/miR-146a复合物在巨噬细胞中的摄取效率高达90%，并且可以从内体逃逸并进入细胞核以抑制炎症基因的表达。因此，所开发的RALA-E肽作为一种安全有效的基因治疗应用载体具有很高的潜力。
