Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory of Embryo Original Diseases, Shanghai Municipal Key Clinical Specialty, Shanghai, China.
Department of Respiratory Medicine, The People's Hospital of Dongtai, Dongtai, Jiangsu, China.
J Pept Sci. 2022 Sep;28(9):e3408. doi: 10.1002/psc.3408. Epub 2022 Feb 15.
Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.
细胞穿透肽 (CPPs) 可辅助细胞内和体内药物递送。然而，CPP 介导的穿透机制仍不清楚，限制了 CPP 的开发和进一步应用。通过流式细胞术和激光共聚焦荧光显微镜检测不同内吞作用抑制剂对 ARPE-19 细胞中 CC12 和 penetratin 内化的影响。通过内吞作用追踪实验检测 CPP 与溶酶体和巨胞饮体的共定位。流式细胞术结果表明，氯丙嗪、渥曼青霉素、细胞松弛素 D 和 ATP 抑制剂寡霉素对 CC12 具有剂量依赖性的内吞抑制作用。激光共聚焦荧光结果表明，寡霉素对 CC12 摄取的抑制作用最显著；CC12 与溶酶体共定位，但与巨胞饮体不共定位。对于 penetratin，细胞松弛素 D 和寡霉素有明显的抑制作用。激光共聚焦荧光结果表明，寡霉素对 penetratin 摄取的抑制作用最显著；穿透肽与溶酶体的共定位高于与巨胞饮体的共定位。非离子依赖型的 CC12 和阳离子型的 penetratin 可能主要通过 caveolae 和网格蛋白介导的内吞作用进入细胞，并且通常依赖于 ATP。穿透肽的转运可能部分通过直接跨膜途径实现，因为穿透肽的正电荷与细胞膜的负电荷相互作用，部分通过内吞作用途径实现。
