Cell‐penetrating peptides (CPPs) have been explored as versatile tools to transport various molecules into cells. The uptake mechanism of CPPs is still not clearly understood and most probably depends on several factors like the nature of the CPP itself, the attached cargo, the investigated cell system, and other experimental conditions, such as temperature and concentration. One of the first steps of internalization involves the interaction of CPPs with negatively charged molecules present at the outer layer of the cell membrane. Recently, thiol‐mediated uptake has been found to support the effective translocation of sulfhydryl‐bearing substances that would actually not be cell‐permeable. Within this work, we aimed to understand the relevance of thiol reactivity for the uptake mechanism of cysteine‐containing CPPs that we have developed previously in our group. Therefore, we compared the two peptides, sC18‐Cys and CaaX‐1, in their single reduced and dimeric disulfide versions. Cytotoxicity, intracellular accumulation, and impact on the internalization process of the disulfides were investigated in HeLa cells. Both disulfide CPPs demonstrated significantly stronger cytotoxic effects and membrane activity compared with their reduced counterparts. Notably, thiol‐mediated uptake could be excluded as a main driver for translocation, showing that peptides like CaaX‐1 are most likely taken up by other mechanisms.

中文翻译：

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研究硫醇反应性和二硫键形成对细胞渗透性肽的细胞摄取的影响

细胞穿透肽（CPP）已被探索作为将各种分子转运到细胞中的多功能工具。 CPP 的吸收机制仍不清楚，很可能取决于多种因素，如 CPP 本身的性质、附着的货物、所研究的细胞系统以及其他实验条件（例如温度和浓度）。内化的第一步涉及 CPP 与细胞膜外层存在的带负电分子的相互作用。最近，发现硫醇介导的摄取支持实际上不具有细胞渗透性的含巯基物质的有效易位。在这项工作中，我们旨在了解硫醇反应性与我们小组之前开发的含半胱氨酸 CPP 的摄取机制的相关性。因此，我们比较了两种肽，sC18-Cys 和 CaaX-1，它们的单一还原和二聚二硫键版本。在 HeLa 细胞中研究了二硫化物的细胞毒性、细胞内积累以及对内化过程的影响。与还原的对应物相比，两种二硫化物 CPP 均表现出明显更强的细胞毒性作用和膜活性。值得注意的是，硫醇介导的摄取可以被排除为易位的主要驱动因素，这表明像 CaaX-1 这样的肽很可能被其他机制摄取。