Cellular Uptake of Cell‐Penetrating Peptides Activated by Amphiphilic p‐Sulfonatocalix[4]arenes

We report the synthesis of a series of amphiphilic p‐sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4‐Cn) and their application as efficient counterion activators for membrane transport of cell‐penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO‐K1, HCT 116, and KTC‐1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4‐Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure‐activity relationships for calixarene‐based counterion activators and show that CX4‐Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4‐C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP‐counterion complexes into the cytosol and highlights the potential benefits of CX4‐Cn for efficient and direct translocation of CPPs and CPP‐conjugated cargo molecules into the cytosol of live cells.