Targeted, receptor‐mediated delivery of a masked d ‐amino acid cell‐penetrating peptide for cell‐specific phototoxicity

Photodynamic therapy (PDT) is an innovative treatment option for cancer, but current approaches are limited by poor tumor selectivity and low uptake. Here, we introduce a novel concept for a targeted phototoxic peptide, in which a lysosomally activatable payload is delivered selectively into the cell by receptor‐mediated endocytosis. For the phototoxic payload, 6‐carboxytetramethylrhodamine (TMR) was attached to a cell‐penetrating peptide (CPP), which is specifically activated after internalization in the endosome. The activity of the CPP was blocked by electrostatic interactions with a poly‐glutamate sequence but could be restored through cleavage by the lysosomal protease cathepsin B, both in vitro and in cells. The unmasked CPP binds to the negatively charged lysosomal membrane and, upon irradiation, TMR generates reactive oxygen species (ROS) that disrupt the integrity of the membrane. This leads to a release of lysosomal contents into the cytosol, which subsequently induces cell death. To achieve targeted delivery, the activatable payload was additionally conjugated to chemerin‐9, a high‐affinity ligand for the chemokine‐like receptor 1 (CMKLR1), a G protein‐coupled receptor overexpressed in various cancers. Through this receptor‐targeted approach, the peptide accumulates only in CMKLR1‐expressing cells while the lysosomal activation completely prevented off‐target toxicity. Notably, this strategy enables even a weak photosensitizer like TMR to achieve potent cytotoxicity through lysosomal targeting. Thus, this approach represents an advancement in the selectivity and efficacy of PDT and holds promise for the development of novel cancer therapies.