Structure-function analysis of Squaraines phototherapeutic activity: in vitro characterization of ROS and intracellular Ca2+ signals interplay in phototoxicity

Photodynamic therapy (PDT) is an emerging, minimally invasive therapeutic modality approved by the U.S Food and Drug Administration for the treatment of several conditions including oncological applications ¹. The approach is based on light-induced photosensitizers (PS) activation which, in turn, promotes the intersystem crossing and triplet state that easily reacts with molecular oxygen, thus generating free radicals and singlet oxygen species which in turn involve intracellular Ca2+ signals as well as other messengers with a final increase in apoptosis, necrosis, and autophagy processes and tumor mass reduction ^1,2. In this context, the development of new PS for the PTD is of great interest to increase the treatment efficacy minimizing side effects^3,4.In the present work, we investigated the structure-functional role of SQ by comparing the phototoxic activity of unsubstituted SQ, carboxyl (COOH)- and bromine (Br)-substituted SQ, as well SQ in which the oxygen atoms of the squaryl ring were replaced with sulfur atoms (S-SQ). Indeed, the presence of heavy atoms such as Br and the replacement of oxygen with sulfur are predicted to shift the emission to the far NIR region and increase the quantum yield of singlet oxygen, thus improving the overall effect in PDT. As expected, Br-SQ and S-SQ demonstrate a higher phototoxicity on MCF-7 cells compared to the COOH-SQ dye, which showed no effect. However, surprisingly, our data reveal that also the unsubstituted SQ has a strong phototoxic activity. To deeper analyze the the molecular mechanisms underlying SQ phototoxicity, we studied the role of Ca2+ signals and ROS generation in light induced PS activation. Our results clearly show that Br-SQ promote ROS production as well as significant increase in cytosolic Ca2+ signals mainly due to Ca2+ release form ER stores which in turn promotes a sustained Ca2+ uptake in the mitochondria while no effect was observed for the COOH-SQ. Our results shed new light on the structure-function relationship of SQ and the intracellular pathways involved in their exploitable photo-activity in PDT, demonstrating a critical role for intracellular Ca²⁺ signals in ROS activation and cell death.