Regioisomeric Engineering of Sterically Hindered Bright Near-Infrared Paraptosis Agents for Chemo-Photodynamic Therapy

Paraptosis emerges as a new promising form of programmed nonapoptotic cell death in chemotherapeutic anticancer therapy. However, current paraptosis agents face critical challenges, including poor targeting specificity, limited imaging capability, and low therapeutic efficacy. To overcome these limitations, we developed a novel approach by functionalizing the tetraphenylethylene (TPE) unit at the meso position of xanthene dyes, enabling the synthesis of two sterically hindered regioisomeric fluorescent paraptosis-inducing agents ( m -TSX and p -TSX) for mitochondria-targeted chemo-photodynamic anticancer therapy. These agents exhibited strong near-infrared (NIR) emissions (∼663 nm) with a quantum yield of up to 82.9%. The TPE substitution, in contrast to the phenyl group, allowed for precise modulation of triplet excited state energy levels, boosting type I/II reactive oxygen species (ROS) generation, and notable enhancement of the paraptotic anticancer activity. Comparative studies of the meta- and para -substituted regioisomers revealed that the meta -substituted m -TSX exhibited superior ROS generation and anticancer behavior. m -TSX effectively induced Alix/ATF4-regulated paraptosis, along with apoptosis and necrosis, while also triggering GPX4/SLC7A11-regulated ferroptosis under low-power 655 nm laser (0.1 W/cm 2 ) irradiation, leading to effective cancer cell growth inhibition. Furthermore, in vivo chemo-photodynamic therapy against HeLa tumor by inducing multiple cell death pathways was successfully achieved. This innovative strategy of steric hindrance regulation represents a breakthrough in developing bright NIR xanthene-based anticancer agents for synergistic cancer therapy.