Oxygen-Dependent Light-Induced Lipid Membrane Perturbation by TLD1433 as a Potential Contributor to Phototoxicity

TLD1433 (Ruvidar®) is a Ru(II) polypyridyl complex currently in phase II clinical trials for the treatment of non-muscle invasive bladder cancer using photodynamic therapy (PDT). Previous studies have demonstrated that TLD1433 exhibits dual Type I/II photosensitization, arising from a combination of a prolonged intrinsic excited state lifetime and the redox-active α-terthienyl group, which together enable both singlet oxygen sensitization and light-driven redox processes. Despite this, molecular determinants contributing to its pronounced phototoxicity are still being elucidated. Herein, we investigated whether light-induced lipid membrane perturbation could represent a contributing photochemical property relevant to cytotoxic activity. To this end, all-atom molecular dynamics simulations and NMR experiments were employed to examine the insertion, partitioning, and residence of TLD1433 within a lipid bilayer in both dark and photoactivated states in normoxia and hypoxia, as well as the resulting effects on membrane structure integrity. Under normoxic conditions, oxidation events from the triplet excited state of TLD1433 promote localized accumulation of oxidized lipids and induce membrane deformations characterized by increased water penetration and transient pore-like features. In contrast, under low ROS-mediated lipid oxidation, membrane perturbations are more limited, although increased permeability and structural disorder are still observed. Taken together, these results suggest that light-induced lipid membrane perturbation, including poration, may represent a contributing factor to the phototoxic effects of TLD1433 under normoxic conditions. This combined computational and experimental study details how the photochemical properties of TLD1433 influence membrane response in a light- and oxygen-dependent manner, offering a framework for understanding how such effects may contribute to PDT efficacy at an unprecedented molecular level.