Bystander effects of a photoactivated ruthenium-based complex in 2D and 3D cell culture models

Photodynamic therapy (PDT) using ruthenium-based photosensitizers (PSs) has attracted increasing interest due to their favorable photophysical properties and their ability to generate reactive oxygen species (ROS) upon irradiation. Although PDT is generally considered a localized treatment, indirect effects on neighboring non-irradiated cells, known as bystander effects, may occur. In this study, we investigated, for the first time, bystander responses induced by the photoactivatable ruthenium nitrosyl complex {TPyP[Ru(NO₂)(bpy)₂]₄}(PF₆)₄ (RuNO₂TPyP) using two-dimensional (2D) and three-dimensional (3D) lung cell culture models. A custom 3D printed device was developed to enable selective irradiation while maintaining controlled communication between irradiated and non-irradiated cells. Photoactivation of RuNO₂TPyP induced pronounced phototoxicity in irradiated cells, whereas non-irradiated cells exhibited measurable reductions in viability and migration while retaining clonogenic capacity. NO-associated fluorescence was predominantly detected in irradiated cells, and pharmacological controls targeting NO signaling, including Nω-Nitro-L-arginine methyl ester (L-NAME) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), suggested a limited contribution of sustained NO signaling to bystander responses. In 3D co-culture models, these effects remained detectable but were attenuated compared to 2D systems, highlighting the influence of microenvironmental complexity. Overall, these findings demonstrate that RuNO₂TPyP primarily affects irradiated cells and induces measurable but attenuated responses in bystander cells. The custom 3D printed device provides a reproducible approach for investigating spatial aspects of photodynamic responses in vitro.