A non-ionic phenothiazine-derived type I photosensitizer

Non-ionic phenothiazine (PTZ) derivatives are widely used in optoelectronic materials and fluorescent probes, yet their potential as Type I photosensitizers (PSs) remains largely unexplored. Herein, we report two PTZ-based donor-acceptor dyes, PTZ-1 and PTZ-2 that exhibit strong charge-transfer absorption beyond 450 nm and pronounced polarity-dependent emission. Photodynamic studies revealed that PTZ-1 and PTZ-2 efficiently generate hydroxyl radicals (•OH) and superoxide (O₂^•-) under 500 nm irradiation while producing negligible singlet oxygen, confirming their Type I photodynamic behavior. Density functional theory calculations show small S₁-T_n energy gaps and appreciable spin-orbit coupling, supporting rapid intersystem crossing and Type I ROS generation. PTZ-1 demonstrates superior solubility, appropriate lipophilicity, and markedly higher phototoxicity toward 4 T1 cancer cells (IC₅₀ = 0.63 μM) compared to PTZ-2, while exhibiting minimal dark toxicity. Importantly, PTZ-1 maintains potent cytocidal effects under hypoxia (2 % O₂), validating its low oxygen dependence. Confocal imaging reveals that PTZ-1 selectively accumulates in lipid droplets and triggers abundant intracellular •OH and O₂^•- production upon irradiation. Mechanistic studies show that PTZ-1-mediated photodynamic therapy induces apoptosis characterized by Annexin V-FITC/PI staining and Western blot analysis, with no evidence of ferroptosis. This work demonstrates a non-ionic PTZ-derived Type I photosensitizer capable of efficient ROS generation under hypoxic conditions and highlights its potential for lipid-droplet-targeted photodynamic cancer therapy.