Structure-dependent reactive oxygen species generation by scintillator-free X-ray-activated porphyrins: insights into charge effects and internal conversion quantum yield.

Radiodynamic therapy enables treatment of deep-seated tumors but typically requires scintillator nanoparticles with associated toxicity concerns. While porphyrin photosensitizers (PS) have been shown to enhance reactive oxygen species (ROS) generation under X-ray irradiation without scintillators, the molecular features governing this effect remain unclear. Here, we systematically examined structure-activity relationships of nine porphyrin derivatives to elucidate the design principles for scintillator-free radiosensitizers. Molecular charge critically influenced ROS modulation: anionic tetrakis (4-carboxyphenyl) porphyrin showed the highest efficacy with approximately 7-fold enhancement over X-ray alone, whereas cationic tetra (N-methyl-4-pyridyl) porphyrin suppressed ROS below control levels. Notably, heavy-atom coordination yielded structure-dependent effects rather than uniform enhancement, distinguishing this process from conventional photodynamic therapy. Using complementary ROS probes, we found that X-ray-mediated ROS generation produces predominantly superoxide and hydrogen peroxide rather than singlet oxygen, suggesting electron transfer rather than energy transfer as the operative pathway. Dose-response analysis demonstrated that ROS generation scales linearly with radiation dose, indicating no saturation within the therapeutic window. Validation with clinically relevant PS showed that Visudyne and protoporphyrin IX-both bearing anionic carboxylate groups-were active. However, the inactivity of anionic but rigid rose bengal suggests that electrostatic properties alone are insufficient. These findings suggest that along with molecular charge, conformational flexibility-potentially facilitating internal conversion from high-energy excited states-represents a key design parameter for scintillator-free radiosensitizers. This approach offers a simplified, biocompatible formulation strategy for deep-tissue cancer therapy without relying on heavy-metal nanoscintillators.