Spectroscopic Evidence of Free Radicals Generated by Indocyanine Green Under Light Irradiation

Reactive oxygen species (ROS), particularly free radicals involved in type I photodynamic mechanisms, are key mediators of photodynamic therapy. Indocyanine green (ICG), a clinically approved near-infrared dye, is increasingly being considered as a photosensitizer. It has attracted considerable interest due to its photophysical properties and affinity for serum albumin, which enhances its stability, circulation time, and tumor accumulation. This study aimed to characterize the formation of free radicals generated by ICG upon light irradiation, with particular emphasis on oxygen radicals involved in type I photodynamic mechanisms. Electron paramagnetic resonance (EPR) spectroscopy combined with the spin trap DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was used to identify radicals formed during irradiation with an OSL2 fiber-optic illuminator providing white light. The detected radical species were identified by analysis of their characteristic hyperfine splitting constants and comparison with simulated EPR spectra corresponding to the DMPO-OH, DMPO-OOH, and DMPO-H adducts. Changes in radical concentrations over time were evaluated using the integral intensity of the EPR signals at two ICG concentrations. Differences in EPR signal intensities for samples containing ICG alone and ICG in the presence of bovine serum albumin (BSA) demonstrated the influence of protein binding on radical generation. Additionally, spectral analysis based on a Hamiltonian spin model was applied to support the reliable identification of the observed radical species. These findings provide insight into the free radical pathways of ICG and contribute to a better understanding of its type I photodynamic activity.