Determination of radical species generated in hexyl-5-aminolevulinate-based photodynamic therapy using electron spin resonance spectroscopy

Generation of radical species in hexyl-5-aminolevulinate (h-ALA)- based photodynamic therapy (PDT) was determined in terms of electron spin resonance (ESR) spectroscopy with N-tert-buthyl-α-phenylnitrone (PBN) as a spin trapping agent. Hexyl-5-aminolevulinate and PBN were administered into murine thymic lymphoma (EL-4) cells, and the ESR spectra of the EL-4 cell dispersions were measured with irradiating light (400-410 nm). There were no ESR signals in the EL-4 dispersion without the irradiation. At 30 min light irradiation we found the specific ESR signals, of which the intensity increased with an irradiation time. The ESR spectra of EL-4 cell dispersion with only PBN (without h-ALA) and of the culture medium with h-ALA and PBN were determined with the same method. The ESR signals in the both systems was similar to that in the system of EL-4 cell dispersion with h-ALA and PBN. Nevertheless, the signal intensity of the ESR signals in the formers was obviously lower than that in the latter. There results indicate, therefore, that some radical species occur in the cell dispersion by h-ALA-based PDT. In addition, the generation of radical species is considered to be related to the release of singlet oxygen by irradiating the light.