Monitoring Singlet Oxygen and Hydroxyl Radical Formation with Fluorescent Probes During Photodynamic Therapy

Singlet oxygen (O-1(2)) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. O-1(2) can give rise to additional reactive oxygen species (ROS) such as the hydroxyl radical ((OH)-O-center dot). The current study was designed to assess 3'-p-(aminophenyl) fluorescein (APF) and 3'-p-(hydroxyphenyl) fluorescein (HPF) as probes for the detection of O-1(2) and (OH)-O-center dot under conditions relevant to PDT. Cell-free studies indicated that both APF and HPF were converted to fluorescent products following exposure to O-1(2) generated by irradiation of a water-soluble photosensitizing agent (TPPS) and that APF was 35-fold more sensitive than HPF. Using the O-1(2) probe singlet oxygen sensor green (SOSG) we confirmed that 1 mm NaN3 quenched O-1(2)-induced APF/HPF fluorescence, while 1% DMSO had no effect. APF and HPF also yielded a fluorescent product upon interacting with (OH)-O-center dot generated from H2O2 via the Fenton reaction in a cell-free system. DMSO quenched the fluorogenic interaction between APF/HPF and (OH)-O-center dot at doses as low as 0.02%. Although NaN3 was expected to quench (OH)-O-center dot-induced APF/HPF fluorescence, co-incubating NaN3 with APF or HPF in the presence of (OH)-O-center dot markedly enhanced fluorescence. Cultured L1210 cells that had been photosensitized with benzoporphyhrin derivative exhibited APF fluorescence immediately following irradiation. Approximately 50% of the cellular fluorescence could be suppressed by inclusion of either DMSO or the iron-chelator desferroxamine. Combining the latter two agents did not enhance suppression. We conclude that APF can be used to monitor the formation of both O-1(2) and (OH)-O-center dot in cells subjected to PDT if studies are performed in the presence and absence of DMSO, respectively. That portion of the fluorescence quenched by DMSO will represent the contribution of (OH)-O-center dot. This procedure could represent a useful means for evaluating formation of both ROS in the context of PDT.