Monitoring singlet oxygen and hydroxyl radical formation with fluorescent probes during photodynamic therapy

Singlet oxygen (1O2) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. 1O2 can give rise to additional reactive oxygen species (ROS) such as the hydroxyl radical (*OH). The current study was designed to assess 3'-p-(aminophenyl) fluorescein (APF) and 3'-p-(hydroxyphenyl) fluorescein (HPF) as probes for the detection of 1O2 and *OH under conditions relevant to PDT. Cell-free studies indicated that both APF and HPF were converted to fluorescent products following exposure to 1O2 generated by irradiation of a water-soluble photosensitizing agent (TPPS) and that APF was 35-fold more sensitive than HPF. Using the 1O2 probe singlet oxygen sensor green (SOSG) we confirmed that 1 mm NaN3 quenched 1O2-induced APF/HPF fluorescence, while 1% DMSO had no effect. APF and HPF also yielded a fluorescent product upon interacting with *OH generated from H2O2 via the Fenton reaction in a cell-free system. DMSO quenched the fluorogenic interaction between APF/HPF and *OH at doses as low as 0.02%. Although NaN3 was expected to quench *OH-induced APF/HPF fluorescence, co-incubating NaN3 with APF or HPF in the presence of *OH 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 1O2 and *OH 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. This procedure could represent a useful means for evaluating formation of both ROS in the context of PDT.