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

Abstract

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.

Figure 1

Structures of HPF and APF and the oxidative reaction that leads to the formation of fluorescein.

Figure 2

Top: fluorogenic interactions between 3 µm SOSG and ¹O₂ during the irradiation at 649 nm of solutions containing 10 µm TPPS. The solvents were D₂O, 100 mm sodium phosphate buffer pH 7.0, or water, using a light flux of 1 mW sq cm⁻¹. Fluorescence was monitored using excitation = 505 nm, emission = 525 nm. Bottom: quenching of fluorescence during irradiation of SOSG + TPPS in phosphate buffer by 1 and 10 m_M sodium NaN₃ and by 1% DMSO.

Figure 3

Fluorogenic interactions of 10 µm TPPS + APF in D₂O, phosphate buffer or water during irradiation (649 nm). Also shown are effects of 1 mm NaN₃ and 1% DMSO in phosphate buffer. For comparison, the fluorogenic reaction with HPF in D₂O is also shown. Inset: the fluorogenic interaction during irradiation between TPPS + APF over 180 s in D₂O. For all studies, excitation = 492 nm, emission = 525 nm.

Figure 4

Fluorescence images of APF fluorescence using L1210 cells photosensitized by BPD. A = dark control (no irradiation), B–E = irradiated cells, B = no additions, C = 1% DMSO, D = 100 µm DFO, E = 1% DMSO + 100 µm DFO, F = 0.1% DMSO. Inhibitors were present during BPD loading, and added back to the culture medium during irradiation.