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Monitoring Singlet Oxygen and Hydroxyl Radical Formation with Fluorescent Probes During Photodynamic Therapy
Author(s) -
Price Michael,
Reiners John J,
Santiago Ann Marie,
Kessel David
Publication year - 2009
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2009.00555.x
Subject(s) - singlet oxygen , fluorescence , chemistry , photodynamic therapy , photochemistry , fluorescein , oxygen , hydroxyl radical , reactive oxygen species , chelation , irradiation , radical , inorganic chemistry , organic chemistry , biochemistry , physics , quantum mechanics , nuclear physics
Singlet oxygen ( 1 O 2 ) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. 1 O 2 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 1 O 2 and ? OH under conditions relevant to PDT. Cell‐free studies indicated that both APF and HPF were converted to fluorescent products following exposure to 1 O 2 generated by irradiation of a water‐soluble photosensitizing agent (TPPS) and that APF was 35‐fold more sensitive than HPF. Using the 1 O 2 probe singlet oxygen sensor green (SOSG) we confirmed that 1 m m NaN 3 quenched 1 O 2 ‐induced APF/HPF fluorescence, while 1% DMSO had no effect. APF and HPF also yielded a fluorescent product upon interacting with ? OH generated from H 2 O 2 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 NaN 3 was expected to quench ? OH‐induced APF/HPF fluorescence, co‐incubating NaN 3 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 1 O 2 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.