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 mM 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.
Photodynamic therapy (PDT) is a minimally invasive, FDA-approved therapy for treatment of endobronchial and esophageal cancers that are accessible to light. Inflammatory breast cancer (IBC) is an aggressive and highly metastatic form of breast cancer that spreads to dermal lymphatics, a site that would be accessible to light. IBC patients have a relatively poor survival rate due to lack of targeted therapies. The use of PDT is underexplored for breast cancers but has been proposed for treatment of subtypes for which a targeted therapy is unavailable. We optimized and used a 3D mammary architecture and microenvironment engineering (MAME) model of IBC to examine the effects of PDT using two treatment protocols. The first protocol used benzoporphyrin derivative monoacid A (BPD) activated at doses ranging from 45 to 540 mJ/cm2. The second PDT protocol used two photosensitizers: mono-l-aspartyl chlorin e6 (NPe6) and BPD that were sequentially activated. Photokilling by PDT was assessed by live–dead assays. Using a MAME model of IBC, we have shown a significant dose–response in photokilling by BPD–PDT. Sequential activation of NPe6 followed by BPD is more effective in photokilling of tumor cells than BPD alone. Sequential activation at light doses of 45 mJ/cm2 for each agent resulted in >90 % cell death, a response only achieved by BPD–PDT at a dose of 360 mJ/cm2. Our data also show that effects of PDT on a volumetric measurement of 3D MAME structures reflect efficacy of PDT treatment. Our study is the first to demonstrate the potential of PDT for treating IBC.
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