Paradoxical potentiation of methylene blue-mediated antimicrobial photodynamic inactivation by sodium azide: Role of ambient oxygen and azide radicals

Sodium azide (NaN3) is widely employed to quench singlet oxygen during photodynamic therapy (PDT), especially when PDT is used to kill bacteria in suspension. We observed that addition of NaN3 (100 mu M or 10 mM) to gram-positive Staphylococcus aureus and gram-negative Escherichia coli incubated with methylene blue (MB) and illuminated with red light gave significantly increased bacterial killing (1-3 logs), rather than the expected protection from killing. A different antibacterial photosensitizer, the conjugate between polyethylenimine and chlorin(e6) (PEI-ce6), showed reduced PDT killing (1-2 logs) after addition of 10 mM NaN3. Azide (0.5 mM) potentiated bacterial killing by Fenton reagent (hydrogen peroxide and ferrous sulfate) by up to 3 logs, but protected against killing mediated by sodium hypochlorite and hydrogen peroxide (considered to be a chemical source of singlet oxygen). The intermediacy of N center dot(3) was confirmed by spin-trapping and electron spin resonance studies in both MB-photosensitized reactions and Fenton reagent with addition of NaN3. We found that N center dot(3) was formed and bacteria were killed even in the absence of oxygen, suggesting the direct one-electron oxidation of azide anion by photoexcited MB. This observation suggests a possible mechanism to carry out oxygen-independent PDT. (C) 2012 Elsevier Inc. All rights reserved.