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 μM 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 N₃ 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 N₃ 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.