Thiocyanate potentiates antimicrobial photodynamic therapy: In situ generation of the sulfur trioxide radical anion by singlet oxygen

Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light excitation of dyes in the presence of O₂, yielding reactive oxygen species including the hydroxyl radical (OH) and singlet oxygen (¹O₂). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN^-) could potentiate the methylene blue (MB) and light-mediated killing of the gram-positive Staphylococcus aureus and the gram-negative Escherichia coli. SCN^- enhanced PDT (10 μM MB, 5 J/cm² 660 nm hv) killing in a concentration-dependent manner of S. aureus by 2.5 log₁₀ to a maximum of 4.2 log₁₀ at 10 mM (P<0.001) and increased killing of E. coli by 3.6 log₁₀ to a maximum of 5.0 log₁₀ at 10 mM (P<0.01). We determined that SCN^- rapidly depleted O₂ from an irradiated MB system, reacting exclusively with ¹O₂, without quenching the MB excited triplet state. SCN^- reacted with ¹O₂, producing a sulfur trioxide radical anion (a sulfur-centered radical demonstrated by EPR spin trapping). We found that MB-PDT of SCN^- in solution produced both sulfite and cyanide anions, and that addition of each of these salts separately enhanced MB-PDT killing of bacteria. We were unable to detect EPR signals of OH, which, together with kinetic data, strongly suggests that MB, known to produce OH and ¹O₂, may, under the conditions used, preferentially form ¹O₂.