Urea enhances the photodynamic efficiency of methylene blue

Methylene blue (MB) is a well-known photosensitizer used mostly for antimicrobial photodynamic therapy (APDT). MB tends to aggregate, interfering negatively with its singlet oxygen generation, because MB aggregates lean towards electron transfer reactions, instead of energy transfer with oxygen. In order to avoid MB aggregation we tested the effect of urea, which destabilizes solute-solute interactions. The antimicrobial efficiency of MB (30 μM) either in water or in 2M aqueous urea solution was tested against a fungus (Candida albicans). Samples were kept in the dark and irradiation was performed with a light emitting diode (λ = 645 nm). Without urea, 9 min of irradiation was needed to achieve complete microbial eradication. In urea solution, complete eradication was obtained with 6 min illumination (light energy of 14.4 J). The higher efficiency of MB/urea solution was correlated with a smaller concentration of dimers, even in the presence of the microorganisms. Monomer to dimer concentration ratios were extracted from the absorption spectra of MB solutions measured as a function of MB concentration at different temperatures and at different concentrations of sodium chloride and urea. Dimerization equilibrium decreased by 3 and 6 times in 1 and 2M urea, respectively, and increased by a factor of 6 in 1M sodium chloride. The destabilization of aggregates by urea seems to be applied to other photosensitizers, since urea also destabilized aggregation of Meso-tetra(4-n-methyl-pyridyl)porphyrin, which is a positively charged porphyrin. We showed that urea destabilizes MB aggregates mainly by causing a decrease in the enthalpic gain of dimerization, which was exactly the opposite of the effect of sodium chloride. In order to understand this phenomenon at the molecular level, we computed the free energy for the dimer association process (ΔG(dimer)) in aqueous solution as well as its enthalpic component in aqueous and in aqueous/urea solutions by molecular dynamics simulations. In 2M-urea solution the atomistic picture revealed a preferential solvation of MB by urea compared with MB dimers while changes in ΔH(dimer) values demonstrated a clear shift favoring MB monomers. Therefore, MB monomers are more stable in urea solutions, which have significantly better photophysics and higher antimicrobial activity. This information can be of use for dental and medical professionals that are using MB based APDT protocols.