ABSTRACT
In photodynamic therapy, intermittent irradiation modes that incorporate an interval between pulses are believed to decrease the effect of hypoxia by permitting an interval of re-oxygenation. The effect of the irradiation intermittency factor (the ratio of the irradiation pulse time to the total irradiation time) on singlet oxygen formation and inflammatory cytokine production was examined using azulene as a photosensitizer. Effects of difference intermittency factor on singlet oxygen formation and inflammatory cytokine were examined. Azulene solutions (1/10 µM) were irradiated with a 638-nm 500 mW diode laser in fractionation (intermittency factor of 5 or 9) or continuous mode using 50 mW/cm2 at 4 or 8 J/cm2. Singlet oxygen measurement was performed using a dimethyl anthracene probe. Peripheral blood mononuclear cells (PBMC) were stimulated by 10 ng/ml rhTNF-α for 6 h, before addition of 1 and 10 µM azulene solutions and irradiation. PGE2 measurement was undertaken using a human PGE2 ELISA kit. Kruskal-Wallis with Dunn Bonferroni test was used for statistical analyses at p < 0.05.Irradiation of 1 µM azulene+4 J/cm2+intermittency factor of 9 increased singlet oxygen 3-fold (p < 0.0001). Irradiation of 10 µM azulene at either 4 J/cm2+intermittency of 9 or 8 J/cm2+intermittency factor of 5 reduced PGE2 expression in PBMCs to non-inflamed levels. Thus, at 50 mW/cm2, 10 µM azulene-mediated photodynamic therapy with a high intermittency factor and a low energy density generated sufficient singlet oxygen to suppress PGE2 in Inflamed PBMCs.
ABSTRACT
BACKGROUND: We aimed to investigate the effect of azulene on peripheral blood mononuclear cells (PBMCs) viability and singlet oxygen formation. METHODS: 1 × 105 PBMCs were cultured in a 96-well plate in RPMI-1640 supplemented with 10% FBS (37 °C, 5% CO2) for 24 h. Each well was treated for 30 min with each azulene concentration between 0-500 µM and activated by a 625 ± 5 nm light emitting diode (power 20-23 mW) at energy densities of 0-200 J/cm2. MTT cell viability was recorded using a spectrophotometer at a 570 nm. 9,10-Dimethylanthracene (DMA) was utilized for the measurement of singlet oxygen, using a fluorescence spectrophotometer at 375 and 436 nm as the excitation and emission wavelengths, respectively. Optical density,ãrelative fluorescence units were compared using one-way ANOVA and a post-hoc test. The correlation between the cell number and singlet oxygen amount was analyzed by the Spearman correlation test. RESUTS: Azulene at all concentrations with 4.2 J/cm2 light significantly induced singlet oxygen formation. 15 µM azulene with 4.2, 100, or 200 J/cm2 light significantly reduced PBMC viability. The inverse relationship between the cell viability and singlet oxygen amount was observed. CONCLUSIONS: An optimum azulene concentration + red light energy density decreased PBMC viability via singlet oxygen formation.
