Effect of intermittency factor on singlet oxygen and PGE2 formation in azulene-mediated photodynamic therapy: A preliminary study

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.

Keywords: Azulene; Inflammation; Intermittency factor; Photodynamic therapy; Pulse mode; Singlet oxygen.

Graphical abstract

Fig. 1

The 638 nm-diode laser irradiation apparatus. The irradiation box is composed of a black acrylic box with an irradiation chamber. Parts of machine composed of 1) electric circuit with laser light bulbs, 2) monitor connected with electric circuit, 3) parameter adjustment button, 4) machine reset hole, 5) operation indicating light, 6) light distraction plate, 7) light projection slit, 8) culture plate holder. This machine is connected with normal AC supply.

Fig. 2

Relative singlet oxygen quantity in 1 μM azulene-mediated PDT using a power density of 50 mW/cm², a) energy density of 4 or b) energy density of 8 J/cm² from a 638 nm red laser and fractionation mode of irradiation (intermittency factor of 5 and 9 and continuous) and using 9,10-dimethylanthracene (DMA) as a singlet oxygen specific fluorescence probe. Positive control = continuous mode, negative control = non irradiation group. n = 12 * = significant difference at p-value < 0.05, a = significant difference from positive control at the same mode of light at p-value < 0.05 (Cont = continuous mode, Int = intermittency factor).

Fig. 3

Relative singlet oxygen quantity in 10 μM azulene-mediated PDT using a power density of 50 mW/cm², a) energy density of 4 or b) energy density of 8 J/cm² from a 638 nm red laser and fractionation mode of irradiation (intermittency factor of 5 and 9 and continuous) and using 9,10-dimethylanthracene (DMA) as a singlet oxygen specific fluorescence probe. Positive control = continuous mode, negative control = non irradiation group. n = 12 * = significant difference at p-value < 0.05, a = significant difference from positive control at the same mode of light at p-value < 0.05 (Cont = continuous mode, Int = intermittency factor).

Fig. 4

PGE₂ quantity after a) 1 μM b) 10 μM azulene-mediated PDT using a power density of 50 mW/cm², energy density of 4 or 8 J/cm² from a 638 nm red laser and fractionation mode of irradiation (intermittency factor of 5 and 9 and continuous) treated rhTNFα-induced inflammatory peripheral blood mononuclear cells and competitive human ELISA kit for detection. Positive control = peripheral blood mononuclear cells pretreated with 50 μg/mL of indomethacin, Negative control = rhTNFα-induced inflammatory peripheral blood mononuclear cells. a = significant difference from Positive control at p-value < 0.05, b = significant difference from Negative control at p-value < 0.05, dot line = basal level PGE2 in non-inflamed cells, a = significant different from (Int = intermittency factor, +ve con = positive control, -ve con = negative control, n = 4).

Fig. 5

Correlation between singlet oxygen and PGE₂ in (a) intermittency factor 5, (b) intermittency factor 9, experiments were performed in triplicate fashion and duplicate for singlet oxygen and PGE2, respectively. Error bar = standard deviation of singlet oxygen amount.