Effects of irradiation modes in photodynamic therapy with azulene on singlet oxygen formation

Differences in the irradiation mode in Photodynamic Therapy result in different quantities of reactive oxygen species. We aimed to quantify the singlet oxygen formation in Photodynamic Therapy among various modes of red LED irradiation. Azulene powder was dissolved in ethanol/deionized water to obtain concentrations of 1, 10, and 100 μM. For all samples (90 μl/well), 10 μl of 10 mM dimethyl anthracene was added to each well of a 96-black well plate along with a singlet oxygen probe. After 30 minutes, irradiation was performed in arbitrary light units (638 nm, 0.5 watts, light bulb-to-well bottom distance = 2 mm) to obtain 4 or 40 J/cm2 by any of the following irradiation modes: 1) a Continuous Mode, 2) a Fractional Mode (4 sessions, each session = 1/4 of final energy density resting 15 minutes between each session), or 3) a Pulse Mode (by alternative irradiation 300 msec with resting 700 msec). Samples were then subjected to singlet oxygen detection in a Varioskan (Thermo Fisher Scientific, USA) fluorescence microplate reader (excitation/emission wavelength = 375/436 nm). Measurements were performed in triplicate and the fluorescence intensity (relative singlet oxygen amount) was recorded. Kruskal Wallis with the Mann Whitney U test was performed, and p < 0.05 was determined as the significant difference. The amounts of relative singlet oxygen from PDT with higher energy density were relatively higher than those with lower energy. The continuous mode of irradiation resulted in a relatively higher singlet oxygen amount (p < 0.05). In conclusion, the continuous mode of red light irradiation with 40 J/cm2 with azulene can produce the highest amount of singlet oxygen using a fluorescence probe.