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Int. J. Mol. Sci. 2018, 19, 1107 5 of 17 Figure 2. Time and concentration dependent toxic effects of MB on Detroit 562 cells, in percentage of viable cells. MB exposure at concentration of 160 μM for 6–8 min caused up to 75% loss of cells compared to control (sham, scattered light in neighboring wells). Detroit 562 cells were exposed to increasing concentrations of MB (Table 1) for 2–8 min, without laser exposure. The time conditions were chosen in order to be also reasonable in a clinical oral tumor surface treating, which also meant that we were able to increase the concentration of MB, but we wanted to limit the exposure time. The μM concentrations of MB have been chosen in pre-experiments completed on more cell lines and also on commercial normal fibroblasts. Increasing concentrations of MB caused decreasing cell viability by 2–8 min exposure. The relationship between MB concentration and percentage of control cell viability was linear by exposure times of 2, 6, and 8 min (r2 = 0.95–0.96), and was not linear by exposure time of 4 min (Figure 2; r2 = 0.78). Table 1. Time and concentration (μM) dependent toxic effects of methylene blue (MB) on Detroit 562 cells, in percentage of viable cells rounded to integer values. % Viability MB-Exposure (min) 2 4 6 8 40 80 120 160 88 69 65 50 73 56 60 46 57 41 34 25 55 44 41 34 Exposure to a diode laser at 660 nm at a fixed area dose of 95 J/cm2 for 2–8 min resulted in a phototoxic effect on top of MB-toxicity (p < 0.001) (Table 2). Estimated marginal means of photoxicity when adjusted for the effects of MB-concentration (ranging from 40 μmol/L to 160 μmol/L) and MB-exposure time (ranging from 2 to 8 min) were up to 52% (95%, CI 50% to 55%), MB at 120 μM concentration for 4 min and laser exposure 8 min. For loss of at least 95% of the viable cells 120–160 μM MB was required for at least 4 min, followed by 6–8 min of 660 nm laser exposure. The highest light-depending effect was measured after 120–160 μmol/L 4 min MB treatment followed by 8 min of laser exposure. The phototoxic effect was not monotonically decreasing with increasing laser exposure times. The laser exposure further increased the MB toxicity, 8 min laser exposure allowed at least 95% tumor cell loss after at least 80 μM MB for 4–6 min. The best specific impact for the combination of MB and laser exposure was found in 4 min MB exposure with the concentration of 160 μM and 8 min laser exposure, which allowed up to 95% reduction of the cell viability in comparison to the control (sham, scattered light in neighboring wells). Interestingly, MB alone at concentrations of 120–160 μmol/liter also achieved a 67% reduction of cell viability, when they were treated for 6–8 min. Therefore, the best specific effects were achieved with high concentration of MB, and as short treatment time as possible,PDF Image | Photodynamic Low Level Laser Squamous Cell Carcinoma
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