Photo-oxidative killing of human colonic cancer cells using indocyanine green and infrared light

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Abstract
Despite of the approval of Photofrin® in various countries, chemically defined sensitizers for photodynamic therapy (PDT) are still needed for the absorption of light in the infrared spectrum, which provides a maximal penetration of light into tissue. Therefore, both the efficacy and the mechanism of action of the clinically approved dye indocyanine green (ICG) and laser irradiation were investigated in vitro. For the investigation of phototoxic effects, HT-29 cells were incubated 24 h prior to irradiation by using different concentrations of ICG (10–500 μM). In each experiment, cells were irradiated using a continuous wave (cw)-diode laser (λex = 805 nm, 30 J cm–2, 40 mW cm–2). After laser irradiation, cell viability of dark control and of cells incubated with 500 μM ICG was 1.27 ± 0.11 or 0.28 ± 0.05 respectively. Using 100 μM ICG and D2O, cell viability was further decreased from 0.46 ± 0.03 (H2O) to 0.11 ± 0.01 (D2O). Using D2O and 100 μM ICG, the concentration of malondialdehyde, a marker of lipid peroxidation, increased from 0.89 ± 0.10 nmol 10–6 cells to 11.14 ± 0.11 nmol 10–6 cells. Using 100 μM ICG and laser irradiation sodium azide or histidine (50 mM), quenchers of singlet oxygen reduced the cell killing significantly. In contrast, when using mannitol, a quencher of superoxide anion and hydroxyl radical, cell killing was not inhibited. According to the present results, photoactivated ICG seems to kill colonic cancer cells due to the generation of singlet oxygen and the subsequent formation of lipid peroxides. Therefore, ICG might present a promising photosensitizer for PDT; first clinical results confirm these findings.

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