Indocyanine green (ICG) and laser irradiation induce photooxidation

The cellular uptake and subcellular localization of indocyanine green (ICG; absorption band 700-850 nm), and cell survival and ultrastructural changes following ICG-mediated phototherapy were investigated in vitro in four different cell lines derived from human skin (SCL1 and SCL2 squamous cell carcinoma, HaCaT keratinocytes and N1 fibroblasts). The cellular uptake of ICG (1-50 microM, incubation times 1, 4, 24 h) was saturable, highly cumulative and could be inhibited by the addition of 250 microM bromosulphophthalein indicating the involvement of the organic anion transporting polypeptide (OATP). For HaCaT cells, the maximum cellular uptake (Vmax) and the Michaelis constant (K(m)) were 9.9 +/- 1.1 mM and 47 +/- 16 microM, respectively, following a 24-h incubation with ICG. Fluorescence microscopy revealed a cytoplasmic distribution of ICG, probably bound to glutathione S-transferase. Following irradiation with a cw-diode laser (805 nm, 80 mW/cm2) at doses of 24 or 48 J/cm2, the phototoxicity was determined using the MTT assay as a measure of cell viability. For all cell lines, ICG concentrations above 25 microM produced a significant phototoxic effect. The EC50, of ICG for HaCaT cells following irradiation at 24 J/cm2 was 20.1 +/- 3.9 microM. Growth curves showed that even HaCaT cells treated at the EC50 were killed within a week following treatment. Electron microscopy 1 h after ICG-mediated phototherapy revealed cytoplasmic vesiculation, dilation of the rough endoplasmic reticulum, the Golgi complex and the perinuclear cisternae and the beginning of chromatin condensation in the nucleus. These ultrastructural findings are not consistent with a photothermal action of ICG-mediated phototherapy. Taken together with those of previous studies by our group these results support photooxidation as a major cell-killing mechanism.