Fluorescence photobleaching, photodynamic therapy (PDT) oxygen consumption and clonogenic cell survival were investigated during 2-(1-hexyloxethyl)-2-devinyl pyropheophoribde-a (HPPH) PDT of MAT-LyLu cells in vitro. Cells were incubated with HPPH concentrations of 0.24, 1.2, 3.6 or 12 μm for 4 h and then treated with 650 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment and photobleaching was quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony forming assay. Intracellular fluorescence lifetime measurements were also performed at each incubation concentration. The photobleaching kinetics did not follow first- or second-order kinetics and the fluorescence lifetime was similar for all intracellular concentrations. As the intracellular concentration of drug was increased, the amount of singlet oxygen and the absorbed quanta per cell required to achieve the same cell kill increased. Singlet oxygen dose was calculated using one- and two-compartment models of HPPH intracellular distribution. It was found that a two-compartment model, in which a PDT-sensitive binding site saturates at low concentrations, accounts for the observed photobleaching, oxygen consumption and cell survival.
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