A photobiological and photophysical-based study of phototoxicity of two chlorins

To understand the fundamental determinants of phototoxic efficacy and absor bed photodynamic dose, the triplet state and photobleaching quantum yields in living cells, cellular uptake, intracellular localization, and correlati on with cell viability were studied for the two purpurins tin ethyl etiopur purin I (SnET2) and tin octaethylbenzochlorin (SnOEBC) in ovarian cancer ce lls (OVCARS), Although the triplet yields of these two photosensitizers wer e not significantly affected by cellular incorporation, the photobleaching yields were shown to be 3 orders of magnitude higher for cellular-bound sen sitizer than for free or albumin-bound photosensitizer and higher for SnET, than for SnOEBC for all of the cases. The intracellular concentration of S nOEBC was half that of SnET, after 3 hand 24 h-incuhation times for both 0. 1 muM and 1.0 phl incubation concentrations. Despite the lower concentratio ns of SnOEBC, the phototoxicity of the two photosensitizers was comparable at 1-muM incubation concentration and was up to 10-fold higher for SnOEBC a t the lower concentration, The subcellular localization established using: confocal microscopy and molecular probes showed that both photosensitizers were primarily lysosomally localized. SnOEBC, however, had an extra-lysosom al, mitochondrial localization component. The photophysical measurements al lowed calculation of the intracellular singlet oxygen production, which ind icated that the photosensitizer-light dose reciprocity was limited by photo bleaching for SnET, but only minimally for SnOEBC, and this was confirmed t hrough cell-survival studies. Taken together, these data indicate that the critical determinant of differences in phototoxicity between the two molecu les was their relative rates of photobleaching and their subcellular locali zation. The study points to the importance of combining photosensitizer upt ake and localization with photophysical measurements in the relevant biolog ical milieu to reasonably interpret and/or predict photosensitization effic acies.