Effects of photosensitizer bleaching and localization on photodynamic oxygen consumption and dosimetry

During photodynamic therapy, tumor eradication is achieved by a series of photochemical reactions resulting from the optical excitation of a photosensitive drug (sensitizer) that localizes preferentially in the tumor. The processes of sensitizer photobleaching and localization are investigated in the context of photodynamic therapy and dosimetry.; Theoretical models describing singlet oxygen (1O 2)- and non-1O2-mediated sensitizer degradation are developed and used for the analysis of ground state oxygen (3O 2) consumption measurements obtained during irradiation of multicell tumor spheroids sensitized with four different drugs: PhotofrinRTM , -aminolevulinic acid-induced protoporphyrin IX (PpIX), Nile Blue Sulfur (EtNBS) and Nile Blue Selenium (EtNBSe). We find that while PhotofrinRTM and PpIX photobleach via a 1O 2-mediated mechanism, EtNBS and EtNBSe are degraded via a non- 1O2-mediated process. We demonstrate that incorporation of sensitizer photobleaching in photodynamic dosimetry models is essential, as it affects significantly the production of 1O2. The spatial and temporal progression of 1O2- and non-1O2-mediated sensitizer degradation occur in distinct ways, influencing the deposition of 1O2 accordingly.; The relationship between sensitizer localization and photodynamic dosimetry is examined on the subcellular level for EtNBS and EtNBSe and on the cellular level for PpIX. Specifically, we present experimental evidence that demonstrates how the subcellular localization and redistribution of EtNBS and EtNBSe might affect 3O2 consumption during irradiation of sensitized spheroids. Our data suggest that sensitizer redistribution is a fluence dependent event that leads to enhanced photodynamic oxygen consumption.; Studies of the dependence of PpIX synthesis on 3O 2 availability in monolayer cultures indicate that hypoxia leads to significant reductions in PpIX production in a manner that is dependent on the cell density and the proliferation status of the population. These results, suggest that PpIX could be synthesized in a non-uniform manner in tumors with significant 3O2 gradients, impairing even further the efficacy of PDT in hypoxic regions.; Finally, we present the design and resolution capabilities of a laser beam scanning confocal microscope constructed for the direct study of sensitizer photobleaching and localization during PDT of tumor spheroids. Preliminary images of optical sections through sensitized spheroids demonstrate the feasibility of such studies.