This proposal explores the limitations set by the local microenvironment in vivo on conventional photosensitizers which act via generation of toxic singlet oxygen. Hypoxia is thus a limiting factor to PDT. The systematic determination of the oxygen dependence of these photosensitizers will facilitate a better understanding of in vivo photosensitization. The development of alternative oxygen-independent sensitization mechanisms of photodamage which overcome existing limitations in PDT is proposed. The 1st specific aim is to investigate the direct effects of the biological microenvironment of the photosensitizer on its mechanism and efficiency. the effects of (i) high local sensitizer concentration; (ii) direct reaction of photosensitizer intermediates with integral biomolecules and (iii) cellular localization sites, on the photophysics and phototoxicity of photosensitizers will be determined in liposomes, erythrocyte ghosts and malignant cell suspensions. The 2nd specific aim is to construct a general relationship to predict efficacy of singlet oxygen photosensitizers to cause cell damage. Diffuse reflectance laser flash photolysis will measure oxygen concentrations in vivo using oxygen-dependent triplet state kinetics. Measured [O2] will be combined with rate constants for natural decay and oxygen quenching of the triplet state to predict photosensitizer efficiency. This relationship will be tested using photosensitizers with different natural triplet lifetimes, varying [O2] through N2/O2 gas mixtures and correlating observed phototoxicity with the efficiency predicted by the photophysical method. The 3rd specific aim is to develop alternative oxygen-independent photosensitization mechanisms for photosensitized damage in biological media which overcome the limitations of hypoxia. Radical species, rather than singlet oxygen, will be generated through bond cleavage and photosensitization processes in related compounds. A correlation of reaction mechanisms with photoionization normal and hypoxic cellular systems will ascertain the potential of radical-based strategies to effect oxygen-independent photodamage.
