Ex vivo quantification of mTHPC concentration in tissue: influence of chemical extraction on the optical properties

A method for the quantification of the concentration of the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) in tissue samples is presented. The technique is an extension of a previously published method based on alkaline hydrolysis of tissue, using Solvable as a tissue solubilizer. mTHPC quantification was achieved by subsequent fluorescence spectroscopy. Since the original extraction method involved multiple steps in which water dilution of the sample was implemented, we studied the spectral characteristics of mTHPC in different Solvable/water mixtures. Using UV-VIS absorption and fluorescence spectroscopy, it was demonstrated that the spectral characteristics of mTHPC vary for different Solvable concentrations. In the range of 20-100% Solvable, the fluorescence intensity of mTHPC did not change, while dramatic changes in the mTHPC fluorescence intensity were observed for lower Solvable concentrations (< 20%) due to increasing hydrophilicity of the environment, combined with pH alterations. We also demonstrated that the absorption and fluorescence spectra of the dissolved tissue were time-dependent. Longer incubation of the samples resulted in a significant increase of the native tissue chromophore fluorescence. This implies that for the correct quantification of photosensitizer concentrations, the fluorescence of native tissue chromophores must be accounted for.