Confocal Raman microspectroscopy and imaging study of theraphthal in living cancer cells

Binary systems combining a transition metal complex and ascorbate have been proposed recently for catalytic therapy of malignant tumors. The killing e ffect on tumor cells is achieved by production of free radicals in the cour se of accelerated oxidation of ascorbate by dioxygen in the presence of tra nsition metal complexes. Further progress in the development of binary cata lytic systems (BCSs) requires a special method for their investigation in c ells and tissues, because neither component of BCSs fluoresces. Here a reso nance Raman confocal spectral imaging (RR CSI) technique was introduced as a unique approach to monitor quantitatively the transition metal complexes within living cells. Intracellular accumulation, localization, and retentio n of theraphthal CTP), a catalyst of the advanced TP/ascorbate BCS, were in vestigated in A549 cells with the RR CSI technique. The cellular analysis w as complemented with the detailed study of molecular interactions of TP in solution and environmental factors affecting the RR spectrum of TP. TP does not penetrate into membranes, it binds very weakly to DNA and RNA, but it readily forms complexes with proteins. Binding with Ca2+ cations and decrea sing pH below 6 induce aggregation of TP. By analyzing RR spectra recorded from every point within a TP-treated cell, three states of the agent were d iscriminated, namely, monomeric TP in polar environment, TP bound to protei ns, and aggregated TP. Their cytoplasmic and nuclear distributions were map ped at different stages of uptake and efflux. By introducing organelle-sele ctive fluorescent probes into drug-treated cells and measuring intracellula r localization of both the probe and the drug, compartmentation of TP was r evealed. Cell growth suppression by the TP/ascorbate system was measured, a nd probable molecular and organelle targets of radical damage were characte rized.