Far-Red Fluorescence Probe for Monitoring Singlet Oxygen during Photodynamic Therapy

Singlet oxygen (O-1(2)), molecular oxygen in the lowest excited state, has a critical role in the cell-killing mechanism of photodynamic therapy (PDT). Although O-1(2) phosphorescence measurement has been mainly used to monitor O-1(2) formation during PDT, its intensity is far insufficient to obtain two-dimensional images of intracellular O-1(2) with the subcellular spatial resolution using the currently available near-IR detector. Here, we propose a new far-red fluorescence probe of O-1(2), namely, Si-DMA, composed of silicon-containing rhodamine and anthracene moieties as a chromophore and a O-1(2) reactive site, respectively. In the presence of O-1(2), fluorescence of Si-DMA increases 17 times due to endoperoxide formation at the anthracene moiety. With the advantage of negligible self-oxidation by photoirradiation (Phi(Delta) < 0.02) and selective mitochondrial localization, Si-DMA is particularly suitable for imaging O-1(2) during PDT. Among three different intracellular photosensitizers (Sens), Si-DMA could selectively detect the O-1(2) that is generated by 5-aminolevulinic acid-derived protoporphyrin IX, colocalized with Si-DMA in mitochondria. On the other hand, mitochondriatargeted KillerRed and lysosomal porphyrins could not induce fluorescence change of Si-DMA. This surprising selectivity of Si-DMA response depending on the Sens localization and photosensitization mechanism is caused by a limited intracellular O-1(2) diffusion distance (similar to 300 rim) and negligible generation of O-1(2) by type-I Sens, respectively. For the first time, we successfully visualized O-1(2) generated during PDT with a spatial resolution of a single mitochondrial tubule.