Development of an Azo-Based Photosensitizer Activated under Mild Hypoxia for Photodynamic Therapy

Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (¹O₂), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate ¹O₂ only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to ¹O₂ generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of ¹O₂ to occur. In PDT <i>in vitro</i>, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O₂), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azo-based strategy should be widely applicable to design a range of activatable photosensitizers

Reversible Off–On Fluorescence Probe for Hypoxia and Imaging of Hypoxia–Normoxia Cycles in Live Cells

We report a fully reversible off–on fluorescence probe for hypoxia. The design employs QSY-21 as a Förster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. At that point, FRET can no longer occur, and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately reoxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia–normoxia cycles in live cells

Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates

Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the NN bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound <b>9</b> reporter system can detect azoreductase-expressing live cells at the single cell level