Singlet Oxygen Release and Cell Toxicity of a Chemiluminescent Squaraine Rotaxane Dye: Implications for Molecular Imaging

The water soluble tetraguanidinium squaraine rotaxane 2 was prepared and photoconverted to its corresponding squaraine rotaxane endoperoxide (SREP), 2EP. As expected, 2EP undergoes a thermal cycloreversion reaction that releases 60 ± 4% singlet oxygen and produces near-infrared emission in aqueous solution. Cell toxicity assays in the dark, using human and bacterial cell cultures, showed that 2EP (up to 20 µM) is no more toxic than its parent 2. This suggests that SREP-derived imaging probes are not likely to exhibit a significant toxicity effect due to the slow release of stoichiometric amounts of singlet oxygen. Additional photosensitization experiments showed that tetraguanidinium squaraine rotaxane 2 is a weak photosensitizer, but nonetheless, red light irradiation of cell cultures that were pre-incubated with 2 (>3 µM) produced moderate phototoxicity. Fluorescence microscopy studies attribute the phototoxicity of 2 to its ability to penetrate into the cell cytosol. The implications of these results are discussed in the context of effective methods to activate SREP as chemiluminescent probes for in vivo optical molecular imaging.