Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance and short-lived nature limits our ability to study them in real time in living subjects with conventional non-invasive imaging methods. Photoacoustic imaging is an emerging modality for in vivo visualization of molecular processes with deep tissue penetration and high spatio-temporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (H2O2). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety and a boronic ester functionality that specifically detects and reacts to H2O2. The optical properties of the probe were characterized using absorption, fluorescence and photoacoustic measurements; upon addition of pathophysiological H2O2 concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed in vitro showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled non-invasive photoacoustic and fluorescence imaging of H2O2. In conclusion, the reported probe shows promise for the in vivo visualization of hydrogen peroxide.

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