Near-Infrared Photoactivatable Nitric Oxide Donors with Integrated Photoacoustic Monitoring

Photoacoustic (PA) tomography is a noninvasive technology that utilizes near-infrared (NIR) excitation and ultrasonic detection to image biological tissue at centimeter depths. While several activatable small-molecule PA sensors have been developed for various analytes, the use of PA molecules for deep-tissue analyte delivery and monitoring remains an underexplored area of research. Herein, we describe the synthesis, characterization, and in vivo validation of photoNOD-1 and photoNOD-2, the first organic, NIR-photocontrolled nitric oxide (NO) donors that incorporate a PA readout of analyte release. These molecules consist of an aza-BODIPY dye appended with an aryl N-nitrosamine NO-donating moiety. The photoNODs exhibit chemostability to various biological stimuli, including redox-active metals and CYP450 enzymes, and demonstrate negligible cytotoxicity in the absence of irradiation. Upon single-photon NIR irradiation, photoNOD-1 and photoNOD-2 release NO as well as rNOD-1 or rNOD-2, PA-active products that enable ratiometric monitoring of NO release. Our in vitro studies show that, upon irradiation, photoNOD-1 and photoNOD-2 exhibit 46.6-fold and 21.5-fold ratiometric turn-ons, respectively. Moreover, unlike existing NIR NO donors, the photoNODs do not require encapsulation or multiphoton activation for use in live animals. In this study, we use PA tomography to monitor the local, irradiation-dependent release of NO from photoNOD-1 and photoNOD-2 in mice after subcutaneous treatment. In addition, we use a murine model for breast cancer to show that photoNOD-1 can selectively affect tumor growth rates in the presence of NIR light stimulation following systemic administration.