Photoacoustic (PA) imaging is an emerging imaging modality that utilizes optical excitation and acoustic detection to enable high resolution at centimeter depths. The development of activatable PA probes can expand the utility of this technology to allow for detection of specific stimuli within live-animal models. Herein, we report the design, development, and evaluation of a series of Acoustogenic Probe(s) for Nitric Oxide (APNO) for the ratiometric, analyte-specific detection of nitric oxide (NO) in vivo. The best probe in the series, APNO-5, rapidly responds to NO to form an N-nitroso product with a concomitant 91 nm hypsochromic shift. This property enables ratiometric PA imaging upon selective irradiation of APNO-5 and the corresponding product, tAPNO-5. Moreover, APNO-5 displays the requisite photophysical characteristics for in vivo PA imaging (e.g., high absorptivity, low quantum yield) as well as high biocompatibility, stability, and selectivity for NO over a variety of biologically relevant analytes. APNO-5 was successfully applied to the detection of endogenous NO in a murine lipopolysaccharide-induced inflammation model. Our studies show a 1.9-fold increase in PA signal at 680 nm and a 1.3-fold ratiometric turn-on relative to a saline control.

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.

Individual cells direct non-equilibrium processes through coordinated signal transduction and gene expression, allowing for dynamic control over multicellular, system-wide behavior. This behavior extends to remodeling the extracellular polymer matrix that encases biofilms and tissues, where constituent cells dictate spatiotemporal network properties including stiffness, pattern formation, and transport properties. The majority of synthetic polymer networks cannot recreate these phenomena due to their lack of autonomous centralized actuators (i.e., cells). In addition, non-living polymer networks that perform computation are generally restricted to a few inputs (e.g., light, pH, enzymes), limiting the logical complexity available to a single network chemistry. Toward synergizing the advantages of living and synthetic systems, engineered living materials leverage genetic and metabolic programming to establish control over material-wide properties. Here we demonstrate that a bacterial metal respiration mechanism, extracellular electron transfer (EET), can control metal-catalyzed radical cross-linking of polymer networks. Linking metabolic electron flux to a synthetic redox catalyst allows dynamic, tunable, and predictable control over material formation and bulk polymer network mechanics using genetic circuits. By programming key EET genes with transcriptional Boolean logic, we rationally design computational networks that sense-and-respond to multiple inputs in biological contexts. Finally, we capitalize on the wide reactivity of EET and redox catalyses to predictably control another class of living synthetic materials using copper(I) alkyne-azide cycloaddition click chemistry. Our results demonstrate the utility of EET as a bridge for controlling abiotic materials and how the design rules of synthetic biology can be applied to emulate physiological behavior in polymer networks.

Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed alkyne–azide cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. The reaction progress and kinetics were manipulated by tailoring the central carbon metabolism. Similarly, EET-CuAAC activity was dependent on specific EET pathways that could be regulated via inducible expression of EET-relevant proteins: MtrC, MtrA, and CymA. EET-driven CuAAC exhibited modularity and robustness in the ligand and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labeling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.

Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the model electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed Alkyne-Azide Cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. In addition, reaction progress and kinetics could be further manipulated by tailoring the central carbon metabolism of S. oneidensis. Similarly, CuAAC activity was dependent on specific EET pathways and could be manipulated using inducible genetic circuits controlling the expression of EET-relevant proteins including MtrC, MtrA, and CymA. EET-driven CuAAC also exhibited modularity and robustness in ligand tolerance and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without requiring regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labelling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.