Photoacoustic (PA) imaging technology, a three-dimensional hybrid imaging modality that integrates the advantage of optical and acoustic imaging, has great application prospects in molecular imaging due to its high imaging depth and resolution. To endow PA imaging with the ability for real-time molecular visualization and precise biomedical diagnosis, numerous activatable molecular PA probes which can specifically alter their PA intensities upon reacting with the targets or biological events of interest have been developed. This review highlights the recent developments of activatable PA probes for precise biomedical applications including molecular detection of the biotargets and imaging of the biological events. First, the generation mechanism of PA signals will be given, followed by a brief introduction to contrast agents used for PA probe design. Then we will particularly summarize the general design principles for the alteration of PA signals and activatable strategies for developing precise PA probes. Furthermore, we will give a detailed discussion of activatable PA probes in molecular detection and biomedical imaging applications in living systems. At last, the current challenges and outlooks of future PA probes will be discussed. We hope that this review will stimulate new ideas to explore the potentials of activatable PA probes for precise biomedical applications in the future.

Chemodynamic therapy is an emerging tumor therapeutic strategy. However, the anticancer effects are greatly limited by the strong acidity requirements for effective Fenton‐like reaction, and the inevitably “off‐target” toxicity. Herein, we develop an acidity‐unlocked nanoplatform (FePt@FeOx@TAM‐PEG) that can accurately perform the high‐efficient and tumor‐specific catalysis for anticancer treatment, through dual pathway of cyclic amplification strategy. Notably, the pH‐responsive peculiarity of tamoxifen (TAM) drug allows for the catalytic activity of FePt@FeOx to be “turn‐on” in acidic tumor microenvironments, while keeping silence in neutral condition. Importantly, the released TAM within cancer cells is able to inhibit mitochondrial complex I, leading to the upregulated lactate content and thereby the accumulated intracellular H+, which can overcome the intrinsically insufficient acidity of tumor. Through the positive feedback loop, large amount of active FePt@FeOx nanocatalyzers are released and able to access to the endogenous H2O2, exerting the improved Fenton‐like reaction within the more acidic condition. Finally, such smart nanoplatform enables self‐boosting generation of reactive oxygen species (ROS) and induces strong intracellular oxidative stress, leading to the substantial anticancer outcomes in vivo, which may provide a new insight for tumor‐specific cascade catalytic therapy and reducing the “off‐target” toxicity to surrounding normal tissues.

Acute pneumonia can greatly increase the vulnerable risk of atherosclerotic plaque and contribute to the mortality of cardiovascular disease. To accurately assess the rupture risk caused by acute pneumonia, we developed a novel kind of ratiometric semiconducting polymer nanoparticle (RSPN) for photoacoustic imaging of vulnerable plaque in apolipoprotein E-deficient mice complicated with pneumonia. Specifically, RSPN can react with O2 •– and exhibit the enhanced photoacoustic signals at about 690 nm, while 800 nm is regarded as an internal photoacoustic reference. As a result, RSPN can provide reliable determination of O2 •– within aortic atherosclerosis by analyzing the ratios of photoacoustic signals, which can successfully reflect the oxidative stress level in vulnerable plaque. Therefore, RSPN enable to specifically distinguish plaque-bearing mice and plaque-bearing mice complicated with pneumonia from healthy mice, which provides a promising tool to predict the vulnerability of plaque for reducing the mortality of atherosclerotic-induced cardiovascular disease.

Nanozymes with intrinsic enzyme‐like characteristics have attracted enormous research interest in biological application. However, there is a lack of facile approach for evaluating the catalytic activity of nanozymes in living system. Herein, we develop a novel manganese‐semiconducting polymer‐based nanozyme (MSPN) with oxidase‐like activity for reporting the catalytic activity of itself in acid‐induced cancer therapy via ratiometric near‐infrared fluorescence (NIRF)‐photoacoustic (PA) molecular imaging. Notably, MSPN possess oxidase‐like activity in tumor microenvironment, owing to the mixed‐valent MnOx nanoparticles, which can effectively kill cancer cells. Because the semiconducting polymer (PFODBT) is conjugated with oxidase‐responsive molecule (ORM), the catalytic activity of nanozyme can be correlated with the ratiometric signals of NIRF (FL695/FL825) and PA (PA680/PA780), which may provide new ideas for predicting anticancer efficacy of nanozymes in living system.

Tumor response to radiotherapy or ferroptosis is closely related to hydroxyl radical (•OH) production. Noninvasive imaging of •OH fluctuation in tumors can allow early monitoring of response to therapy, but is challenging. Here, we report the optimization of a diene electrochromic material (1-Br-Et) as a •OH-responsive chromophore, and use it to develop a near-infrared ratiometric fluorescent and photoacoustic (FL/PA) bimodal probe for in vivo imaging of •OH. The probe displays a large FL ratio between 780 and 1113 nm (FL780/FL1113), but a small PA ratio between 755 and 905 nm (PA755/PA905). Oxidation of 1-Br-Et by •OH decreases the FL780/FL1113 while concurrently increasing the PA755/PA905, allowing the reliable monitoring of •OH production in tumors undergoing erastin-induced ferroptosis or radiotherapy.