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Single-atom nanozymes (SAzymes) with atomically dispersed active sites are potential substitutes for natural enzymes. A systematic study of its multiple functions can in-depth understand SAzymes's nature, which remains elusive. Here, we develop a novel ultrafast synthesis of sputtered SAzymes by in situ bombarding-embedding technique. Using this method, sputtered copper (Cu) SAzymes (CuSA) is developed with unreported unique planar Cu-C 3 coordinated configuration. To enhance the tumor-specific targeting, we employ a bioorthogonal approach to engineer CuSA, denoted as CuSACO. CuSACO not only exhibits minimal off-target toxicity but also possesses exceptional ultrahigh catalase-, oxidase-, peroxidase-like multienzyme activities, resulting in reactive oxygen species (ROS) storm generation for effective tumor destruction. Surprisingly, CuSACO can release Cu ions in the presence of glutathione (GSH) to induce cuproptosis, enhancing the tumor treatment efficacy. Notably, CuSACO's remarkable photothermal properties enables precise photothermal therapy (PTT) on tumors. This, combined with nanozyme catalytic activities, cuproptosis and immunotherapy, efficiently inhibiting the growth of orthotopic breast tumors and gliomas, and lung metastasis. Our research highlights the potential of CuSACO as an innovative strategy to utilize multiple mechanism to enhance tumor therapeutic efficacy, broadening the exploration and development of enzyme-like behavior and physiological mechanism of action of SAzymes., (© 2024 Wiley-VCH GmbH.)

Ratiometric afterglow luminescent (AGL) probes are attractive for in vivo imaging due to their high sensitivity and signal self-calibration function. However, there are currently few ratiometric AGL probes available for imaging enzymatic activity in living organisms. Here, we present an energy diversion (ED) strategy that enables the design of an enzyme-activated ratiometric AGL probe (RAG-RGD) for in vivo afterglow imaging. The ED process provides RAG-RGD with a radiative transition for an 'always on' 520-nm AGL signal (AGL 520 ) and a cascade three-step energy transfer (ET) process for an 'off-on' 710-nm AGL signal (AGL 710 ) in response to a specific enzyme. Using matrix metalloproteinase-2 (MMP-2) as an example, RAG-RGD shows a significant ~11-fold increase in AGL 710 /AGL 520 toward MMP-2. This can sensitively detect U87MG brain tumors through ratiometric afterglow imaging of MMP-2 activity, with a high signal-to-background ratio and deep imaging depth. Furthermore, by utilizing the self-calibration effect of ratiometric imaging, RAG-RGD demonstrated a strong negative correlation between the AGL 710 /AGL 520 value and the size of orthotopic U87MG tumor, enabling accurate monitoring of orthotopic glioma growth in vivo. This ED process may be applied for the design of other enzyme-activated ratiometric afterglow probes for sensitive afterglow imaging., (© 2024 Wiley-VCH GmbH.)

We report here a tumor-pretargted theranostic approach for multimodality imaging-guided synergistic cancer PDT by cascade alkaline phosphatase (ALP)-mediated in situ self-assembly and bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction. Using the enzymatic catalysis of ALP that continuously catalyses the dephosphorylation and self-assembly of trans-cyclooctene (TCO)-bearing P-FFGd-TCO, a high density of fluorescent and magnetic TCO-containing nanoparticles (FMNPs-TCO) can be synthesized and retained on the membrane of tumor cells. They can act as 'artificial antigens' amenable to concurrently capture lately administrated tetrazine (Tz)-decorated PS (775NP-Tz) and carbonic anhydrase (CA) inhibitor (SA-Tz) via the fast IEDDA reaction. This two-step pretargeting process can further induce FMNPs-TCO regrowth into microparticles (FMNPs-775/SA) directly on tumor cell membranes, which is analyzed by bio-SEM and fluorescence imaging. Thus, efficient enrichment of both SA-Tz and 775NP-Tz in tumors can be achieved, allowing to alleviate hypoxia by continuously inhibiting CA activity and improving PDT of tumors. Findings show that subcutaneous HeLa tumors could be completely eradicated and no tumor recurred after irradiation with an 808 nm laser (0.33 W cm -2 , 10 min). This pretargeted approach may be applied to enrich other therapeutic agents in tumors to improve targeted therapy., (© 2023 Wiley-VCH GmbH.)

Tumor-targeted and stimuli-activatable nanosensitizers are highly desirable for cancer theranostics. However, designing smart nanosensitizers with multiple imaging signals and synergistic therapeutic activities switched on is challenging. Herein, we report tumor-targeted and redox-activatable nanosensitizers (1-NPs) for sono-photodynamic immunotherapy of tumors by molecular co-assembly and redox-controlled disassembly. 1-NPs show a high longitudinal relaxivity (r 1 =18.7±0.3 mM -1  s -1 ), but "off" dual fluorescence (FL) emission (at 547 and 672 nm), "off" sono-photodynamic therapy and indoleamine 2,3-dioxygenase 1 (IDO1) inhibition activities. Upon reduction by glutathione (GSH), 1-NPs rapidly disassemble and remotely release small molecules 2-Gd, Zn-PPA-SH and NLG919, concurrently switching on (1) dual FL emission, (2) sono-photodynamic therapy and (3) IDO1 inhibition activities. After systemic injection, 1-NPs are effective for bimodal FL and magnetic resonance (MR) imaging-guided sono-photodynamic immunotherapy of orthotropic breast and brain tumors in mice under combined ultrasound (US) and 671-nm laser irradiation., (© 2023 Wiley-VCH GmbH.)

Multimodal imaging, which harnesses two or more imaging modalities to produce complementary anatomical and molecular information of a living subject, has become as a powerful tool in both basic biomedical research and clinical diagnosis. The progresses in multimodal imaging are paralleled by the advances in multimodal probes, particularly activatable multimodal imaging probes that can generate concurrent switches in different imaging modality signals upon interaction with a molecular target. These probes are extremely promising for in vivo imaging. In this Minireview, we summarize the recent progress in activatable multimodal probes for in vivo imaging and cancer theranostics, focusing on their design principle, signal activation mechanism and biomedical applications. The current challenges and perspectives for future developments of activatable multimodal probes are also briefly discussed. We hope that this Minireview will provide inspiration for the design of other activatable multimodal probes for improving in vivo imaging and theranostics., (© 2022 Wiley-VCH GmbH.)

Reversible imaging probes that allow for the dynamic visualization of the redox cycle between hydroxyl radical (⋅OH) and hydrogen sulfide (H 2 S) are vital to probe the redox imbalance-involved pathological process in vivo. Herein, we report a reversible ratiometric photoacoustic (PA) imaging nanoprobe (1-PAIN) for the real-time imaging of ⋅OH/H 2 S redox cycle in vivo. 1-PAIN displays a low PA ratio between 690 and 825 nm (PA 690 /PA 825 ), which significantly increases by ≈5-fold upon oxidation by ⋅OH, and is switched back to the initially low PA 690 /PA 825 value upon reduction by H 2 S. 1-PAIN could dynamically report on the hepatic ⋅OH production in mice during the lipopolysaccharide (LPS)-induced liver inflammation process, and visualize hepatic H 2 S generation during the N-acetyl cysteine (NAC)-induced anti-inflammation process. 1-PAIN can act as a useful tool to probe the redox state in living biology, beneficial for the study of redox imbalance-related diseases., (© 2022 Wiley-VCH GmbH.)

Enzyme-triggered macrocyclization and in situ self-assembly of small molecules into nanoparticles has shown promise to design activatable probes for molecular imaging. However, controlling macrocyclization and self-assembly to concurrently augment positron emission tomography (PET) and photoacoustic (PA) signals for bimodality imaging is challenging. Herein, we report the engineering of a triazole-IR780 fluorophore as a versatile macrocyclization scaffold for controlling in situ self-assembly and design a caspase-3-activatable PA/PET bimodal probe ([ 18 F]-IR780-1) for in vivo imaging of tumor apoptosis. By leveraging the high-sensitivity whole-body imaging signals offered by PET with the high-resolution imaging signals offered by PA, [ 18 F]-IR780-1 can provide a promising tool for the early evaluation of antitumor efficacy, helpful for optimizing the therapeutic protocol for patients. This scaffold may be adopted to design other activatable bimodal probes for in vivo imaging., (© 2022 Wiley-VCH GmbH.)

Hydrogen sulfide (H 2 S) is an important endogenous gasotransmitter, but the targeted delivery and real-time feedback of exogenous H 2 S are still challenging. With the aid of density functional theory (DFT) calculations, we designed a new 1,3-dithiolium-4-olate (DTO) compound, which can react with a strained alkyne via the 1,3-dipolar cycloaddition and the retro-Diels-Alder reaction to generate carbonyl sulfide (COS) as the precursor of H 2 S, and a thiophene derivative with turn-on fluorescence. Moreover, the diphenylamino substituent in DTO greatly increases the mitochondrial targeting of this H 2 S delivery system. Such a bioorthogonal click-and-release reaction has integrated three functions in one system for the first time: (1) in situ controllable H 2 S release, (2) concomitant fluorescence response, and (3) mitochondria-targeted delivery. In addition, we investigated the mitochondrial membrane potential loss alleviation by using this system in H9c2 cells under oxidative stress., (© 2021 Wiley-VCH GmbH.)

Accurate detection of hepatic hydrogen sulfide (H 2 S) to monitor H 2 S-related enzymes' activity is critical for acute hepatitis diagnosis, but remains a challenge due to the dynamic and transient nature of H 2 S. Here, we report a H 2 S-activatable near-infrared afterglow/MRI bimodal probe F1-GdNP, which shows an "always-on" MRI signal and "off-on" afterglow signal toward H 2 S. F1-GdNP shows fast response, high sensitivity and specificity toward H 2 S, permitting afterglow imaging of H 2 S and evaluation of cystathionine γ-lyase (CSE)'s activity in living mice. We further employ the high spatial-resolution MRI signal of F1-GdNP to track its delivery and accumulation in liver. Importantly, F1-GdNP offers a high signal-to-background ratio (SBR=86.2±12.0) to sensitively report on the increased hepatic H 2 S level in the acute hepatitis mice via afterglow imaging, which correlated well with the upregulated CSE activity in the liver, showcasing the good potential of F1-GdNP for monitoring of acute hepatitis process in vivo., (© 2021 Wiley-VCH GmbH.)

Pretargeted imaging has emerged as a promising approach to advance nuclear imaging of malignant tumors. Herein, we combine the enzyme-mediated fluorogenic reaction and in situ self-assembly with the inverse electron demand Diels-Alder (IEDDA) reaction to develop an activatable pretargeted strategy for multimodality imaging. The trans-cyclooctene (TCO) bearing small-molecule probe, P-FFGd-TCO, can be activated by alkaline phosphatase and in situ self-assembles into nanoaggregates (FMNPs-TCO) retained on the membranes, permitting to (1) amplify near-infrared (NIR) fluorescence (FL) and magnetic resonance imaging (MRI) signals, and (2) enrich TCOs to promote IEDDA ligation. The Gallium-68 ( 68 Ga) labeled tetrazine can readily conjugate the tumor-retained FMNPs-TCO to enhance radioactivity uptake in tumors. Strong NIR FL, MRI, and positron emission tomography (PET) signals are concomitantly achieved, allowing for pretargeted multimodality imaging of ALP activity in HeLa tumor-bearing mice., (© 2021 Wiley-VCH GmbH.)

Stimuli-responsive smart photosensitizer (PS) nanoassemblies that allow enhanced delivery and controlled release of PSs are promising for imaging-guided photodynamic therapy (PDT) of tumors. However, the lack of high-sensitivity and spatial-resolution signals and fast washout of released PSs from tumor tissues have impeded PDT efficacy in vivo. Herein, we report tumor targeting, redox-responsive magnetic and fluorogenic PS nanoassemblies (NP-RGD) synthesized via self-assembly of a cRGD- and disulfide-containing fluorogenic and paramagnetic small molecule (1-RGD) for fluorescence/magnetic resonance bimodal imaging-guided tumor PDT. NP-RGD show high r 1 relaxivity but quenched fluorescence and PDT activity; disulfide reduction by glutathione (GSH) promotes efficient disassembly into a small-molecule probe (2-RGD) and an organic PS (PPa-SH), which could further bind with intracellular albumin, allowing prolonged retention and cascade activation of fluorescence and PDT to ablate tumors., (© 2020 Wiley-VCH GmbH.)

Photoacoustic (PA) imaging shows promise in the sensitive detection of caspase-3 activated in early tumor apoptosis in response to chemotherapy; smart PA probes are thus in high demand. Herein, we report the first smart PA probe (1-RGD) responsive to caspase-3, enabling real-time and high-resolution imaging of tumor apoptosis. 1-RGD is designed to leverage the synergetic effect of active delivery and caspase-3 activation. It is selectively recognized by active caspase-3 to trigger peptide substrate cleavage and biocompatible macrocyclization-mediated self-assembly, leading to an amplified PA imaging signal and prolonged retention in apoptotic tumor cells. Strong, high-resolution PA images are obtained in chemotherapy-induced apoptotic tumors in living mice after intravenous administration with 1-RGD, facilitating sensitive reporting of caspase-3 activity and distribution within tumor tissues., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drug Design: An (18)F-labeled caspase-3-sensitive nanoaggregation positron emission tomography tracer was prepared and evaluated for imaging the caspase-3 activity in doxorubicin-treated tumor xenografts. Enhanced retention of the (18)F activity in apoptotic tumors is achieved through intramolecular macrocyclization and in situ aggregation upon caspase-3 activation (see picture)., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)