Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biological detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of FeO nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase-like catalytic activities. Given the complex interdependence of the physicochemical properties and catalytic characteristics of nanozymes, it is important to establish a standard by which the catalytic activities and kinetics of various nanozymes can be quantitatively compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addition, we describe the detailed experimental procedures for a typical nanozyme strip-based biological detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic standard. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h.

Nanomaterials with intrinsic enzyme-like activities (nanozymes), have been widely used as artificial enzymes in biomedicine. However, how to control their in vivo performance in a target cell is still challenging. Here we report a strategy to coordinate nanozymes to target tumor cells and selectively perform their activity to destruct tumors. We develop a nanozyme using nitrogen-doped porous carbon nanospheres which possess four enzyme-like activities (oxidase, peroxidase, catalase and superoxide dismutase) responsible for reactive oxygen species regulation. We then introduce ferritin to guide nitrogen-doped porous carbon nanospheres into lysosomes and boost reactive oxygen species generation in a tumor-specific manner, resulting in significant tumor regression in human tumor xenograft mice models. Together, our study provides evidence that nitrogen-doped porous carbon nanospheres are powerful nanozymes capable of regulating intracellular reactive oxygen species, and ferritinylation is a promising strategy to render nanozymes to target tumor cells for in vivo tumor catalytic therapy.

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As next-generation artificial enzymes, nanozymes have shown great promise for tumor catalytic therapy. In particular, their peroxidase-like activity has been employed to catalyze hydrogen peroxide (H 2 O 2 ) to produce highly toxic hydroxyl radicals ( • OH) to kill tumor cells. However, limited by the low affinity between nanozymes with H 2 O 2 and the low level of H 2 O 2 in the tumor microenvironment, peroxidase nanozymes usually produced insufficient • OH to kill tumor cells for therapeutic purposes. Herein, we present a pyrite peroxidase nanozyme with ultrahigh H 2 O 2 affinity, resulting in a 4144-and 3086-fold increase of catalytic activity compared with that of classical Fe 3 O 4 nanozyme and natural horseradish peroxidase, respectively. We found that the pyrite nanozyme also possesses intrinsic glutathione oxidase-like activity, which catalyzes the oxidation of reduced glutathione accompanied by H 2 O 2 generation. Thus, the dual-activity pyrite nanozyme constitutes a self-cascade platform to generate abundant • OH and deplete reduced glutathione, which induces apoptosis as well as ferroptosis of tumor cells. Consequently, it killed apoptosis-resistant tumor cells harboring KRAS mutation by inducing ferroptosis. The pyrite nanozyme also exhibited favorable tumor-specific cytotoxicity and biodegradability to ensure its biosafety. These results indicate that the high-performance pyrite nanozyme is an effective therapeutic reagent and may aid the development of nanozyme-based tumor catalytic therapy.

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Nanomaterials with intrinsic enzyme-like activities (nanozymes) have emerged as promising agents for cancer theranostics strategies. However, size-controllable synthesis of nanozymes and their targeting modifications are still challenging. Here, we report a monodispersed ferritin-based cobalt nanozyme (HccFn­(Co3O4)) that specifically targets and visualizes clinical hepatocellular carcinoma (HCC) tissues. The cobalt nanozyme is biomimetically synthesized within the protein shell of the HCC-targeted ferritin (HccFn) nanocage, which is enabled by the display of HCC cell-specific peptide SP94 on the surface of ferritin through a genetic engineering approach. The intrinsic peroxidase-like activity of HccFn­(Co3O4) nanozymes catalyzes the substrates to make color reaction to visualize HCC tumor tissues. We examined 424 clinical HCC specimens and verified that HccFn­(Co3O4) nanozymes distinguish HCC tissues from normal liver tissues with a sensitivity of 63.5% and specificity of 79.1%, which is comparable with that of the clinically used HCC-specific marker α fetoprotein. Moreover, the pathological analysis indicates that the HccFn­(Co3O4) nanozyme staining result is a potential predictor of prognosis in HCC patients. Staining intensity is positively correlated to tumor differentiation degree (P = 0.0246) and tumor invasion (P < 0.0001) and negatively correlated with overall survival (P = 0.0084) of HCC patients. Together, our study demonstrates that ferritin is an excellent platform for size-controllable synthesis and targeting modifications of nanozymes, and the HccFn­(Co3O4) nanozyme is a promising reagent for prognostic diagnosis of HCC.

Tumor hypoxia compromises the effects of photodynamic therapy that consumes oxygen in the therapeutic process.

Extensive efforts are devoted to refining metal sites for optimizing the catalytic performance of single‐atom nanozymes (SANzymes), while the contribution of the defect environment of neighboring metal sites lacks attention. Herein, an iron‐based SANzyme (Fe‐SANzyme) is rationally designed by edge‐site engineering, which intensively exposes edge‐hosted defective Fe–N4 atomic sites anchored in hierarchical mesoporous structures. The Fe‐SANzyme exhibits excellent catalase‐like activity capable of efficiently catalyzing the decomposition of H2O2 into O2 and H2O, with a catalytic kinetic KM value superior to that of natural catalase and reported nanozymes. The mechanistic studies depict that the defects introduce notable charge transfer from the Fe atom to the carbon matrix, making the central Fe more activated to strengthen the interaction with H2O2 and weaken the OO bond. By performing catalase‐like catalysis, the Fe‐SANzyme significantly scavenges reactive oxygen species (ROS) and alleviates oxidative stress, thus eliminating the pathological angiogenesis in animal models of retinal vasculopathies without affecting the repair of normal vessels. This work provides a new way to refine SANzymes by engineering the defect environment and geometric structure around metal sites, and demonstrates the potential therapeutic effects of the nanozyme on retinal vasculopathies.