Mitochondria are crucial sites for biological oxidation and substance metabolism and plays a vital role in maintaining intracellular homeostasis. When mitochondria undergo oxidative damage or dysfunction, they can harm the organism, leading to various reactive oxygen species (ROS)-related diseases. Therefore, therapies targeting mitochondria are a strategy for treating multiple diseases. Many nanozymes can mimic antioxidant enzymes, which enables them to eliminate ROS to mitigate mitochondrial dysfunction. The therapeutic approaches and drugs targeting the mitochondrial electron transport chain (ETC) have emerged as effective treatments for oxidative stress-related diseases resulting from mitochondrial respiratory chain disorders. Therefore, nanozymes that can regulate homeostasis in the mitochondrial ETC have emerged as effective therapeutic agents for treating oxidative stress-related diseases. In addition, benefit from the controllability and modifiability of nanozymes, their modification with TPP, SS-31 peptide, and mitochondrial permeability membrane peptide to eliminate ROS and repair mitochondrial function. The nanozymes that specifically target mitochondria are powerful tools for the treatment of ROS-associated disorders. We discussed the design strategies pertaining to mitochondrion-targeted nanozymes to treat various diseases to develop more efficacious nanozyme tools for the treatment of ROS-related diseases in the future.

Cerium oxide (CeO2) nanospheres have limited enzymatic activity that hinders further application in catalytic therapy, but they have an \"oxidation switch\" to enhance their catalytic activity by increasing oxygen vacancies. In this study, according to the defect-engineering strategy, we developed PtCuOX/CeO2-X nanozymes as highly efficient SOD/CAT mimics by introducing bimetallic copper (Cu) and platinum (Pt) into CeO2 nanospheres to enhance the oxygen vacancies, in an attempt to combine near-infrared (NIR) irradiation to regulate microenvironment for osteoarthritis (OA) therapy. As expected, the Cu and Pt increased the Ce3+/Ce4+ ratio of CeO2 to significantly enhance the oxygen vacancies, and simultaneously CeO2 (111) facilitated the uniform dispersion of Cu and Pt. The strong metal-carrier interaction synergy endowed the PtCuOX/CeO2-X nanozymes with highly efficient SOD/CAT-like activity by the decreased formation energy of oxygen vacancy, promoted electron transfer, the increased adsorption energy of intermediates, and the decreased reaction activation energy. Besides, the nanozymes have excellent photothermal conversion efficiency (55.41%). Further, the PtCuOX/CeO2-X antioxidant system effectively scavenged intracellular ROS and RNS, protected mitochondrial function, and inhibited the inflammatory factors, thus reducing chondrocyte apoptosis. In vivo, experiments demonstrated the biosafety of PtCuOX/CeO2-X and its potent effect on OA suppression. In particular, NIR radiation further enhanced the effects. Mechanistically, PtCuOX/CeO2-X nanozymes reduced ras-related C3 botulinum toxin substrate 1 (Rac-1) and p-p65 protein expression, as well as ROS levels to remodel the inflammatory microenvironment by inhibiting the ROS/Rac-1/nuclear factor kappa-B (NF-κB) signaling pathway. This study introduces new clinical concepts and perspectives that can be applied to inflammatory diseases.

Bioorthogonal chemistry has provided an elaborate arsenal to manipulate native biological processes in living systems. As the great advancement of nanotechnology in recent years, bioorthogonal nanozymes are innovated to tackle the challenges that emerged in practical biomedical applications. Bioorthogonal nanozymes are uniquely positioned owing to their advantages of high customizability and tunability, as well as good adaptability to biological systems, which bring exciting opportunities for biomedical applications. More intriguingly, the great advancement in nanotechnology offers an exciting opportunity for innovating bioorthogonal catalytic materials. In this comprehensive review, the significant progresses of bioorthogonal nanozymes are discussed with both spatiotemporal controllability and high performance in living systems, and highlight their design principles and recent rapid applications. The remaining challenges and future perspectives are then outlined along this thriving field. It is expected that this review will inspire and promote the design of novel bioorthogonal nanozymes, and facilitate their clinical translation.

BACKGROUND: The unique size, physical and chemical properties, and ultra-high stability of nanozymes have attracted extensive attentions in sensing, but improvement of catalytic activity of the nanozymes is still an urgent issue. Given the ultra-high simulated enzyme activity of metal nanoparticles and the advantage of multi-enzyme catalysis, an Au-decorated MoS2 nanosheets (MoS2/Au NS) integrating the double peroxidase-like (POD) activity is developed.
RESULTS: By optimizing and adjusting the density of AuNPs, as well as its morphology and other parameters, a monodisperse and high-density distribution of AuNPs on MoS2 nanosheets was obtained, which can greatly improve the POD-like activity of MoS2/Au NS. Nafion solution was applied to assist the modification of MoS2/Au NS on the electrode surface so as to improved its stability. An electrochemical H2O2 detection platform was constructed by modifying MoS2/Au NS nanozyme on the SPCE using the conductive Nafion solution. And the negatively charged sulfonic acid group can eliminate negatively charged electroactive substances to improve the specificity. Then ascorbic acid was used to stimulate tumor cells to produce H2O2 as therapeutic model, an ultrasensitive chronocoulometry detection for H2O2 in cell lysate was established. The logarithmically of ΔQ and the logarithmically of H2O2 concentration showed a good linear relationship between 1 μM and 500 mM, with a LOD value of 0.3 μM.
CONCLUSIONS: The developed H2O2 sensor has excellent stability, reproducibility (RSD = 2.3 %, n = 6) and selectivity, realized the quantitative detection of H2O2 in cell lysate. Compared with commercial fluorescence detection kits for H2O2 in cell lysate, it is worth mentioning that the electrochemical H2O2 sensor developed in this study is simpler and faster, with higher sensitivity and lower cost. This provides a potential substitute for disease diagnosis and treatment evaluation based on accurate detection of H2O2.

The enzyme-mimicking nature of versatile nanomaterials proposes a new class of materials categorized as nano-enzymes, ornanozymes. They are artificial enzymes fabricated by functionalizing nanomaterials to generate active sites that can mimic enzyme-like functions. Materials extend from metals and oxides to inorganic nanoparticles possessing intrinsic enzyme-like properties. High cost, low stability, difficulty in separation, reusability, and storage issues of natural enzymes can be well addressed by nanozymes. Since 2007, more than 100 nanozymes have been reported that mimic enzymes like peroxidase, oxidase, catalase, protease, nuclease, hydrolase, superoxide dismutase, etc. In addition, several nanozymes can also exhibit multi-enzyme properties. Vast applications have been reported by exploiting the chemical, optical, and physiochemical properties offered by nanozymes. This review focuses on the reported nanozymes fabricated from a variety of materials along with their enzyme-mimicking activity involving tuning of materials such as metal nanoparticles (NPs), metal-oxide NPs, metal-organic framework (MOF), covalent organic framework (COF), and carbon-based NPs. Furthermore, diverse applications of nanozymes in biomedical research are discussed in detail.

As nanoscale materials with the function of catalyzing substrates through enzymatic kinetics, nanozymes are regarded as potential alternatives to natural enzymes. Compared to protein-based enzymes, nanozymes exhibit attractive characteristics of low preparation cost, robust activity, flexible performance adjustment, and versatile functionalization. These advantages endow them with wide use from biochemical sensing and environmental remediation to medical theranostics. Especially in biomedical diagnosis, the feature of catalytic signal amplification provided by nanozymes makes them function as emerging labels for the detection of biomarkers and diseases, with rapid developments observed in recent years. To provide a comprehensive overview of recent progress made in this dynamic field, here an overview of biomedical diagnosis enabled by nanozymes is provided. This review first summarizes the synthesis of nanozyme materials and then discusses the main strategies applied to enhance their catalytic activity and specificity. Subsequently, representative utilization of nanozymes combined with biological elements in disease diagnosis is reviewed, including the detection of biomarkers related to metabolic, cardiovascular, nervous, and digestive diseases as well as cancers. Finally, some development trends in nanozyme-enabled biomedical diagnosis are highlighted, and corresponding challenges are also pointed out, aiming to inspire future efforts to further advance this promising field.

Nanozyme catalytic therapy triggered by the tumor microenvironment (TME)-responsive enzyme-like catalytic activities is an emerging approach for tumor treatment. However, the poor catalytic efficiency of nanozymes in tumors and the toxic side effects on normal tissues limit their further development, primarily due to the limited uptake and penetration depth of nanozyme in tumor tissues. Here, a tumor-targeting TME and electric field stimuli-responsive nanozyme (AgPt@CaCO3-FA) is developed, which is capable of catalyzing the generation of ROS to induce cell death and releasing carbon monoxide (CO) specifically in tumor tissues for on-demand CO therapy and immunotherapy. Benefiting from the endogenous H2S activated NIR-II fluorescence (FL) imaging guidance, AgPt@CaCO3-FA can be delivered into the deeper site of tumor tissues resulted from the TME regulation via generated CO during the electrolysis process to improve the catalytic efficiency of nanozymes in tumors. Moreover, CO effectively relieve immunosuppression TME via reeducating tumor-supportive M2-like macrophages to tumoricidal M1-like macrophages and induce mitochondrial dysfunction by reducing mitochondrial membrane potential, triggering tumor cells apoptosis. The enzyme-like activities combined with CO therapy arouse distinct immunogenic cell death (ICD) effect. Therefore, AgPt@CaCO3-FA permits synergistic CO gas, catalytic therapy and immunotherapy, effectively eradicating orthotopic breast tumors and preventing tumor metastasis and recurrence.

Developing sensor arrays capturing comprehensive fluorescence (FL) spectra from a single probe is crucial for understanding sugar structures with very high similarity in biofluids. Therefore, the analysis of highly similar sugar\' structures in biofluids based on the entire FL of a single nanozyme probe needs more concern, which makes the development of novel alternative approaches highly wanted for biomedical and other applications. Herein, a well-designed deep learning model with intrinsic information of 3D FL of CuO nanoparticles (NPs)\' oxidase-like activity was developed to classify and predict the concentration of a group of sugars with very similar chemical structures in different media. The findings presented that the overall accuracy of the developed model in classifying the nine selected sugars was (99-100 %), which prompted us to transfer the developed model to predict the concentration of the selected sugars at a concentration range of (1-100 μM). The transferred model also gave excellent results (R2 = 97-100 %). Therefore, the model was extended to other more complex applications, namely the identification of mixtures of sugars in serum and the detection of polysaccharides in different media such as serum and lake water. Notably, LOD for fructose was determined at 4.23 nM, marking a 120-fold decrease compared to previous studies. Our developed model was also compared with other deep learning-based models, and the results have demonstrated remarkable progress. Moreover, the identification of other possible coexisting interference substances in lake water samples was considered. This work marks a significant advancement, opening avenues for the widespread application of sensor arrays integrating nanozymes and deep learning techniques in biomedical and other diverse fields.

Iron phosphide/carbon (FeP/C) serving as electrocatalysts exhibit excellent activity in oxygen reduction reaction (ORR) process. H2O2 catalyzed by peroxidase (POD) is similar to the formation of new electron transfer channels and the optimization of adsorption of oxygen-containing intermediates or desorption of products in ORR process. However, it is still a challenge to discover FeP/C with enhanced POD-like catalytic activity in the electrocatalytic database for biocatalysis. The discovery of FeP/carbon dots (FeP/CDs) nanozymes driven by electrocatalytic activity for enhanced POD-like ability is demonstrated. FeP/CDs derived from CDs-Fe3+ chelates show enhanced POD-like catalytic and antibacterial activity. FeP/CDs exhibit enhanced POD-like activities with a specific activity of 31.1 U mg-1 that is double higher than that of FeP. The antibacterial ability of FeP/CDs nanozymes with enhanced POD-like activity is 98.1%. The antibacterial rate of FeP/CDs nanozymes (250 µg mL-1) increased by 5%, 15%, and 36% compared with FeP, Fe2O3/CDs, and Cu3P/CDs nanozymes, respectively. FeP/CDs nanozymes will attract more efforts to discover or screen transition metal phosphide/C nanozymes with enhanced POD-like catalytic activity for biocatalysis in the electrocatalytic database.

Engineering multimetallic nanocatalysts with the entropy-mediated strategy to reduce reaction activation energy is regarded as an innovative and effective approach to facilitate efficient heterogeneous catalysis. Accordingly, conformational entropy-driven high-entropy alloys (HEAs) are emerging as a promising candidate to settle the catalytic efficiency limitations of nanozymes, attributed to their versatile active site compositions and synergistic effects. As proof of the high-entropy nanozymes (HEzymes) concept, elaborate PdMoPtCoNi HEA nanowires (NWs) with abundant active sites and tuned electronic structures, exhibiting peroxidase-mimicking activity comparable to that of natural horseradish peroxidase are reported. Density functional theory calculations demonstrate that the enhanced electron abundance of HEA NWs near the Fermi level (EF) is facilitated via the self-complementation effect among the diverse transition metal sites, thereby boosting the electron transfer efficiency at the catalytic interface through the cocktail effect. Subsequently, the HEzymes are integrated with a portable electronic device that utilizes Internet of Things-driven signal conversion and wireless transmission functions for point-of-care diagnosis to validate their applicability in digital biosensing of urinary biomarkers. The proposed HEzymes underscore significant potential in enhancing nanozymes catalysis through tunable electronic structures and synergistic effects, paving the way for reformative advancements in nano-bio analysis.