Monatomic nano-enzyme as well as preparation and application thereof

Abstract

The invention relates to a monatomic nano-enzyme and preparation and application thereof, the monatomic nano-enzyme comprises alkaline earth metal monoatoms loaded on a carbon material, alkaline earth metal is calcium, magnesium or barium, and the carbon material is a nitrogen-doped carbon material and can be selected from an imidazolate framework structure material (ZIF), carbon fibers, carbon nanotubes, graphene, carbon black and reduced graphene oxide. The invention also provides a method for preparing the monatomic nanoenzyme, and a tumor catalytic treatment method using the monatomic nanoenzyme. Effective catalytic treatment can be provided for glioma and other intractable tumors, and side effects are extremely small.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozymes, and particularly to a single-atom nanozyme. Background Art

[0002] Nanozymes are artificial enzymes engineered at the nanoscale. They mimic the functions of natural enzymes and can catalyze specific chemical reactions. Nanozymes possess unique properties such as high stability, reusability, and enhanced catalytic activity, making them promising for various applications in fields such as medicine, energy, and environmental remediation. They offer potential advantages over natural enzymes, including easier synthesis, modification, and control of their properties.

[0003] Nanozymes exhibit various biological activities due to their unique properties and catalytic capabilities.

[0004] Some biological activities of nanozymes include biocatalysis. Nanozymes can catalyze specific biochemical reactions similar to natural enzymes. They can accelerate chemical reactions, convert substrates into desired products, and facilitate complex biological processes. In drug delivery, nanozymes can be used as carriers for targeted drug delivery. They can encapsulate therapeutic agents and release them at specific sites in the body, thereby enhancing drug efficacy and reducing side effects. Utilizing biosensing, nanozymes can be used in biosensors to detect and quantify biomolecules or analytes. They can catalyze reactions that generate detectable signals, enabling sensitive and selective detection of various substances. In bioimaging, nanozymes can be used in bioimaging techniques to visualize specific biological processes or structures. They can generate signals or contrast agents that enhance imaging resolution and sensitivity. For tissue engineering, nanozymes can play a role in tissue engineering by promoting cell growth, differentiation, and tissue regeneration. They can create a favorable microenvironment for cell adhesion, proliferation, and tissue formation.

[0005] The development of nanozymes is accompanied by several challenges and difficulties. Designing and synthesizing nanozymes with the desired catalytic properties can be complex. It requires in-depth knowledge of the target reaction, substrate specificity, and the required catalytic mechanism. Achieving optimal catalytic activity and stability while maintaining biocompatibility can be challenging. Ensuring the scalability and reproducibility of nanozyme synthesis is crucial for their practical applications. It may be difficult to maintain consistent catalytic performance in different batches or scales of production. To address this challenge, synthesis methods and quality control measures must be standardized. Nanozymes need to exhibit long-term stability and durability to be effective in various applications. They should be resistant to degradation, maintain their catalytic activity under different environmental conditions, and withstand potential interactions with biological systems. Nanozymes for biomedical applications must be biocompatible and non-toxic to ensure their safety. Understanding the potential interactions between nanozymes and biological systems, including cells and tissues, is crucial for minimizing any adverse effects. To enable nanozymes to accurately perform their intended functions, achieving high target specificity and selectivity is necessary. Ensuring that nanozymes only catalyze the desired reaction without interfering with other biological processes can be challenging. The development and commercialization of nanozymes may need to comply with regulatory frameworks. Meeting the necessary safety and efficacy standards and regulatory environment can be time-consuming and resource-intensive.

[0006] Overcoming these difficulties requires interdisciplinary collaboration, advanced characterization techniques, and continuous research efforts to optimize the design, synthesis, and application of nanozymes.

[0007] The present invention seeks to provide a new or otherwise improved nanozyme. Summary of the Invention

[0008] Embodiments of the present invention relate to single-atom nanozymes comprising alkaline earth metal single atoms loaded on a carbon material, wherein the alkaline earth metal is selected from the group consisting of calcium, magnesium, barium, and combinations thereof, wherein the carbon material is a nitrogen-doped carbon material and is selected from the group consisting of zeolitic imidazolate framework materials (ZIFs), ZIF-8, ZIF-67, carbon fibers, carbon nanotubes, graphene, carbon black, reduced graphene oxide, and combinations thereof.

[0009] Without wishing to be bound by theory, the nanozymes according to the present invention can act as immune adjuvants, stimulating cytokine production and promoting the polarization of tumor-associated macrophages to reverse the immunosuppressive microenvironment. These suggest that the nanozymes according to the present invention hold promise as artificial enzymes capable of overcoming the challenges of glioblastoma and other immunosuppressive tumors and providing new treatment methods.

[0010] Embodiments of the present invention relate to a method for preparing the single-atom nanozymes, which comprises the following steps: (A)Providing a carbon material selected from the group consisting of zeolitic imidazolate framework materials (ZIFs), carbon fibers, carbon nanotubes, graphene, carbon black, reduced graphene oxide, and combinations thereof, and (B)Mixing the nitrogen-doped carbon material with an alkaline earth metal source and stirring at room temperature to provide a precursor mixture, the alkaline earth metal source comprising an alkaline earth metal selected from the group consisting of calcium, magnesium, barium, and combinations thereof; (C)Subjecting the precursor mixture to a pyrolysis process to provide the single-atom nanozyme.

[0011] Without wishing to be bound by theory, the preparation method according to the present invention can provide nanozymes with appropriate particle sizes. For example, the nanozymes according to the present invention can have a particle size in the range of about 50 nm to about 100 nm, which is smaller than the particle sizes of previously reported nanoparticles. For example, the nanoparticles previously reported by the inventors of the present invention (Lee et al., US18 / 825,151, City University of Hong Kong) generally have a particle size in the range of about 200 nm to about 300 nm. Without wishing to be bound by theory, this small particle size is beneficial for delivering the nanozyme across the blood-brain barrier (BBB).

[0012] Embodiments of the present invention relate to tumor catalytic therapy, which includes administering to a subject in need a nanozyme comprising a single-atom alkaline earth metal loaded on a nitrogen-doped carbon material, wherein the alkaline earth metal is selected from the group consisting of calcium, magnesium, barium, and combinations thereof, and the carbon material is selected from the group consisting of zeolitic imidazolate framework materials (ZIFs), carbon fibers, carbon nanotubes, graphene, carbon black, reduced graphene oxide, and combinations thereof.

[0013] Without wishing to be bound by theory, the tumor catalytic therapy according to the present invention can provide effective catalytic treatment for gliomas and other refractory tumors with minimal side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will now be described more specifically by way of example with reference to the accompanying drawings, in which:

[0015] Figure 1 is a schematic diagram of the preparation of Ca-SAzyme and its anti-tumor effect;

[0016] Figure 2A shows the HR-TEM image of ZIF-8;

[0017] Figure 2B shows the SEM image of ZIF-8;

[0018] Figure 2C shows the HR-TEM image of Ca / ZIF-8;

[0019] Figure 2DShows the XRD patterns of NC and Ca-SAzyme according to an embodiment of the present invention;

[0020] Figure 3A Shows the HR-TEM image of Ca-SAzyme according to an embodiment of the present invention;

[0021] Figure 3B Shows the HAADF-STEM image of Ca-SAzyme and the corresponding elemental maps (EDS) of carbon (C), nitrogen (N), and calcium (Ca) in Ca-SAzyme according to an embodiment of the present invention;

[0022] Figure 3C Shows the HAADF-STEM image of Ca-SAzyme according to an embodiment of the present invention;

[0023] Figure 3D Shows the electron energy loss spectroscopy (EELS) spectrum of the atomic sites of Ca-SAzyme according to an embodiment of the present invention;

[0024] Figure 3E Shows the nitrogen adsorption data of Ca-SAzyme according to an embodiment of the present invention;

[0025] Figure 3F Shows the pore size distribution diagram of Ca-SAzyme according to an embodiment of the present invention;

[0026] Figure 3G Shows the transmission spectra of NC and Ca-SAzyme according to an embodiment of the present invention;

[0027] Figure 3H Shows the X-ray photoelectron (XPS) full spectrum of Ca-SAzyme according to an embodiment of the present invention;

[0028] Figure 3I Shows the N1s high-resolution XPS spectrum of Ca-SAzyme according to an embodiment of the present invention;

[0029] Figure 3J Shows the Ca 2p high-resolution XPS spectrum of Ca-SAzyme according to an embodiment of the present invention;

[0030] Figure 3K Shows the Ca L-edge X-ray absorption near-edge structure of Ca-SAzyme according to an embodiment of the present invention;

[0031] Figure 3LShows the C K-edge X-ray absorption near-edge structure of Ca-SAzyme according to an embodiment of the present invention;

[0032] Figure 3M Shows the N K-edge X-ray absorption near-edge structure of Ca-SAzyme according to an embodiment of the present invention;

[0033] Figure 4A Shows the Ca K-edge of the XANES curves of CaO and Ca-SAzyme according to an embodiment of the present invention;

[0034] Figure 4B Shows the Ca K-edge FT-EXAFS spectrogram of Ca-SAzyme and CaO in the R-space;

[0035] Figure 4C Shows the Ca K-edge FT-EXAFS spectra of Ca-SAzyme and CaO in the K-space;

[0036] Figure 4D Is the atomic structure simulation diagram of Ca-SAzyme, where the insert is the schematic model of Ca-SAzyme (atom colors: green, Ca; dark gray, C; blue, N);

[0037] Figure 4E Shows the WT analysis results of CaO;

[0038] Figure 4F Shows the WT analysis of Ca-SAzyme according to an embodiment of the present invention;

[0039] Figure 5A Shows the graph of different Ca-SAzyme numbers / ratios of the TMB colorimetric reaction catalyzed by Ca-SAzyme according to an embodiment of the present invention;

[0040] Figure 5B Shows the graph of the peroxidase-like activity of Ca-SAzyme at different TMB concentrations;

[0041] Figure 5C Is the graph of the peroxidase-like activity of Ca-SAzyme at different H2O2 concentrations;

[0042] Figure 5D Shows the reaction-time curve graph of the TMB colorimetric reaction catalyzed by Ca-SAzyme;

[0043] Figure 5E Shows the reaction–time curve graph of the catalyzed H2O2 colorimetric reaction;

[0044] Figure 5F Shows the specific activity (U mg –1 ) of Ca-SAzyme according to one embodiment of the present invention;

[0045] Figure 5G Shows a comparison graph of peroxidase-like activities between blank, Ca-SAzyme, ZIF-8, and ZIF-8(Ca 2+ ) by using the TMB liquid substrate system;

[0046] Figure 5H Shows a comparison graph of peroxidase-like activities between Ca-SAzyme, graphitic carbon nitride, and graphene oxide by using the TMB liquid substrate system;

[0047] Figure 5I Shows a graph of the pH-dependent peroxidase-like activity of Ca-SAzyme according to one embodiment of the present invention;

[0048] Figure 5J Shows a graph of the temperature-dependent peroxidase-like activity of Ca-SAzyme according to one embodiment of the present invention;

[0049] Figure 5K Shows a comparison graph of PL intensities between Ca-SAzymes according to one embodiment of the present invention;

[0050] Figure 6A Shows confocal microscopic images of cells treated with Ca-SAzyme (0, 20, 50, 200 μg mL -1 , illustrated as: i, ii, iii, iv), H2O2 (100 μM), and their combinations, using DCFH-DA cell staining, scale bar: 100 μm;

[0051] Figure 6B Shows confocal microscopic images of cells treated with Ca-SAzyme (0, 20, 50, 200 μg mL -1 , illustrated as i, ii, iii, iv), H2O2 (100 μM), and their combinations, using calcein-AM / PI cell staining;

[0052] Figure 6C Shows the results of annexin V / PI analysis of GBM 21 cells treated with Ca-SAzymes (0, 20, 50, 200 μg mL -1 , illustrated as i, ii, iii, iv) without H2O2 treatment for 24 hours;

[0053] Figure 6DShows the GSEA analysis feedback result graph of Ca-SAzymes generating reactive oxygen species according to an embodiment of the present invention: apoptosis;

[0054] Figure 6E Shows the GSEA analysis feedback result graph of Ca-SAzymes generating reactive oxygen species according to an embodiment of the present invention: inflammation;

[0055] Figure 6F Shows the GSEA analysis feedback result graph of Ca-SAzymes generating reactive oxygen species according to an embodiment of the present invention: enrichment;

[0056] Figure 7A Shows the graph of the cytotoxicity of Ca-SAzymes to astrocytes NHA in a weakly acidic environment (pH = 6);

[0057] Figure 7B Shows the graph of the cytotoxicity of Ca-SAzymes to astrocytes NHA in a neutral environment (pH = 7.4);

[0058] Figure 7C Shows the graph of the in vitro killing ability of Ca-SAzymes to breast tumor cells MDA231;

[0059] Figure 7D Shows the graph of the in vitro killing ability of Ca-SAzymes to lung tumor cells NCl-H520;

[0060] Figure 7E Shows the graph of the cytotoxicity of ZIF-8 to GSC 21 (pH = 7.4);

[0061] Figure 7F Shows the graph of the cytotoxicity of ZIF-8 to GSC 21 (pH = 6);

[0062] Figure 8 Shows the Western blot analysis results of calpain-1 and Clv-caspase 3 in GBM 21 cells after different treatments;

[0063] Figure 9 Shows the enriched chord diagram of the KEGG pathway;

[0064] Figure 10 Shows the box plot of mesenchymal transformation activity inferred across 33 cancer types using ssGSEA based on EMTCGs features;

[0065] Figure 11AShows the GBM 21 cell viability assay results of Ca-SAzymes without H2O2 in neutral medium (pH = 7.4);

[0066] Figure 11B Shows the GBM 22 cell viability assay results of Ca-SAzymes without H2O2 in neutral medium (pH = 7.4);

[0067] Figure 11C Shows the viability assay results of cells pre-incubated with Ferrostatin-1 (200 μM) and 3-MA (200 μM), and then treated with Ca-SAzymes under neutral medium conditions: Ca-SAzymes: 200 μg / mL -1 , pH = 7.4. Values are mean ± standard deviation (the experiment was repeated four times). *P < 0.05, **P < 0.01, ***P < 0.001;

[0068] Figure 12 Shows the graph of the cytotoxicity of Ca-SAzymes in different GBM microenvironments (20C = 20 μg / ml Ca-SAzymes, 50C = 50 μg / ml Ca-SAzymes, 200C = 200 μg / ml Ca-SAzymes);

[0069] Figure 13A Shows the PCA of the ROS metabolism ability of Ca-SAzymes in different tumors and different tumor sites;

[0070] Figure 13B Shows the volcano plot of genes down-regulated or up-regulated in the Ca-SAzymes group compared with the control group;

[0071] Figure 13C Shows the circular plot of GO and KEGG analysis according to an embodiment of the present invention;

[0072] Figure 13D Shows the heat map of certain genes of interest in the GO analysis according to an embodiment of the present invention;

[0073] Figure 13E Shows the differential expression plot of cancer-related genes according to an embodiment of the present invention;

[0074] Figure 13F Shows the quantitative analysis plot of TNF-α and IFN-γ mRNA expression based on PCR results;

[0075] Figure 13G Shows the corresponding quantitative analysis plot of NOS2 and IL6 mRNA expression according to PCR results;

[0076] Figure 13H Images of western blot analysis of MRC1 and NOS2 after different treatments are shown;

[0077] Figure 14A A schematic diagram of the anti-tumor treatment timeline of Ca-SAzymes is shown;

[0078] Figure 14B Fluorescence imaging of nude mice bearing glioblastoma scanned after in-situ injection of Ca-SAzymes is shown;

[0079] Figure 14C A graph showing the survival rates of nude mice bearing glioblastoma under different treatments (log-rank test, n = 5);

[0080] Figure 14D A graph showing the time-dependent body weights of nude mice bearing glioblastoma under different treatments (log-rank test, n = 5);

[0081] Figure 14E Hematoxylin and eosin staining and quantification of the relative maximum cross-sectional area of in-situ GBM tumors are shown; Scale bar: 500 μm;

[0082] Figure 14F A graph showing M1 / M2 of C57BL6 mice bearing glioblastoma under different treatments, with error bars representing mean ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001;

[0083] Figure 14G A graph showing CD4+ / Treg of C57BL6 mice bearing glioblastoma under different treatments, with error bars representing mean ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001;

[0084] Figure 14H A graph showing CD8+ / Treg of C57BL6 mice bearing glioblastoma under different treatments, with error bars representing mean ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001;

[0085] Figure 14I Hematoxylin and eosin staining results of multiple organs collected from nude mice at the end of the treatment are shown. Scale bar: 100 μm;

[0086] Figure 15A A graph showing the OH+OH adsorption structure obtained after HOOH adsorption, where the key distances are marked * represents the absorber adsorbed on the surface;

[0087] Figure 15B Figure showing the differential charge density distribution of CaN3-doped graphene and OH+OH adsorption (blue: accepting electrons, red: donating electrons);

[0088] Figure 15C Figure showing the energy profile of the *OH+*OH+*H→*OH+*H2O reaction occurring at the structure of the transition state (TS) adjacent to the curve;

[0089] Figure 15D Figure showing the energy profile of the *OH+*H→*H2O reaction occurring at the structure of the transition state (TS) adjacent to the curve;

[0090] Figure 15E Figure showing the optimized structure of CaN3-doped graphene;

[0091] Figure 15F Energy diagram for the decomposition of HOOH on CaN3-doped graphene;

[0092] Figure 16 Image showing the TEM analysis of mitochondria, where the red arrow in the picture indicates the position of the mitochondria, scale bar: 500 nm; and

[0093] Figure 17A Figure showing the levels of TNF-α (pg / ml) in GBM cultures after different treatments, Ca-SAzyme: 0, 20, 50, 200 μg mL -1 ;

[0094] Figure 17B Figure showing the levels of IFN-γ (pg / ml) in GBM cultures after different treatments, Ca-SAzyme: 0, 20, 50, 200 μg mL -1 ; and

[0095] Figure 17C Figure showing the levels of IL-1β (pg / ml) in GBM cultures after different treatments, Ca-SAzyme: 0, 20, 50, 200 μg mL -1 .

[0096] The accompanying drawings herein are for illustrative purposes only and need not be drawn to scale. Detailed Description

[0097] It should be understood that, unless specifically stated otherwise, the materials, compounds, chemicals, etc. described herein are generally commercially available products and / or industrial standards obtainable from multiple suppliers worldwide.

[0098] Definitions

[0099] As used herein, "nanozyme" refers to a nanomaterial with intrinsic enzyme-like activity that catalyzes the conversion of substrates under physiological conditions by following the same kinetics and mechanisms as natural enzymes.

[0100] As used herein, "alkaline earth metal single-atom nanozyme" refers to a nanoscale particle or cluster composed of single atoms of alkaline earth metals such as strontium (Sr), radium (Ra), calcium, magnesium, and barium.

[0101] As used herein, "glioblastoma", "GBM" refers to glioblastoma multiforme, the most malignant type of glioma.

[0102] As used herein, "therapeutically effective amount" refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes the regression of a disease. Typical features of such regression are an improvement in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or the prevention of injury or disability caused by the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as in clinical trials involving human subjects, in animal model systems that predict efficacy in humans, or by assessing the activity of the agent in in vitro assays.

[0103] As used herein, "refractory microenvironment immunosuppressive tumor" refers to a tumor that has the ability to regulate the phenotype of immune cells surrounding the tumor, especially the conversion of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, including but not limited to breast cancer, glioma, and some gastrointestinal tumors.

[0104] As used herein, the "tumor immune microenvironment (TIME)" is provided by a variety of immune cells and stromal cells, and its heterogeneity is a major contributing factor to tumor metastasis, recurrence, and drug resistance.

[0105] The "immunosuppressive microenvironment" is a microenvironment that the tumor "creates" to inhibit immune effector cells and promote its own growth.

[0106] Embodiments of the present invention relate to single-atom nanozymes comprising single atoms of alkaline earth metals loaded on carbon materials.

[0107] The disadvantages of natural enzymes are high cost, poor stability, low yield, difficulty in preservation, and short half-life, which severely limit the practical application of enzyme therapy as catalytic therapy. Therefore, it is believed that the emergence of nanozymes as tumor catalytic therapies is of great significance. Without wishing to be bound by theory, the nanozymes according to the present invention can act as immune adjuvants, stimulating cytokine production and promoting the polarization of tumor-associated macrophages to reverse the immunosuppressive microenvironment.

[0108] In some embodiments, the single-atom nanozyme comprises an alkaline earth metal selected from calcium, magnesium, barium, and combinations thereof. In some embodiments, the alkaline earth metal is calcium. Without being bound by theory, metals such as Fe, Zn, Pt, Cu, and Co have been explored in SAzymes, but further improvement in economic feasibility and biocompatibility is needed. Calcium (Ca), the fifth most abundant element in the earth's crust, is one of the most economical and biocompatible metals, making it an attractive candidate for catalytic applications. In addition, single-atom alkaline earth metals have not been explored in nanozymes, and the mechanism by which single-atom doping affects catalytic performance remains unclear in the prior art.

[0109] In some embodiments, the single-atom nanozyme comprises a carbon material, which is nitrogen-doped and selected from zeolitic imidazolate framework materials (ZIF), carbon fiber, carbon nanotube, graphene, carbon black, reduced graphene oxide, and combinations thereof. In some embodiments, the single-atom nanozyme comprises a zeolitic imidazolate framework material selected from ZIF-8, ZIF-67, and combinations thereof. More particularly, the zeolitic imidazolate framework material is ZIF-8.

[0110] Without wishing to be bound by theory, it is believed that zeolitic imidazolate framework materials (ZIF), particularly ZIF-8 and ZIF-67, are generally preferred precursors for single-atom nanozymes (SAzymes) due to their unique structures and chemical properties, for at least the following reasons:

[0111] 1. Porous structure with high surface area

[0112] Advantage: ZIF has a highly porous framework with a large surface area, enabling reactants to better access the active sites of the nanozyme.

[0113] Benefit to the nanozyme: This enhances the catalytic efficiency and activity, making ZIF-derived materials highly effective.

[0114] 2. Uniform metal distribution

[0115] Advantage: Metal ions (e.g., Zn in ZIF-8 or Co in ZIF-67) are uniformly distributed within the framework.

[0116] Benefit to the nanozyme: This uniformity helps form a well-dispersed single-atom catalyst after pyrolysis, which is crucial for maximizing catalytic activity.

[0117] 3. Thermal stability

[0118] Advantage: ZIF has high thermal and chemical stability, enabling them to withstand the conditions required for carbonization.

[0119] Benefits for nanozymes: During the transformation process (e.g., pyrolysis), the framework is converted into a stable carbon structure, effectively embedding single atoms.

[0120] 4. Tailoring properties through composition

[0121] ZIF-8 (Zn-based): Tends to form a nitrogen-doped carbon framework upon pyrolysis, which is beneficial for catalytic activity in oxidation and reduction reactions.

[0122] ZIF-67 (Co-based): Generates cobalt-doped carbon with strong activity and high electronic conductivity in redox reactions.

[0123] 5. Nitrogen-rich framework materials

[0124] Advantages: ZIFs are constructed using nitrogen-rich imidazolate linkers.

[0125] Benefits for nanozymes: Upon pyrolysis, nitrogen atoms form stable metal-N-C (metal-nitrogen-carbon) active sites, which are crucial for the catalytic performance of nanozymes.

[0126] 6. Versatility and tunability

[0127] Advantages: ZIFs offer tunability in structure and composition by changing metal ions or linkers.

[0128] Benefits for nanozymes: This flexibility enables researchers to tailor the properties of nanozymes for specific catalytic applications, such as cancer therapy, environmental remediation, or energy conversion.

[0129] 7. Cost-effectiveness and scalability

[0130] Advantages: ZIFs can be synthesized relatively easily on a large scale and are cost-effective.

[0131] Benefits for nanozymes: This allows them to be used in industrial applications and large-scale production.

[0132] 8. Hierarchical structure precursors

[0133] Advantages: ZIF-derived materials can produce hierarchical porous structures after pyrolysis.

[0134] Benefits for nanozymes: A synergistic effect is generated by combining high surface area, mass transfer efficiency, and accessibility of active sites.

[0135] By selecting ZIF-8 or ZIF-67 as precursors, these combined advantages make them efficient and versatile materials for producing high-performance single-atom nanozymes.

[0136] In one embodiment of the present invention, the single-atom nanozyme comprises calcium, which mimics the active site of natural metalloproteinase. In one embodiment of the present invention, ZIF-8 or ZIF-67 is used as the carbon material. In some embodiments, the alkaline earth metal is calcium and the zeolitic imidazolate framework material is ZIF-8.

[0137] In one embodiment of the present invention, the single-atom nanozyme has a particle size in the range of about 50 nm to about 100 nm. For example, the single-atom nanozyme of the present invention may have a particle size of about 60 nm, about 70 nm, about 80 nm, or about 90 nm. Without wishing to be bound by theory, the small size of the nanozyme provides a high surface area to volume ratio, thereby exposing more catalytic active sites. In particular, single-atom nanozymes (SAzymes) provide excellent catalytic stability and tunability in a series of reactions. Without wishing to be bound by theory, the nanozymes prepared according to the present invention can be delivered across the BBB and delivered to the center of gliomas, and thus have high activity. Compared with existing nanozymes, the nanozymes prepared by the method in the present invention have more stable catalytic activity and biosafety.

[0138] In one embodiment of the present invention, the single-atom nanozyme has a CaN3 structure.

[0139] Referring to the accompanying drawings, the present invention discloses an alkaline earth metal single-atom nanozyme material and its application in biomedicine. The alkaline earth metal single-atom nanozyme refers to nanoscale particles or clusters composed of single atoms of alkaline earth metals such as magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), and radium (Ra). Due to the unique electronic and structural properties of single metal atoms, these nanozymes exhibit enzyme-catalytic activities similar to natural enzymes. Without wishing to be bound by theory, the alkaline earth metal single-atom nanozymes of the present invention have high catalytic efficiency, stability, and selectivity. By the method described in the present invention, the nanozymes according to the present invention can be synthesized while precisely controlling their size, composition, and surface properties, thereby obtaining customized catalytic performance. Without wishing to be bound by theory, the nanozymes according to the present invention can be easily integrated into different systems, such as nanomedicine platforms, biosensing, cancer treatment, water purification, and energy storage.

[0140] Embodiments of the present invention relate to a method for preparing the single-atom nanozyme, which comprises the following steps: (A) providing a carbon material selected from the group consisting of zeolitic imidazolate framework materials (ZIFs), carbon fibers, carbon nanotubes, graphene, carbon black, reduced graphene oxide, and combinations thereof, and (B) mixing the nitrogen-doped carbon material with an alkaline earth metal source and stirring at room temperature to provide a precursor mixture, the alkaline earth metal source comprising an alkaline earth metal selected from the group consisting of calcium, magnesium, barium, and combinations thereof; (C) Subject the precursor mixture to a pyrolysis process to provide the single-atom nanozyme.

[0141] In step (B), the alkaline earth metal is absorbed into the solution of the nitrogen-doped carbon material, for example, by using ion exchange. In some examples, the aqueous solution of the alkaline earth metal (e.g., CaCl2, 1 g mL -1 , 500 μL) is slowly added to the ZIF solution. Then, the mixture is vigorously stirred at room temperature to allow the complete absorption of the salt solution to form a precursor, which is then subjected to the pyrolysis process of step (C). For example, CaCl2 is added and absorbed into the ZIF-8 solution to form Ca / ZIF-8, which is then pyrolyzed.

[0142] In step (C), the organic MOF ligand is converted into a nitrogen-doped carbon structure during the pyrolysis process, and the nitrogen-rich porous carbon captures the alkaline earth metal atoms to produce the nanozyme.

[0143] The present invention relates to a method for preparing alkaline earth metal single-atom nanozymes by a host-guest method. Without being bound by theory, the prepared catalyst exhibits specific and efficient peroxidase-like activity. The preparation method of the present invention can synthesize nanozymes with precise control over their size, composition, and surface properties.

[0144] The single-atom nanozyme material utilizes a carbon source and a calcium source by the host-guest method. As a specific example, carbon-based materials such as carbon nanotubes or graphene and a calcium source are used to generate a host-guest system, which is synthesized by stirring at room temperature and calcining at high temperature, where calcium single atoms are loaded on a nitrogen-doped carbon matrix.

[0145] The carbon-based material acts as the host, providing a stable structure and surface for the guest, which is the single-atom catalyst. Referring to Figure 4C , the host-guest method allows precise control over the dispersibility and stability of the single-atom catalyst, improving its catalytic activity. Without wishing to be bound by theory, the carbon-based host material provides a supporting environment for the single-atom catalyst, preventing aggregation and improving its performance. By utilizing a carbon source and a calcium source in the host-guest method, a single-atom nanozyme material with enhanced catalytic performance can be produced.

[0146] In one embodiment of the present invention, the nitrogen-doped carbon material is a zeolitic imidazolate framework structure material (ZIF), which can be selected from ZIF-8, ZIF-67, and combinations thereof, and the ZIF can be prepared by the following steps: (1) Provide a first mixture containing a Zn source; (2) Provide a second mixture containing 2-methylimidazole; and (3) Mix the first mixture and the second mixture by stirring to provide particles of the zeolitic imidazolate framework structure material.

[0147] The first mixture and the second mixture are solutions comprising an organic solvent such as methanol. For example, a Zn source (e.g., 811 mg of zinc nitrate hexahydrate) can be dissolved in methanol (e.g., 50 mL). 2-Methylimidazole (e.g., 1627 mg) can be dissolved in methanol (e.g., 50 mL). The solution of the second mixture is quickly poured into the solution of the first mixture and stirred vigorously for, for example, 1 hour. Then, the resulting particles are separated by centrifugation and washed several times with methanol to provide ZIF powder, which is then dried overnight at 60 °C.

[0148] In some embodiments, the method further comprises introducing Ca 2+ by an ion exchange method and absorbing it into a ZIF solution such as ZIF-8 to form Ca / ZIF-8.

[0149] In one embodiment of the present invention, the precursor mixture is heated to a temperature of about 900 °C to 1010 °C, such as about 905 °C, about 910 °C, at a rate of greater than 0 to about 10 °C / min in an inert atmosphere to carry out a pyrolysis process. For example, the heating rate can be about 1 °C / min, 2 °C / min, 3 °C / min, 4 °C / min or 5 °C / min.

[0150] In one embodiment of the present invention, the method further comprises centrifuging and drying the precursor mixture overnight in vacuo at 65 °C before the pyrolysis process.

[0151] In one embodiment of the present invention, the method further comprises etching with HCl after the pyrolysis process to purify the single-atom nanozyme.

[0152] In one embodiment of the present invention, the zeolitic imidazolate framework structure material used in the method is ZIF-8 or ZIF-67, and the alkaline earth metal source contains Ca.

[0153] In an embodiment of the present invention, the organic ligand of ZIF-8 or ZIF-67 is converted into an N-doped carbon structure, and Ca atoms are captured by the N-doped carbon structure during the pyrolysis process. Without wishing to be bound by theory, this N-doped carbon structure facilitates the easy anchoring of metals.

[0154] In one embodiment of the present invention, the pyrolysis process is carried out under an Ar atmosphere. In some embodiments, Ar gas is supplied at a rate of about 5 mL / min to about 10 mL / min.

[0156] In some embodiments, alkaline earth metal single-atom nanozymes are prepared by a host-guest method using a carbon source and a calcium source. Alkaline earth metals include calcium, magnesium, barium, etc. The carbon material can be ZIF-8, ZIF-67, carbon fiber, carbon nanotube, graphene, carbon black, reduced graphene oxide, etc. By stirring and mixing the carbon material and the alkaline earth metal, for example, the mixing time is greater than 0.1 min. After stirring, the mixed solution is transferred to a sealed container, such as a stainless-steel autoclave lined with polytetrafluoroethylene. The container is heated to above 10 °C at a rate of 4 °C / min and then cooled to room temperature to obtain a gel-like product with a viscosity of about 2.4 - 3.0 dl / g, and the heating rate of the sealed container is greater than 0.1 °C / min. Then the nanocrystals are separated from the gel by centrifugation at above 100 rpm and then washed several times with methanol. The nanocrystals are heated at a rate greater than 0.1 °C / min above 1 °C and maintained at this temperature for greater than 0.1 h in a tube furnace filled with Ar / N2. The obtained product is washed successively with acid and DI water and finally vacuum-dried overnight at 60 °C and above 1 °C to obtain the final product in powder form.

[0157] The calcium single-atom nanozyme material is obtained by a simple method of stirring at room temperature and high-temperature calcination. The enzyme catalytic system constructed using this material can be used for antibacterial and anti-tumor applications. In terms of catalytic activity, the synthesized calcium single-atom catalyst with a well-defined single structure can mimic the active sites of natural metalloproteases by virtue of its atomically dispersed active sites and well-defined structure, thereby improving its catalytic ability as a nanozyme. Single-atom catalysts have great potential in understanding the catalytic mechanism of nanozymes and narrowing the gap between natural enzymes and nanozymes. The introduction of calcium single atoms more effectively enhances the specific adsorption of the single-atom nanozyme catalyst to hydrogen peroxide, thereby selectively improving their activity as peroxidases and narrowing the gap between the specificity of single-atom nanozymes and natural enzymes, which can play a positive role in achieving effective antibacterial and anti-tumor activities. The prepared material shows a record-high rate constant.

[0158] Embodiments of the present invention relate to tumor catalytic therapy, which includes administering to a subject in need a nanozyme comprising a single-atom alkaline earth metal loaded on a nitrogen-doped carbon material, wherein the alkaline earth metal is selected from calcium, magnesium, barium, and combinations thereof, and wherein the carbon material is selected from the group consisting of zeolitic imidazolate framework structure materials (ZIF), carbon fiber, carbon nanotube, graphene, carbon black, reduced graphene oxide, and combinations thereof.

[0159] As is well known, nanozyme-based tumor catalytic therapy is a revolutionary anti-tumor method, in which nanozymes specifically trigger enzymatic activity at the tumor site to generate toxic reactive oxygen species (ROS) for killing tumor cells. ROS are a class of oxygen-containing atoms or atomic groups with unpaired electron groups and chemical activity, including superoxide anion, H2O2, singlet oxygen, hydroxyl radical (·OH), alkane peroxide radical, and lipid peroxidation radical. ROS kill tumor cells by mediating the damage of DNA, proteins, and other chemicals. The high H2O2, hypoxia, and acidic environment in solid tumors can make tumors resistant and less sensitive to radiotherapy, chemotherapy, and photodynamic therapy. Therefore, regulating the balance between hypoxia and ROS may lead to tumor cell death. For nanozymes, generating ROS through peroxidase (POD) and oxidase (OXD) activities can induce oxidative stress to kill tumor cells. Since 2007, it has been reported that Fe3O4 nanoparticles can mimic the activity of natural peroxidase. In addition, hundreds of nanomaterials have been found to mimic the enzymatic activities of POD, OXD, catalase (CAT), glucose oxidase (GOx), glutathione peroxidase (GPx), superoxide dismutase (SOD), and uricase.

[0160] Without wishing to be bound by theory, the tumor catalytic therapy according to the present invention can provide effective catalytic treatment for glioma and other refractory tumors with minimal side effects. The present invention is mainly applied in the fields of antibacterial and anti-tumor, and utilizes the specific and efficient peroxidase-like activity of calcium single-atom nanozymes to construct a highly selective and sensitive colorimetric sensing platform. See Figure 5F , the present invention makes full use of the unique structure and high enzymatic activity of calcium single-atom nanozymes, overcomes the problems of natural enzymes being easily inactivated, having poor stability, and high cost, and effectively improves the antibacterial and anti-tumor capabilities.

[0161] Without wishing to be bound by theory, the engineered Ca-centered single-atom nanozymes (Ca-SAzymes) of the present invention exhibit peroxidase-like catalytic (POD) activity and kinetics comparable to those of the developed natural enzymes. At the GBM site, Ca-SAzymes can generate a large amount of reactive oxygen species (ROS) through POD catalytic activity. This is also supported by DFT calculations, which show high selectivity for ·OH. In addition, as an exogenous Ca donor, Ca-SAzymes cause mitochondrial Ca overload, which further amplifies oxidative stress. More impressively, Ca-SAzymes can act as an immune adjuvant to wake up innate immunity by stimulating tumors to produce cytokines, which promotes the polarization of tumor-associated macrophages and reverses the immunosuppressive microenvironment. Therefore, the designed Ca-SAzymes show great potential for developing artificial enzymes and can overcome the adverse factors of GBM treatment and other refractory microenvironment immunosuppressive tumors.

[0162] In one embodiment of the present invention, the tumors for catalytic therapy are refractory microenvironment immunosuppressive tumors selected from bladder cancer, bone tumors, brain cancer, breast cancer, and combinations thereof.

[0163] In one embodiment of the present invention, the tumor is brain cancer. Specifically, brain cancer can be selected from acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET), other brain-related disorders, schwannoma, and combinations thereof. In some embodiments, brain cancer can be selected from astrocytoma, ependymoma, glioblastoma, oligodendroglioma, medulloblastoma, and combinations thereof. More specifically, the brain cancer is glioblastoma (GBM). Without wishing to be bound by theory, glioblastoma (GBM) is one of the most lethal malignant nerve tumors, with an average survival time of only 15 months after surgical resection, postoperative radiotherapy, and adjuvant chemotherapy. Despite significant efforts, emerging treatment strategies have had limited success in improving the prognosis. The resistance of GBM to treatment is attributed to several intrinsic characteristics, including the heterogeneity of glioblastoma, the uniqueness of the glioma immune microenvironment, the unlimited self-renewal ability of glioma stem cells (GSC), and the resistance of the blood-brain barrier (BBB). Even the complex glioma microenvironment offers a glimmer of hope for tumor treatment. With the exploration of the tumor microenvironment, it has been found that in glioma tissue, intense aerobic glycolysis leads to a large accumulation of lactic acid and mild local intercellular acidosis (pH ~ 6.0). At the same time, a relatively high concentration of H2O2 (about 50 - 100 μM) was found in tumor cells, which is generated by the disproportionation of superoxide anions by superoxide dismutase (SOD). These inherent tumor characteristics of the glioma microenvironment constitute the prerequisite for tumor-specific catalytic therapy, thus laying a solid foundation for the development of new treatment methods for glioma.

[0164] In one embodiment of the present invention, the nanozyme is administered at a therapeutically effective amount of about 2 mg to about 3 mg per kg of body weight.

[0165] In one embodiment of the present invention, the loading percentage of alkaline earth metals in the nanozyme ranges from greater than 0 to about 2.0 wt%. For example, the loading percentage can be about 0.5 wt%, about 0.8 wt%, about 0.9 wt%, about 1.0 wt%, about 1.5 wt%, about 1.8 wt%.

[0166] In one embodiment of the present invention, the nanozyme has peroxidase-like catalytic (POD) activity and kinetics comparable to those of natural enzymes.

[0167] In one embodiment of the present invention, the nanozyme acts as an immune adjuvant to awaken innate immunity by stimulating the tumor to produce cytokines.

[0168] In one embodiment of the present invention, the nanozyme induces apoptosis of tumor cells by increasing ROS and reversing the glioblastoma immune microenvironment.

[0169] In one embodiment of the present invention, the nanozyme acts as an immune adjuvant to awaken innate immunity by stimulating the tumor to produce cytokines, thereby promoting the polarization of tumor-associated macrophages and reversing the immunosuppressive microenvironment.

[0170] In one embodiment of the present invention, the nanozyme acts as an exogenous Ca donor to cause mitochondrial Ca overload, thereby further amplifying the oxidative stress of the tumor. Examples

[0171] 1. Materials

[0172] All reagents in the examples were used directly without further purification. Anhydrous calcium chloride (CaCl2), concentrated sulfuric acid (H2SO4 ≥ 98%), and potassium permanganate (KMnO4) were purchased from Sinopharm Chemical Reagent (Shanghai, China). Graphite powder, sodium nitrate (NaNO3), and 30% hydrogen peroxide (H2O2) were purchased from Macklin Reagent Co., Ltd. Ammonium sulfite monohydrate ((NH4)2SO3·H2O) was purchased from Aladdin Reagent Co., Ltd. Nafion solution was purchased from Alfa Aesar. Deionized water used for all experiments was prepared using an ultrapure water generator.

[0173] 2. Methods

[0174] Characterization

[0175] Transmission electron microscopy (TEM) images, scanning transmission electron microscopy (STEM) images, and selected area energy-dispersive X-ray elemental mapping spectra (EDS) were performed using a JEM-2100F / HR transmission electron microscope at an acceleration voltage of 200 kV. High-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) images were detected using a JEOL JEM-ARM200F microscope, which incorporates a spherical aberration correction system for STEM. X-ray diffraction (XRD) patterns were recorded on a BRUKER-D8 X-ray diffractometer using Cu Kα radiation (0.15418 nm). Raman spectra were performed using a Lab RAM high-resolution (HR) evolution Raman spectrometer operating at 514 nm. X-ray photoelectron spectroscopy (XPS) analysis was performed using an ESCALAB250 spectrometer equipped with a monochromatized Al Kα (1486.6 eV) source. The measured spectra were recorded at 0.5 eV increments with a pass energy of 140 eV. Detailed scans were recorded at 0.1 eV increments with a pass energy of 140 eV. The elemental spectra were all calibrated with a C1s peak at 284.8 eV. Elemental analysis was performed using an inductively coupled plasma optical emission spectrometer (ICP-OES) Optima 8000. Fluorescence spectroscopy was performed using a fluorescence detector (RF-10A, Shimadzu, Japan). Time-resolved photoluminescence (TRPL) decay spectra were recorded at 475 nm with excitation at 375 nm using an Edinburgh FLS9800. Fourier transform infrared (FT-IR) spectra were recorded in the 500 - 4000 cm -1 wavenumber range using a Nicolet iS10 (Thermo Fisher, USA) infrared spectrometer with a DTGS detector. X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) spectra were performed in transmission mode with beamline XAFCA at the Singapore Synchrotron Light Source (SSLS). The electron storage ring of the SSLS was operated at a maximum current of 200 mA at 700 MeV. A Si(111) double crystal was used to obtain a monochromatic X-ray beam.

[0176] DFT method

[0177] All spin-polarized density functional theory (DFT) calculations were performed using first principles in the generalized gradient approximation (GGA) with the Perdew - Burke - Ernzerhof (PBE) formulation. The projector augmented wave (PAW) potentials were chosen to describe the ionic cores, and a plane-wave basis set with a kinetic energy cutoff of 450 eV was used to account for the valence electrons. Gaussian smearing was used to allow for partial occupation of the Kohn - Sham orbitals with a width of 0.05 eV. When the energy change was less than 10 -5At eV, the electron energy is considered self - consistent. When the energy change is less than , the geometric optimization is considered convergent. The vacuum distance perpendicular to the structural plane is Brillouin zone integration uses a 3×3×1 Monkhorst - Pack k - point sampling method to sample the structure. Finally, the adsorption energy (Eads) is calculated by the formula Eads = Ead / sub - Ead - Esub, where Ead / sub, Ead, and Esub are the total energies of the optimized adsorbent / support system, the adsorbent in the structure, and the clean support, respectively. The free energy is calculated by the formula: G = E+ZPE - TS, where G, E, ZPE, and TS refer to the free energy, the total energy obtained from DFT calculations, the zero - point energy, and the entropy contribution, respectively.

[0178] GSC separation and cell culture

[0179] Briefly, the samples were cut into small pieces, digested into single cells with Accutase (Sigma), and the red blood cells were lysed with red blood cell lysis buffer (Solarbio). Then the cell suspension was passed through a 70 - μm stainless - steel mesh and recultured in serum - free stem cell medium. Primary glioma spheres (GSC21 and GSC22) were cultured in DMEM / F - 12 medium (Gibco) containing 2% (v / v) B27 supplement (Gibco), epidermal growth factor (EGF, 20 ng / ml, Peprotech), basic fibroblast growth factor (bFGF, 20 ng / ml, Peprotech), and heparin (2.5 μg / ml, Sigma). Only early - passage GSC cells were used for the study. Thp - 1 was purchased from the National Certification Cell Culture Collection Center (NCACC, Shanghai, China) and cultured in RPMI1640 (Gibco) containing 10% FBS and 1% penicillin / streptomycin. All the above cells were cultured at 37 °C and 5% CO2.

[0180] Cytotoxicity

[0181] Cytotoxicity was analyzed using the MTS kit and flow cytometry. Briefly, GSC21 and GSC22 cells (glioma stem cells from surgically resected tissues of two patients) were seeded at 5×10 per well 3Cells were seeded at a density in 96-well plates and cultured in 100 μl of medium for 24 hours before adding Ca-SAzymes. Wells with cells treated only with medium were defined as 100% survival, while blank wells with medium were defined as 0% survival. After incubation at 37 °C for different times, the supernatant was removed, and 100 μl of medium and 10 μl of MTS solution were added to each well. After further culturing in the incubator for 2 hours, the absorbance at 490 nm was measured to calculate the survival rate in different environments. For the study of the killing mechanism of Ca-SAzymes on glioma stem cells, GSC21 cells were co-incubated with Fer-1 and MA, and the cell survival rate was equally measured by the MTS method. For flow cytometry analysis, the procedure was similar to the above except that the cells were cultured in 6-well plates and co-incubated with Ca-SAzymes for 48 hours. At the end of the co-incubation, the cells were treated with annexin V / PI for flow cytometry analysis.

[0182] Intracellular ROS measurement

[0183] The fluorescent probe DCFH-DA was used to measure the intracellular production of ROS induced by Ca-SAzymes. GSC21 cells were seeded in 6-well plates with laminin (1×10 6 cells per dish) for adherent growth. Then, in the absence or presence of 100 μM H2O2, GSC21 cells were incubated with different concentrations of Ca-SAzyme for 24 hours. Subsequently, the medium was replaced with fresh medium containing DCFH-DA (10 μM) for 20 min, and then washed with PBS buffer. Finally, the ROS fluorescence signal was observed by fluorescence microscopy. Conditions for DCFH-DA cell staining: Ca-SAzymes: 0, 20, 50, 200 μg mL -1 (corresponding to i, ii, iii, iv in Figure 6A in turn), H2O2: 100 μM, pH = 7.4.

[0184] Western blot

[0185] GBM cells were lysed by vigorous sonication on an ice bath, and protein concentration was determined by Bradford assay. Then, according to the standard protocol, immunoblotting of cell lysates was performed using antibodies against calpain-1 (ab108400, Abcam), Cleaved-caspase 3 (9664, CST), MRC1 (18704-1-AP, Proteintech), NOS2 (AF7281, Beyotime), and GAPDH (60004-1, PTG). The secondary antibody (1:5000) was maintained for an additional 1 hour at room temperature and visualized using the Western blotting luminol reagent (Santa Cruz Biotechnology, CA, USA).

[0186] RNA Extraction and RT-qPCR

[0187] Total RNA was extracted from GSC21 cells and macrophages using Trizol reagent according to the manufacturer's instructions (Invitrogen, USA). mRNA was converted to cDNA using PrimeScrip RT Master mix (RR036A, Takara, Shiga, Japan), and then the expression levels of the analyzed genes were determined using the SYBR Premix ExTaq kit (RR420A, Takara) under amplification conditions. GAPDH was used as a normalization reference gene, and mRNA abundance was quantified by the threshold cycle method. Each reaction was performed in triplicate. The primers are listed in Table 1 below. Table 1. PCR Primers and siRNA Sequences qPCR Primers

[0188] Single-Sample Gene Set Enrichment Analysis (ssGSEA) of ROS Metabolism

[0189] Download the Cancer Genome Atlas (TCGA) transcriptome pan-cancer data (version 2016-12-29) and clinical pan-cancer data (version 2018-09-13) from the Xena website (https: / / xenabrowser.net / ). Thirty-four reactive oxygen species metabolism-related gene ontology biological process signatures (c5.go.bv7.5.1.s1.symbolsrvision) were downloaded from the MSIgDB website (https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp). The ssGSEA method was selected to evaluate the activity of each signature in different TCGA samples using the GSVA R package. The ssGSEA values of GOBP_REACTIVE_OXYGEN_SPECIES_METABOLIC_PROCESS were used as an example, and box plots were used to show the ROS metabolism in different individuals of different cancer types. The t-distributed stochastic neighbor embedding (tSNE) was used to reduce the dimensionality of the 34 ROS-related signature values in the TCGA samples using the Rtsne R package.

[0190] Go analysis

[0191] The DESeq2 R package was used to calculate the differential gene expression (DGE) between the NC group and the Ca-SAzymes group. The ClusterProfiler R package was used for gene ontology (GO) analysis of the DGE between the NC group and the Ca-SAzymes group. The GOplot R package was used to illustrate the results of the GO analysis.

[0192] TEM analysis of mitochondria

[0193] GSC21 cells were first incubated with 50 μg / ml Ca-SAzymes for 24 hours, then fixed in 4% glutaraldehyde after trypsin digestion, rinsed with PBS, and finally centrifuged at 1,200 rpm for 5 min. The collected cells were fixed in 0.1 M PBS containing 2% osmium tetroxide at 4 °C for 1 hour, then dehydrated in ethanol, then dehydrated in acetone, and embedded in LX-112. Ultrathin sections were cut with a Daitome Diamond microtome and observed under a Hitachi transmission electron microscope.

[0194] In vivo anti-cancer evaluation

[0195] Intracranial GBM xenografts were established using immunocompetent female BALB / C mice and C57BL6 (4 - 6 weeks, 18 - 20 g) purchased from Beijing Vital River Laboratory Animal Technology. A total of 2×105 One luciferase-expressing GSC21 cell was intracranially injected into a mouse to generate a tumor-bearing mouse model of GBM. For intratumoral injection studies, three days after tumor implantation, 15 tumor-bearing mice with GSC21 cells were randomly assigned to three groups (n = 5 mice per group). The first group of mice was injected in situ with PBS and ZIF-8, the second group of mice was injected with Ca-SAzymes dissolved in PBS three days after tumor implantation at a dose of 2 mg / kg body weight. The third group of mice was injected in situ with the same Ca-SAzymes starting from day 9. For the group treated with the Ca-SAzymes material, a total of three injections were performed at two-day intervals.

[0196] The body weight of the mice was measured every five days to observe their health status. On day 21, the small animals were imaged in vivo by injecting the substrate. For biosafety analysis, the major organs (heart, liver, spleen, lung, and kidney) were collected after sacrificing the mice for H&E staining, and the brain was collected for H&E staining studies to directly compare tumor sizes. Example 1 Synthesis of Ca-SAzymes

[0197] This example is used to illustrate the preparation of Ca-based nanozymes by atomically capturing Ca (Ca-SAzyme) in nitrogen-doped graphene.

[0198] Specifically, Figure 1 The upper part diagrammatically describes the synthesis of Ca-SAzyme.

[0199] First, zeolitic imidazolate framework material-8 (ZIF-8) was prepared by contacting zinc nitrate hexahydrate with 2-methylimidazole at ambient temperature in the presence of methanol. In a typical synthesis, 811 mg of zinc nitrate hexahydrate was dispersed in 50 mL of methanol solution to provide solution A. Solution B consisted of 1627 mg of 2-methylimidazole dissolved in 50 mL of methanol. After solutions A and B were completely dissolved, B was rapidly poured into the Zn-based solution A and stirred vigorously for 1 hour. The resulting particles were separated from the gel by centrifugation at 4000 rpm and washed four times with methanol. This process was repeated twice. The obtained ZIF-8 powder was dried overnight at 60 °C.

[0200] Then, using the ion exchange method, Ca 2+ was absorbed into the ZIF-8 solution to form Ca / ZIF-8 ( Figure 2C ). Specifically, at room temperature, 0.20 g of ZIF-8 powder was dispersed in water (10 mL) under ultrasound for 5 min. After forming a homogeneous mixture, an aqueous CaCl2 solution (1 g mL -1, 500 μL) was slowly injected into the mixed solution. Then, the mixture was vigorously stirred at room temperature for 1 hour to allow complete absorption of the salt solution. After centrifugation and vacuum drying at 65 °C overnight, the sample was placed in a tubular furnace and pyrolyzed to 900 °C (heating rate 5 °C / min) for 2 hours under an Ar (10 mL / min) atmosphere to obtain Ca-SAzyme. During pyrolysis, the organic MOF ligand was transformed into a nitrogen-doped carbon structure, and the N-rich porous carbon captured Ca atoms, generating Ca-SAzyme.

[0201] High-resolution transmission electron microscopy (HR-TEM) and scanning electron microscopy (SEM) images of ZIF-8 showed a uniform size distribution and a pentagonal morphology ( Figure 2A and Figure 2B ).

[0202] The X-ray diffraction (XRD) pattern of Ca / ZIF-8 was consistent with that of ZIF-8, indicating that the addition of Ca did not affect the crystallization of ZIF-8. The XRD pattern of Ca-SAzyme ( Figure 2D ) was similar to that of nitrogen-doped carbon (NC), and there was no characteristic peak of Ca, indicating poor crystallinity of Ca-SAzyme. Example 2 Characterization of Ca-SAzymes

[0203] Different methods were used to characterize the Ca-SAzymes prepared in Example 1.

[0204] The HR-TEM image of the as-produced Ca-SAzyme showed a polyhedral shape comparable to that of pure ZIF-8, indicating that no Ca-related nanoparticles were found in the material ( Figure 3A ).

[0205] From the aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image, bright spots belonging to isolated Ca atoms distributed on NC could be identified (highlighted by red circles) ( Figure 3C ). The accompanying HAADF-STEM energy-dispersive X-ray spectroscopy (EDS) map ( Figure 3B ) demonstrated that Ca, C, and N were uniformly distributed throughout the matrix. Additionally, the presence of Ca and N components was verified using electron energy loss spectroscopy (EELS). The generation of Ca-Nx bonds was represented by two peaks at approximately 345 eV and 401 eV, respectively, referred to as typical Ca L 2、3 and N K edge ( Figure 3D ). Brunauer–Emmett–Teller (BET) analysis and the corresponding pore size distribution ( Figure 3E and 3F) It is shown that Ca-SAzyme exhibits a porous structure with a large surface area, which is beneficial for exposing the Ca active sites. Figure 3G It indicates that the transmission spectra of Ca-SAzyme and NC are similar.

[0206] The coordination environment and electronic information of Ca single atoms were analyzed by X-ray photoelectron (XPS) and X-ray (XAS) absorption spectra. The measured spectra of the main peak of C and some smaller peaks of N, O, and Ca confirmed the presence of C, N, O, and Ca in Ca-SAzyme ( Figure 3H ). High-resolution N1s spectra showed that the Ca-SAzyme catalyst mainly contains pyridine nitrogen, as well as trace amounts of pyrrole, graphite, and nitrogen oxide ( Figure 3I ). Binding energy peaks at 346.8 eV and 350.3 eV ( Figure 3J ) can be attributed to Ca 2p3 / 2 and Ca2p1 / 2 in the NIST photoelectron spectroscopy database, respectively. The metal-free spectrum of Ca 2p3 / 2 (344.9 eV) indicates that the Ca-cation (shown in the HAADF-STEM image) in Ca-SAzyme is Ca 2+ . The L-edge X-ray absorption near-edge (XANES) structure ( Figure 3K ) can be divided into main peaks at the L3 and L2 edges, which are in complete agreement with HAADF and EELS analyses, respectively. The substances C and N were mainly determined by the fine structure of synchrotron-based (NEXAFS) X-ray absorption. In the C-edge spectrum, three typical resonances were found in the Ca-SAzyme and NC catalysts, at approximately 285.1 eV (π*C═C), 288.5 eV (π*C–N–C), and 293.0 eV (σ*C-C) ( Figure 3L ). In the N K-edge spectrum, three characteristic peaks corresponding to the π*-substance transitions of pyridine N, pyrrole N, and graphite N substances are located at approximately 398.2, 399.1, and 400.5 eV, respectively ( Figure 3M ). Previous studies have shown that substances such as pyridine N or pyrroline N can donate a π electron to the π-conjugated structure, which is generally considered to be the ligand site of the atomically dispersed Ca metal (Sun, J et al., Atomically confined calcium in nitrogen-doped graphene as an efficient heterogeneous catalyst for hydrogen evolution. iScience. 24, 102728 (2021)). In addition, graphite N can affect the electrical and geometric nanostructures of the carbon substrate.

[0207] The Ca K-edge XANES curves of Ca-SAzyme and CaO are shown inFigure 4A 。

[0208] Table 2. EXAFS fitting parameters of various samples at the Ca K-edge (S0 2 = 0.780). a CN, coordination number; b R, distance between the absorbing atom and the backscattering atom; c σ 2 , Debye-Waller factor, used to account for thermal and structural disorder; d ΔE0, inner potential correction; The R factor indicates the goodness of the fit. S0 2 is fixed at 0.780. Fitting range: and and (sample Ca).

[0209] The Ca K-edge XANES spectrum of CaO is used as a calibration reference material because calcium metal is extremely reactive in air. Figure 4A It is described that the Ca-SAzyme XANES adsorption edge position is equal to that of CaO, indicating that the Ca metal atoms in Ca-SAzyme are in the cationic state. The results of the XPS spectrum are consistent with this. For Ca-SAzymes, this visualization is shown separately in Figure 4B and 4C , which can be called the Ca EXAFS R-space and the Ca K-edge EXAFS R-space. Ca-SAzyme has an EXAFS curve different from that of CaO. The Ca-SAzyme R-space diagram shows a strong peak at about , while CaO has two significant bonding features at about and , which are the Ca-O and Ca-O-Ca bonds respectively. The peak of Ca-SAzyme at about is attributed to the formation of the Ca-O bond, indicating that the Ca cation leaves rather than the compound. Atomic structure simulations show that the connected single Ca atoms are located at the defects of NC generated by pyridine-N ( Figure 4D and Figure 2B ). The fitting results reveal that the coordination number (CN) of Ca-N for Ca-SAzyme is 3. This finding is consistent with the DFT simulations, indicating that the single Ca atoms embedded in the pyridine N defects of the carbon are stable. Example 3 Performance of Peroxidase (POD) Activity of Ca-SAzyme

[0210] The product prepared in Example 1 was used to test the POD activity of Ca-SAzyme.

[0211] Method

[0212] Using 3,5,3',5'-tetramethylbenzidine (TMB) as a probe, the peroxidase (POD)-like activity of Ca / NG-SAzyme was evaluated at room temperature. The test was carried out in 0.01 M buffer in the presence of H2O2. All kinetic data were monitored using a Hitachi UV2010 spectrophotometer in time scan mode at 652 nm. To understand the concentration dependence of the POD-like activity of Ca-SAzyme, Ca / NG-SAzyme (different concentrations, 0 - 200 μg / mL) was mixed with various concentrations of H2O2 (1.0 mM), then 1 mM TMB was added, and the final absorbance at 650 nm of the mixture was detected.

[0213] To determine the kinetic parameters, the experiment was carried out in 0.01 M HAc-NaAc buffer containing 50 μg / mL PMCS, 20 mM H2O2 and a series of concentrations of TMB from 0 - 7 mM, or in 2 mM TMB and a series of concentrations of H2O2 from 0 - 100 mM.

[0214] The absorbance of the product was measured within a certain reaction time range. The absorbance data were fitted to a catalytic model through the Michaelis-Menten equation that explains the correlation between the substrate conversion rates of Ca-SAzyme.

[0215] To determine the effects of pH and temperature on the peroxidase activity of Ca-SAzyme, 100 μg Ca-SAzyme, 1 mM H2O2 and 1 mM TMB were added separately to HAc-NaAc buffer solutions with pH from 1.0 to 12.0 and temperature from 10 to 80 °C, with or without PBS buffer. To measure the final absorbance at 650 nm of the mixture, it was monitored using a Hitachi UV2010 spectrophotometer. For comparison, the carbon skeleton (C-N) was doped with nitrogen.

[0216] The generation of hydroxyl radicals was detected using terephthalic acid photoluminescence method. 1 mM H2O2 and 0.5 mM terephthalic acid were incubated in 0.01 M HAc-NaAc buffer (pH 4.0) at 25 °C for 5 min. The mixture was analyzed using a fluorescence spectrophotometer at 410 nm.

[0217] Results

[0218] In the presence of H2O2, the peroxidase (POD) activity of Ca-SAzyme was evaluated in the catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), converting TMB into a blue oxide, and the formation of TMB oxide increased with the concentrations of Ca-SAzyme, TMB, and H2O2( Figures 5A to 5C ). Based on the Michaelis–Menten constant (K M ), the rate of Ca-SAzyme was calculated using the rate of TMB oxidation. For TMB, K M = 0.7 mM and Vmax = 21 μM min -1 , and for H2O2, K M = 1.78 mM and Vmax = 45 μM min -1 ( Figure 5D and 5E ). According to the rate of TMB oxidation when the concentration of Ca-SAzyme changes, Figure 5F the measured specific POD-like activity value (SA) of Ca-SAzyme was shown to be 2.9 U / mg. One nanozyme activity unit (U) is defined as the amount of nanozyme that catalyzes 1 μmol of product per minute. In addition, in qualitative analysis experiments( Figure 5G and Figure 5H ), Ca-SAzyme showed significantly higher peroxidase-like activity than these other carbon materials under the same test reaction conditions for Ca-SAzyme and other carbon materials (graphitic carbon nitride and graphene oxide). More importantly, when Ca 2+ was added to the ZIF-8 solution, there was no effect on the peroxidase-like activity compared to ZIF-8, which supports the view that Ca-SAzyme can be used as a single-atom peroxidase mimic and there are no Ca 2+ nano-particles in the reaction system. Ca-SAzyme exhibited good catalytic activity over a wide temperature range of 10–80 °C and a pH range of 1–12, as Figure 5I and 5J shown, with an optimum temperature of approximately 40 °C and an optimum pH value of approximately 4. In addition, using terephthalic acid (TA) as the ·OH probe, the ·OH production of Ca-SAzyme was evaluated( Figure 5K ). As Figure 5F shown, when Ca-SAzyme was present, the signal intensity increased, confirming that Ca-SAzyme can effectively generate ·OH. Example 4 In Vitro Cytotoxicity and Cellular Distribution of Ca-SAzymes

[0219] In this example, the in vitro cytotoxicity and cellular distribution of Ca-SAzymes prepared according to Example 1 were tested.

[0220] Utilizing the peroxidase-like activity of Ca-SAzymes, Ca-SAzymes can selectively catalyze the production of abundant oxidative substances in glioblastoma-acidic and H2O2 environments ( Figure 6A ), and Ca-SAzymes are used as a therapeutic strategy for glioblastoma cell inhibition ( Figure 6B ) and its effectiveness is evaluated. Figure 6A Images of cells treated with Ca-SAzyme, H2O2, and their combination are shown, with the conditions of DCFH-DA cell staining.

[0221] In the glioblastoma cell viability assay, ZIF-8 without calcium atoms showed limited cytotoxicity. However, when treated with Ca-SAzymes (200 μg ml -1 ), the glioblastoma cells of patient #21 survived only about 25% ( Figure 6C ) after 24 h. To further investigate the multi-level damage caused by Ca-SAzymes, RNA sequencing of glioblastoma cells was performed after treatment with Ca-SAzymes. Through GSEA, ROS- and apoptosis-related genes were also accumulated at the mRNA level based on RNA differences ( Figure 6D and 6E ), which matched the pattern at the cellular level. In addition to the confirmed cellular-level damage, it was also found that the entire glioblastoma immune system was extensively mobilized through GSEA ( Figure 6F ), including cytokines and pathways that activate the immune system and potentially alter tumor fate ( Figures 7A-7F ). In Figures 7A-7F , data are represented as mean ± standard deviation; Student's t-test; * indicates P < 0.05, ** indicates P < 0.01.

[0222] As described in the previous section, the POD-mimicking activity of Ca-SAzymes makes glioblastoma cells prone to severe apoptosis. However, the POD enzyme activity is by no means the only cause of glioblastoma cell death. The initial design was based on the idea that calcium ions can affect mitochondrial homeostasis and lead to programmed glioblastoma to enhance tumor cytotoxicity through the synergistic effect of metal ions and the Fenton reaction. Calpain-1 is a protein associated with calcium enrichment in cells and showed elevated levels after treatment with Ca-SAzymes, demonstrating the enrichment of intracellular calcium ( Figure 8 ). After a series of cascade reactions, it ultimately leads to the high expression of Clv-caspase3 in glioblastoma cells, resulting in the apoptotic pathway and cell destruction. For further mechanistic analysis, the top 10 pathways were constructed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysisFigure 9 ) Genes related to the "TNF signaling pathway", "necroptosis", and some inflammatory pathways were enriched by Ca-SAzymes, indicating that tumor homeostasis might be disrupted by these pathways.

[0223] To investigate the metabolic capacity of glioblastoma, ROS metabolic genes from different cancer patients were integrated, which showed that glioblastoma had a high level of ROS metabolism ( Figure 10 ). The metabolic capacity of ROS was also unequal among patients within the group, which was reflected in the drug sensitivity results, and different patients had different sensitivities to Ca-SAzymes. The in vitro treatment results of patient #21 were significantly better than those of patient #22, indicating that patient #21 might be more suitable for Ca-SAzyme catalytic therapy ( Figure 11A and 11B ). In Figure 11A and 11B , the values were expressed as mean ± s.d (the experiment was repeated four times). *P < 0.05, **P < 0.01, ***P < 0.001. Treatment of human glioblastoma cells and other tumor cells with Ca-SAzymes confirmed concentration-dependent and time-dependent tumor cell death ( Figures 7A-7F and Figure 12 ). To verify the mechanism of glioblastoma cell damage, antioxidants and protein inhibitors were introduced to regulate cell activity. It was found that the oxidative damage caused by Ca-SAzymes could be properly repaired by pre-incubation with α-tocopherol. Since α-tocopherol is lipophilic, it was speculated that in addition to ·OH generated by the Fenton reaction, lipid peroxidation (LPO) was also accompanied by this process ( Figure 11C ). As expected, ferroptosis-1 (a ferroptosis inhibitor) could also prevent cell death caused by Ca-SAzymes, verifying the ferroptosis hypothesis. Example 5 Ca-SAzymes Inhibit Glioblastoma Progression by Activating the Immune Microenvironment

[0224] The glioblastoma progression inhibitory effect of Ca-SAzymes prepared according to Example 1 was tested in this example.

[0225] To identify tumor samples according to the ROS status, unsupervised co-clustering (a mature machine learning method) was used to classify 10,000 samples into 33 cancer types based on the transcriptome expression of 35 ROSCGs. PCA analysis showed that the ROS metabolic capacity of glioblastoma patients was significantly different from that of other cancer patients; the metabolic capacity of central nervous system tumors was also significantly different from that of other system tumors ( Figure 13A)。To further clarify other therapeutic mechanisms of Ca-SAzyme, transcriptome analysis was performed to reveal the messenger RNA (mRNA) changes in GBM21 cells after different treatments. There were 1304 significantly different expressed genes between the Ca-SAzymes group and the control group, involving 1029 upregulated and 275 downregulated mRNAs( Figure 13B )。Based on this difference, gene ontology (GO) analysis and KEGG analysis were performed to elaborate on the biological functions of the altered mRNAs and the corresponding affected pathways( Figure 13C ), demonstrating that the immune response is related to the therapeutic mechanism of Ca-SAzyme, including the tumor necrosis factor (TNF) signaling pathway and the gamma interferon-mediated (IFN-γ) signaling pathway.

[0226] Inspired by the confirmed immune regulation of the GBM microenvironment by the TNF and IFN signaling pathways, related genes rich in the TNF and IFN-γ signaling pathways were further studied( Figure 13D , 13E). It was found that compared with the control group, in the Ca-SAzymes group, a series of inflammatory genes, including the major genes TNFRSF18, ICAM1, CCL2, and VCAM1, had significant expression differences. In addition to high-throughput sequencing, the differential expression of TNF and IFN was identified using mRNA( Figure 13F ), and the results were consistent with the previous ones. Considering that the changes in inflammatory factors are related to the regulation of macrophage fate, the changes in the immune microenvironment after cell polarization at the administration site were confirmed. As expected, with the increase in the concentration of Ca-SAzymes, macrophages gradually polarized towards the inflammatory direction in terms of RNA( Figure 13G ) and protein( Figure 13H ), which can be attributed to the inhibition of the malignant progression of GBM. In Figure 13A –13G, the values are expressed as mean ± s.d (the experiment was repeated four times). *P < 0.05, **P < 0.01, ***P < 0.001. Example 6 In Vivo Tumor Catalytic Therapy of Ca-SAzymes

[0227] The in vivo activity of Ca-SAzymes prepared according to Example 1 was tested in this example.

[0228] Encouraged by the results of in vitro and microenvironment studies, in vivo experiments were conducted to explore the catalytic therapeutic ability of Ca-SAzymes to inhibit the growth of glioblastoma in situ in an intracranial glioblastoma model. Figure 14AShows a schematic diagram of the design and mechanism of action of Ca-SAzymes. Different from the control group that only received PBS containing ZIF-8, the experimental group was divided into the group receiving Ca-SAzymes on day 3 (day 3) and the group receiving Ca-SAzymes on day 9 (day 9). The results showed that compared with the control group after 21 days of conventional administration, glioblastoma growth was significantly inhibited in the mice treated with Ca-SAzymes( Figure 14B ). According to the results of animal imaging, the earlier the administration time, the better the prognosis of the mice may be, which emphasizes again the importance of early administration of drugs for clinical conversion. Corresponding to the previous results, the mice treated with Ca-SAzymes (starting from day 3) reached an 80% survival rate after 50 days, while Ca-SAzymes (starting from day 9) could improve the survival rate to a certain extent( Figure 14C ). During the 50-day treatment period, the body weight of Ca-SAzymes (starting from day 3) did not show obvious changes, suggesting that Ca-SAzymes have no obvious toxicity( Figure 14D ). In contrast, the body weight of untreated mice decreased sharply due to the cachexia depletion of glioblastoma, which further confirmed the success of Ca-SAzymes in catalytic treatment of glioblastoma.

[0229] The immune microenvironment of glioblastoma has always been an important link in the prognosis of glioblastoma, especially because the change of the pro-inflammatory immune environment can inhibit tumor progression. To gain an in-depth understanding of the TME regulation induced by Ca-SAzymes, the tumor-bearing mice (C57BL / 6) with glioblastoma treated with various treatments were euthanized, and the corresponding tumor tissues were analyzed. As expected, a significant increase in the M1 / M2 ratio was observed in the Ca-SAzymes group compared with the control group( Figure 14E ), indicating an improvement in the glioblastoma immune microenvironment. A significant increase in the ratio of CD 4+ T to Treg cells and CD 8+ T to Treg cells was observed in the Ca-SAzymes group, which indicated that the TME was reprogrammed( Figures 14F-14I ). In addition to programmed cell death, the in vivo reprogramming of the peritumoral immunity induced by Ca-SAzymes may be another reason for the shrinkage of solid tumors.

[0230] The biosafety of Ca-SAzymes was further evaluated by pathological analysis of the main organs of healthy mice after intravenous or intratumoral injection of a therapeutic dose of Ca-SAzymes( Figure 14I ). No abnormal organ pathological changes or weight loss were observed in the mice treated with Ca-SAzymes, indicating the safety of Ca-SAzymes treatment in in vivo anti-glioblastoma applications. Example 7 Potential Catalytic Mechanism

[0231] For the DFT calculation, CaN3-doped graphene was constructed in a 5×5 graphene superlattice ( Figure 15A and Figure 15E ). The Ca atom binds to three N and one C atoms, and the bond lengths are about which are higher than the surface The differential charge density distribution reveals that charge migration only exists on the CaN3C part after doping, and there is no charge change in other C atoms, indicating that the CaN3C part is the active site. As Figure 15A shown, before adsorption, the distance of the O-O bond in the free HOOH molecule is Due to the large Coulomb repulsion between the negatively charged O atoms, its net charge is about -1.20|e| ( Figure 15B ), so HOOH dissociates into *OH + *OH simultaneously during adsorption, and the distance between the two O atoms is stretched to However, in the subsequent hydrogenation reaction *OH + *OH + *H → *OH + *H2O ( Figure 15C ), the long distance between the O and H atoms at the TS results in a high energy barrier of 1.94 eV and an endothermic energy of 0.51 eV. After the H2O molecule detaches from the surface, *OH is hydrogenated to *H2O. Although this reaction needs to overcome a smaller energy barrier of 0.72 eV, the endothermic energy of 1.68|e| is much higher, which is also caused by the long distance ( Figure 15D ). The calculation results prove that HOOH dissociates easily due to the high activity of the Ca atom. In addition, the large-sized Ca atom leads to spatial resistance of the high energy barrier and endothermic energy. Thus, *OH can be protected from the subsequent hydrogenation reaction ( Figure 15F ). Therefore, a large amount of OH is released from the surface of CaN3-doped graphene.

[0232] Conclusion

[0233] The present invention develops a microenvironment-responsive single-atom calcium nanozyme for glioblastoma treatment, which induces tumor cell apoptosis by elevating ROS and reversing the glioblastoma immune microenvironment. Ca-SAzymes exhibit high affinity and good activation ability towards H2O2, which is confirmed by experiments and DFT calculations. By inducing the decomposition of H2O2 into ROS, Ca-SAzymes achieve effective killing of tumor cells in vitro. In addition, Ca-SAzymes significantly prolong the survival time of the glioblastoma-bearing mouse model and reverse the glioblastoma immune microenvironment without adverse effects or toxicity. In summary, the present invention provides unprecedented insights into the catalytic treatment and reversal of the immune microenvironment of glioblastoma.

[0234] It should be understood that the above only illustrates and describes examples in which the present invention can be implemented, and modifications and / or changes can be made thereto without departing from the spirit of the present invention.

[0235] It should also be understood that, for clarity, certain features of the present invention are described in the context of separate embodiments, and these features can also be provided in combination in a single embodiment. Conversely, for the sake of brevity, the various features of the present invention described in the context of a single embodiment can also be provided separately, or in any suitable sub-combination.

[0236] All references specifically cited herein are hereby incorporated by reference in their entirety. However, the citation or incorporation of such references does not necessarily imply an admission of their suitability, citable, and / or availability as prior art to the present invention.

Claims

1. A single-atom nanozyme comprising a single alkaline earth metal atom supported on a carbon material, wherein: The alkaline earth metal is selected from the group consisting of calcium, magnesium, barium and combinations thereof, and the carbon material is a nitrogen-doped carbon material and is selected from the group consisting of zeolite imidazolate framework material (ZIF), carbon fiber, carbon nanotube, graphene, carbon black, reduced graphene oxide and combinations thereof.

2. The single-atom nanozyme according to claim 1, wherein: The alkaline earth metal is calcium, which simulates the active site of natural metalloprotease, the carbon material is a zeolite imidazolate framework structure material selected from ZIF-8, ZIF-67 and a combination thereof, and the calcium metal atom in the nanozyme is in a cationic state.

3. The single-atom nanozyme according to claim 1 or 2, having a particle size ranging from about 50 nm to about 100 nm.

4. A method for preparing the single-atom nanozyme according to any one of claims 1 to 3, comprising the following steps: (A) providing a carbon material selected from the group consisting of zeolitic imidazolate framework material (ZIF), carbon fiber, carbon nanotube, graphene, carbon black, reduced graphene oxide and a combination thereof, and (B) mixing a nitrogen-doped carbon material with an alkaline earth metal source comprising an alkaline earth metal selected from the group consisting of calcium, magnesium, barium, and a combination thereof, and stirring at room temperature to provide a precursor mixture; (C) subjecting the precursor mixture to a pyrolysis process to provide the single-atom nanozyme.

5. The method according to claim 4, wherein: The nitrogen-doped carbon material is a zeolite imidazolate framework material, which is prepared by the following steps: (1) providing a first mixture comprising a Zn source; (2) providing a second mixture comprising 2-methylimidazole; and (3) mixing the first mixture and the second mixture by stirring to provide particles of the zeolite imidazolate framework material.

6. The method according to claim 4 or 5, wherein: The pyrolysis process is performed by heating the precursor mixture to a temperature of about 900° C. to about 1010° C. at a rate of greater than 0 to about 10° C. / min in an inert atmosphere.

7. The method according to claim 4 or 5, further comprising drying the precursor mixture by centrifugation and under vacuum at 65°C overnight before the pyrolysis process.

8. The method according to claim 4 or 5, wherein: The zeolite imidazolate framework material is ZIF-8 or ZIF-67, and the alkaline earth metal source comprises calcium.

9. The method according to claim 8, wherein: The organic ligands of ZIF-8 or ZIF-67 are converted into N-doped carbon structures during the pyrolysis process, and calcium atoms are captured by the N-doped carbon structures.

10. The method according to claim 6, wherein: The pyrolysis process was carried out under Ar atmosphere, and Ar gas was supplied at a rate of about 10 mL / min.

11. A use of a single-atom nanozyme according to any one of claims 1 to 3 in the preparation of a drug for tumor catalytic therapy, wherein the single-atom nanozyme comprises an alkaline earth metal single atom supported on a carbon material, wherein: The alkaline earth metal is selected from the group consisting of calcium, magnesium, barium and combinations thereof, and the carbon material is selected from the group consisting of zeolite imidazolate framework material (ZIF), carbon fiber, carbon nanotube, graphene, carbon black, reduced graphene oxide and combinations thereof.

12. The use according to claim 11, wherein The tumor is a refractory microenvironment immunosuppressive tumor selected from the group consisting of bladder cancer, bone tumor, breast cancer, other brain cancer, and combinations thereof.

13. The use according to claim 12, wherein The refractory microenvironment immunosuppressive tumor is selected from a brain cancer in the group consisting of glioblastoma, acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, brain stem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET), other brain-related disorders, schwannoma and combinations thereof.

14. The use according to claim 11, wherein The loading percentage of the alkaline earth metal in the nanozyme is in the range of greater than 0 to about 2.0 wt %.

15. The use according to claim 11, wherein The nanozyme has peroxidase-like catalytic (POD) activity and kinetics comparable to those of natural enzymes.

16. The use according to claim 11, wherein Nanozymes act as immune adjuvants to awaken innate immunity by stimulating tumors to produce cytokines.

17. The use according to claim 11, wherein The nanozyme induced tumor cell apoptosis by increasing ROS and reversing the immune microenvironment of glioblastoma.

18. The use according to claim 11, wherein The nanozyme acts as an immune adjuvant to awaken innate immunity by stimulating tumors to produce cytokines, which promote the polarization of tumor-associated macrophages and reverse the immunosuppressive microenvironment.

19. The use according to claim 11, wherein The nanozyme acts as an exogenous Ca donor to cause mitochondrial Ca overload, further amplifying tumor oxidative stress.

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