A single-atom nanozyme with NADH oxidase-like activity and its application

Abstract

This invention relates to a single-atom nanozyme with NADH oxidase-like activity and its applications, belonging to the field of biomedical technology. The invention provides a single-atom nanozyme, which is a nitrogen-doped carbon-supported single-atom rhodium; this nitrogen-doped carbon-supported single-atom rhodium uses molten cyanamide as a solvent and nitrogen source, and polyethylene glycol as a carbon source. This single-atom nanozyme exhibits NADH oxidase-like activity and can efficiently catalyze the oxidation of NADH to NAD. + It exhibits a higher affinity than natural NADH oxidase; this single-atom nanozyme can consume NADH in cancer cells and inhibit H by mimicking NADH oxidase. + The transport of these nanozymes leads to a series of chain reactions, including increased reactive oxygen species, inhibited oxidative phosphorylation, decreased mitochondrial membrane potential, and suppressed ATP synthesis, ultimately inducing apoptosis in cancer cells. Therefore, this single-atom nanozyme shows great promise in the preparation of drugs for cancer treatment.

Description

Technical Field

[0001] This invention relates to a single-atom nanozyme with NADH oxidase-like activity and its applications, belonging to the field of biomedical technology. Background Technology

[0002] Cancer refers to malignant tumors caused by the malignant proliferation of cells. It is invasive, metastatic, and manifests as a persistent growth of a localized mass that destroys normal tissue structure and can metastasize to other parts of the body. The pathogenesis of cancer is usually due to the long-term combined effects of multiple factors, such as chemical, physical, and viral factors, leading to a series of abnormal gene changes. Surgical resection, radiotherapy, and chemotherapy are the basic treatments for cancer; targeted therapy and biotherapy may be added when necessary. To date, chemotherapy and radiotherapy remain the main methods of cancer treatment. However, long-term chemotherapy can lead to a gradual decrease in the patient's sensitivity to drugs, resulting in drug resistance. Radiotherapy also has limitations. For example, some hypoxic cells exist in tumor tissue, which are insensitive to external radiation and thus resist its effects, making them difficult to eliminate. Furthermore, high-dose radiation, while destroying and killing tumor cells, can also damage normal tissues, ultimately causing serious toxic side effects. Therefore, there is an urgent need to develop more cancer treatment drugs based on different mechanisms of action to compensate for the shortcomings of chemotherapy and radiotherapy in clinical cancer treatment.

[0003] Nanozymes are nanomaterials with enzyme-like activity. Compared to natural enzymes, nanozymes offer advantages such as low cost, high stability, and unique chemical and physical properties characteristic of nanomaterials. Single-atom catalysts (SACs) are materials in which a single metal atom is anchored to a support. Due to their well-defined electronic and geometric structures, SACs hold promise as alternatives to natural enzymes by mimicking the catalytic centers of highly evolved natural enzymes. Inspired by SACs, single-atom nanozymes (SAzymes), as novel nanozymes with dispersed single-atom active sites, possess both the nanomaterial properties and catalytic functions of traditional nanozymes and active sites similar to those of natural metalloenzymes, composed of independent metal atoms. Currently, SAzymes have been successfully applied in cancer treatment, and they have been shown to achieve cancer therapy based on different mechanisms of action.

[0004] For example, some SAzymes contain high-order-number atoms and possess strong X-ray absorption capabilities. They can interact with high-energy ionizing radiation to generate radioactive particles, thereby directly or indirectly causing DNA damage within tumor cells and achieving physical radiosensitization of the tumor exterior. Other SAzymes utilize their unique physicochemical properties to generate a series of biochemical reactions with active substances in the tumor microenvironment through redox reactions, acid stress, depletion of endogenous thiol groups, and radiocatalytic effects, thereby regulating the tumor microenvironment and achieving biochemical radiosensitization within the tumor. Therefore, SAzymes hold promise for developing more cancer therapeutics based on different mechanisms of action to compensate for the shortcomings of chemotherapy and radiotherapy in clinical cancer treatment. However, most reported SAzymes are three-dimensional structures, resulting in a large number of active sites being buried internally and unable to fully contact the substrate. This objectively hinders the efficacy of SAzyme-based cancer therapeutics. Reducing the dimensionality of SAzymes to form ultrathin two-dimensional structures with high specific surface area and porosity is a promising strategy, allowing active sites to be fully exposed during the reaction process and accelerating mass transfer. However, at present, there are still no reports of two-dimensional SAzymes that induce apoptosis in cancer cells by mimicking NADH oxidase. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a single-atom nanozyme with NADH oxidase-like activity, wherein the single-atom nanozyme is nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC); the nitrogen-doped carbon-supported single-atom rhodium uses molten cyanamide (NH2CN) as a solvent and nitrogen source, and polyethylene glycol (PEG) as a carbon source.

[0006] In one embodiment of the present invention, the method for preparing the single-atom nanozyme includes: adding polyethylene glycol and rhodium trichloride (RhCl3) to molten cyanamide to obtain a mixed solution; and pyrolyzing the mixed solution under the protection of an inert gas to obtain the single-atom nanozyme.

[0007] In one embodiment of the present invention, the mass ratio of polyethylene glycol, rhodium trichloride and molten cyanamide is 150-250:0.5-2:4000-5000.

[0008] In one embodiment of the present invention, the mass ratio of polyethylene glycol, rhodium trichloride and molten cyanamide is 200:1:4500.

[0009] In one embodiment of the present invention, the method for preparing the single-atom nanozyme includes: heating and melting cyanamide into a liquid at 50-60°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 850-950°C under the protection of an inert gas and continuing to pyrolyze it at 850-950°C for 1-3 hours to obtain the single-atom nanozyme.

[0010] In one embodiment of the present invention, the method for preparing the single-atom nanozyme includes: heating cyanamide to melt it into a liquid at 55°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 900°C under the protection of an inert gas and then pyrolyzing it at 900°C for 2 hours to obtain the single-atom nanozyme.

[0011] In one embodiment of the present invention, the heating rate is 1 to 3 °C / min.

[0012] In one embodiment of the present invention, the heating rate is 2°C / min.

[0013] In one embodiment of the present invention, the stirring speed is 200-300 rpm and the stirring time is 5-20 min.

[0014] In one embodiment of the present invention, the inert gas includes nitrogen.

[0015] The present invention also provides a method for preparing the above-mentioned single-atom nanozyme, the method comprising: adding polyethylene glycol and rhodium trichloride to molten cyanamide to obtain a mixed solution; and pyrolyzing the mixed solution under the protection of an inert gas to obtain the above-mentioned single-atom nanozyme.

[0016] In one embodiment of the present invention, the mass ratio of polyethylene glycol, rhodium trichloride and molten cyanamide is 150-250:0.5-2:4000-5000.

[0017] In one embodiment of the present invention, the mass ratio of polyethylene glycol, rhodium trichloride and molten cyanamide is 200:1:4500.

[0018] In one embodiment of the present invention, the method includes: heating cyanamide to melt it into a liquid at 50-60°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 850-950°C under the protection of an inert gas and then pyrolyzing it at 850-950°C for 1-3 hours to obtain the above-mentioned single-atom nanozyme.

[0019] In one embodiment of the present invention, the method for preparing the single-atom nanozyme includes: heating cyanamide to melt it into a liquid at 55°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 900°C under the protection of an inert gas and then pyrolyzing it at 900°C for 2 hours to obtain the single-atom nanozyme.

[0020] In one embodiment of the present invention, the heating rate is 1 to 3 °C / min.

[0021] In one embodiment of the present invention, the heating rate is 2°C / min.

[0022] In one embodiment of the present invention, the stirring speed is 200-300 rpm and the stirring time is 5-20 min.

[0023] In one embodiment of the present invention, the inert gas includes nitrogen.

[0024] The present invention also provides the application of the above-mentioned single-atom nanozymes in the preparation of drugs for the prevention and / or treatment of cancer.

[0025] In one embodiment of the invention, the prevention and / or treatment of cancer includes consuming NADH within cancer cells by mimicking NADH oxidase, thereby inhibiting H... + The transport of these substances can induce apoptosis in cancer cells.

[0026] In one embodiment of the invention, the prevention and / or treatment of cancer includes consuming NADH within cancer cells by mimicking NADH oxidase, thereby inhibiting H... + The transport of these substances induces a chain reaction, ultimately leading to apoptosis in cancer cells.

[0027] In one embodiment of the present invention, the chain reaction includes an increase in reactive oxygen species, an inhibition of oxidative phosphorylation, a decrease in mitochondrial membrane potential (ΔΨm), and / or a reduction in ATP synthesis.

[0028] In one embodiment of the present invention, the cancer cells include lung cancer cells.

[0029] In one embodiment of the present invention, the drug components further include a drug carrier and / or pharmaceutical excipients.

[0030] In one embodiment of the present invention, the drug carrier comprises nanoparticles.

[0031] In one embodiment of the present invention, the pharmaceutical excipients include propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, stabilizers, flavoring agents, preservatives, and / or suspending agents.

[0032] The present invention also provides a medicament for the prevention and / or treatment of cancer, wherein the medicament comprises the above-mentioned single-atom nanozyme.

[0033] In one embodiment of the invention, the prevention and / or treatment of cancer includes consuming NADH within cancer cells by mimicking NADH oxidase, thereby inhibiting H... + The transport of these substances can induce apoptosis in cancer cells.

[0034] In one embodiment of the invention, the prevention and / or treatment of cancer includes consuming NADH within cancer cells by mimicking NADH oxidase, thereby inhibiting H... + The transport of these substances induces a chain reaction, ultimately leading to apoptosis in cancer cells.

[0035] In one embodiment of the present invention, the chain reaction includes an increase in reactive oxygen species, an inhibition of oxidative phosphorylation, a decrease in mitochondrial membrane potential (ΔΨm), and / or a reduction in ATP synthesis.

[0036] In one embodiment of the present invention, the cancer cells include lung cancer.

[0037] In one embodiment of the present invention, the drug components further include a drug carrier and / or pharmaceutical excipients.

[0038] In one embodiment of the present invention, the drug carrier comprises nanoparticles.

[0039] In one embodiment of the present invention, the pharmaceutical excipients include propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, stabilizers, flavoring agents, preservatives, and / or suspending agents.

[0040] The technical solution of this invention has the following advantages:

[0041] This invention provides a single-atom nanozyme, wherein the single-atom nanozyme is nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC); the nitrogen-doped carbon-supported single-atom rhodium uses molten cyanamide (NH2CN) as the solvent and nitrogen source, and polyethylene glycol (PEG) as the carbon source. Studies have shown that this single-atom nanozyme possesses NADH oxidase-like activity (NADH is a key electron donor in oxidative phosphorylation) and can efficiently catalyze the oxidation of NADH to NAD. + It exhibits a higher affinity than natural NADH oxidase; simultaneously, research shows that this single-atom nanozyme can consume NADH in cancer cells and inhibit H by mimicking NADH oxidase. +The transport of these nanozymes leads to a series of chain reactions, including increased reactive oxygen species, inhibited oxidative phosphorylation, decreased mitochondrial membrane potential (ΔΨm), and suppressed ATP synthesis, ultimately inducing apoptosis in cancer cells. Therefore, this single-atom nanozyme shows great promise in the preparation of drugs for cancer treatment.

[0042] Further, the preparation method of the single-atom nanozyme includes: heating cyanamide to melt it into a liquid at 50-60°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 850-950°C under the protection of an inert gas, and then pyrolyzing it at 850-950°C for 1-3 hours to obtain the single-atom nanozyme. During the pyrolysis process, when the temperature is below 650°C, cyanamide thermally polymerizes into g-C3N4, while PEG is converted into amorphous carbon. Nitrogen atoms in the g-C3N4 layer can anchor rhodium atoms and prevent rhodium atoms from agglomerating into nanoparticles. Upon further increasing the temperature, g-C3N4 decomposes and volatilizes, while rhodium, nitrogen, and carbon are doped onto the carbon substrate formed by PEG carbonization. PEG further graphitizes with increasing temperature, ultimately yielding a nitrogen-doped carbon-supported single-atom rhodium catalyst (Rh1 / NC). This method utilizes cyanamide, which melts at a relatively low temperature (55°C), as both a solvent and a nitrogen source. It eliminates the need for prolonged heating reactions or centrifugal washing, offering the advantage of simple operation. Furthermore, this method does not consume water or organic solvents, making it environmentally friendly. Attached Figure Description

[0043] Figure 1 Schematic diagram of the preparation process of single-atom nanozymes.

[0044] Figure 2 Schematic diagram of the calcination process of single-atom nanozymes.

[0045] Figure 3 Scanning electron microscope image of a single-atom nanozyme.

[0046] Figure 4 HRTEM image of a single-atom nanozyme.

[0047] Figure 5 : Aberration-corrected HAADF-STEM image of a single-atom nanozyme.

[0048] Figure 6 XRD pattern of single-atom nanozymes.

[0049] Figure 7 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and in-situ energy-dispersive X-ray spectroscopy (EDS) elemental mapping images of single-atom nanozymes.

[0050] Figure 8 X-ray photoelectron spectroscopy (XPS) image of a single-atom nanozyme.

[0051] Figure 9 Schematic diagram of the cascade reaction of NADH oxidase.

[0052] Figure 10 : The UV-Vis absorption spectrum of the reaction solution obtained by reacting single-atom nanozymes with NADH.

[0053] Figure 11 The UV-Vis absorption spectrum of the reaction solution obtained by reacting single-atom nanozymes with NADH after adding HRP and TMB.

[0054] Figure 12 : NADH UV-Vis absorption spectra of reaction solutions obtained from the reaction of different nanoparticles with NADH.

[0055] Figure 13 UV-Vis absorption spectra of reaction solutions obtained by reacting different nanoparticles with NADH after adding HRP and TMB.

[0056] Figure 14 Color changes after adding HRP and TMB to the reaction solution obtained from the reaction of different nanoparticles with NADH.

[0057] Figure 15 NADH dehydrogenation energy barriers of different nanoparticles obtained by DFT calculation.

[0058] Figure 16 The change of NADH content in the reaction solution obtained by reacting different single-atom nanozymes with NADH over time.

[0059] Figure 17 Comparison of specific surface areas of different single-atom nanozymes.

[0060] Figure 18 Michaelis curves of single-atom nanozymes.

[0061] Figure 19 Changes in the activity of single-atom nanozymes after being exposed to air for 12 months.

[0062] Figure 20 Changes in A549 cell viability with single-atom nanozyme concentration.

[0063] Figure 21 : Fluorescent image of reactive oxygen species after co-incubation of A549 cells with single-atom nanozymes.

[0064] Figure 22 : Mitochondrial JC-1 staining fluorescence image after A549 cells were co-incubated with single-atom nanozymes.

[0065] Figure 23 Changes in ATP content in A549 cells after co-incubation with single-atom nanozymes. Detailed Implementation

[0066] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0067] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0068] Example 1: Single-atom nanozymes and their preparation method

[0069] This embodiment provides a single-atom nanozyme, wherein the single-atom nanozyme is nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC); the nitrogen-doped carbon-supported single-atom rhodium uses molten monocyanamide (NH2CN) as solvent and nitrogen source, and polyethylene glycol (PEG) as carbon source.

[0070] The preparation method of the single-atom nanozyme is as follows:

[0071] 4g of cyanamide was melted into a liquid at 55℃ to obtain molten cyanamide. 0.2g of polyethylene glycol and 1mg of rhodium trichloride were added to the molten cyanamide, and the mixture was stirred at 250rpm for 10min to obtain a mixed solution. The mixed solution was poured into an alumina crucible, and under nitrogen atmosphere, the temperature was increased to 900℃ at a rate of 2℃ / min, and then pyrolyzed at 900℃ for 2h to obtain carbon-supported single-atom rhodium (Rh1 / NC) (preparation process see...). Figure 1 The calcination process is shown in the figure. Figure 2 ).

[0072] Comparative Example 1: Single-atom nanozymes and their preparation methods

[0073] This comparative example provides a single-atom nanozyme, which is prepared by means of Example 1 without the addition of polyethylene glycol (calcination process see...). Figure 2 ).

[0074] Although cyanamide contains a large amount of carbon, its synthesis still requires an additional carbon source, namely PEG. Figure 2It is known that when PEG is not added during the synthesis process, the g-C3N4 produced by the thermal polymerization of cyanamide will completely volatilize at temperatures above 650℃, thus failing to obtain the corresponding carbon-containing product.

[0075] Experimental Example 1: Characterization of Single-Atom Nanozymes

[0076] This experimental example characterized the single-atom nanozyme in Example 1. The characterization process is as follows:

[0077] The single-atom nanozyme in Example 1 was observed using a scanning electron microscope. The results are shown in [Figure 1]. Figure 3 The single-atom nanozyme in Example 1 was observed using a transmission electron microscope. The results are shown in [Figure 1]. Figure 4 The single-atom nanozyme in Example 1 was observed using a Titan Themis-corrected scanning / transmission electron microscope. The results are shown in [Figure number missing]. Figure 5 The single-atom nanozyme in Example 1 was detected using XRD diffraction, and the results are shown in [Figure 1]. Figure 6 The single-atom nanozyme in Example 1 was detected using a Talos F200X transmission microscope. The detection results are shown in [Figure 1]. Figure 7 The single-atom nanozyme in Example 1 was detected using Thermo Scientific, and the results are shown in [Figure 1]. Figure 8 .

[0078] Scanning electron microscopy images show that the nitrogen-doped carbon-supported rhodium single-atom (Rh1 / NC) synthesized in Example 1 has a wrinkled nanosheet shape. Figure 3 No obvious rhodium nanoparticles were found in the HRTEM images. Figure 4 ), while the aberration-corrected HAADF-STEM images show that rhodium atoms are dispersed in isolated single-atom form. Figure 5 Meanwhile, the XRD pattern did not reveal the presence of rhodium nanoparticles. Figure 6 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and in-situ energy-dispersive X-ray spectroscopy (EDS) elemental mapping images show that rhodium, nitrogen, and carbon are uniformly distributed on the nanosheets. Figure 7 XPS further confirmed the presence of rhodium. Figure 8 The rhodium loading was found to be approximately 1.18 wt% by inductively coupled plasma atomic emission spectrometry.

[0079] Experimental Example 1: NADH oxidase-like activity of single-atom nanozymes

[0080] Experiment 1: Verification of NADH-like oxidase activity

[0081] NADH and NAD +It is a coenzyme for many oxidoreductases. NADH donates electrons and protons and is oxidized to NAD+ in oxidase-catalyzed reactions. + NAD + As an electron and proton acceptor, NADH participates in dehydrogenase-catalyzed reactions and is reduced to NADH. Therefore, the catalytic oxidation of NADH is of great significance for various cascade reactions (see details). Figure 9 Since the absorption peak at 340 nm disappears after NADH is oxidized, the degree of NADH oxidation can be measured by measuring the change in absorption at 340 nm. Based on this principle, this experimental example verifies the NADH-like oxidase activity of the single-atom nanozyme in Example 1. The verification process is as follows:

[0082] Using no Rh1 / NC as a control, NADH (purchased from Genview) was prepared into a 0.1 mM NADH solution using PBS buffer (pH = 7.4, concentration 0.01 M). Nitrogen-doped carbon-supported rhodium (Rh1 / NC) synthesized in Example 1 was added to 1 mL of the NADH solution until the substrate concentration reached 25 μg / mL. The mixture was then reacted at 25 °C for 20 min to obtain the reaction mixture. The absorption peak of the reaction mixture at 340 nm was detected using a UV-Vis spectrophotometer. The results are shown in [Figure 1]. Figure 10 .

[0083] like Figure 10 As shown, after mixing NADH with the nitrogen-doped carbon-supported rhodium single-atom (Rh1 / NC) synthesized in Example 1 and reacting for 20 min, the characteristic absorption peak at 340 nm disappeared, proving that the nitrogen-doped carbon-supported rhodium single-atom (Rh1 / NC) synthesized in Example 1 has NADH oxidase-like activity.

[0084] Experiment 2: Further Verification of NADH-like Oxidase Activity

[0085] In NADH oxidation-catalyzed reactions, oxygen, the oxidant, can be reduced to H₂O via a four-electron pathway or to H₂O₂ via a two-electron pathway. The resulting H₂O₂ can be further converted into free radicals for oxidizing various substrates. Therefore, using NADH as an electron donor to catalyze the reduction of oxygen to H₂O₂ is of great significance. This experimental example uses a colorimetric method to detect whether the single-atom nanozyme in Example 1 can catalyze the reduction of oxygen to H₂O₂ using NADH as an electron donor to further verify its NADH-like oxidase activity. The verification process is as follows:

[0086] Horseradish peroxidase (HRP) was prepared into a 0.1 mg / mL solution using PBS buffer; 3,3',5,5'-tetramethylbenzidine (TMB) was prepared into a 20 mM solution using a DMSO / EtOH mixed solvent (DMSO:EtOH = 1:9, v / v); 20 μL of HRP solution and 20 μL of TMB solution were added to the reaction mixture of NADH and Rh1 / NC obtained in Experiment 1, and the characteristic absorption peak of the blue substance TMBox in the reaction product at 652 nm was immediately detected using a UV-Vis spectrophotometer. The detection results are shown in [Figure 1]. Figure 11 .

[0087] like Figure 11 As shown, after adding horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) to the reaction mixture of NADH and Rh1 / NC, a characteristic absorption peak of the blue substance TMBox at 652 nm was detected; no absorption peak was detected in the control group containing only NADH. This demonstrates that the nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC) synthesized in Example 1 catalyzes the oxidation of NADH.

[0088] Experiment 3: Verification of the active site of NADH-like oxidase

[0089] Based on Example 1, without the addition of rhodium salt, nitrogen-doped carbon (NC) without Rh was obtained. Using nitrogen-doped carbon (NC) without Rh and Rh nanoparticles (Rh NPs, purchased from Aladdin) as controls, the NADH-like oxidase activity of different nanoparticles was detected according to the methods in Experiments 1 and 2. The experimental results are shown in [Figure 1]. Figures 12-14 .

[0090] like Figure 12 and Figure 13 As shown, comparative experiments revealed that NC and RhNPs exhibited almost no NADH oxidase-like activity. The color change under macroscopic conditions further confirmed this result. Figure 14 (The blue bottle corresponds to Rh1 / NC). Therefore, the highly efficient catalytic ability of the nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC) synthesized in Example 1 mainly comes from the Rh atoms coordinated with N, i.e., the RhNx sites.

[0091] Experiment 4: Further Validation of the Active Site of NADH-like Oxidases

[0092] Building upon Experiment 3, density functional theory (DFT) calculations using DMol3 software were employed to reveal the dehydrogenation of NADH in both Rh1 / NC and RhNPs structures. The energy changes of the NADH dehydrogenation process in RhNPs (Rh clusters) and Rh1 / NC (RhN4) are shown below. Figure 15 As shown, the main difference lies in the following: For Rh NPs, NAD* is adsorbed in a bridging configuration, where the oxygen atom and the pyridine ring are each attached to an Rh atom. This structure is relatively stable, making NAD* difficult to dissociate. For Rh1 / NC, only one carbon atom adsorbs NAD*, making dissociation easier and thus thermodynamically promoting this dehydrogenation process. This result further confirms that the highly efficient catalytic ability of the nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC) synthesized in Example 1 mainly originates from the Rh atom coordinated with N, i.e., the RhNx site.

[0093] Comparative Example 2: Single-atom nanozymes and their preparation methods

[0094] This comparative example provides a single-atom nanozyme, which is a nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC-urea); the nitrogen-doped carbon-supported single-atom rhodium uses molten urea as a solvent and nitrogen source, and polyethylene glycol (PEG) as a carbon source.

[0095] The preparation method of the single-atom nanozyme is as follows:

[0096] 5g of urea was heated to 150℃ and melted into liquid to obtain molten urea. 0.2g of polyethylene glycol and 1mg of rhodium trichloride were added to the molten urea and stirred at 250rpm for 10min to obtain a mixed solution. The mixed solution was poured into an alumina crucible and heated to 900℃ at a rate of 2℃ / min under nitrogen atmosphere protection. The solution was then pyrolyzed at 900℃ for 2h to obtain carbon-supported single-atom rhodium (Rh1 / NC-urea).

[0097] Using the nitrogen-doped carbon-supported rhodium single-atom (Rh1 / NC) synthesized in Example 1 as a control, the NADH oxidase-like activity of the nitrogen-doped carbon-supported rhodium single-atom (Rh1 / NC-urea) was verified according to the method of Experimental Example 1. The specific surface area of ​​the carbon-supported rhodium single-atom (Rh1 / NC-urea) was detected by a specific surface area and porosity analyzer. The detection results are shown in the figure below. Figure 16 and Figure 17 .

[0098] like Figure 16 As shown, Rh1 / NC-urea also possesses NADH-like oxidase activity, but its activity is weaker than that of the cyanamide-derived single-atom rhodium nanozyme (Rh1 / NC). Figure 17 As shown, the specific surface area of ​​Rh1 / NC-urea is 161.4338 m². 2 The specific surface area of ​​Rh1 / NC is 334.0283 m² / g. 2Based on this result, it is speculated that the weaker activity of Rh1 / NC-urea NADH oxidase compared to Rh1 / NC may be related to the higher specific surface area of ​​the cyanamide-derived single-atom rhodium nanozyme.

[0099] Experimental Example 2: Kinetics and Stability of Single-Atom Nanozymes

[0100] Experiment 1: Dynamics Study

[0101] Using natural NADH oxidase (purchased from Genview) as a control, NADH was prepared into a 10 mM NADH solution using PBS buffer (pH = 7.4, concentration 0.01 M). Nitrogen-doped carbon-supported rhodium (Rh1 / NC) synthesized in Example 1 was added to 1 mL of NADH solution to substrate concentrations of 25, 50, 75, 100, 150, 200, and 250 μM, respectively, and the reaction was carried out at 25 °C to obtain the reaction mixture. During the reaction, the reaction mixture was sampled every 20 min, and the NADH oxidation rate under different conditions was detected using a UV-Vis spectrophotometer. Michaelis-Menten curves of the NADH oxidation reaction were plotted based on the measured NADH oxidation rates under different conditions, and the reaction was analyzed using the Michaelis equation V = (V... max ×C) / (K m +C) The maximum reaction rate (V) of the nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC) synthesized in Example 1 was calculated. max ) and Michaelis-Menten constant (K m ).

[0102] The obtained Mie curve is shown below. Figure 18 The calculated maximum reaction rate (V) max ) and Michaelis-Menten constant (K m The values ​​were 21.9 μM min. -1 And 81.1 μM, K of substrate Rh1 / NC m The value is lower than that of natural NADH oxidase (124 μM), indicating that the substrate Rh1 / NC has a higher affinity for NADH.

[0103] Experiment 2: Verification of Stability

[0104] Using the newly synthesized Rh1 / NC as a control, the nitrogen-doped carbon-supported rhodium single-atom (Rh1 / NC) synthesized in Example 1 was stored in air for 12 months. Following the method in Experiment 1, its catalytic activity was detected by comparing the oxidation rate of NADH (i.e., the change in the absorption peak at 340 nm). The results are shown in [Figure 1]. Figure 19 .

[0105] like Figure 19As shown, compared with the catalytic activity of the newly synthesized Rh1 / NC, the catalytic activity of Rh1 / NC after continuous storage in air for 12 months did not decrease significantly. It can be seen that the nitrogen-doped carbon-supported single-atom rhodium (Rh1 / NC) synthesized in Example 1 has excellent stability and its catalytic activity did not decrease significantly after storage in air for 12 months.

[0106] Experimental Example 3: The activity of single-atom nanozymes in inducing apoptosis in cancer cells

[0107] This experimental example validated the apoptosis-inducing activity of the single-atom nanozyme in Example 1. The validation process is as follows:

[0108] A549 lung cancer cells (purchased from Shangen Biotechnology) were used at a rate of 1×10⁻⁶. 4 Inoculate each well with 200 μL of DMEM medium (gibco) containing 10% (v / v) fetal bovine serum (FBS) into 96-well plates and incubate for 24 h at 5% (v / v) CO2 and 37°C. After 24 h of incubation, set up a blank control group and an experimental group in the 96-well plates, with 6 replicates in each group. After setting up the experimental group, add different concentrations (5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL) to the wells of the experimental group. Nitrogen-doped carbon-supported rhodium (Rh1 / NC) synthesized in Example 1 (with concentrations of L, 60 μg / mL, 80 μg / mL, and 100 μg / mL, each concentration treated as a separate group) and a blank control group (0 μg / mL without Rh1 / NC) were co-incubated for 24 h in a 5% (v / v) CO2, 37°C cell culture incubator. After 24 h of co-incubation, cell viability, intracellular ROS levels, changes in intracellular mitochondrial membrane potential, and intracellular ATP content were measured in each group of A549 lung cancer cells.

[0109] Cell viability was determined as follows: 100 μL of CCK8 reagent (purchased from Beyotime) was added to each well of a 96-well plate and incubated at 37°C in the dark for 0.5 h. After incubation, the OD values ​​of each group were measured at 450 nm using a microplate reader. The cell viability was calculated using the formula: Cell viability (%) = [(Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value)] × 100%. The calculation results are shown below. Figure 20 ;

[0110] Intracellular ROS levels were detected as follows: 100 μL of DCFH-DA reagent (purchased from Beyotime) was added to each well of a 96-well plate, and the plates were incubated at 37°C for 10 min for staining. After staining, the fluorescence intensity of each group was measured under a microscope, and the intracellular ROS level was determined based on the fluorescence intensity. The experimental results are shown below. Figure 21 ;

[0111] The changes in intracellular mitochondrial membrane potential were detected as follows: JC-1 reagent (purchased from Beyotime) was added to each well of a 96-well plate at a concentration of 100 μL, and the plates were incubated at 37°C for 10 min for staining. After staining, the intensity of red and green fluorescence in each group was measured under a microscope, and the changes in intracellular mitochondrial membrane potential were obtained based on the relative intensity of red and green fluorescence. The experimental results are shown in [Figure number missing]. Figure 22 ;

[0112] Intracellular ATP levels were detected using an ATP assay kit (ATPAssay Kit, purchased from Beyotime). The experimental results are shown below. Figure 23 .

[0113] like Figure 20 As shown, after treating A549 lung cancer cells with 100 μg / mL Rh1 / NC nanozyme, only 20% of the cell viability was retained. This indicates that the nitrogen-doped carbon-supported rhodium (Rh1 / NC) synthesized in Example 1 can induce apoptosis in cancer cells.

[0114] Rh1 / NC nanozymes not only consume intracellular NADH but also generate large amounts of H2O2 during the catalytic oxidation of NADH, thus promoting intracellular redox imbalance. Figure 21 As shown, after incubation with Rh1 / NC nanozymes, the green fluorescence of DCFH-DA in A549 cells was significantly enhanced, indicating that NADH consumption can generate a large number of reactive oxygen species. Using the JC-1 fluorescent probe to test changes in mitochondrial membrane potential, JC-1 molecules can remain in monomeric form (J-monomers) and exhibit green fluorescence in mitochondria with low membrane potential, while in mitochondria with high membrane potential, they spontaneously assemble into aggregates with red fluorescence (J-aggregates). In the control group A549 cells, the JC-1 dye exhibits typical red fluorescence due to the high membrane potential. Figure 22 As shown, JC-1 exhibited green fluorescence in cells treated with Rh1 / NC nanozymes, indicating a decrease in mitochondrial membrane potential after treatment. This decrease in mitochondrial membrane potential signifies that H... + It is difficult for H to move from the mitochondrial matrix to the intermembrane space due to the presence of H in the intermembrane space. + At low concentrations, ATP synthesis is inhibited. Figure 23 This verifies the phenomenon. These results indicate that Rh1 / NC nanozymes can catalyze the consumption of NADH in cancer cells and inhibit H... + The transport of these substances induces a series of chain reactions, including an increase in reactive oxygen species, impaired oxidative phosphorylation, a decrease in mitochondrial membrane potential (ΔΨm), and inhibition of ATP synthesis, thereby promoting apoptosis in cancer cells.

[0115] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A single-atom nanozyme with NADH oxidase-like activity, characterized in that, The single-atom nanozyme is a nitrogen-doped carbon-supported single-atom rhodium; the nitrogen-doped carbon-supported single-atom rhodium uses molten cyanamide as a solvent and nitrogen source, and polyethylene glycol as a carbon source. The method for preparing the single-atom nanozyme includes: heating and melting cyanamide into a liquid at 50-60°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 850-950°C under the protection of an inert gas and then pyrolyzing it at 850-950°C for 1-3 hours to obtain the single-atom nanozyme; the mass ratio of polyethylene glycol, rhodium trichloride and molten cyanamide is 150-250:0.5-2:4000-5000.

2. The single-atom nanozyme according to claim 1, characterized in that, The heating rate is 1–3 °C / min.

3. A method for preparing the single-atom nanozyme according to claim 1 or 2, characterized in that, The method includes: heating cyanamide to melt it into a liquid at 50-60°C to obtain molten cyanamide; adding polyethylene glycol and rhodium trichloride to the molten cyanamide and stirring to obtain a mixed solution; heating the mixed solution to 850-950°C under the protection of an inert gas and then pyrolyzing it at 850-950°C for 1-3 hours to obtain the single-atom nanozyme according to claim 1 or 2; wherein the mass ratio of polyethylene glycol, rhodium trichloride and molten cyanamide is 150-250:0.5-2:4000-5000.

4. The method as described in claim 3, characterized in that, The heating rate is 1–3 °C / min.

5. The use of the single-atom nanozyme according to claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of lung cancer.

6. A drug for the prevention and / or treatment of lung cancer, characterized in that, The drug comprises a single-atom nanozyme as described in claim 1 or 2.

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