A nanozyme for targeted relief of chemotherapy cardiotoxicity and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and discloses a method for preparing Au-Ru nanozyme, comprising the steps of: 1) dissolving HAuCl4 and ruthenium metal salt in a solvent to obtain a metal salt solution; 2) adding NAC and TA to the metal salt solution, mixing uniformly, and obtaining a mixed solution; 3) adding a NaBH4 aqueous solution to the mixed solution, stirring the reaction at room temperature, and then continuing to stir the reaction at 50°C to 70°C; centrifuging after the reaction is completed, and freeze-drying the precipitate to obtain the Au-Ru nanozyme. The present invention also discloses a method for preparing ATBMzyme nanozyme, specifically: adding EDC and NHS to the Au-Ru nanozyme solution, stirring the reaction, and then adding brain natriuretic peptide to the reaction system, and continuing to stir the reaction; centrifuging after the reaction is completed, and freeze-drying the precipitate to obtain the ATBMzyme nanozyme. The Au-Ru nanozyme and ATBMzyme nanozyme prepared by the present invention can be used to prevent, alleviate or / and treat cardiac toxicity and cardiac damage caused by chemotherapeutic drugs, and do not affect the lethality of chemotherapeutic drugs to tumors, and do not affect the functions of other tissues, and have good safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a nanozyme for targeted relief of chemotherapy cardiotoxicity, and a preparation method and application thereof. Background Art

[0002] The anthracycline drug doxorubicin is currently the main component of clinical anti-cancer treatment. However, earlier studies have pointed out that anthracycline chemotherapy drugs can cause heart-related side effects, including arrhythmia and cardiomyopathy, which has become a limiting factor in their clinical application. How to alleviate chemotherapy-induced cardiotoxicity and explore effective intervention measures is urgent.

[0003] There are many hypotheses for anthracycline-induced cardiac toxicity, including changes in cell death pathways and mitochondrial dysfunction. Oxidative stress is an important mechanism for anthracycline-induced cell death. Nanozymes are a class of mimetic enzymes that have both the unique properties of nanomaterials and catalytic functions. Compared with nanomaterials and natural enzymes, they have many advantages and are widely used in clinical practice. In particular, nanozymes that mimic enzymes with redox activity are mainly involved in the regulation of ROS in vivo and in cells. Whether their ROS-inhibiting effect can be used to alleviate chemotherapy-induced cardiotoxicity and how to provide their targeting in the heart requires further research and exploration. Summary of the invention

[0004] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a nanozyme that targets and alleviates chemotherapy cardiotoxicity and a preparation method and application thereof.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a method for preparing Au-Ru nanozyme, comprising the following steps:

[0007] (1) dissolving HAuCl4 and ruthenium metal salt in a solvent to obtain a metal salt solution;

[0008] (2) adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution, mixing them uniformly to obtain a mixed solution;

[0009] (3) adding NaBH4 aqueous solution to the mixed solution, stirring the reaction at room temperature for 0.5 h to 2 h, and then continuing to stir the reaction at 50°C to 70°C for 2 h to 4 h; after the reaction, centrifuging and collecting the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme.

[0010] According to the above-mentioned method for preparing Au-Ru nanozyme, preferably, the molar ratio of HAuCl4 and ruthenium metal salt is (1.5-6):2; more preferably, the molar ratio of HAuCl4 and ruthenium metal salt is 3:2.

[0011] According to the above-mentioned method for preparing Au-Ru nanozyme, preferably, the molar ratio of N-acetylcysteine ​​to HAuCl4 is (1-10):1; more preferably, the molar ratio of N-acetylcysteine ​​to HAuCl4 is 2.7:1.

[0012] According to the above-mentioned method for preparing Au-Ru nanozyme, preferably, the molar ratio of tannic acid to HAuCl4 is (1-10):(1-10); more preferably, the molar ratio of tannic acid to HAuCl4 is 2:5.

[0013] According to the above-mentioned method for preparing Au-Ru nanozyme, preferably, the molar ratio of NaBH4 to HAuCl4 is (1-8):(0.15-0.6); more preferably, the molar ratio of NaBH4 to HAuCl4 is 1.25:0.15.

[0014] According to the above-mentioned method for preparing Au-Ru nanozyme, preferably, the concentration of the NaBH4 aqueous solution is 0.2-0.4 mol / L. More preferably, the concentration of the NaBH4 aqueous solution is 0.2 mol / L.

[0015] According to the above-mentioned method for preparing Au-Ru nanozymes, preferably, before adding the NaBH4 aqueous solution to the mixed solution in step (3), the NaBH4 aqueous solution needs to be pre-cooled to avoid excessive subsequent reaction, and the temperature of the NaBH4 aqueous solution after pre-cooling is 4°C.

[0016] The second aspect of the present invention provides an Au-Ru nanozyme prepared using the preparation method of the Au-Ru nanozyme described in the first aspect.

[0017] The third aspect of the present invention provides a method for preparing an ATBMzyme nanozyme, comprising: dissolving the Au-Ru nanozyme described in the second aspect in water to obtain an Au-Ru nanozyme solution; adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the Au-Ru nanozyme solution, stirring the reaction, and then adding brain natriuretic peptide (ANP) to the reaction system, and continuing to stir the reaction; after the reaction is completed, centrifuging and collecting the precipitate, and freeze-drying the precipitate to obtain the ATBMzyme nanozyme.

[0018] According to the above-mentioned ATBMzyme nanozyme preparation method, preferably, the mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is (1-20):(1-4); more preferably, the mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 2:1.

[0019] According to the above-mentioned method for preparing ATBMzyme nanozyme, preferably, the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is (5-20):(4-15); more preferably, the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 5:2.

[0020] According to the above-mentioned method for preparing ATBMzyme nanozyme, preferably, the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is (0.2-1):(5-50); more preferably, the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:50.

[0021] According to the above-mentioned method for preparing ATBMzyme nanozyme, preferably, after adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the Au-Ru nanozyme solution, the stirring reaction time is 20 to 90 minutes.

[0022] According to the above-mentioned method for preparing ATBMzyme nanozyme, preferably, the stirring reaction time after adding brain natriuretic peptide to the reaction system is 8 to 16 hours.

[0023] The fourth aspect of the present invention provides an ATBMzyme nanozyme prepared using the preparation method described in the third aspect above.

[0024] The fifth aspect of the present invention provides the use of the Au-Ru nanozyme described in the second aspect in the preparation of a drug for preventing, alleviating or / and treating cardiac toxicity caused by chemotherapeutic drugs.

[0025] According to the above application, preferably, the chemotherapy drug is at least one of antitumor antibiotics, antimetabolites, alkylating agents, antitumor hormones, and antitumor plant component drugs. More preferably, the chemotherapy drug is at least one of DOX (adriamycin), 5-FU (5-fluorouracil), cisplatin, cyclophosphamide, tamoxifen, and paclitaxel.

[0026] The sixth aspect of the present invention provides the use of the ATBMzyme nanozyme described in the fourth aspect in the preparation of a drug for preventing, alleviating or / and treating cardiac toxicity caused by chemotherapeutic drugs.

[0027] According to the above application, preferably, the chemotherapy drug is at least one of antitumor antibiotics, antimetabolites, alkylating agents, antitumor hormones, and antitumor plant component drugs. More preferably, the chemotherapy drug is at least one of DOX (adriamycin), 5-FU (5-fluorouracil), cisplatin, cyclophosphamide, tamoxifen, and paclitaxel.

[0028] The seventh aspect of the present invention provides a drug for preventing, alleviating and / or treating cardiac toxicity caused by chemotherapeutic drugs, wherein the drug contains the Au-Ru nanozyme described in the second aspect or the ATBMzyme nanozyme described in the fourth aspect.

[0029] According to the above-mentioned drugs, preferably, the chemotherapy drug is at least one of antitumor antibiotics, antimetabolites, alkylating agents, antitumor hormones, and antitumor plant component drugs. More preferably, the chemotherapy drug is at least one of DOX (adriamycin), 5-FU (5-fluorouracil), cisplatin, cyclophosphamide, tamoxifen, and paclitaxel.

[0030] According to the above-mentioned medicine, preferably, the medicine also contains a pharmaceutically acceptable carrier / excipient.

[0031] Further, the carrier / auxiliary materials include (but are not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as monosyrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds, sodium lauryl sulfate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; wetting agents such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0032] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0033] (1) The present invention uses NAC, TA, metal ions Au and metal ions Ru as ligands, and through the interaction between ligands, these ligands are firmly bound to the supporting material formed by the action of NAC and TA to prepare and synthesize Au-Ru nanozymes. NAC can stabilize the structure between metal ions Au and metal ions Ru, and TA can not only prolong the circulation time of nanozymes in vivo, but also has a cardiac targeting effect. The Au-Ru nanozymes prepared by the present invention have good antioxidant capacity, can eliminate reactive oxygen species (ROS) and reactive nitrogen species (RNS), and have good cardiac targeting, and can be used to prevent, alleviate or / and treat cardiac toxicity and cardiac damage caused by chemotherapy drugs, and do not affect the lethality of chemotherapy drugs to tumors, and at the same time will not affect the functions of other tissues, and have good safety.

[0034] (2) The present invention connects brain natriuretic peptide (ANP) to Au-Ru nanozyme to further increase its cardiac targeting function, uses 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to catalyze and increase the reaction efficiency of synthesizing ATBMzyme, and synthesizes ATBMzyme nanozyme that can target the heart; the ATBMzyme nanozyme has good cardiac targeting, good antioxidant capacity, can eliminate reactive oxygen species (ROS) and reactive nitrogen species (RNS), can be used to prevent, alleviate or / and treat cardiac toxicity and cardiac damage caused by chemotherapy drugs, and does not affect the lethality of chemotherapy drugs to tumors, and does not affect the functions of other tissues, and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the synthesis route of ATBMzyme nanozyme;

[0036] Figure 2 This is the TEM test result of ATBMzyme nanozyme;

[0037] Figure 3 This is the result of the S, Au, and Ru element localization analysis of ATBMzyme nanozyme;

[0038] Figure 4 The Fourier transform infrared spectroscopy detection results of TBMzyme nanozyme, ATBMzyme nanozyme and BMzyme nanozyme are shown;

[0039] Figure 5 This is the XPS test result diagram of ATBMzyme nanozyme, where A is the element detection of Au element and B is the detection of Ru element;

[0040] Figure 6 The XRD test results of TBMzyme nanozyme and ATBMzyme nanozyme are shown;

[0041] Figure 7 These are the nuclear magnetic proton spectra of TBMzyme nanozyme and ATBMzyme nanozyme;

[0042] Figure 8 The results of UV-visible absorption spectrum analysis of TBMzyme nanozyme, ATBMzyme nanozyme and BMzyme nanozyme;

[0043] Fig. 9 The graph is a test result of the catalase (CAT) activity, superoxide dismutase (SOD) activity, ABTS free radical scavenging ratio, and DPPH free radical scavenging ratio of the Au-Ru nanozyme of the present invention; wherein A is the test result of the catalase (CAT) activity; B is the test result of the superoxide dismutase (SOD) activity; C is the test result of the ABTS free radical scavenging ratio; and D is the test result of the DPPH free radical scavenging ratio.

[0044] Fig.10 Radar chart of various enzyme activities of Au-Ru nanozymes;

[0045] Fig.11 Schematic diagram of the surface configurations of different initial, transition and final states of the Au-Ru nanozyme catalase (CAT) catalytic process;

[0046] Fig.12 The results of the enzyme activity and free radical scavenging ability test of ATBMzyme nanozyme are shown in Figure 1, wherein A is the SOD-like enzyme activity of ATBMzyme nanozyme, B is the specific activity of SOD-like enzyme activity of ATBMzyme nanozyme, C is the CAT enzyme activity of ATBMzyme nanozyme, D is the ABTS free radical scavenging ability test of ATBMzyme nanozyme, and E is the DPPH free radical scavenging ability test of ATBMzyme nanozyme;

[0047] Fig.13 Representative images of immunofluorescence staining of the heart, liver, spleen, lung, kidney, and tumor tissues of tumor-bearing mice; TBMzyme represents mice in the cy5.5-TBMzyme group, and ATBMzyme represents mice in the cy5.5-ATBMzyme group; the scale bar is 50 μm;

[0048] Fig.14 Representative images of HE staining of liver and kidney of tumor-bearing mice; Vehicle represents mice in PBS group, TBMzyme represents mice in cy5.5-TBMzyme group, ATBMzyme represents mice in cy5.5-ATBMzyme group, and the scale bar is 50 μm;

[0049] Fig.15The figure shows the test results of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA) and creatinine (CREA) levels in the serum of tumor-bearing mice; Vehicle represents mice in the PBS group, TBMzyme represents mice in the cy5.5-TBMzyme group, and ATBMzyme represents mice in the cy5.5-ATBMzyme group; *** represents p≤0.001 compared with the vehicle group; ## represents p≤0.01 compared with the TBMzyme group;

[0050] Fig.16 Schematic diagram of the drug treatment process of mice in the DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group;

[0051] Fig.17 Representative echocardiographic images of cardiac function and quantitative analysis results of ejection fraction and short-axis shortening rate, scale bar = 300 milliseconds (n = 6); A is a representative echocardiographic image of cardiac function, B is the statistical analysis result of EF value, C is the statistical analysis result of FS value; *** indicates p≤0.001 compared with vehicle group; # indicates p≤0.05 compared with DOX group; ## indicates p≤0.01 compared with DOX group; ### indicates p≤0.001 compared with DOX group;

[0052] Fig.18 The results of the detection of LDH, cTNT and CKMB, markers of cardiac injury and heart failure in mice; ** indicates p≤0.01 compared with the vehicle group; *** indicates p≤0.001 compared with the vehicle group; # indicates p≤0.05 compared with the DOX group; ## indicates p≤0.01 compared with the DOX group; ### indicates p≤0.001 compared with the DOX group;

[0053] Fig.19 The figures show the test results of the effects of TBMzyme nanozyme and ATBMzyme nanozyme on the tumor killing effect of DOX; A is a schematic diagram of the tumor tissue morphology of four groups of mice; B is a curve chart of the change of tumor volume over time in four groups of mice; C is the tumor volume measured when the four groups of mice began to be injected with DOX; D is the tumor volume measured 24 days after the four groups of mice were injected with DOX; E is a statistical chart of the tumor mass weight of four groups of mice; *** indicates p<0.001 compared with the Vehicle group; # indicates p<0.01 compared with the DOX group; ### indicates p<0.001 compared with the DOX group. DETAILED DESCRIPTION

[0054] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0055] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, components and / or combinations thereof.

[0056] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0058] Embodiment 1:

[0059] A method for preparing Au-Ru nanozyme, the specific steps are as follows:

[0060] (1) HAuCl4 (0.15 mmol) and RuCl3·H2O were dissolved in 10 ml of anhydrous methanol to obtain a metal salt solution; wherein the molar ratio of HAuCl4 to RuCl3·H2O was 3:2.

[0061] (2) Adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution prepared in step (1), mixing evenly, and obtaining a mixed solution; wherein the molar ratio of N-acetylcysteine ​​to HAuCl4 is 2.7:1; and the molar ratio of tannic acid to HAuCl4 is 2:5.

[0062] (3) Adding a 4°C precooled NaBH4 aqueous solution to the mixed solution obtained in step (2), stirring the mixture at room temperature for 1 hour, and then continuing to stir at 60°C for 2 hours; after the reaction, centrifuging at 10000 rpm for 10 minutes to collect the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme (Au-Ru nanozyme is also referred to as TBMzyme nanozyme). The concentration of the NaBH4 aqueous solution is 0.2 mol / L, and the molar ratio of NaBH4 to HAuCl4 is 1.25:0.15.

[0063] Embodiment 2:

[0064] A method for preparing Au-Ru nanozyme, the specific steps are as follows:

[0065] (1) HAuCl4 (0.15 mmol) and RuCl3·H2O were dissolved in 10 ml of anhydrous methanol to obtain a metal salt solution, wherein the molar ratio of HAuCl4 to RuCl3·H2O was 1.5:2.

[0066] (2) Adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution prepared in step (1), mixing evenly, and obtaining a mixed solution; wherein the molar ratio of N-acetylcysteine ​​to HAuCl4 is 1:1; and the molar ratio of tannic acid to HAuCl4 is 1:1.

[0067] (3) Adding a 4°C precooled NaBH4 aqueous solution to the mixed solution obtained in step (2), stirring the reaction at room temperature for 1 hour, and then continuing to stir at 50°C for 4 hours; after the reaction is completed, centrifuging at 10000 rpm for 10 minutes to collect the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme (Au-Ru nanozyme is also referred to as TBMzyme nanozyme). The concentration of the NaBH4 aqueous solution is 0.2 mol / L, and the molar ratio of NaBH4 to HAuCl4 is 1:0.15.

[0068] Embodiment 3:

[0069] A method for preparing Au-Ru nanozyme, the specific steps are as follows:

[0070] (1) HAuCl4 (0.15 mmol) and RuCl3·H2O were dissolved in 10 ml of anhydrous methanol to obtain a metal salt solution; wherein the molar ratio of HAuCl4 to RuCl3·H2O was 3:1.

[0071] (2) Adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution prepared in step (1), mixing evenly, and obtaining a mixed solution; wherein the molar ratio of N-acetylcysteine ​​to HAuCl4 is 10:1; and the molar ratio of tannic acid to HAuCl4 is 10:1.

[0072] (3) Adding a 4°C precooled NaBH4 aqueous solution to the mixed solution obtained in step (2), stirring the reaction at room temperature for 1 hour, and then continuing to stir at 60°C for 4 hours; after the reaction is completed, centrifuging at 10000 rpm for 10 minutes to collect the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme (Au-Ru nanozyme is also referred to as TBMzyme nanozyme). The concentration of the NaBH4 aqueous solution is 0.2 mol / L, and the molar ratio of NaBH4 to HAuCl4 is 8:0.6.

[0073] Embodiment 4:

[0074] A method for preparing Au-Ru nanozyme, the specific steps are as follows:

[0075] (1) HAuCl4 (0.15 mmol) and RuCl3·H2O were dissolved in 10 ml of anhydrous methanol to obtain a metal salt solution; wherein the molar ratio of HAuCl4 to RuCl3·H2O was 2:1.

[0076] (2) Adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution prepared in step (1), mixing evenly, and obtaining a mixed solution; wherein the molar ratio of N-acetylcysteine ​​to HAuCl4 is 5:1; and the molar ratio of tannic acid to HAuCl4 is 1:10.

[0077] (3) Adding a 4°C precooled NaBH4 aqueous solution to the mixed solution obtained in step (2), stirring the reaction at room temperature for 1 hour, and then continuing to stir at 60°C for 3 hours; after the reaction is completed, centrifuging at 10000 rpm for 10 minutes to collect the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme (Au-Ru nanozyme is also referred to as TBMzyme nanozyme). The concentration of the NaBH4 aqueous solution is 0.2 mol / L, and the molar ratio of NaBH4 to HAuCl4 is 3:0.4.

[0078] Embodiment 5:

[0079] A method for preparing Au-Ru nanozyme, the specific steps are as follows:

[0080] (1) HAuCl4 (0.15 mmol) and RuCl3·H2O were dissolved in 10 ml of anhydrous methanol to obtain a metal salt solution; wherein the molar ratio of HAuCl4 to RuCl3·H2O was 1:1.

[0081] (2) Adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution prepared in step (1), mixing evenly, and obtaining a mixed solution; wherein the molar ratio of N-acetylcysteine ​​to HAuCl4 is 8:1; and the molar ratio of tannic acid to HAuCl4 is 5:8.

[0082] (3) Adding a 4°C precooled NaBH4 aqueous solution to the mixed solution obtained in step (2), stirring the mixture at room temperature for 1 hour, and then continuing to stir at 60°C for 2 hours; after the reaction, centrifuging at 10000 rpm for 10 minutes to collect the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme (Au-Ru nanozyme is also referred to as TBMzyme nanozyme). The concentration of the NaBH4 aqueous solution is 0.2 mol / L, and the molar ratio of NaBH4 to HAuCl4 is 5:0.4.

[0083] Embodiment 6:

[0084] A method for preparing Au-Ru nanozyme, the specific steps are as follows:

[0085] (1) HAuCl4 (0.15 mmol) and RuCl3·H2O were dissolved in 10 ml of anhydrous methanol to obtain a metal salt solution; wherein the molar ratio of HAuCl4 to RuCl3·H2O was 5:2.

[0086] (2) Adding N-acetylcysteine ​​(NAC) and tannic acid (TA) to the metal salt solution prepared in step (1), mixing evenly, and obtaining a mixed solution; wherein the molar ratio of N-acetylcysteine ​​to HAuCl4 is 2.7:1; and the molar ratio of tannic acid to HAuCl4 is 3:1.

[0087] (3) Adding a 4°C precooled NaBH4 aqueous solution to the mixed solution obtained in step (2), stirring the mixture for 1 hour at room temperature, and then continuing to stir for 2 hours at 60°C; after the reaction, centrifuging at 10000 rpm for 10 minutes to collect the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme (Au-Ru nanozyme is also referred to as TBMzyme nanozyme). The concentration of the NaBH4 aqueous solution is 0.2 mol / L, and the molar ratio of NaBH4 to HAuCl4 is 1:0.6.

[0088] Embodiment 7:

[0089] A method for preparing ATBMzyme nanozyme (such as Figure 1As shown), specifically: 10mAu-Ru nanozyme is dissolved in 2mL of water to obtain an Au-Ru nanozyme solution; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are added to the Au-Ru nanozyme solution, and the reaction is stirred at room temperature for 20min, and then brain natriuretic peptide (ANP) is added to the reaction system, and the stirring reaction is continued for 8h; after the reaction is completed, the precipitate is collected by centrifugation at 10000rpm for 10min, and the precipitate is freeze-dried to obtain ATBMzyme nanozyme. Among them, the mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 2:1; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 5:2; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:50; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 1.

[0090] Embodiment 8:

[0091] The content of Example 8 is substantially the same as that of Example 7, except that:

[0092] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:20; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 1:3; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 0.2::5; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 1.

[0093] Embodiment 9:

[0094] The content of Example 9 is basically the same as that of Example 7, except that:

[0095] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 4:1; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 5:1; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:5; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 1.

[0096] Embodiment 10:

[0097] The content of Example 10 is substantially the same as that of Example 7, except that:

[0098] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:10; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 1:1; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:30; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 2.

[0099] Embodiment 11:

[0100] The content of Example 11 is substantially the same as that of Example 7, except that:

[0101] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 3:10; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 5:4; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:50; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 3.

[0102] Embodiment 12:

[0103] The content of Example 12 is substantially the same as that of Example 7, except that:

[0104] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 2:1; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 5:2; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:20; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 4.

[0105] Embodiment 13:

[0106] The content of Example 13 is substantially the same as that of Example 7, except that:

[0107] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:4; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 6:5; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:25; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 5.

[0108] Embodiment 14:

[0109] The content of Example 14 is substantially the same as that of Example 7, except that:

[0110] The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:1; the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is 4:3; the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is 1:10; the Au-Ru nanozyme is the Au-Ru nanozyme prepared in Example 6.

[0111] (I) Characterization of Au-Ru nanozymes (also known as TBMzyme nanozymes) and ATBMzyme nanozymes

[0112] The Au-Ru nanozyme (also referred to as TBMzyme nanozyme) prepared in Example 1 and the ATBMzyme nanozyme prepared in Example 7 were characterized. At the same time, for comparison, the present invention also prepared BMzyme nanozyme. The preparation method of BMzyme nanozyme is basically the same as that in Example 1, except that: in step (2), N-acetylcysteine ​​(NAC) is added to the metal salt solution prepared in step (1), mixed evenly, and a mixed solution is obtained.

[0113] 1. Morphological characterization of nanozymes:

[0114] The microscopic morphology of ATBMzyme nanozymes was characterized by transmission electron microscopy (TEM). Figure 1 shown.

[0115] Depend on Figure 2 It can be seen that ATBMzyme is in the form of uniform spherical nanoparticles with an average particle size of 3-5nm.

[0116] 2. Element location analysis of TBMzyme nanozymes and ATBMzyme nanozymes:

[0117] Element Mapping was used to analyze the element location of ATBMzyme nanozyme. The results are as follows: Figure 3 shown.

[0118] Depend on Figure 3 It can be seen that ATBMzyme nanozyme contains S, Au, and Ru elements, which proves the successful synthesis of ATBMzyme nanozyme.

[0119] 3. Fourier transform infrared spectroscopy (FTIR) analysis of TBMzyme nanozyme and ATBMzyme nanozyme:

[0120] Fourier transform infrared spectroscopy (FTIR) was performed on TBMzyme nanozyme, ATBMzyme nanozyme, and BMzyme nanozyme, and the results are as follows: Figure 4 shown.

[0121] Depend on Figure 4 It can be seen that ANP was successfully coupled to TBMzyme nanozyme, proving the synthesis of ATBMzyme.

[0122] 4. ATBMzyme nanozyme X-ray photoelectron spectroscopy (XPS) analysis:

[0123] The ATBMzyme nanozyme was tested by X-ray photoelectron spectroscopy (XPS), and the results are as follows Figure 5 shown.

[0124] Depend on Figure 5It can be seen that the high-resolution XPS spectrum of the Au orbital shows a peak corresponding to zero-valent Au with a binding energy of 83.7 eV; in addition, at a binding energy of 285.4 eV, the XPS spectrum of the Ru orbital shows a peak corresponding to Ru 3d5 / 2, further proving the presence of oxidized Ru on the ATBMzyme.

[0125] 5. X-ray diffraction (XRD) analysis of nanozymes:

[0126] XRD test was performed on TBMzyme nanozyme and ATBMzyme nanozyme, and the test results are as follows Figure 6 shown.

[0127] Depend on Figure 6 It can be seen that TBMzyme nanozyme and ATBMzyme exist in an amorphous form.

[0128] 7. Nuclear magnetic resonance analysis of TBMzyme nanozyme and ATBMzyme nanozyme:

[0129] The results of nuclear magnetic resonance detection of TBMzyme nanozyme and ATBMzyme nanozyme are as follows: Figure 7 shown.

[0130] Depend on Figure 7 It can be seen that the characteristic peak of ANP appeared in the ATBMzym spectrum, proving the successful coupling of ANP.

[0131] 8. UV-visible absorption spectrum analysis of TBMzyme nanozyme and ATBMzyme nanozyme:

[0132] The UV-visible absorption spectra of TBMzyme nanozymes and ATBMzyme nanozymes were analyzed, and the results are as follows: Figure 8 shown.

[0133] Depend on Figure 8 It can be seen that BMzyme, TBMzyme nanozymes and ATBMzyme exhibit good stability and solubility uniformity.

[0134] (II) Detection of enzyme activity and free radical scavenging ability of Au-Ru nanozyme (TBMzyme nanozyme)

[0135] Taking the Au-Ru nanozyme prepared in Example 1 as an example, the enzyme activity and free radical scavenging ability were tested; at the same time, in order to compare with the Au-Ru nanozyme prepared by the present invention, the present invention also prepared Au-Ru nanozyme, Au-Fe nanozyme, Au-Mn nanozyme, Au-Cu nanozyme, Ru-Fe nanozyme, Mn-Fe nanozyme, Fe-Cu nanozyme, Cu-Mn nanozyme, Ru-Mn nanozyme, and Ru-Cu nanozyme; wherein the preparation methods of Au-Ru nanozyme, Au-Fe nanozyme, Au-Mn nanozyme, Au-Cu nanozyme, Ru-Fe nanozyme, Mn-Fe nanozyme, Fe-Cu nanozyme, Cu-Mn nanozyme, Ru-Mn nanozyme, and Ru-Cu nanozyme are basically the same as those in Example 1, except that: HAuCl4 and ruthenium metal salt in step (1) are replaced by metal salts corresponding to the metal elements in the nanozyme, respectively.

[0136] 1. Detection of CAT enzyme activity of Au-Ru nanozyme:

[0137] CAT enzyme, also known as catalase, can decompose H2O2 to produce O2 and water. The method for detecting CAT enzyme activity is as follows: the nanozyme is prepared into a 1 mg / mL aqueous solution and dispersed in a volume of 200 ul into a 10 mL aqueous solution containing 0.1 mM hydrogen peroxide (H2O2), and the generation of O2 is tested using a dissolved oxygen instrument.

[0138] CAT enzyme activity test results Fig. 9 As shown in A.

[0139] Depend on Fig. 9 As shown in Figure A, the CAT enzyme activity of Au-Ru nanozyme is the strongest.

[0140] 2. Detection of SOD enzyme activity of Au-Ru nanozyme:

[0141] SOD enzyme, also known as superoxide dismutase, can dismutate superoxide anions to produce H2O2 and H2O. The xanthine oxidase method was used to determine the SOD mimetic enzyme activity of the sample. The specific operation was as follows: the prepared nanoenzyme solution (0.0015625-0.1 mg / mL) was added to a 96-well plate in a volume of 30 μL, followed by the addition of xanthine, pH 7.4 phosphate buffer, cytochrome C, xanthine oxidase working solution and water. Use an enzyme reader to measure the absorbance of each well at 450 nm 1 minute before and after the addition, and calculate the inhibition percentage. The test results are as follows: Fig. 9 As shown in B

[0142] Depend on Fig. 9 As shown in Figure B, the SOD mimetic enzyme activity of RuCu nanozyme is the highest, while the SOD enzyme activity of Au-Ru nanozyme is relatively low.

[0143] 3. Detection of ABTS free radical scavenging activity of Au-Ru nanozymes:

[0144] ABTS can generate a blue-green ABTS free radical with a stable structure after oxidation, and the ABTS free radical has a maximum absorption peak at 735nm. After ABTS reacts with an oxidant, a blue-green ABTS cation free radical (ABTS·+) is generated, which produces a characteristic absorption peak at a wavelength of 734nm. When antioxidants are present, the generation of ABTS·+ will be inhibited, causing the absorption peak at 735nm to decrease, and the degree of decrease in its absorption peak is proportional to the degree of free radical scavenging. Therefore, the present invention uses the ABTS method to detect the free radical scavenging activity of Au-Ru nanozyme. The specific experimental operation is: first, the ABTS solution (7mM) is incubated with potassium persulfate (2.45mM) 5 overnight to activate the ABTS free radical. Add bimetallic nanozymes (Au-Ru nanozyme, Au-Fe nanozyme, Au-Mn nanozyme, Au-Cu nanozyme, Ru-Fe nanozyme, Mn-Fe nanozyme, Fe-Cu nanozyme, Cu-Mn nanozyme, Ru-Mn nanozyme, Ru-Cu nanozyme) with a final concentration of (1.5625-100 μg / mL) to the ABTS free radical solution. Within 6 minutes, measure the time course absorbance of ABTS free radicals at 734 nm. ABTS clearance percentage = (A0-Asample) / A0*100%. A0 is the absorbance of ABTS at 520 nm when no sample is added; Asample is the absorbance of ABTS at 520 nm after adding the sample. The test results are as follows: Fig. 9 As shown in C.

[0145] Depend on Fig. 9 As shown in Figure C, AuRu nanozyme exhibits the highest ability to scavenge ABTS free radicals.

[0146] 4. Detection of DPPH free radical scavenging activity of Au-Ru nanozyme:

[0147] DPPH is a stable free radical that is easily soluble in polar solvents such as methanol and ethanol, and has a maximum absorption peak at 520nm. When an antioxidant is added, DPPH will undergo a fading reaction. The specific operation of the DPPH free radical scavenging activity assay is as follows: dissolve DPPH in anhydrous ethanol and store it away from light, then mix the prepared DPPH solution with different concentrations of bimetallic nanozymes (Au-Ru nanozyme, Au-Fe nanozyme, Au-Mn nanozyme, Au-Cu nanozyme, Ru-Fe nanozyme, Mn-Fe nanozyme, Fe-Cu nanozyme, Cu-Mn nanozyme, Ru-Mn nanozyme, Ru-Cu nanozyme) at (1.5625~100μg / mL). Incubate in the dark at 37°C for 30 minutes, then use an enzyme reader to record the absorbance at 520nm and calculate the scavenged DPPH. The test results are as follows: Fig. 9 As shown in D.

[0148] Depend on Fig. 9 As shown in Figure 5, RuFe exhibits the strongest ability to scavenge DPPH free radicals, while AuRu also has a strong ability to scavenge DPPH free radicals.

[0149] 5. Radar chart analysis of multiple enzyme activities of metal nanozymes

[0150] The enzyme activity and free radical scavenging ability test data of Au-Ru nanozyme, Au-Fe nanozyme, Au-Mn nanozyme, Au-Cu nanozyme, Ru-Fe nanozyme, Mn-Fe nanozyme, Fe-Cu nanozyme, Cu-Mn nanozyme, Ru-Mn nanozyme and Ru-Cu nanozyme were analyzed by multi-type enzyme activity radar chart, and the results are as follows Fig.10 shown.

[0151] Depend on Fig.10 It can be seen that AuRu bimetallic nanozyme has the best synergistic antioxidant activity.

[0152] The surface configuration of the catalytic process of AuRu nanozyme catalyzing the decomposition of H2O is as follows: Fig.11 shown.

[0153] Depend on Fig.11It can be seen that the decomposition of H2O catalyzed by AuRu nanozymes mainly includes two processes: first, the adsorption of the initial OH-; second, the desorption of O2. The energy barrier for O2 desorption is very high, indicating that the O2 produced during the decomposition process on the pure alloy surface easily oxidizes the alloy. This shows that the alloy has a strong reducibility, making the initial generation of O2 challenging. In the actual reaction, small alloy particles are oxidized by H2O2. Only when they are oxidized to a certain extent will their reducibility weaken, making the generation of O2 easier. The energy barrier for the initial OH-adsorption is not particularly high (even lower for the second OH-adsorption). Electron and proton coupling reactions may also directly produce H2O through the Eley-Rideal mechanism.

[0154] The above studies show that AuRu nanozymes have the potential to eliminate reactive oxygen species (ROS) and reactive nitrogen species (RNS). Therefore, AuRu nanozymes were selected as experimental drugs and their antioxidant effects were further verified in vivo.

[0155] (III) Detection of enzyme activity and free radical scavenging ability of ATBMzyme nanozymes

[0156] Taking the ATBMzyme nanozyme prepared in Example 7 as an example, the enzyme activity and free radical scavenging ability were tested.

[0157] 1. Detection of SOD enzyme activity of ATBMzyme nanozyme:

[0158] SOD enzyme, also known as superoxide dismutase, can dismutate superoxide anions to produce H2O2 and H2O. The xanthine oxidase method was used to determine the SOD mimetic enzyme activity of the sample. The specific operation was as follows: the prepared nanoenzyme solution (0.0015625-0.1 mg / mL) was added to a 96-well plate in a volume of 30 μL, followed by the addition of xanthine, pH 7.4 phosphate buffer, cytochrome C, xanthine oxidase working solution and water. Use an enzyme reader to measure the absorbance of each well at 450 nm 1 minute before and after the addition, and calculate the inhibition percentage. The test results are as follows: Fig.12 As shown in A and B

[0159] Depend on Fig.12As shown in Figures A and B, TBMzyme and ATBMzyme exhibited superoxide dismutase (SOD) mimetic enzyme activity in a phosphate buffer at pH 7.4, and ATBMzyme had the highest SOD enzyme activity, which was quantitatively determined to be approximately 913U / mg by theoretical calculation. The SOD enzyme activities of BMzyme (0.01644U / mg) and TBMzyme (0.065U / mg) were relatively low. This suggests that the inclusion of ANP components may provide more active sites and enhance antioxidant capacity without affecting the ability of nanozymes to eliminate hydrogen peroxide. Therefore, ATBMzyme can remove O2· - , degrade H2O2, and promote the production of O2.

[0160] 2. CAT enzyme activity detection of ATBMzyme nanozyme:

[0161] CAT enzyme, also known as catalase, can decompose H2O2 to produce O2 and water. The method for detecting CAT enzyme activity is as follows: the nanozyme is prepared into a 1 mg / mL aqueous solution and dispersed in a volume of 200 ul into a 10 mL aqueous solution containing 0.1 mM hydrogen peroxide (H2O2), and the generation of O2 is tested using a dissolved oxygen instrument.

[0162] CAT enzyme activity test results Fig.12 As shown in C.

[0163] Depend on Fig.12 As shown in Figure C, ATBMzyme nanozyme, TBMzyme nanozyme, and BMzyme nanozyme exhibited hydrogen peroxide CAT mimetic enzyme-like activity in phosphate buffer at pH 7.4.

[0164] 3. Detection of ABTS free radical scavenging activity of ATBMzyme nanozymes:

[0165] ABTS can generate a blue-green ABTS free radical with a stable structure after oxidation, and the ABTS free radical has a maximum absorption peak at 735nm. After ABTS reacts with an oxidant, a blue-green ABTS cation free radical (ABTS·+) is generated, which produces a characteristic absorption peak at a wavelength of 734nm. When an antioxidant is present, the generation of ABTS·+ will be inhibited, causing the absorption peak at 735nm to decrease, and the degree of decrease in its absorption peak is proportional to the degree of free radical scavenging. Therefore, the present invention uses the ABTS method to detect the free radical scavenging activity of nanozymes. The specific experimental operation is: first, the ABTS solution (7mM) is incubated with potassium persulfate (2.45mM) overnight to activate the ABTS free radical. The nanozyme with a final concentration of (1.5625-100μg / mL) is added to the ABTS free radical solution. Within 6 minutes, the time course absorbance of the ABTS free radical at 734nm is measured. ABTS removal percentage = (A0-Asample) / A0*100%. A0 is the absorbance of ABTS at 520nm without adding sample; Asample is the absorbance of ABTS at 520nm after adding sample. Fig.12 As shown in D.

[0166] Depend on Fig.12 As shown in Figure D, ATBMzyme nanozyme, TBMzyme nanozyme, and BMzyme nanozyme significantly inhibited the production of ABTS free radicals, and ATBMzyme showed the strongest scavenging effect. This result shows that ATBMzyme nanozyme, TBMzyme nanozyme, and BMzyme nanozyme can be used to treat diseases related to nitrogen free radicals.

[0167] 4. Detection of DPPH free radical scavenging activity of ATBMzyme nanozyme:

[0168] DPPH is a stable free radical that is easily soluble in polar solvents such as methanol and ethanol, and has a maximum absorption peak at 520nm. When an antioxidant is added, DPPH will undergo a fading reaction. The specific operation of the DPPH free radical scavenging activity assay is as follows: dissolve DPPH in anhydrous ethanol, store it away from light, and then mix the prepared DPPH solution with nanozymes of different concentrations. Incubate in the dark at 37°C for 30 minutes, then use an enzyme reader to record its absorbance at 520nm and calculate the scavenged DPPH. The test results are as follows: Fig.12 As shown in E.

[0169] Depend on Fig.12 As shown in Figure E, ATBMzyme nanozyme and TBMzyme nanozyme significantly inhibited the production of DPPH hydroxyl radicals, and ATBMzyme nanozyme showed the strongest scavenging effect.

[0170] The above enzyme activity and free radical scavenging ability experimental results show that ANP and TA play a synergistic role in jointly regulating the enzyme activity of ATBMzyme nanozyme, and play an important role in the enzyme activity of ATBMzyme nanozyme.

[0171] (IV) In vivo distribution study and safety assessment of TBMzyme and ATBMzyme nanozymes:

[0172] 1. Experimental animals:

[0173] Male BALB / C mice aged 8 weeks were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China) and were adapted for feeding for one week before the start of the study. All mice were housed in a temperature-controlled environment with a 12-h light / dark cycle at 22 ± 2 °C and provided with sterile food and drinking water. All experiments involving animals were performed in accordance with the Guide for the Use and Care of Laboratory Animals, and the study was approved by the Sub-center Animal Care and Use Committee of the National Center for Cardiovascular Diseases (FZX-IACUCC-2024005).

[0174] 2. Construction of tumor-bearing mouse model:

[0175] Eight-week-old BALB / c mice were injected subcutaneously with 8×10 6 4T1 cells were injected into the mouse cage, and small round and protruding vacuoles were visible at the site of cell inoculation. The mouse was placed back into the cage, and after 3-5 days, obvious white masses were seen at the injection site, indicating that the tumor-bearing mouse model was successfully established.

[0176] 3. Grouping and treatment of experimental animals

[0177] Eighteen tumor-bearing mice were divided into PBS group, cy5.5-TBMzyme group and cy5.5-ATBMzyme group, with 6 mice in each group.

[0178] 4. Experimental drugs:

[0179] The experimental drugs are: Cy5.5-labeled ATBMzyme nanozyme (cy5.5-ATBMzyme nanozyme) and Cy5.5-labeled TBMzyme (cy5.5-TBMzyme nanozyme).

[0180] Synthesis of cy5.5-ATBMzyme nanozyme: Weigh 10 mg of TBMzyme / ATBMzyme and disperse it in 10 ml of deionized water, add 1 mg of Cy5.5-NHS ester, stir for 2 hours, dialyze and freeze-dry to obtain Cy5.5-labeled ATBMzyme nanozyme. The cy5.5-TBMzyme nanozyme is the same as the cy5.5-ATBMzyme nanozyme. The preparation method of the cy5.5-ATBMzyme nanozyme solution is as follows: weigh an appropriate amount of cy5.5-ATBMzyme nanozyme freeze-dried powder, add an appropriate amount of deionized water, and use a probe to prepare a 5 mg / mL cy5.5-ATBMzyme nanozyme solution. The preparation method of the cy5.5-TBMzyme nanozyme solution is the same as that of the cy5.5-ATBMzyme nanozyme solution.

[0181] 5. Study on the in vivo distribution of TBMzyme and ATBMzyme nanozymes:

[0182] PBS buffer, cy5.5-ATBMzyme nanozyme solution, and cy5.5-TBMzyme nanozyme solution were injected into the PBS group, cy5.5-TBMzyme group, and cy5.5-ATBMzyme group tumor-bearing mice by tail vein injection, respectively, with an injection dose of 10 mg / kg body weight. 24 hours after injection, the heart, liver, spleen, kidney, lung, and tumor tissues of the mice were obtained for immunofluorescence frozen section staining.

[0183] The experimental results are as follows Fig.13 shown.

[0184] Depend on Fig.13 It can be seen that ATBMzyme nanozyme and TBMzyme nanozyme can be detected in cardiac tissue, but relatively less in other tissues, and ATBMzyme nanozyme is distributed more in cardiac tissue than TBMzyme nanozyme; this result shows that both ATBMzyme nanozyme and TBMzyme nanozyme can target cardiac tissue.

[0185] 6. Biosafety assessment of TBMzyme and ATBMzyme nanozymes:

[0186] PBS buffer, cy5.5-ATBMzyme nanozyme solution, and cy5.5-TBMzyme nanozyme solution were injected into the PBS group, cy5.5-TBMzyme group, and cy5.5-ATBMzyme group tumor-bearing mice by tail vein injection, respectively, with an injection dose of 10 mg / kg body weight. The injection was performed once a week for 4 weeks. After 4 weeks, the liver and kidney tissues of the mice were taken for HE staining; at the same time, the blood of the mice was taken by eyeball blood sampling, and the blood samples were centrifuged at 3000 rpm for 10 minutes. Then, serum was collected from the supernatant, and the levels of alanine aminotransferase (ALT, elabscience, E-BC-K235-M), aspartate aminotransferase (AST, elabscience, E-BC-K236-M), urea (UREA, elabscience, E-BC-K183-M), and creatinine (CREA, elabscience, E-BC-K188-M) in the serum were evaluated using ELISA kits.

[0187] Among them, the HE staining results of mouse liver and kidney groups are as follows Fig.14 shown.

[0188] Depend on Fig.14 It can be seen that there was no significant difference in the morphology of liver and kidney tissues of tumor-bearing mice in the PBS group, cy5.5-TBMzyme group, and cy5.5-ATBMzyme group.

[0189] The results of the detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA) and creatinine (CREA) levels in the serum of tumor-bearing mice were as follows: Fig.15 shown.

[0190] Depend on Fig.15 It can be seen that there was no significant difference in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA) and creatinine (CREA) in the serum of tumor-bearing mice among the PBS group, cy5.5-TBMzyme group and cy5.5-ATBMzyme group, indicating that TBMzyme nanozymes and ATBMzyme nanozymes do not affect the liver and kidney functions of mice.

[0191] The above results show that TBMzyme nanozymes and ATBMzyme nanozymes can target cardiac tissue to exert their effects, and that they will not affect the functions of other tissues and have good safety.

[0192] (V) Effects of TBMzyme and ATBMzyme nanozymes on myocardial damage caused by DOX and the killing effect of DOX on tumors:

[0193] 1. Experimental animals:

[0194] Male BALB / C mice aged 8 weeks were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China) and were adapted for feeding for one week before the start of the study. All mice were housed in a temperature-controlled environment with a 12-h light / dark cycle at 22 ± 2 °C and provided with sterile food and drinking water. All experiments involving animals were performed in accordance with the Guide for the Use and Care of Laboratory Animals, and the study was approved by the Sub-center Animal Care and Use Committee of the National Center for Cardiovascular Diseases (FZX-IACUCC-2024005).

[0195] 2. Construction of tumor-bearing mouse model:

[0196] The method for constructing the tumor-bearing mouse model is the same as that in Example 6, which will not be described in detail here. After the tumor-bearing mouse model is successfully constructed, the length and width of the mass are measured with a vernier caliper to calculate the volume of the tumor mass.

[0197] 3. Grouping and treatment of experimental animals

[0198] Twenty-four tumor-bearing mice were divided into Vehicle group, DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group, with 6 mice in each group.

[0199] The tumor-bearing mice in the DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group were injected with DOX (injection dose of 5 mg / kg body weight) through the tail vein, and the mice in the Vehicle group were injected with normal saline through the tail vein once a week for a total of four weeks; moreover, 6 hours before each DOX injection, the tumor-bearing mice in the DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group were injected with normal saline (injection dose of 1 ml / kg body weight), TBMzyme nanozyme solution (injection dose of 10 mg / kg body weight), and ATBMzyme nanozyme solution (injection dose of 15 mg / kg body weight) through the tail vein, respectively, and the mice in the Vehicle group were injected with normal saline through the tail vein at the same time period; wherein, the TBMzyme nanozyme solution and ATBMzyme nanozyme solution used for injection were prepared with water as the solvent, and the concentration was 2.5 mg / mL (the schematic diagram of the medication process of the mice in the DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group is shown in Fig.16 shown).

[0200] 4. Echocardiography of mice:

[0201] Transthoracic echocardiography was performed on the mice in the Vehicle group, DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group using the Visual Sonics Vevo 3000 system (Fuji Visual Sonics, Japan). During the examination, the mice were anesthetized and maintained in an environment of 1-2% isoflurane and 2 liters of 20% oxygen per minute. The left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) were analyzed using Vevo analysis software (Fuji Visual Sonics, Japan). The test results are shown in Fig.17 shown.

[0202] Depend on Fig.17 It can be seen that DOX significantly weakened the cardiac ejection function of mice, while TBMzyme and ATBMzyme could effectively alleviate the weakening of cardiac function caused by DOX.

[0203] 5. Myocardial enzyme spectrum detection in mice:

[0204] The cardiac injury and heart failure markers LDH, cTNT and CKMB were detected in the mice in the Vehicle group, DOX group, DOX+TBMzyme group and DOX+ATBMzyme group. LDH, cTNT and CKMB were detected using ELISA kits. The ELISA kit for LDH was No. E-EL-M0419c, purchased from Elabscience; the ELISA kit for cTNT was No. E-EL-M1801c, purchased from Elabscience; and the ELISA kit for CKMB was No. E-EL-M0355c, purchased from Elabscience. The test results are shown in Figure 2. Fig.18 shown.

[0205] Depend on Fig.18 It can be seen that the supplementation of TBMzyme and ATBMzyme can effectively alleviate the increase of myocardial enzyme spectrum caused by DOX and reduce myocardial cell damage.

[0206] 6. Effects of TBMzyme and ATBMzyme nanozymes on the tumor killing effect of DOX:

[0207] To study whether ATBMzyme affects the tumor killing effect of DOX, tumor tissues of tumor-bearing mice in the Vehicle group, DOX group, DOX+TBMzyme group, and DOX+ATBMzyme group were collected to observe the changes in tumor tissue size; at the same time, the tumor volume and weight of the four groups of mice were measured and calculated on the first day (recorded as Day 0) and the 24th day (recorded as Day 24) after DOX injection. Fig.19shown.

[0208] Depend on Fig.19 It can be seen that compared with the DOX group, there was no difference in the size of tumor tissue in the DOX+TBMzyme group and the DOX+ATBMzyme group, while the tumor volume of these three groups was significantly smaller than that of the Vehicle group, indicating that nanozymes did not affect the tumor size after DOX treatment. This result shows that ATBMzyme does not affect the killing effect of DOX on tumors.

[0209] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Au-Ru nanozyme, characterized in that: The following steps are involved: (1) dissolving HAuCl4 and ruthenium metal salt in a solvent to obtain a metal salt solution; (2) adding N-acetylcysteine ​​and tannic acid to the metal salt solution and mixing them evenly to obtain a mixed solution; (3) Adding NaBH4 aqueous solution to the mixed solution, stirring the reaction at room temperature for 0.5 h to 2 h, and then continuing to stir the reaction at 50°C to 70°C for 2 h to 4 h; after the reaction, centrifuging and collecting the precipitate, and freeze-drying the precipitate to obtain Au-Ru nanozyme.

2. The method for preparing Au-Ru nanozyme according to claim 1, characterized in that: The molar ratio of HAuCl4 to ruthenium metal salt is (1.5-6):

2.

3. The method for preparing the Au-Ru nanozyme according to claim 1 or 2, characterized in that: The molar ratio of N-acetylcysteine ​​to HAuCl4 is (1-10):1; the molar ratio of tannic acid to HAuCl4 is (1-10):(1-10); and the molar ratio of NaBH4 to HAuCl4 is (1-8):(0.15-0.6).

4. The method for preparing Au-Ru nanozyme according to claim 3, characterized in that: Before adding the NaBH4 aqueous solution to the mixed solution, the NaBH4 aqueous solution needs to be precooled, and the temperature of the NaBH4 aqueous solution after the precooling treatment is 4°C.

5. An Au-Ru nanozyme prepared by the preparation method of the Au-Ru nanozyme according to any one of claims 1 to 4.

6. A method for preparing ATBMzyme nanozyme, characterized in that: The method comprises: dissolving the Au-Ru nanozyme according to claim 5 in water to obtain an Au-Ru nanozyme solution; adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to the Au-Ru nanozyme solution, stirring the reaction, and then adding brain natriuretic peptide to the reaction system, and continuing to stir the reaction; after the reaction is completed, centrifuging and collecting the precipitate, and freeze-drying the precipitate to obtain the ATBMzyme nanozyme.

7. The method for preparing the ATBMzyme nanozyme according to claim 6, characterized in that: The mass ratio of Au-Ru nanozyme to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is (1-4):(1-20); the mass ratio of Au-Ru nanozyme to N-hydroxysuccinimide is (5-20):(4-15); the mass ratio of brain natriuretic peptide to Au-Ru nanozyme is (0.2-1):(5-20).

8. An ATBMzyme nanozyme prepared using the preparation method according to claim 6 or 7.

9. Use of the Au-Ru nanozyme according to claim 5 or the ATBMzyme nanozyme according to claim 8 in the preparation of a drug for preventing, alleviating or / and treating cardiac toxicity caused by chemotherapeutic drugs; the chemotherapeutic drug is DOX.

10. A drug for preventing, alleviating and / or treating cardiac toxicity caused by chemotherapeutic drugs, wherein the drug contains the Au-Ru nanozyme according to claim 5 or the ATBMzyme nanozyme according to claim 8.

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