Carbon dot nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a RuCDzyme nano-enzyme preparation method, which comprises: S1, dissolving carbon quantum dots (CDs) and ruthenium metal salt in a solvent A to obtain a mixed solution; s2, adjusting the pH value of the mixed solution to be alkaline, adding a sodium borohydride solution into the mixed solution, and stirring to react for 3-6 hours; after the reaction is finished, dialyzing reaction liquid to remove unreacted reactants and the solvent A, and performing freeze-drying treatment on the dialyzed reaction liquid to obtain the RuCDzyme nano-enzyme. The invention also discloses a preparation method of the TPP-coated RuCDzyme nano-enzyme, which comprises the following steps: adding EDC and NHS into a TPP-COOH solution, stirring and reacting for 30-60 minutes at 25-30 DEG C, then adding the RuCDzyme nano-enzyme into a reaction system, continuing stirring and reacting for 16-24 hours, dialyzing a reaction solution after the reaction is finished, removing unreacted reactants in the reaction solution, and freeze-drying the dialyzed reaction solution to obtain the TPP-coated RuCDzyme nano-enzyme. The RuCDzyme nano-enzyme and the TPP coated RuCDzyme nano-enzyme prepared by the invention have similar superoxide dismutase activity, similar catalase activity and similar glutathione peroxidase activity, and can efficiently remove active oxygen to achieve the effect of preventing, relieving or / and treating renal injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a carbon dot nanozyme and its preparation method and application. Background Art

[0002] Acute kidney injury is a common and severe clinical syndrome, characterized by high incidence, high mortality, high medical costs, etc., and has become a health problem that has attracted much attention. The basic clinical diagnostic feature of acute kidney injury is that the serum creatinine level increases by 50% within 1 week or by more than 26 μmol / L every 48 hours. When acute kidney injury occurs, reactive oxygen species react with lipids, nucleic acids, and proteins, causing oxidative stress and inflammation, thus leading to the clinical manifestations of nephrotoxicity. The main source of reactive oxygen species is mitochondria, and its electron transport chain is easily interfered by various factors, resulting in the production of reactive oxygen species. These reactive oxygen species not only directly damage renal cells but also trigger a series of inflammatory and oxidative stress responses, further exacerbating kidney injury. Therefore, developing new drugs or treatment methods that can eliminate reactive oxygen species and relieve oxidative stress is of great significance for the treatment of acute kidney injury. Clinically, in addition to supportive treatment and renal replacement therapy, there seems to be no specific drugs that can improve acute kidney injury. Summary of the Invention

[0003] Aiming at the problems and deficiencies existing in the prior art, the purpose of the present invention is to provide a carbon dot nanozyme and its preparation method and application.

[0004] To achieve the purpose of the invention, the technical scheme adopted by the present invention is as follows:

[0005] In the first aspect of the present invention, a preparation method of a RuCDzyme nanozyme is provided, including the following steps:

[0006] S1: Dissolve carbon quantum dots (CDs) and ruthenium metal salt in solvent A to obtain a mixed solution;

[0007] S2: Adjust the pH of the mixed solution to alkaline, add sodium borohydride solution to the mixed solution, and stir and react for 3 h to 6 h; after the reaction is completed, dialyze the reaction solution to remove unreacted reactants and solvent A, and the dialyzed reaction solution is freeze-dried to obtain RuCDzyme nanozyme.

[0008] According to the above preparation method, preferably, in step S1, the mass ratio of the carbon quantum dots to the ruthenium metal salt is (6 - 15):1; more preferably, the mass ratio of the carbon quantum dots to the ruthenium metal salt is 10:1.

[0009] According to the above preparation method, preferably, in step S1, the mass ratio of the sodium borohydride to the ruthenium metal salt is (2 - 6):1; more preferably, the mass ratio of the sodium borohydride to the ruthenium metal salt is 4:1.

[0010] According to the above preparation method, preferably, the ruthenium metal salt is ruthenium(III) chloride hydrate.

[0011] According to the above preparation method, preferably, the solvent A is water.

[0012] According to the above preparation method, preferably, the concentration of the sodium borohydride solution is 5 mg / ml to 10 mg / ml; more preferably, the concentration of the sodium borohydride solution is 10 mg / ml.

[0013] According to the above preparation method, preferably, in step S2, the dialysis cut-off molecular weight is 8000 - 14000 Da.

[0014] According to the above preparation method, preferably, in step S2, an alkali solution is used to adjust the pH of the mixed solution to be alkaline; more preferably, the alkali solution is sodium hydroxide solution; most preferably, the concentration of the sodium hydroxide solution is 0.5 M.

[0015] According to the above preparation method, preferably, the preparation method of the carbon quantum dots is as follows: glutathione and folic acid are added to solvent B and mixed evenly to obtain a mixture, the mixture is reacted at 140 - 160 °C for 4 - 12 h, the reaction solution is centrifuged, the supernatant is taken, the supernatant is dialyzed to remove unreacted reactants, and the dialyzed supernatant is subjected to ultrafiltration and freeze-drying to obtain carbon quantum dots.

[0016] According to the above preparation method, preferably, the mass ratio of glutathione to folic acid is (15 - 25):1; more preferably, the mass ratio of glutathione to folic acid is 20:1.

[0017] According to the above preparation method, preferably, the dosage of the solvent B is: 30 ml to 70 ml of the solvent B is added per 1 g of glutathione.

[0018] According to the above preparation method, preferably, the solvent B is formamide.

[0019] According to the above preparation method, preferably, when the supernatant is dialyzed, the dialysis cut-off molecular weight is 3500 - 5000 Da.

[0020] According to the above preparation method, preferably, when performing ultrafiltration, the cut-off molecular weight of the ultrafiltration membrane used is 100 kDa.

[0021] The second aspect of the present invention provides a RuCDzyme nanozyme prepared by using the preparation method described in the first aspect above.

[0022] The third aspect of the present invention provides a method for preparing TPP@RuCDzyme nanozyme, comprising: adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to a solution of TPP-COOH (5-carboxypentyl-triphenylphosphonium bromide), stirring and reacting at 25-30 °C for 30-60 minutes, then adding the RuCDzyme nanozyme described in the second aspect above to the reaction system, continuing to stir and react for 16 h-24 h, after the reaction is completed, dialyzing the reaction solution to remove unreacted reactants in the reaction solution, and freeze-drying the dialyzed reaction solution to obtain TPP@RuCDzyme nanozyme.

[0023] According to the above preparation method, preferably, the mass ratio of the RuCDzyme nanozyme to TPP-COOH is (10-20):1; more preferably, the mass ratio of the RuCDzyme nanozyme to TPP-COOH is 15:1.

[0024] According to the above preparation method, preferably, the molar ratio of TPP-COOH to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1-1.5); more preferably, the molar ratio of TPP-COOH to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.2.

[0025] According to the above preparation method, preferably, the molar ratio of TPP-COOH to N-hydroxysuccinimide is 1:(1-1.5); more preferably, the molar ratio of TPP-COOH to N-hydroxysuccinimide is 1:1.

[0026] According to the above preparation method, preferably, the dialysis cut-off molecular weight is 8000-14000 Da.

[0027] The fourth aspect of the present invention provides a TPP@RuCDzyme nanozyme prepared by using the preparation method described in the third aspect above.

[0028] The fifth aspect of the present invention provides an application of the RuCDzyme nanozyme described in the second aspect above or the TPP@RuCDzyme nanozyme described in the fourth aspect above in the preparation of a drug for preventing, alleviating or / and treating kidney injury.

[0029] According to the above application, preferably, the kidney injury is acute kidney injury; more preferably, the acute kidney injury is acute kidney injury caused by oxidative stress; most preferably, the acute kidney injury is acute kidney injury induced by rhabdomyolysis.

[0030] The sixth aspect of the present invention provides a drug for preventing, alleviating or / and treating kidney injury, characterized in that the drug comprises the RuCDzyme nanozyme described in the second aspect above or the TPP@RuCDzyme nanozyme described in the fourth aspect above.

[0031] For the drug according to the above, preferably, the drug further contains a pharmaceutically acceptable carrier / excipient.

[0032] For the drug according to the above, preferably, the carrier / excipient includes (but is not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as simple syrup, glucose solution, starch solution, cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds, sodium dodecyl sulfate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium dodecyl sulfate, monoglyceride stearate, cetyl alcohol, etc.; humidifying agents such as glycerol, starch, etc.; adsorbent carriers such as starch, lactose, bentonite, silica gel, kaolin and saponite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0033] Compared with the prior art, the positive and beneficial effects obtained by the present invention are as follows:

[0034] (1) In the present invention, carbon quantum dots are reacted with ruthenium metal salts to construct RuCDzyme nanozymes. The RuCDzyme nanozymes are spherical in structure, with a particle size of about 85 nm, and have superoxide dismutase-like, catalase-like and glutathione peroxidase-like activities, and can effectively scavenge oxygen-containing and nitrogen-containing free radicals; in addition, the RuCDzyme nanozymes have good water solubility and biocompatibility; moreover, through cell experiments, it is proved that the RuCDzyme nanozymes have the ability to scavenge reactive oxygen species and inhibit apoptosis caused by oxidative stress, and have good therapeutic effects on kidney injury (especially acute kidney injury).

[0035] (2) In the present invention, triphenylphosphonium bromide (TPP) is modified on the surface of RuCDzyme nanozymes, so that the RuCDzyme nanozymes are endowed with the ability of mitochondrial targeting and the ability to overcome the lysosomal barrier in cells. Therefore, the prepared TPP@RuCDzyme nanozymes of the present invention not only have superoxide dismutase-like, catalase-like and glutathione peroxidase-like activities and can effectively scavenge oxygen-containing and nitrogen-containing free radicals; at the same time, they also have mitochondrial targeting properties, can efficiently scavenge reactive oxygen species, protect the biological system from damage induced by reactive oxygen species, and achieve the effect of preventing, alleviating or / and treating kidney injury, especially acute kidney injury induced by rhabdomyolysis.

[0036] (3) The TPP@RuCDzyme nanozyme prepared by the present invention has high - efficient cascade catalytic activity, can scavenge various reactive oxygen species in vivo and in vitro, and thus effectively plays a therapeutic role in acute kidney injury induced by rhabdomyolysis. Description of the Drawings

[0037] Figure 1 Figures showing the morphological, particle size, zeta potential and elemental distribution characterization results of RuCDzyme and TPP@RuCDzyme nanozymes; A is the transmission electron microscope image of CDs; B is the transmission electron microscope image and average particle size of TPP@RuCDzyme nanozyme; C is the zeta potential change detection result graph of CDs, RuCDzyme and TPP@RuCDzyme nanozymes; D is the elemental analysis image of TPP@RuCDzyme;

[0038] Figure 2 Figures showing the structural characterization results of nanozymes; among them, A is the XRD detection result graph; B is the FTIR detection result graph; C is the UV - vis detection result graph; D - G are the XPS detection result graphs; H is the photo of TPP@RuCDzyme nanozyme dispersed in different solvents (where the left photo is for 1 - day dispersion and the right photo is for 7 - day dispersion);

[0039] Figure 3 Figures showing the CAT enzyme activity detection results of RuCDzyme and TPP@RuCDzyme nanozymes;

[0040] Figure 4 Figures showing the SOD enzyme activity detection results of RuCDzyme and TPP@RuCDzyme nanozymes;

[0041] Figure 5 Figures showing the ABTS and DPPH free radical scavenging ability detection results of RuCDzyme and TPP@RuCDzyme nanozymes;

[0042] Figure 6 Figures showing the CCK - 8 proliferation inhibition experiment results of RuCDzyme and TPP@RuCDzyme nanozymes;

[0043] Figure 7 Figures showing the cell viability and death staining results of different drug treatment groups;

[0044] Figure 8 Figures showing the intracellular ROS level detection results of different drug treatment groups;

[0045] Figure 9 Figures showing the detection results of cell apoptosis in different treatment groups;

[0046] Figure 10Detection results of CRE and BUN levels in the sera of mice in different drug treatment groups; among them, A is the blood creatinine value level of mice; B is the blood urea nitrogen value level of mice after 24 hours; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001;

[0047] Figure 11 H&E staining results of organs of mice in different drug treatment groups;

[0048] Figure 12 ROS fluorescence staining map of kidney tissues of mice in different drug treatment groups; in the figure, the red fluorescence is the ROS level, and the blue fluorescence is the cell nucleus;

[0049] Figure 13 Detection results of the expression levels of the heavy KIM-1, HO-1, and 8-OHdG markers in kidney tissues of mice in different drug treatment groups, where *** represents a P value < 0.001, and **** represents a P value < 0.0001;

[0050] Figure 14 H&E staining maps of the main organs of mice; A is the H&E staining map of the heart, liver, spleen, and lungs of mice in different drug treatment groups; B is the H&E staining map of different parts of the kidneys of mice in different drug treatment groups;

[0051] Figure 15 Graphs of the body weight changes and blood biochemical index detection results of mice in different groups. Detailed implementation manners

[0052] 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 combination with specific embodiments.

[0053] The following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0054] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. When the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, components, and / or combinations thereof.

[0055] For the experimental methods without specific conditions noted in the following examples, conventional techniques in the technical field are used, or the conditions recommended by the manufacturer are followed; for the reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0056] Example 1:

[0057] A preparation method of RuCDzyme nanozyme, the specific steps are as follows:

[0058] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) chloride hydrate in water to obtain a mixed solution. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) chloride hydrate is 10:1. The preparation method of the carbon quantum dots (CDs) is as follows: Add glutathione and folic acid to formamide, mix well to obtain a mixture, where the mass ratio of glutathione to folic acid is 20:1, and the dosage of formamide is 35 ml of formamide per 1 g of glutathione; React the mixture at 160 °C for 12 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500-5000 Da) to remove unreacted reactants. The supernatant after dialysis is ultrafiltered (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-dried to obtain carbon quantum dots.

[0059] S2: Adjust the pH of the mixed solution to alkaline with NaOH solution (0.5 M), add a 10 mg / ml sodium borohydride solution to the mixed solution, stir and react for 3 h, and the mass ratio of sodium borohydride to ruthenium(III) chloride hydrate in the mixed solution is 4:1; After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000-14000 Da) to remove unreacted reactants and water, and the reaction solution after dialysis is freeze-dried to obtain RuCDzyme nanozyme.

[0060] Example 2:

[0061] A preparation method of RuCDzyme nanozyme, the specific steps are as follows:

[0062] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) chloride hydrate in water to obtain a mixed solution. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) chloride hydrate is 6:1. The preparation method of the carbon quantum dots (CDs) is as follows: Add glutathione and folic acid to formamide, mix well to obtain a mixture, where the mass ratio of glutathione to folic acid is 15:1, and the dosage of formamide is 30 ml of formamide per 1 g of glutathione; React the mixture at 140 °C for 12 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500-5000 Da) to remove unreacted reactants. The supernatant after dialysis is ultrafiltered (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-dried to obtain carbon quantum dots.

[0063] S2: Adjust the pH of the mixture to alkaline with a NaOH solution (0.5 M), add a sodium borohydride solution at 5 mg / ml to the mixture, stir and react for 5 h, where the mass ratio of sodium borohydride to ruthenium(III) hydrate in the mixture is 2:1; after the reaction, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants and water, and the dialyzed reaction solution is freeze-dried to obtain RuCDzyme nanozyme.

[0064] Example 3:

[0065] A method for preparing RuCDzyme nanozyme, the specific steps are as follows:

[0066] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) hydrate in water to obtain a mixture. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) hydrate is 15:1. The preparation method of the carbon quantum dots (CDs) is: add glutathione and folic acid to formamide, mix well to obtain a mixture, where the mass ratio of glutathione to folic acid is 25:1, and the dosage of formamide is 70 ml of formamide added per 1 g of glutathione; react the mixture at 150 °C for 10 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500 - 5000 Da) to remove unreacted reactants, and the dialyzed supernatant is ultrafiltered (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-dried to obtain carbon quantum dots.

[0067] S2: Adjust the pH of the mixture to alkaline with a NaOH solution (0.5 M), add a sodium borohydride solution at 8 mg / ml to the mixture, stir and react for 6 h, where the mass ratio of sodium borohydride to ruthenium(III) hydrate in the mixture is 6:1; after the reaction, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants and water, and the dialyzed reaction solution is freeze-dried to obtain RuCDzyme nanozyme.

[0068] Example 4:

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

[0070] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) hydrate in water to obtain a mixed solution. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) hydrate is 8:1. The preparation method of the carbon quantum dots (CDs) is as follows: Add glutathione and folic acid to formamide and mix well to obtain a mixture. Among them, the mass ratio of glutathione to folic acid is 18:1, and the dosage of formamide is 50 ml of formamide added per 1 g of glutathione; React the mixture at 150 °C for 8 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500 - 5000 Da) to remove unreacted reactants. The supernatant after dialysis is subjected to ultrafiltration (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-drying to obtain carbon quantum dots.

[0071] S2: Use a NaOH solution (0.5 M) to adjust the pH of the mixed solution to alkaline, add a 10 mg / ml sodium borohydride solution to the mixed solution, and stir and react for 4 h. The mass ratio of sodium borohydride to ruthenium(III) hydrate in the mixed solution is 5:1; After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants and water. The reaction solution after dialysis is subjected to freeze-drying to obtain RuCDzyme nanozyme.

[0072] Example 5:

[0073] A preparation method of RuCDzyme nanozyme, the specific steps are as follows:

[0074] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) hydrate in water to obtain a mixed solution. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) hydrate is 12:1. The preparation method of the carbon quantum dots (CDs) is as follows: Add glutathione and folic acid to formamide and mix well to obtain a mixture. Among them, the mass ratio of glutathione to folic acid is 20:1, and the dosage of formamide is 40 ml of formamide added per 1 g of glutathione; React the mixture at 140 °C for 10 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500 - 5000 Da) to remove unreacted reactants. The supernatant after dialysis is subjected to ultrafiltration (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-drying to obtain carbon quantum dots.

[0075] S2: Adjust the pH of the mixture to alkaline with a NaOH solution (0.5 M), add a sodium borohydride solution at 5 mg / ml to the mixture, stir and react for 6 h, and the mass ratio of sodium borohydride to ruthenium(III) hydrate in the mixture is 3:1; after the reaction, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants and water, and the dialyzed reaction solution is freeze-dried to obtain RuCDzyme nanozyme.

[0076] Example 6:

[0077] A preparation method of RuCDzyme nanozyme, the specific steps are as follows:

[0078] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) hydrate in water to obtain a mixture. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) hydrate is 9:1. The preparation method of the carbon quantum dots (CDs) is: add glutathione and folic acid to formamide, mix evenly to obtain a mixture, where the mass ratio of glutathione to folic acid is 22:1, and the dosage of formamide is 60 ml of formamide added per 1 g of glutathione; react the mixture at 160 °C for 4 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500 - 5000 Da) to remove unreacted reactants, and the dialyzed supernatant is ultrafiltered (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-dried to obtain carbon quantum dots.

[0079] S2: Adjust the pH of the mixture to alkaline with a NaOH solution (0.5 M), add a sodium borohydride solution at 8 mg / ml to the mixture, stir and react for 5 h, and the mass ratio of sodium borohydride to ruthenium(III) hydrate in the mixture is 5:1; after the reaction, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants and water, and the dialyzed reaction solution is freeze-dried to obtain RuCDzyme nanozyme.

[0080] Example 7:

[0081] A preparation method of RuCDzyme nanozyme, the specific steps are as follows:

[0082] S1: Dissolve carbon quantum dots (CDs) and ruthenium(III) chloride hydrate in water to obtain a mixed solution. Among them, the mass ratio of the carbon quantum dots to ruthenium(III) chloride hydrate is 10:1. The preparation method of the carbon quantum dots (CDs) is as follows: Add glutathione and folic acid to formamide, mix well to obtain a mixture, where the mass ratio of glutathione to folic acid is 18:1, and the dosage of formamide is 40 ml of formamide added per 1 g of glutathione; React the mixture at 150 °C for 8 h, centrifuge the reaction solution, take the supernatant, and dialyze the supernatant (the dialysis cut-off molecular weight is 3500 - 5000 Da) to remove unreacted reactants. The supernatant after dialysis is subjected to ultrafiltration (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa) and freeze-drying to obtain carbon quantum dots.

[0083] S2: Adjust the pH of the mixed solution to alkaline with a NaOH solution (0.5 M), add a 6 mg / ml sodium borohydride solution to the mixed solution, and stir and react for 6 h. The mass ratio of sodium borohydride to ruthenium(III) chloride hydrate in the mixed solution is 3:1; After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants and water. The reaction solution after dialysis is subjected to freeze-drying to obtain RuCDzyme nanozyme.

[0084] Example 8:

[0085] A preparation method of TPP@RuCDzyme nanozyme, the specific steps are as follows: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the TPP-COOH solution. The molar ratio of TPP-COOH, EDC and NHS is 1:1.2:1, and stir and react at 25 °C for 45 minutes; Then add the RuCDzyme nanozyme prepared in Example 1 to the reaction system. The mass ratio of RuCDzyme nanozyme to TPP-COOH is 15:1, and continue to stir and react for 24 h. After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000 - 14000 Da) to remove unreacted reactants in the reaction solution. The reaction solution after dialysis is subjected to freeze-drying to obtain TPP@RuCDzyme nanozyme.

[0086] Example 9:

[0087] A preparation method of TPP@RuCDzyme nanozyme, the specific steps are as follows: Add carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the TPP-COOH solution, and the molar ratio of TPP-COOH, EDC and NHS is 1:1:1. Stir and react at 25 °C for 30 minutes; then add the RuCDzyme nanozyme prepared in Example 1 to the reaction system, and the mass ratio of RuCDzyme nanozyme to TPP-COOH is 10:1. Continue to stir and react for 16 h. After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000-14000 Da) to remove the unreacted reactants in the reaction solution. The dialyzed reaction solution is freeze-dried to obtain TPP@RuCDzyme nanozyme.

[0088] Example 10:

[0089] A preparation method of TPP@RuCDzyme nanozyme, the specific steps are as follows: Add carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the TPP-COOH solution, and the molar ratio of TPP-COOH, EDC and NHS is 1:1.5:1.5. Stir and react at 30 °C for 30 minutes; then add the RuCDzyme nanozyme prepared in Example 1 to the reaction system, and the mass ratio of RuCDzyme nanozyme to TPP-COOH is 20:1. Continue to stir and react for 20 h. After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000-14000 Da) to remove the unreacted reactants in the reaction solution. The dialyzed reaction solution is freeze-dried to obtain TPP@RuCDzyme nanozyme.

[0090] Example 11:

[0091] A preparation method of TPP@RuCDzyme nanozyme, the specific steps are as follows: Add carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the TPP-COOH solution, and the molar ratio of TPP-COOH, EDC and NHS is 1:1.2:1.5. Stir and react at 28 °C for 40 minutes; then add the RuCDzyme nanozyme prepared in Example 1 to the reaction system, and the mass ratio of RuCDzyme nanozyme to TPP-COOH is 16:1. Continue to stir and react for 18 h. After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000-14000 Da) to remove the unreacted reactants in the reaction solution. The dialyzed reaction solution is freeze-dried to obtain TPP@RuCDzyme nanozyme.

[0092] Example 12:

[0093] A method for preparing TPP@RuCDzyme nanozyme, the specific steps are as follows: Add carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the TPP-COOH solution, and the molar ratio of TPP-COOH, EDC and NHS is 1:1.2:1. Stir and react at 30 °C for 45 minutes; then add the RuCDzyme nanozyme prepared in Example 1 to the reaction system, and the mass ratio of RuCDzyme nanozyme to TPP-COOH is 16:1. Continue to stir and react for 22 h. After the reaction is completed, dialyze the reaction solution (the dialysis cut-off molecular weight is 8000-14000 Da) to remove the unreacted reactants in the reaction solution. The dialyzed reaction solution is freeze-dried to obtain TPP@RuCDzyme nanozyme.

[0094] Example 13:

[0095] The content of Example 13 is basically the same as that of Example 8, and the difference is that: the RuCDzyme nanozyme is the RuCDzyme nanozyme prepared in Example 2.

[0096] Example 14:

[0097] The content of Example 14 is basically the same as that of Example 8, and the difference is that: the RuCDzyme nanozyme is the RuCDzyme nanozyme prepared in Example 3.

[0098] Example 15:

[0099] The content of Example 15 is basically the same as that of Example 8, and the difference is that: the RuCDzyme nanozyme is the RuCDzyme nanozyme prepared in Example 4.

[0100] Example 16:

[0101] The content of Example 16 is basically the same as that of Example 8, and the difference is that: the RuCDzyme nanozyme is the RuCDzyme nanozyme prepared in Example 5.

[0102] Example 17:

[0103] The content of Example 17 is basically the same as that of Example 8, and the difference is that: the RuCDzyme nanozyme is the RuCDzyme nanozyme prepared in Example 6.

[0104] Example 18:

[0105] The content of Example 18 is basically the same as that of Example 8, and the difference is that: the RuCDzyme nanozyme is the RuCDzyme nanozyme prepared in Example 7.

[0106] (1) Characterization of RuCDzyme nanozyme and TPP@RuCDzyme

[0107] Taking the RuCDzyme nanozyme prepared in Example 1 and the TPP@RuCDzyme nanozyme prepared in Example 8 as examples, the RuCDzyme nanozyme and TPP@RuCDzyme nanozyme were characterized.

[0108] 1. Morphology and particle size characterization of nanozyme:

[0109] The morphology and particle size of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme were characterized by transmission electron microscopy (TEM), and the results are shown in Figure 1 A, B, and D therein.

[0110] As Figure 1 can be seen, CDzyme has a spherical structure with a size of about 2 nm and good dispersibility. The size of TPP@RuCDzyme nanozyme is about 85. As Figure 1 shown by the elemental analysis in D therein, Ru element is uniformly distributed in the TPP@RuCDzyme nanozyme, and P element is uniformly distributed on the surface of TPP@RuCDzyme, indicating the successful synthesis of TPP@RuCDzyme nanozyme.

[0111] 2. Potential characterization of nanozyme:

[0112] The potentials of CDs, RuCDzyme nanozyme, and TPP@RuCDzyme nanozyme were detected using dynamic light scattering (DLS), and the potential change results are shown in Figure 1 C therein.

[0113] As Figure 1 shown in C therein, the zeta potentials of CDs and RuCDzyme nanozyme are -14.98 mV and -39.39 mV, respectively. Moreover, compared with RuCDzyme nanozyme, the zeta potential of TPP@RuCDzyme decreased from -39.39 of RuCDzyme nanozyme to about -29.36, indicating the successful coupling of TPP on RuCDzyme and the successful synthesis of TPP@RuCDzyme nanozyme.

[0114] 3. Structure characterization of nanozyme:

[0115] RuCDzyme nanozyme and TPP@RuCDzyme nanozyme were analyzed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, and X-ray photoelectron spectroscopy (XPS) respectively, and the detection results are shown in Figure 2 A - G therein.

[0116] Among them, the XRD detection patterns of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme are as shown in Figure 2 A in it. It can be seen from Figure 2 A in it that TPP@RuCDzyme nanozyme shows diffraction peaks similar to those of RuCDzyme nanozyme, indicating the successful construction of TPP@RuCDzyme nanozyme.

[0117] The FTIR detection patterns of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme are as shown in Figure 2 B in it. It can be seen from Figure 2 B in it that the characteristic peak heights of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme are highly consistent, and this result further proves that the TPP modification does not change the original structural characteristics of RuCDzyme nanozyme.

[0118] The ultraviolet-visible spectra of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme are as shown in Figure 2 C in it. It can be seen from Figure 2 C in it that RuCDzyme nanozyme and TPP@RuCDzyme nanozyme have similar absorption peaks, indicating that the electronic structure of RuCDzyme nanozyme has not changed significantly due to TPP modification.

[0119] The XPS detection results of TPP@RuCDzyme nanozyme are as shown in Figure 2 D-G in it. From Figure 2 D-G in it, obvious Ru 3d binding energy peaks can be observed, indicating that the existence form of ruthenium element in TPP@RuCDzyme is in the oxidized state.

[0120] 4. Solubility characterization of nanozyme:

[0121] TPP@RuCDzyme nanozyme (5 mg / mL) was added to double-distilled water, 0.9% sodium chloride solution, phosphate buffered saline (PBS), and Dulbecco's modified Eagle medium (DMEM) respectively, and ultrasonic treatment (power 500 W) was carried out for 10 min to ensure uniform dispersion, obtaining four kinds of dispersions. The four kinds of dispersions were placed statically in transparent EP tubes to observe the sedimentation situation after 24 h and 7 days. The results are as shown in Figure 2 H in it.

[0122] It can be seen from Figure 2 H in it that TPP@RuCDzyme shows good solubility and stability in double-distilled water, 0.9% sodium chloride solution, phosphate buffered saline (PBS), and Dulbecco's modified Eagle medium (DMEM) on the 1st day and the 7th day.

[0123] (2) Enzyme activity detection of RuCDzyme nanozyme and TPP@RuCDzyme:

[0124] 1. Detection of CAT (catalase) activity of nanozyme:

[0125] CAT, also known as catalase, can decompose H 2 O 2 to produce O 2 and water. The specific operation for CAT activity detection is as follows: Add 200 μL of H 2 O 2 solution with a concentration of 100 mM and 200 μL of RuCDzyme or TPP@RuCDzyme solution into 10 mL of PBS buffer solution with a concentration of 0.01 M respectively. After mixing, immediately mix by vibration, and use a dissolved oxygen meter to monitor the change of dissolved oxygen content in the system in real time. The detection results are as Figure 3 shown.

[0126] As Figure 3 can be seen, the TPP@RuCDzyme nanozyme exhibits significant catalase-like activity, and the activity is close to that of the RuCDzyme nanozyme, indicating that the catalase-like activity of the RuCDzyme nanozyme is not affected after being modified by TPP.

[0127] 2. Detection of SOD (superoxide dismutase) activity of nanozyme:

[0128] SOD, also known as superoxide dismutase, can dismutate superoxide anions to produce H 2 O 2 and H 2 O. The xanthine oxidase method is used to determine the superoxide dismutase (SOD)-like activity of RuCDzyme and TPP@RuCDzyme nanozymes. The specific operation for SOD activity detection is as follows: Transfer 30 μL of the prepared nanozyme solution to a 96-well plate, and sequentially add xanthine, pH 7.4 phosphate buffer, cytochrome C, and xanthine oxidase working solution. After mixing evenly, use an enzyme-labeling instrument to measure the absorbance change of each well before and after the reaction at 450 nm. Each group is measured in parallel three times, and each measurement lasts for 2 minutes. The detection results are as Figure 4 shown.

[0129] As Figure 4 can be seen, both RuCDzyme and TPP@RuCDzyme nanozymes have excellent superoxide dismutase (SOD)-like activity; indicating that after being modified by TPP, the retention amount of antioxidant groups in the RuCDzyme nanozyme is still relatively sufficient.

[0130] 3. Detection of ABTS and DPPH free radical scavenging ability of nanozyme

[0131] ABTS can be oxidized to form a structurally stable blue-green ABTS radical, which has a maximum absorption peak at 735 nm. After ABTS reacts with an oxidant, a blue-green ABTS cation radical (ABTS·+) is formed, which produces a characteristic absorption peak at a wavelength of 734 nm. When antioxidant substances are present, the formation of ABTS·+ is inhibited, causing the absorption peak at 735 nm to decrease. The degree of decrease in its absorption peak is proportional to the degree of free radical scavenging. In this invention, the ABTS radical scavenging ability of RuCDzyme and TPP@RuCDzyme nanozymes at different concentrations was quantitatively determined by the ABTS assay method, and the detection results are as Figure 5 shown in A below.

[0132] As Figure 5 can be seen from A below, as the dosages of RuCDzyme and TPP@RuCDzyme nanozymes increase, the formation of ABTS radicals is significantly inhibited; indicating that RuCDzyme and TPP@RuCDzyme nanozymes have excellent ABTS radical scavenging ability.

[0133] 4. Detection of the DPPH radical scavenging ability of nanozymes

[0134] DPPH is a stable free radical that is soluble in polar solvents such as methanol and ethanol and has a maximum absorption peak at 520 nm. When an antioxidant is added, DPPH will undergo a fading reaction. In this invention, the DPPH radical scavenging ability of RuCDzyme and TPP@RuCDzyme nanozymes at different concentrations was determined by the DPPH radical assay method, and the detection results are as Figure 5 shown in B below.

[0135] As Figure 5 can be seen from B below, as the dosages of RuCDzyme and TPP@RuCDzyme nanozymes increase, the inhibition rate gradually increases, indicating that RuCDzyme and TPP@RuCDzyme nanozymes can effectively scavenge DPPH radicals.

[0136] (III) In vitro antioxidant experiments of nanozymes

[0137] At the cellular level, the efficacy of the nanozymes of this invention was studied through cell proliferation inhibition, Live / Dead cell staining experiments, intracellular ROS scavenging, and apoptosis experiments.

[0138] 1. Cell proliferation inhibition experiment

[0139] The CCK8 method was used to evaluate the effect in H 2 O 2Proliferation inhibition and protection of RuCDzyme and TPP@RuCDzyme nanozymes on HK-2 cells under injury conditions.

[0140] The experimental grouping and treatment methods were as follows:

[0141] Experimental grouping: Four groups were set up, namely: (1) Control group; (2) H 2 O 2 injury group; (3) RuCDzyme nanozyme + H 2 O 2 group; (4) TPP@RuCDzyme nanozyme + H 2 O 2 group.

[0142] After digesting, centrifuging and resuspending HK-2 cells, they were seeded at 8000 cells / well in a 96-well plate and cultured for 24 hours. RuCDzyme and TPP@RuCDzyme nanozymes were diluted to appropriate concentrations using complete medium. Carefully aspirate the original medium in each well of the 96-well plate; then, the RuCDzyme nanozyme + H 2 O 2 group was added with 100 μL of complete medium containing RuCDzyme nanozyme, and the TPP@RuCDzyme nanozyme + H 2 O 2 group was added with 100 μL of complete medium containing TPP@RuCDzyme nanozyme. The Control group and the H 2 O 2 injury group were both added with 100 μL of complete medium without drugs. The 96-well plate treated as above was placed in a cell culture incubator and co-incubated for 30 min; then, to the RuCDzyme nanozyme + H 2 O 2 group, the TPP@RuCDzyme nanozyme + H 2 O 2 group and the H 2 O 2 injury group, H 2 O 2 diluted with complete medium was added to make the final concentration of H 2 O 2 in each group 400 μM, and the concentration of nanozyme in the RuCDzyme nanozyme + H 2 O 2 group and the TPP@RuCDzyme nanozyme + H 2 O 2 group was 50 μg / mL. Subsequently, the 96-well plate was placed back into the cell culture incubator and cultured for another 24 hours.

[0143] Aspirate the culture medium in each well of the 96-well plate treated by the above "experimental grouping and treatment method", wash the cells in each well with PBS buffer, and then add 100 μL of 10% CCK-8 working solution to each well of the 96-well plate according to the operation instructions of the CCK8 kit (Beyotime, product number C0038). After incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value. The cell survival rate is calculated according to the following formula:

[0144] Percentage of cell survival = [(A - C) / (B - C)] × 100%

[0145] A: Absorbance value of the experimental group (the absorbance value containing culture medium, cells, drug to be tested, and CCK-8 Solution); B: Absorbance value of the control group (the absorbance value containing culture medium, cells, and CCK-8 Solution)

[0146] C: Absorbance value of the blank group (the absorbance value containing culture medium and CCK-8 Solution).

[0147] The detection results of the cell survival rate are as Figure 6 shown.

[0148] It can be seen from Figure 6 that the cell viability of the RuCDzyme nanozyme + H 2 O 2 group and the TPP@RuCDzyme nanozyme + H 2 O 2 group is significantly higher than that of the H 2 O 2 injury group, and the cell viability of the RuCDzyme nanozyme + H 2 O 2 group is better than that of the RuCDzyme nanozyme + H 2 O 2 group. This result indicates that the RuCDzyme nanozyme and TPP@RuCDzyme nanozyme prepared in the present invention have excellent antioxidant capacity and can effectively scavenge excessive intracellular reactive oxygen species.

[0149] 2. Live / Dead cell staining experiment

[0150] In this experiment, the Calcein-PI Cell Viability and Cytotoxicity Detection Kit (Beyotime, product number C2015M) was used to evaluate the protective effects of RuCDzyme and TPP@RuCDzyme nanozymes on HK-2 cells under H 2 O 2 injury conditions.

[0151] The experimental grouping and treatment method are as follows: Set 4 groups, which are in turn: (1) Control group; (2) H2 O 2 Injury group; (3) RuCDzyme nanozyme + H 2 O 2 group; (4) TPP@RuCDzyme nanozyme + H 2 O 2 group.

[0152] After digesting and centrifuging HK-2 cells, resuspend the cells with complete medium at a concentration of 1.0×10 5 cells / mL, and then seed 2 mL of the cell suspension into each well of a six-well plate and continue culturing for 24 hours. Dilute RuCDzyme and TPP@RuCDzyme nanozymes with complete medium, discard the medium in each well of the six-well plate, and wash each well once with 1 mL of PBS; then, add 2 mL of drug-free complete medium to the Control group and the H 2 O 2 injury group respectively, add 2 mL of complete medium containing RuCDzyme nanozyme to the RuCDzyme nanozyme + H 2 O 2 group, add 2 mL of complete medium containing TPP@RuCDzyme nanozyme to the TPP@RuCDzyme nanozyme + H 2 O 2 group, place the six-well plate treated as above in a cell culture incubator and co-incubate for 30 min; then add 20 μL of 40 mM H 2 O 2 diluted with complete medium to the RuCDzyme nanozyme + H 2 O 2 group, the TPP@RuCDzyme nanozyme + H 2 O 2 group and the H 2 O 2 injury group, and do not treat the Control group; then place the six-well plate in a cell culture incubator and culture for 24 hours; aspirate the medium in each well, wash the cells once with PBS, and add 1 mL of Calcein AM / PI detection working solution to each well. Incubate at 37 °C in the dark for 30 min. After the incubation, observe and record the staining effect under an inverted fluorescence microscope. The staining results are as Figure 7 shown.

[0153] Calcein AM stains living cells, showing green fluorescence; while propidium iodide (PI) stains dead cells, showing red fluorescence. As Figure 7 can be seen, compared with the Control group, the H 2 O 2 injury group showed significantly enhanced red fluorescence, indicating that H2 O 2 can cause apoptosis by directly damaging cells; compared with the H 2 O 2 damage group, the red fluorescence in the RuCDzyme nanozyme + H 2 O 2 group and the TPP@RuCDzyme nanozyme + H 2 O 2 group was significantly weakened, and the green fluorescence increased significantly. Moreover, the red fluorescence in the TPP@RuCDzyme nanozyme + H 2 O 2 group was further reduced compared with that in the RuCDzyme nanozyme + H 2 O 2 group. These results indicate that both RuCDzyme nanozyme and TPP@RuCDzyme nanozyme have strong antioxidant capabilities and can effectively reduce H 2 O 2 -mediated oxidative damage to cells.

[0154] 3. Intracellular ROS Scavenging

[0155] The reactive oxygen species (ROS) detection kit (Beyotime, catalog number S0033S) was used to detect the ROS scavenging ability of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme

[0156] under H 2 O 2 damage conditions. The specific experimental method was as follows: The medium in each well of the six-well plate treated according to the "experimental grouping and treatment method" described in step 2 "Live / Dead cell staining experiment" above was aspirated, and the cells in each well were washed with PBS buffer. Then, 1 mL of DCFH-DA probe (10 μM) was added to each well, and the cells were incubated in a cell culture incubator for 20 min. After washing three times with serum-free medium, 2 mL of serum-free medium was added, and the staining results were observed using a laser confocal microscope. The staining results are as Figure 8 shown.

[0157] Under normal circumstances, as a natural by-product of oxygen metabolism, ROS is at a low level in the body. When the body is stimulated, the ROS level will increase sharply, exceeding the body's own scavenging and processing ability, resulting in an imbalance between oxidation and antioxidant effects in the body and oxidative stress. The ROS level is an important indicator of the normal physiological function of cells and cell oxidative damage caused by environmental factors. Detecting the intracellular ROS level is of great significance for understanding cell signal transduction and studying the potential mechanisms of diseases. As Figure 8 can be seen, the H 2 O 2 damage group showed extremely strong green fluorescence, indicating that H2 O 2 A large amount of ROS was generated in the cells after treatment. Compared with the H 2 O 2 -damaged group, the intracellular green fluorescence level in the RuCDzyme nanozyme + H 2 O 2 group and the TPP@RuCDzyme nanozyme + H 2 O 2 group decreased significantly, and there was no significant difference in the intracellular green fluorescence level between the TPP@RuCDzyme nanozyme + H 2 O 2 group and the Control group. These results indicate that the TPP@RuCDzyme nanozyme constructed by modifying RuCDzyme with TPP further enhances its ability to scavenge intracellular ROS. Therefore, the experimental results show that both RuCDzyme nanozyme and TPP@RuCDzyme nanozyme can significantly scavenge the excessive ROS generated due to H 2 O 2 damage, showing excellent antioxidant ability.

[0158] 4. Apoptosis assay

[0159] Apoptosis is a normal physiological process in embryonic development and maintaining homeostasis in the body. To further prove the protective effect of TPP@RuCDzyme nanozyme on HK-2 cells, flow cytometry was used to quantitatively analyze the antioxidant ability of RuCDzyme and TPP@RuCDzyme nanozyme and the effect of reducing apoptosis. Annexin V is a Ca 2+ -dependent phospholipid-binding protein that can bind to the cell membrane of early apoptotic cells. Therefore, Annexin V can be used as one of the sensitive indicators for detecting early apoptosis of cells. Propidium Iodide (PI) is a nucleic acid dye that can bind to the DNA of late apoptotic cells or necrotic cells and can be used to distinguish early apoptotic cells from late apoptotic cells or necrotic cells.

[0160] The apoptosis of each group of HK-2 cells treated according to the "Experimental grouping and treatment methods" recorded in step 2 "Live / Dead cell staining experiment" above was detected by Annexin V-FITC and PI, and then analyzed using a flow cytometer. The results are as Figure 9 shown.

[0161] As Figure 9 can be seen, the average percentage of apoptosis in the Control group was 4.96%, and H 2 O 2The apoptosis rate of the injury group was 21.19%, and that of the RuCDzyme nanozyme + H 2 O 2 group was 9.44%, and that of the TPP@RuCDzyme nanozyme + H 2 O 2 group was 5.95%. These results indicate that both RuCDzyme nanozyme and TPP@RuCDzyme nanozyme can effectively inhibit H 2 O 2 -induced apoptosis, and the inhibitory effect of TPP@RuCDzyme nanozyme is more significant.

[0162] (IV) Efficacy verification test of nanozyme in a mouse model of acute kidney injury

[0163] To study the efficacy of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme in mice with acute kidney injury, the present invention constructed a mouse model of acute kidney injury and explored the effects of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme on the renal function, kidney tissue, and kidney injury of the mouse model of acute kidney injury.

[0164] 1. Establishment of a glycerol-induced mouse model of acute kidney injury: Female Balb / c mice (6 - 8 weeks old, 17 - 21 g) were prefasted for 15 h, and then 50% glycerol was evenly injected into the bilateral hind limb muscles of the mice at a dose of 8 mL / Kg.

[0165] 2. Experimental grouping and treatment methods:

[0166] The glycerol-induced mice with acute kidney injury were randomly divided into three groups, namely: AKI group, RuCDzyme nanozyme group, and TPP@RuCDzyme nanozyme group, with 6 mice in each group; at the same time, normal mice (female Balb / c mice, 6 - 8 weeks old, 17 - 21 g) were used as the Control group (n = 6).

[0167] Two hours after injecting 50% glycerol, the mice in the RuCDzyme nanozyme group were injected with RuCDzyme nanozyme (dissolved in PBS buffer before injection) via the tail vein at a dose of 20 mg / kg body weight; two hours after injecting 50% glycerol, the mice in the TPP@RuCDzyme nanozyme group were injected with TPP@RuCDzyme nanozyme (dissolved in PBS buffer before injection) via the tail vein at a dose of 20 mg / kg body weight; two hours after injecting 50% glycerol, the mice in the AKI group were injected with the same volume of sterile PBS buffer via the tail vein; the mice in the Control group were directly injected with the same volume of sterile PBS buffer.

[0168] 3. Detection of renal function indicators:

[0169] The specific operation is as follows: Euthanize the mice in each group 24 hours after being treated according to the "experimental grouping and treatment method" described in step 2 above, and collect blood for detection. Place the whole blood in a 1.5 mL sterile EP tube, let it stand at 4°C for 1 - 2 hours, then centrifuge at 4°C (3000 rpm, 15 min), extract the upper serum and place it in a new EP tube, and use the creatinine (CRE) assay kit (C011 - 2 - 1) and blood urea nitrogen (BUN) assay kit (C013 - 2 - 1) from Nanjing Jiancheng Bioengineering Institute to detect the CRE value and BUN value in the serum. The detection results are as Figure 10 shown.

[0170] As Figure 10 can be seen, the CRE and BUN values of the mice in the AKI group were significantly higher than those of the mice in the Control group, confirming the successful construction of the glycerol - induced AKI model. For the mice with acute kidney injury treated with RuCDzyme and TPP@RuCDzyme nanozymes, different degrees of reduction in CRE and BUN were observed. In addition, compared with the RuCDzyme nanozyme group, the CRE and BUN in the TPP@RuCDzyme nanozyme group decreased most significantly, and there was no significant difference from the normal CRE and BUN values of the Control group, indicating that RuCDzyme nanozyme and TPP@RuCDzyme nanozyme have excellent effects in protecting kidney tissues and renal tubular cells from acute kidney injury caused by glycerol in vivo.

[0171] 4. H&E staining of kidney tissues

[0172] H&E staining is the most intuitive experimental method for observing kidney tissue damage. To further evaluate the therapeutic effects of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme on kidney organ damage in mice with acute kidney injury, euthanize the mice in each group 24 hours after being treated according to the "experimental grouping and treatment method" described in step 2 above, dissect and remove the kidneys of the mice, fix them with paraformaldehyde (4% PBS), embed them in paraffin, then section and stain the cytoplasm and nucleus with hematoxylin - eosin respectively. Finally, use an inverted fluorescence microscope from Zeiss to observe and take pictures. The results are as Figure 11 shown.

[0173] As Figure 11It can be seen that compared with the normal renal tubular structure in the Control group, obvious vacuolization of renal tubules, enlarged renal tubular lumen, and protein denaturation deposition in renal tubules were observed in the AKI group of mice. In the acute kidney injury mice treated with RuCDzyme and TPP@RuCDzyme nanozymes, varying degrees of alleviation of renal tubular morphological pathology were observed. In addition, compared with the RuCDzyme treatment group, the morphological pathology of renal tubules in the TPP@RuCDzyme nanozyme group changed most significantly, showing no obvious difference from the normal renal tubular structure in the Control group. This indicates that RuCDzyme nanozyme and TPP@RuCDzyme nanozyme have excellent effects in protecting kidney tissues and renal tubular cells from acute kidney injury caused by glycerol in vivo.

[0174] 5. ROS staining of kidney tissues

[0175] To further verify and analyze the ability of RuCDzyme nanozyme and TPP@RuCDzyme nanozyme to scavenge excessive ROS generated in kidney tissues, the mice in each group after being treated according to the "experimental grouping and treatment method" described in step 2 above for 24 hours were euthanized, and the kidneys of the mice were dissected and taken out. After the kidneys were taken out, they were stored in a refrigerator at -80°C. Using a cryostat (Thermo Cryotome E), cryosections were made at -20°C with a thickness of about 5 μm. Then the cryosections of renal tissues were slightly dried, stained with DHE, and incubated in a light-proof incubator at 37°C for 30 min. Subsequently, the slides of cryosections of renal tissues were placed in PBS (pH 7.4) and shaken on a shaker for washing 3 times, 5 min each time. After the sections were slightly dried, DAPI staining solution was added dropwise, and incubated at room temperature in the dark for 10 min, and then sealed with an anti-fluorescence quenching mounting medium. Observation and photography were carried out using a fluorescence microscope (Zeiss). The detection results are as Figure 12 shown.

[0176] As Figure 12 can be seen, the level of red fluorescence in the kidneys of the AKI group of mice was significantly higher than that in the Control group, further proving the successful establishment of the AKI mouse model induced by glycerol. In the acute kidney injury mice treated with RuCDzyme and TPP@RuCDzyme nanozymes, varying degrees of weakening of the red fluorescence level were observed. In addition, compared with the RuCDzyme nanozyme group, the red fluorescence level in the TPP@RuCDzyme nanozyme group weakened most significantly, showing no obvious difference from the red fluorescence level in the normal Control group. It was confirmed that RuCDzyme nanozyme and TPP@RuCDzyme nanozyme can protect mouse kidney tissues by scavenging excessive ROS in the mouse kidneys, and thus play an excellent role in protecting kidney tissues in vivo.

[0177] 6. Analysis of renal injury molecules and markers

[0178] Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein in renal proximal tubular epithelial cells, which is slightly expressed in normal liver, kidney and spleen, while its expression is significantly enhanced in regenerated proximal tubular epithelial cells after injury; heme oxygenase-1 (HO-1) is a cytoprotective enzyme, which is induced in cell injury and stress response and highly expressed in acute kidney injury, playing a protective role on the kidney; 8-hydroxydeoxyguanosine (8-OHDG) is an oxidative adduct produced by reactive oxygen free radicals attacking the 8th carbon atom of guanine base in DNA molecule, and is the most commonly used biomarker in DNA oxidative damage. Therefore, in this invention, the mice in each group after being treated according to the "experimental grouping and treatment method" described in step 2 above for 24 hours were euthanized, and the kidneys of the mice were dissected out. The expressions of biomarkers KIM-1 (Jiangsu Enzyme Immunoassay Industry Co., Ltd., MM-0318M2), HO-1 (Jiangsu Enzyme Immunoassay Industry Co., Ltd., MM-0473M2) and 8-OHdG (Jiangsu Enzyme Immunoassay Industry Co., Ltd., MM-0221M2) in kidney tissues were detected by ELISA method to further evaluate the protective effect of TPP@RuCDzyme nanozyme on the kidneys of mice with acute kidney injury. The detection results are as Figure 13 shown.

[0179] As Figure 13 can be seen, the values of KIM-1, HO-1 and 8-OHdG in the AKI group mice were significantly higher than those in the Control group mice, further verifying the successful construction of the AKI model. For the mice with acute kidney injury treated with RuCDzyme and TPP@RuCDzyme nanozymes, different degrees of decrease in the biomarkers KIM-1, HO-1 and 8-OHdG were observed. In addition, compared with the RuCDzyme nanozyme group, the levels of the biomarkers KIM-1, HO-1 and 8-OHdG in the TPP@RuCDzyme nanozyme group decreased most significantly, and there was no significant difference from the normal biomarker values in the Control group. It shows that TPP@RuCDzyme nanozyme exerts a therapeutic effect of protecting kidney tissues by protecting renal tubular cells in vivo, and can also protect intracellular DNA from oxidative stress damage.

[0180] (V) Safety evaluation of nanozyme

[0181] Histopathological examination observes the tissue samples of animals under a microscope to evaluate the effects of drugs on the structure and function of animal tissues. This is a very important part in the safety evaluation of drugs, which helps to discover tissue damage or lesions that drugs may cause. In order to evaluate the biosafety of TPP@RuCDzyme nanozyme, in this invention, the safety of the nanozyme on organs was investigated by H&E staining, and the body weight and blood biochemical indexes of mice

[0182] 1. Experimental grouping and treatment methods:

[0183] Balb / c female mice (6 - 8 weeks old, 17 - 21 g) were randomly divided into two groups, the TPP@RuCDzyme nanozyme group and the Control group, with 5 mice in each group. Mice in the TPP@RuCDzyme nanozyme group were injected with TPP@RuCDzyme nanozyme via the tail vein (after dissolving the TPP@RuCDzyme nanozyme in PBS buffer for injection), and the injection dose was 20 mg / kg body weight, injected twice a week with a 3 - day interval between each injection; mice in the Control group were injected with the same dose of sterile PBS buffer, twice a week with a 3 - day interval between each injection. Mice in both the TPP@RuCDzyme nanozyme group and the Control group were marked as day 1 starting from the first injection, and were normally raised after injection. On the 14th day, the mice were euthanized, and the organ tissues and blood of the mice were taken for detection and treatment.

[0184] 2. Investigation of the safety of major organs by H&E staining

[0185] After euthanizing the mice on the 14th day, the hearts, livers, spleens, lungs, and kidneys of the mice in the TPP@RuCDzyme nanozyme group and the Control group were taken for H&E staining, and the staining results are as Figure 14 shown.

[0186] As Figure 14 can be seen, the TPP@RuCDzyme nanozyme did not cause obvious pathological damage to the major tissues, indicating that the TPP@RuCDzyme nanozyme prepared in the present invention and the injection dose can be safely used in mice.

[0187] 2. Investigation of the biological safety of the drug by body weight and blood biochemistry

[0188] To evaluate the biological safety of the TPP@RuCDzyme nanozyme in mice, the changes in body weight of the mice in the TPP@RuCDzyme nanozyme group and the Control group were continuously monitored starting from the first day of injection, and the results are as Figure 15 shown in A.

[0189] As Figure 15 can be seen from A, there was no significant difference in the body weight change of the mice in the TPP@RuCDzyme nanozyme group compared with the Control group.

[0190] On the 14th day after treating the mice, blood samples of the mice in the TPP@RuCDzyme nanozyme group and the Control group were collected, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), blood urea nitrogen (BUN), white blood cells (WBC), lymphocytes (LYM), red blood cells (RBC), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), platelet distribution width (PDW), mean platelet volume (MPV), and red blood cell distribution width (RDW) in the blood were detected. The detection results are as Figure 15 shown in B-H in

[0191] As can be seen from Figure 15 B-H in, the examination of blood biochemical markers, including BUN, CRE, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), indicates that the liver and kidneys show normal functions, and the remaining biochemical indexes suggest that there is no significant difference between the TPP@RuCDzyme nanozyme group and the control group. This result indicates that the TPP@RuCDzyme nanozyme exhibits minimal toxicity and good biocompatibility in organisms.

[0192] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing RuCDzyme nanozyme, characterized in that: The following steps are involved: S1: dissolving carbon quantum dots and ruthenium metal salt in solvent A to obtain a mixed solution; S2: The pH of the mixed solution is adjusted to alkaline, sodium borohydride solution is added to the mixed solution, and the mixture is stirred for reaction for 3 h to 6 h. After the reaction is completed, the reaction solution is dialyzed to remove unreacted reactants and solvent A, and the dialyzed reaction solution is freeze-dried to obtain RuCDzyme nanozyme.

2. The preparation method according to claim 1, characterized in that The mass ratio of the carbon quantum dots to the ruthenium metal salt is (6-15):1; the mass ratio of the sodium borohydride to the ruthenium metal salt is (2-6):1; the ruthenium metal salt is ruthenium (III) chloride hydrate; and the solvent A is water.

3. The preparation method according to claim 1 or 2, characterized in that: The preparation method of the carbon quantum dots is as follows: glutathione and folic acid are added to solvent B, mixed to obtain a mixture, the mixture is reacted at 140-160° C. for 4-12 hours, the reaction solution is centrifuged, the supernatant is taken, the supernatant is dialyzed to remove unreacted reactants, and the dialyzed supernatant is ultrafiltered and freeze-dried to obtain carbon quantum dots.

4. The preparation method according to claim 3, characterized in that: The mass ratio of glutathione to folic acid is (15-25):1; the amount of solvent B is: 30ml-70ml of solvent B is added for every 1g of glutathione; and solvent B is formamide.

5. A RuCDzyme nanozyme prepared by the preparation method according to any one of claims 1 to 4.

6. A method for preparing TPP@RuCDzyme nanozyme, characterized in that: include: Add carbodiimide hydrochloride and N-hydroxysuccinimide to the TPP-COOH solution, stir and react at 25-30° C. for 30-60 minutes, then add the RuCDzyme nanozyme according to claim 7 to the reaction system, continue to stir and react for 16h-24h, dialyze the reaction solution after the reaction to remove unreacted reactants in the reaction solution, and freeze-dry the dialyzed reaction solution to obtain TPP@RuCDzyme nanozyme.

7. The preparation method according to claim 8, characterized in that: The mass ratio of the RuCDzyme nanozyme to TPP-COOH is (10-20):1; the molar ratio of the TPP-COOH to carbodiimide hydrochloride is 1:(1-1.5); and the molar ratio of the TPP-COOH to N-hydroxysuccinimide is 1:(1-1.5).

8. A TPP@RuCDzyme nanozyme prepared by the preparation method according to claim 8 or 9.

9. Use of the RuCDzyme nanozyme according to claim 7 or the TPP@RuCDzyme nanozyme according to claim 10 in the preparation of a drug for preventing, alleviating or / and treating renal injury.

10. A drug for preventing, alleviating or / and treating renal injury, characterized in that: The drug comprises the RuCDzyme nanozyme according to claim 5 or the TPP@RuCDzyme nanozyme according to claim 8.

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