Trimetal nano enzyme as well as preparation method and application thereof

Abstract

The invention provides a trimetal nano enzyme as well as a preparation method and application thereof. The ABC trimetal nano-enzyme disclosed by the invention has multi-enzyme activities of glucose oxidase, peroxidase, catalase, glutathione peroxidase, nicotinamide adenine dinucleotide phosphate oxidase and the like, and can be used for enhancing the catalytic treatment on tumors. In a tumor microenvironment, the component A has glucose oxidase-like activity, and can catalyze endogenous glucose to generate hydrogen peroxide to enhance the generation of hydroxyl radicals; the component B has peroxidase-like and glutathione-like peroxidase activity, and can generate hydroxyl radicals and consume glutathione; the component C has peroxidase-like activity and catalase-like activity and can generate hydroxyl radicals, and generated oxygen can improve the glucose oxidase-like activity of the component A. In addition, the component BC has the activity of nicotinamide adenine dinucleotide phosphate oxidase, and oxygen can be catalyzed to generate superoxide anions by utilizing the excellent electron transmission characteristic of the interface of the component BC.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozymes, and in particular to a trimetallic nanozyme and a preparation method and application thereof. Background Art

[0002] Nanozymes are a class of nanomaterials with enzyme-like catalytic activity. In particular, precious metal nanozymes can catalyze hydrogen peroxide to produce toxic hydroxyl radicals. They have the advantages of high catalytic activity and stable performance, and have potential application prospects in tumor catalytic therapy. However, traditional precious metal nanozymes have a single enzyme activity, and the excess glutathione and insufficient hydrogen peroxide in the tumor microenvironment severely limit the production of hydroxyl radicals. If precious metal nanozymes with multi-enzyme activity can be constructed, it can not only increase the concentration of hydrogen peroxide in the tumor microenvironment, but also consume excess glutathione, thereby improving the catalytic therapeutic performance. Summary of the invention

[0003] The purpose of the present invention is to provide a trimetallic nanozyme and a preparation method and application thereof, so as to solve the problem that the catalytic therapeutic performance is limited due to the single enzyme-like activity of a single metal nanozyme.

[0004] The present invention is achieved in that:

[0005] A trimetallic nanozyme, the structure of the nanozyme is ABC, wherein A is gold (Au), B is iron (Fe), copper (Cu), molybdenum (Mo) or manganese (Mn), and C is platinum (Pt), palladium (Pd) or iridium (Ir).

[0006] The trimetallic nanozyme provided by the present invention is a trimetallic nanozyme having multiple enzyme activities such as glucose oxidase-like, peroxidase-like, catalase-like, glutathione peroxidase-like and nicotinamide adenine dinucleotide phosphate oxidase-like.

[0007] The trimetallic nanozyme is prepared according to the following method:

[0008] 0.01-0.1mmol chloroauric acid, 0.01-0.1mmol copper nitrate (or ferric nitrate, or manganese chloride, or sodium molybdate) and 0.01-0.1mmol chloroplatinic acid (or sodium chloropalladate, or chloroiridic acid) are dissolved in 50mL deionized water; under stirring conditions, 0.05-0.2g potassium bromide is added, and the pH value of the system is adjusted to between 5-6 with hydrochloric acid; then, 0.01-0.1g ascorbic acid (or tannic acid) is added, and the reaction is stirred at 80-150°C for 1-4 hours; finally, the product is cooled to room temperature, centrifuged, washed with water and dispersed in 50mL deionized water to obtain the trimetallic nanozyme.

[0009] Furthermore, the amount of chloroauric acid used is preferably 0.02-0.04 mmol; the amount of copper nitrate (or ferric nitrate, or manganese chloride, or sodium molybdate) used is preferably 0.05-0.08 mmol; the amount of chloroplatinic acid (or sodium chloropalladate, or chloroiridic acid) used is preferably 0.02-0.04 mmol.

[0010] Furthermore, the dosage of potassium bromide is preferably 0.05-0.12 g, and the dosage of ascorbic acid (or tannic acid) is preferably 0.02-0.05 g.

[0011] Furthermore, the reaction temperature is preferably 90-120° C., and the reaction time is preferably 2-3 hours.

[0012] The ABC trimetal nanozyme of the present invention has multi-enzyme activities such as glucose oxidase, peroxidase, catalase, glutathione peroxidase and nicotinamide adenine dinucleotide phosphate oxidase, which can enhance the catalytic treatment of tumors. In the tumor microenvironment, component A has glucose oxidase activity, which can catalyze endogenous glucose to produce hydrogen peroxide to enhance the production of hydroxyl radicals; component B has peroxidase and glutathione peroxidase activities, which can produce hydroxyl radicals, and can consume glutathione to reduce the removal of hydroxyl radicals; component C has peroxidase and catalase activities, which can produce hydroxyl radicals, and the generated oxygen can increase the glucose oxidase activity of component A and then enhance the peroxidase and catalase activities. In addition, component BC has the activity of nicotinamide adenine dinucleotide phosphate oxidase, and the excellent electron transfer characteristics of the BC component interface can catalyze oxygen to produce superoxide anions, and the production of superoxide anions does not depend on the concentration of hydrogen peroxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a transmission electron microscope image of AuCuIr prepared in Example 1.

[0014] Figure 2 This is the UV-visible absorption spectrum of AuMoPd prepared in Example 2 after incubation with potassium permanganate in the presence of glucose.

[0015] Figure 3 This is the UV-visible absorption spectrum of AuMnPt prepared in Example 3 after incubation with methylene blue in the presence of hydrogen peroxide.

[0016] Figure 4 This is the oxygen concentration variation curve of AuFeIr prepared in Example 4 in the presence of hydrogen peroxide.

[0017] Figure 5This is a UV-visible absorption spectrum of AuMoIr prepared in Example 5 after incubation with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of glutathione.

[0018] Figure 6 This is a UV-visible absorption spectrum of AuCuPd prepared in Example 6 after incubation with 3,3',5,5'-tetramethylbenzenediamine in the absence of hydrogen peroxide. DETAILED DESCRIPTION

[0019] Example 1 Preparation of AuCuIr

[0020] 0.02 mmol chloroauric acid, 0.05 mmol copper nitrate and 0.02 mmol chloroiridic acid were dissolved in 50 mL deionized water; under stirring conditions, 0.05 g potassium bromide was added, and the pH value of the system was adjusted to between 5 and 6 with hydrochloric acid; then, 0.02 g ascorbic acid was added, and the reaction was stirred at 90°C for 2 hours; finally, the product was cooled to room temperature, centrifuged, washed with water and dispersed in 50 mL deionized water to obtain AuCuIr trimetallic nanozyme.

[0021] The prepared AuCuIr nanozyme was characterized by transmission electron microscopy. Figure 1 .from Figure 1 As can be seen from the figure, AuCuIr is spherical with an average size of about 40 nanometers.

[0022] Example 2 Preparation of AuMoPd

[0023] 0.03mmol chloroauric acid, 0.08mmol sodium molybdate and 0.03mmol sodium chloropalladate were dissolved in 50mL deionized water; under stirring conditions, 0.08g potassium bromide was added, and the pH value of the system was adjusted to between 5-6 with hydrochloric acid; then, 0.04g tannic acid was added, and the reaction was stirred at 120°C for 3 hours; finally, the product was cooled to room temperature, centrifuged, washed with water and dispersed in 50mL deionized water to obtain AuMoPd trimetallic nanozyme.

[0024] 100 μL of glucose solution (2.5 mg / mL) and 100 μL of potassium permanganate solution (0.5 mg / mL) were added to 1 mL of phosphate buffer (pH = 6.5) with different concentrations of AuMoPd (0, 50, 100, 150 and 200 μg / mL); after incubation for 30 minutes, the mixture was centrifuged to obtain the supernatant, and the UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The results are as follows: Figure 2 .from Figure 2It can be seen that with the increase of AuMoPd concentration, the absorbance of potassium permanganate incubated with it gradually decreases, indicating that AuMoPd can catalyze glucose to produce hydrogen peroxide and undergo redox reaction with potassium permanganate, thereby reducing the absorbance of potassium permanganate.

[0025] Example 3 Preparation of AuMnPt

[0026] 0.04 mmol chloroauric acid, 0.06 mmol manganese chloride and 0.04 mmol chloroplatinic acid were dissolved in 50 mL deionized water; under stirring conditions, 0.12 g potassium bromide was added, and the pH value of the system was adjusted to between 5 and 6 with hydrochloric acid; then, 0.05 g ascorbic acid was added, and the reaction was stirred at 100°C for 3 hours; finally, the product was cooled to room temperature, centrifuged, washed with water and dispersed in 50 mL deionized water to obtain AuMnPt trimetallic nanozyme.

[0027] 100 μL of hydrogen peroxide solution (10 mmol / L) and 100 μL of methylene blue solution (0.5 mg / mL) were added to 1 mL of phosphate buffer (pH = 6.5) with different concentrations of AuMnPt (0, 50, 100, 150 and 200 μg / mL); after incubation for 30 minutes, the mixture was centrifuged to obtain the supernatant, and the UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The results are as follows: Figure 3 .from Figure 3 It can be seen that with the increase of AuMnPt concentration, the absorbance of methylene blue incubated with it gradually decreases, indicating that AuMnPt can catalyze hydrogen peroxide to produce hydroxyl radicals and react with methylene blue, thereby reducing the absorbance of methylene blue.

[0028] Example 4 Preparation of AuFeIr

[0029] 0.02 mmol chloroauric acid, 0.07 mmol ferric nitrate and 0.02 mmol chloroiridic acid were dissolved in 50 mL deionized water; under stirring conditions, 0.08 g potassium bromide was added, and the pH value of the system was adjusted to between 5 and 6 with hydrochloric acid; then, 0.03 g ascorbic acid was added, and the reaction was stirred at 110°C for 2.5 hours; finally, the product was cooled to room temperature, centrifuged, washed with water and dispersed in 50 mL deionized water to obtain AuFeIr trimetallic nanozyme.

[0030] 100 microliters of hydrogen peroxide solution (10 mmol / L) was added to 1 ml of phosphate buffer (pH = 6.5) with different concentrations of AuFeIr (0, 50, 100, 150 and 200 μg / mL); the change in oxygen concentration within 30 minutes was monitored using a dissolved oxygen meter. The results are as follows: Figure 4 .from Figure 4It can be seen that with the increase of AuFeIr concentration, the oxygen concentration gradually increases, indicating that AuFeIr can catalyze hydrogen peroxide to produce oxygen.

[0031] Example 5 Preparation of AuMoIr

[0032] 0.04 mmol chloroauric acid, 0.08 mmol sodium molybdate and 0.04 mmol chloroiridic acid were dissolved in 50 mL deionized water; under stirring conditions, 0.1 g potassium bromide was added, and the pH value of the system was adjusted to between 5 and 6 with hydrochloric acid; then, 0.05 g tannic acid was added, and the reaction was stirred at 120°C for 3 hours; finally, the product was cooled to room temperature, centrifuged, washed with water and dispersed in 50 mL deionized water to obtain AuMoIr trimetallic nanozyme.

[0033] 1 ml of glutathione (1 mmol / L) was added to 1 ml of phosphate buffer (pH = 6.5) with different concentrations of AuMoIr (0, 50, 100, 150 and 200 μg / mL); after incubation for 30 minutes, the mixture was centrifuged to obtain the supernatant, and 10 ml of 5,5'-dithiobis(2-nitrobenzoic acid) (0.1 mmol / L) was added; the UV-visible absorption spectrum was measured using a UV-visible spectrophotometer, and the results were as follows: Figure 5 .from Figure 5 It can be seen that with the increase of AuMoIr concentration, the absorbance of 2-nitro-5-thiobenzoic acid gradually decreases, indicating that AuMoIr can consume glutathione and reduce the absorbance of 2-nitro-5-thiobenzoic acid.

[0034] Example 6 Preparation of AuCuPd

[0035] 0.03 mmol chloroauric acid, 0.06 mmol copper nitrate and 0.03 mmol sodium chloropalladate were dissolved in 50 mL deionized water; 0.07 g potassium bromide was added under stirring, and the pH value of the system was adjusted to between 5 and 6 with hydrochloric acid; then, 0.04 g tannic acid was added, and the reaction was stirred at 110°C for 2 hours; finally, the product was cooled to room temperature, centrifuged, washed with water and dispersed in 50 mL deionized water to obtain AuCuPd trimetallic nanozyme.

[0036] 100 microliters of 3,3',5,5'-tetramethylbenzidine (0.5 mg / mL) was added to 1 ml of phosphate buffer (pH = 6.5) with different concentrations of AuCuPd (0, 50, 100, 150 and 200 μg / mL); after incubation for 30 minutes, the mixture was centrifuged to obtain the supernatant, and the UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The results are as follows: Figure 6 .from Figure 6It can be seen that with the increase of AuCuPd concentration, the absorbance of 3,3',5,5'-tetramethylbenzidine incubated with it gradually increases, indicating that AuCuPd can catalyze oxygen to produce superoxide anions and react with 3,3',5,5'-tetramethylbenzidine, increasing the absorbance of 3,3',5,5'-tetramethylbenzidine.

Claims

1. A trimetallic nanozyme, characterized in that: The structure of the trimetallic nanozyme is ABC, wherein A is gold, B is iron, copper, molybdenum or manganese, and C is platinum, palladium or iridium.

2. The trimetallic nanozyme according to claim 1, characterized in that: The trimetallic nanozyme is a trimetallic nanozyme having glucose oxidase-like, peroxidase-like, catalase-like, glutathione peroxidase-like and nicotinamide adenine dinucleotide phosphate oxidase-like properties.

3. The method for preparing the trimetallic nanozyme according to claim 1 or 2, characterized in that: The steps include: (1) preparing raw materials containing A, B, and C, that is, preparing 0.01-0.1 mmol chloroauric acid, 0.01-0.1 mmol copper nitrate, ferric nitrate, manganese chloride or sodium molybdate, and 0.01-0.1 mmol chloroplatinic acid, sodium chloropalladate or chloroiridic acid, and dissolving the three in 50 mL of deionized water; (2) stirring the mixed solution in step (1), adding 0.05-0.2 g of potassium bromide under stirring conditions, and adjusting the pH value of the system to between 5 and 6 with hydrochloric acid; (3) adding 0.01-0.1 g of ascorbic acid or tannic acid, stirring and reacting at 80-150° C. for 1-4 hours; (4) Finally, the product was cooled to room temperature, centrifuged, washed and dispersed in deionized water to obtain trimetallic nanozymes.

4. The method for preparing the trimetallic nanozyme according to claim 3, characterized in that: In step (1), the amount of chloroauric acid is 0.02-0.04 mmol; the amount of copper nitrate, ferric nitrate, manganese chloride or sodium molybdate is 0.05-0.08 mmol; the amount of chloroplatinic acid, sodium chloropalladate or chloroiridic acid is 0.02-0.04 mmol.

5. The method for preparing the trimetallic nanozyme according to claim 3, characterized in that: In step (2), the amount of potassium bromide used is 0.05-0.12 g.

6. The method for preparing the trimetallic nanozyme according to claim 3, characterized in that: In step (3), the amount of ascorbic acid or tannic acid used is 0.02-0.05 g.

7. The method for preparing the trimetallic nanozyme according to claim 3, characterized in that: In step (3), the reaction temperature is 90-120° C. and the reaction time is 2-3 hours.

8. Use of the trimetallic nanozyme according to claims 1 to 2 and the trimetallic nanozyme prepared by the method according to any one of claims 3 to 7 in tumor catalytic therapy.

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