High-entropy nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a high-entropy nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of functional materials. Five metal complexes including ferric acetylacetonate, aluminum acetylacetonate, nickel acetylacetonate, manganese acetylacetonate and copper acetylacetonate are subjected to a pyrolytic oxidation-reduction reaction in an oleylamine solution containing a surfactant and glucose, and then the high-entropy nano material is obtained. And the prepared high-entropy nano material has activities similar to peroxidase, glutathione and catalase, and has a good medical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of functional materials technology, specifically relating to a high-entropy nanozyme, its preparation method, and its applications. Background Technology

[0002] High-entropy alloys are a novel type of alloy material, typically composed of five or more metals, each with a content ranging from 5% to 35%. Due to the synergistic effect among the various metal elements, high-entropy alloys often exhibit unique physicochemical properties, such as high strength, high hardness, good corrosion resistance, and thermal stability. In the biomedical field, high-entropy alloy nanomaterials have gradually attracted attention due to their excellent catalytic properties. They can serve as mimics of various natural enzymes, demonstrating superior performance in biosensing, antibacterial applications, and tumor treatment.

[0003] Nanozymes, as nanomaterials with catalytic activity similar to natural enzymes, have so far been mainly prepared based on high-entropy alloys using noble metal elements. Since noble metal elements themselves possess excellent catalytic activity, these nanozymes can achieve higher catalytic effects. However, traditional preparation methods (such as high-energy ball milling and vacuum casting) still face technical drawbacks such as easy phase separation, nanoparticle agglomeration, and unclear catalytic mechanisms, limiting their precise application in the field of functional materials. Therefore, developing new methods for preparing high-entropy nanozymes is not only of great value for the preparation of high-entropy alloys but also plays a crucial role in the development of nanozyme science. Summary of the Invention

[0004] To address the aforementioned shortcomings in the prior art, this invention provides a high-entropy nanoenzyme, its preparation method, and its applications. This invention involves subjecting five metal complexes—iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone—to a pyrolysis-redox reaction in an oleylamine solution containing a surfactant and glucose, thereby obtaining high-entropy nanomaterials. The prepared high-entropy nanomaterials possess peroxidase-like, glutathione-like, and catalase-like activities.

[0005] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a method for preparing high-entropy nanozymes, comprising the following steps: (1) Add the surfactant to the solvent, dissolve it by sonication, then add a mixture of iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone and copper acetylacetone, sonicate in a water bath for 1-3 hours, then add the reducing agent and continue sonicating for 1-2 hours. (2) In a protective gas atmosphere, the solution after sonication in step (1) is replaced with air, then stirred at 80~100℃ for 30~50min, then heated to 200~220℃, and continued to react for 5~8h, and then cooled to room temperature; (3) Wash and centrifuge with ethanol-cyclohexane mixed solution, collect the precipitate and dry it, then disperse it in buffer solution, sonicate it and add dopamine solution, stir and react in the dark for 2-4 hours, wash and centrifuge to collect the precipitate, and dry it to obtain dopamine-encapsulated high-entropy nanomaterials. (4) Disperse the dopamine-encapsulated high-entropy nanomaterials in a buffer solution, sonicate, add SH-PEG-NH2 solution, stir in the dark for 10-12 h, wash, centrifuge, collect the precipitate, and dry to obtain PEG-encapsulated high-entropy nanomaterials. (5) Disperse the dopamine-encapsulated high-entropy nanomaterials in a solvent, then add the activated indomethacin solution, stir in the dark for 10-12 hours, wash and centrifuge to collect the precipitate, and dry to obtain indomethacin-modified high-entropy nanomaterials.

[0006] Furthermore, the surfactant used in step (1) is hexadecyltrimethylammonium bromide, and the solvent is oleylamine.

[0007] Furthermore, in step (1), the mass ratio of iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone and copper acetylacetone in the solution is 0.5~1:0.5~1:1:0.1~0.5:0.1~0.5.

[0008] Furthermore, in step (1), an excess of reducing agent glucose is added, the amount of which is 10 times the total molar amount of the metal.

[0009] Furthermore, in step (3), the mass concentration of the dispersed high-entropy nanomaterial is 0.5~1 mg / mL, and the volume and mass concentration of the added dopamine solution are the same.

[0010] Furthermore, the dopamine is dopamine hydrochloride.

[0011] Furthermore, in step (3), ethanol and cyclohexane form a mixed solution in a volume ratio of 8 to 10:1.

[0012] Further, in step (4), the mass concentration of the high-entropy nanomaterials encapsulated with dopamine dispersed in the solution is 0.2~0.5 mg / mL, and then an SH-PEG-NH2 solution with the same volume and mass concentration is added.

[0013] Furthermore, the buffer solution used in steps (3) and (4) is Tris-HCl with a pH of 8 to 8.5.

[0014] Furthermore, the mass concentration of the PEG-encapsulated high-entropy nanomaterial solution obtained in step (4) is 8~10 mg / mL.

[0015] Furthermore, in step (5), indomethacin is activated using EDC / NHS, and the mass concentration of the activated indomethacin solution is 2~5 mg / mL; its volume-to-mass ratio with the PEG-encapsulated high-entropy nanomaterial is 2:1.

[0016] Another object of the present invention is to provide a high-entropy nanozyme prepared by the above method.

[0017] Another object of the present invention is to provide the use of the above-mentioned high-entropy nanozymes in the preparation of tumor therapeutic drugs.

[0018] The beneficial effects of this invention are: The method described in this invention has yielded a uniform high-entropy nanozyme. The high-entropy nanozyme prepared using this method has advantages such as simplicity, a nanoparticle size of 120 nm, and adjustable nanoalloy density. Furthermore, the reaction system does not require the addition of excess solvent, achieving the requirements of green and environmentally friendly practices. In addition, the high-entropy nanomaterials prepared by this method exhibit excellent peroxidase-like and glutathionease-like activities. While catalyzing the generation of highly cytotoxic hydroxyl radicals from hydrogen peroxide, they can also consume glutathione to induce ferroptosis. The prepared high-entropy nanozyme has the potential for tumor treatment, thus showing great promise for medical applications and further promoting research related to nanozymes and high-entropy alloys.

[0019] Furthermore, due to the unique electron orbital arrangement of transition metals, they possess a large number of unpaired electrons, making them highly efficient in electron transfer and well-suited for magnetic resonance imaging. Therefore, this invention selects four transition metals—iron, nickel, manganese, and copper—emphasizing iron content as the element source for catalysis and imaging, and aluminum as the element to enhance alloy strength, thus constructing a high-entropy nanozyme. However, tumor tissues exhibit considerable immune resistance and oxidative stress defenses. To reduce unnecessary damage to normal tissues caused by the high-entropy nanozyme, it was further modified. This modification improves biosafety, and the indomethacin modification reduces COX-2 production in tumor tissues due to oxidative stress, thereby improving immunosuppression in tumor tissues. This, in turn, promotes immunogenic ferroptosis, further killing tumor cells. Attached Figure Description

[0020] Figure 1 This is a scanning electron microscope (SEM) image of the high-entropy nanozyme prepared in this invention. Figure 2 This is a transmission electron microscope (TEM) image of the high-entropy nanozyme prepared in this invention; Figure 3X-ray energy dispersive spectroscopy (EDS) image of the high-entropy nanozyme prepared in this invention; Figure 4 Study on the peroxidase-like enzyme activity of the high-entropy nanozyme prepared in this invention; Figure 5 Study on the glutathionein-like enzyme activity of the high-entropy nanozyme prepared in this invention; Figure 6 Magnetic resonance T2 imaging effects of different high-entropy nanozymes prepared in this invention; Figure 7 The effect of the high-entropy nanozyme prepared in this invention on the generation of reactive oxygen species in HUCCT1 cells; Figure 8 The consumption of glutathione in HUCCT1 cells by the high-entropy nanozyme prepared in this invention; Figure 9 The high-entropy nanozyme prepared in this invention demonstrates its magnetic resonance T2 imaging capability in subcutaneous xenograft tumors. Detailed Implementation

[0021] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0022] Example 1 A method for preparing a high-entropy nanozyme includes the following steps: (1) First, the surfactant cetyltrimethylammonium bromide was added to oleylamine and treated in a 250W water bath ultrasonic machine for 30 minutes until fully dissolved, which was used as the first solution. Then, five compounds, iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone, were added to the first solution. The mass ratio of the mixed solution was: iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone = 0.72:0.78:1:0.23:0.23. The solution was treated in a 250W water bath ultrasonic machine for 1 hour, and then excess glucose was added as a reducing agent and ultrasonication was continued for 1 hour to obtain a homogeneous solution.

[0023] (2) Transfer the above homogeneous solution to a double-necked flask, replace the air three times using a double-row tube, and protect it with argon gas; then place the double-necked flask on a heatable magnetic stirrer, stir for 30 minutes at 80°C and 400 rpm, then raise the temperature to 220°C and continue the reaction for 6 hours.

[0024] (3) After the reaction was completed, the product was allowed to cool naturally to room temperature. Then, it was washed with a mixed solution of ethanol:cyclohexane = 9:1 and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60°C and dried for 12 hours to obtain high-entropy nanomaterials.

[0025] (4) The obtained high-entropy material was dispersed in Tris-HCl with pH 8.5 to make its mass-volume concentration 0.5 mg / mL. It was treated in a water bath ultrasonic machine with a power of 250 W for 1 hour. Then it was transferred to a single-necked flask and the same mass-volume dopamine hydrochloride solution was added. The mixture was stirred open and protected from light at 400 rpm for 2 hours. Then it was washed with deionized water and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain dopamine-encapsulated high-entropy nanomaterials.

[0026] (5) The dopamine-encapsulated high-entropy nanomaterials were dispersed in Tris-HCl at pH 8.5 to a mass-volume concentration of 0.5 mg / mL. The mixture was treated in a water bath ultrasonic machine with a power of 250 W for 10-20 minutes. Then, it was transferred to a single-necked flask and 5 times the mass-volume of SH-PEG-NH2 (molecular weight 2000 MW) solution was added. The mixture was stirred at 400 rpm in the dark for 12 hours. The mixture was then washed with deionized water and centrifuged at 10000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain PEG-encapsulated high-entropy nanomaterials.

[0027] (6) 25 mg indomethacin, 30 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 25 mg N-hydroxysuccinimide were dissolved in 10 ml of anhydrous dimethylformamide and stirred at 400 rpm in the dark for 4 hours to activate the carboxyl group of indomethacin as the second solution. The PEG-coated high-entropy nanomaterials were dispersed in anhydrous dimethylformamide to a mass-volume concentration of 10 mg / mL as the third solution. The third solution was added to the second solution and stirred at 400 rpm in the dark for 12 hours. Then, the mixture was washed with anhydrous ethanol and deionized water and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain indomethacin-modified high-entropy nanomaterials (HE-IMC).

[0028] Example 2 A method for preparing a high-entropy nanozyme includes the following steps: (1) First, the surfactant cetyltrimethylammonium bromide was added to oleylamine and treated in a 250W water bath ultrasonic machine for 30 minutes until fully dissolved, which was used as the first solution. Then, five compounds, iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone, were added to the first solution. The mass ratio of the mixed solution was: iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone = 0.5:0.5:1:0.1:0.1. The solution was treated in a 250W water bath ultrasonic machine for 1 hour, and then excess glucose was added as a reducing agent and ultrasonication was continued for 1 hour to obtain a homogeneous solution.

[0029] (2) Transfer the above homogeneous solution to a double-necked flask, replace the air three times using a double-row tube, and protect it with argon gas; then place the double-necked flask on a heatable magnetic stirrer, stir for 30 minutes at 80°C and 400 rpm, then raise the temperature to 220°C and continue the reaction for 6 hours.

[0030] (3) After the reaction was completed, the product was allowed to cool naturally to room temperature. Then, it was washed with a mixed solution of ethanol:cyclohexane = 9:1 and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60°C and dried for 12 hours to obtain high-entropy nanomaterials.

[0031] (4) The obtained high-entropy material was dispersed in Tris-HCl with pH 8.5 to make its mass-volume concentration 0.7 mg / mL. It was treated in a water bath ultrasonic machine with a power of 250 W for 1 hour. Then it was transferred to a single-necked flask and the same mass-volume dopamine hydrochloride solution was added. The mixture was stirred open and protected from light at 400 rpm for 2 hours. Then it was washed with deionized water and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain dopamine-encapsulated high-entropy nanomaterials.

[0032] (5) The dopamine-encapsulated high-entropy nanomaterials were dispersed in Tris-HCl at pH 8.5 to a mass-volume concentration of 0.3 mg / mL. The mixture was treated in a water bath ultrasonic machine with a power of 250 W for 10-20 minutes. Then, it was transferred to a single-necked flask and 5 times the mass-volume of SH-PEG-NH2 (molecular weight 2000 MW) solution was added. The mixture was stirred at 400 rpm in the dark for 12 hours. The mixture was then washed with deionized water and centrifuged at 10000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain PEG-encapsulated high-entropy nanomaterials.

[0033] (6) 20 mg indomethacin, 30 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 25 mg N-hydroxysuccinimide were dissolved in 10 ml of anhydrous dimethylformamide and stirred at 400 rpm in the dark for 4 hours to activate the carboxyl group of indomethacin as the second solution. The PEG-coated high-entropy nanomaterials were dispersed in anhydrous dimethylformamide to a mass-volume concentration of 8 mg / mL as the third solution. The third solution was added to the second solution and stirred at 400 rpm in the dark for 12 hours. Then, the mixture was washed with anhydrous ethanol and deionized water and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain indomethacin-modified high-entropy nanomaterials (HE-IMC).

[0034] Example 3 A method for preparing a high-entropy nanozyme includes the following steps: (1) First, the surfactant cetyltrimethylammonium bromide was added to oleylamine and treated in a 250W water bath ultrasonic machine for 30 minutes until fully dissolved, which was used as the first solution. Then, five compounds, iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone, were added to the first solution. The mass ratio of the mixed solution was: iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone, and copper acetylacetone = 0.8:0.82:1:0.5:0.5. The solution was treated in a 250W water bath ultrasonic machine for 1 hour, and then excess glucose was added as a reducing agent and ultrasonication was continued for 1 hour to obtain a homogeneous solution.

[0035] (2) Transfer the above homogeneous solution to a double-necked flask, replace the air three times using a double-row tube, and protect it with argon gas; then place the double-necked flask on a heatable magnetic stirrer, stir for 30 minutes at 80°C and 400 rpm, then raise the temperature to 220°C and continue the reaction for 6 hours.

[0036] (3) After the reaction was completed, the product was allowed to cool naturally to room temperature. Then, it was washed with a mixed solution of ethanol:cyclohexane = 9:1 and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60°C and dried for 12 hours to obtain high-entropy nanomaterials.

[0037] (4) The obtained high-entropy material was dispersed in Tris-HCl with pH 8.5 to a mass-volume concentration of 1 mg / mL. It was then treated in a water bath ultrasonic machine with a power of 250 W for 1 hour. Subsequently, it was transferred to a single-necked flask and the same mass-volume dopamine hydrochloride solution was added. The mixture was stirred open and protected from light at 400 rpm for 2 hours. Then, it was washed with deionized water and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain dopamine-encapsulated high-entropy nanomaterials.

[0038] (5) The dopamine-encapsulated high-entropy nanomaterials were dispersed in Tris-HCl at pH 8.5 to a mass-volume concentration of 0.5 mg / mL. The mixture was treated in a water bath ultrasonic machine with a power of 250 W for 10-20 minutes. Then, it was transferred to a single-necked flask and 5 times the mass-volume of SH-PEG-NH2 (molecular weight 2000 MW) solution was added. The mixture was stirred at 400 rpm in the dark for 12 hours. The mixture was then washed with deionized water and centrifuged at 10000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain PEG-encapsulated high-entropy nanomaterials.

[0039] (6) 25 mg indomethacin, 30 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 25 mg N-hydroxysuccinimide were dissolved in 10 ml of anhydrous dimethylformamide and stirred at 400 rpm in the dark for 4 hours to activate the carboxyl group of indomethacin as the second solution. The PEG-coated high-entropy nanomaterials were dispersed in anhydrous dimethylformamide to a mass-volume concentration of 10 mg / mL as the third solution. The third solution was added to the second solution and stirred at 400 rpm in the dark for 12 hours. Then, the mixture was washed with anhydrous ethanol and deionized water and centrifuged at 10,000 rpm to obtain the precipitate. The product was then transferred to a vacuum drying oven at 60 °C and dried for 12 hours to obtain indomethacin-modified high-entropy nanomaterials (HE-IMC).

[0040] Test case 1. The TEM image of the high-entropy nanozyme prepared in Example 1 of this invention is shown below. Figure 1 As shown, the STEM diagram is as follows. Figure 2 As shown in the figure, the ESD diagram is as follows Figure 3 As shown.

[0041] Figure 1 and Figure 2 The high-entropy nanozyme is shown to have a size of approximately 120 nm. For example... Figure 3 As shown, ESD characterization indicates that this high-entropy nanozyme contains five elements: iron, aluminum, nickel, and manganese.

[0042] 2. High-entropy nanozymes were dispersed in ultrapure water to achieve a mass-volume concentration of 1 mg / mL. The solution was then sonicated at 250 W for 10–30 min to obtain a high-entropy nanozyme solution. Performance tests, including peroxidase-like activity and glutathionease-like activity, were then performed as follows: (1) Peroxidase-like activity test 20 μL of a 5 mg / mL solution of 3,3',5,5'-tetramethylbenzidine (TMB) was added to a phosphate-buffered saline (PBS) buffer solution at pH 5.4. Then, 5–25 μL of a high-entropy nanozyme solution was added, followed by 20 μL of hydrogen peroxide. The UV absorbance changes of the reaction solution were then measured using a UV spectrophotometer to determine the enzyme's catalytic activity. The results are shown below. Figure 4 .

[0043] like Figure 4 As shown, the absorbance of TMB at 525 nm increases with the concentration of hydrogen peroxide, indicating that this nanozyme has a good POD-like effect and can rapidly catalyze the production of reactive oxygen species from hydrogen peroxide.

[0044] (2) Glutathioneinase activity test 10 μL of high-entropy nanozyme solution was added to 30 μL of PBS buffer solution with a pH of 5.4, followed by 20 μL of 5 mM glutathione solution. After reacting for different times, 10 μL of 10 mM DTNB was added. The solution was then diluted 10-fold with PBS, and the absorbance change was measured using a UV spectrophotometer. The enzyme catalytic activity was measured by measuring the UV absorption change of the reaction solution using a UV spectrophotometer. The results are shown in [Figure number missing]. Figure 5 .

[0045] like Figure 5 As shown, the amount of glutathione remaining gradually decreases with increasing reaction time, indicating that this nanozyme has good GPx-like enzyme activity.

[0046] (3) Magnetic resonance T2 relaxation rate test Due to their strong ferromagnetism, high-entropy nanozymes were dispersed in 4% xanthan gum. The mass-volume concentrations of the high-entropy nanozymes were set at 0, 25, 50, 100, 150, 200, and μg / ml, and then packaged in EP tubes. Imaging was then performed using a clinical 3.0T MRI scanner. Furthermore, products obtained by replacing iron acetylacetone with vanadium acetylacetone, chromium acetylacetone, and platinum acetylacetone, respectively, were prepared at the same concentrations and imaged using a clinical 3.0T MRI scanner. The results are shown in [Figure number missing]. Figure 6 .

[0047] like Figure 6As shown, when iron is replaced by platinum, vanadium, or chromium, its magnetic resonance T2 imaging capability decreases significantly, indicating that iron plays an important role in nanozyme T2 imaging.

[0048] Based on the above tests, the high-entropy nanozyme material prepared by the method of this invention exhibits highly efficient peroxidase-like and glutathionease-like catalytic activities, enabling it to rapidly catalyze hydrogen peroxide and generate hydroxyl radicals. Furthermore, due to the unique element selection, it can significantly shorten the magnetic resonance T2 relaxation time, resulting in a significant negative T2 enhancement contrast.

[0049] 3. Cell validation (1) HUCCT1 cells were seeded in 6-well plates and cultured for 24 h. The culture medium was then removed, and the cells were gently washed three times with PBS. The cells were then divided into four groups: control group, laser group, HE-IMC (100 μg / ml) group, and HE-IMC (100 μg / ml) + laser group. All cells were co-cultured for another 6 h. After 6 h, the light-illuminated groups were irradiated with a 1064 nm (1 W / cm²) laser for 5 min, followed by staining with medium containing DCFH-DA (10 μM) for 30 min. The culture medium was then removed, and the cells were gently washed three times with PBS. Intracellular ROS generation was observed using the Olympus imaging system. Quantitative analysis of the obtained images was performed using ImageJ software. The results are shown in [Figure 1]. Figure 7 The scale bar in the figure is 100μm.

[0050] like Figure 7 As shown, compared with the control group, HE-IMC showed enhanced green fluorescence, indicating the generation of a large amount of reactive oxygen species. This was even more pronounced after laser irradiation, which fully demonstrates that the nanozyme still possesses good POD-like catalytic activity within the cell.

[0051] (2) HUCCT1 cells were seeded in 10cm culture dishes. When the HUCCT1 cells grew to about 80%, the cells were divided into 4 groups: control group, laser group, HE-IMC (100μg / ml) group, and HE-IMC (100μg / ml) + laser group. They were co-cultured for another 6h. After 6h, the light group was treated with 1064nm (1W / cm) light. 2 After laser irradiation for 5 minutes, cells were digested and counted. 1 ml of extraction buffer was added to each dish of cells, and the cells were sonicated using a probe (3 seconds operation, 10 seconds pause, repeated 10 times). The cells were then centrifuged at 12000G for 10 minutes at 4°C. Various detection reagents were added to the supernatant, and the absorbance was measured on a microplate reader after 2 minutes. The relative intracellular GSH or MDA content was calculated using the control group as a baseline. The results are shown in [Figure number missing]. Figure 8 .

[0052] like Figure 8As shown, the intracellular glutathione level in the HE-IMC group was significantly reduced, and this was even more pronounced after laser irradiation, which fully demonstrates that the nanozyme still possesses good GPx-like enzyme catalytic activity in the cell.

[0053] (3) HUCCT1 cells were subcutaneously inoculated into nude mice to form a subcutaneous xenograft model. Subsequently, 200 μL of HE-IMC (8 mg / mL) was injected via the tail vein. Imaging monitoring was then performed using a Siemens 3.0T MRI scanner. The results are shown in [Figure number missing]. Figure 9 .

[0054] like Figure 9 As shown, the nanozymes gradually accumulate in the tumor site within 2 hours, forming a significant negative contrast effect, which is strongest at 8 hours. The tumor area gradually recovers after 24 hours, indicating that the nanozymes can be rapidly metabolized and have high safety.

[0055] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a high-entropy nanozyme, characterized in that, Includes the following steps: (1) Add the surfactant to the solvent, dissolve it by sonication, then add a mixture of iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone and copper acetylacetone, sonicate in a water bath for 1-3 hours, then add the reducing agent and continue sonicating for 1-2 hours. (2) In a protective gas atmosphere, the solution after sonication in step (1) is replaced with air, then stirred at 80~100℃ for 30~50min, then heated to 200~220℃, and continued to react for 5~8h, and then cooled to room temperature; (3) Wash and centrifuge with ethanol-cyclohexane mixed solution, collect the precipitate and dry it to obtain high-entropy nanomaterials. Then disperse it in buffer solution, sonicate it and add dopamine solution. Stir the reaction in the dark for 2-4 hours, wash and centrifuge to collect the precipitate, and dry it to obtain dopamine-encapsulated high-entropy nanomaterials. (4) Disperse the dopamine-encapsulated high-entropy nanomaterials in a buffer solution, sonicate, add SH-PEG-NH2 solution, stir in the dark for 10-12 h, wash, centrifuge, collect the precipitate, and dry to obtain PEG-encapsulated high-entropy nanomaterials. (5) Disperse the PEG-encapsulated high-entropy nanomaterials in a solvent, then add the activated indomethacin solution, stir in the dark for 10-12 hours, wash and centrifuge to collect the precipitate, and dry to obtain the indomethacin-modified high-entropy nanomaterials.

2. The preparation method according to claim 1, characterized in that, The surfactant used in step (1) is hexadecyltrimethylammonium bromide, and the solvent is oleylamine.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of iron acetylacetone, aluminum acetylacetone, nickel acetylacetone, manganese acetylacetone and copper acetylacetone in the solution is 0.5~1:0.5~1:1:0.1~0.5:0.1~0.

5.

4. The preparation method according to claim 1, characterized in that, The reducing agent in step (1) is glucose.

5. The preparation method according to claim 1, characterized in that, In step (3), the mass concentration of the dispersed high-entropy nanomaterial is 0.5~1 mg / mL, and the volume and mass concentration of the added dopamine solution are the same.

6. The preparation method according to claim 1, characterized in that, In step (4), the mass concentration of the high-entropy nanomaterials encapsulated with dopamine dispersed in the solution is 0.2~0.5 mg / mL, and then an SH-PEG-NH2 solution with the same volume and mass concentration is added.

7. The preparation method according to claim 1, characterized in that, The buffer solution used in steps (3) and (4) is Tris-HCl with a pH of 8 to 8.

5.

8. The preparation method according to claim 1, characterized in that, In step (5), indomethacin is activated using EDC / NHS. The mass concentration of the activated indomethacin solution is 2~5 mg / mL, and its volume-to-mass ratio with the PEG-encapsulated high-entropy nanomaterial is 2:

1.

9. A high-entropy nanozyme, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

10. Use of the high-entropy nanozyme of claim 9 in the preparation of tumor therapeutic drugs.

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