Preparation method and application of Ru-NiB nanoscale enzyme

Abstract

The application discloses a preparation method of Ru-NiB nano-enzyme and application thereof. The method comprises the following steps: dissolving NiCl2.6H2O and RuCl3 in ultrapure water at room temperature, and then dropping an aqueous solution of NaBH4 in 2-3 min; the reaction is kept at room temperature for 3-8 h to form a black precipitate, i.e. an amorphous nano-enzyme. The preparation process is simple, the production cycle is short, the raw materials are cheap and easy to obtain, the production cost is saved, and the method is suitable for large-scale production; and the ultrafine Ru clusters in the obtained material exhibit excellent peroxidase (POD) and catalase (CAT) activities.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine technology in nanomaterials and wound repair, specifically a method for preparing nanoenzymes that treat diabetic ulcers by killing bacteria at the wound site, consuming glucose, and generating oxygen. Background Technology

[0002] The incidence of diabetes is increasing year by year, becoming one of the major diseases threatening human health. The most common diabetic syndrome is diabetic foot ulcer (DFU). The 5-year mortality rate for DFU patients is 50%, rising to 80% after amputation. The main characteristic of DFU is extremely slow wound healing. Hyperglycemia, hypoxia, and bacterial infection in the wound area exacerbate the difficulty of DFU treatment. Generally, excessive glucose oxidation increases oxygen consumption, thus aggravating the hypoxia of the wound microenvironment. The function of skin-related cells (fibroblasts, vascular cells, etc.) is inhibited, and the limited formation of microvessels in the wound further leads to insufficient oxygen and nutrient supply, thus hindering wound repair. Worse still, bacteria can colonize non-healing wounds, secreting polysaccharides, proteins, and nucleic acids to form extracellular polymers and bacterial biofilms. Antibiotic resistance caused by bacterial biofilms inhibits the bactericidal effect of antibiotics, exacerbates bacterial infection, and further endangers diabetic ulcer patients. Most researchers are dedicated to promoting diabetic wound healing through methods such as lowering blood sugar, continuous oxygen supply, and antibacterial agents. However, these adverse factors do not exist independently, and the effect of a single treatment method is not satisfactory. Therefore, a synergistic treatment strategy that takes a multi-faceted approach and the regulation of the microenvironment is crucial for the healing of diabetic wounds.

[0003] Nanozymes are a class of nanomaterials with enzyme-like activities that have attracted widespread attention in the field of medical biology. Of particular interest are noble metal nanozymes, which exhibit significant multi-enzyme activities. For example, ruthenium (Ru) has been widely used in antibacterial applications due to its peroxidase (POD) and catalase (CAT) activities. The antibacterial effect of nanozymes is based on their POD activity: catalyzing hydrogen peroxide (H₂O₂) to form toxic hydroxyl radicals (·OH), which then disrupt bacterial structure, achieving an antibacterial effect. However, catalase activity can decompose H₂O₂ to generate oxygen (O₂), which is used for glucose oxidation. Since the enzyme activity of most nanozymes is still too low, the generation of ·OH is severely limited. Obtaining highly active nanozymes often requires loading high doses of noble metals, which makes it difficult to balance the relationship between dosage and biosafety.

[0004] The paper *Chemical Engineering Journal* 2023, 466, 143292 (doi:10.1016 / j.cej.2023.143292) reports a gold / platinum bimetallic nanocluster enzyme (Au / PtNCs@GOx) modified with glucose oxidase (GOx) for cascade catalysis. GOx effectively consumes glucose to produce H₂O₂, while Au / Pt simultaneously mimics peroxidase to convert H₂O₂ into highly cytotoxic ·OH, thus achieving satisfactory antibacterial and anti-biofilm effects. However, the O₂ required for glucose oxidation is neglected, and insufficient substrate significantly hinders the cascade catalytic reaction. Furthermore, this technology involves loading GOx, necessitating consideration of the complex processes of GOx dosage, loading, and release. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of current technologies by providing a method for preparing Ru-NiB nanozymes and their applications. This method uses RuCl3 as a noble metal substrate and leverages the multiple valence states and electronic configurations of Ru to achieve high catalytic activity of the prepared nanozyme; specifically, Ru is reduced by adding an aqueous solution of NaBH4 to a mixed solution of NiCl2 and RuCl3. 3+ Furthermore, by controlling the dripping rate of NaBH4 to regulate the size of the Ru clusters, ultrafine Ru clusters were deposited on the surface of the more easily prepared amorphous NiB alloy, thus obtaining the desired Ru-NiB nanozyme in a simple one-step synthesis method. This invention features a simple preparation process, short production cycle, and readily available and inexpensive raw materials, saving production costs and making it suitable for large-scale production. The ultrafine Ru clusters in the obtained material exhibit excellent peroxidase-like (POD) and catalase-like (CAT) activities.

[0006] The technical solution of this invention is:

[0007] A method for preparing Ru-NiB nanozymes, the method comprising the following steps:

[0008] The first step is to synthesize amorphous nanozymes.

[0009] NiCl2·6H2O and RuCl3 were dissolved in ultrapure water at room temperature, and then NaBH4 aqueous solution was added dropwise over 2-3 minutes. The reaction was allowed to stand at room temperature for 3-8 hours to form a black precipitate, which is the crude amorphous nanozyme.

[0010] For every 28 mL to 32 mL of ultrapure water, add 126 to 130 mg NiCl2·6H2O, 14 to 18 mg RuCl3, 0.2 to 0.3 mL of 0.5 to 1.5 M HCl solution, and 8 to 12 mL of NaBH4 aqueous solution; the concentration of the NaBH4 aqueous solution is 0.8 to 1.2 M.

[0011] The second step is to refine amorphous nanoenzymes.

[0012] The above-mentioned black precipitate was rinsed 4 to 6 times with ultrapure water. The washed precipitate was then freeze-dried for 6 to 10 hours to obtain clean Ru-NiB, which is the Ru-NiB nanozyme used to treat diabetic ulcers.

[0013] The resistivity of the ultrapure water is 17.5-18.2 MΩ·cm.

[0014] The nanozyme comprises an amorphous NiB alloy and ultrafine Ru clusters, with the Ru clusters loaded on the surface of the amorphous NiB alloy at an ultrafine size of 0.40–0.45 nm; and a three-dimensional continuous network formed by interconnected nanoparticles with an average diameter of 30 nm.

[0015] The Ru-NiB nanozyme obtained by the method is used in combination with glucose oxidase in drugs for treating diabetic ulcers, as an oxygen-supplying agent and bactericide.

[0016] In diabetic ulcers, firstly, Ru-NiB nanozymes can utilize H2O2, the product of the reaction between GOx and glucose, to generate O2, promoting the cascade reaction of glucose oxidation and supplementing the oxygen supply to the wound microenvironment; secondly, Ru-NiB nanozymes react with H2O2 to generate toxic ·OH, which kills bacteria at the wound site, achieving a highly effective antibacterial effect.

[0017] The above-mentioned method for preparing Ru-NiB nanozymes for treating diabetic ulcers uses raw materials and equipment that are obtained through known means, and the operating process can be mastered by those skilled in the art.

[0018] The amorphous nanozyme obtained by this invention has Fenton-like reaction performance, and the generated highly oxidizing reactive oxygen species can rapidly oxidize many organic compounds, thus making it applicable to wastewater degradation treatment.

[0019] The essential features of this invention are:

[0020] First, the Ru metal prepared in current technologies, whether in the form of single particles or loaded on a carrier, typically has a size between 2-20 nm. In contrast, the Ru-NiB nanozyme prepared in this invention has Ru loaded on an amorphous NiB alloy with an ultrafine size of 0.42 nm. Second, the ultrafine Ru clusters and amorphous core have multiple enzyme-like activities, which can effectively respond to the microenvironment of diabetic wounds and efficiently drive cascade catalytic reactions, thereby improving the wound repair effect. Third, the prepared nanozyme ensures high catalytic activity by forming ultrafine noble metal clusters, while minimizing the amount of noble metal used.

[0021] The essential features of this invention in its preparation method are: first, it employs a one-step reduction method to directly synthesize Ru-NiB nanozymes, avoiding unnecessary raw material losses, resulting in a short production cycle and high yield; second, unlike previous methods that used organic solvents such as ethylene glycol, dimethylformamide, or dimethyl sulfoxide, this invention uses ultrapure water as the solvent, which is non-toxic, abundant, readily available, and inexpensive; and third, the entire operation process does not release toxic or harmful chemical reagents (such as chloroform), ensuring a high safety factor.

[0022] The beneficial effects of this invention are:

[0023] This invention employs a one-step synthesis method to prepare nanoenzymes with ultrafine Ru clusters supported on amorphous NiB alloy. These nanoenzymes are suitable for in-situ triggering of cascade catalytic reactions at diabetic ulcer sites, achieving highly efficient and synergistic wound repair effects through oxygen replenishment and antibacterial action. Specifically, this is reflected in:

[0024] (1) This invention discloses a method for preparing Ru-NiB nanozymes and their applications. Ultrafine Ru clusters are uniformly distributed on the surface of amorphous NiB alloys, providing a high density of active sites. The ultrafine Ru clusters exhibit excellent peroxidase-like (POD) and catalase-like (CAT) activities. Specifically, the POD performance of 1 μg / mL Ru-NiB nanozymes is 26 times that of 1 μg / mL amorphous NiB alloy; similarly, the CAT performance of 50 μg / mL Ru-NiB nanozymes is 4.8 times that of 50 μg / mL amorphous NiB alloy.

[0025] (2) The present invention provides a method for preparing Ru-NiB nanozymes and their application. The ultrafine Ru clusters are highly efficient cascade catalysts. In diabetic ulcers, the addition of GOx can catalyze the oxidation of high blood sugar at the wound site to generate gluconic acid and H2O2 in the presence of O2. The generated H2O2 is further catalyzed by the ultrafine Ru clusters through POD and CAT activity to generate ·OH and O2, which can play a highly efficient antibacterial role and alleviate the hypoxic microenvironment of the wound. This achieves a multi-strategy combined wound repair, effectively avoids the toxic side effects of GOx, and can achieve highly efficient antibacterial treatment. This provides a new idea for developing a new highly efficient wound repair combined treatment strategy.

[0026] (3) The present invention provides a method for preparing Ru-NiB nanozymes and its application. It adopts a one-step synthesis method, which is simple in preparation process, has a short production cycle, uses inexpensive and readily available raw materials, saves production costs, and is suitable for large-scale production.

[0027] (4) The present invention provides a method for preparing Ru-NiB nanozymes and its application. No highly toxic raw materials or volatile chemical reagents are used in the production process, and the production process is safe and environmentally friendly.

[0028] (5) In terms of treatment, this invention adds GOx in situ to the infected wound to avoid the problems of loading and release and to precisely control the dosage, thus avoiding excessive consumption of natural enzymes. For example, in the existing CN114470177A technology, 100 μL of GOx at 1 mg / mL needs to be added to every 10 mg of raw material, while we only need 10 μL of GOx at 0.5 mg / mL to treat each mouse, saving 20 times the amount of GOx used. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] Figure 1 Transmission electron microscopy (TEM) image of the surface of the Ru-NiB nanozyme prepared in Example 1.

[0031] Figure 2 High-angle annular dark-field image and energy dispersive spectroscopy elemental analysis diagram of Ru-NiB nanozyme prepared in Example 1.

[0032] Figure 3 High-magnification transmission electron microscopy image of the surface of the Ru-NiB nanozyme prepared in Example 1.

[0033] Figure 4 The UV-Vis spectrum of the Ru-NiB nanozyme POD activity prepared in Example 1.

[0034] Figure 5 The image shows the ·OH signal detected by electron spin resonance (ESR) spectroscopy of the Ru-NiB nanozyme prepared in Example 1.

[0035] Figure 6 The image shows the CAT activity test results of the Ru-NiB nanozyme prepared in Example 1.

[0036] Figure 7 Schematic diagram of in vitro antibacterial effect

[0037] Figure 8 Schematic diagram of wound healing effect in diabetic mice

[0038] Figure 9 Schematic diagram of weight changes in diabetic mice

[0039] Figure 10 Schematic diagram of hematoxylin-eosin (H&E), Masson, and Giemsa staining analysis of skin in diabetic mice. Detailed Implementation

[0040] Example 1

[0041] The first step is to synthesize amorphous nanozymes.

[0042] 128 mg NiCl2·6H2O and 16.6 mg RuCl3 were dissolved in 30 mL of ultrapure water at room temperature. 0.25 mL of 1 M HCl was added, followed by the continuous dropwise addition of 10 mL of 1 M NaBH4 aqueous solution (67 μL / s) while stirring. After the addition was complete, stirring was stopped, and the reaction solution was allowed to stand undisturbed at room temperature for 6 hours, resulting in the formation of a black precipitate. This precipitate is the crude amorphous nanozyme.

[0043] The second step is to refine amorphous nanoenzymes.

[0044] The above black precipitate was rinsed five times with ultrapure water. The washed precipitate was then freeze-dried (at -40 degrees Celsius) for 8 hours to obtain clean Ru-NiB, which is the Ru-NiB nanozyme used to treat diabetic ulcers.

[0045] The resistivity of the ultrapure water used is 18.0 MΩ·cm.

[0046] like Figure 1 As shown, Ru-NiB nanozymes are composed of interconnected nanoparticles with an average diameter of 30 nm.

[0047] like Figure 2 As shown, Ru is uniformly distributed throughout the amorphous NiB.

[0048] like Figure 3 As shown, the Ru metal on this nanozyme is distributed in the form of clusters with an average diameter of 0.42 nm on the amorphous NiB alloy. Examples 2 and 3 both possess the structural features described above.

[0049] This embodiment prepared a Ru-NiB nanozyme for treating diabetic ulcers, and tested its enzyme activity, in vitro and in vivo antibacterial properties, and wound repair efficacy (other embodiments have the same test results as this embodiment). The method is as follows:

[0050] (1) POD-like activity test

[0051] The POD-like activity of Example 1 was determined colorimetrically using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate in 0.1 M acetate-sodium acetate (HAc-NaAc) buffer (pH = 4.0). Nanozyme solution (60 μL, 1 mg / mL), hydrogen peroxide (H₂O₂) aqueous solution (40 μL, 10 mM), and TMB solution (100 μL, 10 mM) were added sequentially to 0.8 mL of HAc-NaAc buffer and mixed thoroughly. The mixture was incubated in the dark at 37 °C for 10 minutes. The final concentrations of nanozyme, hydrogen peroxide, and TMB were fixed at 60 μg / mL, 0.4 mM, and 1 mM, respectively. The UV-Vis absorption spectra of the test solutions were recorded using a UV spectrophotometer (Shimadzu UV-2600).

[0052] The results are as follows Figure 4 As shown, TMB molecules are oxidized by ·OH, with a maximum absorbance of 652 nm. In addition to the colorimetric method described above, short-lived ·OH molecules were directly captured using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) by ESR detection, with results as shown... Figure 5 As shown, a strong characteristic ·OH signal was observed in the presence of H2O2. All the above tests demonstrate that Example 1 exhibits significant POD-like activity.

[0053] (2) CAT-like activity test

[0054] The concentration of dissolved oxygen (DO) was measured at room temperature using a portable dissolved oxygen meter to evaluate the CAT-like activity of the obtained nanozyme. The oxygen electrode was immersed in a test solution containing nanozyme (60 μg / mL) and H₂O₂ (0.4 mM), and the dissolved oxygen level was recorded. In Example 1, 600 μL (1 mg / mL) and H₂O₂ (400 μL, 10 mM) were added to 9 mL of ultrapure water and mixed thoroughly. The results are as follows. Figure 6 As shown, the concentration of DO gradually increased over time, with a limiting DO concentration of 13.7 mg / L, exhibiting significant CAT-like activity.

[0055] (3) In vitro antibacterial effect test in high-sugar environment

[0056] Gram-negative *Pseudomonas aeruginosa* (PA) single colonies were transferred to tryptone soybean broth (TSB) and incubated at 37°C in a shaker for 12 hours. After the bacteria reached the logarithmic growth phase, the bacterial suspension was collected by two centrifugations, washed with 0.9% physiological saline, and diluted. To simulate the in vitro environment of diabetes, 1 mg / mL glucose was added to the TSB. The bacterial concentration was 1 × 10⁻⁶. 8The CFU / mL PA was divided into four groups: control group, GOx group, Example 1 group, and Example 1 + GOx group. The final concentrations of the reagents for bacterial treatment were: Example 1 (60 μg / mL) and GOx (0.4 μg / mL). After incubating the bacterial suspension of each group on a shaker at 37°C for 4 hours, 100 μL of each suspension was spread onto an agar plate and then incubated at 37°C for 12 hours. The number of colony-forming units in each group was then counted using the plate count method to evaluate the in vitro antibacterial effect.

[0057] The results are as follows Figure 7 As shown, the treatment with Example 1 and GOx showed a significant sterilization rate of up to 95% against PA, which far exceeded the effect of using Example 1 and GOx alone.

[0058] (4) Antibacterial and wound healing effects in diabetic mice

[0059] Female BALB / c laboratory mice (6 weeks old, 20g) were selected as the animal model. Animal experiments in this study were strictly conducted according to the protocol approved by the Ethics Committee of Guangxi Medical University Cancer Hospital (Approval No.: KY-2022-272). First, streptozotocin (40mg / kg) was injected continuously for 5 days to induce a diabetic mouse model. The model was considered successfully established when the blood glucose concentration of the mice exceeded 11.1mM for 3 consecutive days. A cavity with an area of ​​approximately 80mm² was cut from the back of each diabetic mouse. 2 The wound was treated, and a suspension of Pseudomonas aeruginosa (10 μL, 2 × 10⁻⁶ ml) was instilled into the wound. 9 The mice with infected wounds were randomly divided into four groups (n=6 per group): a blank control group, a GOx group, the Example 1 group, and the Example 1 + GOx group. Treatment was administered on days 1, 3, and 5 post-infection according to the group, with the treatment dose being Example 1 (10 μL, 6 mg / mL) and GOx (10 μL, 0.5 mg / mL). The mice were then observed for 14 days, during which time body weight and wound area were measured and photographed every other day.

[0060] The results are as follows Figure 8 As shown, in stark contrast to the control group, mice treated with Example 1 and GOx exhibited significantly enhanced wound healing capabilities, with wounds essentially healed by day 14. Combined with nanozyme activity testing, we can conclude that the excellent therapeutic effect of Example 1 stems from the highly efficient cascade catalytic reaction activated by glucose. Figure 9 As shown, the body weight of mice in each group remained basically unchanged over 14 days, which further illustrates that the toxicity and side effects of these synthetic nanozymes on mice are negligible.

[0061] Next, histopathological analysis of the skin tissue was performed. The repair capacity of the nanozyme on the skin tissue structure was evaluated by hematoxylin and eosin (H&E) staining; the amount of collagen fibers at the wound site was assessed by Masson staining; and the number of bacteria at the infected wound site was observed by Giemsa staining.

[0062] The results are as follows Figure 10 H&E staining showed that the epidermal cell layer in the control group was significantly thickened, exhibiting crusting characteristics and severe skin structure damage. However, after treatment with Example 1 + GOx, the mice showed a more intact skin structure, such as thickened and thinned epidermis, with newly formed hair follicles, sebaceous glands, and blood vessels. Figure 10 (Top row). As shown by Masson staining, after treatment with GOx in Example 1, the number of collagen fibers increased significantly and they were arranged more tightly and regularly. Figure 10 (Second line in the middle). Giemsa staining analysis showed that the number of bacteria in the infected skin of mice treated with Example 1+GOx was significantly reduced, almost undetectable. Figure 10 (Last line). In summary, Example 1+GOx continuously generates ·OH through a highly efficient cascade catalytic reaction activated by glucose, exhibiting good antibacterial and wound repair properties in vivo.

[0063] Example 2

[0064] The first step is to synthesize amorphous nanozymes.

[0065] 126 mg NiCl2·6H2O and 14 mg RuCl3 were dissolved in 28 mL of ultrapure water at room temperature. Then, 0.2 mL of 0.5 M HCl and 8 mL of 1 M NaBH4 aqueous solution were successively added to the solution. The reaction was allowed to proceed undisturbed at room temperature for 3 hours, resulting in the formation of a black precipitate. This precipitate is the crude amorphous nanozyme.

[0066] The second step is to refine amorphous nanoenzymes.

[0067] The black precipitate obtained from the above steps was washed four times with ultrapure water with a resistivity of 17.5 MΩ·cm. The washed precipitate was then freeze-dried for 6 hours to obtain clean Ru-NiB, which is the Ru-NiB nanozyme used to treat diabetic ulcers.

[0068] Example 3

[0069] The first step is to synthesize amorphous nanozymes.

[0070] 130 mg NiCl₂·6H₂O and 18 mg RuCl₃ were dissolved in 32 mL of ultrapure water at room temperature. Then, 0.3 mL of 1.5 M HCl and 12 mL of 1 M NaBH₄ aqueous solution were successively added to the solution. The reaction was allowed to proceed undisturbed at room temperature for 8 hours, resulting in the formation of a black precipitate. This precipitate is the crude amorphous nanozyme.

[0071] The second step is to refine amorphous nanoenzymes.

[0072] The black precipitate obtained from the above steps was washed six times with ultrapure water with a resistivity of 18.2 MΩ·cm. The product was then freeze-dried for 10 hours to obtain clean Ru-NiB, which is the Ru-NiB nanozyme used to treat diabetic ulcers.

[0073] Comparative Example 1

[0074] The amount of RuCl3 was set at 4.15 mg, and other conditions were the same as in Example 1. The surface morphology of the obtained samples was observed, and Ru clusters could not be observed distributed on NiB. The POD-like activity of the obtained nanozyme was 13% of that in Example 1; the CAT-like activity was only 28% of that in Example 1.

[0075] Comparative Example 2

[0076] The amount of RuCl3 was set at 8.3 mg, and other conditions were the same as in Example 1. The surface morphology of the obtained samples was observed, and only a very small amount of Ru clusters were observed distributed on NiB. The POD-like activity of the obtained nanozyme was 27% of that in Example 1; the CAT-like activity was 56% of that in Example 1.

[0077] Comparative Example 3

[0078] The amount of RuCl3 was set at 12.45 mg, and 10 mL of 1M NaBH4 aqueous solution was rapidly added (160 μL / s). The surface morphology of the sample was observed, and it was found that Ru formed large clusters that were unevenly distributed on NiB. The POD-like activity of the obtained nanozyme was only 49% of that of Example 1; the CAT-like activity of the obtained nanozyme was only 77% of that of Example 1.

[0079] In Comparative Examples 1 and 2, due to insufficient RuCl3, uniformly distributed ultrafine Ru clusters could not be observed, and the POD and CAT enzyme activities were far lower than those in Example 1. In Comparative Example 3, because the NaBH4 aqueous solution was added rapidly, the reduction rate of RuCl3 was too fast, causing Ru to aggregate into larger clusters that could not be uniformly distributed on NiB. Therefore, the performance of the obtained nanozyme was also far inferior to that of Example 1.

[0080] As can be seen from the above embodiments, a method for preparing nanozymes involves a nanozyme (abbreviated as Ru-NiB) with ultrafine Ru clusters loaded on an amorphous NiB alloy, which, in conjunction with GOx, constructs a glucose-activated cascade catalytic reaction. In this reaction, Ru-NiB utilizes H2O2 generated from glucose oxidation by GOx to sustainably produce ·OH for antibacterial purposes. Furthermore, Ru-NiB possesses catalase activity, providing oxygen for glucose oxidation, and the consumption of glucose further prevents bacterial proliferation and infection. The invented Ru-NiB nanozyme exhibits an interconnected network of nanoparticles with an average diameter of 30 nm. The presence of Ru is mainly observed in the form of ultrafine clusters with an average size of 0.42 nm on the amorphous NiB, thus generating abundant active sites. Its preparation method is similar to the prior art of CN115645436A, "A method for preparing an amorphous nanozyme with synergistic effects of inducing apoptosis and ferroptosis," both utilizing the differences in the electrochemical reduction potentials of metal salts. This invention utilizes the difference in the electrochemical reduction potentials of NiCl2 and RuCl3. Secondly, this invention improves upon previous methods by adjusting the dripping rate of NaBH4 aqueous solution to control the size of Ru clusters. This allows for the deposition of ultrafine Ru clusters on the surface of an easier-to-prepare amorphous NiB alloy, resulting in a simple one-step synthesis of the desired Ru-NiB nanozyme. Compared to previous materials, the nanozyme prepared by this invention has a simpler preparation process and uses raw materials with lower toxicity (harm can be avoided with simple protection) or no toxicity. Studies have shown that smaller particle size results in higher enzyme activity, and boron (B) can alter the electron charge distribution around Ru, thereby enhancing its catalytic activity. Therefore, ultrafine Ru clusters make its structural and performance advantages in antibacterial applications more prominent. Furthermore, the in-situ addition of GOx avoids GOx loss and activity reduction during loading and release, allowing for precise control of GOx dosage.

[0081] The above examples and comparative examples illustrate a method for preparing Ru-NiB nanozymes for treating diabetic ulcers. This method involves continuously experimenting with different amounts of raw materials to ensure that the resulting nanozyme exhibits high catalytic efficiency within the minimum range of raw material usage. Through strict control of each process step and numerous trials, a synthetic nanozyme capable of treating diabetic ulcers has finally been developed.

[0082] Matters not covered in this invention are common knowledge.

Claims

1. A method for preparing Ru-NiB nanozymes, characterized in that the method includes the following steps: The first step is to synthesize amorphous nanozymes. NiCl2•6H2O and RuCl3 were dissolved in ultrapure water at room temperature, and then NaBH4 aqueous solution was added dropwise. The reaction was allowed to stand at room temperature for 3-8 h to form a black precipitate, which is the crude amorphous nanozyme. For every 28 mL to 32 mL of ultrapure water, add 126 to 130 mg NiCl2•6H2O, 14 to 18 mg RuCl3, 0.2 to 0.3 mL of 0.5 to 2.0 M HCl solution, and 8 to 12 mL of NaBH4 aqueous solution; the concentration of the NaBH4 aqueous solution is 0.8 to 1.2 M. The second step is to refine amorphous nanoenzymes. The above black precipitate was rinsed 4-6 times with ultrapure water, and the washed precipitate was freeze-dried for 6-10 h to obtain Ru-NiB nanozyme. The resistivity of the ultrapure water is 17.5-18.2 MΩ•cm; The NaBH4 aqueous solution is added dropwise over a period of 2 to 3 minutes in the first step. The nanozyme comprises an amorphous NiB alloy and ultrafine Ru clusters, with the Ru clusters loaded on the surface of the amorphous NiB alloy at an ultrafine size of 0.40~0.45 nm; and a three-dimensional continuous network formed by interconnected nanoparticles with an average diameter of 30 nm.

2. The application of the Ru-NiB nanozyme obtained by the method described in claim 1 in the preparation of drugs, characterized in that, It is used in combination with glucose oxidase in medications for the treatment of diabetic ulcers, as an oxygen-providing agent and bactericide.

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