Preparation method of magnesium-doped monatomic metal-loaded ceria nanoscale enzyme and application thereof in diabetic wound repair materials

Abstract

The application discloses a preparation method of magnesium-doped monatomic metal-loaded cerium oxide and application thereof in a diabetic wound repairing material, and belongs to the technical field of nanomaterials and biomedical materials. The prepared multi-enzyme activity nanenzyme material takes cerium oxide as a carrier, is subjected to magnesium doping, and is selectively etched by acetic acid to realize partial removal and proper reservation of magnesium components and loading of a monatomic metal Ru active center, and can be used as an antibacterial, antioxidant, local oxygen supply and tissue repairing material for a diabetic wound.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and biomedical materials technology, specifically relating to a method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes and their application in diabetic wound repair materials. Background Technology

[0002] Diabetic wounds are a common and difficult-to-treat type of chronic wound. The local area is typically situated in a pathological microenvironment influenced by multiple factors, including high glucose levels, bacterial infection, persistent inflammation, excessive accumulation of reactive oxygen species (ROS), and impaired tissue repair, leading to prolonged difficulty in healing. Compared to ordinary wounds, diabetic wounds are more prone to bacterial colonization and infection in the early stages, and subsequent repair is often accompanied by persistent oxidative stress, slow cell migration, insufficient angiogenesis, and limited collagen remodeling. Therefore, how to simultaneously address early infection control and subsequent oxidative stress regulation and tissue regeneration promotion within the same treatment system is a crucial issue that needs to be addressed in diabetic wound repair materials.

[0003] Metal oxide nanozymes, capable of mimicking the catalytic behavior of some natural enzymes, have attracted widespread attention in the field of wound repair. Some metal oxides exhibit peroxidase (POD)-like activity, catalyzing the generation of active species in the presence of H₂O₂, thereby exerting antibacterial effects. Others possess superoxide dismutase (SOD)-like and catalase (CAT)-like activities, which can be used to scavenge excess reactive oxygen species (ROS), decompose H₂O₂, and improve local oxidative stress and hypoxic environments. Therefore, metal oxide nanozymes with multi-enzyme-like activities hold promise for the continuous microenvironmental regulation of diabetic wounds.

[0004] Among various metal oxides, cerium oxide is distinguished by its Ce content. 3+ / Ce 4+Cerium oxide nanozymes exhibit reversible oxidation capabilities, offering certain advantages in ROS regulation. However, the limited number of surface active sites and relatively singular enzyme-like activities of ordinary cerium oxide materials hinder their effectiveness in early antibacterial activity, subsequent ROS scavenging, and H2O2 decomposition for oxygen supply in infectious diabetic wounds. To further improve the catalytic efficiency of cerium oxide nanozymes, current technologies typically employ strategies such as elemental doping, defect engineering, noble metal modification, or single-atom construction. However, these approaches still suffer from the following shortcomings: Firstly, existing materials often struggle to simultaneously achieve both antibacterial and antioxidant functions. Some nanozyme materials prioritize enhancing POD-like catalytic antibacterial activity, but their SOD-like and CAT-like activities are relatively insufficient, making it difficult to sustainably improve the oxidative stress and hypoxia present in diabetic wounds. Other materials emphasize ROS scavenging, but their early-stage catalytic antibacterial activity is insufficient, making it difficult to promptly inhibit bacterial proliferation in the early stages of wound infection. Secondly, existing single-atom construction methods mostly employ direct loading or simple defect construction routes. While these methods can improve metal atom utilization and the number of surface active sites to some extent, they generally lack synergistic regulation of the carrier defect structure and the fixed positions of single atoms, resulting in limited single-atom dispersion stability and a limited increase in multi-enzyme-like activity. Thirdly, in existing doping control methods, dopants are mostly used to assist in material forming or induce defect generation, and are usually removed in subsequent processing. Although this method can create certain defects, if the removal degree is too high, it can easily affect the stability of the carrier structure and the distribution of active sites, and is also not conducive to the stable loading of single-atom active sites and the balanced development of multi-enzyme-like activity.

[0005] In the prior art, patent CN118078856A discloses a degradable dual-enzyme active nanozyme for treating diabetic foot ulcers. This nanozyme has POD-like and CAT-like activities, which can reduce inflammation, alleviate hypoxia, and promote angiogenesis. However, it does not adequately consider SOD-like ROS scavenging and the balance of POD / SOD / CAT multi-enzyme activities. Patent CN113492004A constructs oxygen vacancies on the cerium oxide surface through acid etching and annealing, which improves catalytic activity. However, it does not address the appropriate retention of dopant components, nor does it solve the problem of synergistic fixation of oxygen vacancies and single-atom active sites. Therefore, it is difficult for the prior art to simultaneously achieve a balance of multi-enzyme activities, stable dispersion of single atoms, and a defective structure with controlled retention of dopants in the same material system. Consequently, it cannot simultaneously meet the comprehensive needs of early antibacterial activity, ROS scavenging, oxygen supply, and tissue repair in diabetic wounds. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing magnesium-doped single-atom metal-loaded cerium oxide nanozymes and their application in diabetic wound repair materials. This method balances carrier defect construction, stable loading of single-atom active sites, and multi-enzyme-like activity, enabling it to simultaneously meet the application requirements of early infection control, excessive ROS removal, local oxygen supply, and tissue repair promotion in diabetic wounds.

[0007] To address the aforementioned technical problems, this invention provides a method for preparing single-atom metal-supported cerium oxide nanozyme materials based on magnesium doping and selective etching, comprising the following steps: (1) Cerium nitrate hexahydrate (Ce(NO3)3·6H2O), magnesium nitrate hexahydrate (Mg(NO3)2·6H2O) and polyvinylpyrrolidone (PVP, MW≈8000) were added to ethylene glycol and ultrasonically dispersed until completely dissolved; then hydrochloric acid solution was added and ultrasonic dispersion was continued to obtain a precursor solution; the precursor solution was transferred to a reaction vessel for solvothermal reaction; after the reaction was completed, the precipitate was collected by centrifugation and washed three times with deionized water and anhydrous ethanol in sequence, and after vacuum drying and calcination, the first solid was obtained; (2) Weigh the first solid and disperse it in acetic acid solution and stir. After the treatment is completed, centrifuge to collect the solid, wash it with deionized water until neutral, wash it with anhydrous ethanol, and dry it under vacuum to obtain the second solid. (3) Weigh the second solid and disperse it in a mixture of deionized water and anhydrous ethanol. After stirring and dispersing, slowly add RuCl3 aqueous solution and continue stirring at room temperature. Then, evaporate the obtained mixture at 80°C to remove the solvent and transfer it into a muffle furnace for calcination to obtain Ru / Mg-vCeO2 multi-enzyme active nanomaterial, which is magnesium-doped single-atom metal-supported cerium oxide nanoenzyme.

[0008] Further, in step (1), the ratio of cerium nitrate hexahydrate, magnesium nitrate hexahydrate, polyvinylpyrrolidone, hydrochloric acid solution, and ethylene glycol is 1 mmol: 0.10-0.12 mmol: 0.02-0.028 mmol: 1.5-2.5 mL: 25-40 mL; the concentration of the hydrochloric acid solution is 0.8-1.2 mol / L.

[0009] Further, in step (1), the solvothermal reaction temperature is 140-200℃, the reaction time is 3-6h; the drying temperature is 50-70℃, the drying time is 8-15h; the calcination temperature is 300-450℃, the calcination time is 1-4h, and the calcination heating rate is 1-5℃ / min.

[0010] Further, in step (2), the ratio of the first solid to the acetic acid solution is 1g:70-130mL; the volume fraction of the acetic acid solution is 50-80 vol.

[0011] Furthermore, in step (2), the stirring temperature is 45-80℃ and the stirring time is 4-10h; the drying temperature is 70-120℃ and the drying time is 8-13h.

[0012] Furthermore, in step (3), the ratio of the amount of the second solid, deionized water, anhydrous ethanol and RuCl3 aqueous solution used is 1g:0.8-2mL:0.8-1.5mL:28-32mL, and the concentration of the RuCl3 aqueous solution is 0.95-1.05mg / mL.

[0013] Furthermore, in step (3), the first stirring time is 0.5-1.5h; the second stirring time is 3-6h; the calcination temperature is 250-400℃, and the calcination time is 2-6h.

[0014] The second solid has oxygen vacancy sites on its surface, which can anchor the Ru precursor and form single-atom or atomically dispersed Ru active sites after calcination. The resulting Ru / Mg-vCeO2 multi-enzyme active nanomaterial has CeO2 as the main crystal phase and contains residual Mg components and atomically dispersed Ru active sites. It has the structural characteristics of residual Mg, oxygen vacancy sites and Ru active centers coexisting, and exhibits POD, SOD and CAT-like enzyme activities.

[0015] Another objective of this invention is to provide an application of the magnesium-doped single-atom metal-supported cerium oxide nanozyme prepared by the above method in a material for repairing diabetic wounds.

[0016] Compared with the prior art, the beneficial effects of this invention are as follows: This invention constructs a CeO2 support rich in oxygen defect sites through Mg doping and selective acetic acid etching. Mg participates in the construction of the CeO2 support during material formation, and is partially removed and appropriately retained after acetic acid etching. The partial removal of Mg promotes the formation of defect sites, while the appropriate amount of residual Mg helps maintain the stability of the CeO2 crystal phase and avoids structural damage caused by excessive etching. XRD, XPS, and EPR test results show that the nanoenzyme material of this invention maintains the main CeO2 crystal phase and has high CeO2 content. 3+ The correlation signals between the ratio and the defect oxygen ratio indicate that this method can effectively increase the number of defect sites while maintaining the stability of the carrier structure. The defect oxygen ratio in the prepared Ru / Mg-vCeO2 nanozyme is 32.9-35.1%.

[0017] This invention further introduces single-atom Ru active sites into the aforementioned defect-enriched CeO2 support to prepare Ru / Mg-vCeO2 materials. The defect sites on the support surface provide a stable environment for Ru, making it less prone to significant migration and aggregation during loading and subsequent calcination, thus maintaining an atomically dispersed state. Aberration-corrected electron microscopy results show numerous isolated bright spots in the material; XRD patterns do not show obvious Ru or RuO2 particle characteristic peaks, indicating that Ru does not form large particles and mainly exhibits atomically dispersed characteristics. Inductively coupled plasma atomic emission spectrometry (ICP-OES) results show that the Ru loading in the material can be controlled at 1.28-1.61 wt%, providing a structural basis for the synergistic activity of POD, SOD, and CAT multi-enzyme-like activities. The prepared Ru / Mg-vCeO2 nanozyme material has a specific surface area of ​​126.1-145.5 m². 2 / g, with a residual Mg content of 2.1-2.9at%.

[0018] The Ru / Mg-vCeO2 material prepared in this invention exhibits synergistic enhancement of POD, SOD, and CAT-like enzyme activities, making it suitable for use as a wound repair material for diabetic wounds. In the early stages of wound healing, the material catalyzes the production of active species from H2O2 through POD-like activity, exerting an antibacterial effect. As infection subsides and repair progresses, the material scavenges excess superoxide anions through SOD-like activity and decomposes H2O2 to generate oxygen through CAT-like activity, reducing oxidative stress and alleviating local hypoxia. Results show that the material prepared in this invention achieves a bactericidal rate of 96.8-98.9% against Staphylococcus aureus and 98.2-99.9% against Escherichia coli. The SOD-like superoxide anion scavenging rate reaches 81.5-90.4%, and the CAT-like H2O2 decomposition efficiency (CAT decomposition rate) reaches 75.8-82.7%. In a diabetic wound healing experiment, the wound closure rate (day 14) reached 96.1-99.7%. Therefore, the nanozymes prepared in this invention can effectively inhibit bacteria and dynamically regulate oxygen supply at different repair stages, thereby improving the wound microenvironment and promoting tissue repair.

[0019] This invention combines Mg doping, selective etching, and single-atom metal loading to enable the Mg component to participate in the structural regulation of the CeO2 support during material formation. After etching, the Mg component remains partially retained within the support, thus constructing a composite structure where residual Mg, oxygen vacancies, and single-atom metal active centers coexist. This structure is beneficial for increasing the number of defect sites on the support, enhancing the stable immobilization of single-atom metal active centers, and promoting the synergistic activity of multiple enzymes such as POD, SOD, and CAT. Attached Figure Description

[0020] Figure 1The X-ray diffraction (XRD) pattern of the sample from Example 1; Figure 2 The images show transmission electron microscopy (TEM) and elemental distribution (EDS) diagrams of the sample from Example 1. Figure 3 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the sample from Example 1. Figure 4 The electron paramagnetic resonance (EPR) spectrum of the sample from Example 1 is shown below. Figure 5 These are representative plate photographs of the in vitro antibacterial experiment in Example 1; (a) Staphylococcus aureus, (b) Escherichia coli Figure 6 A representative photograph of the healing process of the diabetic wound in Example 2; Figure 7 This is a statistical chart showing the wound closure rate in diabetic patients. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are used to further illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all raw materials used are commercially available, and all methods used are conventional methods in the art.

[0022] Example 1: (1) Weigh 2.07 mmol of cerium nitrate hexahydrate (Ce(NO3)3∙6H2O), 0.23 mmol of magnesium nitrate hexahydrate (Mg(NO3)2∙6H2O), and 0.05 mmol of polyvinylpyrrolidone (PVP, MW≈8000), add them to 60 mL of ethylene glycol, and ultrasonically disperse until completely dissolved; then add 3.1 mL of 1 mol / L hydrochloric acid solution, and continue ultrasonic dispersion to obtain a homogeneous precursor solution. Transfer the above precursor solution to a polytetrafluoroethylene-lined reactor and react at 160 °C for 3 h. After the reaction, cool naturally to room temperature, centrifuge to collect the precipitate, and wash it three times each with deionized water and anhydrous ethanol, and vacuum dry it at 60 °C for 10 h. Then, place the obtained precursor in a muffle furnace, heat it to 350 °C at a heating rate of 2 °C / min under air atmosphere, and calcine it for 2 h to obtain the first solid.

[0023] (2) Weigh 1g of the first solid and disperse it in 100mL of 60vol% acetic acid solution. Stir at 60℃ for 6h. After treatment, collect the solid by centrifugation, wash it with deionized water until neutral, wash it once with anhydrous ethanol, and dry it under vacuum at 100℃ for 8h to obtain the second solid.

[0024] (3) Weigh 1g of the second solid and disperse it in a mixture of 1mL deionized water and 1mL anhydrous ethanol. After stirring for 1h (first stirring), take 31.3mL of RuCl3 aqueous solution with a concentration of 1mg / mL and slowly add it dropwise to the above dispersion. After the addition is complete, continue stirring at room temperature for 4h (second stirring). Subsequently, evaporate the solvent at 80℃ to remove the solvent from the resulting mixture and transfer it to a muffle furnace. Calcine it at 300℃ for 4h to obtain Ru / Mg-vCeO2-1 material.

[0025] Example 2: (1) Weigh 2.07 mmol of cerium nitrate hexahydrate (Ce(NO3)3∙6H2O), 0.207 mmol of magnesium nitrate hexahydrate (Mg(NO3)2∙6H2O), and 0.0414 mmol of polyvinylpyrrolidone (PVP, MW≈8000), add them to 51.75 mL of ethylene glycol, and ultrasonically disperse until completely dissolved; then add 5.17 mL of 0.8 mol / L hydrochloric acid solution, and continue ultrasonic dispersion to obtain a homogeneous precursor solution. Transfer the above precursor solution to a polytetrafluoroethylene-lined reactor and react at 140 °C for 3 h. After the reaction, allow it to cool naturally to room temperature, centrifuge to collect the precipitate, and wash it three times each with deionized water and anhydrous ethanol, and vacuum dry it at 50 °C for 8 h. Then, place the obtained precursor in a muffle furnace, heat it to 300 °C at a heating rate of 1 °C / min under air atmosphere, and calcine it for 1 h to obtain the first solid.

[0026] (2) Weigh 1g of the first solid and disperse it in 70mL of 50vol% acetic acid solution. Stir at 45℃ for 4h. After treatment, collect the solid by centrifugation, wash it with deionized water until neutral, wash it once with anhydrous ethanol, and dry it under vacuum at 70℃ for 8h to obtain the second solid.

[0027] (3) Weigh 1g of the second solid and disperse it in a mixture of 0.8mL deionized water and 0.8mL anhydrous ethanol. After stirring for 0.5h, take 28mL of RuCl3 aqueous solution with a concentration of 0.95mg / mL and slowly add it dropwise to the above dispersion. After the addition is complete, continue stirring at room temperature for 3h. Then, evaporate the solvent at 80℃ and transfer the mixture to a muffle furnace and calcine at 250℃ for 2h to obtain Ru / Mg-vCeO2-2 material.

[0028] Example 3: 2.07 mmol of cerium nitrate hexahydrate (Ce(NO3)3∙6H2O), 0.2484 mmol of magnesium nitrate hexahydrate (Mg(NO3)2∙6H2O), and 0.058 mmol of polyvinylpyrrolidone (PVP, MW≈8000) were weighed and added to 82.8 mL of ethylene glycol. The mixture was ultrasonically dispersed until completely dissolved. Then, 4 mL of 1.2 mol / L hydrochloric acid solution was added, and ultrasonic dispersion was continued to obtain a homogeneous precursor solution. The precursor solution was transferred to a polytetrafluoroethylene-lined reactor and reacted at 200 °C for 6 h. After the reaction, the mixture was naturally cooled to room temperature, and the precipitate was collected by centrifugation. The precipitate was washed three times each with deionized water and anhydrous ethanol, and then vacuum dried at 70 °C for 15 h. Subsequently, the obtained precursor was placed in a muffle furnace and heated to 450 °C at a heating rate of 5 °C / min under air atmosphere, and calcined for 4 h to obtain the first solid.

[0029] (2) Weigh 1g of the first solid and disperse it in 130mL of 80vol% acetic acid solution. Stir at 80℃ for 10h. After treatment, collect the solid by centrifugation, wash it with deionized water until neutral, wash it once with anhydrous ethanol, and dry it under vacuum at 120℃ for 13h to obtain the second solid.

[0030] (3) Weigh 1g of the second solid and disperse it in a mixture of 2mL deionized water and 1.5mL anhydrous ethanol. After stirring for 1.5h, take 32mL of RuCl3 aqueous solution with a concentration of 1.05mg / mL and slowly add it dropwise to the above dispersion. After the addition is complete, continue stirring at room temperature for 6h. Then, evaporate the solvent at 80℃ to remove the solvent from the resulting mixture and transfer it to a muffle furnace. Calcine it at 400℃ for 6h to obtain Ru / Mg-vCeO2-3 material.

[0031] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that magnesium nitrate hexahydrate was not added in step (1), and the treatments in steps (2) and (3) were omitted. All other steps were the same as in Example 1, and CeO2 material was obtained.

[0032] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that steps (2) and (3) are omitted. All other steps are the same as in Example 1, and Mg-CeO2 material is obtained.

[0033] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that magnesium nitrate hexahydrate was not added in step (1) and the treatment in step (2) was omitted. All other steps were the same as in Example 1, and Ru / CeO2 material was obtained.

[0034] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that step (2) is omitted. All other steps are the same as in Example 1, and Ru / Mg-CeO2 material is obtained.

[0035] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that the volume fraction of the acetic acid solution used in step (2) is 90 vol%, the stirring temperature is 80°C, and the stirring time is 16 h. All other steps are the same as in Example 1, and Ru / vCeO2 material is obtained.

[0036] 1. Nanozyme Activity Assay: POD, SOD, and CAT Nanozyme Activity Assay: The sample from the example and the comparative sample were prepared according to the instructions of Solarbio Peroxidase (POD) Activity Assay Kit (BC0095), Solarbio Superoxide Dismutase (SOD) Activity Assay Kit (BC0170), and Catalase (CAT) Activity Assay Kit (BC4780), respectively. The reaction systems were then analyzed using an enzyme-linked immunosorbent assay (ELISA) reader, and the POD-like activity, SOD-like activity, and CAT-like activity of each sample were calculated.

[0037] 2. Structural characterization and performance analysis: such as Figure 1The XRD patterns of the sample from Example 1 show typical CeO2 diffraction peaks near 2θ of 28.6°, 33.1°, 47.5°, and 56.3°, indicating that the material obtained after Mg doping, selective etching with acetic acid, and Ru loading still retains the CeO2 main crystalline phase without significant crystalline phase destruction. Simultaneously, no obvious Ru particles or RuO2 crystalline phase characteristic peaks were observed in the patterns, indicating that Ru did not form large grains detectable by XRD. Combined with the XRD phase composition and ICP test results in Table 1, it can be seen that samples from Examples 1 to 3 all retain the CeO2 main crystalline phase, and residual Mg and Ru components were detected simultaneously. In Examples 1, 2, and 3, the residual Mg contents were 2.4 at%, 2.9 at%, and 2.1 at%, respectively, and the Ru loadings were 1.50 wt%, 1.28 wt%, and 1.61 wt%, respectively. This indicates that after Mg doping and selective etching, the Mg component was not completely removed but retained in a certain amount in the CeO2 support, which is beneficial for Ru species anchoring and atomic-level dispersion, reducing the generation of Ru particles and agglomerates. Comparative Example 1 was CeO2, with no detected Mg or Ru; Comparative Example 2 was Mg-CeO2, with a residual Mg content of 8.2 at%, but no Ru loading; Comparative Example 3 was Ru / CeO2, with no detected Mg and a Ru loading of 1.50 wt%; Comparative Example 4 was Ru / Mg-CeO2, with a residual Mg content of 7.8 at% and a Ru loading of 1.50 wt%; Comparative Example 5 was Ru / vCeO2, with a residual Mg content of less than 0.2 at% and a Ru loading of 1.50 wt% due to excessive etching. The above results indicate that the absence of Mg, insufficient etching, or excessive etching of Mg are all detrimental to the stable dispersion of Ru species, easily leading to the formation of Ru particles or agglomerates. The residual Mg content refers to the percentage of Mg atoms in the total number of metallic atoms in the final sample. Ru loading refers to the percentage of Ru element mass measured by ICP-OES in the total mass of the final sample.

[0038] like Figure 2As shown in Figure a, the sample from Example 1 exhibits a near-spherical nanoparticle morphology with good particle dispersion. Elemental distribution results show that Ce, Mg, and Ru are relatively uniformly distributed within the sample area, indicating that Mg doping, selective etching, and Ru introduction processes did not significantly damage the basic morphology of the CeO2 support and are conducive to the formation of Ru / Mg-vCeO2 nanomaterials with a relatively uniform compositional distribution. Combined with the TEM particle size statistics in Table 2, the average particle sizes of the samples from Examples 1, 2, and 3 are 50 nm, 53 nm, and 55 nm, respectively, with small size differences; while the average particle sizes of Comparative Examples 1, 3, and 5 are 58 nm, 60 nm, and 59 nm, respectively, all higher than the Example samples, indicating that Mg doping and selective acetic acid etching help obtain nanoparticles with smaller particle sizes and better dispersion. Further... Figure 2 The TEM results for b in Example 1 show numerous isolated bright spots on the sample surface. Combined with the absence of Ru or RuO2 crystal phase characteristic peaks observed by XRD, indicating the absence of obvious Ru clusters or large particles, it suggests that Ru primarily exists as single atoms on the CeO2 support surface. Table 1, showing the phase composition, main species, residual Mg content, and Ru loading of each sample, reveals that a suitable amount of residual Mg, along with the etching-induced defect structures, can provide stable anchoring points for Ru species, thereby improving the dispersion stability of Ru on the CeO2 support surface.

[0039] like Figure 3 As shown, after XPS analysis of the sample from Example 1, corresponding characteristic signals were observed in the high-resolution spectra of Ce3d, O1s, Ru3p, and Mg2p, indicating that the sample contains Ce, O, Ru, and Mg elements. Figure 3 a. Combined with Table 2, it can be seen that Ce in each sample 3+ The proportions, from highest to lowest, are: Example 1 (43.34%) > Example 3 > Example 2 > Comparative Example 5 > Comparative Example 4 > Comparative Example 2 > Comparative Example 3 > Comparative Example 1. Figure 3 b. Combined with Table 2, it can be seen that the defect-related oxygen ratio in the sample of Example 1 is 35.1%, and the trend of oxygen defect ratio in Example 1 and Comparative Examples 1-5 is similar to that of Ce. 3+ The fact that the proportions are basically consistent indicates that Mg doping and selective etching are beneficial for promoting Ce in the CeO2 support. 3+ And the formation of oxygen vacancy structures. Further by Figure 3 c indicates that the appearance of the Ru3p characteristic peak shows that Ru has been successfully introduced into the material surface; Figure 3 As shown in d, Mg signals can still be detected in the Mg2p spectrum, indicating that the Mg component was not completely removed during selective etching, but partially retained in the material. The oxygen defect ratio refers to the percentage of defect-related oxygen content relative to the total oxygen content of the sample, calculated based on the XPS O1s spectrum fitting results. Figure 4 As shown, the samples of Example 1 and Comparative Examples 1-5 all showed a relatively obvious EPR signal at g≈2.001. This signal is usually related to unpaired electrons associated with oxygen vacancies in CeO2, indicating that a certain number of oxygen vacancies were formed in the above samples. Figure 4 In the comparison, the EPR signal intensity of each sample, from highest to lowest, was: Example 1 > Comparative Example 5 > Comparative Example 4 > Comparative Example 2 > Comparative Example 3 > Comparative Example 1. The sample in Example 1 showed the strongest signal, indicating a high oxygen defect content. These results suggest that selective etching after Mg doping removes some Mg components while retaining an appropriate amount, which is beneficial for adjusting the local structure and surface defect state of the CeO2 support, thereby promoting the formation of oxygen defect sites.

[0040] As shown in Table 2, the BET specific surface areas of the samples in Examples 1-3 were 145.5, 126.1, and 133.7 m², respectively. 2 / g, all significantly higher than comparative examples 1-4 (67.6, 74.1, 69.4 and 78.3m). 2 / g), indicating that Mg doping and selective etching are beneficial for improving the pore structure, increasing the specific surface area and surface exposure of the material. Comparative Example 5 (109.4m), where Mg was almost completely removed, shows this. 2 Compared to the BET sample ( / g), the specific surface area of ​​the example sample is similar, indicating that the BET specific surface area cannot alone reflect the distribution of defect structures and active sites. Further analysis combining Ru dispersion state and Ce... 3+ The proportions and EPR defect signals show that although the specific surface areas of the example samples and Comparative Example 5 are similar, there are significant differences in residual Mg, oxygen vacancies, and Ru dispersion. These results indicate that the formation of active sites in the material is closely related to the synergistic effect of residual Mg, oxygen vacancies, and atomically dispersed Ru.

[0041] As shown in Table 3, Example 1 exhibited superior performance in the POD, SOD, and CAT enzyme activity tests. In the TMB-H2O2 system, the relative activity of the POD enzyme in Example 1 was 97.5%, approximately 6.1 times that of pure CeO2 in Comparative Example 1; its SOD enzyme superoxide anion scavenging rate was 90.4%, an increase of 50.8 percentage points compared to Comparative Example 1; and its CAT enzyme H2O2 decomposition efficiency was 82.7%, an increase of 51.2 percentage points compared to Comparative Example 1. These results indicate that Example 1 did not simply improve the activity of a single enzyme, but rather demonstrated significant improvements in the activities of POD, SOD, and CAT enzymes. Combined with the aforementioned structural characterization results, this performance improvement is mainly related to the synergistic effect of appropriate residual Mg, oxygen defect sites, and atomically dispersed Ru, which collectively improved the catalytic environment and active site distribution on the CeO2 support surface.

[0042] Further comparison of Examples 1 to 3 shows that, under different Mg doping levels and etching conditions, the samples in each example formed certain defect structures and exhibited POD, SOD, and CAT-like activities. Example 1 showed a better balance between residual Mg content, defect degree, Ru dispersion state, and multi-enzyme-like activity, thus exhibiting superior overall performance. Example 2 had a higher residual Mg content, which may have reduced defect site exposure or affected the effective immobilization of Ru species to some extent. Therefore, although the Mg retention was higher, the overall enzyme-like activity was lower than that of Example 1. This result indicates that the role of Mg is not simply dependent on its retention amount, but is closely related to the degree of selective etching, the carrier defect environment, and the Ru immobilization state. When Mg retention is insufficient or etching is excessive, the stability of the defect structure and the Ru anchoring environment are affected; when Mg retention is excessive, it may limit the exposure of active sites, hindering the full realization of multi-enzyme-like activity. Only under suitable conditions can residual Mg, oxygen defects, and Ru active sites form an effective synergy, thereby obtaining superior structure and performance.

[0043] In summary, combining Figures 1 to 4 As shown in Tables 1 to 3, Example 1 constructed a metal oxide composite structure containing residual Mg, oxygen defects, and highly dispersed Ru active sites through Mg doping, selective etching, and Ru introduction. While maintaining the stability of the CeO2 main crystal phase, this material exhibits a strong defect signal and high Ce content. 3+ The material exhibits a balanced and well-dispersed Ru content, demonstrating a synergistic enhancement of POD, SOD, and CAT-like activities. Comparative results further indicate that the aforementioned structure and properties are difficult to achieve without the introduction of Mg, with ineffective Mg removal, or with near-complete Mg removal. Therefore, the improved performance of the material in this invention primarily stems from the synergistic construction of residual Mg, defect sites, and Ru active centers, rather than a simple superposition of single factors.

[0044] Application Example 1: In vitro antibacterial experiment: 1. Experimental method: In vitro bactericidal experiment of Staphylococcus aureus (S. aureus): A glycerol-preserved strain of Staphylococcus aureus was inoculated into TSB liquid medium and cultured overnight at 37°C and 200 rpm. Subsequently, the bacterial solution was diluted 1:100 and activated for 2 hours to obtain a bacterial solution in the logarithmic growth phase for the antibacterial experiment. The activated S. aureus bacterial solution was adjusted to 1×10⁻⁶. 6CFU / mL was mixed with different nanozyme materials to achieve a final concentration of 600 µg / mL; H₂O₂ was added simultaneously to achieve a final concentration of 0.05 mM. The resulting mixtures were incubated at 37°C for 2 h. After incubation, the mixtures were serially diluted with sterile PBS, and 10 µL was evenly spread onto TSA solid medium. After incubation at 37°C for 24 h, colony counting was performed. Each group was repeated three times. The sterilization rate (%) = [1 - number of colonies in the experimental group / number of colonies in the control group] × 100%.

[0045] In vitro bactericidal experiment of *Escherichia coli* (E. coli): *E. coli* culture was inoculated into TSB liquid medium and incubated overnight at 37°C and 200 rpm. Subsequently, the bacterial suspension was diluted 1:100 and activated for 2 hours to obtain a suspension in the logarithmic growth phase for the antibacterial experiment. The activated *E. coli* bacterial suspension was adjusted to 1×10⁻⁶. 6 CFU / mL was mixed with different nanozyme materials to achieve a final concentration of 600 µg / mL; H₂O₂ was added simultaneously to achieve a final concentration of 0.05 mM. The mixture was incubated at 37°C for 2 h. After incubation, the mixture was serially diluted with sterile PBS, and 10 µL was evenly spread onto TSA solid medium. After incubation at 37°C for 24 h, colony counting was performed. Each group was repeated three times. The sterilization rate (%) = [1 - number of colonies in the experimental group / number of colonies in the control group] × 100%.

[0046] 2. Results and Analysis: such as Figure 5 As shown in Table 4, the sample of Example 1 exhibited good bactericidal effects against Staphylococcus aureus and Escherichia coli, with bactericidal rates reaching 98.9% and 99.9%, respectively, slightly higher than that of Examples 2 and 3, and significantly higher than that of the tested comparative samples. The results indicate that the sample of Example 1 possesses strong POD-like catalytic antibacterial ability in the presence of H2O2, effectively reducing bacterial survival rates. Combined with structural characterization and multi-enzyme-like activity analysis, its superior antibacterial effect is mainly attributed to the synergistic effect of partial removal of Mg, appropriate retention of residual Mg, formation of defect sites, and atomically dispersed Ru construction. This synergistic structure facilitates the exposure of active sites and promotes catalytic reactions, thereby enhancing the antibacterial ability of the material in the presence of H2O2.

[0047] Application Example 2: Diabetic Wound Healing Experiment: 1. Experimental Methods: Eight-week-old mice were selected and a diabetic model was established by intraperitoneal injection of streptozotocin (STZ, 150 mg / kg). Random blood glucose levels were monitored periodically after injection. Mice with two consecutive blood glucose levels not lower than 16.7 mmol / L were considered to have successfully established a diabetic model. Animal experiments were conducted in accordance with ethical requirements for experimental animals. Before the experiment, mice were anesthetized with 2% isoflurane, their back hair was shaved, and their skin was disinfected with 75% ethanol. Subsequently, a full-thickness skin wound with a diameter of 8 mm was prepared on the back of the mice using a skin biopsy piercing device. The materials prepared in Examples 1-3 and Comparative Examples 1-5 were dispersed in sodium hyaluronate / gelatin composite hydrogels to a final concentration of 0.8 mg / mL. The blank control group used only sodium hyaluronate / gelatin composite hydrogel dressings. The hydrogel dressings were applied topically to the wounds and changed every two days. Wound photographs were taken on days 0, 3, 7, 10, and 14, and the wound area was measured. The wound closure rate was calculated using the following formula: Wound closure rate (%) = (Initial wound area - Wound area at the corresponding time point) / Initial wound area × 100%. The results of the wound closure rates for each group are summarized in Table 4.

[0048] 2. Results and Analysis: such as Figure 6 and Figure 7 As shown, the Example 1 group exhibited a rapid wound shrinkage trend throughout the observation period. Combined with the statistical results in Table 4, it can be seen that on day 10, the wound closure rate of the Example 1 group reached 89.7%, higher than that of the blank control group and all comparative samples; on day 14, the wound closure rate of the Example 1 group further increased to 99.7%, significantly higher than the tested comparative samples. These results indicate that the material of Example 1 has a good wound repair effect in the context of diabetic wounds.

[0049] Based on the aforementioned structural characterization and multi-enzyme-like activity analysis, it is evident that the superior wound repair effect observed in Example 1 is related to the optimal equilibrium state of its material structure. On one hand, this sample exhibits strong POD-like activity, which is beneficial for inhibiting bacterial infection in the early stages of wound healing; on the other hand, it simultaneously possesses high SOD-like and CAT-like activities, which are beneficial for regulating oxidative stress in the wound and alleviating local hypoxia. In summary, this material system can achieve continuous regulation from early infection control to later wound repair through the synergistic effect of multi-enzyme-like activities.

[0050] Comprehensive comparison and summary analysis of the examples and comparative examples: To facilitate comparison of differences in composition, structural characteristics, enzyme activity, and application effects among different samples, this invention compares the main components and species, residual Mg content, Ru form, average particle size, specific surface area, and Ce content of each sample. 3+The results regarding proportion, defect-related oxygen ratio, POD, SOD and CAT-like activities, in vitro antibacterial rate, and diabetic wound closure rate are summarized in Tables 1 to 4. Based on the comprehensive structural characterization, enzyme-like activity test, in vitro antibacterial experiment, and animal wound repair experiment results, it can be seen that the sample of Example 1, while maintaining the stability of the CeO2 main crystal phase, exhibits high CeO2 content. 3+ The proportion of deficient oxygen and the EPR defect signal indicate that Ru has a good dispersion state and exhibits superior POD, SOD and CAT-like activity and wound repair effect, suggesting a good correspondence between its composition, structure and function.

[0051] Further comparison of Examples 1 to 3 shows that, under different Mg doping amounts and etching conditions, the samples in each example formed certain defect structures and exhibited multi-enzyme-like activities. Among them, Example 1 achieved a better match between residual Mg content, defect degree, Ru dispersion state, and multi-enzyme-like activity, thus showing better overall performance in in vitro antibacterial and diabetic wound repair experiments. This result indicates that the role of Mg is not simply dependent on the amount incorporated or retained, but is closely related to the degree of selective etching, the carrier defect environment, and the immobilization state of Ru active sites. When there is too little residual Mg or excessive etching, the stability of the defect structure and the Ru immobilization environment are affected; when there is too much residual Mg or insufficient etching, the exposure of active sites and the exertion of multi-enzyme-like activities are also limited.

[0052] The results of the comparative examples further illustrate the role of each step in the technical solution of the present invention. Comparative Examples 1 and 3 show that relying solely on the CeO2 support, or loading Ru without introducing Mg regulation, makes it difficult to form a stable defect structure and efficiently dispersed Ru active sites. Comparative Examples 2 and 4 show that simply performing Mg doping, or directly loading Ru without selective etching after Mg doping, makes it difficult to effectively increase the number of defect sites and the exposure of active sites. Comparative Example 5 shows that although excessive etching can increase the specific surface area and defect level to a certain extent and bring about a certain improvement in enzyme-like activity, when the residual amount of Mg is too low, its synergistic effect on the stability of the defect structure and the fixation of Ru active sites is weakened, and the overall performance is still lower than that of Example 1.

[0053] In summary, the core of this invention lies in achieving selective removal and appropriate retention of Mg components through Mg doping and selective etching, and then introducing Ru active sites to construct a metal oxide composite structure in which residual Mg, oxygen vacancies, and highly dispersed Ru active centers coexist. This structure can regulate the defect environment on the CeO2 support surface, stabilize Ru active sites, and promote the synergistic effect of POD, SOD, and CAT-like activities, enabling the material to possess early antibacterial, ROS-regulating, oxygen-supplying, and tissue repair-promoting effects in diabetic wound repair. Compared with technical routes that completely remove dopants or construct single defects, this invention has more clearly defined structural regulation characteristics and application advantages.

[0054] Table 1 shows the XRD, TEM, and ICP-OES characterization results of Examples 1-3 and Comparative Examples 1-5, presented in the form of major components, residual Mg content (at%), and Ru loading (wt%).

[0055] Table 2. TEM, BET, and XPS characterization results of Examples 1-3 and Comparative Examples 1-5. TEM characterization results are presented as average particle size, BET characterization results as specific surface area, and XPS characterization results as Ce. 3+ Display of ratio and defect oxygen ratio

[0056] Table 3. Relative activities of POD, SOD, and CAT enzymes in Examples 1-3 and Comparative Examples 1-5

[0057] Table 4. In vitro antibacterial and diabetic wound repair effects of each embodiment and comparative example.

Claims

1. A method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes, characterized in that, Includes the following steps: (1) Cerium nitrate hexahydrate, magnesium nitrate hexahydrate and polyvinylpyrrolidone were added to ethylene glycol and ultrasonically dispersed until completely dissolved; then hydrochloric acid solution was added and ultrasonic dispersion was continued to obtain a precursor solution; the precursor solution was transferred to a reaction vessel for solvothermal reaction; after the reaction was completed, the precipitate was collected by centrifugation and washed with deionized water and anhydrous ethanol in sequence, and after vacuum drying and calcination, the first solid was obtained; (2) Weigh the first solid and disperse it in acetic acid solution and stir. After the treatment is completed, centrifuge to collect the solid, wash it with deionized water until neutral, wash it with anhydrous ethanol, and dry it under vacuum to obtain the second solid. (3) Weigh the second solid and disperse it in a mixture of deionized water and anhydrous ethanol. After stirring and dispersing, slowly add RuCl3 aqueous solution and continue stirring at room temperature. Then, evaporate the obtained mixture to remove the solvent and calcine it to obtain Ru / Mg-vCeO2 material, which is magnesium-doped single-atom metal-supported cerium oxide nanozyme.

2. The method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes according to claim 1, characterized in that, In step (1), the ratio of cerium nitrate hexahydrate, magnesium nitrate hexahydrate, polyvinylpyrrolidone, hydrochloric acid solution, and ethylene glycol is 1 mmol: 0.10-0.12 mmol: 0.02-0.028 mmol: 1.5-2.5 mL: 25-40 mL; the concentration of the hydrochloric acid solution is 0.8-1.2 mol / L.

3. The method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes according to claim 1, characterized in that, In step (1), the solvothermal reaction temperature is 140-200℃ and the reaction time is 3-6h; the drying temperature is 50-70℃ and the drying time is 8-15h; the calcination temperature is 300-450℃ and the calcination time is 1-4h, and the calcination heating rate is 1-5℃ / min.

4. The method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes according to claim 1, characterized in that, In step (2), the ratio of the first solid to the acetic acid solution is 1g:70-130mL; the volume fraction of the acetic acid solution is 50-80 vol.

5. The method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes according to claim 1, characterized in that, In step (2), the stirring temperature is 45-80℃ and the stirring time is 4-10h; the drying temperature is 70-120℃ and the drying time is 8-13h.

6. The method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes according to claim 1, characterized in that, In step (3), the ratio of the amount of the second solid, deionized water, anhydrous ethanol and RuCl3 aqueous solution used is 1g:0.8-2mL:0.8-1.5mL:28-32mL, and the concentration of the RuCl3 aqueous solution is 0.95-1.05mg / mL.

7. The method for preparing magnesium-doped single-atom metal-supported cerium oxide nanozymes according to claim 1, characterized in that, In step (3), the first stirring time is 0.5-1.5h; the second stirring time is 3-6h; the calcination temperature is 250-400℃ and the calcination time is 2-6h.

8. An application of a magnesium-doped single-atom metal-supported cerium oxide nanozyme prepared by any one of the preparation methods of claims 1-7, applied to a wound repair material for diabetes.

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