A ceria multifunctional nanoscale enzyme material, a preparation method and application thereof

Abstract

The application discloses a ceria multifunctional nanoscale enzyme material and a preparation method and application thereof. The ceria multifunctional nanoscale enzyme material comprises mesoporous ceria, the mesoporous ceria is internally loaded with artesunate, and the mesoporous ceria loaded with the artesunate is coated with a hyaluronic acid layer. The preparation method comprises the following steps: mixing a cerium source with a mesoporous template, and then performing high-temperature calcination to obtain the mesoporous ceria; loading the artesunate in the mesoporous ceria through electrostatic adsorption; and forming a stable hyaluronic acid coating layer on the surface of the mesoporous ceria loaded with the artesunate through coordination of carboxyl groups of the hyaluronic acid with cerium. The ceria multifunctional nanoscale enzyme material is a safe and effective material with good cell biocompatibility, can promote healing of an infected diabetic wound, and can catalyze a peroxide bond in the artesunate through the enzyme-like activity of the ceria nanoscale enzyme, does not depend on H2O2, and spontaneously generates ROS to remove bacteria.

Description

Technical Field

[0001] This invention relates to the field of antibacterial nanomaterials, specifically to a cerium dioxide multifunctional nanoenzyme material, its preparation method, and its application. Background Technology

[0002] Bacterial infections remain a major public health problem globally, causing a variety of human diseases. With the decline in the development of new antibiotics and the long-term overuse of antibiotics, multidrug-resistant (MDR) bacteria are rapidly spreading worldwide. Bacteria develop resistance to antibiotics through various pathways, including expressing enzymes that can degrade, modify, or inactivate various antibiotics (such as β-lactamases), modifying antibiotic targets (e.g., through amino acid mutations or post-translational modifications), altering cell wall composition, and limiting drug uptake and efflux. Besides multidrug resistance, bacterial biofilm formation is also a major challenge in antimicrobial therapy. Biofilm formation often leads to recurrent or chronic infections, and even the formation of refractory infections, such as infective endocarditis, diabetic wound infections, and osteomyelitis. During the body's fight against bacterial infection, small amounts of reactive oxygen species (ROS) produced in a short period can combat bacteria. However, when infections persist for a long time or diseases such as sepsis produce large amounts of ROS and reactive nitrogen species (RNS) in a short period, exceeding the body's clearance capacity, excessive ROS can damage the host through oxidative damage, the initiation of inflammatory cascades, and systemic disorders. Among them, a representative refractory infection is diabetic wound infection. Due to high blood sugar, diabetic wounds are prone to mixed infection by multiple bacteria. In addition, the chemotaxis and phagocytosis of leukocytes in diabetic patients are defective, and bacteria in the wound can easily form a biofilm that is difficult to remove. Long-term use of antibiotics can easily lead to the development of drug-resistant bacteria. Bacterial infection in the wound and high concentrations of advanced glycation end products in the blood further lead to the generation of a large amount of reactive oxygen species and reactive nitrogen species, thereby preventing wound healing.

[0003] To address the above issues, current research has explored the application of nanozymes in antibacterial therapy. Nanozymes, with their enzyme-like properties, can disrupt bacteria and biofilms. Their antibacterial mechanisms differ significantly from those of traditional antibiotics, effectively circumventing antibiotic resistance mechanisms and targeting multidrug-resistant bacteria. Cerium dioxide (CeO2) nanoparticles are a novel type of nanozyme, with their surface containing Ce... 3+ and Ce 4+ The rapid and reversible conversion process endows cerium dioxide nanozymes (CeO2NPs) with both antioxidant and pro-oxidative properties. This dual nature makes CeO2NPs potentially useful in the treatment of diseases such as cancer, inflammation, and tissue damage. Furthermore, compared to other metallic materials, CeO2NPs exhibit relatively low toxicity to mammalian cells, are simple to prepare, possess excellent biocompatibility, and have regenerable catalytic performance, giving them significant advantages in biological applications.

[0004] Although CeO2NPs possess good enzyme-like activity, the amount of H2O2 in the human body, including infected tissues, is limited, and nanozymes cannot generate sufficient ROS to eliminate bacteria at the lesion site. Therefore, it is necessary to find a substrate that can replace H2O2, enabling it to exert peroxidase (POD) activity, undergo a Fenton-like reaction to generate hydroxyl radicals (·OH), and achieve antibacterial function. Summary of the Invention

[0005] In view of the above-mentioned shortcomings, this invention provides a cerium dioxide multifunctional nanozyme material, its preparation method, and its applications. The cerium dioxide multifunctional nanozyme material of this invention utilizes the porous structure of mesoporous cerium dioxide nanoparticles loaded with artesunate, and the artesunate-loaded mesoporous cerium dioxide nanoparticles encapsulate hyaluronic acid. Artesunate provides a peroxy-bridge structure, serving as a substrate for the Fenton reaction; hyaluronic acid can be degraded by hyaluronidase secreted by bacteria at the lesion site, increasing the concentration of artesunate (ACH) at the infection site, thereby initiating a cascade reaction to generate ROS and eliminate bacteria; on the other hand, it can improve the chemical stability and biocompatibility of the drug. Under acidic conditions at bacterial infection and biofilm sites, ACH can generate ROS to kill bacteria through POD-like enzyme activity and generate hypohalous acid to inhibit quorum sensing through halogen peroxidase (HPO)-like activity, thereby achieving antibacterial and anti-biofilm effects. Under physiological conditions, ACH exerts its antioxidant effect through superoxide dismutase (SOD) and catalase (CAT)-like activities. While killing bacteria, it effectively removes excess reactive oxygen species, generates oxygen, achieves antioxidant function, and promotes tissue healing.

[0006] To achieve the above objectives, the present invention provides a cerium dioxide multifunctional nanoenzyme material, wherein the cerium dioxide multifunctional nanoenzyme material comprises mesoporous cerium dioxide, wherein artesunate (ART) is loaded inside the mesoporous cerium dioxide, and the mesoporous cerium dioxide loaded with artesunate is coated with a hyaluronic acid layer.

[0007] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned cerium dioxide multifunctional nanoenzyme material, comprising the following steps:

[0008] Step 1: The gel ligand and Ce source are ultrasonically dispersed under the action of a mesoporous template, and then a sol is formed at a certain temperature. After calcination, mesoporous cerium dioxide is obtained.

[0009] Step 2: Dissolve mesoporous cerium dioxide in solvent A, add artesunate, and dissolve by ultrasonication, stir, centrifuge, and collect the precipitate to obtain mesoporous cerium dioxide loaded with artesunate;

[0010] Step 3: Dissolve hyaluronic acid in solvent B, and add mesoporous cerium dioxide loaded with artesunate to it. After stirring, centrifugation, precipitation, washing and drying, the hyaluronic acid is coated with mesoporous cerium dioxide loaded with artesunate, thus obtaining cerium dioxide multifunctional nanoenzyme material.

[0011] According to one aspect of the present invention, in step 1, the gel ligand is any one or more of sodium citrate, EDTA, and sodium trimetaphosphate; the Ce source is any one or more of Ce(NO3)3, CeCl3, Ce(OH)3, Ce2(C2O4)3, and CeH2SO4; and the mesoporous template is any one or more of CTAB, PVP, and F127.

[0012] According to one aspect of the present invention, in step 1, the temperature for forming the sol is 65-85°C and the time is 6-10 h; the calcination temperature is 250-300°C and the time is 2-3 h.

[0013] According to one aspect of the present invention, step 1 specifically involves: dispersing the gel ligand, Ce source, and a small amount of Ni, Co, or Pt precursor compound under the action of a mesoporous template by ultrasonication, forming a sol at a certain temperature, and then calcining it to obtain mesoporous cerium dioxide; wherein the mass of Ni, Co, or Pt in the small amount of Ni, Co, or Pt precursor compound is 3-5% of the mass of Ce in the Ce source.

[0014] It should be noted that adding a small amount of Ni, Co, or Pt precursor compounds can prepare oxygen-vacancy-deficient mesoporous cerium dioxide nanoparticles, which can improve the catalytic efficiency of cerium dioxide.

[0015] According to one aspect of the present invention, the precursor compound of Ni is Ni(NO3)2·6H2O, the precursor compound of Co is Co(NO3)2·6H2O, and the precursor compound of Pt is H2PtCl6.

[0016] According to one aspect of the present invention, in step 2, solvent A includes any one or more of anhydrous ethanol, methanol, DMSO, and DMF.

[0017] According to one aspect of the present invention, in step 3, the solvent B includes any one or more of ddH2O, ethanol, and DMF.

[0018] Based on the same inventive concept, this invention also discloses the application of the above-mentioned cerium dioxide multifunctional nanozyme material or the cerium dioxide multifunctional nanozyme material prepared by the above preparation method in the preparation of antibacterial therapeutic drugs.

[0019] According to one aspect of the present invention, the antibacterial therapeutic agent is a therapeutic agent for diabetic infected wounds.

[0020] Preparation mechanism of the present invention:

[0021] In this invention, cerium dioxide is mixed with a mesoporous template and then calcined at high temperature (burning off the mesoporous template) to obtain mesoporous cerium dioxide; artesunate is loaded inside the mesoporous cerium dioxide through electrostatic adsorption; a stable hyaluronic acid coating is formed by the coordination of the carboxyl groups of hyaluronic acid with metallic cerium.

[0022] The beneficial effects of this invention are:

[0023] (1) The cerium dioxide multifunctional nanozyme material of the present invention is produced by CeO 2-X NPs-type enzymes catalyze the peroxide bond (-OO-) in Art, independently of H2O2 and spontaneously generate ROS to eliminate bacteria;

[0024] (2) The cerium dioxide multifunctional nanoenzyme material (ACH) of the present invention is a safe and effective material with good cell biocompatibility;

[0025] (3) The cerium dioxide multifunctional nanozyme material (ACH) of the present invention can promote the healing of infectious diabetic wounds. Attached Figure Description

[0026] Figure 1 a is CeO prepared in Example 1 of this invention. 2-X Transmission electron micrographs of NPs and ACH; where a is CeO 2-X b is a transmission electron microscope image of NPs; b is a transmission electron microscope image of ACH.

[0027] Figure 2 The ACH thermogravimetric analysis diagram prepared in Example 1 of this invention;

[0028] Figure 3 This diagram illustrates the peroxidase-like (POD) activity of ACH as described in Example 2 of the present invention; where a represents the generation of hydroxyl radicals (·OH) by ACH under neutral (pH 7.2) and acidic (pH 6.0) conditions at different time points; b represents the generation of hydroxyl radicals (·OH) at different ACH concentrations; Note: The color order of the lines in the diagram is consistent with the color order of the upper right corner of each diagram;

[0029] Figure 4 The CeO as described in Embodiment 2 of the present invention 2-X The catalase-like (CAT) activity and degradation rate of NPs;

[0030] Figure 5This is the halogenated peroxidase (HPO) activity of ACH as described in Example 2 of the present invention; wherein, a is a schematic diagram of the principle of using phenol red as a probe to evaluate the conversion of phenol red to tetrabromophenol blue; b is the effect of different reaction times on the conversion of phenol red to tetrabromophenol blue; c is the effect of different ACH concentrations on the conversion of phenol red to tetrabromophenol blue;

[0031] Figure 6 The ACH and CeO described in Embodiment 2 of the present invention 2-X The ability of NPs to scavenge ROS; where a represents the reduction of DPPH to DPPH· by different concentrations of ACH; b represents the scavenging kinetics of DPPH free radicals by ACH; c represents the reduction of ABTS to ABTS· by different concentrations of ACH; and d represents the scavenging kinetics of ABTS free radicals by ACH.

[0032] Figure 7 This document describes the antibacterial effect of ACH on CREC as described in Example 2 of the present invention; wherein: a is a growth curve of carbapenem-resistant Escherichia coli (CREC) after 6 hours of treatment with ACH at different concentrations and pH values; b is a growth curve of CREC after 6 hours of treatment with different material combinations; c is a colony diagram of bacterial growth plates of CREC after 6 hours of treatment with different material combinations; d is a scanning electron microscope image of CREC after 6 hours of treatment with different material combinations.

[0033] Figure 8 This document describes the antibacterial effect of ACH on MRSA bacteria as described in Example 2 of the present invention; wherein: a is a growth curve of methicillin-resistant Staphylococcus aureus (MRSA) after 6 hours of treatment with ACH at different concentrations and pH values; b is a growth curve of MRSA after 6 hours of treatment with different material combinations; c is a colony diagram of MRSA bacteria on a plate after 6 hours of treatment with different material combinations; d is a scanning electron microscope image of MRSA bacteria after 6 hours of treatment with different material combinations.

[0034] Figure 9 The specific effects of ACH on CREC and MRSA bacterial biofilms described in Example 2 of this invention are shown below; where a represents the specific effect of ACH on CREC bacterial biofilm; and b represents the specific effect of ACH on MRSA bacterial biofilm.

[0035] Figure 10 This refers to the cellular biocompatibility of ACH and its components as described in Example 2 of the present invention; wherein, a represents the biocompatibility of ACH and its components with human microvascular endothelial cells; b represents the biocompatibility of ACH and its components with human vascular smooth muscle cells; c represents the biocompatibility of ACH and its components with human skin fibroblasts; and d represents the biocompatibility of ACH and its components with mouse macrophages RAW264.7.

[0036] Figure 11 The ACH, control group, and CeO2 described in Example 2 of this invention 2-x The effects of subcutaneous injection of ART, vancomycin (Van), and penicillin (PG) on MRSA-infected skin wounds in diabetic mice. Detailed Implementation

[0037] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0038] To address the technical problems mentioned in the background of this application, the present invention provides a cerium dioxide multifunctional nanoenzyme material, wherein the cerium dioxide multifunctional nanoenzyme material comprises mesoporous cerium dioxide, the interior of the mesoporous cerium dioxide is loaded with artesunate, and the mesoporous cerium dioxide loaded with artesunate is coated with a hyaluronic acid layer.

[0039] To address the technical problems mentioned in the background section of this application, the present invention also provides a method for preparing the above-mentioned cerium dioxide multifunctional nanoenzyme material, comprising the following steps:

[0040] Step 1: The gel ligand and Ce source are ultrasonically dispersed under the action of a mesoporous template, and then a sol is formed at a certain temperature. After calcination, mesoporous cerium dioxide is obtained. Preferably, the gel ligand is any one or more of sodium citrate, EDTA, and sodium trimetaphosphate.

[0041] Preferably, the Ce source is any one or more of Ce(NO3)3, CeCl3, Ce(OH)3, Ce2(C2O4)3, and CeH2SO4. Preferably, the mesoporous template is any one or more of CTAB, PVP, and F127. Preferably, the sol-forming temperature is 65-85℃, and the time is 6-10h. Preferably, the calcination temperature is 250-300℃, and the time is 2-3h. Preferably, the gel ligand, Ce source, and a small amount of Ni, Co, or Pt precursor compound are ultrasonically dispersed under the action of the mesoporous template, and then a sol is formed at a certain temperature, followed by calcination to obtain mesoporous cerium dioxide; wherein the mass of Ni, Co, or Pt in the small amount of Ni, Co, or Pt precursor compound is 3-5% of the mass of Ce in the Ce source. Preferably, the precursor compound of Ni is Ni(NO3)2·6H2O, the precursor compound of Co is Co(NO3)2·6H2O, and the precursor compound of Pt is H2PtCl6.

[0042] Step 2: Dissolve mesoporous cerium dioxide in solvent A, add artesunate, and dissolve by ultrasonication, stir, centrifuge, and collect the precipitate to obtain mesoporous cerium dioxide loaded with artesunate;

[0043] Preferably, solvent A includes any one or more of anhydrous ethanol, methanol, DMSO, and DMF.

[0044] Step 3: Dissolve hyaluronic acid in solvent B, and add mesoporous cerium dioxide loaded with artesunate to it. After stirring, centrifugation, precipitation, washing and drying, the hyaluronic acid is coated with mesoporous cerium dioxide loaded with artesunate, thus obtaining cerium dioxide multifunctional nanoenzyme material.

[0045] Preferably, solvent B includes any one or more of ddH2O, ethanol, and DMF.

[0046] Example 1

[0047] A method for preparing cerium dioxide multifunctional nanoenzyme material:

[0048] Step 1: Synthesis of mesoporous cerium dioxide nanoparticles (CeO) 2-X The specific steps for NPs are as follows: 260 mg sodium citrate and 900 mg CTAB are added to a reaction flask, followed by 1.077 mg Ce(NO3)3, 360 mg hexamethylenetetramine (to provide an alkaline environment, which is more conducive to the reaction), and 900 mL of water. The mixture is dissolved by sonication and then reacted at 85°C for 8 hours. The product is then washed twice with anhydrous ethanol, centrifuged, dried, and calcined at 300°C for 2 hours to obtain a yellow powder product.

[0049] Step 2: Synthesis of artesunate@cerium dioxide nanoparticles (ART@CeO) 2-X The specific steps (NPs) include: taking 50 mg of CeO2 obtained in step 1 above. 2-X NPs were dissolved in 50 ml of anhydrous ethanol, 15 mg of artesunate (Art) was added, the mixture was sonicated to dissolve, stirred overnight, and then centrifuged to collect the precipitate to obtain the product.

[0050] Step 3: Prepare Artesunate@Cerium Dioxide@Hyaluronic Acid Nanozyme (ART@CeO) 2-X @HANPs, ACH), specifically including the following steps: dissolve 50mg of hyaluronic acid in 50ml of ddH2O, and then add the Art@CeO obtained in step 2. 2-X NPs were dissolved in the solution, stirred for 2 hours, centrifuged to collect the precipitate, and finally washed with ddH2O and dried to obtain the final artesunate@cerium dioxide@hyaluronic acid nanozyme (ACH).

[0051] The artesunate@cerium dioxide@hyaluronic acid nanozyme (ACH) prepared above was analyzed by transmission electron microscopy and thermogravimetric analysis, and the results are as follows: Figure 1 , Figure 2 Among them, the transmission electron microscopy image shows CeO 2-X NPs are hollow spherical structures with a size distribution of 100-300 nm. Figure 1 a) Transmission electron microscopy images show that ACH has a spherical structure with a size distribution of 100-300 nm, and is uniform in size and well dispersed. Figure 1 b). Thermogravimetric analysis showed that the loading rate of Art was 25%, the loading rate of HA was 35%, and the loading rate of CeO2 was... 2-X The load rate is 40% ( Figure 2 ).

[0052] Example 2

[0053] Application of a cerium dioxide multifunctional nanozyme material in the treatment of diabetic infected wounds:

[0054] The peroxidase-like activity, catalase-like activity, halogenated peroxidase-like activity, antioxidant properties, in vitro antibacterial and anti-biofilm properties, and biocompatibility of ACH were investigated.

[0055] It should be noted that the ACH used in this embodiment was prepared by the method in Example 1.

[0056] It should be noted that this embodiment uses CeO2 without the substrate Art. 2-X NPs were obtained in step 1 of Example 1.

[0057] To evaluate the peroxidase-like (POD) activity of the ACH nanocomposite, we used TMB as a probe to assess its efficiency in generating hydroxyl radicals (·OH) under acidic conditions (pH 6.0). TMB can be oxidized by ·OH to generate oxidized TMB (oxTMB), changing from colorless to blue. Figure 3 As shown in Figure a, under pH 6.0 conditions, after reacting with 400 μg / mL ACH for different times (5, 10, 20, 30, 40, 50, 60 min), TMB can be oxidized by the generated ·OH to form oxidized TMB (oxTMB). The solution gradually changes from colorless to blue, and exhibits a characteristic absorption peak of oxTMB at 652 nm, showing a time-dependent change. Under neutral conditions (pH 7.2), the solution color did not change significantly after reacting with 400 μg / mL ACH for 60 min. These experimental phenomena indicate that ACH has good POD-like enzyme activity in a pH 6.0 environment. We further evaluated the colorimetric efficiency of TMB catalyzed by different concentrations of ACH, and the results are shown in Figure a. Figure 3 As shown in b, the characteristic absorption peak of oxTMB increases synchronously with increasing ACH concentration, indicating that the POD-like enzyme activity of ACH is concentration-dependent. These results demonstrate that ACH can spontaneously generate hydroxyl radicals under acidic conditions.

[0058] To investigate whether ACH possesses catalase-like activity (CAT) and its degradation rate under neutral conditions, we used CeO2 without the substrate Art. 2-X NPs were incubated with 100 mM H2O2, and the remaining H2O2 concentration was measured at different times. The results are as follows: Figure 4 As shown, 400 μg / mL CeO 2-X NPs can decompose over 85% of H2O2 within 120 min, and during this process, the amount of H2O2 in the solution gradually decreases with increasing time, exhibiting a time-dependent effect, confirming the synthesis of CeO2. 2-X NPs exhibit good catalase (CAT) activity.

[0059] To evaluate the activity of halogen-like peroxidases (HPO), we used phenol red (PR) as a probe to assess the efficiency of its conversion to tetrabromophenol blue. The principle is as follows: Figure 5 As shown in a, PR in Br - In the presence of hydrogen peroxide, it can be converted to tetrabromophenol blue (TBPB) via HPO catalysis. Co-incubation with 400 μg / mL ACH, 50 μM PR, and 200 mM NH4Br for different times (0, 6, 12, 24, 30 h) yielded the following results: Figure 5As shown in b, with increasing reaction time, the characteristic absorption peak of PR (430 nm) gradually decreases, while the characteristic absorption peak of TBPB (590 nm) gradually increases, indicating that ACH has HPO activity. With increasing ACH concentration, the UV characteristic absorption peak of TBPB gradually increases, exhibiting a concentration-dependent relationship. Figure 5 c). The above results indicate that ACH exhibits good HPO-like enzyme activity.

[0060] Under acidic conditions (pH 6.0), ACH kills bacteria by generating ROS through peroxidase-like activity. However, under normal physiological pH (pH 7.2), ACH can scavenge ROS as an antioxidant. Therefore, we further evaluated the ROS scavenging ability of ACH. DPPH is a stable purple free radical that, when reduced, transforms into a stable covalent structure (NH₄), changing its color to pale yellow and exhibiting a characteristic absorption peak at 520 nm. ABTS is generated by the reaction of ABTS with the oxidant (NH₄)₂S₂O₈. + It appears green and has a characteristic absorption peak at 734 nm. We used the above two probes to evaluate the overall antioxidant capacity of ACH. Figure 6 a. As the concentration of ACH increases, DPPH· is gradually reduced, and the solution color gradually changes from purple to yellow. The characteristic absorption peak of DPPH· at 520 nm decreases in dependence on the concentration of ACH. Figure 6 c indicates that as the ACH concentration increases, the green color of the solution gradually lightens, indicating that free radicals are scavenged, and ABTS... + The absorption peak at 734 nm also gradually decreased. We measured the scavenging kinetics of free radicals. Figure 6 (b) and (d) show that 400 μg / mL ACH can scavenge approximately 50% of free radicals within 10 hours. These experiments demonstrate that ACH possesses excellent antioxidant capacity.

[0061] To investigate the bactericidal effect of ACH, we evaluated the antibacterial effects of different materials. Figure 7 a and 7b are growth curves of carbapenem-resistant Escherichia coli (CREC) after 6 hours of exposure to various materials. Figure 7 c shows the colony diagram of CREC bacteria on a plate 6 hours after drug treatment. Figure 7 Scanning electron microscopy images of CREC bacteria 6 hours after drug treatment; results show that under pH 6.0 conditions, 200 μg / mL and 400 μg / mL ACH and 200 μg / mL CeO2... 2-X NPs + H₂O₂ inhibited the growth of CRECs, resulting in few or no colonies on plates, and scanning electron microscopy showed that the bacterial structure was destroyed. At pH 6.0, CeO₂... 2-X NPs cannot inhibit CREC growth in the absence of H2O2 substrate. Figure 7 b) Under pH 7.2 conditions, ACH and CeO 2-X NPs have no antibacterial effect. Figure 8 a and 8b are growth curves of methicillin-resistant Staphylococcus aureus (MRSA) after 6 hours of treatment with various materials. Figure 8 c shows the colony diagram of MRSA bacteria on a plate 6 hours after drug treatment. Figure 8 Image d shows a scanning electron microscope image of MRSA bacteria 6 hours after drug treatment; the results show that under pH 6.0 conditions, 200 μg / mL and 400 μg / mL ACH and 200 μg / mL CeO2... 2-X NPs + H₂O₂ inhibited the growth of MRSA; only a small number of colonies grew on the plate, and scanning electron microscopy showed that the bacterial structure was destroyed. At pH 6.0, CeO₂... 2-X NPs cannot inhibit MRSA growth in the absence of H2O2 substrate. Figure 8 b) Under pH 7.2 conditions, ACH and CeO 2-X NPs have no antibacterial effect. Figure 8 (a, b, c, d). The above results show that CeO2 only produces CeO2 under acidic conditions. 2-X NPs generate hydroxyl radicals in H2O2 solution through POD activity, and the ACH self-supplying peroxy bridge structure generates hydroxyl radicals through POD activity, which is necessary to eliminate bacteria. CeO 2-X NPs may kill some bacteria by utilizing the H2O2 produced by the bacteria's own metabolism, but the limited H2O2 produced by the body's metabolism cannot produce a significant bactericidal effect.

[0062] In conclusion, 400 μg / mL ACH treatment for 6 hours demonstrated potent antibacterial effects against both MRSA and CREC. Furthermore, the antibacterial effect of the ACH group was significantly superior to that of CeO. 2-X The antibacterial effects of the NPs group and the Art group used alone indicate that the ACH material complex, through CeO2, demonstrates its antibacterial properties. 2-X NPs-type enzymes catalyze the peroxide bond (-OO-) in Art, spontaneously generating ROS to eliminate bacteria without relying on H2O2.

[0063] We have already successfully demonstrated that ACH has good HPO activity. We further investigated the specific effects of ACH on the biofilm formation of two bacteria, CREC and MRSA. Figure 9 The formation of biofilms was quantitatively detected using crystal violet staining, and the results are as follows: Figure 9 As shown, the amount of CREC and MRSA biofilms formed was significantly reduced after the addition of ACH, indicating that ACH can significantly inhibit the formation of CREC and MRSA biofilms.

[0064] To assess the cell biocompatibility of ACH and its components, ACH and CeO2 were used. 2-X NPs and ART are associated with four types of cells: human microvascular endothelial cells (NPs and ART, respectively). Figure 10 a) Human vascular smooth muscle cells ( Figure 10 b), human skin fibroblasts ( Figure 10 c), mouse macrophages RAW264.7 ( Figure 10 d) After co-incubation for 24 hours, cell viability was assessed using the CCK-8 assay to determine cytotoxicity. The experimental results are shown in Figure 10. The ACH group and CeO2 group... 2-X Both the NPs and ART groups maintained high cell viability, indicating that 400 μg / mL ACH and CeO2 were effective. 2-X NPs are not toxic to the four cell types mentioned above. ACH is a safe, effective material with good cell biocompatibility.

[0065] The ACH nanozyme prepared in Example 1 was used to treat MRSA-infected skin wounds in diabetic mice via subcutaneous injection. Male C57BL / 6 mice were randomly divided into a control group (PBS), an ACH treatment group, and a cerium dioxide (CeO) group. 2-x Mice were divided into four groups: NPs (anti-inflammatory drugs), artesunate (ART), vancomycin (Van), and penicillin (PG), with 16 mice in each group. Subcutaneous injections of the drugs were administered on days 2, 5, 8, and 11 post-infection. Mice were euthanized on day 15, and organs were harvested for histopathological examination. Figure 11 It can be seen that, compared with the control group, the ACH group showed the best wound repair effect, indicating that the ACH nanozyme prepared in this invention can promote the healing of infectious diabetic wounds.

[0066] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A cerium dioxide multifunctional nanoenzyme material, characterized in that, The cerium dioxide multifunctional nanoenzyme material includes mesoporous cerium dioxide, the mesoporous cerium dioxide is internally loaded with artesunate, and the mesoporous cerium dioxide loaded with artesunate is coated with a hyaluronic acid layer.

2. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 1, characterized in that, Includes the following steps: Step 1: The gel ligand and Ce source are ultrasonically dispersed under the action of a mesoporous template, and then reacted at 65-85℃ for 6-10h to form a sol. After calcination, mesoporous cerium dioxide is obtained. Step 2: Dissolve mesoporous cerium dioxide in solvent A, add artesunate, and dissolve by ultrasonication, stir, centrifuge, and collect the precipitate to obtain mesoporous cerium dioxide loaded with artesunate; Step 3: Dissolve hyaluronic acid in solvent B, and add mesoporous cerium dioxide loaded with artesunate to it. After stirring, centrifugation, precipitation, washing and drying, the hyaluronic acid is coated with mesoporous cerium dioxide loaded with artesunate, thus obtaining cerium dioxide multifunctional nanoenzyme material.

3. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 2, characterized in that, In step 1, the gel ligand is any one or more of sodium citrate, EDTA, and sodium trimetaphosphate; the Ce source is any one or more of Ce(NO3)3, CeCl3, Ce(OH)3, Ce2(C2O4)3, and CeH2SO4; and the mesoporous template is any one or more of CTAB, PVP, and F127.

4. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 2, characterized in that, In step 1, the calcination temperature is 250-300℃ and the time is 2-3 hours.

5. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 2, characterized in that, Step 1 specifically involves: dispersing the gel ligand, Ce source, and a small amount of Ni, Co, or Pt precursor compound under the action of a mesoporous template by ultrasonication, reacting at 65-85℃ for 6-10 hours to form a sol, and then calcining it to obtain mesoporous cerium dioxide; wherein the mass of Ni, Co, or Pt in the small amount of Ni, Co, or Pt precursor compound is 3-5% of the mass of Ce in the Ce source.

6. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 5, characterized in that, The precursor compound for Ni is Ni(NO3)2·6H2O, the precursor compound for Co is Co(NO3)2·6H2O, and the precursor compound for Pt is H2PtCl6.

7. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 2, characterized in that, In step 2, solvent A includes any one or more of anhydrous ethanol, methanol, DMSO, and DMF.

8. The method for preparing cerium dioxide multifunctional nanoenzyme material according to claim 2, characterized in that, In step 3, solvent B includes any one or more of ddH2O, ethanol, and DMF.

9. The use of a cerium dioxide multifunctional nanozyme material as described in claim 1 or a cerium dioxide multifunctional nanozyme material prepared by any of the preparation methods described in claims 2-8 in the preparation of therapeutic drugs against carbapenem-resistant Escherichia coli and / or methicillin-resistant Staphylococcus aureus.

10. The application according to claim 9, characterized in that, The treatment drug is a drug for treating diabetic infected wounds.

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