A mo o 3-x nano-enzyme composite hydrogel as well as a preparation method and application thereof

Abstract

The application relates to the field of biomedical technology, and discloses a MoO 3‑X Nanoplasma composite hydrogel, a preparation method and application thereof, and a preparation method and application thereof. 3‑X The composite hydrogel is prepared after the nanoplasma, and a photothermal-catalysis-immune regulation synergistic treatment system is constructed, which can be applied to a multiple drug-resistant bacteria infected wound as a functional dressing, and solves the comprehensive technical problems that the existing wound dressing is difficult to simultaneously overcome multiple drug-resistant bacteria infection, a biofilm barrier, wound hypoxia and excessive inflammation, and lacks intelligent response capability to an infected microenvironment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a MoO 3-X Nanozyme composite hydrogels, their preparation methods, and applications. Background Technology

[0002] Multidrug-resistant (MDR) bacterial infections and the resulting biofilms pose a common challenge to clinical wound healing. Traditional antibiotics are limited in efficacy due to drug resistance and also have poor permeability to the biofilm matrix, easily leading to prolonged infections. In recent years, nanozymes have attracted attention in antibacterial therapy due to their enzyme-like catalytic activity. Among them, peroxidase-like (POD) nanozymes can utilize the overexpressed hydrogen peroxide (H₂O₂) at the infection site to catalyze the generation of hydroxyl radicals (·OH), thereby causing oxidative damage to bacteria.

[0003] However, existing nanozymes still face some challenges in practical applications. For example, they tend to aggregate in physiological environments, which not only reduces their catalytic activity but also makes it difficult to distribute them evenly at the site of infection. Their residence time on the wound surface is short, making it difficult for them to exert a sustained effect in the infection microenvironment, and their penetration into deep biofilms is also limited. Furthermore, relying solely on the chemical catalysis or photothermal effects of nanozymes is often insufficient to completely eliminate multidrug-resistant bacteria and their biofilms, and may even induce bacterial tolerance. The cytotoxicity and inflammatory risks of some nanozymes also need attention, and their in vivo metabolic pathways are still unclear, requiring further research into their long-term safety.

[0004] To address these issues, researchers have attempted to load nanozymes onto hydrogels to improve their stability and local retention capacity. However, currently used hydrogel supports still have some shortcomings. For example, most are static network structures, lacking adaptability to dynamic changes in the wound microenvironment; the interaction between the support and the nanozyme is weak, easily leading to burst release or uneven loading. Functionally, they are also relatively singular, making it difficult to achieve synergistic effects such as antibacterial, anti-biofilm, pro-angiogenic, and anti-inflammatory properties.

[0005] In summary, existing solutions are often functionally limited, focusing only on one aspect such as antibacterial activity, wound healing promotion, or drug loading. Even when attempting to combine multiple functions, they are mostly simple additives, lacking synergistic effects at the mechanistic level. Furthermore, most materials are not sensitive enough to changes in the wound microenvironment, making it difficult to dynamically adjust according to the infection status. Therefore, a material system that can comprehensively address multiple issues such as biofilm barriers, hypoxia, and excessive inflammation, and intelligently adjust treatment behavior according to different stages of the wound, is still lacking. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a MoO 3-XNanoenzyme composite hydrogels, their preparation methods, and applications. A gel framework was formed by grafting L-glutamine with chitosan (CG) and oxidized dextran (PDA) prepared by sodium periodate oxidation, and MoO2 was loaded onto the gel. 3-X The preparation of composite hydrogels using nanozymes has created a photothermal, catalytic, and immunomodulatory synergistic therapeutic system that can be used as a functional dressing for wounds infected with multidrug-resistant bacteria. This solves the comprehensive technical challenges of existing wound dressings, which are unable to simultaneously overcome multidrug-resistant bacterial infections, biofilm barriers, wound hypoxia and excessive inflammation, and lack the ability to intelligently respond to the infection microenvironment.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A MoO 3-X The preparation method of nanozyme composite hydrogel includes the following steps: Step (1): Dissolve L-glutamine in water and activate it with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to obtain an L-glutamine solution; L-glutamine solution was slowly added dropwise to chitosan solution, and the reaction was carried out. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain L-glutamine-grafted chitosan (CG), which was then prepared into an aqueous solution. Step (2): Dissolve sodium periodate (NaIO4) in water and add it to the dextran aqueous solution. After the reaction is complete, dialyze and freeze dry to obtain ODex; ODex was prepared into an aqueous solution, 3-aminophenylboronic acid was added, and the reaction continued. After the reaction was completed, the solution was dialyzed and freeze-dried to obtain oxidized dextran (PDA), which was then prepared into an aqueous solution. Step (3): (NH4)6Mo7O 24 ·4H2O (ammonium molybdate tetrahydrate) reacts with N-acetyl-L-cysteine ​​dissolved in water. After the reaction is complete, the mixture is cooled, centrifuged, washed, and freeze-dried to obtain MoO. 3-X Nanozymes, and formulated into MoO 3-X Nanoenzyme aqueous solution; L-glutamine-grafted chitosan aqueous solution, oxidized dextran aqueous solution, MoO 3-X The nanozyme aqueous solution was mixed and gelled to obtain MoO. 3-X Nanoenzyme composite hydrogel.

[0008] Preferably, in step (1): the chitosan solution is prepared by dispersing 2g of chitosan in 70mL of 1%-1.5% (v / v) acetic acid aqueous solution; the molar ratio of L-glutamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(2-4):(0.8-1.2); the activation conditions are: after adjusting the pH value to 8.5, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added for activation for 2-3h.

[0009] Preferably, in step (1), the molar ratio of chitosan to L-glutamine is 1:(0.8-1.2), preferably 1:1; the reaction conditions are: reaction at room temperature for 2-2.5 h.

[0010] Preferably, in step (2): sodium periodate is prepared into an 8%-12% (w / v) aqueous solution and added to a 3%-5% (w / v) dextran aqueous solution, wherein the mass ratio of sodium periodate to dextran is 1:(1.2-1.4); the reaction conditions are: reacting at room temperature for 5-6 hours.

[0011] Preferably, in step (2): the concentration of ODex in the ODex aqueous solution is 5% (w / v); 3-aminophenylboronic acid is added to the solution, wherein the mass ratio of ODex to 3-aminophenylboronic acid is 1:(0.10-0.14), preferably 1:0.12; the reaction is continued under the following conditions: stirring at room temperature for 11-12 hours.

[0012] Preferably, in step (3), MoO is prepared 3-X Nanozymes: (NH4)6Mo7O 24 The molar ratio of 4H2O to N-acetyl-L-cysteine ​​is 1:(12-18), preferably 1:15; in (NH4)6Mo7O 24 In a mixed aqueous solution of 4H2O / N-acetyl-L-cysteine, (NH4)6Mo7O 24 The concentration of 4H2O was 0.008-0.012 mol / L; the reaction conditions were: reaction at 25-27℃ for 10-12 h.

[0013] Preferably, in step (3), MoO is prepared 3-X When making nanoenzyme composite hydrogels: L-glutamine-grafted chitosan aqueous solution, oxidized dextran aqueous solution, and MoO2 are added. 3-X The nanozyme aqueous solution is uniformly mixed at a volume ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2), preferably 1:1:1; wherein the concentration of L-glutamine-grafted chitosan aqueous solution is 5wt%, the concentration of oxidized dextran aqueous solution is 5wt%, and the concentration of MoO2 is...3-X The concentration of the nanozyme aqueous solution was 10 wt%; the gelation reaction conditions were: gelation reaction at room temperature, and the reaction time was within 3 minutes.

[0014] Preferably, a MoO2 solution as described above is used. 3-X Preparation method of nanoenzyme composite hydrogel to obtain MoO 3-X Nanoenzyme composite hydrogel.

[0015] Preferably, a MoO2 solution as described above is used. 3-X Application of nanozyme composite hydrogel in wound dressings.

[0016] Compared with the prior art, the beneficial effects of the present invention are reflected in: 1. This invention uses MoO 3-X The highly efficient photothermal conversion capacity and peroxidase-like activity of nanozymes, combined with the healing-promoting function of L-glutamine-grafted chitosan, have enabled the construction of a synergistic therapeutic system encompassing physical ablation (photothermal), chemical catalysis (·OH generation / O2 supply), and immune microenvironment regulation (VEGF upregulation / TNF-α downregulation). By constructing this synergistic therapeutic system of photothermal, catalysis, and immune regulation, multiple pathological processes, including drug-resistant bacterial infections, biofilm formation, tissue hypoxia, and excessive inflammation, can be addressed simultaneously.

[0017] 2. This invention constructs a hydrogel network based on the dynamic Schiff base bonds between CG and PDA, which can sensitively respond to the weakly acidic microenvironment of infected wounds, achieving MoO2. 3-X The nanozyme enables on-demand, controlled release of L-glutamine. Simultaneously, this dynamic bond endows the hydrogel with excellent self-healing capabilities, allowing it to rapidly restore structural integrity after damage, thus maintaining a durable and stable physical barrier and therapeutic function on dynamic wound surfaces.

[0018] 3. In this invention, MoO 3-X Nanozymes are prepared by mixing ammonium molybdate and N-acetyl-L-cysteine ​​in water, dialysis purification, and freeze-drying. They can simultaneously achieve near-infrared photothermal response and peroxidase-like activity, providing a multifunctional basis for wound treatment, specifically manifested in MoO2. 3-X The local photothermal effect generated by nanozymes under near-infrared light can physically destroy the biofilm matrix and enhance nanozyme penetration; the O2 produced by its catalysis can also alleviate the oxidative stress faced by persistently infected wounds, showing a highly efficient effect in resisting biofilms and eliminating drug-resistant bacteria.

[0019] N-acetyl-L-cysteine, acting as a reducing agent and structure-directing agent, reduces molybdate under mild conditions, thereby imparting MoO₂ to the structure. 3-XThe unique surface properties of nanozymes are due to the fact that the thiol (-SH) and amide groups in the N-acetyl-L-cysteine ​​molecule can not only effectively reduce the molybdenum precursor, but also, either themselves or their decomposition and coordination products, can be in situ modified on the nanozyme surface, introducing additional coordination sites, negatively charged groups, or improving biocompatibility. Compared with nanozymes prepared by hydrothermal reduction of molybdate, which suffer from problems such as limited surface properties, uneven dispersion, easy aggregation, and weak interaction with gel networks, the MoO2 nanozymes of this invention... 3-X The preparation method of nanozymes optimizes the performance of nanozymes from the source to better adapt them to smart hydrogel systems.

[0020] 4. The composite hydrogel prepared by this invention has injectability and good thixotropy, can closely conform to the contour of irregular wounds, and can be stably molded at body temperature, thus having excellent wound adaptability; its three-dimensional porous structure can absorb exudate and maintain a moist environment, while also having a certain degree of adhesion and not easily falling off, providing long-term physical protection and a therapeutic base for wounds.

[0021] 5. All components (chitosan, dextran, L-glutamine) used in the preparation of the composite hydrogel in this invention have good biocompatibility and degradability, good biosafety, and the preparation process is carried out in an aqueous phase under mild conditions, with clear steps and easy standardization. Attached Figure Description

[0022] Figure 1 The MoO prepared in Example 1 of this invention 3-X TEM morphology and EDS analysis of nanozymes; in the figures, a represents MoO2. 3-X TEM images of nanozymes on a 100 nm scale, image b shows the morphology of MoO. 3-X TEM morphology of nanozymes at a 50 nm scale; c represents MoO 3-X EDS analysis chromatogram of nanozymes, element is Mo; d is MoO 3-X EDS analysis chromatogram of nanozymes, element is O; Figure 2 The MoO prepared in Example 1 of this invention 3-X FTIR spectrum of nanozymes; Figure 3 The MoO prepared in Example 1 of this invention 3-X Particle size distribution of nanozymes; Figure 4 The MoO prepared in Example 1 of this invention 3-X X-ray photoelectron spectroscopy (XPS) image of nanozymes; Figure 4 In the middle, a represents MoO 3-X XPS elemental map of nanozymes, b is the elemental distribution of oxygen, and c is the elemental distribution of molybdenum. Figure 5 The MoO prepared in Example 1 of this invention 3-X O2 production of nanozymes under dissolved oxygen measurement; Figure 6 The MoO prepared in Example 1 of this invention 3-X Graph of enzyme-catalyzed reaction rate of nanozymes under fixed H2O2 content; Figure 7 The MoO prepared in Example 1 of this invention 3-X Graph of enzyme-catalyzed reaction rate under TMB immobilization; Figure 8 This is the MoO prepared in Comparative Example 1 of the present invention. 3-X Graph of enzyme-catalyzed reaction rate of nanozymes under fixed H2O2 content; Figure 9 This is the MoO prepared in Comparative Example 1 of the present invention. 3-X Graph of enzyme-catalyzed reaction rate under TMB immobilization; Figure 10 The MoO prepared in Example 2 of this invention 3-X Physical image of the @CG-PDA composite hydrogel; Figure 10 In the diagram, 'a' represents MoO₂ at pH = 6. 3-X @CG-PDA composite hydrogel physical image; b is MoO at pH=7.5 3-X @CG-PDA composite hydrogel physical image; c is MoO at pH=8 3-X Physical image of the @CG-PDA composite hydrogel; Figure 11 The MoO prepared in Example 2 of this invention 3-X Scanning electron microscope (SEM) image and elemental distribution energy dispersive spectroscopy (EDS) image of the @CG-PDA composite hydrogel; Figure 11 In the image, 'a' represents a scanning electron microscope (SEM) image, where a1 and a3 are SEM images of different sections of the hydrogel at a 200 μm scale, and a2 and a4 are SEM images of different sections of the hydrogel at a 50 μm scale; 'b' represents elemental energy dispersive spectroscopy (EDS) spectra, where b1 is the EDS spectrum of oxygen, b2 is the EDS spectrum of molybdenum, b3 is the EDS spectrum of carbon, and b4 is the EDS spectrum of nitrogen. Figure 12 The MoO prepared in Example 2 of this invention 3-X @CG-PDA composite hydrogel and CS (chitosan), CG (L-glutamine-grafted chitosan), MoO 3-X Fourier transform infrared (FTIR) spectrum of nanozymes; Figure 13 The MoO prepared in Example 2 of this invention 3-XFrequency scan, amplitude scan, and composite viscosity scan of the @CG-PDA composite hydrogel, as well as MoO2. 3-X Comparison of mechanical characterization of @CG-PDA composite hydrogel compared to CS-ODP hydrogel and CGCS hydrogel; Figure 13 In the diagram, a is the frequency scan of the hydrogel, b is the amplitude scan of the hydrogel, c is the composite viscosity scan of the hydrogel, and d is the MoO2 viscosity scan. 3-X Comparison of mechanical characterization of @CG-PDA composite hydrogel compared to CS-ODP hydrogel and CGCS hydrogel; Figure 14 The MoO prepared in Example 1 of this invention 3-X Temperature-time curves of nanozymes at different concentrations under 808nm near-infrared laser irradiation; Figure 15 The MoO prepared in Example 1 of this invention 3-X Temperature-time curves of nanozymes under different laser powers; Figure 16 The MoO prepared in Example 1 of this invention 3-X Photothermal cycling stability test results for nanozymes; Figure 17 The MoO prepared in Example 1 of this invention 3-X Fitting curve of photothermal conversion efficiency of nanozymes; Figure 18 The control group, CG group, CG-PDA group, and MoO group in Experimental Example 3 of this invention are... 3-X Group, MoO 3-X @CG-PDA group colony forming unit (CFU) count diagram of drug-resistant bacteria; Figure 18 In the diagram, A is the CFU count experimental graph of drug-resistant Staphylococcus aureus, B is the CFU count experimental graph of drug-resistant Escherichia coli, C is the CFU count statistical graph of drug-resistant Staphylococcus aureus, and D is the CFU count statistical graph of drug-resistant Escherichia coli. Figure 19 The control group, CG group, CG-PDA group, and MoO group in Experimental Example 3 of this invention are... 3-X Group, MoO 3-X @CG-PDA group biofilm removal effect image Figure 19 In the diagram, A represents the control group, CG group, CG-PDA group, and MoO2 group. 3-X Group, MoO 3-X @CG-PDA group's crystal violet staining effect on the removal of biofilms from drug-resistant Staphylococcus aureus and drug-resistant Escherichia coli. B represents the control group, CG group, CG-PDA group, and MoO2 group. 3-X Group, MoO 3-XLaser confocal images of the @CG-PDA group's effect on biofilm removal by drug-resistant Staphylococcus aureus and drug-resistant Escherichia coli; Figure 20 This refers to the control group, CG treatment group, CG-PDA treatment group, and MoO in Experimental Example 4 of this invention. 3-X Treatment group, MoO 3-X @CG-PDA treatment group wound healing trajectory on days 0, 3, 6, 9, and 12 in a mouse model of drug-resistant bacterial infection; Figure 21 This refers to the control group, CG treatment group, CG-PDA treatment group, and MoO in Experimental Example 4 of this invention. 3-X Treatment group, MoO 3-X Masson trichrome staining and hematoxylin-eosin (H&E) staining images of wound tissues from mice infected with drug-resistant bacteria in the @CG-PDA treatment group; Figure 21 In the diagram, 'a' represents the control group, CG treatment group, CG-PDA treatment group, and MoO2 group. 3-X Treatment group, MoO 3-X @CG-PDA treatment group mouse wound tissue infected with drug-resistant bacteria smeared with Masson's trichrome staining, b represents control group, CG treatment group, CG-PDA treatment group, MoO 3-X Treatment group, MoO 3-X Hematoxylin-eosin staining of wound tissue from mice infected with drug-resistant bacteria in the CG-PDA treatment group; Figure 22 This refers to the control group, CG treatment group, CG-PDA treatment group, and MoO in Experimental Example 4 of this invention. 3-X Treatment group, MoO 3-X VEGF factor ELISA images of wound tissues from mice infected with drug-resistant bacteria in the @CG-PDA treatment group on days 4 and 8; Figure 23 This refers to the control group, CG treatment group, CG-PDA treatment group, and MoO in Experimental Example 4 of this invention. 3-X Treatment group, MoO 3-X TNF-α factor ELISA images of wound tissues from mice infected with drug-resistant bacteria in the @CG-PDA treatment group on days 4 and 8; Figure 24 This refers to the control group, CG treatment group, CG-PDA treatment group, and MoO in Experimental Example 4 of this invention. 3-X Treatment group, MoO 3-X Hematoxylin-eosin (H&E) staining images of major organs (heart, liver, spleen, lung, and kidney) in wound tissues of mice infected with drug-resistant bacteria in the @CG-PDA treatment group. Detailed Implementation

[0023] The present invention will be further illustrated below through specific embodiments. The following embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the following embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and are included within the protection scope of the present invention.

[0024] Example 1 This embodiment discloses a MoO 3-X The preparation method of nanozymes includes the following steps: 0.5g (0.405mmol) of (NH4)6Mo7O 24 • 4H₂O and 0.98 g (6.0 mmol) of N-acetyl-L-cysteine ​​were dissolved in 100 mL of deionized water to form (NH₄)₆Mo₇O. 24 The concentration of ·4H2O was 0.01 mol / L. The reaction was carried out at 25 °C for 12 h. After the reaction was completed, the mixture was cooled, the precipitate was collected by centrifugation, washed with water and ethanol, and then freeze-dried under vacuum at -40 °C to obtain powdered MoO2. 3-X Nanozymes.

[0025] Example 2 This embodiment discloses a MoO 3-X The preparation method of nanozyme composite hydrogel includes the following steps: Step (1): Disperse 2g of chitosan in 70mL of 1% (v / v) acetic acid aqueous solution to obtain chitosan solution; L-glutamine was dissolved in deionized water at a ratio of 0.5 g to 20 mL. The pH was adjusted to 8.5, and EDC and NHS were added to activate the solution for 2 hours to obtain an L-glutamine solution. L-glutamine solution was slowly added dropwise to chitosan solution and reacted at room temperature for 2 hours. After the reaction was completed, small molecule impurities were removed by dialysis using a dialysis membrane with a molecular weight cut-off limit of 14 kDaltons. The mixture was then replaced with deionized water and freeze-dried to obtain L-glutamine-grafted chitosan, denoted as CG. The molar ratio of chitosan to L-glutamine is 1:1; the molar ratio of L-glutamine, EDC and NHS is 1:3:1. Step (2): Sodium periodate (NaIO4) was dissolved in deionized water to prepare a 10% (w / v) aqueous solution, which was then added to a 4% (w / v) dextran aqueous solution. The mass ratio of sodium periodate to dextran was 1:1.3. After reacting at room temperature for 6 hours, ethylene glycol was added and stirred for 2 hours to terminate the reaction and obtain the reaction product. The reaction product was dialyzed in deionized water for 48 hours using a dialysis bag (molecular weight cutoff of 3500 Da) and then freeze-dried to obtain ODex. ODex was dissolved in deionized water at a concentration of 5% (w / v), and 3-aminophenylboronic acid was added, wherein the mass ratio of ODex to 3-aminophenylboronic acid was 1:0.12. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed in deionized water for 48 h using a dialysis bag (molecular weight cutoff of 3500 Da). The mixture was then freeze-dried to obtain oxidized dextran, denoted as PDA. Step (3): Prepare a 5 wt% CG aqueous solution from the CG prepared in step (1); prepare a 5 wt% PDA aqueous solution from the PDA prepared in step (2); prepare the MoO2 prepared in Example 1... 3-X Nanozymes were formulated with 10 wt% MoO 3-X Nanoenzyme aqueous solution; Mix 5 wt% CG aqueous solution, 5 wt% PDA aqueous solution, and 10 wt% MoO 3-X The nanozyme aqueous solution was mixed in a volume ratio of 1:1:1 and subjected to a gelation reaction at room temperature. A pale yellow gel was formed within 3 minutes to obtain MoO. 3-X @CG-PDA composite hydrogel, i.e., MoO 3-X Nanoenzyme composite hydrogel.

[0026] Example 3 The difference from Example 2 is that the molar ratio of chitosan to L-glutamine in step (1) is changed to "the molar ratio of chitosan to L-glutamine is 1:0.8", while the other parameters and conditions are the same as in Example 2.

[0027] Example 4 The difference from Example 2 is that in step (3), "5wt% CG aqueous solution, 5wt% PDA aqueous solution, and 10wt% MoO" are used. 3-X The phrase "nanozyme aqueous solution mixed in a volume ratio of 1:1:1" is changed to "5 wt% CG aqueous solution, 5 wt% PDA aqueous solution, and 10 wt% MoO2". 3-X The nanozyme aqueous solution was mixed at a volume ratio of 1:1.2:1. All other parameters and conditions were the same as in Example 2.

[0028] Comparative Example 1 This comparative example discloses a MoO 3-XThe preparation method of nanozymes includes the following steps: Mix 1.5g of glucose and 0.5g of (NH4)6Mo7O 24 • 4H₂O was dissolved in 30 mL of deionized water and stirred for 20 min until completely dissolved. Hydrochloric acid was added dropwise to adjust the pH to 4, and the mixture was stirred for another 5 min. The reaction was carried out at 80 °C for 12 h. After the reaction was complete, the mixture was dialyzed for 48 h and then freeze-dried to obtain MoO₂. 3-X Nanozymes.

[0029] Comparative Example 2 The difference from Example 2 is that in step (3), "5wt% CG aqueous solution, 5wt% PDA aqueous solution, and 10wt% MoO" are used. 3-X The phrase "mixing nanozyme aqueous solution in a volume ratio of 1:1:1" was changed to "mixing 5 wt% CG aqueous solution and 5 wt% PDA aqueous solution in a volume ratio of 1:1". All other parameters and conditions were the same as in Example 2, and CG-PDA composite hydrogel was obtained.

[0030] Experimental Example 1 The MoO prepared in Example 1 3-X The structure, composition, and enzymatic properties of nanozymes were systematically characterized using transmission electron microscopy (TEM), elemental distribution spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), dissolved oxygen measurement, and enzyme-catalyzed reaction kinetics. The results are as follows: Figures 1 to 7 As shown; the MoO prepared in Comparative Example 1 3-X The enzymatic reaction kinetics of nanozymes were systematically characterized, and the results are as follows: Figures 8 to 9 As shown; Figure 1 TEM morphology and EDS analysis showed that the MoO prepared in Example 1 3-X The nanozyme exhibits a stacked nanosheet structure with a size of approximately 250 nm and good dispersion; the EDS elemental mapping clearly shows that Mo and O elements are uniformly distributed in the nanostructure.

[0031] Figure 2 The FTIR spectrum showed that the MoO prepared in Example 1 3-X Nanozymes at 3450 cm - A distinct Mo appeared at ¹ The characteristic tensile vibration peak of OH is located at 990 cm⁻¹. - The characteristic peak of the Mo=O bond at position ¹ is relatively weakened, which provides direct chemical evidence for the successful introduction of oxygen vacancies into the material.

[0032] Figure 3The particle size distribution results, with an average hydrated particle size of 240 nm measured by dynamic light scattering, further validate the MoO prepared in Example 1. 3-X The size of nanozymes.

[0033] Figure 4 The XPS spectra of the material show that the high-resolution 3d spectrum of Mo indicates the presence of characteristic Mo. 5+ The peaks, and the O 1s spectrum showing an increased proportion of hydroxyl oxygen, together confirm the presence of oxygen vacancies and the variable valence state of Mo. Specifically, in the chemical formula MoO... 3-X In this context, "3-X" indicates that the stoichiometric coefficient of oxygen is less than 3, meaning the material is in an oxygen-deficient state. Standard molybdenum trioxide (MoO3) is a stoichiometric compound with molybdenum having a +6 oxidation state and oxygen having 3 atoms. When some oxygen atoms are missing from the crystal, oxygen vacancies are formed, and the overall chemical formula changes to MoO. 3-X , where x represents the number of missing oxygen atoms in each chemical formula unit. The MoO prepared in Example 1 of this invention... 3-X In nanozymes, the value of x ranges from 0.1 to 0.3.

[0034] Figures 5 to 7 Systematic enzymatic evaluation showed that the MoO2 prepared in Example 1 3-X Nanozymes can effectively catalyze the production of oxygen from H2O2; among them, Figure 6 , Figure 7 As shown, the MoO prepared in Example 1 3-X Nanozymes exhibit higher catalytic efficiency and affinity for the substrates TMB and H2O2; while Figure 8 , Figure 9 This shows the MoO prepared in Comparative Example 1. 3-X The catalytic reaction rate of nanozymes is relatively low under the same test system. Therefore, the MoO prepared using the method of Example 1 of this invention... 3-X Nanozymes exhibited peroxidase-like catalytic performance significantly superior to glucose reduction. This significant improvement in catalytic activity is directly attributed to the optimization of surface chemistry and electronic structure brought about by the introduction of N-acetyl-L-cysteine, which in turn enables the final composite hydrogel to have superior efficiency in catalyzing endogenous H2O2 oxygen production, sterilization, and regulating oxidative stress.

[0035] In summary, the experimental results confirm that MoO2 possesses a unique nanosheet morphology, abundant oxygen vacancies, and highly efficient enzymatic activity. 3-X Nanozymes have been successfully prepared.

[0036] Experimental Example 2 MoO prepared in Example 2 3-XThe macroscopic morphology, microstructure, and chemical composition of the @CG-PDA composite hydrogel were characterized by digital photographs, scanning electron microscopy (SEM), elemental distribution energy dispersive spectroscopy (EDS), and Fourier transform infrared spectroscopy (FTIR). Simultaneously, the MoO2 prepared in Example 2 was also characterized. 3-X The mechanical properties of the @CG-PDA composite hydrogel were characterized compared to hydrogels with other components. The results are as follows: Figures 10 to 13 As shown.

[0037] Figure 10 MoO prepared in Example 2 3-X The image shows the physical sample of the @CG-PDA composite hydrogel. The material appears as a uniform pale yellow color. The gel remains intact in a buffer solution with a pH of 7.4, but it swells significantly and even disintegrates in a weakly acidic buffer solution (pH 6) that simulates an infection microenvironment. This also demonstrates its self-supporting gel properties.

[0038] Figure 11 For MoO 3-X SEM microstructure and EDS elemental distribution of the @CG-PDA composite hydrogel. The SEM images clearly show that the hydrogel has a continuous three-dimensional porous network structure with uniform pore size. EDS elemental mapping further confirms that Mo (representing the nanozyme) and C, N, and O (representing the polymer matrix) elements are uniformly distributed throughout the gel cross-section, indicating that MoO2... 3-X Nanozymes were successfully and uniformly loaded into the hydrogel network without significant aggregation.

[0039] Figure 12 The FTIR spectra provided evidence of chemical recombination, while simultaneously preserving the characteristic amide bands of CG-PDA (such as at 1650 cm⁻¹). - ¹C=O stretching vibration) and MoO 3-X The characteristic Mo-OH peaks indicate that the components have successfully recombinated through physical interactions.

[0040] Figure 13 This indicates that cyclic amplitude scanning ( Figure 13 c) shows that MoO 3-X The @CG-PDA composite hydrogel maintained a stable solid structure within a broad linear viscoelastic region, reaching approximately 200%. Composite viscosity (η*) scan ( Figure 13 a) shows that MoO 3-X The composite viscosity of the @CG-PDA composite hydrogel decreases with increasing frequency.

[0041] Furthermore, considering the crucial role of hydrogel self-healing properties in tissue adhesion, especially in highly deformable areas, oscillatory step strain tests were performed to evaluate self-healing capabilities. Figure 13in b). The results show that within the strain range of 1% to 300%, the reversible transition between structural damage (G′<G″) and structural recovery (G′>G″) can be repeatedly achieved, showing excellent self-healing properties.

[0042] In addition, through Figure 13 d, it can be concluded that in the present invention, the gel framework formed by crosslinking L-glutamine modified chitosan with 3-aminophenylboronic acid grafted oxidized dextran has better mechanical strength of the grid structure compared with hydrogels of other components (such as CS-ODP hydrogel and CGCS hydrogel). The stress peak of MoO prepared in Example 2 3-X @CG-PDA composite hydrogel is about 135kPa, while the CS-ODP hydrogel (see patent CN119978437A) is 85kPa, and the stress peak of CGCS hydrogel (see reference Ma Y, Liu C, Yan. A bacteriostatic hemostatic dressing prepared from L-glutamine-modified chitosan, tannic acid-modified gelatin and oxidized dextran[J]. International Journal of Biological Macromolecules: Structure, Function and Interactions, 2023, 242(Pt.1). DOI: 10.1016 / j.ijbiomac.2023.124669.) is only 37kPa. The reason is that the gel framework of Example 2 (MoO 3-X @CG-PDA) is formed by crosslinking L-glutamine grafted chitosan (CG) and 3-aminophenylboronic acid grafted oxidized dextran (PDA) via dynamic Schiff base bonds. The dynamic covalent bond not only endows the hydrogel with self-healing ability, but more importantly, forms a uniform, dense and stable three-dimensional network structure. The preparation method of CS-ODP hydrogel adopts the reaction of oxidized dextran with 1-(2-aminoethyl)piperidine or piperazin-1-amine, and then self-crosslinks with 3-aminophenylboronic acid grafted oxidized dextran. Crosslinking in this system mainly depends on the dynamic interaction between tertiary amine groups and phenylboronic acid. This interaction force is weaker than that of Schiff base bonds, and the crosslinking density may be lower, resulting in insufficient mechanical strength of its grid structure. CGCS hydrogel is prepared by mixing CG with GTA (gallic acid grafted chitosan) and then crosslinking with ODEX (oxidized dextran). In this system, there is a lack of strong covalent crosslinking between CG and GTA, and it relies more on physical mixing or weak chemical interactions, resulting in a relatively loose network structure and therefore the worst mechanical properties.

[0043] In summary, the experimental results validate the development of a MoO2 nanozyme with a uniform porous structure and uniform nanozyme loading. 3-X @CG-PDA composite hydrogel has been successfully prepared.

[0044] Experimental Example 3 MoO prepared in Example 1 3-X Nanozymes and MoO prepared in Example 2 3-X The photothermal properties and in vitro antibacterial and anti-biofilm efficacy of the @CG-PDA composite hydrogel were evaluated, and the results are as follows: Figures 14 to 19 As shown.

[0045] Figure 14 and Figure 15 MoO₂ under irradiation with different concentrations and powers of near-infrared laser (808nm) 3-X Temperature-time curve and photothermal imaging of nanozyme dispersion. Experimental method: Take 1 mL of MoO2... 3-X Nanoenzyme aqueous solution (containing different concentrations of MoO) 3-X Nanoparticles (50, 100, 200, and 300 μg / mL) were placed in a container and irradiated with an 808 nm near-infrared (NIR) laser, with temperature changes recorded in real time. The temperature response under different laser powers (0.5, 0.73, 1, and 1.25 W) was simultaneously tested. The results showed that the temperature rise was positively correlated with the nanozyme concentration and the laser power, confirming the reliability and controllability of its photothermal effect.

[0046] Figure 16 and Figure 17 MoO 3-X The photothermal cycling stability test and photothermal conversion efficiency fitting curve of the nanozyme are shown in the figure. It can be seen that after four laser on / off cycles, the material's heating capacity did not show a significant decrease, exhibiting good photothermal stability. Based on the cooling curve data, its photothermal conversion efficiency is as high as 49.7%, proving that MoO2... 3-X Nanozymes have excellent light-to-heat energy conversion capabilities.

[0047] like Figure 18 As shown, drug-resistant Escherichia coli and drug-resistant Staphylococcus aureus were used as model strains for in vitro antibacterial experiments. After pre-culturing the bacteria in LB nutrient medium for 12 hours, 200 μL of bacterial solution was aliquoted and mixed with different concentrations of MoO2. 3-XCo-incubation was performed at 37°C for 30 min. To analyze the photothermal effect, two different treatment groups were set up: the non-irradiated control group (-NIR) was placed in a dark environment, and the near-infrared laser treatment group (+NIR) was irradiated with an 808 nm laser (power density 0.75 W / cm²) for 5 min. The plate colony counting method (CFU) was used to count the bacteria in the control group, CG group (using 5 wt% CG aqueous solution from Example 2), CG-PDA group (using the CG-PDA composite hydrogel prepared in Comparative Example 2), and MoO2 group according to the above procedure. 3-X Group (using MoO prepared in Example 1) 3-X Nanozymes), MoO 3-X @CG-PDA group (using MoO prepared in Example 2) 3-X Group experiments were conducted using @CG-PDA composite hydrogel. After treatment, 100 μL of standardized bacterial suspension was taken from each group and evenly spread onto solid LB agar plates using a sterile spreader. After standing for 30 min for adsorption, the plates were inverted and incubated at 37℃ for 24 h. The antibacterial effect was finally evaluated by the standard colony forming unit (CFU) counting method. Quantitative colony counting showed that MoO2... 3-X @CG-PDA hydrogel under near-infrared light irradiation, MDR E. col i and S. aureus The sterilization rate exceeded 99%, and the relative survival rate of bacteria was reduced to an extremely low level. The same material also showed a significant antibacterial effect (survival rate of about 25-27%) under the condition of no light, or due to the continuous catalytic effect of nanozymes, but it was still far from the effect of photothermal synergy.

[0048] Figure 19 For the control group, CG group, CG-PDA group, and MoO 3-X Group, MoO 3-X The CG-PDA group's effect on the removal of biofilms from the above-mentioned drug-resistant bacteria (quantitatively quantified by crystal violet staining), MoO 3-X The CG-PDA+NIR treatment group showed the most significant removal effect on mature biofilms, with a reduction of over 80% in biofilm compared to the blank control group. Fluorescent staining also confirmed the mass death of bacteria within the biofilm. This is attributed to the MoO2 provided in this invention. 3-X Nanozymes possess excellent photothermal capabilities and synergistic effects among their components.

[0049] In summary, this embodiment confirms the effectiveness of the prepared MoO2. 3-X Nanozymes and composite hydrogels possess efficient, stable, and controllable photothermal properties, and can achieve efficient removal of multidrug-resistant bacteria and their biofilms in vitro through photothermal-catalytic synergy.

[0050] Test Example 4 To verify the MoO prepared in this invention3-X @CG-PDA composite hydrogel exhibits excellent wound-healing effects against drug-resistant bacterial infections. A mouse wound infection model was established using four-week-old mice (purchased from the Institute of Model Animals, Nanjing University). The mice were housed in a suitable environment with constant temperature (20±2℃) and humidity (50-60% relative humidity), with free access to sterile drinking water and specialized feed. Animals of similar weight were randomly divided into groups (n=7 per group). A circular full-thickness skin defect approximately 1 cm in diameter was surgically created on the back of the mice, and inoculated with a specified concentration of methicillin-resistant Staphylococcus aureus (MRSA) solution to establish the infected wound model. Subsequently, the mice were divided into a control group (PBS only), a CG treatment group (administered with 5 wt% CG aqueous solution from Example 2), a CG-PDA treatment group (administered with the CG-PDA composite hydrogel prepared in Comparative Example 2), and a MoO2 group. 3-X Treatment group (administered MoO prepared in Example 1) 3-X Nanozymes), MoO 3-X @CG-PDA treatment group (administered MoO2 prepared in Example 2) 3-X (@CG-PDA composite hydrogel), each treatment group received an additional 5 minutes of 808nm laser irradiation daily. Wound healing was quantitatively and qualitatively assessed by continuous measurement and photography on postoperative days 0, 3, 6, 9, and 12. Mice were euthanized after the experiment, and the wound and surrounding tissue were completely excised. Tissue samples were fixed, paraffin-embedded, and sectioned for hematoxylin-eosin (H&E) staining and Masson's trichrome staining. Histopathological examination was conducted to further evaluate the material's impact on mouse wound healing, primarily focusing on the integrity of tissue structure and the morphology and density of collagen deposition. Results are as follows: Figure 20 , Figure 21 As shown.

[0051] Figure 20 The results of wound healing experiments showed that in the drug-resistant bacterial infection wound model, MoO2... 3-X The wound closure rate in the @CG-PDA treatment group was close to 100% by day 12, and the healing speed was significantly faster than other groups. Quantitative analysis further confirmed the MoO2 treatment effect. 3-X The wound area in the @CG-PDA treatment group decreased rapidly over time, significantly better than that in the control groups.

[0052] Figure 21 Tissue sections (H&E and Masson staining) showed that MoO 3-X The @CG-PDA treatment group achieved complete wound re-epithelialization, with dense and orderly collagen fiber arrangement, resulting in the best repair quality.

[0053] In summary, the experimental results verify the MoO2 content. 3-X@CG-PDA composite hydrogel can achieve efficient healing of infected wounds by eliminating drug-resistant bacteria, regulating the wound microenvironment, and promoting tissue regeneration through the synergistic effect of photothermal and nanoenzyme properties.

[0054] Experimental Example 5 To verify the MoO prepared in this invention 3-X The CG-PDA composite hydrogel exhibits excellent anti-inflammatory and immunomodulatory capabilities. At specified time points (day 4 and day 8), the control group (using only PBS), the CG treatment group (using 5 wt% CG aqueous solution from Example 2), the CG-PDA treatment group (using the CG-PDA composite hydrogel prepared in Comparative Example 2), and the MoO2 group were respectively tested. 3-X Treatment group (administered MoO prepared in Example 1) 3-X Nanozymes), MoO 3-X @CG-PDA treatment group (administered MoO2 prepared in Example 2) 3-X Wound tissue samples were collected from mice using a CG-PDA composite hydrogel. After fixation, the tissue samples were embedded in an optimal cutting temperature (OCT) compound and then sectioned to prepare thin sections. The tissue sections were first blocked with an appropriate buffer, then incubated overnight at 4°C with primary antibodies targeting vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) for immunofluorescence analysis. After washing, the sections were incubated with fluorescently labeled secondary antibodies. Cell nuclei were counterstained with DAPI. The stained sections were imaged using a fluorescence microscope or a confocal laser scanning microscope. Results are as follows: Figures 22 to 23 As shown.

[0055] Figure 22 Fluorescent staining of vascular endothelial growth factor (VEGF) in mouse wounds revealed significantly different expression patterns among the treatment groups. MoO 3-X The @CG-PDA treatment group exhibited the strongest and most extensive VEGF fluorescence signal, indicating that angiogenesis was significantly promoted at the wound site.

[0056] Figure 23 Fluorescent staining of mouse wounds for the pro-inflammatory factor TNF-α showed that MoO2... 3-X The @CG-PDA treatment group exhibited the weakest TNF-α fluorescence intensity, indicating an effective inhibition of excessive inflammatory response. This controlled inflammatory state is crucial for normal wound healing, as persistent inflammation delays tissue repair. The control group maintained strong TNF-α signaling throughout the observation period, reflecting that unresolved inflammation hinders the healing process.

[0057] In summary, this aspect of MoO 3-X The mechanism of @CG-PDA composite hydrogel in promoting wound healing and angiogenesis was supported by specific experimental data. The experimental results verified the MoO2 mechanism.3-X The @CG-PDA composite hydrogel effectively reduces inflammatory responses and synergistically promotes tissue regeneration by regulating the interaction of two key factors: VEGF, which promotes angiogenesis, and TNF-α, which inhibits pro-inflammatory activity, creating an optimal microenvironment for wound repair.

[0058] Experimental Example 6 The MoO provided in this embodiment 3-X @CG-PDA hydrogel exhibits good in vivo biocompatibility. Its safety was characterized by histological staining experiments of major organs in mice infected with drug-resistant bacteria, and the results are as follows: Figure 24 As shown.

[0059] The results of histological analysis of major organs showed that: compared with the control group (PBS only), the CG treatment group (administered 5 wt% CG aqueous solution from Example 2), the CG-PDA treatment group (administered CG-PDA composite hydrogel prepared in Comparative Example 2), and MoO 3-X Treatment group (administered MoO prepared in Example 1) 3-X Nanozymes), MoO 3-X @CG-PDA treatment group (administered MoO2 prepared in Example 2) 3-X H&E staining was performed on the heart, liver, spleen, lungs, and kidneys of mice using the @CG-PDA composite hydrogel. The tissue morphology of the organs in each experimental group was consistent with that in the control group. The organ tissue structures of all mice in the treatment groups were clear and intact, with normal cell morphology, and no observable pathological changes such as cell edema, degeneration, necrosis, inflammatory cell infiltration, or fibrous tissue hyperplasia were observed. This directly confirms that the material has good systemic biocompatibility at effective therapeutic doses.

[0060] In summary, the experimental results verify the MoO2 content. 3-X The @CG-PDA composite hydrogel showed no significant toxic damage to the major organs of mice and demonstrated good in vivo biocompatibility.

[0061] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A MoO 3-X A method for preparing nanozyme composite hydrogels, characterized in that, Includes the following steps: Step (1): Dissolve L-glutamine in water, activate it with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then react it with chitosan solution to obtain L-glutamine-grafted chitosan, and prepare an aqueous solution of L-glutamine-grafted chitosan. Step (2): (NH4)6Mo7O 24 ·4H2O reacts with N-acetyl-L-cysteine ​​dissolved in water to produce MoO 3-X Nanozymes, and configured as MoO 3-X Nanoenzyme aqueous solution; Step (3): Add L-glutamine-grafted chitosan aqueous solution and MoO 3-X A mixture of nanozyme aqueous solution and oxidized dextran aqueous solution undergoes a gelation reaction to obtain MoO2. 3-X Nanoenzyme composite hydrogel; The oxidized dextran aqueous solution is prepared by the following steps: Sodium periodate was dissolved in water and added to a dextran aqueous solution. The reaction was carried out, dialyzed, and freeze-dried to obtain ODex. ODex was prepared into an ODex aqueous solution, 3-aminophenylboronic acid was added, the reaction was continued, dialyzed, and freeze-dried to obtain oxidized dextran, which was then prepared into an oxidized dextran aqueous solution.

2. A MoO 3-X The preparation method of the nanoscale enzyme composite hydrogel, characterized in that, In step (1): the chitosan solution is prepared by dispersing 2g of chitosan in 70mL of 1%v / v-1.5%v / v acetic acid aqueous solution; the molar ratio of L-glutamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(2-4):(0.8-1.2); the activation conditions are: after adjusting the pH value to 8.5, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added for activation for 2-3h.

3. A MoO 3-X The preparation method of the nanoscale enzyme composite hydrogel, characterized in that, In step (1), the molar ratio of chitosan to L-glutamine is 1:(0.8-1.2); the reaction conditions are: reacting at room temperature for 2-2.5 h.

4. A MoO according to claim 1 3-X A method for preparing nanozyme composite hydrogels, characterized in that, In step (2): (NH4)6Mo7O 24 The molar ratio of 4H₂O to N-acetyl-L-cysteine ​​is 1:(12-18); in (NH₄)₆Mo₇O 24 In a mixed aqueous solution of 4H2O / N-acetyl-L-cysteine, (NH4)6Mo7O 24 The concentration of 4H2O was 0.008-0.012 mol / L; the reaction conditions were: reaction at 25-27℃ for 10-12 h.

5. A MoO according to claim 1 3-X A method for preparing nanozyme composite hydrogels, characterized in that, In step (3): sodium periodate is prepared into an 8% w / v-12% w / v aqueous solution and added to a 3% w / v-5% w / v dextran aqueous solution, wherein the mass ratio of sodium periodate to dextran is 1:(1.2-1.4); the reaction conditions are: react at room temperature for 5-6 hours; the concentration of ODex in the ODex aqueous solution is 5% w / v; the mass ratio of ODex to 3-aminophenylboronic acid is 1:(0.10-0.14); the reaction is continued at room temperature with stirring for 11-12 hours.

6. A MoO according to claim 1 3-X A method for preparing nanozyme composite hydrogels, characterized in that, In step (3): L-glutamine-grafted chitosan aqueous solution, oxidized dextran aqueous solution, and MoO2 are added. 3-X The nanozyme aqueous solution was uniformly mixed at a volume ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2).

7. A MoO 3-X The preparation method of the nanoscale enzyme composite hydrogel, characterized in that, In step (3): the concentration of L-glutamine-grafted chitosan aqueous solution is 5 wt%, the concentration of oxidized dextran aqueous solution is 5 wt%, and the concentration of MoO2 is... 3-X The concentration of the nanozyme aqueous solution was 10 wt%; the gelation reaction conditions were: gelation reaction at room temperature, and the reaction time was within 3 minutes.

8. A MoO 3-X The preparation method of the nanoscale enzyme composite hydrogel The prepared MoO 3-X The nanoscale enzyme composite hydrogel.

9. A method using MoO as described in claim 8 3-X Application of nanozyme composite hydrogels in the preparation of wound dressings.

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