Nano-enzyme compound with enhanced antibacterial activity as well as preparation method and application of nano-enzyme compound

Abstract

The invention relates to a nano-enzyme compound with enhanced antibacterial activity. A preparation method of the nano-enzyme compound comprises the following steps: (1) synthesizing hollow ferroferric oxide nano-particles; and (2) constructing a glucose oxidase-hollow ferroferric oxide nano-particle compound. The invention provides a preparation method of the nano-enzyme compound and application of the nano-enzyme compound in treatment of drug-resistant bacterial infection and biofilm-related infection, and further provides biological safety data of the nano-enzyme compound, so that support is provided for popularization of the nano-enzyme compound in clinical application. The diameter of the compound is about 200nm, the biocompatibility is good, the hollow structure endows the material with a larger surface area, and more enzyme immobilization sites and higher catalytic activity are provided. Through an optimized enzyme immobilization technology, glucose oxidase and ferroferric oxide are perfectly combined to form a sequential enzyme system, hydrogen peroxide can be accurately generated in a glucose oxidation reaction, and a large amount of active oxygen is further generated through catalysis of ferroferric oxide, so that the antibacterial effect is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nanoenzyme complex with enhanced antibacterial activity and a preparation method and application thereof. Background Art

[0002] Bacteria usually tend to form single or multiple microbial aggregates at interfaces rather than exist in a single cell form. They form complex biofilm structures by colonizing and secreting various biopolymers. Biofilms not only serve as physical barriers to limit drug penetration, but also help bacteria resist attacks from the immune system, further exacerbating the threat of drug-resistant bacterial infections.

[0003] Traditionally, antibiotics are the main means for humans to deal with bacterial infections. However, over time, the adaptability of bacteria has gradually emerged, the number of resistant strains has continued to increase, and the sensitivity of these strains to conventional antibiotics has been significantly reduced. In particular, some pathogens have developed multidrug resistance and are resistant to multiple antibiotics, which poses a great challenge to medical institutions in treating drug-resistant bacterial infections. In addition, antibiotics cannot effectively destroy or penetrate the biofilm structure, further weakening their therapeutic effect. In addition to the decline in therapeutic efficacy, antibiotics may also cause a series of side effects, including adverse effects on the host's gastrointestinal system, allergic reactions, and liver and kidney damage. Long-term or improper use of antibiotics may lead to intestinal microecological imbalance, and even cause super infection or further evolution of resistant strains, posing potential risks to patient health. Finally, the development of antibiotics requires high technical requirements, involving complex chemical synthesis and clinical trials, with a long development cycle and high costs. These factors not only increase the difficulty of developing new antibiotics, but also limit their widespread application and promotion speed in the market.

[0004] In addition to antibiotics, phage-based therapy is also an effective treatment for bacterial infections. Phages can specifically infect and kill specific bacteria, so they have potential advantages in dealing with drug-resistant bacterial infections. They have strong specificity and can self-add value to enhance the therapeutic effect, but phages can only target specific bacterial strains and have poor broad-spectrum, which limits their widespread application. In addition, the host may produce an immune response to phages, resulting in reduced therapeutic efficacy or even allergic reactions. The extraction and production process of phages is complicated, and it is difficult to ensure their quality and consistency.

[0005] Antimicrobial peptide therapy is a newly developed method for treating bacterial infections in recent years, which uses natural or synthetic antimicrobial peptides (AMPs) to treat bacterial infections. These antimicrobial peptides are composed of short-chain amino acids and have broad-spectrum antimicrobial activity, which can effectively fight against a variety of bacteria, fungi and viruses. The unique mechanism of antimicrobial peptides enables them to interfere with the integrity of bacterial cell membranes and quickly kill target bacteria. However, the synthesis and purification process of antimicrobial peptides is complicated and costly, which limits their widespread application. Many antimicrobial peptides are easily degraded by enzymes in the body, have a short biological half-life, and may require frequent administration. At the same time, some antimicrobial peptides may be toxic to host cells and cause side effects, especially when used in high concentrations. In addition, the broad-spectrum characteristics of antimicrobial peptides may affect non-target bacteria and disrupt normal flora. Therefore, although antimicrobial peptides show good potential in combating drug-resistant bacteria, many challenges still need to be overcome in clinical applications.

[0006] In the context of many limitations faced by current treatment methods, it is urgent to develop new antibacterial strategies that can effectively deal with drug-resistant bacterial infections. Driven by this demand, nanotechnology and enzyme catalysis have gradually become research hotspots. Nanomaterials have unique physicochemical properties, such as large specific surface area, adjustable surface charge and good biocompatibility, which enable them to penetrate biofilms and act on bacteria; at the same time, enzymes, as natural catalysts, can act on the metabolic process of bacteria through specific biochemical reactions, thereby achieving efficient sterilization. However, single nanomaterials or enzymes are easily disturbed in complex infection environments, and have poor stability and durability, which limits their practical application effects.

[0007] To overcome these problems, the present invention has created a new nanozyme complex based on glucose oxidase and ferroferric oxide. The complex uses glucose oxidase to catalyze the production of hydrogen peroxide, and at the same time, with the help of the peroxidase (POD) activity of ferroferric oxide, it generates highly active hydroxyl radicals, effectively destroying the biofilm structure and killing drug-resistant bacteria. This synergistic effect not only enhances the antibacterial activity, but also improves the stability and durability of the system, providing an efficient and safe alternative for the treatment of drug-resistant bacterial infections. Summary of the invention

[0008] The purpose of the present invention is to provide a nanozyme complex with enhanced antibacterial activity and a preparation method and application thereof in view of the deficiencies in the prior art.

[0009] In a first aspect, the present invention provides

[0010] A nanozyme complex with enhanced antibacterial activity, wherein the nanozyme complex is prepared by the following method:

[0011] (I) Synthesis of hollow Fe3O4 nanoparticles

[0012] a: Preparation of precursor solution: Dissolve iron nitrate nonahydrate in ethylene glycol, stir and add urea to obtain a precursor solution;

[0013] b: Hydrothermal reaction: The precursor solution is transferred to a reactor and the reaction is carried out under a magnetic field to generate magnetite nanocrystals;

[0014] c: Formation of nanoclusters: The concentration of generated magnetite nanocrystals increases and they aggregate in the supersaturated solution to form magnetite nanoclusters;

[0015] d: Carboxylation modification: adding sodium citrate solution to the obtained hollow ferroferric oxide nanoparticle solution, stirring the mixed solution to achieve carboxylation modification of the nanoparticles;

[0016] (II) Construction of Glucose Oxidase-Hollow Fe3O4 Nanoparticle Complex

[0017] a. Preparation of EDC / NHS activation solution: dissolve EDC and NHS in PBS buffer, add the EDC / NHS solution to the carboxyl-modified HMN suspension, and mix;

[0018] b. Activation of HMN carboxyl groups: Stirring the HMN and EDC / NHS mixture to activate the carboxyl groups on the surface of HMN and react them with the amino groups of the GOx enzyme;

[0019] c. Remove excess EDC / NHS: Centrifuge the activated HMN, discard the supernatant, and wash the precipitate with PBS to remove unreacted EDC / NHS;

[0020] d. Coupling with glucose oxidase: Add the GOx solution to the activated HMN suspension and incubate with stirring to form an amide bond between the HMN carboxyl group and the GOx amino group;

[0021] e. Purification of GOx-HMN conjugate: The reaction mixture was centrifuged to separate the GOx-HMN conjugate and free GOx, and washed with PBS or deionized water to remove unbound GOx.

[0022] As a preferred example, in the step of preparing a. EDC / NHS activation solution: the pH of the PBS buffer is 7.4, the final concentration of EDC is 5 mg / mL, and the final concentration of NHS is 5 mg / mL.

[0023] As a preferred example, in the step d. coupling with glucose oxidase: adding 2 mg / mL GOx solution to the activated HMN suspension.

[0024] As a preferred example, the synthesis step of hollow ferroferric oxide nanoparticles also includes washing and collecting: after carboxylation modification, the nanoparticles are washed with deionized water to remove unbound sodium citrate molecules, and then the surface carboxylated HMN is dried and collected for subsequent use.

[0025] As a preferred example, the nanozyme complex with enhanced antibacterial activity is a glucose oxidase-hollow ferrosoferric oxide nanoparticle complex with a diameter of about 200 nm.

[0026] In a second aspect, the present invention provides

[0027] A method for preparing a nanozyme complex with enhanced antibacterial activity, the method comprising the following steps:

[0028] (I) Synthesis of hollow Fe3O4 nanoparticles

[0029] a: Preparation of precursor solution: Dissolve iron nitrate nonahydrate in ethylene glycol, stir and add urea to obtain a precursor solution;

[0030] b: Hydrothermal reaction: The precursor solution is transferred to a reactor and the reaction is carried out under a magnetic field to generate magnetite nanocrystals;

[0031] c: Formation of nanoclusters: The concentration of generated magnetite nanocrystals increases and they aggregate in the supersaturated solution to form magnetite nanoclusters;

[0032] d: Carboxylation modification: adding sodium citrate solution to the obtained hollow ferroferric oxide nanoparticle solution, stirring the mixed solution to achieve carboxylation modification of the nanoparticles;

[0033] (II) Construction of Glucose Oxidase-Hollow Fe3O4 Nanoparticle Complex

[0034] a. Preparation of EDC / NHS activation solution: dissolve EDC and NHS in PBS buffer, add the EDC / NHS solution to the carboxyl-modified HMN suspension, and mix;

[0035] b. Activation of HMN carboxyl groups: Stirring the HMN and EDC / NHS mixture to activate the carboxyl groups on the surface of HMN and react them with the amino groups of the GOx enzyme;

[0036] c. Remove excess EDC / NHS: Centrifuge the activated HMN, discard the supernatant, and wash the precipitate with PBS to remove unreacted EDC / NHS;

[0037] d. Coupling with glucose oxidase: Add the GOx solution to the activated HMN suspension and incubate with stirring to form an amide bond between the HMN carboxyl group and the GOx amino group;

[0038] e. Purification of GOx-HMN conjugate: The reaction mixture was centrifuged to separate the GOx-HMN conjugate and free GOx, and washed with PBS or deionized water to remove unbound GOx.

[0039] In a third aspect, the present invention provides the use of nanozyme complexes in drugs for treating drug-resistant bacterial infections and biofilm-related infections.

[0040] As a preferred example, the bacterial infection is Staphylococcus aureus infection.

[0041] As a preferred example, the drug-resistant bacteria is methicillin-resistant Staphylococcus aureus.

[0042] The invention has the advantages of providing a glucose oxidase-hollow ferroferric oxide nanoparticle complex with a diameter of about 200 nm, which has good biocompatibility, and the hollow structure increases the surface area of ​​the material, provides more sites for enzyme fixation, and thus improves the catalytic activity. Through the optimized enzyme fixation technology, glucose oxidase and ferroferric oxide form a "sequential enzyme" system, which can accurately generate hydrogen peroxide in the glucose oxidation reaction, and then catalyze the production of a large amount of reactive oxygen (ROS) by ferroferric oxide, significantly improving the antibacterial effect. The complex can quickly destroy the structure of the bacterial biofilm, prevent the formation of the biofilm and its protective effect. It has excellent biosafety, ensuring safety and tolerance when used in the human body or on the surface of medical devices. In addition, the complex shows a wide range of application prospects in the treatment of drug-resistant bacterial infections and biofilm-related infections, especially in antibacterial treatment of medical device surfaces, wound care and new antibacterial therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Characterization of HMN and GHC. (a) Scanning electron microscope (SEM) image of HMN; (b) Transmission electron microscope (TEM) image of HMN; (c) Hydrodynamic diameter measurement results of HMN and GHC; (d) Zeta potential measurement results of HMN and GHC; (e) Electrophoretograms of HMN and GHC.

[0044] Figure 2 :Characterization of GHC. (a) Protease stability of GOx free enzyme and immobilized enzyme (GHC); (b) Relative activity comparison of GOx+HMN complex and GHC; (c) POD activity of GHC at different pH.

[0045] Figure 3:In vitro antibacterial effect of GHC. (a) Representative images of the spread plate method; (b) Statistical analysis results of the spread plate method; (c) Representative images of crystal violet staining; (d) Quantitative results of crystal violet staining; (e) Results of ONPG hydrolysis experiment; (f) Results of MDA concentration determination.

[0046] Figure 4 :GHC biosafety evaluation. (a) Cell survival rate of L929 cells after incubation with different concentrations of nanozyme complexes; (b) Cell survival rate of RAW 264.7 cells after incubation with different concentrations of nanozyme complexes; (c) HE sections of the main organs of mice after injection of nanozyme complexes. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0048] Example 1

[0049] 1. Synthesis and characterization of nanozyme complexes:

[0050] 1.1 Synthesis of Hollow Magnetite Nanoparticles (HMN)

[0051] a. Preparation of precursor solution: Weigh 1.782g of iron nitrate nonahydrate (Fe(NO3)3·9H2O) and dissolve it in 20mL of ethylene glycol, place it in a 20mL beaker, and stir it with a magnetic stirrer for 30 minutes. Then add 0.792g of urea and continue stirring for 30 minutes to obtain a uniform precursor solution.

[0052] b. Hydrothermal reaction: The precursor solution was transferred to a 20 mL Teflon-lined stainless steel reactor, and the reaction was carried out under an external 0.5 T magnetic field. The reactor was heated to 200°C, maintained for 48 hours, and then naturally cooled to room temperature. During this process, the complex of iron nitrate and urea completely decomposed at high temperature to generate magnetite (Fe3O4) nanocrystals.

[0053] c. Formation of nanoclusters: As the reaction proceeds, the concentration of generated magnetite nanocrystals increases and they spontaneously aggregate in the supersaturated solution to form magnetite nanoclusters.

[0054] d. Construction of hollow structure (Ostwald ripening): Under the action of an external magnetic field, the outer layer of magnetite nanocrystals of the magnetite nanocluster continues to grow through the Ostwald ripening process, gradually consuming the smaller particles inside. The magnetic field accelerates the dissolution and outward migration of the internal particles, and eventually forms a cavity inside, thereby obtaining hollow ferroferric oxide nanoparticles (HMN).

[0055] e. Carboxylation modification: Add sodium citrate solution to the obtained hollow ferroferric oxide nanoparticle (HMN) solution, stir the mixed solution at room temperature for 2 hours, so that the sodium citrate molecules are adsorbed on the surface of HMN, and the carboxylation modification of the nanoparticles is achieved through electrostatic interaction or coordination bonds.

[0056] f. Washing and collection: After carboxylation, the nanoparticles were washed thoroughly with deionized water to remove unbound sodium citrate molecules. The surface carboxylated HMNs were then dried in a vacuum oven and collected for subsequent applications.

[0057] 1.2 Construction of Glucose Oxidase-Hollow Magnetite Nanoparticle Composite (GHC):

[0058] a. Preparation of EDC / NHS activation solution: Dissolve EDC (final concentration: 5 mg / mL) and NHS (final concentration: 5 mg / mL) in PBS buffer (pH 7.4), add the EDC / NHS solution to the carboxyl-modified HMN suspension, and mix thoroughly.

[0059] b. Activation of HMN carboxyl groups: Gently stir the HMN and EDC / NHS mixture at room temperature for 30 min to activate the carboxyl groups on the surface of HMN so that they can react with the amino groups of the GOx enzyme.

[0060] c. Remove excess EDC / NHS: Centrifuge the activated HMN at 10,000 rpm for 10 minutes, carefully discard the supernatant, and wash the precipitate with PBS to remove unreacted EDC / NHS.

[0061] d. Coupling with glucose oxidase: GOx solution (2 mg / mL) was added to the activated HMN suspension and incubated at room temperature for 4 hours with gentle stirring to form an amide bond between the HMN carboxyl group and the GOx amino group. e. Purification of GOx-HMN conjugate: The reaction mixture was centrifuged at 10,000 rpm for 10 minutes to separate the GOx-HMN conjugate and free GOx, and the conjugate was washed three times with PBS or deionized water to remove unbound GOx.

[0062] 1.3 Characterization of HMN and GHC:

[0063] First, HMN ( Figure 1 In Figure a), we can see that the surface of the nanoparticles is rough. This structure not only increases the surface area, but also provides abundant binding sites for subsequent immobilization, ensuring that more glucose oxidase (GOx) can be firmly attached to it. This design is to increase the enzyme loading and thus increase the overall catalytic activity of the complex. Furthermore, TEM observation of HMN ( Figure 1 In b), the hollow structure inside can be clearly seen. This hollow design greatly increases the reaction space of the enzyme and provides an ideal microenvironment for the catalytic reaction. The hollow core allows the substrate and generated ROS to efficiently enter and exit the particle, avoiding the accumulation of reaction products, while maximizing the use of the enzyme's active sites and optimizing the catalytic efficiency. Figure 1 Medium c and Figure 1 Figure d shows the particle size and surface potential of HMN and GHC. The results of DLS measurement show that the particle size of HMN is 201.39nm, which slightly increases to 212.29nm after enzyme fixation, indicating that the enzyme is successfully bound to the particle surface. In addition, the zeta potential measurement results show that HMN and GHC have negative potentials of -29.64mV and -23.89mV, respectively. This negative potential helps their dispersibility and stability in water, thereby maintaining a good state during the reaction process and ensuring that the complex can play a continuous and efficient catalytic role in the liquid phase. Finally, the electrophoresis results ( Figure 1 The immobilization effect of the enzyme was further confirmed in Figure e). HMN without enzyme immobilization had no obvious bands, while GHC after enzyme immobilization showed protein bands, confirming the successful binding of GOx to nanoparticles.

[0064] The immobilized GHC showed significant advantages in applications. Figure 2 In (a), the activity of free GOx decreased rapidly in a short period of time, reflecting its instability. However, even after three hours of incubation, the immobilized GHC was still able to maintain more than 80% of the enzyme activity, showing the great advantage of immobilization in improving enzyme stability. This result not only lays the foundation for the application of GHC in complex in vivo environments, but also shows that immobilization can effectively resist the degradation of proteases, allowing the enzyme to function for a long time. Next, we further compared the POD activity of GHC with equal amounts of free GOx and HMN ( Figure 2b). The results showed that the immobilized GHC, as a "sequential enzyme" complex, exhibited higher overall catalytic activity. This significant improvement was mainly due to the increase in reaction sites and optimization of the catalytic environment brought about by immobilization, which enabled the enzyme to generate reactive oxygen species (ROS) more efficiently. This further emphasizes the importance of the immobilization process and demonstrates its potential in improving catalytic efficiency and enhancing antibacterial effects. In addition, the POD activity of GHC is pH-dependent ( Figure 2 In c), the activity of GHC is significantly enhanced, especially in an acidic environment, while it is almost inactive under neutral conditions. This property is crucial for targeted antibacterial therapy, because the bacterial infection site usually presents an acidic environment, and the activity of the immobilized enzyme complex is fully exerted at the infection site without causing damage to normal tissues.

[0065] 2. In vitro antibacterial effect of nanozyme complex:

[0066] 2.1 Methods:

[0067] a. Spread plate method: Bacteria cultured to the logarithmic growth phase are diluted to about 1×10^6 CFU / mL, mixed with different concentrations of GHC solution, and incubated at 37°C for 2 hours. Then, a series of dilutions are performed, spread on agar plates and cultured for 24 hours, the number of colonies on the plates is counted, and the number of viable bacteria at each concentration is calculated to evaluate the killing effect of GHC on bacteria.

[0068] b. Crystal violet staining method: After inoculating bacteria in a 24-well plate and culturing for 24 hours to form a biofilm, different concentrations of GHC solution were added and incubated for 2 hours. After the incubation was completed, crystal violet was used for staining at room temperature for 15 minutes, and the unbound dye was removed by washing. The absorbance was measured at 590 nm using a microplate reader to quantify the formation of the biofilm.

[0069] c. ONPG experiment: After the bacteria were mixed and incubated with different concentrations of GHC, ONPG was added and the incubation continued, and then the absorbance change at 420 nm was measured.

[0070] d. MDA content detection: After the bacteria are incubated with GHC, trichloroacetic acid and thiobarbituric acid are used to react, and the absorbance of the reaction product is measured to calculate the MDA concentration.

[0071] 2.2 Results:

[0072] A series of in vitro antibacterial experiments were conducted using methicillin-resistant Staphylococcus aureus (MRSA) as a representative strain to evaluate the antibacterial effect of the nanozyme complex and its possible mechanism of action. MRSA is a widely spread drug-resistant strain, and effective antibacterial strategies against it have important clinical significance. First, in the plate coating experiment ( Figure 3In (a), we can observe the effect of different concentrations of nanozyme complex on bacterial survival. Statistics show ( Figure 3 b), at a concentration of 25 μg / mL, the complex treatment significantly reduced the number of surviving bacteria, with an antibacterial effect of more than 50%; when the concentration increased to 100 μg / mL, the number of surviving bacteria was further significantly reduced, indicating that the nanozyme complex has a strong concentration dependence and good antibacterial effect. In order to evaluate its effect on MRSA biofilm, a crystal violet staining experiment was performed. The results show ( Figure 3 (c), after the complex treatment, the biofilm structure was obviously destroyed. Further quantitative analysis ( Figure 3 Figure d) shows that the nanozyme complex has a significant inhibitory effect on bacteria and can also effectively destroy biofilms. This property is of great significance for the treatment of stubborn drug-resistant bacterial infections. Next, to explore the antibacterial mechanism of the nanozyme complex, an ONPG hydrolysis experiment was conducted ( Figure 3 (e) As the concentration of the nanozyme complex increases, the absorbance increases significantly, indicating that the complex significantly increases the permeability of the bacterial membrane. This increase in permeability may disrupt the physiological balance of bacteria and further lead to bacterial death. Finally, the MDA content was determined ( Figure 3 In Figure f), the MDA concentration of bacteria was significantly increased after treatment with the nanozyme complex, indicating that the bacterial cell membrane underwent lipid peroxidation. As one of the important signs of ferroptosis, lipid peroxidation suggests that the nanozyme complex may achieve its antibacterial effect by inducing a "ferroptosis-like" mechanism in bacteria.

[0073] 3. Biosafety of Nanozyme Complex:

[0074] 3.1 Methods:

[0075] a. Cytotoxicity analysis (CCK-8 method): The CCK-8 kit was used to evaluate the in vitro biosafety of the nanozyme complex on L929 cells and Raw264.7 cells. L929 and Raw264.7 cells were inoculated in 96-well plates and cultured at a density of 1×10^4 cells per well for 24 hours. Nanozyme complexes of different concentrations were then added and cultured for another 24 hours. After adding CCK-8 solution and incubating at 37°C for 1-4 hours, the absorbance was measured at a wavelength of 450nm using a microplate reader. The experiment was repeated three times, and the cell survival rate was calculated to evaluate the cytotoxicity of the nanozyme complex.

[0076] b. In vivo toxicity analysis: In order to evaluate the in vivo biosafety of the nanozyme complex, healthy adult mice were selected as experimental subjects. 20 mg / kg of the nanozyme complex was administered by tail vein injection. The behavior and signs of the mice were observed after injection, and the mice were killed at the designated time point (3 days later). The heart, liver, spleen, lungs, and kidneys were removed and immediately fixed with 4% paraformaldehyde for 24 hours. The fixed tissues were routinely paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE), and the pathological changes of each tissue were observed under a microscope. The biosafety of the nanozyme complex in vivo was evaluated by comparing the histological results. 3.1 Results:

[0077] In the evaluation of the biosafety of the nanozyme complex GHC, cell viability analysis was first performed on L929 and Raw264.7 cells ( Figure 4 The results showed that even at a concentration as high as 200 μg / mL, L929 cells ( Figure 4 a) and Raw264.7 cells ( Figure 4 The survival rate of b) remains above 90%. This indicates that the nanozyme complex has less toxicity to cells at high concentrations, further confirming its good biosafety. In addition, considering its excellent effect in antibacterial experiments, a concentration of 100 μg / mL is sufficient for use.

[0078] Next, we evaluated the in vivo safety of the nanozyme complex GHC. The mice in the experimental group were injected with 20 mg / kg of the nanozyme complex, while the control group was injected with PBS. The results showed that both groups of mice behaved normally, with no significant changes in body weight, and their physiological indicators were also within the normal range. Three days after the injection, we removed the main organs for HE staining observation, and the results showed that there was no significant difference in the pathological characteristics of the two groups of tissues. These results collectively indicate that the safety of the nanozyme complex in vivo is also satisfactory and suitable for further application research.

[0079] In this study, a new type of glucose oxidase-hollow ferroferric oxide nanozyme complex (GHC) was successfully synthesized and systematically characterized and evaluated. The complex exhibited excellent antibacterial properties in vitro, especially against the widely spread drug-resistant bacteria MRSA, and was able to effectively destroy bacterial biofilms. This provides a new idea and method for the treatment of drug-resistant bacterial infections. In addition, we also explored the biosafety of the nanozyme complex in depth. The results of cell experiments and animal experiments showed that even at high concentrations, the nanozyme complex had extremely low toxicity to cells, and after in vivo injection, there were no obvious abnormalities in the physiological indicators and behaviors of mice, indicating that the complex has good biocompatibility and safety in practical applications.

[0080] By developing this highly efficient glucose oxidase-hollow ferroferric oxide nanozyme complex (GHC), we have provided a new solution for fighting drug resistance, significantly reducing the frequency of use of traditional antibiotics, thereby alleviating medical expenses and the economic burden on patients. In addition, good biosafety further enhances its feasibility in clinical applications. This research not only promotes the application of nanomaterials in the field of biomedicine, but also promotes scientific research progress in related fields, provides new product development directions for related companies, and contains broad commercial prospects.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A nanozyme complex with enhanced antibacterial activity, characterized in that: The nanozyme complex is prepared by the following method: (I) Synthesis of hollow Fe3O4 nanoparticles a: Preparation of precursor solution: Dissolve iron nitrate nonahydrate in ethylene glycol, stir and add urea to obtain a precursor solution; b: Hydrothermal reaction: The precursor solution is transferred to a reactor and the reaction is carried out under a magnetic field to generate magnetite nanocrystals; c: Formation of nanoclusters: The concentration of generated magnetite nanocrystals increases and they aggregate in the supersaturated solution to form magnetite nanoclusters; d: Carboxylation modification: adding sodium citrate solution to the obtained hollow ferroferric oxide nanoparticle solution, stirring the mixed solution to achieve carboxylation modification of the nanoparticles; (II) Construction of Glucose Oxidase-Hollow Fe3O4 Nanoparticle Complex a. Preparation of EDC / NHS activation solution: dissolve EDC and NHS in PBS buffer, add the EDC / NHS solution to the carboxyl-modified HMN suspension, and mix; b. Activation of HMN carboxyl groups: Stirring the HMN and EDC / NHS mixture to activate the carboxyl groups on the surface of HMN and react them with the amino groups of the GOx enzyme; c. Remove excess EDC / NHS: Centrifuge the activated HMN, discard the supernatant, and wash the precipitate with PBS to remove unreacted EDC / NHS; d. Coupling with glucose oxidase: Add the GOx solution to the activated HMN suspension and incubate with stirring to form an amide bond between the HMN carboxyl group and the GOx amino group; e. Purification of GOx-HMN conjugate: The reaction mixture was centrifuged to separate the GOx-HMN conjugate and free GOx, and washed with PBS or deionized water to remove unbound GOx.

2. The nanozyme complex according to claim 1, characterized in that In the preparation step of a. EDC / NHS activation solution: the pH of PBS buffer is 7.4, the final concentration of EDC is 5 mg / mL, and the final concentration of NHS is 5 mg / mL.

3. The nanozyme complex according to claim 1, characterized in that In the coupling step d. with glucose oxidase: 2 mg / mL GOx solution was added to the activated HMN suspension.

4. The nanozyme complex according to claim 1, characterized in that: The synthesis steps of hollow ferroferric oxide nanoparticles also include washing and collection: after carboxylation modification, the nanoparticles are washed with deionized water to remove unbound sodium citrate molecules, and then the surface carboxylated HMN is dried and collected for subsequent use.

5. The nanozyme complex according to claim 1, characterized in that The nanozyme complex with enhanced antibacterial activity is a glucose oxidase-hollow ferroferric oxide nanoparticle complex with a diameter of about 200 nm.

6. A method for preparing a nanozyme complex with enhanced antibacterial activity, characterized in that: The method comprises the following steps: (I) Synthesis of hollow Fe3O4 nanoparticles a: Preparation of precursor solution: Dissolve iron nitrate nonahydrate in ethylene glycol, stir and add urea to obtain a precursor solution; b: Hydrothermal reaction: The precursor solution is transferred to a reactor and the reaction is carried out under a magnetic field to generate magnetite nanocrystals; c: Formation of nanoclusters: The concentration of generated magnetite nanocrystals increases and they aggregate in the supersaturated solution to form magnetite nanoclusters; d: Carboxylation modification: adding sodium citrate solution to the obtained hollow ferroferric oxide nanoparticle solution, stirring the mixed solution to achieve carboxylation modification of the nanoparticles; (II) Construction of Glucose Oxidase-Hollow Fe3O4 Nanoparticle Complex a. Preparation of EDC / NHS activation solution: dissolve EDC and NHS in PBS buffer, add the EDC / NHS solution to the carboxyl-modified HMN suspension, and mix; b. Activation of HMN carboxyl groups: Stirring the HMN and EDC / NHS mixture to activate the carboxyl groups on the surface of HMN and react them with the amino groups of the GOx enzyme; c. Remove excess EDC / NHS: Centrifuge the activated HMN, discard the supernatant, and wash the precipitate with PBS to remove unreacted EDC / NHS; d. Coupling with glucose oxidase: Add the GOx solution to the activated HMN suspension and incubate with stirring to form an amide bond between the HMN carboxyl group and the GOx amino group; e. Purification of GOx-HMN conjugate: The reaction mixture was centrifuged to separate the GOx-HMN conjugate and free GOx, and washed with PBS or deionized water to remove unbound GOx.

7. Use of the nanozyme complex according to any one of claims 1-5 in the preparation of drugs for treating drug-resistant bacterial infections and biofilm-related infections.

8. The use according to claim 7, characterized in that: The bacterial infection is a Staphylococcus aureus infection.

9. The use according to claim 8, characterized in that: The drug-resistant bacteria are methicillin-resistant Staphylococcus aureus.

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