Nanogel for activating metronidazole through starvation therapy to co-treat non-anaerobic infection

Abstract

The invention discloses nanogel for activating metronidazole to co-treat non-anaerobic bacteria infection through starvation therapy. The nanogel takes composite nano hydrogel which is formed by compounding sodium alginate SA, a natural polyphenol compound and high-valence metal ions and has a three-dimensional network structure as a main carrier; glucose oxidase GOx and anaerobic bacteria antibiotics required by starvation therapy are loaded in composite nanogel, meanwhile, natural polyphenol and high-valence metal ions in the composite nanogel can be self-assembled to form a metal-polyphenol network structure MPNs, and the nanogel loaded with GOx and MNZ is packaged to prepare the composite nanogel. A new cocktail therapy is constructed, a new thought is provided for research and development of antibacterial treatment drugs, and great scientific research significance and clinical application value are achieved for collaborative treatment of tolerant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials technology, and more specifically, it relates to a nanogel that activates metronidazole for synergistic treatment of non-anaerobic bacterial infections using starvation therapy, its preparation method, and its application. Background Technology

[0002] Bacterial infections have long posed a serious threat to human life and health, and are one of the leading causes of death. Antibiotics, due to their high efficacy and broad-spectrum antibacterial properties, have been widely used to treat bacterial infections in humans and animals since their discovery. Therefore, there is an urgent need to develop novel antibacterial materials and seek new antibacterial strategies.

[0003] Starvation therapy, by blocking the nutrient supply to bacteria through various means, "starves" the bacteria to achieve therapeutic goals, rather than directly removing or killing them. It offers the advantages of being non-invasive and environmentally friendly, providing a new approach to bacterial treatment. Glucose oxidase (GOx) is a biological enzyme widely present in nature and within organisms. In the presence of oxygen, GOx catalyzes the production of gluconic acid and hydrogen peroxide (H2O2) from glucose, depleting the oxygen and glucose necessary for the microenvironment or bacterial metabolism, thus "starving" the bacteria and achieving therapeutic goals. Although GOx-mediated starvation therapy has shown excellent performance in antibacterial treatment, GOx can catalyze reactions in both bacterial and normal tissue cells. Non-specific administration may lead to systemic toxicity and lack of targeted delivery. Furthermore, GOx consumes intracellular oxygen during glucose catalysis, causing intracellular hypoxia in bacteria, which limits the catalytic reaction and hinders the full effectiveness of starvation therapy. Therefore, it is necessary to design reasonable nano-drug delivery systems with specific functions so that GOx can be specifically released at the site of bacterial infection to exert its effects, reduce systemic toxicity, and work in conjunction with other treatment regimens to achieve the ideal antibacterial effect at a lower concentration, thus overcoming the shortcomings of using single drugs for antibacterial treatment.

[0004] Anaerobic antibiotics are antibiotics used to treat various infectious diseases caused by anaerobic bacteria. These are antibiotics that possess high activity and strong antibacterial effects against various bacteria that can survive and reproduce in anaerobic or severely hypoxic environments. After starvation therapy, the oxygen in the bacterial microenvironment is gradually depleted, creating a severely hypoxic environment. This hypoxic environment can further activate anaerobic antibiotics, causing them to produce cytotoxicity and subsequently kill non-anaerobic bacteria with lower metabolic activity under hypoxic conditions.

[0005] Due to the complex interactions between bacteria and their hosts, bacterially infected tissues typically possess a unique slightly acidic environment, i.e., a low pH of 4.5–6.5. Therefore, the pH difference between the bacterial infection site and normal tissue can stimulate a "gatekeeper" response, enabling nanoparticles to intelligently release drugs, achieving controlled drug release and reducing drug toxicity. Polyphenols are a class of secondary metabolites containing multiple phenolic hydroxyl groups, widely found in various plants. They can rapidly self-assemble with various metal ions in aqueous solutions through coordination chelation to form metal-polyphenol networks (MPNs). MPNs are pH-responsive; when the pH is above 7, each metal ion on the MPN structure tightly binds to the three parts of the polyphenol in a stable network structure. However, when the pH is below 6, most of the phenolic hydroxyl groups on the polyphenol are protonated under acidic conditions, weakening their chelation with metal ions and leading to the decomposition of the metal-polyphenol network. Furthermore, MPNs have a simple synthesis process, good biocompatibility and biodegradability, and strong adhesion to various carrier surfaces. Therefore, MPNs can be used as gating mechanisms for drug release, controlling drug release at the bacterial infection site. Summary of the Invention

[0006] The technical problem this invention aims to solve is to activate anaerobic antibiotics, enabling them to exert their effects in non-anaerobic bacteria, reducing antibiotic resistance in bacterial infections, and allowing GOx and anaerobic antibiotics to specifically exert their effects at the site of bacterial infection. This invention provides a nanogel for synergistic treatment of non-anaerobic bacterial infections using a starvation therapy to activate metronidazole. The nanogel uses a composite nanohydrogel with a three-dimensional network structure, formed by sodium alginate (SA), natural polyphenolic compounds, and high-valence metal ions, as its main carrier. Glucose oxidase (GOx) and anaerobic antibiotics required for starvation therapy are loaded into the composite nanogel. Simultaneously, the natural polyphenols and high-valence metal ions in the composite nanogel can self-assemble to form metal-polyphenol network structures (MPNs), encapsulating the GOx and anaerobic antibiotic-loaded nanogel. The components of this nanogel drug delivery system work synergistically. This drug delivery system is pH-responsive; only in the slightly acidic environment of bacterial infection does the MPN structure formed by the outer natural polyphenolic compounds and high-valence metal ions decompose, releasing GOx and anaerobic antibiotics. GOx can consume glucose in bacteria, blocking the cell's energy source and causing the cell to starve to death due to the "starvation effect." Simultaneously, GOx's consumption of intracellular O2 exacerbates hypoxia in the bacterial microenvironment. This hypoxic environment can further activate anaerobic antibiotics, causing them to produce cytotoxicity and ultimately killing non-anaerobic bacteria with low metabolic activity under hypoxic conditions. Furthermore, with the decomposition of MPNs in the nanogel drug delivery system, some of the released natural polyphenolic compounds can react with the H2O2 produced by the starvation therapy, thereby clearing the generated H2O2 and exerting an antioxidant effect. At the same time, excess natural polyphenolic compounds can repolarize M1 phenotype macrophages to the M2 phenotype, exerting an anti-inflammatory effect, improving the inflammatory response at the bacterial infection site, and promoting tissue healing. This nanogel combines the advantages of antibiotics in treating bacterial infections while avoiding the problem of drug resistance. Moreover, the complementary advantages of various treatment methods construct a new "cocktail therapy," providing new ideas for antibacterial drug development and possessing significant scientific research and clinical application value for the synergistic treatment of resistant bacteria.

[0007] The objective of this invention can be achieved through the following technical solutions: A nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole activated by starvation therapy is prepared. The nanogel uses a composite nanohydrogel with a three-dimensional network structure formed by sodium alginate (SA), natural polyphenolic compounds, and high-valence metal ions as the main carrier. The glucose oxidase (GOx) and anaerobic antibiotics required for starvation therapy are loaded into the composite nanogel. At the same time, the natural polyphenols and high-valence metal ions in the composite nanogel can self-assemble to form metal-polyphenol network structures (MPNs). The nanogel loaded with GOx and MNZ is then encapsulated.

[0008] As a further aspect of the present invention: the natural polyphenolic compounds include epigallocatechin gallate EGCG, epigallocatechin EGC, epicatechin gallate ECG, and epicatechin EC.

[0009] As a further aspect of the present invention: the anaerobic antibiotics include metronidazole MNZ, ornidazole ONZ, tinidazole TNZ, dimetronidazole DMZ, etc.

[0010] As a further aspect of the present invention: the high-valence metal ions include metal ions such as iron, calcium, aluminum, vanadium, chromium, manganese, copper, and zinc.

[0011] The preparation method of a nanogel that activates metronidazole for synergistic treatment of non-anaerobic bacterial infections via starvation therapy specifically includes the following steps: Weigh 24 mg SA and 12 mg natural polyphenol compound and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of anaerobic antibiotic (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% high-valence metal ion solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

[0012] The application of a starvation therapy-activated metronidazole synergistic treatment nanogel for non-anaerobic bacterial infections, specifically the application of the starvation therapy-activated metronidazole synergistic treatment nanogel in the synergistic killing of bacteria.

[0013] The beneficial effects of this invention are: The starvation therapy activates the synergistic effect of various components of the nanogel used in the synergistic treatment of non-anaerobic bacterial infections with metronidazole. This drug delivery system is pH-responsive; it only decomposes the MPNs structure formed by the outer natural polyphenolic compounds and high-valence metal ions in the slightly acidic environment of bacterial infection, releasing GOx and anaerobic antibiotics. GOx consumes glucose in bacteria, blocking the cell's energy source and causing cell death due to a "starvation effect." Simultaneously, GOx's consumption of intracellular O2 exacerbates the hypoxia in the bacterial microenvironment. This hypoxic environment further activates the anaerobic antibiotics, making them cytotoxic and killing non-anaerobic bacteria with low metabolic activity under hypoxic conditions. Furthermore, with the decomposition of MPNs in the nanogel drug delivery system, some of the released natural polyphenolic compounds can react with the H2O2 generated by the starvation therapy, thereby clearing the generated H2O2 and exerting an antioxidant effect. At the same time, excess natural polyphenolic compounds can repolarize M1 phenotype macrophages to the M2 phenotype, exerting an anti-inflammatory effect, improving the inflammatory response at the bacterial infection site, and promoting tissue healing. This invention enables the treatment of non-anaerobic bacterial infections with the anaerobic antibiotic MNZ. This repurposing of an existing drug leverages the advantages of antibiotics in treating bacterial infections while avoiding the problem of drug resistance. Furthermore, the complementary advantages of various treatment methods create a novel "cocktail therapy," providing new insights for antibacterial drug development and possessing significant scientific research and clinical application value for the synergistic treatment of resistant bacteria. Attached Figure Description

[0014] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0015] Figure 1 This is a schematic diagram illustrating the preparation process and antibacterial properties of a nanogel that activates metronidazole for synergistic treatment of non-anaerobic bacterial infections using a starvation therapy, as described in this invention.

[0016] Figure 2 Transmission electron microscopy (TEM) image of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention.

[0017] Figure 3 The antioxidant level of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention was tested.

[0018] Figure 4 The GOx reactivity in the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention was tested.

[0019] Figure 5Plate and bar charts showing the effect of GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention on killing Staphylococcus aureus and Escherichia coli.

[0020] Figure 6 This is a live-dead staining confocal image showing the effect of GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of the present invention on killing Staphylococcus aureus and Escherichia coli.

[0021] Figure 7 The image shows the cytotoxicity of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel at different concentrations prepared in Example 1 of this invention.

[0022] Figure 8 The image shows hemolysis experiments at different concentrations of GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0024] like Figure 1-3 As shown, a starvation therapy activates a metronidazole-assisted nanogel for non-anaerobic bacterial infections. The nanogel uses a three-dimensional network structure formed by sodium alginate (SA), natural polyphenolic compounds, and high-valence metal ions as the main carrier. Glucose oxidase (GOx) and anaerobic antibiotics required for starvation therapy are loaded into the composite nanogel. Simultaneously, the natural polyphenols and high-valence metal ions in the composite nanogel can self-assemble to form metal-polyphenol network structures (MPNs), which encapsulate the GOx and MNZ-loaded nanogel. The natural polyphenolic compounds include epigallocatechin gallate (EGCG), epigallocatechin gallate (EGC), epicatechin gallate (ECG), and epicatechin (EC). The anaerobic antibiotics include metronidazole (MNZ), ornidazole (ONZ), tinidazole (TNZ), and dimetronidazole (DMZ). The high-valence metal ions include iron, calcium, aluminum, vanadium, chromium, manganese, copper, and zinc.

[0025] The preparation method of a nanogel that activates metronidazole for synergistic treatment of non-anaerobic bacterial infections via starvation therapy specifically includes the following steps: Weigh 24 mg SA and 12 mg natural polyphenol compound and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of anaerobic antibiotic (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% high-valence metal ion solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

[0026] Application of nanogels that activate metronidazole synergistically for non-anaerobic bacterial infections through starvation therapy; application of nanogels that activate metronidazole synergistically for non-anaerobic bacterial infections through starvation therapy in synergistic bacterial killing.

[0027] Example 1: A starvation therapy-activated metronidazole synergistic treatment for non-anaerobic bacterial infections using a nanogel GOx / MNZ@SA-Fe-EGCG (GMSFE) The preparation of the GMSFE nanogel for synergistic treatment of non-anaerobic bacterial infections by metronidazole activated by starvation therapy specifically includes the following steps: S. Weigh 24 mg SA and 12 mg EGCG and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of MNZ (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% FeCl3 solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

[0028] Example 2: A starvation therapy to activate metronidazole for synergistic treatment of non-anaerobic bacterial infections: GOx / MNZ@SA-Fe-EGC nanogel The preparation of the starvation therapy-activated metronidazole synergistic treatment for non-anaerobic bacterial infections nanogel GOx / MNZ@SA-Fe-EGC specifically includes the following steps: S. Weigh 24 mg SA and 12 mg EGC and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of MNZ (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% FeCl3 solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

[0029] Example 3: A starvation therapy to activate metronidazole for synergistic treatment of non-anaerobic bacterial infections: GOx / ONZ@SA-Fe-EGCG nanogel The preparation of the aforementioned starvation therapy-activated metronidazole synergistic treatment for non-anaerobic bacterial infections nanogel GOx / ONZ@SA-Fe-EGCG specifically includes the following steps: S. Weigh 24 mg SA and 12 mg EGCG and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of ONZ (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% FeCl3 solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

[0030] Example 4: A starvation therapy to activate metronidazole for synergistic treatment of non-anaerobic bacterial infections: GOx / MNZ@SA-Ca-EGCG nanogel The preparation of the starvation therapy-activated metronidazole synergistic treatment for non-anaerobic bacterial infections nanogel GOx / MNZ@SA-Ca-EGCG specifically includes the following steps: S. Weigh 24 mg SA and 12 mg EGCG and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of MNZ (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% CaCl2 solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4℃.

[0031] Comparative Example 1: A starvation therapy-activated metronidazole synergistic treatment for non-anaerobic bacterial infections using the nanogel MNZ@SA-Fe-EGCG (MSFE). The preparation of the GMSFE nanogel for synergistic treatment of non-anaerobic bacterial infections by metronidazole activated by starvation therapy specifically includes the following steps: S. Weigh 24 mg SA and 12 mg EGCG and dissolve them in 20 mL of deionized water, then stir magnetically for 30 minutes. Next, slowly add 1 mL of MNZ (4.5 mg / mL, DMSO as solvent) solution. Then, use a syringe pump to add 8 mL of 0.1% FeCl3 solution dropwise at a rate of 40 mL / h. After the addition is complete, continue magnetic stirring for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

[0032] Figure 2 This is a transmission electron microscope (TEM) image of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention. As can be seen from the image, the nanogel material exhibits a regular spherical structure with a particle size of approximately 100-150 nm.

[0033] Figure 3This study tested the antioxidant level of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention. We evaluated the antioxidant level of the GMSFE nanogel by examining its ability to scavenge hydrogen peroxide. 950 μL of GMSFE nanogel at different concentrations (0, 20, 40, 80, 100, 200 μg / mL) was mixed thoroughly with 10 μL of 3% H2O2 solution and incubated on a shaker for 2 hours. Then, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The concentration of unremoved hydrogen peroxide was determined using a hydrogen peroxide scavenging kit. The ability of different concentrations of GMSFE nanogel to scavenge hydrogen peroxide was evaluated by the scavenging rate of the nanogel. In addition, we mixed 950 μL of 200 μg / mL GMSFE nanogel with 10 μL of 3% H2O2 solution and incubated them on a shaker for different times (0, 20, 40, 60, 120, 180 min), then centrifuged at 8000 rpm for 10 min, and used a hydrogen peroxide kit to analyze the supernatant. Figure 3 As shown in a and 3b, the scavenging rate of hydrogen peroxide gradually increases with the increase of GMSFE nanogel concentration, reaching 47.6% when the concentration reaches 200 μg / mL. Figure 3 As shown in c and 3d, the scavenging rate of hydrogen peroxide gradually increased with the extension of incubation time, reaching 43.6% after 180 min of incubation. This indicates that GMSFE nanogel has a certain antioxidant capacity.

[0034] Figure 4This invention relates to the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of the present invention, which tested the GOx reactivity. In the presence of oxygen, GOx catalyzes the formation of gluconic acid and hydrogen peroxide (H2O2) from glucose. The generated gluconic acid increases the acidity of the reaction system. Therefore, we evaluated the GOx reactivity by measuring the change in pH and the H2O2 content of the system. Glucose solutions of different concentrations were mixed thoroughly with deionized water, GOx solution, and GMSFE solution, respectively, and then incubated in a shaker for 24 hours. The pH value was measured using a pH meter, and the hydrogen peroxide content was measured using a hydrogen peroxide kit to evaluate the glucose oxidase activity. As shown in the figure, compared to the control group, the pH value of the reaction system in the GOx group and the GMSFE nanogel group gradually decreased with increasing glucose concentration, indicating that the GOx catalysis of glucose to gluconic acid in the GOx group and the GMSFE nanogel group led to a decrease in the pH value of the reaction system. Furthermore, the concentration of H2O2 in the GOx group increased with increasing glucose concentration, indicating that GOx catalyzed the production of H2O2 from glucose, leading to an increase in H2O2 concentration. In contrast, the H2O2 concentration in the GMSFE nanogel group was very low, close to that of the control group. This is because the H2O2 produced by the GMSFE nanogel group was scavenged by the released natural polyphenolic compounds. Therefore, the changes in pH and the H2O2 content in the system demonstrate that GOx in the GMSFE nanogel exhibits good reactivity.

[0035] Figure 5This image shows a plate and bar graph illustrating the effectiveness of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention in killing Staphylococcus aureus and Escherichia coli under normoxic and anaerobic conditions. We inoculated 50 μL of bacterial suspension (Staphylococcus aureus or Escherichia coli) at a concentration of 2×10⁶ CFU / mL into 96-well plates, and then added 50 μL of PBS solution. The final concentration of glucose in the glucose treatment group was 5 mM. After adding 100 μL of different materials (PBS solution, 3 μg / mL MNZ solution, 3 μg / mL MSFE nanogel, or 3 μg / mL GMSFE nanogel), the plates were incubated at 37 °C for 12 h under normoxic or anaerobic conditions (5% oxygen). The bacterial suspensions treated under different conditions were diluted with physiological saline and then plated onto petri dishes. After incubation at 37 °C for a certain period, the bacterial survival rate was calculated by counting the number of bacterial colonies in each group. The survival rate was calculated using the following formula: Survival rate (%) = Colony count (sample) / Colony count (control) × 100%. From the smears and bar charts, it can be seen that under normoxic conditions, the PBS, MNZ, and MSFE groups showed no significant bactericidal effect in the presence or absence of glucose. The GMSFE group showed no significant bactericidal effect without glucose, but with glucose, it exhibited a significant bactericidal effect, with survival rates of 0% for *S. aureus* and 47.0% for *E. coli* in the GMSFE group. This indicates that under normoxic conditions and in the presence of glucose, GOx can kill bacteria through starvation therapy. Under anaerobic conditions, MNZ mainly exerted its bactericidal effect. Therefore, under anaerobic conditions, the survival rates of *S. aureus* and *E. coli* in the MNZ, MSFE, and GMSFE groups, due to the presence of MNZ, showed a decreasing trend compared to the PBS group. Furthermore, in the GMSFE group, the addition of glucose, due to the GOx in the GMSFE nanogel material killing bacteria through starvation in the presence of glucose and oxygen, and the simultaneous consumption of oxygen, created a more anaerobic environment compared to other groups, thus further stimulating the bactericidal effect of MNZ. Under anaerobic conditions, after the addition of glucose, the bacterial survival rates of *S. aureus* and *E. coli* in the GMSFE group were 0% and 36.3%, respectively. Therefore, the addition of glucose to the GMSFE group resulted in a better bactericidal effect.

[0036] Figure 6This image shows a confocal micrograph of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention, illustrating its effectiveness against Staphylococcus aureus and Escherichia coli under both normoxic and anaerobic conditions. The inhibitory effects of different material groups (PBS, MNZ, MSFE, GMSFE) on Staphylococcus aureus and Escherichia coli under normoxic and anaerobic conditions were verified by confocal microscopy after staining bacteria with a PI / SYTO9 mixed dye. The SYTO9 dye labeled all bacteria, producing green fluorescence (Ex / Em: 485 / 498 nm) after laser irradiation; while the PI dye could only penetrate damaged cell membranes, thus only labeling dead bacteria, producing red fluorescence (Ex / Em: 535 / 617 nm) after laser irradiation. As shown in the figure, under normoxic conditions, without glucose, the bacteria in the PBS, MNZ, MSFE, and GMSFE groups all exhibited strong green fluorescence (live bacteria) but no red fluorescence (dead bacteria), indicating that the dark toxicity of the material was negligible. However, under normoxic conditions, with the addition of glucose, GOx, in the presence of oxygen and glucose, could kill bacteria through starvation therapy. Therefore, under normoxic conditions, the GMSFE group showed significant red fluorescence after the addition of glucose, indicating that the bacteria were completely killed. Under anaerobic conditions, MNZ primarily exerted its bactericidal effect. Therefore, under anaerobic conditions, without glucose, the MNZ, MSFE, and GMSFE groups, containing MNZ, had a bactericidal effect. Thus, the bacteria in these three groups showed a small amount of red fluorescence (dead bacteria). However, after the addition of glucose, the bacteria in the GMSFE group showed significant red fluorescence. This is because the GOx in the GMSFE nanogel material undergoes starvation therapy in the presence of glucose and a small amount of oxygen, consuming oxygen in the process. Its environment is more anaerobic than the other groups, thus further stimulating the bactericidal effect of MNZ. Therefore, the addition of glucose under anaerobic conditions enhances the bactericidal effect.

[0037] Figure 7This figure shows the cytotoxicity of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention at different concentrations. We evaluated the cytotoxicity of different concentrations of GMSFE nanogel to L929 cells or RAW 264.7 cells using the CCK8 assay. We used DMEM complete medium as the control group. MNZ-3 represents 3 μg / mL of MNZ, MSFE-3 represents 3 μg / mL of MNZ per mL of MSFE, and GMSFE-1, GMSFE-2, GMSFE-3, and GMSFE-4 represent 1 μg / mL, 2 μg / mL, 3 μg / mL, and 4 μg / mL of MNZ per mL of GMSFE, respectively. As can be seen from the figure, when the concentration of GMSFE nanogel increased to 4 μg / mL, the cell viability was still greater than 80%, thus demonstrating its good biocompatibility.

[0038] Figure 8 This figure shows the hemolysis experiment of the GOx / MNZ@SA-Fe-EGCG (GMSFE) nanogel prepared in Example 1 of this invention at different concentrations. We used water as a positive control group and measured the hemolysis rate of GMSFE nanogel at different concentrations (1, 2, 3, 4, 5 μg / mL). As can be seen from the figure, when the mass concentration is not higher than 5 μg / mL, the hemolysis rate of the GMSFE nanogel is lower than the 5% required by the international standard. This further demonstrates that the GMSFE nanogel has good biocompatibility and safety at a mass concentration not higher than 5 μg / mL.

[0039] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole activated by starvation therapy, characterized in that, The nanogel uses a three-dimensional network structure formed by sodium alginate (SA), natural polyphenolic compounds, and high-valence metal ions as the main carrier. Glucose oxidase (GOx) and anaerobic antibiotics required for starvation therapy are loaded into the composite nanogel. At the same time, the natural polyphenols and high-valence metal ions in the composite nanogel can self-assemble to form metal-polyphenol network structures (MPNs), which encapsulate the nanogel loaded with GOx and MNZ.

2. The nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole activated by starvation therapy according to claim 1, characterized in that, The natural polyphenolic compounds include epigallocatechin gallate ester EGCG, epigallocatechin EGC, epigallocatechin gallate ester ECG, and epigallocatechin EC.

3. The nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole activated by starvation therapy according to claim 1, characterized in that, The anaerobic antibiotics include metronidazole (MNZ), ornidazole (ONZ), tinidazole (TNZ), and dimetronidazole (DMZ).

4. The nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole activated by starvation therapy according to claim 1, its preparation method and application, characterized in that, The high-valence metal ions include iron, calcium, aluminum, vanadium, chromium, manganese, copper, zinc, and other metal ions.

5. The method for preparing the nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole activated by starvation therapy according to claim 1, characterized in that, Specifically, the following steps are included: Weigh 24 mg SA and 12 mg natural polyphenol compound and dissolve them in 20 mL of deionized water and stir magnetically for 15 minutes. Then add 1 mL of GOx (6 mg / mL) aqueous solution and continue stirring magnetically for 15 minutes. Next, slowly add 1 mL of anaerobic antibiotic (4.5 mg / mL, DMSO as solvent) solution. Finally, use a syringe pump to add 8 mL of 0.1% high-valence metal ion solution dropwise at a rate of 40 mL / h. After the addition is complete, continue stirring magnetically for 2 hours. After the reaction is complete, wash the obtained nanogel with deionized water until the supernatant is colorless, centrifuge, discard the supernatant, and obtain the nanogel. Finally, disperse the nanogel in deionized water and store at 4 °C.

6. The application of the nanogel for synergistic treatment of non-anaerobic bacterial infections with metronidazole using starvation therapy according to claim 1, characterized in that, The starvation therapy activates the application of metronidazole in synergistic treatment of non-anaerobic bacterial infections in the synergistic killing of bacteria in nanogels.

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