Nano-drug preparation for tumor treatment and preparation method and application of nano-drug preparation

Abstract

The invention provides a nano medicinal preparation for tumor treatment as well as a preparation method and application thereof, and relates to the technical field of medicinal preparations. The preparation method of the nano medicinal preparation comprises the following steps: S1, adding metal salt and oxidase into water, uniformly stirring and mixing to form a compound, then adding antibiotics, and continuously stirring and uniformly mixing for reaction to obtain a mixed solution; and S2, centrifuging the mixed solution, collecting the precipitate, washing, and dispersing into water for storage to obtain the nano-drug preparation. The nano medicinal preparation disclosed by the invention can intelligently respond to a tumor microenvironment, efficiently remove fusobacterium nucleatum in tumors and kill tumor cells through synergistic antibacterial and anticancer effects, so that tumor development is inhibited. Moreover, the complex prepared by the invention can enhance the stability of a medicinal preparation, can realize targeted release of the medicine in a tumor microenvironment, and improves the action effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a nanomedicine formulation for tumor treatment, its preparation method, and its application. Background Technology

[0002] Cancer poses a serious threat to human life and health, and current drug treatments have the following limitations: (1) Traditional antibiotics: When treating intracellular bacteria in tumors, they face a double barrier of the tumor microenvironment. First, the intracellular permeability barrier makes it difficult for drugs to reach the bacterial colonization site. Second, the physicochemical properties of the tumor microenvironment (TME) significantly weaken the antibacterial efficacy and cannot effectively eliminate nucleated Fusobacterium nucleatum in the tumor.

[0003] (2) Existing nano-drug delivery systems: Most of them use inert carriers such as liposomes and polymers for physical encapsulation. Although this can improve bioavailability, the “skeleton-load” model has inherent defects, such as low drug loading, easy premature drug leakage, and complicated synthesis steps, which seriously limit clinical application.

[0004] (3) Existing related treatment technologies: The three points of the core method of constructing nanocarriers by utilizing antibiotic-metal complex, simultaneously loading glucose oxidase (GOX) to achieve synergistic treatment, and utilizing the high glutathione (GSH) properties in TME to achieve intelligent drug release are not effectively integrated. They are either simple physical loading or lack intelligent response design to the key features of TME, making it difficult to achieve the dual effect of efficiently and synergistically clearing Fusobacterium nucleatum in tumors and treating tumors.

[0005] (4) GOX delivery related technologies: GOX faces challenges such as poor stability and off-target toxicity during in vivo delivery. The lack of efficient targeted delivery systems affects its therapeutic efficacy and safety. Summary of the Invention

[0006] In view of this, the present invention proposes a nanomedicine formulation for tumor treatment, its preparation method and application.

[0007] The fundamental difference of the nanomedicine formulation of this invention lies in abandoning the traditional "inert carrier + drug loading" model. It innovatively uses the therapeutic components themselves (antibiotics and metal ions) to construct nanocarriers through complexation, which fundamentally solves the problem of low drug loading capacity of traditional nanocarriers and achieves precise control of drug release at the target site, greatly reducing systemic toxic side effects.

[0008] The technical solution of this invention is implemented as follows: A method for preparing a nanomedicine formulation for tumor treatment includes the following steps: S1. Add metal salt and oxidase to water and stir to form a complex. Then add antibiotic and continue stirring to react and obtain a mixture. S2. Centrifuge the mixture, collect the precipitate, wash it, and disperse it in water for storage to obtain the nanomedicine formulation.

[0009] Preferably, the water should just cover the sediment during storage.

[0010] Preferably, the metal salt is selected from at least one of soluble salts of copper, iron, and manganese; the oxidase is selected from at least one of GOX and lactate oxidase; and the antibiotic is selected from at least one of ampicillin (AMP), metronidazole, and ciprofloxacin.

[0011] Preferably, in step S1, the pH value is controlled at 6.5-7.5 and the temperature is 20-30℃ during the stirring process.

[0012] Preferably, in step S1, the pH value is controlled at 7.0 and the temperature is 25°C during the stirring process.

[0013] Preferably, in step S2, the centrifugation is performed at a speed of 12000-15000 rpm for 9-12 minutes.

[0014] Preferably, the process further includes a surface modification process, which is as follows: the mixed solution obtained in S1 and the biological cell membrane or the targeting molecule are added to a buffer solution and mixed to obtain a surface-modified mixture.

[0015] Preferably, the biological cell membrane is selected from at least one of erythrocyte membrane, macrophage membrane, and tumor cell membrane; the targeting molecule is selected from at least one of hyaluronic acid, arginylglycyl aspartate peptide (RGD peptide), and folic acid; and the buffer solution is at least one of pure water, PBS solution, and NaCl solution.

[0016] Preferably, arginylglycylaspartic peptide is a short peptide containing arginine-glycine-aspartic acid.

[0017] Preferably, the PBS solution has a pH of 7.2-7.4 and a concentration of 1×PBS; the NaCl solution has a concentration of 0.9% (w / v).

[0018] Preferably, the bio-cell membrane or targeting molecule accounts for 1% to 10% of the total mass.

[0019] Preferably, the mixing process can be carried out by liposome extrusion, ultrasonic mixing, or stirring.

[0020] Preferably, the stirring is carried out at 25℃-30℃ for 1-3 hours.

[0021] A nanomedicine formulation for tumor treatment comprises metal salts, oxidases, and antibiotics in weight percentages of 20%–50%, 1%–15%, and 30%–70%, respectively.

[0022] The application of the nanomedicine formulation of the present invention in the preparation of drugs for eliminating Fusobacterium nucleatum in tumors and for treating tumors.

[0023] Preferably, the tumor includes at least one of colorectal cancer cells, gastric cancer cells, and breast cancer cells.

[0024] Compared with the prior art, the beneficial effects of the present invention are: (1) Innovative drug loading paradigm with high drug loading capacity: Antibiotics are transformed from "loaded material" to "carrier material", which fundamentally solves the problem of low drug loading capacity of traditional nanocarriers.

[0025] (2) The synthesis process is simple: the one-pot synthesis process is simple, has good reproducibility, is easy to scale up production, and has significant industrial advantages.

[0026] (3) Intelligent and precise release with high safety: By using the tumor-specific high GSH environment as a trigger switch, the drug is released precisely at the target site, greatly reducing systemic toxic side effects.

[0027] (4) Multiple synergistic treatments with significant effects: It integrates antibiotic treatment, enzyme-catalyzed starvation therapy and metal ion chemokinetics, which promote each other and produce a synergistic effect of "1+1+1>3". It shows a far greater effect than traditional methods in clearing Fusobacterium nucleatum and killing tumors.

[0028] Furthermore, the components in this invention are not simply mixed, but rather in the form of Cu 2+ Metal ions and oxidases such as GOX are first mixed via electrostatic adsorption, and then covalently coordinated with antibiotics such as AMP to form complexes, thereby forming a multifunctional nanomedicine formulation (Cu-Amp@GOX). This nanomedicine formulation can intelligently respond to the tumor microenvironment, efficiently eliminating nucleated Fusobacterium within tumors and killing tumor cells through synergistic antibacterial and anticancer effects, thereby inhibiting tumor development. Furthermore, the complexes prepared in this invention enhance the stability of the drug formulation, enabling targeted release of the drug within the tumor microenvironment and improving its efficacy. Attached Figure Description

[0029] Figure 1 Electron micrographs of Cu-AMP@GOX nanoparticles prepared in Example 1 and Cu-Amp prepared in Example 2.

[0030] Figure 2The figures show the dimensions of Cu-AMP@GOX nanoparticles prepared in Example 1 and Cu-Amp nanoparticles prepared in Example 2; the hydrodynamic diameter in the figures represents the hydrodynamic diameter of the nanoparticles.

[0031] Figure 3 The figures show the Zeta potential diagrams for Cu-AMP@GOX nanoparticles prepared in Example 1 and Cu-Amp prepared in Example 2; the Zeta potential in the figures represents the electrokinetic potential.

[0032] Figure 4 The figure shows the release effect of Cu-AMP@GOX nanoparticles prepared in Example 1 in GSH solutions of different concentrations; in the figure, GOX cumulative release represents the cumulative release rate of GOX, and Time represents time.

[0033] Figure 5 The graph shows the evaluation of the ability of Cu-AMP@GOX nanoparticles prepared in Example 1 to generate ROS in glucose solutions of different concentrations; in the figure, Absorbance (652 nm) represents the absorbance at 652 nm, and Glucose represents the glucose concentration.

[0034] Figure 6 The graph shows the reactive oxygen species (ROS) generation of Cu-AMP@GOX nanoparticles prepared in Example 1 in the colorectal cancer cell line HCT116; in the graph, concentration represents concentration and RFU represents relative fluorescence units.

[0035] Figure 7 Colony images and bacterial viability diagrams of *Fusobacterium nucleatum* after treatment with AMP (100 μg / mL), GOX (50 μg / mL), CuSO4 (100 μg / mL), Cu-AMP (100 μg / mL), and Cu-AMP@GOX nanoparticles (100 μg / mL) prepared in Example 1 for 72 h are shown. In the figures, 7(a) is a colony image and 7(b) is a bacterial viability diagram. In the figures, the bacterial survival rate represents the bacterial survival rate.

[0036] Figure 8 The images show colony images and bacterial viability diagrams of *Fusobacterium nucleatum* after being treated with different concentrations of Cu-AMP@GOX nanoparticles prepared in Example 1 for 72 hours. In the images, 8(a) is a colony image and 8(b) is a bacterial viability diagram. In the figures, the bacterial survival rate represents the bacterial survival rate and the concentration represents the concentration.

[0037] Figure 9The image shows cell viability of the colorectal cancer cell line HCT116 after treatment with different concentrations of Cu-AMP@GOX nanoparticles prepared in Example 1. In the image, Cell viability represents cell viability, Concentration represents concentration, and BLANK represents blank.

[0038] Figure 10 The image shows the effect of treating colorectal cancer organoids with different concentrations of Cu-AMP@GOX nanoparticles prepared in Example 1 for 24 hours; in the image, BLANK represents the blank group.

[0039] Figure 11 The figures show cell viability of colorectal cancer organoids after treatment with different concentrations of Cu-AMP@GOX nanoparticles prepared in Example 1; in the figures, Cell viability represents cell viability, Concentration represents concentration, and BLANK represents blank.

[0040] Figure 12 The image shows the blood compatibility of Cu-AMP@GOX nanoparticles from Example 1. In the image, the hemolytic rate represents the hemolysis rate.

[0041] Figure 13 Blood routine and blood biochemistry images of mice 7 days after tail vein injection of Cu-AMP@GOX nanoparticles prepared in Example 1; in the figure, saline solution represents physiological saline.

[0042] Figure 14 HE staining results of various organs of mice 7 days after tail vein injection of Cu-AMP@GOX nanoparticles prepared in Example 1; in the figure, saline solution represents the physiological saline group. Detailed Implementation

[0043] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0044] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0046] In this invention, the concentration of PBS solution was 1×PBS throughout the experimental process.

[0047] Example 1 A method for preparing nanomedicine formulations for tumor treatment includes the following steps: S1. Copper sulfate and GOX were added to pure water at a pH of 7.0 and mixed thoroughly at room temperature. A Cu-GOX complex was formed through electrostatic adsorption. AMP was then added and the mixture was stirred for 25 minutes under the same temperature and pH conditions to allow for a complete reaction and form a preliminary Cu-AMP@GOX mixture. The mass percentages of copper sulfate, GOX, AMP, and pure water were 0.5%, 0.15%, 1%, and 98.35%, respectively. S2. Centrifuge the Cu-AMP@GOX mixture at 12000 rpm for 10 min to remove unreacted free drug and salt. Collect the precipitate, wash it, and obtain Cu-AMP@GOX nanoparticles. Disperse the Cu-AMP@GOX nanoparticles in pure water for storage to obtain Cu-AMP@GOX nanomedicine formulation.

[0048] Example 2 Preparation of Cu-AMP complex: Copper sulfate and AMP were added to pure water and mixed evenly at room temperature with the pH controlled at 7.0 (the mass ratios of copper sulfate, AMP and pure water were 0.5%, 1% and 98.5%, respectively) to obtain a solution containing the complex. The solution was then centrifuged at 12000 rpm for 10 min, the precipitate was collected and washed to obtain Cu-AMP, which was then dispersed in pure water for storage.

[0049] The performance of the Cu-AMP@GOX nanoparticle material of Example 1 and the Cu-AMP material of Example 2 were tested. The Cu-AMP@GOX concentrations mentioned in the figure below refer to the concentrations of copper in the material.

[0050] (1) Morphology and characteristics The particle size distribution and zeta potential of the Cu-AMP@GOX nanoparticles in Example 1 and the Cu-AMP material in Example 2 were determined by dynamic light scattering (DLS). Their morphology was observed using transmission electron microscopy (TEM), and their structure was observed using scanning electron microscopy (SEM). The results are as follows. Figure 1-3 As shown.

[0051] Depend on Figure 1-3 As can be seen, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) show that Cu-AMP exhibits a porous cluster structure, with particles agglomerating to form porous aggregates. Cu-AMP@GOX, on the other hand, shows an irregular aggregate structure formed after GOX encapsulation. Due to the coating effect of GOX, the material assembles into composite aggregate particles with irregular contours, and the overall morphology exhibits more complex assembly characteristics. Figure 1 Dynamic light scattering (DLS) showed a slight increase in the hydrated particle size of the material after GOX encapsulation. Figure 2However, the GOX's enclosure did not change its charge, and the entire GOX remains negatively charged. Figure 3 This indicates that the material still possesses good colloidal stability in aqueous solutions.

[0052] (2) GSH-responsive drug release test The fluorescent labeling scheme for glucose oxidase (GOX) with fluorescein isothiocyanate (FITC) was as follows: FITC was dissolved in DMSO (50 mM concentration), and FITC and GOX were added at a molar ratio of 1:20. The mixture was then dialyzed in a dialysis bag at 4°C for 24 h, followed by concentration using a 30 KD ultrafiltration tube to obtain FITC-labeled GOX. Cu-AMP@FITC-GOX was synthesized using the labeled FITC-GOX. 0 mM, 2 mM, 5 mM, and 10 mM GSH solutions were prepared. The Cu-AMP@FITC-GOX material was added to the GSH solution (Cu-AMP@FITC-GOX concentration was 100 μg / mL, added in 10 μL) at these times. The fluorescence value of the supernatant was then measured at 5 min, 0.5 h, 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, and 24 h to estimate the GOX release rate in the GSH solution. The results are as follows. Figure 4 As shown.

[0053] In a release medium simulating high GSH concentrations (10 mM) within tumor cells, the cumulative GOX release rate over 24 hours was significantly higher than that in the group without GSH. Furthermore, the cumulative GOX release rate increased in a dose-dependent manner with increasing GSH concentration (0-10 mM). Figure 4 This study confirms that Cu-AMP@GOX possesses excellent GSH-responsive smart release properties, enabling targeted drug release within the tumor microenvironment.

[0054] (3) Detection of reactive oxygen species The reactive oxygen species (ROS) generation capacity of the Cu-AMP@GOX material in Example 1 at different glucose concentrations was evaluated by the 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric method. TMB was oxidized by ROS to generate a blue oxidation product, and its absorbance was positively correlated with the ROS content. 10 μL of Cu-AMP@FITC-GOX material with a concentration of 10 μg / mL was added to glucose solutions of different concentrations.

[0055] Experimental results showed that the absorbance of the reaction system gradually increased with increasing glucose concentration (0-0.25 mM). Test tube photographs clearly showed that the high glucose concentration group exhibited a distinctly deep blue color, while the low concentration group showed a lighter color. Figure 5 This demonstrates that increased glucose concentration promotes the generation of more ROS in Cu-AMP@GOX materials.

[0056] At the cellular level, the 2',7'-dichlorofluorescein diacetate (DCFH-DA) ROS detection kit was used for further validation: after treatment with different concentrations of Cu-AMP, no significant changes in fluorescence signal were observed in HCT116 cells, indicating that it could not effectively trigger ROS generation; while Cu-AMP@GOX showed an increase in fluorescence intensity at low concentrations, and the fluorescence signal significantly increased with increasing concentration. Figure 6 This study confirmed that Cu-AMP@GOX can efficiently generate ROS in cells, and that its ROS generation capacity is concentration-dependent.

[0057] (4) In vitro antibacterial effect test Using *Fusobacterium nucleatum* as the target bacterium, the in vitro antibacterial activity of different drug formulations was evaluated using the plate count method. A concentration of 2 × 10⁻⁶ was used. 5 A suspension of *Fusobacterium nucleatum* at CFU / mL was inoculated onto Columbia blood agar plates containing 5% defibrinated sheep blood. Different concentrations of AMP (100 μg / mL), GOX (50 μg / mL), CuSO4 (100 μg / mL), Cu-AMP (100 μg / mL), Cu-AMP@GOX (1-100 μg / mL), and PBS buffer solution (blank control) were then added. The plates were incubated at 37°C under anaerobic conditions for 72 h. The experimental results are as follows: Figure 7 and Figure 8 As shown.

[0058] from Figure 7 (a) and Figure 7 As shown in (b), the GOX group alone had a low inhibition rate against *Fusobacterium nucleatum*, with no significant inhibitory effect; the copper ion group showed some antibacterial activity (inhibition rate of approximately 30%). Plate counting experiments showed that no *Fusobacterium nucleatum* colonies were observed on the surface of the culture media in the AMP group, Cu-AMP group, and Cu-AMP@GOX group, while the blank control group had a higher colony count. Figure 8 (a) and Figure 8 (b) It can be seen that as the concentration of Cu-AMP@GOX increases, the number of surviving colonies further decreases, confirming that Cu-AMP@GOX has an excellent inhibitory effect on Fusobacterium nucleatum, and the antibacterial activity is concentration-dependent.

[0059] (5) In vitro antitumor effect test In the HCT116 colorectal cancer cell line and a colorectal cancer organoid model, the in vitro antitumor activity of different concentrations of Cu-AMP@GOX was evaluated using the CCK8 assay and ATP assay, respectively. 5FU (thymidine synthase inhibitor) was used as a positive control group, and PBS buffer solution was used as a blank control group. Results Figure 9-11 As shown.

[0060] The results showed that Cu-AMP@GOX had a significant inhibitory effect on HCT116 cells. Figure 8 In a colorectal cancer organoid model, after Cu-AMP@GOX treatment for 24 h, the organoid volume was significantly smaller than that of the blank control group. Figure 10 Furthermore, as the material concentration increases, cell viability decreases significantly. Figure 11 This study confirmed that Cu-AMP@GOX exhibited good antitumor activity at both the cellular and organoid levels.

[0061] (6) In vivo safety testing ① Hemolysis test The blood compatibility of Cu-AMP@GOX formulation was assessed using a hemolysis assay. Fresh mouse blood was centrifuged and washed with PBS until the supernatant was clear. The resulting red blood cells were then prepared into an 8% suspension with PBS for testing. In 1.5 mL centrifuge tubes, PBS solution was added to each tube to prepare an 8% suspension. Different concentrations of Cu-AMP@GOX particles (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL) were then added as sample solutions. Water was used as a positive control, and PBS buffer as a negative control. The tubes were incubated at 37°C for 2 h, centrifuged at 3000 g / min for 10 min, and the absorbance of the supernatant was measured at 540 nm. Each treatment group was tested in triplicate. The hemolysis rate (%) was calculated using the following formula: (A sample group) = (A sample group) / (A sample group) * ... (A negative control) / (A positive control) A negative control) × 100%. Results are as follows: Figure 12 As shown.

[0062] ②Complete blood count, blood biochemical indicators, and hematoxylin-eosin (HE) staining in mice Balb / c mice were injected intravenously with Cu-AMP@GOX at a concentration of 1.9 mg / kg. After a single administration, a 7-day interval was observed. 100 μL of blood was collected from each mouse, and ethylenediaminetetraacetic acid (EDTA) was added as an anticoagulant for routine blood tests to determine white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts. Whole blood was placed in 1.5 mL centrifuge tubes and allowed to stand at room temperature for 1 h. The tubes were then centrifuged at 3000 × g for 30 min at 4°C. The supernatant was transferred to new centrifuge tubes and stored at -80°C for the determination of alanine aminotransferase (AST), aspartate aminotransferase (ALT), uric acid (UA), creatinine (CR), urea (UREA), and blood urea nitrogen (BUN) levels. The mice were then sacrificed, and their hearts, livers, spleens, lungs, and kidneys were stained using the hematoxylin-eosin (HE) staining method for histopathological examination. Balb / c mice injected with an equal volume of physiological saline served as a control group. The results are as follows. Figure 13-14 As shown.

[0063] The results showed that when the Cu-AMP@GOX concentration was as high as 100 μg / mL, the hemolysis rate was <5% (meeting the blood safety standards for biomaterials), and no obvious hemolysis was observed. Figure 12 Further, Cu-AMP@GOX was injected into the tail vein of Balb / c mice. Seven days after administration, the mice's complete blood count and blood biochemical parameters were measured. The results showed that all parameters were within the normal physiological range. Figure 13 Hematoxylin-eosin (HE) staining results showed that the tissue structure of major organs such as the heart, liver, spleen, lungs, and kidneys of mice was intact, with no pathological damage such as inflammatory infiltration or cell necrosis, confirming that Cu-AMP@GOX has good in vivo biocompatibility. Figure 14 ).

[0064] Example 3 A method for preparing nanomedicine formulations for tumor treatment includes the following steps: S1. Copper chloride and glucose oxidase (GOX) were added to pure water at a pH of 7.0 and mixed thoroughly at room temperature. A Cu-GOX complex was formed through electrostatic adsorption. AMP was then added and the mixture was stirred for 25 minutes under the same temperature and pH conditions to allow for a complete reaction, forming a preliminary Cu-AMP@GOX mixture. The mass percentages of copper chloride, glucose oxidase, AMP, and pure water were 0.45%, 0.15%, 1%, and 98.4%, respectively. S2. Cu-AMP@GOX mixture and red blood cell membrane were added to PBS solution and ultrasonically mixed to obtain a surface-modified mixture. The mass percentages of Cu-AMP@GOX mixture, red blood cell membrane, and PBS solution were 1% and 98% respectively. The surface-modified mixture was centrifuged at 14,000 rpm for 12 min to remove unreacted free drug and salt. The precipitate was collected, washed, and Cu-AMP@GOX nanoparticles were obtained. The Cu-AMP@GOX nanoparticles were dispersed in pure water for storage to obtain the Cu-AMP@GOX nanomedicine formulation.

[0065] Example 4 A method for preparing nanomedicine formulations for tumor treatment includes the following steps: S1. Ferric chloride and GOX were added to pure water at a pH of 7.0 and mixed thoroughly at room temperature. An Fe-GOX complex was formed through electrostatic adsorption. AMP was then added and the mixture was stirred for 30 minutes under the same temperature and pH conditions to ensure a complete reaction and form a preliminary Fe-AMP@GOX mixture. The mass percentages of ferric chloride, glucose oxidase, AMP, and pure water were 0.5%, 0.15%, 1%, and 98.35%, respectively. S2. The Fe-AMP@GOX mixture and hyaluronic acid were added to a 0.9% (w / v) NaCl solution and stirred at room temperature for 2 hours to obtain a surface-modified mixture. The Fe-AMP@GOX mixture accounted for 1% of the mass, the hyaluronic acid accounted for 1% of the mass, and the NaCl solution accounted for 99% of the mass. The surface-modified mixture was centrifuged at 15,000 rpm for 9 min to remove unreacted free drugs and salts. The precipitate was collected, washed, and Fe-AMP@GOX nanoparticles were obtained. The Fe-AMP@GOX nanoparticles were dispersed in pure water for storage to obtain the Fe-AMP@GOX nanomedicine formulation.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nanomedicine formulation for tumor treatment, characterized in that, Includes the following steps: S1. Add metal salt and oxidase to water and stir to form a complex. Then add antibiotic and continue stirring to react and obtain a mixture. S2. Centrifuge the mixture, collect the precipitate, wash it, and disperse it in water for storage to obtain the nanomedicine formulation.

2. The method for preparing the nanomedicine formulation for tumor treatment according to claim 1, characterized in that, The metal salt is selected from at least one of soluble salts of copper, iron, and manganese; the oxidase is selected from at least one of glucose oxidase and lactate oxidase; and the antibiotic is selected from at least one of ampicillin, metronidazole, and ciprofloxacin.

3. The method for preparing the nanomedicine formulation for tumor treatment according to claim 1, characterized in that, In step S1, the pH value is controlled at 6.5-7.5 and the temperature is 20-30℃ during the stirring process; in step S2, the centrifugation is carried out at a speed of 12000-15000 rpm for 9-12 minutes.

4. The method for preparing the nanomedicine formulation for tumor treatment according to claim 1, characterized in that, It also includes a surface modification process, the specific process of which is as follows: the mixed solution obtained in S1 and the biological cell membrane or target molecule are added to a buffer solution for mixing to obtain a surface-modified mixture.

5. The method for preparing the nanomedicine formulation for tumor treatment according to claim 4, characterized in that, The biological cell membrane is selected from at least one of erythrocyte membrane, macrophage membrane, and tumor cell membrane; the targeting molecule is selected from at least one of hyaluronic acid, arginylglycyl aspartate peptide, and folic acid; the buffer solution is at least one of pure water, PBS solution, and NaCl solution.

6. The method for preparing the nanomedicine formulation for tumor treatment according to claim 1, characterized in that, The biocellular membrane or targeting molecule accounts for 1% to 10% of the total mass.

7. The method for preparing nanomedicine formulations for tumor treatment according to claim 1, characterized in that, The mixing process can be carried out by liposome extrusion, ultrasonic mixing, or stirring.

8. A nanomedicine formulation for tumor treatment, characterized in that, This includes metal salts, oxidases, and antibiotics, which account for 20%–50%, 1%–15%, and 30%–70% of the total mass, respectively.

9. The use of the nanomedicine formulation prepared by any one of claims 1-7 or the nanomedicine formulation of claim 8 in the preparation of drugs for eliminating Fusobacterium nucleatum in tumors and for treating tumors.

10. The application according to claim 9, wherein the tumor comprises at least one of colorectal cancer cells, gastric cancer cells, and breast cancer cells.

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