Preparation and application of a core-shell structure metal organic framework composite material with both macroporous and microporous sizes

Abstract

The application discloses a kind of core-shell structure metal organic framework composite material with macropore and micropore size preparation and application, belong to biomedical and material chemistry field, the hollow mesoporous prussian blue metal organic framework material of macromolecular glucose oxidase supported is synthesized by acid etching strategy in the application, shell ZIF-8 is grown on the surface of hollow mesoporous prussian blue by heterojunction epitaxial growth method, shell ZIF-8 can effectively prevent the leakage of glucose oxidase and can provide loading space for small molecule 5-fluorouracil drug, can realize the core-shell structure metal organic framework composite material of two anticancer drugs simultaneously loaded.The composite material synthesized in the application is uniform in size, realizes the channel grading strategy of MOF-on-MOF, realizes the effective sealing of size guest molecules, expands the practical application field, utilizes the combined action of two anticancer drugs, and further realizes the demand of treating cancer, has certain clinical application potential.

Description

Technical Field

[0001] This invention relates to the preparation and application of a core-shell structured metal-organic framework composite material with both macropore and micropore sizes, belonging to the fields of biomedicine and materials chemistry. Background Technology

[0002] Cancer poses a serious threat to human life and health, impacting quality of life and becoming a major global public health challenge, with its incidence rate continuing to rise. In recent years, glucose oxidase has been widely used in cancer treatment. Glucose oxidase catalyzes the oxidation of glucose into gluconic acid and toxic hydrogen peroxide, starving and killing tumor cells—a process known as starvation therapy. Hydrogen peroxide can be further converted into highly toxic ·OH for chemodynamic therapy. The combination of starvation therapy and chemodynamic therapy achieves a superadditive antitumor effect. 5-Fluorouracil is a thymidylate synthase inhibitor, converting to 5-fluorouracil deoxynucleotide (5F-dUMP) within cells, while inhibiting deoxythymidylate synthase, preventing the methylation of deoxyuridine acid (dUMP) to deoxythymidine acid (dTMP), thereby affecting DNA synthesis. Studies have shown that starvation therapy combined with chemotherapy has a highly effective antitumor effect.

[0003] Metal-organic frameworks (MOFs) are one-dimensional, two-dimensional, or three-dimensional periodic porous materials formed by the self-assembly of organic ligands and metal ions. They possess advantages such as porosity, extremely large specific surface area, tunable pore size and porosity, ease of functionalization, and catalytic activity. These significant advantages make MOFs highly promising for applications in tumor therapy. Mesoporous MOFs, with their large pore size, can serve as excellent carriers for large protein drugs; however, the problem of premature leakage during drug delivery in single mesoporous MOFs remains unresolved. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides a method for preparing and applying a core-shell metal-organic framework composite material with both macropore and micropore sizes. The preparation method is simple, easy to operate, and the process is stable and reliable. The HMPB@ZIF-8 composite material simultaneously loads two anticancer drugs: glucose oxidase and 5-fluorouracil. This invention achieves combined tumor therapy by hierarchically classifying the pores of the mesoporous metal-organic framework and loading it with a large-molecule glucose oxidase protein and a small-molecule chemotherapeutic drug, 5-fluorouracil.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A core-shell metal-organic framework composite material with both macropore and micropore sizes is disclosed, in which the anticancer drugs glucose oxidase and 5-fluorouracil are loaded. The molecular formula of the framework composite material is HMPB@GOx@ZIF-8@5-Fu@HA, wherein the glucose oxidase is encapsulated in the mesoporous HMPB in the composite material, and the 5-fluorouracil is adsorbed into the shell of ZIF-8.

[0007] Furthermore, in the above-mentioned core-shell structured metal-organic framework composite material, the glucose oxidase loading can reach 15.51 wt%, and the 5-fluorouracil loading can reach 19.33 wt%; the HMPB uses polyvinylpyrrolidone as a ligand and iron ions as metal ions, and ZIF-8 uses 2-methylimidazole as a ligand and zinc ions as metal ions, and is a nano-sized core-shell structured metal-organic framework material synthesized by hydrothermal method.

[0008] The preparation method of the above-mentioned core-shell structured metal-organic framework material includes the following steps: synthesizing mesoporous HMPB by solvothermal method, encapsulating glucose oxidase inside the mesoporous cavity, epitaxially growing a shell layer ZIF-8 as a controlled release switch, then adsorbing 5-fluorouracil into the shell layer of ZIF-8, and using hyaluronic acid as a targeting agent to deliver the drug to the tumor tissue, finally obtaining a core-shell structured metal-organic framework composite material loaded with glucose oxidase and 5-fluorouracil, which can also be called HMPB@GOx@ZIF-8@5-Fu@HA.

[0009] Furthermore, the HMPB is a metal-organic framework (MOF) material, which can be prepared using methods disclosed in the prior art.

[0010] Furthermore, during the synthesis of the metal-organic framework composite material (HMPB@GOx) loaded with glucose oxidase, the reaction was carried out under stirring at room temperature, with a stirring speed of 500 r / min and a stirring time of 12 hours.

[0011] Furthermore, when synthesizing the core-shell structured metal-organic framework composite material (HMPB@GOx@ZIF-8), the concentration of the poly(4-styrene sulfonate) modification solution was 0.3 wt%.

[0012] Furthermore, in the synthesis of the core-shell structured metal-organic framework composite material (HMPB@GOx@ZIF-8@5-Fu@HA) loaded with 5-fluorouracil, the concentrations of 5-fluorouracil and hyaluronic acid were 2 mg / mL and 1.5 mg / mL, respectively.

[0013] The metal-organic framework composite material (HMPB@GOx) loaded with glucose oxidase obtained by the above method is epitaxially grown with ZIF-8. The shell ZIF-8 can further adsorb 5-fluorouracil by physical adsorption, and finally obtain a core-shell structure metal-organic framework composite material (HMPB@GOx@ZIF-8@5-Fu@HA) simultaneously loaded with glucose oxidase and 5-fluorouracil.

[0014] This invention uses ZIF-8 to divide the pore space of mesoporous structures. The small molecule 5-FU occupies the pore space of the ZIF-8 shell through physical adsorption, enabling it to respond to pH and release glucose accordingly. This largely satisfies the synergistic effect of two anticancer drugs in treating tumors, which can further enhance the anti-tumor efficacy and reduce the development of drug resistance, and has a very good prospect for clinical application.

[0015] This invention is based on a hydrothermal method. First, a metal-organic framework composite material (HMPB@GOx) loaded with glucose oxidase is synthesized via a solvothermal method. Then, utilizing the porous adsorption properties of the MOF-On-MOF shell material, the small molecule 5-fluorouracil is physically adsorbed into the pores of ZIF-8. Compared to a metal-organic framework composite material loaded with only one anticancer drug, the core-shell structure metal-organic framework composite material synthesized in this invention makes better use of the pores of the MOF material, exhibits better antitumor effects, and reduces premature drug release. Furthermore, experiments have confirmed that this material exhibits good pH-controlled release effects during the release of both drugs, greatly advancing the application of synergistic effects of multiple different types of anticancer drugs in the treatment of cancer tissues.

[0016] The specific beneficial effects of this invention are as follows:

[0017] (1) This invention employs a strategy of first synthesizing mesoporous materials via a solvothermal method followed by physical adsorption to synthesize a core-shell metal-organic framework composite material loaded with two anticancer drugs. The preparation method is simple and the process is stable and reliable. By epitaxially growing ZIF-8 as the shell structure, effective encapsulation of two guest molecules of different sizes is achieved, which not only greatly enhances the pore utilization rate of the mesoporous metal-organic framework material but also increases the overall drug loading capacity. Verification showed that the drug loading capacity of glucose oxidase can reach 15.51 wt%, and the drug loading capacity of 5-fluorouracil can reach 19.33 wt%.

[0018] (2) The core-shell structured metal-organic framework composite material loaded with two anticancer drugs in this invention exhibits good controlled release effect during drug release. The macromolecular glucose oxidase is encapsulated in the mesopores, while the small molecule 5-fluorouracil is adsorbed into the shell divided by ZIF-8. Due to the spatial confinement effect, a strong host-guest interaction is caused, which can avoid premature release of glucose oxidase.

[0019] (3) This invention utilizes a core-shell structured metal-organic framework material to load two different types of anticancer drugs: glucose oxidase and 5-fluorouracil, to achieve a comprehensive and synergistic anticancer effect, improve the anticancer activity of glucose oxidase, reduce the toxic side effects of 5-fluorouracil, and achieve the characteristics of long-term and sustained-release anticancer effect, providing a new direction for the application of antitumor drugs and providing potential possibilities for their clinical application. Attached Figure Description

[0020] Figure 1 X-ray diffraction (XRD) pattern of HMPB@ZIF-8;

[0021] Figure 2 The images are transmission electron microscope (TEM) images and SEM elemental mapping images, where a is a TEM image of HMPB@GOx@ZIF-8@5-Fu, b and c are TEM images of HMPB, d, e, and f are TEM images of a single HMPB@GOx@ZIF-8@5-Fu particle, and h is an elemental mapping image of HMPB@GOx@ZIF-8@5-Fu.

[0022] Figure 3 XPS spectra of HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, and HMPB@GOx@ZIF-8@5-Fu;

[0023] Figure 4 The graph shows the UV absorption curves of GOx and 5-Fu standard solutions and the changes in UV absorption curves of the supernatant before and after loading GOx and 5-Fu.

[0024] Figure 5 This is a drug release curve diagram, where a represents the GOx release curve of HMPB@GOx@ZIF-8@5-Fu@HA in different pH buffer solutions, b represents the 5-Fu release curve of HMPB@GOx@ZIF-8@5-Fu@HA in different pH buffer solutions, and c represents the Zn release curve of HMPB@GOx@ZIF-8@5-Fu@HA in different pH buffer solutions. 2+ Release curves of Zn in glucose solutions at different pH values ​​using DHMPB@GOx@ZIF-8@5-Fu@HA 2+ Release curve;

[0025] Figure 6Figure 1 shows the enzyme activity of HMPB@GOx@ZIF-8@5-Fu@HA in glucose solution. Figure 2 shows the pH change curves of equal amounts of enzyme, HMPB@GOx, and HMPB@GOx@ZIF-8@5-Fu@HA in glucose solution. Figure 3 shows the glucose consumption curves of different concentrations of HMPB@GOx@ZIF-8@5-Fu@HA in glucose solution. Figure 4 shows the EPR spectra of HMPB@GOx@ZIF-8@5-Fu@HA in glucose solution or buffer solution at pH 7.4. Figure 5 shows the reaction curves of HMPB@GOx@ZIF-8@5-Fu@HA with TMB at different concentrations. Figure 6 shows the reaction curves of HMPB@GOx@ZIF-8@5-Fu@HA with MB at different concentrations.

[0026] Figure 7 The figures show the cytotoxicity of the materials. Figure a shows the cytotoxicity of HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA to 4T1 cells at different concentrations. Figure b shows the cytotoxicity of HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA to 3T3 cells at different concentrations.

[0027] Figure 8 Cell uptake diagrams of HMPB@GOx@ZIF-8@5-Fu@HA and 4T1 cells at different times after co-incubation;

[0028] Figure 9 Cell viability and mortality staining images of HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, HMPB@GOx@ZIF-8@5-Fu@HA and 4T1 cells co-incubated for 6 hours;

[0029] Figure 10 The diagram shows the ROS generation in 4T1 cells after co-incubation with HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA for 6 hours.

[0030] Figure 11 JC-1 staining image of changes in mitochondrial membrane potential in HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, HMPB@GOx@ZIF-8@5-Fu@HA cells after co-incubation with 4T1 cells for 6 hours.

[0031] Figure 12 Flow cytometry apoptosis images of HMPB, HMPB@GOx, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, HMPB@GOx@ZIF-8@5-Fu@HA and 4T1 cells co-incubated for 6 hours. Detailed Implementation

[0032] The present invention will be further illustrated below with examples, but these examples do not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

[0033] Example 1

[0034] (1) Using a solvothermal method, 3.8 g of PVP and 110 mg of K3Fe(CN)6 were added to 50 mL of 0.1 M HCl and sonicated for 30 min to disperse them fully. The solution was placed in an 80 °C oven for 24 h, and the solution changed from a transparent yellow to a dark blue turbid liquid. PB NPs were separated by centrifugation at 14000 rpm / min for 15 min, washed three times with distilled water and once with ethanol, and dried overnight at 50 °C to obtain PB NPs. Then, 20 mg of PB NPs and 100 mg of PVP were ultrasonically mixed and dispersed in 20 mL of 1 M HCl. The mixture was etched in a high-pressure reactor at 140 °C for 2 h. Finally, it was centrifuged at 10000 rpm / min for 12 min, washed three times with distilled water and once with ethanol, and dried overnight at 50 °C to obtain 5.45 mg of cubic Prussian blue nanoparticles, abbreviated as HMPB.

[0035] (2) 10 mg of glucose oxidase and 10 mg of HMPB prepared above were ultrasonically dissolved in 10 mL of ultrapure water. The mixture was stirred at 600 r / min for 12 hours at room temperature. The mixture was collected, centrifuged, washed three times with deionized water, and freeze-dried to obtain 10.6 mg of mesoporous Prussian blue material loaded with glucose oxidase, abbreviated as HMPB@GOx.

[0036] (3) 10 mg of the above HMPB@GOx and 120 mg of poly(4-styrene sulfonate) were uniformly mixed in 40 mL of ultrapure water. The mixture was stirred at 600 r / min for 0.5 h at room temperature, centrifuged at 10000 rpm for 12 min, and then ultrasonically dispersed in 15 mL of ultrapure water. 1 mL of 0.0672 M zinc nitrate hexahydrate was added to the mixture, and the mixture was stirred at 600 r / min for 0.5 h at room temperature. Then, 2 mL of 0.1096 M 2-methylimidazole was added to the solution, and the mixture was stirred at 600 r / min for 0.5 h at room temperature. The mixture was collected, centrifuged, washed three times with deionized water, and freeze-dried to obtain 10.3 mg of mesoporous Prussian blue material with ZIF-8 as the shell and glucose oxidase loaded, abbreviated as HMPB@GOx@ZIF-8.

[0037] (4) 10 mg of the above HMPB@GOx@ZIF-8 and 20 mg of 5-fluorouracil were uniformly mixed in 10 mL of ultrapure water, and 8 mg of glucose oxidase was added to it. The mixture was stirred at 600 r / min for 12 h at room temperature. The mixture was collected, centrifuged, washed three times with deionized water, and freeze-dried to obtain 10.1 mg of mesoporous Prussian blue material with ZIF-8 as the shell and glucose oxidase and 5-fluorouracil loaded on it, abbreviated as HMPB@GOx@ZIF-8@5-Fu.

[0038] (5) 10 mg of the above HMPB@GOx@ZIF-8, 20 mg of 5-fluorouracil, and 15 mg of hyaluronic acid were uniformly mixed in 10 mL of ultrapure water, and 8 mg of glucose oxidase was added to it. The mixture was stirred at 600 r / min for 12 h at room temperature. The mixture was collected, centrifuged, washed three times with deionized water, and freeze-dried to obtain 10.2 mg of mesoporous Prussian blue material with ZIF-8 as the shell and loaded with glucose oxidase and 5-fluorouracil, abbreviated as HMPB@GOx@ZIF-8@5-Fu@HA.

[0039] Comparative Example 1

[0040] A one-pot hydrothermal method was used to uniformly mix 10 mg of the above-mentioned HMPB with 120 mg of poly(4-styrene sulfonate) in 40 mL of ultrapure water. The mixture was stirred at 600 rpm for 0.5 h at room temperature, centrifuged at 10000 rpm for 12 min, and then ultrasonically dispersed again in 15 mL of ultrapure water. 1 mL of 0.0672 M zinc nitrate hexahydrate was added to the mixture, and the mixture was stirred at 600 rpm for 0.5 h at room temperature. Then, 2 mL of 0.1096 M 2-methylimidazole was added to the solution, and the mixture was stirred at 600 rpm for 0.5 h at room temperature. The mixture was collected, centrifuged, washed three times with deionized water, and freeze-dried to obtain 10.3 mg of mesoporous Prussian blue material with ZIF-8 as the shell, abbreviated as HMPB@ZIF-8.

[0041] Verification Example

[0042] Experimental Example 1

[0043] X-ray diffraction experiment

[0044] Experimental procedure: X-ray diffraction measurements were performed on HMPB@ZIF-8 obtained in Example 1 using an X-ray diffractometer.

[0045] Experimental results:

[0046] Depend on Figure 1 It can be seen that the X-ray diffraction curve has characteristic peaks of both ZIF-8 and HMPB, indicating that ZIF-8 nucleates and grows on the mesoporous Prussian blue surface.

[0047] Experimental Example 2

[0048] Transmission electron microscopy experiments and elemental mapping experiments

[0049] Experimental procedure: Transmission electron microscopy was used to perform transmission electron microscopy on HMPB and HMPB@GOx@ZIF-8@5-Fu obtained in the examples, and elemental mapping experiments were performed on HMPB@GOx@ZIF-8@5-Fu.

[0050] Experimental results:

[0051] Depend on Figure 2 It can be seen that HMPB exhibits a hollow structure. After loading GOx and 5-FU, the morphology and size are not significantly different from those of HMPB, with the size of both around 120 nm. HMPB@GOx@ZIF-8@5-FU contains C, N, F, Zn, P, K, and Fe elements.

[0052] Experimental Example 3

[0053] X-ray photoelectron spectroscopy

[0054] Experimental procedure: Take 2mg of powder sample and stick it onto double-sided carbon conductive adhesive or ordinary double-sided adhesive. Press it into small pieces directly using a tablet press and test the full spectrum and Fe and Zn spectra of the sample.

[0055] Experimental results:

[0056] Depend on Figure 3 (a)(b) XPS full spectrum and Fe partial spectrum show that HMPB has 2p3 / 2 and 2p1 / 2 Fe at 705 and 718 eV. 3+ The peak indicates that HMPB@GOx@ZIF-8@5-Fu uses an iron-based MOF as its core template. Figure 3 (c) It can be seen that after the epitaxial growth of the shell ZIF-8, both HPGZ and HPGZF showed 2p3 / 2 and 2p1 / 2 Zn at 1020 and 1043 eV, respectively. 2+ The peak indicates that the core-shell structured metal-organic framework material has been successfully prepared.

[0057] Experiment Example 4

[0058] Drug loading determination of the product

[0059] Experimental steps:

[0060] 10 mg of HMPB from Example 1 was mixed with 10 mg of GOx solution and stirred for 12 h. After centrifugation, the supernatant was obtained without GOx loading. The absorbance at 595 nm was measured using a UV spectrophotometer using the Bradford method. The drug loading was then calculated based on the standard curve of GOx.

[0061] 10 mg of HMPB@GOx@ZIF-8 from Example 1 was mixed with 20 mg of 5-Fu solution and stirred for 12 h. After centrifugation, the supernatant containing unloaded 5-Fu was obtained, and the absorbance at 256 nm was measured using a UV spectrophotometer. The drug loading was then calculated based on the 5-Fu standard curve.

[0062] The formula for calculating drug loading is as follows:

[0063] (wt%)=((m-cv)) / M

[0064] Wherein, (wt%) is the drug loading, m is the content of GOx or 5-Fu drug added in mg, c is the concentration of supernatant in mg / mL, v is the volume of supernatant in mL, and M is the total mass of the product prepared in the example.

[0065] Experimental results:

[0066] Depend on Figure 4Calculations based on the UV absorption spectrum after hydrochloric acid degradation show that the GOx loading in HMPB@GOx@ZIF-8@5-Fu is 13.04 wt%, and the 5-FU loading is 26.02 wt%.

[0067] Experimental Example 5

[0068] The product's pharmaceuticals and Zn 2+ Release determination

[0069] Experimental steps:

[0070] The HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 were subjected to sustained-release experiments in PBS at pH 7.4, 6.5, and 5.0. Specifically, 2 mg of each sample from the example was taken and subjected to sustained-release experiments in 4 mL of PBS. At different time points (0 min, 30 min, 60 min, 120 min, 240 min, 360 min, 1080 min, 1200 min, and 1440 min), a certain volume of supernatant was aspirated, centrifuged to remove any possible trace precipitates, and then PBS at the corresponding pH was added. Any precipitates that appeared were transferred back to the sustained-release solution. The absorbance was measured at a wavelength of 595 nm, and the drug release amount was calculated based on the GOx standard curve.

[0071] The HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 were subjected to sustained-release experiments in PBS at pH 7.4, 6.5, and 5.0. Specifically, 2 mg of each sample from the example was taken and subjected to sustained-release experiments in 4 mL of PBS. At different time points (0 min, 30 min, 60 min, 120 min, 240 min, 360 min, 1080 min, 1200 min, and 1440 min), a certain volume of supernatant was aspirated, centrifuged to remove any possible trace precipitates, and then PBS at the corresponding pH was added. Any precipitates that appeared were transferred back to the sustained-release solution. The absorbance was measured at a wavelength of 256 nm, and the drug release amount was calculated based on the 5-Fu standard curve.

[0072] The HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 was subjected to Zn treatment in PBS at pH 7.4, 6.5, and 5.0, or in 1 mg / mL glucose solution. 2+Release experiment. Specifically, 2 mg of the sample from each example was taken and subjected to a sustained-release experiment in 4 mL of PBS. At different time points (0 min, 30 min, 60 min, 120 min, 240 min, 360 min, 1080 min, 1200 min, 1440 min), a certain volume of supernatant was taken, centrifuged to remove any possible trace precipitates, and then PBS of the corresponding pH was added. The Zn content in the solution was determined by ICP-MS, and the Zn concentration was calculated based on the total Zn content in the material. 2+ The amount released.

[0073] Experimental results:

[0074] Depend on Figure 5 (a) It can be seen that GOx, when encapsulated in a core-shell material, exhibits pH-responsive controlled-release properties. From Figure 5 (b) It can be seen that 5-FU encapsulated in the ZIF-8 shell has poor drug release control, but more drug is released under acidic conditions at pH 5.0 and pH 6.5, and less under neutral conditions. Figure 5 From (c) and (d), we can see that Zn 2+ The release of HMPB@GOx@ZIF-8@5-Fu@HA is greatly affected by the glucose solution. This is because glucose oxidase catalyzes glucose to produce acid, which further lowers the pH of the reaction system, thereby intensifying the release effect. This indicates that HMPB@GOx@ZIF-8@5-Fu@HA has glucose-responsive Zn 2+ Release characteristics.

[0075] Experimental Example 6

[0076] Enzyme activity assay

[0077] Experimental steps:

[0078] 1 mg of HMPB@GOx and HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 were dispersed in a 1 mg / mL glucose solution. The pH value of the system was measured at different time points (0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min), and the pH change after GOx consumed glucose to produce acid was plotted.

[0079] The HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 was dispersed in a 1 mg / mL glucose solution to prepare different material concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). After mixing and reacting for 12 h, a curve of GOx consuming glucose content was plotted.

[0080] The HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 was dispersed in a 1 mg / mL glucose solution or PBS to prepare a material concentration of 1 mg / mL. After mixing and reacting for 6 h, the ·OH generated in the solution was measured using an electron paramagnetic resonance spectrometer.

[0081] The HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 was dispersed in a 1 mg / mL glucose solution to prepare different material concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). After mixing and reacting for 6 h, the ·OH generation capacity in the solution was tested using TMB.

[0082] HMPB@GOx@ZIF-8@5-Fu@HA from Example 1 was dispersed in a 1 mg / mL glucose solution to prepare different material concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) and reacted with MB for 12 h. The ·OH generation capacity in the solution was then tested.

[0083] Experimental results:

[0084] Depend on Figure 6 (a) It can be seen that both HMPB@GOx and HMPB@GOx@ZIF-8@5-Fu@HA reached enzyme catalytic reaction equilibrium in about 2 hours, and the pH of the reaction system could be reduced from 7.4 to about 3.5, indicating that the encapsulated GOx still maintained enzyme activity.

[0085] Depend on Figure 6 (b) It can be seen that the glucose consumption of HMPB@GOx@ZIF-8@5-Fu@HA at different concentrations within 12h is concentration-dependent. 100 μg / mL of material is sufficient to consume all the glucose in the system within 12h, indicating that the encapsulated GOx has good enzyme catalytic activity.

[0086] Depend on Figure 6 (c) It can be seen that the generation of ·OH by HMPB@GOx@ZIF-8@5-Fu@HA varies slightly in different reaction systems. The glucose reaction system generates more ·OH than the reaction system without glucose, indicating that the glucose oxidase in the reaction system consumes glucose to produce H2O2, which can further react with Fe. 3+ The reaction produces toxic ·OH. This indicates that HMPB@GOx@ZIF-8@5-Fu@HA, after encapsulation with GOx, possesses potential chemical kinetic properties.

[0087] Further by Figure 6As shown in (d)(e), HMPB@GOx@ZIF-8@5-Fu@HA at different concentrations can generate toxic ·OH in a glucose-free reaction system, indicating that HMPB@GOx@ZIF-8@5-Fu@HA encapsulated with GOx has concentration-dependent chemical kinetic properties.

[0088] Experimental Example 7

[0089] Cytotoxicity assay

[0090] Experimental steps:

[0091] HMPB experimental solution preparation: HMPB was dissolved in 1640 medium, and HMPB experimental solutions with concentrations of 0, 0.78125, 1.5625, 3.125, 6.25, and 12.5 μg / mL were prepared using 1640 medium;

[0092] Preparation of HMPB@GOx experimental solutions: HMPB@GOx was dissolved in 1640 medium, and HMPB@GOx experimental solutions with concentrations of 0, 0.78125, 1.5625, 3.125, 6.25, and 12.5 μg / mL were prepared using 1640 medium;

[0093] Preparation of HMPB@GOx@ZIF-8 experimental solutions: HMPB@GOx@ZIF-8 was dissolved in 1640 medium, and HMPB@GOx@ZIF-8 experimental solutions with concentrations of 0, 0.78125, 1.5625, 3.125, 6.25, and 12.5 μg / mL were prepared using 1640 medium;

[0094] Preparation of HMPB@GOx@ZIF-8@5-Fu experimental solutions: HMPB@GOx@ZIF-8@5-Fu was dissolved in 1640 medium, and HMPB@GOx@ZIF-8@5-Fu experimental solutions with concentrations of 0, 0.78125, 1.5625, 3.125, 6.25, and 12.5 μg / mL were prepared using 1640 medium.

[0095] Preparation of experimental solutions for HMPB@GOx@ZIF-8@5-Fu@HA: HMPB@GOx@ZIF-8@5-Fu@HA was dissolved in 1640 medium, and experimental solutions of HMPB@GOx@ZIF-8@5-Fu@HA with concentrations of 0, 0.78125, 1.5625, 3.125, 6.25, and 12.5 μg / mL were prepared using 1640 medium.

[0096] Collect 4T1 or 3T3 cells in the logarithmic growth phase and in good growth condition, and perform cell digestion, removing the original culture medium. First, wash twice with 2 mL of PBS solution, then add 1 mL of trypsin and incubate for 4 min. After digestion, add 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) to terminate digestion. Centrifuge at 1200 rpm for 5 min, remove the supernatant, add 9 mL of complete culture medium, mix well, and perform cell counting. Prepare a cell suspension according to experimental requirements and distribute 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates and incubated for 24 hours. After cell attachment, the original culture medium was removed, and 200 μL of 1640 medium solution, HMPB experimental solution, HMPB@GOx experimental solution, HMPB@GOx@ZIF-8 experimental solution, HMPB@GOx@ZIF-8@5-Fu experimental solution, and HMPB@GOx@ZIF-8@5-Fu@HA experimental solution were added to each well, respectively. After drug addition, the plates were incubated for another 6 hours. After incubation, the drug-containing medium was aspirated, and 100 μL of 10% CCK-8 reagent was added to each well, which was then incubated at 37°C for another 2 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and the data were recorded. The corresponding cell viability was calculated using the cell viability calculation formula, and a line graph was plotted with cell viability as the y-axis and drug concentration as the x-axis.

[0097] The cell survival rate formula is as follows:

[0098]

[0099] Experimental results:

[0100] Depend on Figure 6 (a) It is evident that at the maximum concentration of 12.5 μg / mL, after 6 hours of co-incubation with HMPB, almost no 4T1 or 3T3 cells died, indicating that HMPB possesses a certain degree of biocompatibility. After drug loading, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA exhibited significant dose-dependent inhibition of cell death, and showed stronger killing ability against 4T1 cells compared to 3T3 cells. This is because the intracellular environment of 4T1 cells becomes acidic, accelerating the catalytic activity of glucose oxidase, leading to increased Zn... 2+ The large release of ·OH produces strong cytotoxicity.

[0101] Experimental Example 8

[0102] Cell uptake experiment

[0103] Experimental steps:

[0104] Preparation of experimental solution for Rhodamine B-modified HMPB@GOx@ZIF-8@5-Fu@HA group: 5 mg of Rhodamine B was dissolved in 10 mL of ultrapure water by sonication. 10 mg of HMPB@GOx@ZIF-8@5-Fu@HA was added, and the mixture was magnetically stirred for 12 h. The solution was centrifuged at 10000 r / min for 10 min, the supernatant was discarded, and the solution was washed with ultrapure water. Centrifugation and washing were repeated until the supernatant became colorless and transparent. The solution was then dialyzed for 12 h. Finally, the solution was centrifuged, the supernatant was discarded, and the solution was freeze-dried to obtain the Rhodamine B-modified HMPB@GOx@ZIF-8@5-Fu@HA sample. Rhodamine B-modified HMPB@GOx@ZIF-8@5-Fu@HA was dissolved in 1640 medium, and an experimental solution of Rhodamine B-modified HMPB@GOx@ZIF-8@5-Fu@HA with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0105] 4T1 cells in the logarithmic growth phase and in good growth condition were collected and digested, and the original culture medium was removed. The cells were washed twice with 2 mL of PBS solution, followed by 1 mL of trypsin and incubation for 4 min. After digestion, 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 min, the supernatant was removed, and 8 mL of complete culture medium was added and mixed thoroughly. Cell counting was performed, and a cell suspension was prepared according to experimental requirements, with 5 × 10⁶ cells per well. 4 Cells were seeded into 12-well plates and incubated for 24 hours. After cell attachment, the original culture medium was removed, and 1 mL of Rhodamine B-modified HMPB@GOx@ZIF-8@5-Fu@HA experimental solution was added to each well. After drug addition, the plates were incubated again for 0, 1, 2, 4, and 6 hours. After incubation, the drug-containing culture medium was aspirated, and the cells were washed three times with 1 mL of PBS. Then, 1 mL of 4% paraformaldehyde solution was added to each well to fix the cells at room temperature for 20 minutes. After fixation, the cells were washed twice with 1 mL of PBS, and 1 mL of DAPI staining solution (100 μL) was added to each well. The cells were stained at 37°C in the dark for 15 minutes, and then washed twice with PBS. Finally, the phagocytosis of the nanomaterials at different time points was observed using a fluorescence inverted microscope.

[0106] Experimental results:

[0107] Depend on Figure 8 As time progressed, the red fluorescence in 4T1 cells gradually increased, verifying the effective uptake of HMPB@GOx@ZIF-8@5-Fu@HA by 4T1 cells, which greatly satisfied the effect of releasing drugs into cells after uptake of the material.

[0108] Experimental Example 9

[0109] Live and dead cell imaging experiments

[0110] Experimental steps:

[0111] HMPB experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0112] HMPB@GOx experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0113] HMPB@GOx@ZIF-8 experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8 experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium;

[0114] HMPB@GOx@ZIF-8@5-Fu experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0115] HMPB@GOx@ZIF-8@5-Fu@HA experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu@HA experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0116] 4T1 cells in the logarithmic growth phase and in good growth condition were collected and digested, and the original culture medium was removed. The cells were washed twice with 2 mL of PBS solution, followed by 1 mL of trypsin and incubation for 4 min. After digestion, 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 min, the supernatant was removed, and 8 mL of complete culture medium was added and mixed thoroughly. Cell counting was performed, and a cell suspension was prepared according to experimental requirements, with 5 × 10⁶ cells per well. 4Cells were seeded into small dishes and incubated for 24 hours. After cell attachment, the original culture medium was removed, and 200 μL of 1640 medium solution, HMPB experimental solution, HMPB@GOx experimental solution, HMPB@GOx@ZIF-8 experimental solution, HMPB@GOx@ZIF-8@5-Fu experimental solution, and HMPB@GOx@ZIF-8@5-Fu@HA experimental solution were added to each well. After drug addition, the cells were incubated again for 6 hours. After incubation, the drug-containing medium was aspirated, and 100 μL of a mixed fluorescent dye of Calcein-AM (staining live cells green) and PI (staining dead cells red) was added to each well. The cells were stained at 37°C in the dark for 15 minutes, washed twice with PBS, and observed under a fluorescence microscope for cell fluorescence imaging.

[0117] Experimental results:

[0118] Depend on Figure 9 It can be seen that, compared with the HMPB and HMPB@GOx groups, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA exhibited effective cell-killing effects, as evidenced by the strong red fluorescence emitted by the stained dead cells. This is due to the activity of GOx and Zn. 2+ This is due to the synergistic anticancer effect of 5-FU.

[0119] Experimental Example 10

[0120] Intracellular ROS Imaging Experiment

[0121] Experimental steps:

[0122] HMPB experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0123] HMPB@GOx experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0124] HMPB@GOx@ZIF-8 experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8 experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium;

[0125] HMPB@GOx@ZIF-8@5-Fu experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0126] HMPB@GOx@ZIF-8@5-Fu@HA experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu@HA experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0127] 4T1 cells in the logarithmic growth phase and in good growth condition were collected and digested, and the original culture medium was removed. The cells were washed twice with 2 mL of PBS solution, followed by 1 mL of trypsin and incubation for 4 min. After digestion, 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 min, the supernatant was removed, and 8 mL of complete culture medium was added and mixed thoroughly. Cell counting was performed, and a cell suspension was prepared according to experimental requirements, with 5 × 10⁶ cells per well. 4 Cells were seeded into small dishes and incubated for 24 hours. After cell attachment, the original culture medium was removed, and 200 μL of 1640 medium solution, HMPB experimental solution, HMPB@GOx experimental solution, HMPB@GOx@ZIF-8 experimental solution, HMPB@GOx@ZIF-8@5-Fu experimental solution, and HMPB@GOx@ZIF-8@5-Fu@HA experimental solution were added to each well. After drug addition, the cells were incubated again for 6 hours. After incubation, the drug-containing medium was aspirated, and 200 μL of DCFH-DA probe (1:1000) was prepared according to the kit test requirements. The probe was added to each well, incubated at 37°C for 30 minutes, washed twice with PBS, and ROS generation was recorded using an inverted fluorescence microscope.

[0128] Experimental results:

[0129] Depend on Figure 10 It can be seen that, compared with the HMPB and HMPB@GOx groups, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA exhibited a strong intracellular ROS aggregation effect, which proves that ZIF-8 dissociates and releases Zn intracellularly. 2+ It can induce strong ROS-induced cell killing, resulting in a chemokinetic therapeutic effect.

[0130] Experimental Example 11

[0131] Mitochondrial membrane potential depolarization experiment

[0132] Experimental steps:

[0133] HMPB experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0134] HMPB@GOx experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0135] HMPB@GOx@ZIF-8 experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8 experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium;

[0136] HMPB@GOx@ZIF-8@5-Fu experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0137] HMPB@GOx@ZIF-8@5-Fu@HA experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu@HA experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0138] 4T1 cells in the logarithmic growth phase and in good growth condition were collected and digested, and the original culture medium was removed. The cells were washed twice with 2 mL of PBS solution, followed by 1 mL of trypsin and incubation for 4 min. After digestion, 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 min, the supernatant was removed, and 8 mL of complete culture medium was added and mixed thoroughly. Cell counting was performed, and a cell suspension was prepared according to experimental requirements, with 5 × 10⁶ cells per well. 4Cells were seeded in small dishes and incubated for 24 hours. After cell attachment, the original culture medium was removed, and 200 μL of 1640 medium solution, HMPB experimental solution, HMPB@GOx experimental solution, HMPB@GOx@ZIF-8 experimental solution, HMPB@GOx@ZIF-8@5-Fu experimental solution, and HMPB@GOx@ZIF-8@5-Fu@HA experimental solution were added to each well. After drug addition, the cells were incubated for another 6 hours. After incubation, the drug-containing medium was aspirated, and JC-1 working solution was prepared according to the JC-1 mitochondrial membrane potential detection kit instructions. 1 mL of JC-1 reagent was added to each well, and the cells were incubated at 37°C in the dark for 20 minutes. The cells were washed twice with JC-1 washing buffer to remove the dye, and then 0.5 mL of whole culture was added. The cell nuclei were stained with DAPI dye, and fluorescence changes were recorded using an inverted fluorescence microscope.

[0139] Experimental results:

[0140] Depend on Figure 11 It can be seen that, compared with the HMPB and HMPB@GOx groups, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA exhibited strong mitochondrial membrane damage, which also proves that ZIF-8 dissociates and releases Zn intracellularly. 2+ It can cause mitochondrial damage, leading to the death of cancer cells.

[0141] Experimental Example 12

[0142] Flow cytometry apoptosis experiment

[0143] Experimental steps:

[0144] HMPB experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0145] HMPB@GOx experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0146] HMPB@GOx@ZIF-8 experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8 experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium;

[0147] HMPB@GOx@ZIF-8@5-Fu experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0148] HMPB@GOx@ZIF-8@5-Fu@HA experimental solution preparation: HMPB was dissolved in 1640 medium, and an HMPB@GOx@ZIF-8@5-Fu@HA experimental solution with a concentration of 12.5 μg / mL was prepared using 1640 medium.

[0149] 4T1 cells in the logarithmic growth phase and in good growth condition were collected and digested, and the original culture medium was removed. The cells were washed twice with 2 mL of PBS solution, followed by 1 mL of trypsin and incubation for 4 min. After digestion, 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 min, the supernatant was removed, and 8 mL of complete culture medium was added and mixed thoroughly. Cell counting was performed, and a cell suspension was prepared according to experimental requirements, with 5 × 10⁶ cells per well. 4 Cells were seeded into small dishes and incubated for 24 hours. After cell attachment, the original culture medium was removed, and 200 μL of 1640 medium solution, HMPB experimental solution, HMPB@GOx experimental solution, HMPB@GOx@ZIF-8 experimental solution, HMPB@GOx@ZIF-8@5-Fu experimental solution, and HMPB@GOx@ZIF-8@5-Fu@HA experimental solution were added to each well. After drug addition, the cells were incubated again for 6 hours. After incubation, the drug-containing medium was aspirated, cells were collected, and stained with 5 μL Annexin V-FITC and 10 μL LPI at room temperature in the dark for 15 minutes. Apoptosis was quantified using flow cytometry (CytoFLEX).

[0150] Experimental results:

[0151] Depend on Figure 12 It can be seen that, compared with the HMPB and HMPB@GOx groups, HMPB@GOx@ZIF-8, HMPB@GOx@ZIF-8@5-Fu, and HMPB@GOx@ZIF-8@5-Fu@HA exhibited strong apoptosis effects, which proves that HMPB@GOx@ZIF-8@5-Fu@HA has a very effective anti-cancer effect.

[0152] The experimental results above show that the core-shell metal-organic framework material for loading two anticancer drugs, prepared by the present invention using pore hierarchical methods, exhibits high GOx loading capacity (13.04 wt%) and 5-FU loading capacity (26.02 wt%). Furthermore, by rationally selecting ZIF-8 to divide the mesoporous channels of HMPB, it possesses glucose and pH-responsive co-release capabilities, enabling the co-release of GOx and Zn. 2+ It has a synergistic anticancer effect with 5-fluorouracil, enhancing the therapeutic effect on cancerous tissue sites and showing certain potential for clinical application.

Claims

1. A core-shell structured metal-organic framework composite material with both macropore and micropore sizes, characterized in that, The molecular formula of the framework composite material is HMPB@GOx@ZIF-8@5-FU@HA; the loaded anticancer drugs are glucose oxidase and 5-fluorouracil; wherein the glucose oxidase in the composite material is encapsulated in the mesopores of HMPB, a thin layer of ZIF-8 is grown on the surface of HMPB, and the 5-fluorouracil is adsorbed into the shell of ZIF-8. The preparation method of the core-shell structured metal-organic framework composite material with both macropore and micropore sizes is as follows: (1) Synthesize cubic Prussian blue nanoparticles and etch them with hydrochloric acid to form HMPB with a hollow mesoporous structure; (2) The hollow mesoporous HMPB prepared in step (1) was stirred together with glucose oxidase solution to obtain metal-organic framework composite material HMPB@GOx; (3) The HMPB@GOx prepared in step (2) was dispersed in a poly(4-styrene sulfonate) solution for modification. The modified HMPB@GOx was dispersed in an aqueous solution, and zinc nitrate solution and 2-methylimidazole solution were added to it in sequence. After stirring, the core-shell structure metal-organic framework composite material HMPB@GOx@ZIF-8 was obtained. (4) Disperse the HMPB@GOx@ZIF-8 prepared in step (3) in an aqueous solution of 5-fluorouracil and hyaluronic acid and stir to obtain a core-shell structure metal-organic framework composite material HMPB@GOx@ZIF-8@5-FU@HA loaded with glucose oxidase and 5-fluorouracil.

2. The core-shell structured metal-organic framework composite material according to claim 1, characterized in that, The glucose oxidase had a drug loading of 13.04 wt%, and the 5-fluorouracil had a drug loading of 26.02 wt%.

3. The core-shell structured metal-organic framework composite material according to claim 1, characterized in that, The HMPB core structure is a nanoscale metal-organic framework material synthesized by hydrothermal method using polyvinylpyrrolidone as ligand and iron ions as metal ions; the ZIF-8 shell structure is grown in situ on the surface of HMPB by epitaxial growth method using 2-methylimidazole as ligand and zinc ions as metal ions.

4. A method for preparing a core-shell structured metal-organic framework composite material according to any one of claims 1-3, characterized in that, The following steps are adopted: (1) Synthesize cubic Prussian blue nanoparticles and etch them with hydrochloric acid to form HMPB with a hollow mesoporous structure; (2) The hollow mesoporous HMPB prepared in step (1) was stirred together with glucose oxidase solution to obtain metal-organic framework composite material HMPB@GOx; (3) The HMPB@GOx prepared in step (2) was dispersed in a poly(4-styrene sulfonate) solution for modification. The modified HMPB@GOx was dispersed in an aqueous solution, and zinc nitrate solution and 2-methylimidazole solution were added to it in sequence. After stirring, the core-shell structure metal-organic framework composite material HMPB@GOx@ZIF-8 was obtained. (4) Disperse the HMPB@GOx@ZIF-8 prepared in step (3) in an aqueous solution of 5-fluorouracil and hyaluronic acid and stir to obtain a core-shell structure metal-organic framework composite material HMPB@GOx@ZIF-8@5-FU@HA loaded with glucose oxidase and 5-fluorouracil.

5. The preparation method according to claim 4, characterized in that, The cubic Prussian blue nanoparticles obtained in step (1) are prepared by a hydrothermal method; the specific steps are as follows: 1) Potassium ferricyanide and polyvinylpyrrolidone were ultrasonically dissolved in 0.1 mM hydrochloric acid solution, and the product was collected and washed after hydrothermal reaction at 80℃ for 24 h to obtain cubic Prussian blue nanoparticles. 2) The cubic Prussian blue nanoparticles prepared in step 1) were ultrasonically dispersed with polyvinylpyrrolidone in a 1 mM hydrochloric acid solution and reacted at 140 °C for 2 h. After the reaction was completed, the product was collected and washed to obtain cubic Prussian blue nanoparticles.

6. The preparation method according to claim 4, characterized in that, The preparation method of the metal-organic framework composite material HMPB@GOx in step (2) is as follows: 10 mg of cubic Prussian blue nanoparticles and 10 mg of glucose oxidase are sonicated in 10 mL of ultrapure water for 10 min and stirred at 500 r / min for 12 h. After the reaction is completed, the product is collected and washed to obtain HMPB@GOx.

7. The preparation method according to claim 4, characterized in that, The preparation method of the core-shell structure metal-organic framework composite material HMPB@GOx@ZIF-8 in step (3) is as follows: 10 mg HMPB@GOx and 120 mg poly(4-styrene sulfonate) are ultrasonically dissolved in 30 mL of ultrapure water for 10 min and stirred at 500 r / min for 30 min. After the reaction is completed, the precipitate is obtained by centrifugation and then dispersed again in 15 mL of ultrapure water. 1 mL of 0.067 M zinc nitrate hexahydrate is added and stirred at 500 r / min for 30 min. 2 mL of 0.1096 M dimethylimidazole is added and stirred at 500 r / min for 30 min. After the reaction is completed, the product is collected and washed to obtain HMPB@GOx@ZIF-8.

8. The preparation method according to claim 4, characterized in that, The core-shell structured metal-organic framework composite material HMPB@GOx@ZIF-8@5-Fu@HA described in step (4) is as follows: 10 mg of HMPB@GOx@ZIF-8, 20 mg of 5-fluorouracil, and 15 mg of HA are dispersed in 10 mL of ultrapure water and stirred at 500 r / min for 12 h at room temperature. After the reaction is completed, the product is collected and washed to obtain HMPB@GOx@ZIF-8@5-Fu@HA.

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