A composite hydrogel loaded with copper-manganese sulfide nanoszyme and a preparation method and application thereof

Abstract

This invention provides a composite hydrogel encapsulating copper-manganese sulfide nanozymes, its preparation method, and its applications. The preparation method includes: firstly, preparing polyetherimide-modified copper-manganese sulfide nanoparticles via solvation pyrolysis; secondly, preparing an amphiphilic polymer of polyethylene glycol monomethyl ether and cinnamaldehyde bonded to adipic acid dihydrazide as a pH-responsive center via a two-step acylation reaction; thirdly, self-assembling these polymers into nano-prodrug micelles in an aqueous solution; and finally, forming a supramolecular hydrogel based on the host-guest interaction between α-cyclodextrin and these micelles. The nanoparticles are then uniformly loaded into the hydrogel to form a composite supramolecular hydrogel. Direct injection of the gel into the tumor site, followed by near-infrared irradiation at a specific time, can achieve synergistic photothermal-photodynamic-chemodynamic therapy. Notably, the micelles can exhibit pH-responsive drug release under the acidic environment of the tumor, thereby increasing intracellular hydrogen peroxide levels, making this invention promising for applications in tumor treatment.

Description

Technical Field

[0001] This invention belongs to the fields of polymer materials and biomedical engineering technology, specifically relating to a composite hydrogel loaded with copper manganese sulfide nanozymes, its preparation method, and its application. Background Technology

[0002] Various cancer treatments have been developed, with chemotherapy still playing a dominant role in clinical practice. Its efficacy depends on the drug's ability to accumulate at the tumor site and kill cancer cells. However, drugs administered intravenously, subcutaneously, or via interventional perfusion undergo metabolism in the bloodstream, resulting in insufficient drug concentrations reaching the tumor site and causing significant toxic side effects on normal tissue cells. Furthermore, repeated administration can lead to multidrug resistance, severely impacting treatment outcomes. In recent years, various nanomedicine delivery platforms (such as micelles, vesicles, and microspheres) have been fabricated through nano-self-assembly to address the issues of chemotherapy targeting and drug utilization. However, these drug carriers still face the challenge of dilution and clearance within the complex human circulatory system. Therefore, designing novel drug carrier materials that avoid circulation within the body is of great significance for achieving precise tumor treatment. Injectable hydrogels not only share similarities with the extracellular matrix (ECM) and possess good biocompatibility, but can also deliver drug molecules or functional nanoparticles to the lesion site via minimally invasive injection into the tumor, achieving effective accumulation at the tumor site. Compared with passive delivery through diffusion or degradation mechanisms, constructing stimulus-responsive hydrogels using endogenous or exogenous stimuli (such as temperature, pH, light, ultrasound, etc.) can achieve controlled release of therapeutic agents, providing a reliable strategy for cancer treatment. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a composite hydrogel encapsulating copper manganese sulfide nanozymes and its application in tumor therapy and drug delivery. The main highlight is the first-ever preparation of an injectable hydrogel in which cinnamaldehyde (CA) is linked to a gel backbone, while simultaneously loading copper manganese sulfide nanozymes to achieve multi-modal synergistic tumor therapy and achieve good therapeutic effects. This invention uses adipic acid dihydrazide (ADH) as a pH-responsive center to link polyethylene glycol monomethyl ether (mPEG) and CA to form an amphiphilic prodrug polymer chain, which self-assembles into prodrug micelles (PAC micelles) in aqueous solution. A pH-responsive injectable hydrogel is formed based on the host-guest interaction between α-cyclodextrin (α-CD) and PAC micelles. Polyetherimide (PEI) modified copper manganese sulfide nanozymes (PCMS NPs) are prepared using a solvothermal method, which can be uniformly dispersed in the hydrogel to form a composite hydrogel. In use, this composite hydrogel is directly injected into the tumor site, and near-infrared light is applied at a specific time to achieve a good photothermal-photodynamic-chemodynamic synergistic therapeutic effect. In the acidic environment of a tumor, pH-responsive hydrazone bonds break, slowly releasing hydroxyl radicals (CA) and increasing hydrogen peroxide (H2O2) levels. PCMS NPs exhibit peroxidase-like (POD) and catalase-like (CAT) activities, converting H2O2 into hydroxyl radicals (·OH) and oxygen (O2), alleviating intratumoral hypoxia and inducing apoptosis. Simultaneously, they exert glutathione peroxidase (GPX)-like activity to consume glutathione (GSH), further enhancing the efficacy of chemodynamic therapy (CDT). They utilize multiple enzyme-like catalytic activities to achieve chemodynamic therapy. Under near-infrared (NIR) irradiation, PCMS NPs exhibit excellent photothermal conversion properties, rapidly raising tumor temperature to above 42°C for photothermal therapy (PTT), and exert oxidase-like (OXD) activity to convert O2 into superoxide anions (·O2). - This invention enables photodynamic therapy (PDT), combining photothermal and photodynamic treatments, which holds great potential for tumor therapy. Furthermore, the composite supramolecular hydrogel prepared in this invention exhibits excellent in-situ injectability and biocompatibility.

[0004] To achieve the above objectives, the solution of the present invention is:

[0005] A method for preparing a composite hydrogel loaded with copper manganese sulfide nanozymes includes the following steps:

[0006] (1) Copper chloride dihydrate (CuCl2·2H2O), manganese chloride tetrahydrate (MnCl2·4H2O), and polyethyleneimine (PEI) were fully dissolved in ethylene glycol (EG) to obtain solution A. Thiourea ((NH4)2S) was dissolved in EG to obtain solution B. Then, solution B was added dropwise to solution A, and after thorough stirring, the mixture was transferred to a stainless steel autoclave and reacted at high temperature. After cooling to room temperature, the product was collected by centrifugation, washed twice, and dried to obtain dark green powdered polyetherimide modified copper manganese sulfide nanoparticles (PCMS NPs).

[0007] (2) Polyethylene glycol monomethyl ether (mPEG) was dissolved in anhydrous dichloromethane (DCM), and then succinic anhydride and 4-dimethylaminopyridine (DMAP) were added to the above solution. The mixture was stirred and reacted under nitrogen (N2) protection. After precipitation, filtration, dialysis, freezing, drying and other post-treatment, a white product, polyethylene glycol monomethyl ether acid (mPEG-COOH), was obtained.

[0008] (3) Adipic acid dihydrazide (ADH) was dissolved in deionized water, and then CA was added dropwise to the solution while stirring at room temperature. After filtration, precipitation, washing, and drying, a milky white product, adipic acid dihydrazide cinnamaldehyde (ADH-CA), was obtained.

[0009] (4) mPEG-COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were dissolved in N,N-dimethylformamide (DMF) and reacted under N2 protection with stirring. Subsequently, ADH-CA was added to the above solution and the reaction continued. After dialysis, freezing, drying and other post-treatments, a white solid powder amphiphilic polymer of polyethylene glycol monomethyl ether and cinnamaldehyde (mPEG-ADH-CA) was obtained.

[0010] (5) Dissolve mPEG-ADH-CA in an organic solvent and obtain self-assembled nano prodrug micelles (PAC micelles) by dialysis;

[0011] (6) Dissolve PAC micelles and α-cyclodextrin (α-CD) in deionized water to form a solution. Disperse PCMS NPs in the PAC micelle solution and mix with the α-CD solution at a volume ratio of 1:1. After sonication for 5-10 minutes, let stand at room temperature to form a composite hydrogel loaded with copper sulfide manganese nanozymes.

[0012] Furthermore, in step (1), the molar ratio of CuCl2·2H2O, MnCl2·4H2O and (NH4)2S is 1:1:3-2:1:6.

[0013] Furthermore, in step (1), the stirring time is 0.5-1h.

[0014] Furthermore, in step (1), the high temperature is 120-180℃ and the reaction time is 6-12h.

[0015] Furthermore, in step (1), the washing solution used is ethanol and deionized water.

[0016] Furthermore, in step (2), the molar ratio of mPEG, succinic anhydride and DMAP is 1:5:1-1:6:1.

[0017] Furthermore, in step (2), the stirring reaction time is 24-48 hours.

[0018] Furthermore, in step (2), icy diethyl ether is used as the precipitant during precipitation.

[0019] Furthermore, in step (2), the dialysis time is 48-72 hours, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis solution is deionized water.

[0020] Furthermore, in step (3), the molar ratio of ADH to CA is 4:1 to 6:1.

[0021] Furthermore, in step (3), the stirring reaction time is 2-4 hours.

[0022] Furthermore, in step (3), the washing solution is deionized water and methanol.

[0023] Furthermore, in step (4), the molar ratio of mPEG-COOH, EDC·HCl, NHS and ADH-CA is 1.7:2:2.5:2.4.

[0024] Furthermore, in step (4), the stirring time under nitrogen is 0.5-2h.

[0025] Furthermore, in step (4), the reaction continues for 24-48 hours.

[0026] Furthermore, in step (4), the dialysis time is 48-72 hours, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis solution is deionized water.

[0027] Further, in step (5), the organic solvent is selected from any one of tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0028] Furthermore, in step (5), the ratio of mPEG-ADH-CA to organic solvent is (50-100) mg: (5-25) mL.

[0029] Furthermore, in step (5), the dialysis time is 48-72 hours, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis solution is deionized water.

[0030] Further, in step (6), the weight ratio of PAC micelles, α-CD and PCMS NPs is 30:(140-170):(0.125-0.5).

[0031] A composite hydrogel encapsulating copper manganese sulfide nanozymes is obtained by the above preparation method.

[0032] An application of the aforementioned composite hydrogel containing copper-manganese sulfide nanozymes in the preparation of materials for tumor therapy and drug delivery. Direct injection of the composite gel into the tumor site, followed by near-infrared irradiation at a specific time, enables synergistic photothermal-photodynamic-chemodynamic therapy. Specifically, the micelles can exhibit pH-responsive drug release in the acidic environment of the tumor, thereby increasing intracellular hydrogen peroxide levels, demonstrating promising applications in tumor therapy.

[0033] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0034] First, the composite hydrogel material prepared by this invention has good injectability and biocompatibility, enabling in situ injection at tumor sites without toxic side effects on normal tissue cells.

[0035] Secondly, the composite gel prepared by this invention has a better anti-cancer effect than traditional chemotherapy, and exerts the integrated advantages of synergistic treatment.

[0036] Third, the raw materials used in this invention are widely available, such as α-CD, polyethylene glycol, solvents, and precipitants, all of which can be industrially produced. The preparation method is simple and easy to implement, and has good application value. Attached Figure Description

[0037] Figure 1 The images show the physical sample, X-ray diffraction pattern, and scanning electron microscope image of the composite gel prepared in Example 1.

[0038] Figure 2 This is a diagram illustrating the injectability of the composite gel in Example 1.

[0039] Figure 3 The figure shows the results of the in vitro biocompatibility and cytotoxicity experiments of the composite gel in Example 1.

[0040] Figure 4 This is a diagram showing the in vivo antitumor effect of the composite hydrogel obtained in Example 1. Detailed Implementation

[0041] The present invention will be further described below with reference to embodiments, but this is not intended to limit the scope of the invention.

[0042] Example 1:

[0043] (1) CuCl2·2H2O (0.1705 g, 1 mmol), MnCl2·4H2O (0.1979 g, 1 mmol), and PEI (0.2000 g) were fully dissolved in EG (20 mL) to obtain solution A. (NH4)2S (0.2284 g, 3 mmol) was dissolved in EG (10 mL) to obtain solution B. Then, solution B was added dropwise to solution A, stirred for 30 min, transferred to a stainless steel autoclave, and reacted at 180 °C for 6 h. After cooling to room temperature, the product was collected by centrifugation, washed twice with deionized water and ethanol, and dried to obtain dark green powder PCMS NPs.

[0044] (2) mPEG (4.0 g, 2 mmol) was dissolved in DCM (30 mL), and then succinic anhydride (1.0 g, 10 mmol) and DMAP (0.244 g, 2 mmol) were added to the above solution. The mixture was stirred for 48 h under N2 protection. After the reaction was completed, the product was precipitated several times in cold diethyl ether. The precipitate was filtered and dissolved in water for dialyzing (dialysis bag molecular weight cutoff: 1000 Da) for 48 h. The precipitate was then freeze-dried to obtain a white solid mPEG-COOH.

[0045] (3) ADH (3.3 g, 18 mmol) was dissolved in deionized water (20 mL), and then CA (0.5 mL, 3 mmol) was slowly added dropwise to the solution. The mixture was stirred at room temperature for 2 h. The precipitate was filtered and washed three times with deionized water and methanol, respectively. The product was collected and dried in a vacuum oven at room temperature to constant weight to obtain a milky white powder, ADH-CA.

[0046] (4) mPEG-COOH (3.6 g, 1.7 mmol), EDC·HCl (0.38 g, 2 mmol), and NHS (0.28 g, 2.5 mmol) were dissolved in DMF (30 mL) and stirred for 2 h under N2 protection. Subsequently, ADH-CA (0.7 g, 2.4 mmol) was dissolved in the above solution, and stirring was continued for 24 h. After the reaction was completed, the mixture was dialyzed (molecular weight cutoff of 1000 Da) for 48 h, and then freeze-dried to obtain a white solid powder mPEG-ADH-CA.

[0047] (5) Dissolve mPEG-ADH-CA (300mg) in DMSO (30mL) to prepare a solution. Dialyze the solution with distilled water (dialysis bag molecular weight cutoff: 1000Da) for 48h to completely remove DMSO. Change the water 3 times a day to obtain PAC micelles.

[0048] (6) PAC micelles and α-CD were dissolved in deionized water to obtain PAC micelle solution and α-CD solution. PCMS NPs (500 μg / mL) were dispersed in PAC solution (3 wt%) by ultrasonic dispersion, and then mixed with α-CD solution (16 wt%) at a volume ratio of 1:1. The mixture was ultrasonicated for 5 min and allowed to stand at room temperature for a period of time to form a composite hydrogel loaded with copper manganese sulfide nanozyme.

[0049] Figure 1 The formation process of the composite hydrogel was demonstrated by dispersing (i) PCMS NPs (500 μg / mL) in a PAC micelle solution (3 wt%), then mixing it with (ii) an α-CD solution (16 wt%) to form (iii) a mixed solution. After several minutes, (iv) a composite supramolecular hydrogel was obtained. Figure 1 (a) in the image). XRD pattern ( Figure 1 In (b) of the diagram, the diffraction peaks at 19.00° and 23.18° represent the crystallization peaks of the PEG chain. These peaks disappear in the gel, and a new diffraction peak appears at 19.8°, representing the crystallization peak of the quasi-polypropylene (PPR) assembly. Hydrogen bonding between adjacent PPRs leads to microcrystal aggregation, thereby promoting physical cross-linking and gel formation. This was confirmed by SEM (Sequencing). Figure 1 (c) The morphology of the gel was observed, confirming that the gel has a distinct three-dimensional network structure, providing space for encapsulating nano-therapeutic agents. Figure 2 The injectability of the gel was demonstrated. The composite gel was drawn into a 1 mL syringe and injected into a mold through the syringe tip. The shear force applied during injection transformed the gel into a low-viscosity injectable fluid. Once the shear force disappeared, the gel rapidly formed in situ. This proves the gel's good injectability and provides feasibility for intratumoral injection.

[0050] Cytotoxicity assay of the composite hydrogel: NIH 3T3 cells were co-cultured with gel extracts of different concentrations. Cell viability was assessed using CCK-8 assay after 24 h and 48 h. Results are shown below. Figure 3 As shown in (a) of the figure, compared with the control group, the cell viability of all experimental groups remained above 80%, indicating that the composite supramolecular hydrogel did not cause significant damage to somatic cells and had good biocompatibility. The viability of 4T1 cells was determined by CCK-8 assay after co-culturing different experimental groups with 4T1 cells for 24 h. The results are as follows: Figure 3As shown in (b), the increase in cytotoxicity of free PCMS NPs to 4T1 cells was limited with increasing light exposure time; the cytotoxicity of the PCMS+PAC group increased significantly; while the Gel@PCMS group showed reduced cytotoxicity compared to the PCMS+PAC group. It is speculated that this is because the hydrogel has a sustained-release effect on CA and PCMS NPs, which can show a significant advantage in long-term treatment by avoiding the rapid metabolism of therapeutic agents.

[0051] In vivo tumor treatment experiment: The average tumor volume in the right axilla was 100 mm. 3 Nude mice with tumors were randomly divided into 5 groups. Each group of mice received an intratumoral injection of 50 μL of a specified solution or hydrogel. On days 1, 3, 5, and 7 post-injection, mice in groups 3 and 5 were subjected to near-infrared light (808 nm, 1 W / cm²). 2 Irradiation for 5 minutes. Thirteen days after treatment, the tumor tissue was removed and photographed. Figure 4 As shown in (a) and (b), the tumors in the control group mice grew rapidly and were not inhibited, with a final volume approximately 16.87 times the initial tumor size. The PCMS+PAC+NIR group, due to rapid dilution in body fluids, only inhibited tumor growth in the early stages of treatment, with a final volume approximately 14.16 times the initial tumor size. Compared to free PCMS and PAC, the Gel@PCMS group showed a sustained and slow release of PCMS and PAC from the gel, resulting in a more significant inhibition of tumor growth, with a final volume approximately 8.47 times the initial tumor size. The Gel@PCMS+NIR group exhibited the best tumor inhibition effect compared to other groups, with the smallest final tumor volume and lightest weight. NIR-triggered photothermal conversion enhanced the catalytic efficiency of PCMS NPs, amplified intracellular oxidative stress levels, induced apoptosis, and achieved a synergistic therapeutic effect of PTT-PDT-CDT.

[0052] Example 2:

[0053] (1) CuCl2·2H2O (0.1705 g, 1 mmol), MnCl2·4H2O (0.1979 g, 1 mmol), and PEI (0.2000 g) were fully dissolved in EG (20 mL) to obtain solution A. (NH4)2S (0.2284 g, 3 mmol) was dissolved in EG (10 mL) to obtain solution B. Then, solution B was added dropwise to solution A, stirred for 60 min, transferred to a stainless steel autoclave, and reacted at 180 °C for 12 h. After cooling to room temperature, the product was collected by centrifugation, washed twice with deionized water and ethanol, and dried to obtain dark green powder PCMS NPs.

[0054] (2) mPEG (4.0 g, 2 mmol) was dissolved in DCM (30 mL), and then succinic anhydride (1.0 g, 10 mmol) and DMAP (0.244 g, 2 mmol) were added to the above solution. The mixture was stirred vigorously for 24 h under N2 protection. After the reaction was completed, the product was precipitated several times in cold diethyl ether. The precipitate was filtered and dissolved in water for dialyzing (dialysis bag molecular weight cutoff: 1000 Da) for 72 h. The precipitate was then freeze-dried to obtain a white solid mPEG-COOH.

[0055] (3) ADH (3.3 g, 18 mmol) was dissolved in deionized water (20 mL), and then CA (0.5 mL, 3 mmol) was slowly added dropwise to the solution. The mixture was stirred at room temperature for 4 h. The precipitate was filtered and washed three times with deionized water and methanol, respectively. The product was collected and dried in a vacuum oven at room temperature to constant weight to obtain a milky white powder, ADH-CA.

[0056] (4) mPEG-COOH (3.6 g, 1.7 mmol), EDC·HCl (0.38 g, 2 mmol), and NHS (0.28 g, 2.5 mmol) were dissolved in DMF (30 mL) and stirred for 2 h under N2 protection. Subsequently, ADH-CA (0.7 g, 2.4 mmol) was dissolved in the above solution, and stirring was continued for 24 h. After the reaction was completed, the mixture was dialyzed (molecular weight cutoff of 1000 Da) for 72 h, and then freeze-dried to obtain a white solid powder mPEG-ADH-CA.

[0057] (5) Dissolve mPEG-ADH-CA (100mg) in tetrahydrofuran (25mL) to prepare a solution. Dialyze the solution with distilled water (dialysis bag molecular weight cutoff: 1000Da) for 72h to completely remove tetrahydrofuran. Change the water 3 times a day to obtain PAC micelles.

[0058] (6) PAC micelles and α-CD were dissolved in deionized water to obtain PAC micelle solution and α-CD solution. PCMS NPs (125 μg / mL) were dispersed in PAC solution (3 wt%) by ultrasonic dispersion, and then mixed with α-CD solution (17 wt%) at a volume ratio of 1:1. The mixture was ultrasonicated for 5 min and allowed to stand at room temperature for a period of time to form a composite hydrogel loaded with copper manganese sulfide nanozyme.

[0059] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for preparing a composite hydrogel encapsulating copper-manganese sulfide nanoszyme, characterized by comprising the following steps: It includes the following steps: ​ (1) Dissolve copper chloride dihydrate, manganese chloride tetrahydrate and polyethyleneimine in ethylene glycol to obtain solution A, dissolve thiourea in ethylene glycol to obtain solution B, then add solution B dropwise to solution A, stir thoroughly and transfer to a stainless steel autoclave, and react at 120-180℃. After cooling to room temperature, collect the product by centrifugation, wash and dry to obtain polyethyleneimine modified copper manganese sulfide nanoparticles. (2) Polyethylene glycol monomethyl ether was dissolved in anhydrous dichloromethane. Succinic anhydride and 4-dimethylaminopyridine were added to the solution and stirred under nitrogen protection. After precipitation, filtration, dialysis and freeze drying, polyethylene glycol monomethyl ether acid was obtained. (3) Dissolve adipic dihydrazide in deionized water, then add cinnamaldehyde dropwise to the solution, stir the reaction at room temperature, filter, precipitate, wash and dry to obtain adipic dihydrazide cinnamaldehyde; (4) The polyethylene glycol monomethyl ether acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in N,N-dimethylformamide and stirred under nitrogen protection. Then, the adipic acid dihydrazide cinnamaldehyde was added to the solution and the reaction continued. After dialysis and freeze drying, the amphiphilic polymer of polyethylene glycol monomethyl ether and cinnamaldehyde was obtained. (5) The amphiphilic polymer of polyethylene glycol monomethyl ether and cinnamaldehyde was dissolved in an organic solvent and dialyzed to obtain self-assembled nanoprodrug micelles. (6) The self-assembled nanoprodrug micelles and α-cyclodextrin are dissolved in deionized water to form a solution. Polyethyleneimine-modified copper manganese sulfide nanoparticles are dispersed in the self-assembled nanoprodrug micelle solution and mixed with the α-cyclodextrin solution. After sonication for 5-10 minutes, the mixture is allowed to stand at room temperature to form a composite hydrogel loaded with copper manganese sulfide nanoenzymes.

2. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of copper chloride dihydrate, manganese chloride tetrahydrate, and thiourea is 1:1:3-2:1:6; and / or, In step (1), the time for stirring until homogeneous is reached is 0.5-1 hour; and / or, In step (1), the reaction time is 6-12 hours; and / or, In step (1), the washing solution used is ethanol and deionized water.

3. The preparation method according to claim 1, characterized in that: In step (2), the molar ratio of polyethylene glycol monomethyl ether, succinic anhydride, and 4-dimethylaminopyridine is 1:5:1 to 1:6:1; and / or, In step (2), the stirring reaction takes 24-48 hours; and / or, In step (2), during precipitation, the precipitant is icy diethyl ether; and / or, In step (2), the dialysis time is 48-72 hours, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis solution is deionized water.

4. The preparation method according to claim 1, characterized in that: In step (3), the molar ratio of adipic acid dihydrazide to cinnamaldehyde is 4:1-6:1; and / or, In step (3), the stirring reaction takes 2-4 hours; and / or, In step (3), the washing solution is deionized water and methanol.

5. The preparation method according to claim 1, characterized in that: In step (4), the molar ratio of polyethylene glycol monomethyl ether acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and adipate dihydrazide cinnamaldehyde is 1.7:2:2.5:2.4; and / or, In step (4), the stirring time under nitrogen is 0.5-2 hours; and / or, In step (4), the continued reaction time is 24-48 hours; and / or, In step (4), the dialysis time is 48-72 hours, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis solution is deionized water.

6. The preparation method according to claim 1, characterized in that: In step (5), the organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide; and / or, In step (5), the ratio of the amphiphilic polymer of polyethylene glycol monomethyl ether and cinnamaldehyde to the organic solvent is (50-100) mg: (5-25) mL; and / or, In step (5), the dialysis time is 48-72 hours, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis solution is deionized water.

7. The preparation method according to claim 1, characterized in that: In step (6), the weight ratio of the self-assembled nanoprodrug micelles, α-cyclodextrin and polyethyleneimine-modified copper manganese sulfide nanoparticles is 30:(140-170):(0.125-0.5).

8. A composite hydrogel encapsulating copper manganese sulfide nanozymes, characterized in that: It is obtained by the preparation method described in any one of claims 1-7.

9. The application of a composite hydrogel containing copper manganese sulfide nanozymes as described in claim 8 in the preparation of delivery materials for tumor therapeutic drugs.

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