A method for preparing and application of copper ion dynamic coordination injectable hydrogel loaded with lactic acid oxidase

Abstract

The application relates to a preparation method and application of a copper ion dynamic coordination lactic acid oxidase loaded injectable hydrogel, and belongs to the technical field of biomedical materials and tumor treatment. A natural biological macromolecule containing a phenolic hydroxyl compound, a solution formed by a low eutectic solvent formed by a hydrogen bond donor and a hydrogen bond acceptor and water, a copper death induction core (Cu 2+ ) and a metabolic regulation and synergistic unit LOx. Cu 2+ Through the cooperation of metal coordination and hydrogen bond, a crosslinked network is formed with the prepared solution, and then LOx is loaded in the hydrogel. For the first time, lactic acid oxidase LOx and copper ions Cu 2+ are integrated in the same injectable hydrogel system to construct a cascade amplification type metabolic reprogramming path. The design not only overcomes the shortcomings of a single copper death inducer, but also fundamentally destroys the metabolic barrier and antioxidant defense of a tumor through LOx driven lactic acid metabolic remodeling, so that the synergy of metabolic intervention and cell death induction is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tumor treatment technology, specifically relating to an injectable hydrogel system based on metabolic regulation. This system achieves highly efficient synergistic treatment of solid tumors such as breast cancer by simultaneously intervening in tumor lactate metabolism and copper death pathway. Background Technology

[0002] Cancer cells often resist traditional apoptosis pathways, posing a significant challenge to effective cancer treatment. Copper death, a novel cell death mechanism independent of classical apoptosis signals, offers a new approach to tumor therapy. This process relies on copper ions and active mitochondrial respiration: copper directly targets esterified proteins in the tricarboxylic acid cycle, inducing their abnormal oligomerization while simultaneously reducing iron-sulfur cluster protein levels, ultimately leading to metabolic collapse and cell death. However, the therapeutic efficacy of copper death is limited by two key factors: first, high intracellular concentrations of glutathione (GSH, 1-10 mM) can effectively chelate free copper ions, significantly weakening their cytotoxicity; second, tumor cells typically rely heavily on glycolysis (the Warburg effect) rather than mitochondrial respiration for energy and biosynthetic precursors, resulting in generally lower sensitivity to copper death.

[0003] Rapidly proliferating tumor cells rely on aerobic glycolysis to meet their energy and biosynthetic needs, leading to a large accumulation of lactate. Studies have shown that lactate is not only a metabolic byproduct but also an important tumor-promoting factor: it creates a favorable microenvironment for tumor growth through multiple mechanisms, including inducing angiogenesis and inhibiting immune cell function. The reduction of pyruvate to lactate catalyzed by LDH is accompanied by the conversion of NADH to NAD. + The transformation helps maintain cytoplasmic NAD. + It supplies and supports the continuous execution of high-throughput glycolysis; at the same time, the lactate / pyruvate ratio can serve as a cytoplasmic NADH / NAD ratio. + Indirect indicators of tumor cell redox regulation demonstrate the crucial role of lactate metabolism. Recent studies have revealed that lactate is not only a key metabolite of glycolysis in tumor cells but also acts as a signaling molecule, influencing the stability or activity of HIF-1α in certain tumors or related cell types, thereby promoting the expression of glycolysis-related genes and contributing to the maintenance of metabolic adaptation. Therefore, targeting the core aspects of lactate metabolism to break the vicious cycle driven by lactate, while simultaneously disrupting tumor redox homeostasis, could be an effective strategy for enhancing copper death sensitivity, potentially leading to a promising new therapeutic paradigm.

[0004] Currently, regulating lactate levels in the tumor microenvironment has become a research hotspot, with main strategies including inhibiting lactate production and secretion and promoting lactate clearance. Among these, enzyme-catalyzed therapies have shown unique advantages. Lactate oxidase (LOx) can directly use lactate accumulated in tumor tissue as a substrate, catalyzing its conversion into pyruvate and generating H2O2. This process gives LOx a unique advantage in lactate metabolism intervention. On the one hand, LOx can directly deplete local lactate, weakening lactate-related metabolic adaptation and immunosuppression; on the other hand, the generated H2O2 can serve as an additional source of oxidative stress, synergistically amplifying the tumor cell damage effect with other treatments. The decrease in lactate levels and the surge in ROS jointly weaken tumor cell metabolic adaptation, forcing a shift in their metabolic patterns. Therefore, combining LOx with metal ions (such as Cu) is a promising approach. 2+ By combining these pathways, a cascade regulatory pathway of "lactic acid metabolism - metal metabolism" can be constructed, providing a new approach to enhancing copper death.

[0005] In recent years, various biomaterials and hydrogel systems have been developed for copper death therapy. While systemically administered nanomaterials possess some tumor-targeting capabilities, studies have shown that only a small number of nanoparticles accumulate in tumor tissue after intravenous injection, and off-target effects may pose toxic risks to normal tissues. In contrast, injectable hydrogels have attracted significant attention due to their unique local delivery advantages. These materials achieve controlled and sustained drug release through their three-dimensional network structure, maintaining effective therapeutic concentrations at the lesion site while significantly reducing the toxic side effects of systemic exposure. In particular, their minimally invasive injection and local retention characteristics provide an ideal platform for personalized tumor treatment. Currently, hydrogels based on natural polymers such as hyaluronic acid, alginate, and chitosan have become important drug carriers, showing great promise in local tumor treatment. However, existing hydrogel systems still have limitations in achieving drug spatial gradient and release kinetic regulation, lacking a comprehensive biomimetic delivery system capable of simultaneously regulating lactate metabolism, enhancing copper death sensitivity, and achieving sustained local treatment. Therefore, developing a multifunctional hydrogel system that can respond to the tumor microenvironment and integrate metabolic regulation and metal death induction functions has become the key to improving the efficacy of tumor treatment. Summary of the Invention

[0006] Based on the aforementioned research background, this invention successfully constructed an injectable hydrogel system for synergistic therapy. This system significantly enhances the copper death effect by simultaneously disrupting the antioxidant defense mechanisms of tumor cells and inhibiting the glycolytic metabolic pathway, and synergizes with the apoptosis pathway, providing a new approach for achieving highly efficient anti-tumor therapy.

[0007] The above-mentioned method for preparing an injectable hydrogel system based on lactate metabolism regulation to sensitize copper death includes the following steps:

[0008] (1) The EDC / NHS activation coupling method was used to condense the carboxyl group (-COOH) of natural biomacromolecules with the amino group (-NH2) on the compound containing amino and hydroxyl groups to form an amide bond (-CONH-), thereby synthesizing a covalent polymer. After purifying the obtained solution, it was pre-frozen at -80℃ to allow it to solidify completely. Then, the pre-frozen solidified sample was quickly transferred to a freeze dryer and freeze-dried according to the preset program to obtain a white spongy solid, which was then sealed and stored in a dry environment.

[0009] (2) Weigh the hydrogen bond donor and hydrogen bond acceptor according to a certain molar ratio, and mix them initially at room temperature by magnetic stirring to form a uniform suspension; then place the mixture in an oil bath, and continue to heat and stir under stirring until the system becomes a completely transparent colorless viscous liquid, thus obtaining the eutectic solvent DES.

[0010] (3) Prepare a DES / water mixed solvent according to a certain volume ratio; weigh the freeze-dried powder obtained in step (1), and gradually add it to the DES / water mixed solvent under stirring conditions. After the addition is completed, continue stirring under light-protected conditions for 2 to 4 hours until the freeze-dried powder is completely dissolved and a homogeneous solution is obtained.

[0011] (4) Take the lyophilized lactate oxidase (LOx) powder and prepare a lactate oxidase LOx solution with PBS buffer pre-cooled to 4°C. Mix it initially by gently vortexing and let it stand on ice for 30 minutes to fully dissolve it. Transfer the dissolved LOx solution to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze it in PBS buffer at 4°C for 12 hours. Change the dialysate 3 times during the process to remove small molecule impurities and salts.

[0012] (5) Mix the LOx solution treated in step (4) with the HD solution prepared in step (3) in a predetermined ratio, incubate at 37°C for 15 minutes, and then add Cu-containing compounds to the resulting mixture. 2+ The solution induces rapid gelation, and after standing and solidification, a composite hydrogel loaded with lactate oxidase is obtained.

[0013] This invention provides an injectable hydrogel system for enhancing copper death based on lactate metabolism regulation. The system comprises: a natural biomacromolecule modified with a phenolic hydroxyl compound; a solution formed by a eutectic solvent (composed of hydrogen bond donors and acceptors) and water; and a copper death-inducing core (Cu). 2+ ) and metabolic regulation and enhancement units (LOx). Among them, Cu 2+ Through the combined effects of metal coordination and hydrogen bonding, a cross-linked network is formed with the prepared solution, and LOx is subsequently loaded into the hydrogel.

[0014] In further step (1), the natural biomolecule is one or more of gelatin, hyaluronic acid, chitosan, sodium alginate, and silk protein; preferably, the selected biomolecule is hyaluronic acid.

[0015] Further, the amino and hydroxyl-containing compound in step (1) is one or more of dopamine hydrochloride and tannic acid; preferably, the selected compound is dopamine hydrochloride.

[0016] Further, in step (1), the activation reaction time of the carboxyl group in the EDC / NHS activation coupling method is in the range of 15 min-1 h, and the reaction temperature is in the range of 20-25 °C; preferably, the reaction temperature is 25 °C and the reaction time is 1 h.

[0017] Further, in step (1), the coupling reaction time in the EDC / NHS activation coupling method is in the range of 2-4h, and the reaction temperature is in the range of 20-25℃; preferably, the reaction temperature is 25℃ and the reaction time is 2h.

[0018] Further, in step (1), the molar ratio of EDC: NHS: carboxyl substrate in the EDC / NHS activation coupling method is in the range of (2.5 - 5): (2.5 - 5): 1; preferably, the selected ratio is EDC: NHS: carboxyl substrate molar ratio = 2.5: 2.5: 1.

[0019] Further, in step (2), the hydrogen bond donor is a molecule containing functional groups such as amide group, carboxylic acid group or polyol, which can provide hydrogen atoms to participate in the formation of hydrogen bonds; preferably, the selected hydrogen bond donor is glycerol.

[0020] Further, the hydrogen bond acceptor in step (2) is one or more of choline, quaternary ammonium salt, betaine and metal chloride; preferably, the selected hydrogen bond acceptor is choline chloride.

[0021] Further, in step (2), the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is in the range of 1-5:1; preferably, the selected molar ratio is 1:1.

[0022] The reaction temperature range of the eutectic solvent formed in further step (2) is 60-100℃, and the reaction time range is 0.5-4 h; preferably, the reaction temperature of the eutectic solvent is 90℃, and the reaction time is 0.5h.

[0023] Further, in step (3), the volume ratio of the eutectic solvent to water in the eutectic solvent aqueous solution is in the range of 1-2:1-2; preferably, the volume ratio between the selected eutectic solvent and water is 1.5:1;

[0024] Further step (3) involves 0.5-10 ml of DES / water mixed solvent for every 50 mg of HD lyophilized powder; preferably, 1 ml of DES / water mixed solvent for every 50 mg of HD lyophilized powder.

[0025] Further, in step (4), the concentration of the lactate oxidase (LOx) solution is 10 mg / mL; after dialysis, the protein concentration of the obtained LOx solution is determined by the standard curve method using bovine serum albumin (BSA) as a standard.

[0026] Further step (5) Cu 2+ It can be provided by copper sulfate solution or copper chloride solution; preferably, copper chloride solution is used.

[0027] Further step (5) involves Cu-containing... 2+ Cu in solution 2+ The mass percentage is 2%-20%; preferably, a copper chloride solution with a mass percentage of 10% is used.

[0028] Further, in step (5), the volume ratio of HD solution to LOx solution is in the range of 100:1-5; preferably, the ratio of HD solution (μL): LOx solution (μL) is 100:1.

[0029] Further step (5) involves the HD solution and Cu 2+ The volume ratio of the solution is in the range of 100:1-5; preferably, HD solution (μL): Cu 2+ The solution (μL) ratio is 100:1.

[0030] The hydrogel system obtained in this invention is used to prepare a drug for treating tumors. This drug is an injectable drug that is injected locally into the tumor.

[0031] In summary, the technical solutions conceived by this invention have the following advantages compared with the prior art:

[0032] (1) Innovative dual intervention mechanism of synergistic metabolism-copper death pathway

[0033] Traditional copper death therapy often relies on a single copper ion carrier, which is susceptible to the clearance effect of high concentrations of glutathione (GSH) in the tumor microenvironment and the dominance of glycolytic metabolism, thus limiting its efficacy. This invention innovatively combines lactate oxidase (LOx) with copper ions (Cu). 2+ Integrating these components into a single injectable hydrogel system, a cascaded large-scale metabolic reprogramming pathway was constructed. This design not only overcomes the shortcomings of single copper death inducers but also fundamentally disrupts the metabolic barrier and antioxidant defense of tumors through LOx-driven lactate metabolic remodeling, achieving a synergistic effect of metabolic intervention and cell death induction.

[0034] (2) Precise and controllable local drug delivery and long-lasting sustained release performance

[0035] Compared to the problems of low targeting efficiency and high off-target toxicity inherent in systemic drug delivery nanosystems, the injectable hydrogel platform used in this invention exhibits excellent local retention and biocompatibility. Its three-dimensional network structure can achieve Cu… 2+ With its long-lasting and controllable release of LOx, it maintains high concentrations of therapeutic drugs at the tumor site while minimizing systemic exposure to normal tissues. The hydrogel system's shear-thinning and self-healing properties allow for easy injection via fine needles and the formation of stable gels in situ. This facilitates convenient clinical operation, ensures high compliance, and provides an ideal dosage form for precise local treatment of solid tumors.

[0036] (3) Good biosafety and promising clinical translation prospects

[0037] Comprehensive in vitro and in vivo safety assessments showed that the hydrogel system, at effective therapeutic doses, did not cause significant pathological damage to major organs (heart, liver, spleen, lungs, and kidneys), and mouse weight remained stable, demonstrating good biocompatibility. Its raw materials are readily available, relatively cost-effective, and the preparation process is simple and reproducible. These characteristics collectively endow this invention with high clinical translational potential and market application value. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation process of the injectable hydrogel system of the present invention.

[0039] Figure 2 This is a physical image of the injectable hydrogel prepared according to the present invention.

[0040] Figure 3 This is a scanning electron microscope image of the injectable hydrogel of the present invention.

[0041] Figure 4 This invention relates to the rheological properties and injectability properties of the injectable hydrogel.

[0042] Figure 5 This is a flow cytometry analysis of the apoptosis-inducing effect of the injectable hydrogel of the present invention on 4T1 cells.

[0043] Figure 6 This invention provides fluorescence microscopy images and flow cytometry quantitative analysis of ROS generated in 4T1 cells after treatment with injectable hydrogel.

[0044] Figure 7 The results show the lactate and pyruvate levels in the culture medium of 4T1 cells after treatment with the injectable hydrogel of this invention.

[0045] Figure 8This is the result of detecting energy metabolism in 4T1 cells using the injectable hydrogel of this invention.

[0046] Figure 9 These are the results of immunofluorescence and Western blot analysis of copper death induced by the injectable hydrogel of this invention.

[0047] Figure 10 To demonstrate the antitumor effect of the hydrogel of this invention in 4T1 tumor-bearing mice: tumor growth curve, in vitro tumor photographs, mouse weight changes, and tumor quality statistics.

[0048] Figure 11 ROS staining of tumor tissue after hydrogel treatment according to the present invention.

[0049] Figure 12 This invention relates to the changes in lactic acid, GSH, and ATP levels in tumor tissue after hydrogel treatment.

[0050] Figure 13 The results show the staining of glycolysis-related proteins in tumor tissue after hydrogel treatment according to the present invention.

[0051] Figure 14 The results of copper death-related protein staining in tumor tissue after hydrogel treatment according to the present invention.

[0052] Figure 15 HE staining results of major organs (heart, liver, spleen, lung, and kidney) in mice after hydrogel treatment according to this invention. Detailed Implementation

[0053] The following description, in conjunction with specific implementation examples and accompanying drawings, further illustrates the present invention, but it is not limited to the following embodiments.

[0054] Example 1

[0055] This embodiment provides a method for preparing an injectable hydrogel based on lactate metabolism regulation to enhance copper death, including:

[0056] 1. Sodium hyaluronate was dissolved in buffer solution, and its carboxyl groups on the molecular chain were activated using an EDC / NHS activation coupling method, wherein the molar ratio of carboxyl groups, EDC, and NHS was 1:2.5:2.5. Subsequently, dopamine hydrochloride was added, causing the activated carboxyl groups to undergo a condensation reaction with the amino groups on the dopamine hydrochloride molecule to form amide bonds, thus preparing a hyaluronic acid-dopamine covalent polymer (HD). After the reaction, the resulting HD solution was dialyzed, and after dialysis, it was pre-frozen at -80°C for 12 hours to allow it to completely solidify, obtaining a white, spongy HD solid, which was then sealed and stored in a dry environment.

[0057] 2. Weigh choline chloride (ChCl) and glycerol (Gly) in a molar ratio of 1:2 and place them in a round-bottom flask. Mix them initially at room temperature using magnetic stirring to form a homogeneous suspension. Then, place the mixture in a 90°C oil bath and heat and stir continuously at 300 rpm until the system becomes a completely transparent, colorless, viscous liquid, thus obtaining the eutectic solvent (DES).

[0058] 3. Prepare a DES / water mixed solvent at a volume ratio of 1.5:1.

[0059] 4. Accurately weigh 50 mg of the HD lyophilized powder obtained in step 1, and gradually add it to 1 mL of a DES / water (1.5:1) mixed solvent under magnetic stirring (300 rpm, 25℃). After the addition is complete, continue stirring under light-protected conditions for 2 to 4 hours until HD is completely dissolved, obtaining a homogeneous solution with a concentration of HD.

[0060] 5. Take the lyophilized lactate oxidase (LOx) powder and prepare a 10 mg / mL solution with pre-cooled PBS buffer (4℃). Gently vortex to initially mix the LOx and incubate on ice for 30 minutes to allow it to fully dissolve. Transfer the dissolved LOx solution to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in PBS buffer at 4℃ for 12 hours, changing the dialysate three times to remove small molecule impurities and salts. After dialysis, determine the protein concentration of the obtained LOx solution using a standard curve method with bovine serum albumin (BSA) as the standard.

[0061] 6. Mix 5 μL of the LOx solution treated in step 5 with 500 μL of the HD solution prepared in step 4, and incubate at 37°C for 15 minutes. Then add 5 μL of 10% CuCl2 solution to the mixture to initiate rapid gelation. After static curing, the HD-Cu / LOx composite hydrogel loaded with lactate oxidase is obtained. Figure 1 , Figure 2 )

[0062] The structure of the resulting injectable hydrogel was characterized. Scanning electron microscopy revealed that the HD-Cu / LOx gel exhibited a loose porous network structure; energy dispersive spectroscopy analysis showed that Cu elements were uniformly distributed in the gel, indicating that copper ions successfully participated in the construction of the gel network. Figure 3 Further rheological property tests were conducted on the hydrogel. The results showed that, under the test conditions, the storage modulus G' was higher than the loss modulus G'', indicating that it has a stable gel network structure. Figure 4 (A) In high and low strain cycle tests, the mechanical properties of the gel can recover relatively quickly after strain switching, indicating that it has a certain self-healing ability. Figure 4(B) Injection experiments show that the overall injection force of this gel is in the range of 9-12N, with suitable injection resistance, which can meet the basic requirements for ease of operation and safety during injection. Figure 4 (C)

[0063] Example 2: In vitro antitumor effect

[0064] The cells used in Example 2 were 4T1 (mouse breast cancer cell line) purchased from Beina Biotechnology. These cells were cultured in DMEM medium according to the ATCC cell culture protocol, containing 1% penicillin-streptomycin antibiotic solution and 10% fetal bovine serum. The cell culture incubator was humidified, containing 5% carbon dioxide, and the culture temperature was 37°C.

[0065] 1. Apoptosis detection: Cells were divided into PBS, HD, LOx, HD-Cu Gel, and HD-Cu / LOx gel groups. After 12 h of treatment, Annexin V-FITC / PI double staining was used for detection. Results are as follows: Figure 5 As shown: the apoptosis rate in the PBS group was only about 7%, while the total apoptosis rate in the HD-Cu / LOx Gel group was as high as 95.45%, which was higher than that in other treatment groups. Figure 5 ).

[0066] 2. Reactive oxygen species (ROS) detection: After 6 hours of treatment in each group, the ROS were detected using the DCFH-DA probe. Results are as follows: Figure 6 As shown in the fluorescence images, the green fluorescence of HD-Cu / LOx Gel was significantly enhanced; FCM results indicated that the green fluorescence intensity of cells in the HD-Cu / LOx Gel group was approximately 6.7 times that of the HD-Cu / LOx Gel group, suggesting that the prepared hydrogel could significantly promote the accumulation of ROS in tumor cells, implying that it could disrupt intracellular redox homeostasis by enhancing oxidative stress. Figure 6 ).

[0067] 3. Glycolysis metabolism assay: Cell culture media from each group were collected, and lactic acid and pyruvate levels were measured. Results showed that lactic acid concentrations in the free LOx group and the HD-Cu / LOx Gel group decreased to 67% and 70% of the control group, respectively, while pyruvate concentrations increased to 1.75-fold and 1.83-fold, respectively. Figure 7 Seahorse metabolic analysis showed that HD-Cu / LOx Gel treatment significantly reduced extracellular acidification rate (ECAR) and OCR value.

[0068] 4. Detection of copper death-related proteins: After 24 h of treatment, immunofluorescence showed that DLAT in the HD-Cu / LOx Gel group exhibited significant green fluorescence accumulation around the cell nucleus, while the fluorescence intensity of FDX1 was significantly reduced. Western blot ( Figure 8The results showed that the expression level of DLAT protein in the HD-Cu / LOx Gel group was approximately 60% of that in the control group, while the expression level of FDX1 protein decreased to 16.7% of that in the control group, exhibiting typical characteristics of copper death molecules. Figure 9 ).

[0069] Example 3: In vivo antitumor experiment

[0070] In this embodiment, female Balb / c mice (6 weeks old, 16-19 g) were subcutaneously injected with 4T1 cells (1×10⁻⁶) on the right side of their backs. 6 A subcutaneous breast cancer tumor model was established. When the tumor volume reached 80-100 mm³, mice were randomly divided into 5 groups (n=5): PBS group, HD group, LOx group, HD-Cu Gel group, and HD-Cu / LOx Gel group. Administered intratumorally on days 1, 3, 5, and 7, with each injection volume being 25 μL. The Cu dose was 1.25 mg / kg, and the LOx dose was 0.5 mg / kg. Tumor volume and mouse weight were measured every two days during treatment.

[0071] (1) Tumor growth inhibition: such as Figure 10 As shown, after 14 days of treatment (with no further medication administered for the last 7 days), the average tumor volume in the PBS group was approximately 800 mm. 3 The HD-Cu Gel group is 415 mm. 3 The HD-Cu / LOx Gel group is only 288 mm. 3 (P<0.0001). Ex vivo tumor weighing showed that the HD-Cu / LOx Gel group had the lowest average tumor mass. There were no significant changes in mouse body weight during treatment, indicating no significant systemic toxicity.

[0072] (2) Metabolic indicators of tumor tissue: DHE staining showed that the ROS signal was strongest in the HD-Cu / LOx Gel group ( Figure 11 Biochemical tests showed that GSH and lactate levels were significantly decreased in this group, while ATP levels increased by approximately 1.9 times. Figure 12 ).

[0073] (3) Glycolysis and expression of copper death proteins: Immunohistochemistry and immunofluorescence showed that the expression of PKM2 and HIF-1α in tumor tissues of the HD-Cu / LOx Gel group was significantly downregulated. Figure 13 FDX1 immunofluorescence showed that its expression decreased to 22% in the PBS group, and DLAT immunohistochemistry showed significant oligomerization, demonstrating that it can effectively activate the copper death pathway in vivo. Figure 14 ).

[0074] (4) Biosafety evaluation: HE staining of major organs (heart, liver, spleen, lung, kidney) of mice after treatment ( Figure 15The results showed that no significant pathological abnormalities were observed in any of the drug administration groups compared to the PBS group, indicating that the hydrogel system has good tissue compatibility and in vivo safety.

Claims

1. A method for preparing an injectable hydrogel system based on lactate metabolism regulation to sensitize copper death, characterized in that, Includes the following steps: (1) The EDC / NHS activation coupling method was used to condense the carboxyl group (-COOH) of natural biomacromolecules with the amino group (-NH2) on the compound containing amino and hydroxyl groups to form an amide bond (-CONH-), thereby synthesizing a covalent polymer; after purifying the obtained solution, it was pre-frozen at -80℃ to allow it to solidify completely; then the pre-frozen solidified sample was quickly transferred to a freeze dryer and freeze-dried according to the preset program to obtain a white spongy solid, which was then sealed and stored in a dry environment; (2) Weigh the hydrogen bond donor and hydrogen bond acceptor according to a certain molar ratio, and mix them initially at room temperature by magnetic stirring to form a uniform suspension; then place the mixture in an oil bath, and continue to heat and stir under stirring until the system becomes a completely transparent colorless viscous liquid, thus obtaining the eutectic solvent DES. (3) Prepare a DES / water mixed solvent according to a certain volume ratio; weigh the freeze-dried powder obtained in step (1), and gradually add it to the DES / water mixed solvent under stirring conditions. After the addition is completed, continue stirring under light-protected conditions for 2 to 4 hours until the freeze-dried powder is completely dissolved and a homogeneous solution is obtained. (4) Take the lyophilized lactate oxidase (LOx) powder and prepare a lactate oxidase LOx solution with PBS buffer pre-cooled to 4°C. Mix it initially by gently vortexing and let it stand on ice for 30 minutes to fully dissolve it. Transfer the dissolved LOx solution to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze it in PBS buffer at 4°C for 12 hours. Change the dialysate 3 times during the process to remove small molecule impurities and salts. (5) The LOx solution treated in step (4) and the eutectic solvent DES prepared in step (2) are mixed uniformly in a predetermined ratio and incubated at 37°C for 15 minutes. Then, Cu-containing solvents are added to the resulting mixture. 2+ The solution induces rapid gelation, and after standing and solidification, a composite hydrogel loaded with lactate oxidase is obtained.

2. The method according to claim 1, characterized in that, The natural biomolecules mentioned in step (1) are one or more of gelatin, hyaluronic acid, chitosan, sodium alginate, and silk protein; The amino and hydroxyl-containing compound in step (1) is one or more of dopamine hydrochloride and tannic acid; In step (1), the activation reaction time for the carboxyl group in the EDC / NHS activation coupling method is 15 min to 1 h, and the reaction temperature is 20 to 25 °C; preferably, the reaction temperature is 25 °C and the reaction time is 1 h. Further, in step (1), the coupling reaction time in the EDC / NHS activation coupling method ranges from 2 to 4 hours, and the reaction temperature ranges from 20 to 25°C; preferably, the reaction temperature is 25°C and the reaction time is 2 hours. In step (1), the molar ratio of EDC: NHS: carboxyl substrate in the EDC / NHS activation coupling method is in the range of (2.5 - 5): (2.5 - 5): 1; preferably, the selected ratio is EDC: NHS: carboxyl substrate molar ratio = 2.5: 2.5:

1.

3. The method according to claim 1, characterized in that, The hydrogen bond donor in step (2) is a molecule containing functional groups such as amide group, carboxylic acid group or polyol, which can provide hydrogen atoms to participate in the formation of hydrogen bonds; preferably, the selected hydrogen bond donor is glycerol; The hydrogen bond acceptor in step (2) is one or more of choline, quaternary ammonium salt, betaine and metal chloride; preferably, the selected hydrogen bond acceptor is choline chloride. The molar ratio of hydrogen bond donor to hydrogen bond acceptor in step (2) is in the range of 1-5:1; preferably, the selected molar ratio is 1:

1. The reaction temperature range of the eutectic solvent formed in step (2) is 60-100℃, and the reaction time range is 0.5-4 h; preferably, the reaction temperature of the eutectic solvent is 80℃, and the reaction time is 0.5h.

4. The method according to claim 1, characterized in that, In step (3), the volume ratio of the eutectic solvent to water in the aqueous solution is in the range of 1-2:1-2; preferably, the volume ratio between the selected eutectic solvent and water is 1.5:

1. Step (3) 0.5-10 ml of DES / water mixed solvent is used for every 50 mg of HD lyophilized powder; preferably, 1 ml of DES / water mixed solvent is used for every 50 mg of HD lyophilized powder.

5. The method according to claim 1, characterized in that, The concentration of the lactate oxidase (LOx) solution in step (4) is 10 mg / mL; after dialysis, the protein concentration of the obtained LOx solution is determined by the standard curve method using bovine serum albumin (BSA) as a standard.

6. The method according to claim 1, characterized in that, Step (5) Cu 2+ It can be provided by copper sulfate solution or copper chloride solution; Step (5) contains Cu 2+ Cu in solution 2+ The mass percentage is 2%-20%; preferably, a copper chloride solution with a mass percentage of 10% is used. The eutectic solvent DES (mg):LOx (U) in step (5) is in the range of 1:10-100; preferably, DES (mg):LOx (U) is 1:

80.

7. The eutectic solvent DES (mg) mentioned in step (5): Cu 2+ (mg) range: 1:10-200; preferred, eutectic solvent DES (mg): Cu 2+ The concentration (mg) is 1:

50.

8. The hydrogel system prepared according to any one of claims 1-6.

9. The application of the hydrogel system prepared according to any one of claims 1-6 for the preparation of drugs for treating antitumor diseases.

10. The application according to claim 8, wherein the drug is an injectable drug for injection into the local area corresponding to the tumor.

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