Preparation method and application of multifunctional bimetallic nano-enzyme

Abstract

The invention provides a preparation method and application of a multifunctional bimetallic nano enzyme, and belongs to the technical field of inorganic nano therapeutic agents. The preparation method comprises the following steps: performing hydrothermal reaction on tyrosine, ferric trichloride, cobalt nitrate and sodium hydroxide to synthesize CoFe2O4; the method comprises the following steps: adding a dimethyl sulfoxide solution of mPEG into pure polylipoic acid, dialyzing, and freeze-drying to obtain PALA-mPEG; adding a dimethyl sulfoxide solution of JUG into the PALA-mPEG, carrying out dialysis, and carrying out freeze-drying, so as to obtain PALA-mPEG-JUG; the preparation method comprises the following steps: dispersing CoFe2O4 in deionized water, adding PALA-mPEG-JUG, and carrying out a reaction so as to obtain CFO-coated PPJ; the CFO coated PPJ prepared by the invention is a tumor microenvironment self-adaptive material combining a modified polymer chain and a bimetallic nano-enzyme, and can be used in the fields of enhanced chemical kinetics therapy, multifunctional antitumor agents, diagnosis and treatment integration and the like.

Description

Technical Field

[0001] The present application relates to a preparation method and application of a multifunctional bimetallic nanozyme, and belongs to the technical field of inorganic nanotherapeutic agents. Background Art

[0002] Reactive oxygen species (ROS) have significant biological activity and the ability to induce intracellular oxidative stress, which makes them a powerful tool for tumor treatment. Several treatments have been developed based on ROS, including photodynamic therapy (PDT), sonodynamic therapy (SDT), chemodynamic therapy (CDT), etc. Among them, unlike other dynamic therapies, CDT relies on Fenton chemistry to inhibit tumor growth by converting endogenous hydrogen peroxide (H2O2) in the TME into highly cytotoxic hydroxyl radicals (⋅OH), allowing it to operate independently of external stimuli (such as light, sound, and electricity) and does not rely on a continuous oxygen supply. As the most active ROS, ⋅OH has the inherent ability to cause oxidative damage to cancer cells. These characteristics of CDT enable it to overcome and penetrate the challenges associated with deep and hypoxic microenvironments, making it a promising approach for cancer treatment.

[0003] To mitigate the harmful consequences of excessive ROS, cancer cells upregulate the adaptive antioxidant defense system (ADS) for self-protection. By maintaining redox homeostasis, cancer cells are able to limit ROS to a comfortable range, thereby promoting cancer cell proliferation and differentiation. Glutathione (GSH), which is highly expressed in the TME as the main antioxidant, significantly enhances the tolerance of tumor cells to oxidative stress, thereby weakening the therapeutic effect of CDT. Therefore, achieving GSH consumption may become a key direction to improve the therapeutic effect.

[0004] Alpha lipoic acid (LA or ALA) is a sulfur-containing fatty acid naturally produced by plant and animal cells. When LA is exposed to high melting temperatures, the autonomous and dynamic exchange of disulfide bonds in its five-membered ring can form linear covalent polymer polylipoic acid chains (PALA) with disulfide bonds. Although PALA has excellent biological activity, its hydrophobicity makes it unable to dissolve and release in water, which limits the construction of its aqueous system and the full utilization of its biological activity. Therefore, it remains a challenge to construct an aqueous PALA system and fully utilize the biological activity of LA-based small molecules. In addition, due to the limited supply of endogenous H2O2, it is difficult to maintain a sustained Fenton reaction as the sole substrate for chemodynamic therapy (CDT). This limitation significantly restricts the continuous generation of highly toxic hydroxyl radicals (•OH), thereby reducing the oxidative damage effect of CDT on cancer cells. To overcome this limitation, supplementing exogenous H2O2 can effectively increase the generation of •OH, thereby enhancing the therapeutic effect. Juglone (5-hydroxy-1,4-naphthoquinone, JUG), a natural phenolic compound isolated from Juglans mandshurica, has been considered as a promising anticancer drug candidate. Previous studies have shown that the anticancer effect of juglone is closely related to its induced upregulation of intracellular H2O2 levels. This mechanism not only effectively increases intracellular oxidative stress, but also provides a persistent substrate for the Fenton reaction, thereby enhancing the generation of •OH, making it exhibit a unique synergistic effect in CDT. Therefore, the application of juglone is expected to play an important role in enhancing the therapeutic effect of CDT and provide new ideas for the development of CDT catalytic systems with self-supply of H2O2. However, the poor solubility of juglone in aqueous solution and the existing nano drug delivery systems with complex synthetic routes and low drug loading have considerably limited its further application. Summary of the invention

[0005] In view of this, the present application first provides a method for preparing a multifunctional bimetallic nanozyme, wherein the multifunctional bimetallic nanozyme prepared by the method has both glutathione (GSH) consumption ability and H2O2 generation ability.

[0006] Specifically, the present application is implemented through the following scheme: A method for preparing a multifunctional bimetallic nanozyme, comprising the following steps: Step 1: After tyrosine, ferric chloride, cobalt nitrate and sodium hydroxide are mixed, the mixture is synthesized by hydrothermal reaction, washed and dried at room temperature to obtain iron cobalt acid nanozyme, which is recorded as CoFe2O4; Step 2: polylipoic acid (PALA) is dissolved in N,N-dimethylformamide (DMF), and monomethyl polyethylene glycol (mPEG), an activator and a catalyst dissolved in dimethyl sulfoxide (DMSO) are added, stirred, dialyzed with deionized water, and freeze-dried to obtain a polylipoic acid chain modified with monomethyl polyethylene glycol, which is recorded as PALA-mPEG; Step 3, dissolving pure PALA-mPEG in N,N-dimethylformamide (DMF), adding juglone (JUG), coupling agent and catalyst dissolved in DMSO, stirring, dialyzing with deionized water, and freeze-drying to obtain JUG-modified water-soluble polylipoic acid chains, recorded as PALA-mPEG-JUG; Step 4: Disperse the CoFe2O4 obtained in step 1 in deionized water, add the PALA-mPEG-JUG obtained in step 3, stir the reaction, centrifuge and wash, and collect the precipitate to obtain a multifunctional bimetallic nanozyme of iron cobalt acid nanozyme coated with a modified polylipoic acid chain, recorded as CFO@PPJ.

[0007] In the above scheme, iron cobalt acid bimetallic nanozymes were synthesized by hydrothermal reaction, and then the modified polylipoic acid chain was wrapped on the surface of the nanozyme. Not only the nanozyme is endowed with disulfide bonds for the consumption of GSH and the grafted JUG for the generation of intracellular H2O2, but also the material is endowed with high biocompatibility and stability. The above structural characteristics realize the construction of bimetallic nanozymes with tumor microenvironment adaptation, so that these nanoparticles can consume reducing substances in tumors while efficiently generating ROS, breaking the redox balance in tumor cells and inducing oxidative stress, so that it can be used for enhanced tumor chemokinetic therapy.

[0008] Further, as a preference: In step one, The molar ratio of tyrosine, ferric chloride, cobalt nitrate and sodium hydroxide is 1-6:1:1:9.

[0009] More preferably, ethylene glycol is further added to form an ethanol solution of tyrosine, ferric chloride, cobalt nitrate and sodium hydroxide, and then a hydrothermal reaction is carried out.

[0010] In step 2, The polylipoic acid is prepared by the following method: alpha lipoic acid is polymerized by heating to obtain polylipoic acid (PALA), PALA is dissolved in tetrahydrofuran (THF) and purified by ether precipitation method, and then dried at room temperature to obtain pure polylipoic acid.

[0011] Preferably, the reaction temperature of the thermal polymerization is 90-150° C., and the reaction time is 30-100 min.

[0012] The molar mass ratio of polylipoic acid, monomethyl polyethylene glycol, activator and catalyst is 1:3~6:0.36:0.03.

[0013] The activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL).

[0014] The catalyst is 4-dimethylaminopyridine (DMAP).

[0015] In step three, The molar ratio of PALA-mPEG, juglone, coupling agent, and catalyst is 1:2~4:0.36:0.03.

[0016] The coupling agent is N,N'-diisopropylcarbodiimide (DIC).

[0017] The catalyst is 4-dimethylaminopyridine (DMAP).

[0018] In step four, The mass ratio of CoFe2O4 to PALA-mPEG-JUG is 1:0.5-2.0. More preferably, the mass ratio of CoFe2O4 to PALA-mPEG-JUG is 1:0.9-1.2.

[0019] The above scheme successfully constructed a multifunctional bimetallic nanozyme (CFO@PPJ) based on GSH consumption and H2O2 generation. This multifunctional bimetallic nanozyme not only has good biocompatibility and excellent ROS generation ability, but also has good adaptability to the complex tumor microenvironment. More importantly, the inherent superparamagnetism of CFO@PPJ can realize the tracing of nanozymes and real-time monitoring of tumor sites. In the tumor microenvironment, the disulfide bonds and JUG on the outer polymer chains can induce oxidative stress, break the redox balance in tumor tissues, and accelerate the apoptosis of tumor cells. This has a very high reference value for the biomedical application of inorganic diagnosis and treatment integrated composite systems and the design and development of efficient tumor chemodynamic therapy materials.

[0020] Therefore, the second purpose of the applicant is to provide an application of the above-mentioned multifunctional bimetallic nanozyme. Based on the GSH consumption and ROS generation ability of the multifunctional bimetallic nanozyme prepared by the above-mentioned method, it can be used to prepare anti-tumor nanomaterials, such as: anti-tumor nanomaterials, enhanced chemodynamic therapy materials, multifunctional anti-tumor agents, etc.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) A multifunctional bimetallic nanozyme CFO@PPJ prepared by the present invention has high GSH consumption capacity, significant H2O2 generation capacity, and excellent ·OH generation capacity, and has good adaptability to the complex tumor microenvironment.

[0022] (2) The multifunctional bimetallic nanozyme CFO@PPJ, in vitro and in vivo experiments showed that the composite material achieved GSH consumption and H2O2 generation in tumor tissues, had a certain killing effect on tumor cells, and had a relatively significant ability to inhibit tumor growth.

[0023] (3) The multifunctional bimetallic nanozyme CFO@PPJ exhibited a significant ability to generate ·OH in vitro, and the generated ROS had a high killing efficiency against tumor cells.

[0024] (4) The multifunctional bimetallic nanozyme CFO@PPJ prepared by the present invention consumes GSH in the tumor microenvironment and promotes the generation of intracellular H2O2 through the released JUG, significantly enhancing the generation of ROS to further kill tumor cells, inducing oxidative stress in tumor cells, and achieving efficient tumor treatment by breaking the redox homeostasis.

[0025] (5) The present invention has low requirements for experimental instruments, the method is simple and easy to operate, and the obtained nanocomposite material has excellent performance in biocompatibility, ROS generation performance and anti-tumor performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a transmission electron microscope image of CFO@PPJ obtained in Example 1; Figure 2 This is the UV-visible spectrum of the product CFO@PPJ obtained in Example 1; Figure 3 is the potential diagram of the product CFO@PPJ obtained in Example 1; Figure 4 is the XRD curve of the product CFO@PPJ obtained in Example 1; Figure 5 This is the XPS spectrum of the product CFO@PPJ obtained in Example 1; Figure 6 This is the ·OH generation curve of the product CFO@PPJ obtained in Example 1; Figure 7 This is the GSH consumption curve of the product CFO@PPJ obtained in Example 1; Figure 8 Confocal images of intracellular H2O2 of the product CFO@PPJ obtained in Example 1 were generated; Fig. 9 Cytotoxicity analysis of the product CFO@PPJ obtained in Example 1 co-cultured in L929 and HUVEC cells; Fig.10 This is the MRI imaging picture of the product CFO@PPJ obtained in Example 1 in mice; Fig.11 The anti-tumor experimental results of the product CFO@PPJ obtained in Example 1 on CT26. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below in conjunction with the drawings in the embodiments of this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit the technical solutions of this application. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0029] It should be noted that the following examples provide those of ordinary skill in the art with information on how to make and evaluate the present invention, and the examples are merely exemplary of the present disclosure and do not define the scope of limitation. Although every effort has been made to ensure the accuracy of numerical values ​​(e.g., amounts, temperatures, etc.), some errors and deviations should be considered. Unless otherwise stated, temperatures are in ° C or at ambient temperature.

[0030] Example 1

[0031] This embodiment provides a method for preparing a multifunctional bimetallic nanozyme, and the process is as follows: First, prepare iron cobalt oxide nanoparticles: Tyrosine (6 mmol), ferric chloride (1 mmol), cobalt nitrate (1 mmol), sodium hydroxide (9 mmol), and ethylene glycol (60 mL) were mixed and added into a reactor for high-temperature heating reaction at 120 °C. The reaction was completed after 12 hours of reaction. The product was washed and freeze-dried to obtain iron cobalt oxide nanoparticles, recorded as CoFe2O4.

[0032] Secondly, prepare JUG-modified water-soluble polylipoic acid chains: ALA (19.42 mmol) was added to the flask and stirred at 100 °C for 1.5 hours. The crude product was dissolved in 30.0 mL of THF and then precipitated twice in 500 mL of ether to obtain a sticky white solid. THF and ether were removed in vacuo and dried at room temperature to obtain dry PALA.

[0033] PALA (1.00 g) was dissolved in 30.0 mL of DMF. Then mPEG (0.80 mmol), EDC·HCl (1.20 mmol) and DMAP (0.30 mmol) were dissolved in 30.0 mL of DMSO and mixed with PALA, and then stirred at room temperature for 48 hours. The mixture was dialyzed in deionized water for 2 days. PALA-PEG was obtained after lyophilization.

[0034] PALA-PEG (0.15 mmol), DMAP (0.30 mmol) and JUG (1.00 mmol) were dissolved in 20.0 mL of dry DMF, and then DIC (1.20 mmol) was added and stirred at room temperature for 24 hours. The reaction mixture was precipitated into ether, redissolved in DMF, dialyzed with distilled water, and finally lyophilized to obtain PALA-PEG-JUG.

[0035] Finally, the multifunctional bimetallic nanozyme was prepared: CoFe2O4 (100 mg) was dispersed in deionized water (15 mL). PALA-g-mPEG-JUG (120 mg) was then added to the above solution and stirred for 12 hours. The resulting multifunctional bimetallic nanozyme was recorded as CFO@PPJ.

[0036] The performance test of CFO@PPJ is as follows: Figures 1 to 5 shown.

[0037] Depend on Figure 1 It can be seen from the transmission electron microscopy image that CFO@PPJ is spherical with uniform size, and the particle size is basically maintained at around 100nm.

[0038] Depend on Figure 2 It can be seen from the UV-visible spectrum that JUG is effectively grafted onto PALA-mPEG.

[0039] Depend on Figure 3 It can be seen from the potential diagram that PALA-mPEG-JUG is successfully wrapped in the outer layer of CFO.

[0040] Depend on Figure 4 It can be seen from the XRD curve that the crystal form of CFO did not change significantly after it was encapsulated with PALA-mPEG-JUG.

[0041] Depend on Figure 5 It can be seen from the XPS spectrum that the main valence states of Co and Fe in CFO are +2 and +3, respectively.

[0042] Depend on Figure 6 It can be seen from the ·OH generation curve that CFO@PPJ exhibits a relatively significant ·OH generation ability in vitro, and the generated ROS has a high killing efficiency on tumor cells.

[0043] Depend on Figure 7 , Figure 8 The GSH consumption curve and confocal images of intracellular H2O2 generation show that CFO@PPJ has a high efficiency in GSH consumption and a significant ability to promote H2O2 generation in CT26 cells. Fig. 9 The safety experiment results showed that when the CFO@PPJ concentration reached 50μg / ml, the survival rate of tumor cells dropped to about 85%. When it continued to increase to 200μg / ml, the survival rate of tumor cells dropped to about 80%. This also confirmed that the multifunctional bimetallic nanozyme does have a certain killing effect on tumor cells, but the change in CFO@PPJ concentration has no effect on normal cells L929.

[0044] The obtained multifunctional bimetallic nanozyme CFO@PPJ was subjected to anti-tumor experiments. Figures 10-11 As shown: MRI images show that after tail vein injection of CFO@PPJ, the material is significantly enriched in the tumor site over time. In addition, after 11 days of treatment, the size of the tumor was significantly inhibited, indicating that the material CFO@PPJ consumes GSH in the tumor microenvironment and promotes the generation of intracellular H2O2 through the released JUG, significantly enhancing the generation of ROS to further kill tumor cells, inducing oxidative stress in tumor cells, and achieving efficient tumor treatment and tumor inhibition by breaking the redox homeostasis.

[0045] Example 2

[0046] This embodiment provides a method for preparing a multifunctional bimetallic nanozyme, and the process is as follows: First, prepare iron cobalt oxide nanoparticles: Tyrosine (6 mmol), ferric chloride (1 mmol), cobalt nitrate (1 mmol), sodium hydroxide (9 mmol), and ethylene glycol (60 mL) were mixed and added into a reactor for high-temperature heating reaction at 120 °C. The reaction was completed after 12 hours of reaction. The product was washed and freeze-dried to obtain iron cobalt oxide nanoparticles, recorded as CoFe2O4.

[0047] Secondly, prepare JUG-modified water-soluble polylipoic acid chains: ALA (19.42 mmol) was added to the flask and stirred at 120 °C for 1.0 h. The crude product was dissolved in 30.0 mL of THF and then precipitated twice in 500 mL of ether to obtain a sticky white solid. THF and ether were removed in vacuo and dried at room temperature to obtain dry PALA.

[0048] PALA (1.20 g) was dissolved in 30.0 mL of DMF. Then mPEG (0.90 mmol), EDC·HCl (1.50 mmol) and DMAP (0.30 mmol) were dissolved in 30.0 mL of DMSO and mixed with PALA, and then stirred at room temperature for 48 hours. The mixture was dialyzed in deionized water for 2 days. PALA-PEG was obtained after lyophilization.

[0049] PALA-PEG (0.3 mmol), DMAP (0.50 mmol) and JUG (2.00 mmol) were dissolved in 20.0 mL of dry DMF, and then DIC (1.20 mmol) was added and stirred at room temperature for 24 hours. The reaction mixture was precipitated into ether, redissolved in DMF, dialyzed with distilled water, and finally lyophilized to obtain PALA-PEG-JUG.

[0050] Finally, the multifunctional bimetallic nanozyme was prepared: CoFe2O4 (100 mg) was dispersed in deionized water (15 mL). PALA-g-mPEG-JUG (110 mg) was then added to the above solution and stirred for 12 h.

[0051] Example 3

[0052] This embodiment provides a method for preparing a multifunctional bimetallic nanozyme, and the process is as follows: First, prepare iron cobalt oxide nanoparticles: Tyrosine (6 mmol), ferric chloride (1 mmol), cobalt nitrate (1 mmol), sodium hydroxide (9 mmol), and ethylene glycol (60 mL) were mixed and added into a reactor for high-temperature heating reaction at 120 °C. The reaction was completed after 12 hours of reaction. The product was washed and freeze-dried to obtain iron cobalt oxide nanoparticles, recorded as CoFe2O4.

[0053] Secondly, prepare JUG-modified water-soluble polylipoic acid chains: ALA (25.0 mmol) was added to the flask and stirred at 110 °C for 1.0 h. The crude product was dissolved in 30.0 mL of THF and then precipitated twice in 500 mL of ether to obtain a sticky white solid. THF and ether were removed in vacuo to obtain dry PALA.

[0054] PALA (1.00 g) was dissolved in 30.0 mL of DMF. Then mPEG (0.80 mmol), EDC·HCl (1.0 mmol) and DMAP (0.30 mmol) were dissolved in 30.0 mL of DMSO and mixed with PALA, and then stirred at room temperature for 48 hours. The mixture was dialyzed in deionized water for 2 days. PALA-PEG was obtained after lyophilization.

[0055] PALA-PEG (0.2 mmol), DMAP (0.30 mmol) and JUG (1.00 mmol) were dissolved in 20.0 mL of dry DMF, and then DIC (1.10 mmol) was added and stirred at room temperature for 48 hours. The reaction mixture was precipitated into ether, redissolved in DMF, dialyzed with distilled water, and finally lyophilized to obtain PALA-PEG-JUG.

[0056] Finally, the multifunctional bimetallic nanozyme was prepared: CoFe2O4 (100 mg) was dispersed in deionized water (15 mL). PALA-g-mPEG-JUG (90 mg) was then added to the above solution and stirred for 12 h.

[0057] Example 2 and Example 3 respectively provide the preparation processes of different mixing mass ratios of CoFe2O4 (100 mg) and PALA-g-mPEG-JUG. The results are consistent with those of Example 1. When the mass ratio of CoFe2O4 (100 mg) to PALA-g-mPEG-JUG is 1:0.9~1.2, the obtained multifunctional bimetallic nanozyme has stable performance, efficient GSH consumption ability, significant H2O2 generation ability, and excellent ·OH generation ability. It can realize GSH consumption and H2O2 generation in tumor tissue, has a certain killing effect on tumor cells, and has a relatively significant ability to inhibit tumor growth.

[0058] The above-mentioned embodiments only express several feasible implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention, and the embodiments are not used to limit the scope of protection in the claims of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and all equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A method for preparing a multifunctional bimetallic nanozyme, characterized in that: Here are the steps: Step 1, tyrosine, ferric chloride, cobalt nitrate and sodium hydroxide are mixed and subjected to hydrothermal reaction to synthesize iron cobalt acid nanozyme, recorded as CoFe2O4; Step 2, polylipoic acid is dissolved in N,N-dimethylformamide, monomethyl polyethylene glycol, an activator, a catalyst, and dimethyl sulfoxide are added, stirred, dialyzed with deionized water, and lyophilized to obtain a monomethyl polyethylene glycol-modified polylipoic acid chain, which is recorded as PALA-mPEG; Step 3, dissolving PALA-mPEG in N,N-dimethylformamide, adding juglone, coupling agent, catalyst, and dimethyl sulfoxide, stirring, dialyzing with deionized water, and freeze-drying to obtain a water-soluble polylipoic acid chain, recorded as PALA-mPEG-JUG; Step 4: Disperse the CoFe2O4 obtained in step 1 in deionized water, add the PALA-mPEG-JUG obtained in step 3, stir the reaction, centrifuge and wash, collect the precipitate, and obtain a multifunctional bimetallic nanozyme, recorded as CFO@PPJ.

2. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 1, the molar ratio of tyrosine, ferric chloride, cobalt nitrate and sodium hydroxide is 1-6:1:1:

9.

3. The method for preparing a multifunctional bimetallic nanozyme according to claim 2, characterized in that: In step 1, ethylene glycol is also added, and tyrosine, ferric chloride, cobalt nitrate, sodium hydroxide and ethylene glycol form a mixed solution and then a hydrothermal reaction is carried out.

4. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 2, the polylipoic acid is prepared by the following method: α-lipoic acid is heated and polymerized to obtain a crude product, the crude product is dissolved in tetrahydrofuran and purified by ether precipitation method, and then dried at room temperature to obtain polylipoic acid, the reaction temperature of the heating polymerization is 90-150°C, and the reaction time is 30-100 min.

5. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 2, the molar mass ratio of polylipoic acid, monomethyl polyethylene glycol, activator and catalyst is 1:3-6:0.36:0.

03.

6. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 2, the activating agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

7. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 3, the molar ratio of PALA-mPEG, juglone, coupling agent, and catalyst is 1:2~4:0.36:0.

03.

8. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 3, the coupling agent is N,N'-diisopropylcarbodiimide.

9. The method for preparing a multifunctional bimetallic nanozyme according to claim 1, characterized in that: In step 4, the mass ratio of CoFe2O4 to PALA-mPEG-JUG is 1:0.5-2.

0.

10. An application of a multifunctional bimetallic nanozyme, characterized in that: The multifunctional bimetallic nanozyme prepared by the method of claim 1 is used to prepare any one of anti-tumor nanomaterials, enhanced chemodynamic therapeutic materials, and multifunctional anti-tumor agents.

Images