Composite nanoscale enzyme, preparation method and application thereof

Abstract

This invention discloses a composite nanozyme, its preparation method, and its application. The composite nanozyme uses Prussian blue nanoparticles as a carrier, with a phospholipid bilayer formed by dimyristoylphosphatidylcholine coated on the surface of the Prussian blue nanoparticles. The phospholipid bilayer contains (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole. This invention is the first to disclose a composite nanozyme formed by modifying the surface of Prussian blue nanoparticles with DOTAP and imidazole groups. The obtained composite nanozyme can effectively catalyze ester hydrolysis, producing at least two orders of magnitude acceleration, which provides a possibility for the direct hydrolysis of cholesterol esters in atherosclerotic plaques by nanomedicines. This invention expands the enzyme-like activity of the Prussian blue nanozyme through special modification, providing a solution for simultaneously achieving multifunctional composite nanozymes with ester hydrolytic enzyme function, catalase-like properties, and superoxide dismutase-like properties.

Description

Technical Field

[0001] This invention relates to a composite nanozyme, its preparation method, and its application, belonging to the field of biomedicine. Background Technology

[0002] In the process of atherosclerosis, plaque rupture leading to thrombus formation is a major cause of serious diseases such as coronary heart disease and cerebral infarction. Foam cells, which develop from macrophages, accumulate large amounts of cholesterol esters (CE) and protrude into the lumen of blood vessels to form plaques. Lipid-rich plaques are prone to rupture. Intracellularly, (neutral) cholesterol esterase (nCEH) is responsible for hydrolyzing cholesterol esters to generate free cholesterol. Only when intracellular cholesterol esters are degraded into free cholesterol by nCEH can they be transported out of the cell; therefore, the hydrolysis by nCEH is considered a key step in the reverse cholesterol transport process.

[0003] The main function of nCEHs is to hydrolyze cholesterol esters into cholesterol and fatty acids, thereby promoting the efflux of cholesterol and reducing the accumulation of cholesterol esters in cells. nCEHs also have a relatively good effect on inhibiting inflammation. Therefore, in atherosclerotic cells, high expression of cholesterol hydrolase can promote CE hydrolysis and the efflux of intracellular free cholesterol, inhibit the development of foam cells and plaque formation, effectively delaying and inhibiting atherosclerotic lesions. This helps prevent further plaque development and the onset of diseases such as coronary heart disease and cerebral infarction, thus curbing the development of the disease at its source.

[0004] Nanozymes represent an important frontier research area in the field of biomedical nanotechnology and a significant advancement and innovation in enzyme engineering. Compared with natural enzymes, nanozymes offer advantages such as simple preparation, large-scale production capability, strong environmental tolerance, low preparation and storage costs, and reusability.

[0005] In the field of artificial hydrolases, short peptide-gold, carbon nanocomposites, and metal micelles have been repeatedly demonstrated to hydrolyze lipids. Gold nanocomposites typically use Au-S bonds to link gold nanoparticles to related alkyl groups with specific functional groups, which then self-assemble with short peptides to form the final complex. The functional groups on the short peptides in the complex contribute to hydrolysis, partly through the synergistic effect of multiple functional groups and partly through the local microenvironment created by the gold nanoparticles to further accelerate catalysis. Carbon nanocomposites are similar, replacing gold nanoparticles with rigid carbon nanomaterials. These carbon nanomaterials can create a hydrophobic microenvironment to promote proton transfer and enhance catalytic activity. Metal micelles, on the other hand, combine functional ligands with ionic complexes to form colloids. These colloids contain both catalytic centers and mimic hydrophobic microenvironments, exhibiting strong hydrolytic activity towards esters.

[0006] In the aforementioned and other artificially simulated ester hydrolases, the imidazole group plays a key role in hydrolysis. Histidine residues, including cholesterol ester hydrolases, contain histidine in their active sites, and the imidazole group they carry is precisely the site of hydrolysis. In catalytic triads, the imidazole group also plays an important role in catalytic hydrolysis. Furthermore, we found that most artificial hydrolases utilize metal or other material carriers to create a local microenvironment to mimic natural enzymes, thereby enhancing catalytic activity. Currently, the main clinical treatment for atherosclerosis is cholesterol-lowering drugs, but there is no effective method to directly reduce lipid content within plaques, and there are no nanomedicines based on the hydrolysis of cholesterol esters within plaques. Prussian blue nanozymes possess catalase-like (CAT) and superoxide dismutase-like (SOD) functions and have been shown to regulate the inflammatory microenvironment within plaques, alleviating atherosclerosis. Currently, there are no reports on whether Prussian blue nanozymes possess hydrolytic activity; therefore, Prussian blue nanozymes have not been used to prepare composite nanozymes with ester hydrolase properties for the treatment of atherosclerosis. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a composite nanozyme with ester hydrolase properties, its preparation method and application.

[0008] Technical solution: To solve the above technical problems, the present invention provides a composite nanoenzyme, which uses Prussian blue nanoparticles as carriers and coats their surface with a phospholipid bilayer formed by dimyristoylphosphatidylcholine. Due to the influence of hydrophilic and hydrophobic interactions, (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole are embedded in the phospholipid bilayer.

[0009] The present invention also provides a method for preparing the aforementioned composite nanozyme, comprising the following steps: rotary evaporation of a mixed acetonitrile solution of myristoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole, addition of an aqueous solution of Prussian blue nanoparticles, and purification to obtain the aforementioned composite nanozyme.

[0010] The concentration ratio of dimyristoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole in the mixed solution is 10:(5-10):(5-10).

[0011] The volume ratio of dimyristoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole in the mixed solution is 975:(25-50):(25-50).

[0012] The volume ratio of the mixed solution to the Prussian blue nanoparticle aqueous solution is 1050:(300-400).

[0013] The Prussian blue nanoparticles have a size of 120–160 nm.

[0014] The composite nanozyme has a size of 160–200 nm.

[0015] The potential of the Prussian blue nanoparticles is -35 to -25 mV.

[0016] The potential of the composite nanozyme is +5 to +15 mV.

[0017] The iron concentration in the Prussian blue nanoparticle aqueous solution is 700–800 ug / ml.

[0018] The preparation method of Prussian blue nanoparticles includes the following steps: FeCl3·6H2O is dissolved in polyvinylpyrrolidone aqueous solution, K4[Fe(CN)6]·3H2O aqueous solution is added dropwise, and Prussian blue nanoparticles are obtained after purification.

[0019] The mass-to-volume ratio of FeCl3·6H2O to the polyvinylpyrrolidone aqueous solution is (25-30):80 mg / mL.

[0020] The dropping rate of the K4[Fe(CN)6]·3H2O aqueous solution was 30-50 mL / h.

[0021] The molar ratio of FeCl3·6H2O to K4[Fe(CN)6]·3H2O is (3.5~4.5):1.

[0022] The concentration of the FeCl3·6H2O aqueous solution was 0.01–0.015 g / mL.

[0023] The concentration of the K4[Fe(CN)6]·3H2O aqueous solution is 2.0–2.5 mg / mL.

[0024] This invention also provides the application of the aforementioned composite nanozyme in the preparation of reagents or drugs with ester hydrolysis function.

[0025] The present invention also provides the application of the aforementioned composite nanozyme in the preparation of reagents or drugs for treating atherosclerosis.

[0026] The principle of this invention: Prussian blue nanozymes possess oxidoreductase activities such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), but they do not inherently possess the functional characteristics of hydrolases. Furthermore, the imidazole group, acting as a proton donor, is difficult to catalyze hydrolysis when present alone. This invention modifies the surface of Prussian blue nanoparticles with DOTAP and imidazole groups to obtain a composite nanozyme with ester hydrolase function. Specifically, DOTAP adsorbs OH- ions on the nanozyme surface, creating locally alkaline conditions for hydrolysis. The imidazole group deprotonates in this locally alkaline microenvironment, accelerating the catalytic hydrolysis of lipid bonds. Simultaneously, the Prussian blue nanoparticles, due to their antioxidant enzyme activity, can potentially exert anti-inflammatory effects.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to develop a composite nanozyme formed by modifying the surface of Prussian blue nanoparticles with DOTAP and imidazole groups; 2. DOTAP adsorbs OH groups on the surface of the nanozyme. - 1. By creating conditions for localized alkaline hydrolysis, the imidazole group deprotonates in the locally alkaline microenvironment, accelerating the catalytic hydrolysis of lipid bonds. In this way, the composite nanozyme mimics some key characteristics of natural enzymes. 2. The obtained composite nanozyme can effectively catalyze ester hydrolysis and produces at least two orders of magnitude acceleration, which provides the possibility for direct hydrolysis of cholesterol esters in atherosclerotic plaques by nanomedicines. 3. Prussian blue nanozymes possess catalase (CAT) and superoxide dismutase (SOD) functions, and can play a role in regulating the inflammatory microenvironment in plaques and alleviating atherosclerosis. 4. Single-component nanozymes have limited functions, restricting their application in biomedicine, production, and other fields. Furthermore, Prussian blue nanoparticles themselves are difficult to possess the characteristic conditions of hydrolytic enzymes. 5. This invention expands the enzyme-like activity of Prussian blue nanozymes through special modifications, providing a solution for simultaneously realizing multifunctional composite nanozymes with ester hydrolytic enzyme functions and redox enzyme-like properties. Attached Figure Description

[0028] Figure 1 The microstructure of the Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazol composite nanoenzyme;

[0029] Figure 2 To obtain the particle size distribution curve of Prussian blue nanoparticles and Prussian blue@DMPC@DOTAP@undecylimidazolium composite nanozymes;

[0030] Figure 3 To compare the zeta potential of Prussian blue nanoparticles with that of Prussian blue@DMPC@DOTAP@undecylimidazolium composite nanozymes;

[0031] Figure 4 Three sets of absorbance curves for the Prussian blue@dimyristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazol composite nanozyme and the hydrolysis substrate Cbz-Phe-Onp of single Prussian blue nanoparticles and the self-hydrolysis blank control of Cbz-Phe-Onp.

[0032] Figure 5 The absorbance change curve of Cbz-Phe-Onp after self-hydrolysis is shown as a blank control.

[0033] Figure 6 The absorbance curves of the Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium complex nanoenzyme hydrolysis substrate Cbz-Phe-Onp under different pH conditions are shown.

[0034] Figure 7 The second-order reaction rate constant curves of the Prussian blue@dimyristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanoenzyme hydrolysis substrate Cbz-Phe-Onp under different pH conditions are shown. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0036] Example 1: Preparation and analysis of Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanozyme (hereinafter referred to as: composite nanozyme)

[0037] The raw materials used are polyvinylpyrrolidone (PVP-K30, Sinopharm Chemical Reagent Co., Ltd.), FeCl3·6H2O, K4[Fe(CN)6]·3H2O, chloroform, myristoylphosphatidylcholine (DMPC), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), and undecylimidazole.

[0038] 1. Preparation of Prussian blue nanoparticles:

[0039] 1.11 g (10 mmol) of PVP was ultrasonically dispersed in 80 mL of ultrapure water. After homogeneity, 27.0 mg (0.1 mmol) of FeCl3·6H2O was added to the PVP aqueous solution and ultrasonicated for 10 min. The solution was then transferred to a 250 mL three-necked flask in a 60 °C constant temperature water bath and stirred at 1200 rpm for 30 min to obtain a FeCl3·6H2O solution. Separately, 42.2 mg (0.1 mmol) of K4[Fe(CN)6]·3H2O was dissolved in 20 mL of ultrapure water and ultrasonicated for 5 min to ensure homogeneity. The solution was then transferred to a 20 mL syringe, fixed to a dual-channel microfluidic injection pump, and the K4[Fe(CN)6]·3H2O aqueous solution was added dropwise to the FeCl3·6H2O solution. After the addition was complete, the mixture was stirred at 1200 rpm for 1 h in a 60 °C constant temperature water bath. After the reaction was complete, the water bath was closed and the mixture was slowly cooled to room temperature. After the reaction was completed, the aggregates were filtered off using a 0.45 μm filter membrane, the supernatant was removed by centrifugation at 12000 rpm, and 50 mL of pure water was added to redissolve the aggregates to obtain a solution of Prussian blue nanoparticles (PBNP) with a hydrodynamic size of 120–160 nm and a PDI of 0.021.

[0040] 2. Preparation of Prussian Blue@Myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanozyme:

[0041] DMPC, DOTAP, and undecylimidazole were dissolved in chloroform to obtain 975 μL of DMPC solution (10 mg / mL), 50 μL of DOTAP solution (5 mg / mL), and 25 μL of undecylimidazole solution (10 mg / mL). These three solutions were then transferred to 2 mL of chloroform in a round-bottom flask and mixed thoroughly. The mixture was rotary evaporated at 60 °C at 70 rpm until the chloroform was completely evaporated, and then continued to evaporate for 10 min. 349 μL of PBNP solution (iron content 713 μg / mL) and 4.651 mL of pure water were added to a centrifuge tube and mixed thoroughly to redissolve the ester membrane, obtaining a hydrated solution. This hydrated solution was added to a round-bottom flask, shaken until the membrane was completely dissolved, sealed with plastic wrap, and incubated in an ultrasonic water bath for 30 min. The solution was filtered through a 0.45 μm filter three times. Finally, it was centrifuged at 10,000 rpm for 15 min. The supernatant was discarded, and 5 mL of pure water was added to reconstitute the solution. The mixture was then sonicated in a water bath for 5 min to obtain a Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazole composite nanozyme (composite nanozyme) with a hydrodynamic size of 160–200 nm. The composite nanozyme uses Prussian blue nanoparticles as a carrier, with a phospholipid bilayer formed by myristoylphosphatidylcholine coated on the surface of the Prussian blue nanoparticles. Since both (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole possess both hydrophilic and hydrophobic ends, they are embedded within the phospholipid bilayer due to hydrophilic-hydrophobic interactions. The PDI of the composite nanozyme is 0.050.

[0042] Prussian blue nanoparticles (PBNPs) and a Prussian blue@dimyristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanozyme (composite nanozyme) were subjected to kinetic and zeta potential assays, respectively. The particle size distribution curves are shown below. Figure 2 As shown, the Zeta potential results are as follows: Figure 3 As shown, the hydrodynamic dimensions of the obtained Prussian blue nanoparticles and composite nanozymes are 120–160 nm and 160–200 nm, respectively, both with relatively high particle sizes, and their potentials are approximately -30 mV and 10 mV, respectively. The Prussian blue nanoparticles increased in size by approximately 20 nm and their potential increased by approximately 40 mV after coating, indicating that the (2,3-dioleoyl-propyl)-trimethylamine DOTAP, undecylimidazole, and DMPC formed a bilayer that successfully coated the surface of the Prussian blue particles, yielding the Prussian blue@DMPC@DOTAP@undecylimidazole composite nanozyme.

[0043] Example 2: Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanozyme and single Prussian blue nanoparticle hydrolysis substrate Cbz-Phe-Onp

[0044] The raw materials used were Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanozyme prepared in Example 1, Prussian blue nanoparticles, N-benzyloxycarbonyl-L-phenylalanine p-nitrobenzene ester (Cbz-Phe-Onp), acetonitrile, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), sodium hydroxide, and hydrochloric acid.

[0045] It is known that the lipid bond of Cbz-Phe-Onp breaks to form p-nitrophenol (PNP). The product PNP exhibits an absorption peak around 400 nm in an aqueous solution at pH 7-8 under room temperature conditions. Therefore, the hydrolysis status of the substrate Cbz-Phe-Onp can be determined by measuring the absorbance change at 400 nm. However, the substrate Cbz-Phe-Onp is easily decomposed by light, so the experiment should be conducted in the dark throughout.

[0046] Prepare a 0.2 mM Cbz-Phe-Onp acetonitrile solution under light-protected conditions and store it in the dark. Prepare a 10 mmol / L HEPES buffer solution and adjust the pH to 7.5 using NaOH and hydrochloric acid. Prepare HEPES solutions with a Fe element concentration of 10 μg / mL, including a Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium complex nanozyme and a single Prussian blue nanoparticle, respectively. Both solutions have a pH of 7.5. Take 1.8 mL of each solution, including the complex nanozyme group and the Prussian blue nanoparticle group. The single HEPES solution serves as a blank control. The groups are as follows:

[0047] (1) Composite nanozyme group: Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazol composite nanozyme HEPES solution with Fe element concentration of 10 μg / ml, pH=7.5;

[0048] (2) Prussian blue nanoparticle group: HEPES solution of single Prussian blue nanoparticles with Fe element concentration of 10 μg / ml, pH=7.5;

[0049] (3) Blank group: 10 mmol / L HEPES solution, pH=7.5.

[0050] Because the substrate Cbz-Phe-Onp is easily decomposed by light, 0.2 ml of 0.2 mM Cbz-Phe-Onp acetonitrile solution was rapidly added to each group (composite nanozyme group, Prussian blue nanoparticle group, or blank group) to obtain three solutions (water to acetonitrile volume ratio of 9:1). The absorbance change at 400 nm was immediately observed using a multi-functional microplate reader, with measurement intervals of 10 s. The absorbance changes of the three groups over time are shown below. Figure 4The absorbance of a single blank group changes over time as follows: Figure 5 .

[0051] The pseudo-first-order kinetic degradation equation is as follows:

[0052]

[0053] Where, k obs It is the apparent rate constant (s) -1 C0 is the initial concentration of the substrate (mol / L), and C is the concentration of the substrate during degradation (mol / L).

[0054] The second-order reaction rate equation is given below:

[0055]

[0056] Where k2 is the rate constant of the second-order reaction (M) -1 s -1 C is the concentration of the substrate (mol / L) during the degradation process.

[0057] The PNP concentration was calculated using Lambert-Beer's law, and the substrate concentration change was then obtained.

[0058] A = ε × L × C

[0059] Where A is the absorbance value, and ε is the molar absorptivity (L·mol⁻¹). -1 ·cm -1 L is the distance the light travels through the solution (cm), and C is the concentration of the sample (mol / L). By default, L = 1 cm. The molar absorptivity of PNP at 400 nm is known to be approximately 1.0 × 10⁻⁶. 4 L·mol -1 ·cm -1 .

[0060] Referring to the above formulas, use the following equations to fit the kinetic curve and calculate the apparent rate constant k. obs :

[0061] A t =(A0-A ∞ )exp(-k obs t)+A ∞

[0062] Calculate the rate constant k2 of the second-order reaction by fitting the kinetic curve using the following equation:

[0063]

[0064] Where, A, A0, A ∞These are the absorbance values ​​at times t, 0, and ∞, respectively.

[0065] The apparent rate constant k was calculated. obs The velocity acceleration k relative to the background obs / k cat (k cat =k obs-Blank The second-order reaction rate constant k2 is shown in the table below.

[0066] Table 1

[0067]

[0068] like Figure 4 , Figure 5 As shown in Table 1, the decomposition rate of Cbz-Phe-Onp was accelerated by at least 100 times under the catalysis of the composite nanozyme, proving that the composite nanozyme can effectively catalyze the hydrolysis of Cbz-Phe-Onp. Furthermore, the decomposition rate of Cbz-Phe-Onp was accelerated by approximately 40 times under the catalysis of PBNPs, indicating that PBNPs also possess a certain ability to catalyze substrate hydrolysis without any surface modification. By comparing the data from the composite nanozyme group and the Prussian blue nanoparticle (PBNP) group, it can be concluded that the imidazole group improves the catalytic efficiency to some extent in the local microenvironment.

[0069] Example 3: Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium composite nanoenzyme hydrolysis substrate Cbz-Phe-Onp under different pH conditions

[0070] The raw materials used were Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazolium complex nanozyme prepared in Example 1, Cbz-Phe-Onp, acetonitrile, HESPES, sodium hydroxide, and hydrochloric acid.

[0071] It is known that the lipid bond of Cbz-Phe-Onp breaks to form p-nitrophenol (PNP). The product PNP exhibits an absorption peak around 400 nm in an aqueous solution at pH 7-8 under normal temperature conditions. Therefore, the hydrolysis status of the substrate Cbz-Phe-Onp can be determined by measuring the absorbance change at 400 nm. However, the substrate Cbz-Phe-Onp is easily decomposed by light, so the experiment should be conducted in the dark throughout.

[0072] Prepare a 0.2 mM Cbz-Phe-Onp acetonitrile solution under light-protected conditions and store it in the dark. Prepare a 10 mmol / L HEPES buffer solution and adjust the pH to 7.0, 7.5, 8.0, and 8.5 using NaOH and hydrochloric acid, respectively, to obtain four HEPES buffer solutions with different pH values. Prepare four Prussian blue@myristoylphosphatidylcholine@(2,3-dioleoyl-propyl)-trimethylamine@undecylimidazole HEPES solutions (pH = 7.0, 7.5, 8.0, and 8.5) with a Fe concentration of 10 μg / ml. Take 1.8 mL of each solution and quickly add 0.2 mL of the prepared Cbz-Phe-Onp acetonitrile solution to each solution, mixing to obtain four water-acetonitrile solutions with a volume ratio of 9:1. Immediately observe the absorbance change at 400 nm using a multi-mode microplate reader, with measurement intervals of 10 s, and obtain the absorbance changes of the four solutions over time as shown below. Figure 6 As shown. The calculated second-order reaction rate constant k2 is as follows: Figure 7 As shown.

[0073] like Figure 6 As shown, PNP release accelerates with increasing solution pH, indicating that the catalytic efficiency of the composite nanozyme increases with increasing solution pH. This is based on the second-order rate constant (…). Figure 7 It can be seen that as the solution pH gradually increases, the reaction rate constant generally shows a rapid increase, with the rate constant increasing approximately threefold as the solution pH increases from 7.0 to 8.5. This indicates that the stronger the alkalinity of the solution, the higher the catalytic efficiency of the composite nanozyme. It is evident that the synthesized composite nanozyme carries a large number of positrons on its surface, enabling it to adsorb hydroxide ions (OH-) from the solution. - This provides a localized alkaline environment and reaction sites for the ester hydrolysis reaction. As the pH of the solution increases, the OH groups adsorbed on the surface of the composite nanozyme... - As the density gradually increases, more reaction catalytic sites are provided, which improves the catalytic efficiency of the composite nanozyme and further illustrates the contribution of the local alkaline microenvironment.

Claims

1. A composite nanozyme, characterized in that, Using Prussian blue nanoparticles as a carrier, a phospholipid bilayer formed by dimyristoyl phosphatidylcholine is coated on the surface of the Prussian blue nanoparticles. The phospholipid bilayer contains (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole.

2. A method for preparing the composite nanozyme according to claim 1, characterized in that, Includes the following steps: A mixed acetonitrile solution of myristoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, and undecylimidazole was rotary evaporated, and then purified by adding an aqueous solution of Prussian blue nanoparticles to obtain the composite nanozyme. The concentration ratio of myristoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, and undecylimidazole in the mixed acetonitrile solution was 10:(5-10):(5-10); the volume ratio of myristoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, and undecylimidazole in the mixed acetonitrile solution was 975:(25-50):(25-50); and the volume ratio of the mixed acetonitrile solution to the aqueous solution of Prussian blue nanoparticles was 1050:(300-400).

3. The method according to claim 2, characterized in that, The preparation method of Prussian blue nanoparticles includes the following steps: FeCl3•6H2O is dissolved in polyvinylpyrrolidone aqueous solution, K4[Fe(CN)6]•3H2O aqueous solution is added dropwise, and Prussian blue nanoparticles are obtained after purification.

4. The method according to claim 3, characterized in that, The mass-to-volume ratio of FeCl3•6H2O and polyvinylpyrrolidone aqueous solution is (25-30):80 mg / mL.

5. The method according to claim 3, characterized in that, The dropping rate of the K4[Fe(CN)6]•3H2O aqueous solution was 30–50 mL / h.

6. The method according to claim 3, characterized in that, The molar ratio of FeCl3•6H2O and K4[Fe(CN)6]•3H2O is (3.5~4.5):

1.

7. The use of the composite nanozyme according to claim 1 in the preparation of reagents or drugs for hydrolyzing Cbz-Phe-Onp.

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