Artificial multienzyme nanoflower composite hydrogel wound dressing and preparation method thereof

Abstract

The invention relates to an artificial multienzyme nanoflower composite hydrogel wound dressing and a preparation method thereof. According to the preparation method of the artificial multienzyme nanoflower composite hydrogel wound dressing, the nanoflower loaded with glucose oxidase and hemoglobin and an NO donor are jointly doped into a matrix to prepare the artificial multienzyme nanoflower composite hydrogel wound dressing. According to the artificial multienzyme nanoflower composite hydrogel wound dressing, the diabetes wound microenvironment can be remodeled by reducing the local blood glucose concentration and pH, relieving oxygen deficit and removing excessive active oxygen; nO of different doses is released under different blood glucose levels to play the antibacterial and angiogenesis promoting functions, and then healing of methicillin-resistant staphylococcus aureus infected diabetes wounds is effectively promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel wound dressings, and particularly relates to an artificial multi-enzyme nanoflower composite hydrogel wound dressing and a preparation method thereof. Background Art

[0002] Diabetes is one of the most common complex metabolic diseases in the world, characterized by persistent high blood sugar levels. About 19%-34% of diabetic patients will develop diabetic chronic wounds, which are common and difficult to cure. Diabetic wounds have typical microenvironments such as high blood sugar levels, high pH, ​​hypoxia, persistent inflammation, imbalance between oxidation and antioxidant reactions, and lack of angiogenesis. In addition, high blood sugar and high pH are prone to pathogen invasion. These factors can cause the four orderly physiological processes of coagulation, inflammation, proliferation and remodeling in the conventional wound repair process to become chaotic, thus turning diabetic wounds into chronic non-healing wounds. Wound dressings, as a physical barrier between the wound and the external environment, can prevent further damage and infection of the wound surface. Among them, hydrogel dressings with a three-dimensional network structure similar to the extracellular matrix and good biocompatibility can not only play a role in physical isolation and maintain a moist environment on the wound surface, but also show great application potential in wound healing due to the addition of active ingredients. Therefore, the development of wound dressings that can effectively regulate the wound microenvironment and provide efficient antibacterial activity is crucial to promote the healing of diabetic chronic wounds.

[0003] Glucose oxidase (GOx) can catalyze the oxidation of glucose (Glu) in the presence of oxygen to produce gluconic acid and hydrogen peroxide (H 2 O 2 ), some GOx-based hydrogel dressings have been developed to regulate local blood glucose concentration and pH value in diabetic wounds. However, the hypoxic microenvironment of diabetic wounds limits the function of GOx. Antibiotics are the first choice for the treatment of bacterial infections. However, the healing cycle of diabetic wounds is long, and long-term use of antibiotics can induce the production of multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Therefore, it is of great significance to develop "green therapies" that are not easy to induce bacterial resistance to deal with bacterial infections in wounds. Although GOx catalyzes the oxidation of glucose to produce H 2 O 2 It can be used to kill bacteria, but due to the presence of oxidases (such as superoxide dismutase, etc.) in bacteria, bacteria are sensitive to H 2 O 2 Resistant, can weaken H 2 O 2 In addition, excessive H 2 O 2Can hinder wound healing. Nitric oxide (NO) is an endogenous biological signal molecule that participates in a variety of physiological processes, and the physiological function of NO is concentration-dependent. High concentrations of NO have a broad-spectrum antibacterial effect. High concentrations of NO cause bacterial lipid peroxidation, membrane protein nitration, and DNA degradation by producing hydrophobic free radicals, and will not induce bacterial resistance. Low concentrations of NO can accelerate wound healing by promoting angiogenesis, cell proliferation, and migration. The activity of biological enzymes is easily affected by temperature and pH fluctuations, and is easily degraded in the biological environment, thereby limiting their function. Therefore, it is crucial to maintain the activity and stability of GOx and Hb in the microenvironment of diabetic wounds and to increase the rate of cascade catalytic reactions.

[0004] The invention patent with application publication number CN113262296A discloses a nanoreactor loaded with glucose oxidase and hemoglobin, and its preparation method and application. The structure of the nanoreactor loaded with glucose oxidase and hemoglobin is a zeolite imidazolate skeleton structure, and glucose oxidase and hemoglobin are loaded inside; its morphology is a regular dodecahedron with a diameter of 100-1000 nanometers. The nanoreactor loaded with glucose oxidase and hemoglobin can efficiently kill multidrug-resistant bacteria and destroy bacterial biofilms, and can cooperate with starvation therapy and chemodynamic therapy to control diabetic wound infection. However, the hypoxic microenvironment of diabetic wounds will limit the function of GOx, and the presence of oxidase in bacteria can weaken H 2 O 2 The bactericidal properties of the nanoreactor are reduced, and the antibacterial function of the nanoreactor is reduced. In addition, the excessive H generated by the nanoreactor at the wound site 2 O 2 It can hinder the healing of diabetic wounds. Summary of the invention

[0005] The purpose of the present invention is to provide an artificial multi-enzyme nanoflower composite hydrogel wound dressing to solve the technical problem that the existing nanoreactor fails to solve the effect of the diabetic wound microenvironment on the activity and stability of GOx and Hb; at the same time, it solves the problem of H produced by the existing nanoreactor. 2 O 2 The bactericidal effect is poor and affects the speed and quality of wound healing.

[0006] The second object of the present invention is to provide a method for preparing an artificial multi-enzyme nanoflower composite hydrogel wound dressing.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] A method for preparing an artificial multi-enzyme nanoflower composite hydrogel wound dressing is prepared by co-incorporating nanoflowers loaded with glucose oxidase and hemoglobin and an NO donor into a matrix.

[0009] Furthermore, the NO donor is hydroxyurea.

[0010] Furthermore, the matrix is ​​a mixed matrix of an oxidized sodium alginate aqueous solution and a carboxymethyl chitosan aqueous solution.

[0011] Furthermore, the preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing comprises the following steps: mixing hydroxyurea with a carboxymethyl chitosan aqueous solution to obtain a hydroxyurea-carboxymethyl chitosan aqueous solution, and mixing nanoflowers loaded with glucose oxidase and hemoglobin, an oxidized sodium alginate aqueous solution, and a hydroxyurea-carboxymethyl chitosan aqueous solution to obtain.

[0012] Furthermore, 0.0020-0.0030 g of hydroxyurea is added to each mL of the carboxymethyl chitosan aqueous solution, 250-350 μL of oxidized sodium alginate aqueous solution is added to each 100 μL of the nanoflowers loaded with glucose oxidase and hemoglobin; 550-650 μL of hydroxyurea-carboxymethyl chitosan aqueous solution is added to each 100 μL of the nanoflowers loaded with glucose oxidase and hemoglobin; the mass fraction of the oxidized sodium alginate aqueous solution is 2-6%, and the mass fraction of the carboxymethyl chitosan aqueous solution is 4-10%.

[0013] Furthermore, the preparation method of the nanoflower loaded with glucose oxidase and hemoglobin is: adding glucose oxidase and hemoglobin to a phosphate buffered saline solution, then adding a calcium chloride aqueous solution for incubation to obtain a crude product solution, centrifuging the crude product solution to obtain a precipitate, and resuspending the precipitate to obtain the nanoflower.

[0014] Furthermore, the mass ratio of the glucose oxidase to the hemoglobin is 1:1-2, 0.5-3 mL of the phosphate buffered saline solution is added for every mg of the glucose oxidase, and 10-50 μL of the calcium chloride aqueous solution is added for every mg of the glucose oxidase; the concentration of the phosphate buffered saline solution is 1-10 mM, and the concentration of the calcium chloride aqueous solution is 50-500 nM.

[0015] Furthermore, the incubation is carried out at room temperature for 6-24 hours.

[0016] Furthermore, the resuspension is to resuspend the precipitate in 0.25-1 mL of phosphate buffered saline solution.

[0017] An artificial multi-enzyme nanoflower composite hydrogel wound dressing is prepared by adopting the above-mentioned artificial multi-enzyme nanoflower composite hydrogel wound dressing preparation method.

[0018] Beneficial effects of the present invention:

[0019] The artificial multi-enzyme nanoflower composite hydrogel wound dressing (COH-GB) of the present invention can reshape the diabetic wound microenvironment by reducing local blood glucose concentration and pH, alleviating hypoxia and removing excessive reactive oxygen species (ROS); and by releasing different doses of NO at different blood glucose levels, it exerts antibacterial and angiogenesis-promoting functions, thereby effectively promoting the healing of MRSA-infected diabetic wounds.

[0020] In the present invention, glucose oxidase and hemoglobin are prepared into nanoflowers loaded with glucose oxidase and hemoglobin through calcium ions, which can significantly enhance enzyme activity and stability, while improving the cascade reaction efficiency, thereby more effectively improving the microenvironment regulation performance, antibacterial activity, angiogenesis performance and wound healing effect of COH-GB hydrogel.

[0021] The artificial multi-enzyme nanoflower composite hydrogel wound dressing of the present invention releases NO and regulates the microenvironment through glucose activation. The GOx in the COH-GB hydrogel catalyzes the oxidation of glucose, which can reduce the local blood sugar level and pH value, improve the diabetic wound microenvironment, and provide H for the generation of NO in the presence of Hb. 2 O 2 Subsequently, the cascade reaction triggered by high blood sugar makes COH-GB hydrogel have efficient antibacterial and biofilm removal functions. When the local blood sugar of the wound drops to normal level, hydroxyurea (HU) will release a small amount of NO to promote angiogenesis. In addition, Hb can deliver O 2 , relieve the hypoxic state of MRSA-infected diabetic wounds, thereby accelerating the consumption of glucose and H 2 O 2 The generation of NO further promotes the rapid release of NO and increases the microenvironment regulation performance of COH-GB hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The preparation process and mechanism of action of artificial multi-enzyme nanoflower composite hydrogel wound dressing;

[0023] Figure 2 Characterization of GB nanoflowers, wherein A is the SEM image of the GB nanoflowers in Example 1, B is the TEM image of the GB nanoflowers in Example 1, C is the size distribution image of the GB nanoflowers in Example 1, D is the Hb content image in the GB nanoflowers in Examples 1-4, E is the GOx activity image in the GB nanoflowers in Examples 1-4, and F is the peroxidase activity verification image of the GB nanoflowers in Example 1;

[0024] Figure 3The results of the evaluation of GOx activity in GB nanoflowers in Example 1 are shown in Figure 1, where A is the GOx enzyme kinetics diagram in GB nanoflowers, B is the free GOx enzyme kinetics diagram, C is the enzyme activity of GB nanoflowers and free GOx at different temperatures, and D is the change in enzyme activity of GB nanoflowers and free GOx stored at room temperature for 15 days, *P<0.5, **P<0.01, ***P<0.001;

[0025] Figure 4 This is a graph of the oxygen carrying capacity of GB nanoflowers in Example 1;

[0026] Figure 5 The performance characterization diagrams of the COH-GB hydrogel in Example 1, wherein A is the gelation diagram of the COH-GB hydrogel, B is the injectability and shape adaptability result diagram of the COH-GB hydrogel, and C is the self-healing result diagram of the COH-GB hydrogel;

[0027] Figure 6 The internal microscopic morphology and pore size diagrams of the hydrogels in Comparative Example 1 and Examples 1-4, wherein A is the COH hydrogel in Comparative Example 1, B is the COH-GB1 hydrogel in Example 1, C is the COH-GB2 hydrogel in Example 2, D is the COH-GB3 hydrogel in Example 3, and E is the COH-GB4 hydrogel in Example 4;

[0028] Figure 7 Figures 1 and 2 are graphs showing the air permeability and water content of the hydrogels in Comparative Example 1 and Examples 1-4, wherein A is the air permeability graph and B is the water content graph;

[0029] Figure 8 This is a graph showing the adhesion test results of the COH-GB hydrogel in Example 1;

[0030] Fig. 9 Glucose consumption performance graph and microenvironment regulation performance graph of the hydrogels in Comparative Example 1 and Examples 1-4, wherein A is a glucose consumption performance graph and B is a graph of the ability to induce a decrease in pH;

[0031] Fig.10 : NO release performance graph, wherein A is a NO release performance graph of the hydrogels in Comparative Example 1 and Examples 1-4 under 16.7 mM glucose, B is a NO release performance graph of the hydrogels in Comparative Examples 1-4 and Example 3 under 16.7 mM glucose, and C is a NO release performance graph of the hydrogels in Comparative Examples 1-4 and Example 3 under 4.33 mM glucose;

[0032] Fig.11 Graphs showing oxygen supply performance of the hydrogels in Comparative Examples 1-4 and Example 3 to L929 cells, wherein A is a fluorescence image and B is a relative fluorescence intensity graph;

[0033] Fig.12 The ability of the hydrogel to remove excess reactive oxygen species in cells, where A is the fluorescence image and B is the relative fluorescence intensity image;

[0034] Fig.13 Graphs showing the in vitro antibacterial performance of the hydrogels in Comparative Examples 1-5 and Example 3, wherein A and B are graphs showing the antibacterial performance against Escherichia coli, C and D are graphs showing the antibacterial performance against Staphylococcus aureus, and E and F are graphs showing the antibacterial performance against MRSA;

[0035] Fig.14 The performance diagram of the hydrogel in Example 3 and Comparative Example 2 in promoting cell migration, wherein A is a cell migration diagram and B is a cell migration rate diagram;

[0036] Fig.15 Graphs showing the in vitro angiogenesis ability of the hydrogels in Example 3 and Comparative Example 2, wherein A is a graph showing in vitro angiogenesis, B is a graph showing a quantitative analysis of the corresponding number of nodes, C is a graph showing a quantitative analysis of the number of blood vessels, and D is a graph showing a quantitative analysis of the vascular network;

[0037] Fig.16 The biocompatibility graphs of COH-GB hydrogels of Examples 1-4, wherein A is a cytotoxicity graph, B is a blood compatibility graph, and C is an in vivo biological toxicity graph;

[0038] Fig.17 The figures are the treatment diagrams of the hydrogels in Comparative Examples 1-4 and Example 3 on MRSA-infected diabetic wounds in mice, wherein A is the animal experiment flow chart, B is the wound diagram at different days, and C is the wound area statistical analysis diagram;

[0039] Fig.18 H&E and Masson staining of wound tissues after 14 days of treatment with the hydrogels in Comparative Examples 1-4 and Example 3 (scale bar: 2 mm; 200 μm);

[0040] Fig.19 Figures 1-4 and 3 show the NO release and antibacterial performance of the hydrogels in vivo, wherein A is the Gram staining image of the diabetic wound tissue after 2 hours of treatment, and B is the NO fluorescence image and the corresponding relative fluorescence intensity image of the wound tissue after 24 hours of treatment;

[0041] Fig. 20 The graphs show the in vivo anti-inflammatory and angiogenic properties of the hydrogels in Comparative Examples 1-4 and Example 3. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0043] Example 1

[0044] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 1 comprises the following steps:

[0045] S1: 1 mg of GOx and 1 mg of Hb were added to 1 mL of phosphate buffered saline (PBS) and then 20 μL of calcium chloride (CaCl 2 ) aqueous solution and incubated at room temperature for 12 h to obtain a crude product solution, the crude product solution was centrifuged at 14000 rpm for 8 min to remove excess calcium ions and unbound GOx and Hb, the precipitate after centrifugation was resuspended in 1 mL of PBS, and the reaction was repeated three times to obtain nanoflowers loaded with glucose oxidase and hemoglobin, named GB1; the concentration of PBS was 3 mM, and the concentration of calcium chloride solution was 200 mM;

[0046] S2: 0.0072 g of hydroxyurea solid and 3 mL of carboxymethyl chitosan (CMCs) aqueous solution were mixed to obtain a hydroxyurea-carboxymethyl chitosan aqueous solution, and then 100 μL of GB1, 300 μL of oxidized sodium alginate (OSA) aqueous solution, and 600 μL of hydroxyurea-carboxymethyl chitosan aqueous solution were mixed to obtain the artificial multi-enzyme nanoflower composite hydrogel wound dressing (COH-GB1) of Example 1. The mass fraction of the oxidized sodium alginate aqueous solution was 4%, and the mass fraction of the carboxymethyl chitosan aqueous solution was 6%.

[0047] Example 2

[0048] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 2 is roughly the same as that of Example 1. The difference between the preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 2 and Example 1 is that in Example 2, the precipitate is resuspended in 0.5 mL of PBS, and the process is repeated three times to obtain nanoflowers loaded with glucose oxidase and hemoglobin, which are named GB2; the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 2 is named COH-GB2.

[0049] Example 3

[0050] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 3 is roughly the same as that of Example 1. The difference between the preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 3 and Example 1 is that in Example 3, the precipitate is resuspended in 0.333 mL of PBS, and the process is repeated three times to obtain nanoflowers loaded with glucose oxidase and hemoglobin, which are named GB3; the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 3 is named COH-GB3.

[0051] Example 4

[0052] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 4 is roughly the same as that of Example 1. The difference between the preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 4 and Example 1 is that in Example 4, the precipitate is resuspended in 0.25 mL of PBS, and the process is repeated three times to obtain nanoflowers loaded with glucose oxidase and hemoglobin, which are named GB4; the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Example 4 is named COH-GB4.

[0053] Comparative Example 1

[0054] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Comparative Example 1 comprises the following steps:

[0055] 0.0072 g of hydroxyurea was mixed with 3 mL of CMCs aqueous solution to obtain a hydroxyurea-carboxymethyl chitosan aqueous solution; 300 μL of OSA aqueous solution and 600 μL of hydroxyurea-carboxymethyl chitosan aqueous solution were mixed to obtain the artificial multi-enzyme nanoflower composite hydrogel of Comparative Example 1, named COH hydrogel. The mass fraction of OSA aqueous solution was 4%, and the mass fraction of CMCs aqueous solution was 6%.

[0056] Comparative Example 2

[0057] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Comparative Example 2 comprises the following steps:

[0058] 0.0072g of hydroxyurea was mixed with 3mL of CMCs aqueous solution to obtain hydroxyurea-carboxymethyl chitosan aqueous solution, 1mL of glucose oxidase solution was mixed with 1mL of hemoglobin solution to form a free GOx and Hb mixed solution, 100μL of free GOx and Hb mixed solution, 300μL of OSA aqueous solution, and 600μL of hydroxyurea-carboxymethyl chitosan aqueous solution were mixed to obtain the artificial multi-enzyme nanoflower composite hydrogel of Comparative Example 2, named COH-GOx-Hb hydrogel. The concentration of GOx solution was 1mg / mL, the concentration of Hb solution was 1mg / mL, the mass fraction of OSA aqueous solution was 4%, and the mass fraction of CMCs aqueous solution was 6%.

[0059] Comparative Example 3

[0060] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Comparative Example 3 comprises the following steps:

[0061] 0.0072 g of hydroxyurea was mixed with 3 mL of CMCs aqueous solution to obtain a hydroxyurea-carboxymethyl chitosan aqueous solution; 100 μL of Hb solution, 300 μL of OSA aqueous solution, and 600 μL of hydroxyurea-carboxymethyl chitosan aqueous solution were mixed to obtain the artificial multi-enzyme nanoflower composite hydrogel of Comparative Example 3, named COH-Hb hydrogel. The concentration of the Hb solution was 1 mg / mL, the mass fraction of the oxidized sodium alginate aqueous solution was 4%, and the mass fraction of the carboxymethyl chitosan aqueous solution was 6%.

[0062] Comparative Example 4

[0063] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Comparative Example 4 comprises the following steps:

[0064] 0.0072 g of hydroxyurea was mixed with 3 mL of CMCs aqueous solution to obtain a hydroxyurea-carboxymethyl chitosan aqueous solution, and 100 μL of GOx solution, 300 μL of OSA aqueous solution, and 600 μL of hydroxyurea-carboxymethyl chitosan aqueous solution were mixed to obtain the artificial multi-enzyme nanoflower composite hydrogel of Comparative Example 4, named COH-GOx hydrogel. The concentration of GOx solution was 1 mg / mL, the mass fraction of oxidized sodium alginate aqueous solution was 4%, and the mass fraction of carboxymethyl chitosan aqueous solution was 6%.

[0065] Comparative Example 5

[0066] The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of Comparative Example 5 comprises the following steps:

[0067] 100 μL PBS solution, 300 μL OSA aqueous solution, and 600 μL CMCs aqueous solution were mixed to obtain the artificial multi-enzyme nanoflower composite hydrogel of Comparative Example 5, named CO hydrogel. The mass fraction of the oxidized sodium alginate aqueous solution was 4%, the mass fraction of the carboxymethyl chitosan aqueous solution was 6%, and the concentration of the PBS solution was 3 mM.

[0068] Figure 1 The preparation process and working principle diagram of the artificial multi-enzyme nanoflower composite hydrogel wound dressing of the present invention. GOx-catalyzed glucose oxidation in COH-GB hydrogel can reduce local blood sugar levels and pH values, improve the microenvironment of diabetic wounds, and provide H for the generation of NO in the presence of Hb. 2 O 2 Subsequently, the cascade reaction triggered by high blood sugar makes COH-GB hydrogel have efficient antibacterial and biofilm removal functions. When the local blood sugar of the wound drops to normal level, HU will release a small amount of NO to promote angiogenesis. In addition, Hb can deliver O 2 , relieve the hypoxic state of MRSA-infected diabetic wounds, thereby accelerating the consumption of glucose and H 2 O2 The COH-GB hydrogel prepared by the present invention can reshape the diabetic wound microenvironment by reducing the local blood glucose concentration and pH, relieving hypoxia and removing excessive ROS; and release different doses of NO at different blood glucose levels to exert antibacterial or angiogenic functions, thereby effectively promoting the healing of MRSA-infected diabetic wounds.

[0069] from Figure 2 A- Figure 2 C It can be seen that GB nanoflowers are formed by the aggregation and assembly of many nanoclusters, showing a flower-like appearance with a particle size of about 3.5 μm. Subsequently, the hemoglobin colorimetric kit and the micro-glucose oxidase activity kit were used to detect the Hb content and GOx activity in GB nanoflowers. The results are shown in Figure 2 D- Figure 2 As shown in E, both Hb concentration and GOx activity increased with the increase of GB nanoflower concentration. In the presence of glucose, tetramethylbenzidine (TMB) was used as a chromogenic substrate to evaluate the peroxidase activity of Hb in GB nanoflowers. Figure 2 F, These results demonstrate the successful preparation of GB nanoflowers.

[0070] The Michaelis-Menten kinetic model was used to evaluate the activity of GOx in GB nanoflowers. Figure 3 A- Figure 3 As shown in Figure B, compared with free GOx, GB nanoflowers have a lower Michaelis constant (Km) and a higher maximum reaction rate (Vmax), which means that GOx in GB nanoflowers acts as an anti-competitive inhibitor and shows better affinity for the substrate glucose. Figure 3 C- Figure 3 As can be seen in D, the formation of GB nanoflowers can improve the tolerance of GOx to extreme environments and prolong the storage time at room temperature.

[0071] Since GOx-catalyzed glucose oxidation is an oxygen-consuming reaction, O is provided in the process. 2 Hemoglobin is a special protein that transports oxygen in red blood cells. 2 , and releases O according to the surrounding oxygen concentration 2 Therefore, the oxygen carrying capacity of GB nanoflowers was investigated, and the results are as follows Figure 4 As shown, O 2 The concentration gradually decreased over time, confirming that O 2 It is an indispensable participant in the reaction between glucose and GOx; while O 2The concentrations gradually increased, verifying the oxygen-releasing behavior of Hb under hypoxic conditions.

[0072] A hydrogel with a loose porous structure was selected as a carrier of GB nanoflowers and HU to prepare wound dressings. COH-GB hydrogel can be prepared in about 100 seconds by simply mixing OSA, CMCs, HU and GB together. The gelation time of COH-GB hydrogel is negatively correlated with the concentration of GB nanoflowers. In addition, the injectability and shape adaptability of COH-GB hydrogel were also verified. The results are as follows Figure 5 As shown in Figure B, COH-GB hydrogel can be conveniently used to fill irregular wounds through minimally invasive injection. Hydrogels with self-healing ability can prolong the service life, so the self-healing property of COH-GB hydrogel was evaluated. The results are shown in Figure 2. Figure 5 As shown in Figure C, two COH-GB hydrogels dyed with methylene blue and one without methylene blue dye were cut into two halves respectively, and then the two halves of the hydrogels of different colors were put together and placed at ambient temperature. The COH-GB hydrogels fused into one within 1.0 h without any external intervention. The healed COH-GB hydrogel can bear its own weight and can be stretched without breaking. The good self-healing performance of COH-GB hydrogel is attributed to the dynamic Schiff base bonds between OSA and CMCs.

[0073] from Figure 6 It can be seen that the internal morphology of COH-GB hydrogel shows a uniform honeycomb porous structure. The average pore sizes of COH, COH-GB1, COH-GB2, COH-GB3 and COH-GB4 hydrogels are 110.24±69.05μm, 83.48±38.83μm, 62.11±27.49μm, 49.95±21.17μm and 36.11±16.49μm, respectively. The average pore sizes of COH, COH-GB1, COH-GB2, COH-GB3 and COH-GB4 hydrogels decrease with the increase of GB nanoflower concentration, which may be due to the increase in cross-linking density caused by the added GB nanoflowers.

[0074] According to the "moisture healing theory", keeping the wound moderately moist is conducive to wound healing, which requires the wound dressing to have a certain water content and water vapor transmission rate (WVYR). Figure 7 It can be seen from A that the WVYR of COH-GB hydrogels of Examples 1-4 is about 2900 g / (m 2 ·d), compared with the ideal medical dressing WVYR (2500g / (m 2 ·d)) No statistical difference; Figure 7B It can be seen that the water content of COH-GB hydrogels of Examples 1-4 is above 96%, which is similar to the water content of natural extracellular matrix. In addition, COH-GB hydrogels of Examples 1-4 reach swelling equilibrium in PBS (pH=7.4) at 37°C for 120 minutes, and the swelling rate is above 252%. These results prove that COH-GB hydrogels of Examples 1-4 can quickly absorb wound exudate and maintain a moist environment at the wound site, which is mainly due to the loose and porous internal structure of COH-GB hydrogels. In addition, COH-GB hydrogels of Examples 1-4 can maintain a gel state for more than 21 days under simulated physiological conditions, meeting the long-term needs of wound healing.

[0075] As a physical barrier between the wound and the external environment, wound dressings need to have good adhesion properties. Figure 8 It can be seen that from a macroscopic point of view, COH-GB hydrogels show good adhesion properties in non-biological objects such as glass, plastic, wood and biological tissues such as mouse skin, heart, liver, spleen, lung, and kidney. The adhesion strength of COH-GB hydrogels increases with the increase of GB nanoflower content, which may be due to the fact that GB nanoflowers enhance the cross-linking density of COH-GB hydrogels and further improve their cohesion. In addition, rheological experiments confirmed that COH-GB hydrogels have good elasticity and structural stability.

[0076] The complex microenvironment in diabetic wounds, such as high blood sugar, high pH (7.0-8.9), hypoxia and excessive reactive oxygen species, seriously hinder the healing process. Therefore, the COH-GB hydrogels of Examples 1-4 and the COH hydrogel of Comparative Example 1 were studied for glucose consumption, pH reduction, O 2 The production of NO and ROS scavenging ability were used to demonstrate its microenvironment regulation ability. Fig. 9 A and Fig. 9 B It can be seen that compared with COH hydrogel, COH-GB hydrogel of Examples 1-4 can effectively consume glucose and reduce the pH value. The acidic microenvironment can reduce the microbial load on the wound surface and inhibit bacterial colonization.

[0077] Conventional Griess analysis confirmed the effective release of NO from COH-GB hydrogels via a glucose-activated cascade reaction. Fig. 9 As can be seen from A and 10A, COH-GB3 hydrogel exhibited the best hypoglycemic effect and NO generation, inducing glucose to decrease from 16.7mM to 4.33mM and pH to decrease from 8.1 to 6.04 within 48h. In the presence of 16.7mM glucose, the NO generated by the hydrogels of Comparative Examples 1-4 and Example 3 was detected, and the results are as follows: Fig.10As shown in B, there is no significant difference in the NO produced by COH, COH-Hb and COH-GOx hydrogels compared with the Control group (containing only 16.7 mM glucose aqueous solution), while the NO concentrations produced by COH-GOx-Hb and COH-GB hydrogels gradually increased; among them, the amount of NO released by COH-GB hydrogel was significantly higher than that of COH-GOx-Hb hydrogel, indicating that GB nanoflowers, as artificial multi-enzyme complexes, can effectively improve the efficiency of cascade reactions. As the cascade reaction proceeds, the initial concentration of 16.7 mM glucose is consumed to 4.33 mM. The NO release of hydrogels with different components was further tested in the presence of 4.33 mM glucose, and the results are shown in the figure. Fig.10 As shown in Figure C, the release behavior of NO in each group of hydrogels was similar to that in the presence of 16.7 mM glucose, but the NO concentration was significantly reduced. The above results show that COH-GB hydrogels can quickly release high-concentration NO in a hyperglycemic environment to exert antibacterial effects, and continue to produce low-concentration NO to promote angiogenesis when blood sugar drops to normal levels, thereby effectively accelerating wound healing. Fig.10 Con in C is an aqueous solution containing only 16.7 mM glucose.

[0078] The hypoxic microenvironment in infected diabetic wounds will significantly limit the reaction between glucose and GOx. 2 Therefore, the hypoxia indicator probe [Ru(dpp) 3 ]Cl 2 The oxygen supply performance of COH-GB hydrogel to L929 cells was further investigated. 3 ]Cl 2 In anoxic environment, it shows red fluorescence. Fig.11 As can be seen in the figure, the red fluorescence intensity observed in L929 cells treated with COH-GOx hydrogel was the strongest, proving that the reaction between glucose and GOx requires the consumption of O 2 The red fluorescence intensity of COH-GOx-Hb and COH-GB hydrogel groups was significantly reduced, indicating that the cell hypoxia caused by glucose oxidation has been effectively alleviated. 2 The efficiency of glucose-activated cascade reactions will be increased, thereby more effectively improving the wound microenvironment regulation and NO release performance of COH-GB hydrogels.

[0079] Appropriate levels of ROS in the body can effectively promote antibacterial and wound healing, while excessive ROS production in diabetic wounds can lead to oxidative stress, causing the wound to enter a chronic inflammatory stage. Therefore, wound dressings with good antioxidant properties are of great significance. 2 O 2A cell oxidative stress model was constructed, and the DCFH-DA fluorescent probe was used as an indicator of intracellular ROS to investigate the scavenging ability of COH-GB hydrogel on intracellular ROS. Fig.12 As shown, compared with the positive control (1 mM H 2 O 2 Compared with CO hydrogels formed by matrix materials alone, the green fluorescence intensity of the COH hydrogel group was significantly reduced, indicating that HU has certain antioxidant properties. Compared with COH hydrogels, COH-GOx hydrogels showed an increase in green fluorescence, which was attributed to the H produced by the reaction of glucose and GOx. 2 O 2 Almost no green fluorescence was detected in the COH-GOx-Hb hydrogel and COH-GB hydrogel groups, which may be due to the antioxidant properties of HU and the effect of HU and Hb on H 2 O 2 COH-GB hydrogel can effectively consume the excess H generated during glucose oxidation. 2 O 2 Converted into NO, thereby alleviating intracellular oxidative stress and promoting wound healing.

[0080] The complex microenvironment of diabetic wounds makes them susceptible to bacterial infection, which can cause persistent inflammation and hinder the healing process. Therefore, inhibiting bacterial proliferation plays a key role in diabetic wound healing. The present invention selects the most common bacteria that cause wound infections, including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and MRSA to investigate the antibacterial activity of COH-GB hydrogel. E. coli, S. aureus and MRSA were incubated with the hydrogels of Example 3 and Comparative Examples 1-5 for 2h and 4h, and the bactericidal rate was calculated based on the absorbance of the bacterial suspension at 600nm, and the surviving bacteria were counted by the plate count method. The results are shown in the figure. Fig.13 As shown in the figure, there is no significant difference in the antibacterial effect of COH and COH-Hb hydrogels, and both are higher than that of CO hydrogel, which is attributed to the antibacterial activity of HU. 2 O 2With the decrease of pH value, COH-GOx hydrogel showed more effective antibacterial effect than COH and COH-Hb hydrogel. After 2h of co-incubation, the antibacterial activities of COH-GOx-Hb hydrogel against E.coli, S.aureus and MRSA were 81.083±4.247%, 78.733±0.905% and 80.243±0.294%, respectively, and the corresponding bactericidal rates of COH-GB hydrogel were 99.227±0.168%, 85.157±1.199% and 90.227±0.373%, respectively, indicating that COH-GB hydrogel has effective antibacterial activity. This may be attributed to the cascade reaction between glucose, GOx, Hb and HU, as well as the synergistic antibacterial effect of NO and pH reduction. In addition, a higher bacterial killing rate was observed in COH-GB hydrogel, which once again proved that the formation of GB nanoflowers can induce a higher cascade reaction efficiency than free GOx and Hb, thus facilitating the release of NO and antibacterial properties. In addition, after 4 h of co-incubation, there was no significant difference in the bactericidal rates of COH-GOx-Hb and COH-GB hydrogels against E. coli and MRSA, except for S. aureus. The antibacterial performance of COH-GB hydrogel against E. coli was superior to that of S. aureus and MRSA, which should be due to the differences in the cell wall structure or composition of the two bacteria. Fig.13 The control was a treatment without adding any hydrogel.

[0081] Lack of angiogenesis is one of the main reasons that hinder diabetic wound healing. NO has been widely shown to promote angiogenesis. HUVECs were used to evaluate the angiogenic effect of COH-GB hydrogel in vitro. Cell migration is the basis of angiogenesis and fibroblast proliferation. The cell scratch test was used to evaluate the migration ability of HUVECs. Fig.14 It can be seen that compared with the Control (no hydrogel treatment), the scratch area of ​​the COH-GOx-Hb and COH-GB hydrogel groups gradually decreased over time, and the smallest scratch area was observed in the COH-GB hydrogel group. After 12 hours of co-culture, the cell migration rates of the COH-GOx-Hb group (31.657±8.191%) and the COH-GB group (35.697±1.286%) were higher than those of the Control (3.24±2.037%). After 24 hours, the cell migration rate of the COH-GB group (62.480±7.905%) was significantly higher than that of the COH-GOx-Hb group (42.960±4.386%), indicating that the motility of HUVECs was significantly enhanced after treatment with COH-GB hydrogel. The in vitro pro-angiogenic effect of COH-GB hydrogel was further studied using the HUVECs tube formation experiment. Fig.15It can be seen that compared with the Control (no hydrogel treatment), both the COH-GOx-Hb and COH-GB hydrogel groups showed a more developed tubular network, and semi-quantitative analysis further verified the results. Among them, COH-GB hydrogel has efficient in vitro tubular ability, with the largest number of knots (1720±43.55), blood vessels (354.3±79.75), and vascular networks (187±51.03). These results indicate that COH-GB hydrogel can significantly accelerate cell migration and enhance the ability of endothelial cells to form tubes, thereby helping to promote angiogenesis, which may be due to the fact that GB nanoflowers in COH-GB hydrogels produce NO more effectively than free GOx and Hb, thereby more effectively promoting angiogenesis and wound repair at the wound site.

[0082] Biocompatibility is a prerequisite for the application of hydrogel dressings in clinical wound management. The cell compatibility, blood compatibility and in vivo biosafety of COH-GB hydrogel were evaluated. Fibroblasts play a vital role in wound repair. L929 cells were used to evaluate the cytotoxicity of COH-GB hydrogel. According to the CCK-8 test results, Fig.16 As can be seen from A, the survival rates of L929 cells after incubation with the COH-GB hydrogel extract of Examples 1-4 for 24 hours and 48 hours were 93.673±2.163% and 90.640±0.485%, respectively, which meets the international standard for biocompatibility evaluation of biomaterials, that is, the relative cell proliferation rate should be greater than 70% (ISO10993:2009), proving the excellent cell compatibility of COH-GB hydrogel. The blood compatibility of COH-GB hydrogel was evaluated by the mouse fresh blood hemolysis test. Fig.16 B shows that the positive control group (ddH 2 O) was noticeably red in supernatant due to rupture of red blood cells and release of hemoglobin; while the normal saline (NaCl) group and the COH-GB hydrogel group showed a transparent state. In addition, the hemolysis rates of the COH-GB hydrogel groups were significantly lower than 5% (internationally recognized standard), indicating that COH-GB gel has excellent blood compatibility. In addition, the in vivo biosafety of COH-GB hydrogel was also evaluated. COH-GB hydrogel was implanted into mice by subcutaneous injection, and major organs (including heart, liver, spleen, lungs, kidneys) and muscles in contact with COH-GB hydrogel were obtained on days 0, 3, 7, and 14, respectively. Fig.16 As can be seen in Figure C, no obvious histological abnormalities were observed in the H&E stained tissue sections of the main organs, indicating that COH-GB hydrogel has good tissue compatibility. The above results show that COH-GB hydrogel has excellent biocompatibility and can be used as wound dressing.

[0083] Diabetic ulcer is a typical chronic non-healing wound, which is prone to persistent microbial infection, leading to excessive inflammatory cells and cytokine aggregation, destroying angiogenesis and tissue remodeling, and ultimately leading to about 41% of amputations. Diabetic ulcer is a huge challenge in the biomedical field. COH-GB hydrogel has the characteristics of glucose response, hypoxia relief, anti-oxidation, antibacterial, enhanced cell migration and angiogenesis in vitro. The in vivo therapeutic effect of COH-GB hydrogel was evaluated by constructing a MRSA-infected diabetic mouse wound model. First, streptozotocin (STZ) was intraperitoneally injected into healthy mice. When the fasting blood glucose level exceeded 16.7mM for one week and was accompanied by weight loss, the diabetic mouse model was successfully constructed. Then, a full-thickness skin defect model with a diameter of 0.8cm was constructed on the back of the mouse, and then MRSA infection was used for 24h to establish a MRSA-infected diabetic full-thickness wound. The animals were randomly divided into 7 groups (n=10), and treated with 3M film dressings (3M Film) and the hydrogels in Comparative Examples 1-4 and Example 3, respectively, and the untreated mice were used as controls. Fig.17 In A and 17B, Control is the control group of mice that did not receive treatment.

[0084] Fig.17 B is a representative photograph of the wounds on days 0, 3, 7, 10, and 14 of treatment. Image J was used to quantitatively analyze the wound area. Fig.17 As shown in C, the wound area gradually decreased over time, and the COH-GB group showed a significantly faster healing speed. On the 10th day, the remaining wound area in the COH-GB group was 13.200±1.758%, which was significantly lower than that in the Control (no treatment) (40.577±6.516%), 3M Film group (37.640±7.544%), COH group (28.917±12.092%), COH-Hb group (27.307±7.041%), COH-GOx group (26.160±9.058%) and COH-GOx-Hb group (17.497±2.813%). On day 14, the infected wounds in the COH-GB group were almost completely healed, with a wound remaining rate of 1.093±1.89%, and the unhealed wounds in the Control, 3M Film, COH, COH-Hb, COH-GOx, and COH-GOx-Hb groups were 20.737±2.755%, 18.693±1.167%, 12.3±0.926%, 9.650±3.309%, 6.853±2.004%, and 4.940±0.159%, respectively. These results indicate that the therapeutic effect of COH-GB hydrogel on infected diabetic wounds is significantly better than that of commercial 3M film dressings (3M Film) and COH-GOx-Hb hydrogels made with free GOx and Hb.

[0085] Subsequently, H&E and Masson staining were performed on the regenerated skin tissue to further evaluate the wound healing quality. Fig.18 As shown in the figure, after 14 days of treatment, residual scabs (dry blood and exudate scabs) were still observed in the Control (no treatment), 3M Film group, COH group, COH-Hb group and COH-GOx group, indicating that the chronic wound healing process was delayed; no intermediate scabs were found in the COH-GOx-Hb group and COH-GB group. In addition, a large number of dead inflammatory cells wrapped in scabs could be observed in the Control, 3M Film, COH and COH-Hb groups, indicating that a severe inflammatory response occurred in the early stage of healing (marked by yellow circles); while a large number of inflammatory cells were present in the dermis of the COH-GOx group. In contrast, the COH-GB group and COH-GOx-Hb group had a milder inflammatory response that was beneficial to wound healing. At the same time, Masson staining showed that in the COH-GB and COH-GOx-Hb groups, the formation of granulation tissue was significantly enhanced, the organizational structure of the regenerated skin was better, and there were more newly formed skin appendages (hair follicles and sebaceous glands). In addition, compared with the COH-GOx-Hb group, more new collagen deposition and smaller diameter of the dermis without appendages were observed in the COH-GB group. The above results indicate that COH-GB hydrogel can promote epidermal formation, increase collagen deposition, and promote skin tissue regeneration, thereby accelerating the healing process of infected diabetic wounds.

[0086] The high blood sugar level in diabetic patients makes skin wounds more susceptible to bacterial infection, so the in vivo antibacterial ability of wound dressings is crucial. Gram staining images of infected wound tissues after 2 hours of different treatments are shown in Figure 2. Fig.19 As shown in A, the in vivo antibacterial properties of hydrogels with different compositions were similar to those in vitro, and the COH-GB group almost completely eliminated bacteria. In addition, the number of bacteria in the wound was positively correlated with the residual wound area, confirming that eliminating MRSA infection will promote diabetic wound healing.

[0087] Since the antibacterial activity of COH-GB hydrogels mainly depends on the release of NO, the in vivo NO release behavior of COH-GB hydrogels was investigated by fluorescence imaging of wound tissues after staining with the NO intracellular probe DAF-FM DA (green fluorescence). After 24 h of treatment, high levels of green fluorescence were observed only in the COH-GB group, as shown in Figure 2. Fig.19B. The relative fluorescence intensity showed that the NO produced by the COH-GB group was significantly higher than that of the COH-GOx-Hb group, proving that the preparation of GB nanoflowers as an artificial multi-enzyme complex can significantly improve the cascade catalytic efficiency compared with free enzymes. The above results confirm that the endogenous blood glucose concentration in the skin tissue of diabetic mice is sufficient to activate the cascade reaction mediated by GB nanoflowers, thereby triggering the release of NO to the surrounding wound tissue, thereby helping the wound to effectively resist bacterial infection.

[0088] Blood vessels can deliver nutrients, oxygen, and bioactive factors required for wound healing, so promoting angiogenesis is beneficial to accelerate wound healing. Since COH-GB hydrogel can induce angiogenesis in vitro, its in vivo vascular remodeling performance was further evaluated. CD31 is a transmembrane protein that is usually used to prove the presence of endothelial cells, and VEGF is a vascular endothelial growth factor. Therefore, immunofluorescence staining analysis of CD31 and VEGF in new skin tissue was used to evaluate the in vivo angiogenesis of COH-GB hydrogel, and the results are shown in Figure 2. Fig. 20 As shown, no obvious red fluorescence of CD31 and VEGF was observed in the Control and 3M Film groups, while the fluorescence intensity corresponding to CD31 and VEGF in COH-GB hydrogel was significantly higher than that in other groups, indicating that COH-GB gel has superior angiogenesis-promoting ability.

[0089] In addition, the activation of inflammatory factors is a key process in wound healing. However, excessive inflammation can cause tissue damage and delay the healing process. Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation, mainly produced by activated macrophages and lymphocytes, among which TNF-β is a cytokine secreted by lymphocytes, which can mediate various inflammatory, immune stimulation and antiviral responses. Interleukin (IL)-4 is an important anti-inflammatory cytokine that not only widely inhibits the expression of other proinflammatory factors and inflammatory mediators, but also promotes the secretion of anti-inflammatory factors, thereby maintaining the balance between proinflammatory and anti-inflammatory factors. Therefore, the expression of TNF-β and IL-4 was used to evaluate the inflammatory response in the wound area. The TNF-β levels in the Control (no treatment) and 3M Film groups were significantly increased, while almost no TNF-β expression was found in the COH-GOx-Hb and COH-GB hydrogel groups. On the contrary, the levels of IL-4 gradually increased in the Control (no treatment), 3M Film group, COH group, COH-Hb group, COH-GOx-Hb group, and COH-GB group, among which the expression level in the COH-GB group was the highest, indicating that COH-GB hydrogel can promote the balance of inflammatory response at the wound site by downregulating the expression of pro-inflammatory factors and upregulating the expression of anti-inflammatory factors.

Claims

1. A method for preparing an artificial multi-enzyme nanoflower composite hydrogel wound dressing, characterized in that: The nanoflowers loaded with glucose oxidase and hemoglobin and NO donors are co-incorporated into a matrix to prepare the nanoflowers.

2. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 1, characterized in that: The NO donor is hydroxyurea.

3. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 2, characterized in that: The matrix is ​​a mixed matrix of oxidized sodium alginate aqueous solution and carboxymethyl chitosan aqueous solution.

4. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 3, characterized in that: The preparation method of the artificial multi-enzyme nanoflower composite hydrogel wound dressing comprises the following steps: mixing hydroxyurea with a carboxymethyl chitosan aqueous solution to obtain a hydroxyurea-carboxymethyl chitosan aqueous solution, and mixing nanoflowers loaded with glucose oxidase and hemoglobin, an oxidized sodium alginate aqueous solution, and a hydroxyurea-carboxymethyl chitosan aqueous solution to obtain the wound dressing.

5. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 4, characterized in that: For every mL of the carboxymethyl chitosan aqueous solution, 0.0020-0.0030 g of hydroxyurea is added, and for every 100 μL of the nanoflowers loaded with glucose oxidase and hemoglobin, 250-350 μL of the oxidized sodium alginate aqueous solution is added; for every 100 μL of the nanoflowers loaded with glucose oxidase and hemoglobin, 550-650 μL of the hydroxyurea-carboxymethyl chitosan aqueous solution is added; the mass fraction of the oxidized sodium alginate aqueous solution is 2-6%, and the mass fraction of the carboxymethyl chitosan aqueous solution is 4-10%.

6. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 1, characterized in that: The preparation method of the nanoflower loaded with glucose oxidase and hemoglobin is as follows: glucose oxidase and hemoglobin are added into a phosphate buffered saline solution, then a calcium chloride aqueous solution is added for incubation to obtain a crude product solution, the crude product solution is centrifuged to obtain a precipitate, and the precipitate is resuspended to obtain the nanoflower.

7. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 6, characterized in that: The mass ratio of the glucose oxidase to the hemoglobin is 1:1-2, 0.5-3 mL of the phosphate buffered saline solution is added for every mg of the glucose oxidase, and 10-50 μL of the calcium chloride aqueous solution is added for every mg of the glucose oxidase; The concentration of the phosphate buffered saline solution is 1-10 mM, and the concentration of the calcium chloride aqueous solution is 50-500 nM.

8. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 6, characterized in that: The incubation is carried out at room temperature for 6-24 hours.

9. The method for preparing the artificial multi-enzyme nanoflower composite hydrogel wound dressing according to claim 6, characterized in that: The resuspension is to resuspend the pellet in 0.25-1 mL of phosphate buffered saline.

10. An artificial multi-enzyme nanoflower composite hydrogel wound dressing, characterized in that: The artificial multi-enzyme nanoflower composite hydrogel wound dressing is prepared by the preparation method of any one of claims 1 to 9.

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