Hydrogel, preparation thereof and medical dressing containing hydrogel

Abstract

The invention provides hydrogel, preparation of the hydrogel and medical dressing containing the hydrogel, CMC-PBA and HA-DA are used as hydrogel matrixes and are connected through boric acid ester bonds, and copper nano-enzyme (Cu NCs) chelated by EGCG is added into the hydrogel matrixes, so that the hydrogel (EGCG-Cu NPs) loaded with the copper nano-enzyme (injectable) is obtained. Wherein the CMC-PBA is a product obtained by reacting carboxymethyl chitosan (CMC) with carboxyl activated 4-carboxyphenylboronic acid (PBA), and the HA-DA is a product obtained by reacting carboxyl activated hyaluronic acid (HA) with dopamine hydrochloride (DA). The hydrogel can deeply promote wound recovery through in-vivo use, has good antioxidant activity and antibacterial activity, has extremely high active oxygen removal performance on necrotic cells of the wound, greatly accelerates the healing speed of the wound, brings clinical welfare to the wound, especially a chronic disease wound, and promotes the development of chronic wound diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a hydrogel and a preparation method thereof, and a medical dressing containing the hydrogel and application thereof. Background Art

[0002] The current clinical application technologies of hydrogels in wound treatment mainly include the following aspects:

[0003] 1. Wound dressing

[0004] Moisturizing and barrier effect: Hydrogel maintains a moist environment on the wound surface through its high water content, promotes cell migration and epithelial regeneration, and isolates external pollutants.

[0005] Autolytic debridement: It softens necrotic tissue by absorbing exudate, accelerates the decomposition of necrotic material by autologous enzymes, and reduces the need for mechanical debridement.

[0006] Indications: Commonly used for superficial burns, chronic ulcers (such as diabetic foot), donor site wounds and postoperative incisions.

[0007] 2. Drug delivery vehicles

[0008] Antimicrobial loading: silver ions, antibiotics (such as gentamicin) or natural antimicrobial ingredients (such as chitosan) are integrated to achieve local sustained release and control infection.

[0009] Controlled release of growth factors: loaded with EGF (epidermal growth factor), bFGF (basic fibroblast growth factor), etc. to promote angiogenesis and tissue repair.

[0010] Anti-inflammatory and analgesic: Delivers nonsteroidal anti-inflammatory drugs (such as ibuprofen) or local anesthetics to reduce pain and inflammation.

[0011] 3. Functional design

[0012] Responsive hydrogels: Temperature / pH-responsive materials (such as poly (N-isopropylacrylamide)) can regulate drug release rates according to changes in the wound environment.

[0013] Conductive hydrogels: Incorporating conductive polymers (such as polypyrrole) for electrical stimulation to assist chronic wound healing.

[0014] 3D printing customization: Use bioprinting technology to prepare personalized structures to fit the complex wound morphology.

[0015] 4. Combined therapy technology

[0016] Stem cell therapy: as a carrier of mesenchymal stem cells or adipose stem cells to enhance tissue regeneration capacity.

[0017] Photothermal therapy: Combined with photosensitizers (such as black phosphorus nanosheets), near-infrared light is used to trigger antibacterial and healing effects.

[0018] Negative pressure wound therapy (NPWT): used in conjunction with negative pressure devices to enhance exudate management and granulation tissue formation.

[0019] 5. Material Type

[0020] Natural hydrogels: hyaluronic acid, alginate, collagen, etc., have high biocompatibility but low mechanical strength.

[0021] Synthetic / semi-synthetic hydrogels: polyethylene glycol (PEG), carboxymethyl cellulose (CMC), etc., which can regulate degradability and mechanical properties.

[0022] The hydrogel used for wound treatment is mainly used externally, and is applied externally to patients to treat wounds by forming a hydrogel dressing or protective film. For example, CN111939308B discloses a medical hydrogel non-porous breathable film for wound healing, which comprises: a hydrogel formed by methacrylated gelatin-gN-isopropylacrylamide (GelMA-g-NIPAAm); copper-based MOF particles embedded in the hydrogel, wherein the copper-based MOF particles and the hydrogel form a functionalized medical hydrogel; a non-porous vapor barrier film or film laminate prepared from a thermoplastic resin composition; wherein the surface of the non-porous vapor barrier film or film laminate is modified and connected to the medical hydrogel or its surface. The medical hydrogel non-porous breathable film of the present invention has the advantages of high antibacterial properties, good waterproof vapor permeability, and non-adhesion, and is suitable for difficult-to-heal wounds such as common wounds, large-area burns, and diabetic hand and foot ulcers. Although these topical hydrogel products can have a certain therapeutic effect, they cannot treat deep wound damage deep below the body surface (cell tissue deep under the skin). Therefore, their therapeutic significance is not great, and they cannot promote cell growth from inside the wound and accelerate the treatment efficiency of the wound. Summary of the Invention

[0023] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0024] In one aspect, a hydrogel is provided, comprising the following components:

[0025] The first hydrogel matrix (CMC-PBA) is obtained by reacting carboxymethyl chitosan (CMC) with carboxyl-activated 4-carboxyphenylboronic acid (PBA);

[0026] as well as,

[0027] A second hydrogel matrix (HA-DA) is obtained by reacting hyaluronic acid (HA) with activated carboxyl groups with dopamine hydrochloride (DA);

[0028] as well as,

[0029] Copper nanozymes (Cu NCs) chelated by epigallocatechin gallate (EGCG): loaded polyphenol-copper-derived nanozymes (EGCG-CuNCs).

[0030] In another aspect, a method for preparing a hydrogel is provided, comprising the following steps:

[0031] EGCG-CuNCs powder was added to HA-DA solution and dissolved by ultrasonication;

[0032] Prepare HA-DA / Cu-EGCG solution;

[0033] Take the prepared CMC-PBA solution and add it into the centrifuge tube, add OH - solution, and then adding the HA-DA / EGCG-CuNCs solution, shaking and mixing to obtain the hydrogel (EGCG-Cu NPs) loaded with copper-derived nanozymes.

[0034] On the other hand, a medical dressing comprising the hydrogel is also provided.

[0035] In another aspect, a method for using the hydrogel by injection is also provided.

[0036] On the other hand, a use of the hydrogel in wound treatment is also provided.

[0037] On the other hand, the present invention also provides a use of the hydrogel in treating chronic diabetic wounds.

[0038] The beneficial effects brought about by the technical solution provided by the embodiment of the present invention include at least:

[0039] The hydrogel proposed in this invention is an injectable hydrogel loaded with copper metalloenzymes. Its basic characteristics are that it uses CMC-PBA and HA-DA as the hydrogel matrix, connected by boronate bonds, and copper nanozymes (Cu NCs) chelated with epigallocatechin gallate (EGCG) are added to the hydrogel, resulting in an injectable copper metalloenzyme-loaded hydrogel (EGCG-Cu NPs). CMC-PBA is the product of the reaction between carboxymethyl chitosan (CMC) and carboxyl-activated 4-carboxyphenylboronic acid (PBA), while HA-DA is the product of the reaction between carboxyl-activated hyaluronic acid (HA) and dopamine hydrochloride (DA). This hydrogel can profoundly promote wound healing through in vivo use. It exhibits excellent antioxidant and antibacterial activity, and is highly effective in scavenging reactive oxygen species (ROS) against necrotic wound cells, significantly accelerating wound healing. This provides clinical benefits for wounds, especially those with chronic diseases, and promotes the progression of chronic wound diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 is a transmission electron microscope (TEM) image of SalB-CuNCs provided in an embodiment of the present invention;

[0042] Figure 2 This is a local X-ray photoelectron spectrum of EGCG-CuNCs provided by an embodiment of the present invention;

[0043] Figure 3 This is an infrared absorption spectrum of EGCG and EGCG-CuNCs provided in an embodiment of the present invention;

[0044] Figure 4 This is a full-wavelength ultraviolet scanning curve diagram of EGCG and EGCG-CuNCs provided in an embodiment of the present invention;

[0045] Figure 5 5A and 5B show the appearance and injectability of a hydrogel provided in an embodiment of the present invention.

[0046] Figure 6 is a SEM image of a different hydrogel provided in an embodiment of the present invention;

[0047] Figure 7This is a diagram showing the appearance of a hemolyzed hydrogel incubated for 4 hours provided by an embodiment of the present invention (from left to right: PBS, CMC-PBA / HA-DA / EGCG-CuNCs, CMC-PBA / HA-DA / EGCG-CuNCs, and H2O);

[0048] Figure 8 This is a schematic diagram of the antibacterial activity cultivation of a hydrogel provided by an embodiment of the present invention;

[0049] Figure 9 This is a biocompatibility experiment provided by an embodiment of the present invention: electron micrographs of cell activity visualization analysis performed using a confocal microscope at 1 day, 2 days, and 3 days;

[0050] Figure 10 This is an intracellular ROS scavenging assay provided by an embodiment of the present invention: a visualization of the cell number measured by the DCFH-DA ROS probe;

[0051] Figure 11 This is a flow cytometric analysis of apoptotic HUVECs after treatment with different materials provided in an embodiment of the present invention;

[0052] Figure 12 This is a representative diagram of a chronic diabetic wound treated with different dressings provided in an embodiment of the present invention;

[0053] Figure 13 This is a performance diagram of the morphological characterization and enzyme-like activity evaluation of a nanozyme EGCG-CuNCs provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is described below in conjunction with the accompanying drawings.

[0055] In the embodiments of the present invention, words such as "exemplarily" and "for example" are used to indicate examples, illustrations, or explanations. Any embodiment or design described as an "exemplary" in the present invention should not be interpreted as being preferred or advantageous over other embodiments or designs. Rather, the use of the word "exemplary" is intended to present concepts in a concrete manner. Furthermore, in the embodiments of the present invention, "and / or" can mean both or either of the two.

[0056] In the embodiments of the present invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, when the distinction is not emphasized, the meanings they convey are the same. The terms "of," "corresponding," and "corresponding" may sometimes be used interchangeably. It should be noted that, when the distinction is not emphasized, the meanings they convey are the same.

[0057] In the embodiments of the present invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meanings to be expressed are the same.

[0058] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0059] In this embodiment, the experimental process, equipment and hydrogel component ratio involved can be determined according to laboratory standards or hydrogel models (divided into different grades, qualities, etc.), and the relevant preparation raw materials and preparation process equipment can be provided by the laboratory or purchased on the market.

[0060] The preparation and clinical validation of the hydrogel will be described in detail below.

[0061] An injectable hydrogel loaded with copper metalloenzymes (EGCG-Cu NPs) is characterized by using CMC-PBA and HA-DA as the hydrogel matrix, connected by boronate bonds, and then adding copper nanozymes (Cu NCs) chelated with epigallocatechin gallate (EGCG) to form the hydrogel matrix. CMC-PBA is the product of the reaction between carboxymethyl chitosan (CMC) and carboxyl-activated 4-carboxyphenylboronic acid (PBA), while HA-DA is the product of the reaction between carboxyl-activated hyaluronic acid (HA) and dopamine hydrochloride (DA).

[0062] 1. Hydrogel Preparation and In Vivo Experiments

[0063] 1.1 Materials

[0064] Epigallocatechin gallate (EGCG, purity ≥98.0%), carboxymethyl chitosan (CMC, degree of substitution: ≥80%), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, analytical grade), N-hydroxysuccinimide (NHS, analytical grade), and anhydrous copper sulfate (CuSO4, purity ≥99.9%) were all provided by MacLean Biotechnology Co., Ltd. Hyaluronic acid (HA, MW = 100,000) and dopamine hydrochloride (DA, purity ≥98.0%) were provided by Yuanye Biotechnology Co., Ltd. Tetracarboxyphenylboronic acid (PBA, purity ≥98%) was purchased from Bidex Pharmaceutical Technology Co., Ltd. Phosphate buffered saline (PBS) was purchased from Sewell Biotechnology Co., Ltd.

[0065] 1.2 Synthesis and characterization of CMC-PBA, HA-DA, and EGCG-Cu NPs

[0066] Synthesis of CMC-PBA: First, 4-carboxyphenylboronic acid (PBA) (465 mg, 2.8 mmol), EDC (546 mg, 2.8 mmol), and NHS (330 mg, 2.8 mmol) were weighed, sequentially added to 30 mL of DMSO, and activated at room temperature for 4 hours with stirring. 3 g of CMCS was dissolved in 400 mL of deionized water and stirred for half an hour to fully dissolve. Next, the activated PBA solution was slowly added to the CMCS solution using a 5 mL pipette. After addition, the pH of the solution was adjusted to approximately 8 with 0.1 M sodium hydroxide. The solution was stirred at room temperature for 48 hours. The resulting solution was then adjusted to approximately pH 9 with 0.1 M sodium hydroxide. The solution was transferred to a dialysis bag (MW: 3500) and dialyzed for three days, changing the water every four hours. After dialysis, it was freeze-dried for later use. The successful synthesis of CMCS-PBA was verified using NMR and IR.

[0067] Synthesis of HA-DA: First, dissolve hyaluronic acid (HA) (1 g) in 100 mL of deionized water. Under nitrogen, slowly add 1.48 g of EDC-HCl and 0.9 g of NHS. After vigorous stirring for 20 minutes, add 1480 mg of DA. Adjust the pH of the solution to 5.0-5.5 with 0.1 M hydrochloric acid and allow to react for 8 hours. Transfer the final product to a dialysis bag (8000-14000 Da). Dialyze for 48 hours to remove unreacted reagents and salts. After dialysis, freeze-dry and set aside. Successful synthesis of HA-DA was verified by NMR and IR.

[0068] Synthesis of EGCG-Cu NPs: EGCG-Cu NPs were prepared using a one-pot method. Briefly, 0.1 mL of CuSO4 solution (mW: 160) (0.1 mL, 0.1 M, prepared in 1 mL, weighing 16 mg) and 0.25 mL of EGCG solution (mW: 458.37) (0.25 mL, 16 mM, prepared in 10 mL, weighing 73 mg) were mixed and stirred at room temperature for 5 minutes. Subsequently, 0.1 mL of ascorbic acid solution (AA, 0.1 mL, 1 M, prepared in 1 mL, weighing 176 mg) was added. The mixture was stirred at 50°C for 24 hours to obtain a pale yellow solution. The solution was dialyzed using a dialysis bag (MW: 3500 Da) for 48 hours. After dialysis, the solution was stored at 4°C until use.

[0069] The morphology and shape of the nanoparticles were determined by TEM (JEM-2100) imaging. Fourier transform infrared spectroscopy (FT-IR) determined the composition of the nanoparticles. X-ray photoelectron spectroscopy (XPS) was performed using an X-ray diffractometer equipped with a copper sealed tube. The size distribution and zeta potential of the nanoparticles were analyzed using a Malvern Zetasizer Nano ZS dynamic light scattering analyzer (DLS).

[0070] 1.3 Construction and characterization of hydrogels

[0071] 1.31 Hydrogel Construction

[0072] First, 5 mg of EGCG-CuNCs powder was added to 5 mL of 4% HA-DA solution and sonicated for 3 minutes to dissolve the solution. This prepared a HA-DA / Cu-EGCG solution. Then, 200 μL of the prepared 4% CMC-PBA solution was added to a 2 mL centrifuge tube. 10 μL of 1M NaOH solution was added dropwise, followed by 200 μL of the HA-DA / EGCG-CuNCs solution. After oscillation and mixing, the CMC-PBA / HA-DA / EGCG-CuNCs composite hydrogel loaded with copper-derived nanozymes (EGCG-Cu NPs) was obtained. The hydrogel was named CH@EGCG-Cu.

[0073] Therefore, CMC-PBA / HA-DA hydrogel (CH@EGCG-Cu) was synthesized without the addition of EGCG-CuNCs.

[0074] The above preparation process and proportion are only one of those provided in this embodiment. In actual application, the user can set the proportion according to the required quality, grade and other standards.

[0075] 1.32 Characterization of hydrogels

[0076] To observe the internal microstructure of the hydrogel, 400 μL of the prepared hydrogel was refrigerated in a -20°C refrigerator, freeze-dried in a freeze dryer, and fixed to the electron microscope stage with conductive glue. After 90 s of gold spraying, its surface morphology was observed using a scanning electron microscope.

[0077] The swelling ratio (SR) of the hydrogel was determined using a swelling test. The hydrogel was placed in a sealed test tube, added to 20 mL of PBS (pH 7.4 or 5.0), and incubated at 37°C. After predetermined time intervals, the hydrogel was removed from the solution and weighed. SR was calculated using the following equation: SR = Wt / W0 × 100%, where W0 and Wt are the initial weight of the hydrogel and its weight after swelling at the predetermined time, respectively. This test was repeated three times.

[0078] In a DHR-2 rotational rheometer (TA Instruments, USA), a parallel plate (40 mm) measuring fixture was used to perform time sweep, steady-state shear, amplitude sweep, frequency sweep, step sweep, and creep recovery in sequence.

[0079] First, 200 μL of hydrogel was placed between the pigskins to be tested. The pigskins were overlapped and the hydrogel was squeezed evenly in the overlapping area. Subsequently, the breaking force (F) of the two pigskins was tested using a universal testing machine at a tensile speed of 10 mm / min. The lap shear joint between the experimental materials covered an area (S: 25 mm long × 12.5 mm wide × 1 mm thick). Three parallel samples were tested in each group (n=3). The adhesion force was calculated using the following formula:

[0080] First, 200 μL of hydrogel was placed between the pigskins to be tested. The pigskins were overlapped and the hydrogel was squeezed evenly in the overlapping area. Subsequently, the breaking force (F) of the two pigskins was tested using a universal testing machine at a tensile speed of 10 mm / min. The lap shear joint between the experimental materials covered an area (S: 25 mm long × 12.5 mm wide × 1 mm thick). Three parallel samples were tested in each group (n=3). The adhesion force was calculated using the following formula:

[0081]

[0082] PBS solution with pH=7.4 was used to simulate human body fluids, and the in vitro degradation performance of the hydrogel was measured at 37°C. The degradation status of the hydrogel was evaluated by the weight retention percentage.

[0083] 1.4 In vitro activity assay

[0084] 1.41 Hemolysis test

[0085] To evaluate the compatibility of the hydrogel with blood, the hydrogel was added to whole blood, incubated at 37°C for 60 minutes, and then the absorbance was measured at a wavelength of 545 nm. The negative and positive controls consisted of PBS and deionized water, respectively. The hemolysis rate was calculated as follows:

[0086]

[0087] Where: H is the hemolysis rate, %; Dt, Dnc and Dpc are the absorbance of the sample, negative control group and positive control group respectively.

[0088] 1.42 Antioxidant activity

[0089] To investigate the antioxidant activity of the hydrogels, their ability to scavenge ABTS+ and DPPH free radicals was evaluated. The hydrogel extracts were added to 3 mL of ABTS+ and DPPH working solutions, respectively, and incubated in the dark. Absorbance was measured in the corresponding nanometer wavelength range using ABTS+ and DPPH solutions without the hydrogel extracts as controls.

[0090] 1.42 Antimicrobial activity

[0091] 100 μL of activated Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) at a concentration of 1 × 108 CFU / mL was mixed with different hydrogels in a 24-well plate. After overnight, 100 μL of the corresponding airborne bacterial suspension was diluted 105-fold under sterile conditions and then plated onto LB agar. The plates were incubated at 37°C for 24 hours, and the colonies on the LB agar plates were observed and counted.

[0092] 1.43 Cell culture

[0093] Human umbilical vein endothelial HUVEC cells (purchased from ATCC) were cultured in DMEM complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. The cell culture medium was changed every 2-3 days.

[0094] 1.44 Cell viability

[0095] To evaluate the cell viability of the hydrogels, the cell viability was detected by CCK8 kit after corresponding treatment.

[0096] 2. Results

[0097] 2.1 Synthesis and Characterization of EGCC-Cu NCs (Self-Cascading Copper Cluster Nanozymes Constructed by Polyphenol-Copper Coordination Strategy, a New Bioactive Material with Multiple Biological Functions, Especially Excellent in Tissue Regeneration and Hypoxia Alleviation)

[0098] Figure 1 Transmission electron microscopy (TEM, see 13A for details on TEM of EGCG NCs) images showed that the average diameter of SalB-CuNCs was 2.99 nm with a uniform particle size distribution. Figure 2The electron binding energy (Binding energy) is a function of intensity. The main peaks of Cu 2p are located at 932.3 and 952.62 eV, with a spin-orbit splitting of 20 eV, which is attributed to Cu 2p3 / 2 and Cu 2p1 / 2. Interestingly, the typical copper peak (≈934 eV) oxidation state at ≈943 eV and the strong satellite feature at ≈944 eV are not observed, confirming the absence of divalent copper in EGCG-CuNCs. Since the binding energy difference between Cu (0) and Cu (I) is only 0.1 eV, charge transfer usually occurs in the Cu-polyphenol bond, so the Cu oxidation state in the unprepared SalB-CuNCs may be between 0 and +1.

[0099] At 3500–3200 cm -1 and 1689 cm -1 The typical spectral bands correspond to Ar—OH and C═O groups in EGCG. The red shift of the Ar—OH peak in the EGCG-CuNCs spectrum (3454 cm -1 ) and C═O absorption shift (1779 cm -1 ) indicates the interaction between copper and phenolic hydroxyl groups, oxidized semiquinones or quinones. In addition, the characteristic absorption of the benzene ring in EGCG is located at 1614, 1518 and 1443 cm -1 , also changed in the spectra of EGCG-CuNCs, which may be caused by the changes in the phenyl ring substituents caused by the cation-interaction and the coordination bond between copper and phenolic hydroxyl groups ( Figure 3 , in the figure, Wavenumber represents the wave number).

[0100] Figure 4 (The relationship between wavelength and absorbance) is that the characteristic peak of EGCG in EGCG-CuNCs in the UV-visible absorption spectrum shifts from 272 nm to around 270 nm, representing the -* electron transition in the free phenolic hydroxyl group of EGCG. Interestingly, the n- -* The shoulder peak in the 300–350 nm range of the electronic transition is weakened, which may be due to the effect of electron transfer between Cu and phenolic ligands. In addition, the absorption peak corresponding to large-sized CuNPs is not seen in the visible spectrum, further confirming the uniform formation of ultrasmall EGCG-CuNCs.

[0101] 2.2 Appearance and injectability of hydrogel

[0102] As attached Figure 5 As shown, Figure A is a schematic diagram of hydrogel gelation, and Figure B is a diagram of hydrogel injectability performance.

[0103] 2.3 Microstructure of hydrogel

[0104] like Figure 6 As shown, scanning electron microscopy (SEM) results reveal the hydrogel's microstructure, including pore size and arrangement, as well as the traffic between pores. All hydrogels exhibit an irregular three-dimensional network structure, which facilitates nutrient transport and gas exchange during cell culture.

[0105] 2.4 Hemolytic properties of hydrogels

[0106] like Figure 7 As shown, the hemolysis test results of the hydrogel samples showed that the hemolysis rates of the two hydrogel samples were lower than 2% of the ASTM F756-08 industry standard, indicating that the hydrogels have good blood compatibility.

[0107] 2.5 Antibacterial properties

[0108] In order to evaluate the ability of the material in the hydrogel to inhibit bacteria, the present invention selected two common bacteria as agar plate experimental subjects, namely Staphylococcus aureus and Escherichia coli ( Figure 8 ).

[0109] 3. In vitro experiments

[0110] 3.1 Biocompatibility test

[0111] like Figure 9 The live / dead staining assay, shown here, visually assessed cell biocompatibility. First, 5,000 RAW264.7 cells were plated onto a 28 mm confocal laser scanning microscopy dish. After the cells attached, the hydrogel extract was added and incubated for 1, 2, and 3 days. Cell viability was visualized by confocal microscopy using a calcein-AM / propidium iodide (PI) live / dead staining kit at 1, 2, and 3 days, respectively. As shown in Figure 9, after 7 days of co-culture, the majority of cells were viable, with only a few cells dead, indicating that the hydrogel exhibited little cytotoxicity. Therefore, the hydrogel demonstrated cytocompatibility and potential for tissue repair.

[0112] 3.2 Intracellular ROS scavenging ability test

[0113] like Figure 10The intracellular antioxidant properties of the hydrogels were investigated in H₂O₂-stimulated RAW264.7 cells. DCFH-DA staining was used to analyze their intracellular ROS scavenging capacity. RAW264.7 cells were incubated at a density of 40,000 cells / well for 12 hours. The cells were then treated with PBS + hydrogen peroxide, CH₂ + hydrogen peroxide, or CH₂@EGCG-Cu + hydrogen peroxide for 4 hours. The final hydrogen peroxide concentration was 200 μM. The cells were then washed three times with PBS and incubated with 10 μM DCFH-DA at 37°C in the dark for 30 minutes. Intracellular ROS levels were measured using confocal microscopy. Intracellular reactive oxygen species (ROS) levels were assessed using a fluorescent probe (2′,7′-dichlorofluorescein diacetate, DCFH-DA). After the addition of hydrogen peroxide, strong intracellular green fluorescence was observed, indicating a significant increase in ROS. In contrast, the fluorescence intensity of the CH₂ and CH₂@EGCG-Cu hydrogel groups decreased, demonstrating their excellent ROS scavenging capacity.

[0114] 3.3 Analysis of cell apoptosis inhibition effect

[0115] like Figure 11 As shown in Figure 2, the accumulation of reactive oxygen species (ROS) usually leads to strong oxidative stress in cells, which eventually leads to cell dysfunction and apoptosis. The FITC Annexin V apoptosis detection kit (BD, USA) was used to detect the apoptosis of HUVECs. Briefly, HUVECs were seeded in six-well plates at a concentration of 200,000 cells / well and cultured for 24 h. Hydrogen peroxide (300 μM) was added together with EGCG-CuNCs, CH or CH@EGCG-Cu hydrogels and incubated for another 24 h. After treatment, the cells were collected, washed three times with ice-cold PBS, and then stained with Annexin V and PI, and then detected by flow cytometry. Therefore, the effect of CH and CH@EGCG-Cu hydrogels in reducing cell apoptosis under oxidative stress conditions was further studied. As Figure 11 As shown in the results, after hydrogen peroxide stimulation, cell apoptosis increased significantly, with the percentage of apoptotic cells reaching 53.9%. In contrast, the percentages of apoptotic cells in the CH and CH@EGCG-Cu hydrogel-treated groups decreased to 19.27% ​​and 11.01%, respectively, further demonstrating their excellent ability to scavenge intracellular reactive oxygen species and protect cells.

[0116] 4. Clinical diabetic wound research

[0117] like Figure 12The hydrogel's wound healing effect was evaluated using a rat full-thickness skin defect model. In vivo diabetic wound healing assessment: All animal experiments were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Animals and approved by the Animal Ethics Committee of Shaoxing People's Hospital (Approval No. 2024Z089). Male Sprague-Dawley rats (6-8 weeks, 220-250 g) were purchased from Hangzhou Qizhen Laboratory Animal Technology Co., Ltd. (Hangzhou, China) for establishment of the diabetic wound model. All mice were fed a high-fat and high-sugar diet and maintained under a specific pathogen-free environment. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in healthy mice according to previous literature. All treated mice with blood glucose levels ≥16.7 mmHg were considered diabetic. Animals were maintained in a diabetic state during the wound healing experiments. After anesthesia and hair removal, two full-thickness skin wounds with a diameter of 10 mm were made on the dorsal skin of each rat using surgical scissors. The mice were then randomly divided into four groups: control, 3M, CH hydrogel, and COC@EGCG-Cu hydrogel. Change the hydrogel dressing every 3 days. Figure 12 Representative images of wounds in different groups at predetermined time intervals are shown. Visual observation showed that the wound healing efficiency of CH and CH@EGCG-Cu hydrogels was much higher than that of the other two groups.

[0118] 5. Morphological Characterization and Enzyme-like Activity Evaluation of Nanozyme EGCG-CuNCs

[0119] EGCG and CuSO4 were synthesized into nanozymes EGCG-CuNCs using a one-pot method. The morphology of EGCG-CuNCs was observed by transmission electron microscopy (TEM). The particle size distribution of EGCG-CuNCs was uniform, with an average diameter of 2.99 nm (as shown in Figure 13A).

[0120] In addition, different concentrations of EGCG-CuNCs were added to 300mM H2O2 solution. In the presence of EGCG-CuNCs, obvious oxygen bubbles were observed, indicating that hydrogen peroxide was decomposed into oxygen. As the concentration of EGCG-CuNCs increased, the dissolved oxygen concentration gradually increased, indicating that EGCG-CuNCs showed a dose-dependent pattern in oxygen generation (e.g. Figure 13 B).

[0121] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A hydrogel, characterized in that The hydrogel comprises the following components: The first hydrogel matrix (CMC-PBA) is obtained by reacting carboxymethyl chitosan (CMC) with carboxyl-activated 4-carboxyphenylboronic acid (PBA); as well as, A second hydrogel matrix (HA-DA) is obtained by reacting hyaluronic acid (HA) with activated carboxyl groups with dopamine hydrochloride (DA); as well as, Copper nanozymes (Cu NCs) chelated by epigallocatechin gallate (EGCG): loaded polyphenol-copper-derived nanozymes (EGCG-CuNCs).

2. The method for preparing the hydrogel according to claim 1, characterized in that: The method comprises the following steps: EGCG-CuNCs powder was added to HA-DA solution and dissolved by ultrasonication; Prepare HA-DA / Cu-EGCG solution; Take the prepared CMC-PBA solution and add it into the centrifuge tube, add OH - solution, and then adding the HA-DA / EGCG-CuNCs solution, shaking and mixing to obtain the hydrogel (EGCG-Cu NPs) loaded with copper-derived nanozymes.

3. A medical dressing comprising the hydrogel according to claim 1 or prepared according to claim 2.

4. A method for using the hydrogel according to claim 1 or prepared according to claim 2 by injection.

5. Use of the hydrogel according to claim 1 or prepared by claim 2 in wound treatment.

6. Use of the hydrogel according to claim 1 or prepared by claim 2 in the treatment of chronic diabetic wounds.

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