Preparation method and application of composite hydrogel dressing based on antibacterial activity nano-enzyme

Abstract

The invention relates to a preparation method and application of a composite hydrogel dressing based on antibacterial activity nano-enzyme. Arg-CDs are obtained through dissolution and hydrothermal reaction of arginine and ethidene diamine, Zn (NO3) 26H2O mixed liquid and 2-methylimidazole mixed liquid are mixed and washed after reaction to obtain ZIF-8, the Arg-CDs and the ZIF-8 are added into DMF-methanol to be stirred, washing is performed after reaction, and Arg-CDs / ZIF-8 is obtained; the preparation method comprises the following steps: adjusting a sodium alginate solution to be alkaline, reacting the sodium alginate solution with methacrylic anhydride in an ice bath, dialyzing and freeze-drying to obtain AlgMA, re-suspending the AlgMA in a PBS buffer solution containing a photoinitiator, adding CaO2 and Arg-CDs / ZIF-8, uniformly mixing, carrying out ultraviolet irradiation, and spraying a CaCl2 solution to prepare the ACZA hydrogel. The prepared ACZA hydrogel can reduce potential toxicity, efficiently resist bacteria, promote healing and improve biological safety.

Description

Technical Field

[0001] This invention relates to the technical field of nanozymes and biomedicine, and in particular to a method for preparing and using an antibacterial active nanozyme composite hydrogel dressing. Background Technology

[0002] Wound infection is one of the major challenges faced in clinical practice, especially bacterial infection accompanying full-thickness skin defects, which often leads to delayed wound healing or even systemic infection.

[0003] Hydrogels, due to their high water content, good biocompatibility, and tunable physicochemical properties, have become ideal carriers for nanoenzyme delivery. Their high water retention and adhesion help retain and release active ingredients locally on the wound surface.

[0004] In existing technologies, traditional antibacterial dressings (such as silver ion dressings and antibiotic-loaded dressings) suffer from problems such as easy development of drug resistance and poor biocompatibility, making it difficult to meet the treatment needs of complex wound microenvironments. In recent years, nanozyme technology has attracted much attention due to its highly efficient catalytic antibacterial activity. It can exert antibacterial effects by catalyzing the generation of reactive oxygen species (ROS) by mimicking natural enzymes (such as peroxidase, POD). However, free nanozymes face many limitations in wound applications: 1) Wound exudate will dilute the concentration of nanozymes and reduce their antibacterial efficiency; 2) Nanoparticles are prone to agglomeration and inactivation, and it is difficult to distribute them evenly in deep or irregular wounds; 3) The lack of a continuous reactive oxygen species supply mechanism leads to short-lived antibacterial effects. Summary of the Invention

[0005] This invention addresses the technical problems of poor antibacterial properties and self-toxicity of existing wound dressings by providing a method for preparing an antibacterial active nanoenzyme-based composite hydrogel dressing with good antibacterial properties and low self-toxicity.

[0006] The objective of this invention is achieved by providing a method for preparing an antibacterial active nanoenzyme composite hydrogel dressing, comprising the following steps: (1) Preparation of Arg-CDs: Arginine and ethylenediamine were dissolved in ultrapure water, transferred to a hydrothermal reactor, and reacted fully to obtain Arg-CDs; (2) Preparation of ZIF-8: Weigh Zn(NO3)2·6H2O and dissolve it in DMF-methanol to obtain a Zn(NO3)2·6H2O mixture. Dissolve 2-methylimidazole in DMF-methanol to obtain a 2-methylimidazole mixture. Mix the Zn(NO3)2·6H2O mixture and the 2-methylimidazole mixture. After the mixture has reacted fully, centrifuge to remove the sediment, wash with methanol and dry to obtain ZIF-8. (3) Preparation of Arg-CDs / ZIF-8: The Arg-CDs prepared in step (1) and the ZIF-8 prepared in step (2) are added to DMF-methanol and stirred. The precipitate is obtained by centrifugation and the precipitate is washed with methanol multiple times to obtain Arg-CDs / ZIF-8. (4) Preparation of AlgMA: After adjusting the sodium alginate solution to alkaline, react it with methacrylic anhydride in an ice bath, and obtain methacrylated sodium alginate, i.e. AlgMA, by dialysis and freeze drying. (5) Preparation of ACZA hydrogel: The AlgMA prepared in step (4) is resuspended in PBS buffer containing photoinitiator, CaO2 and Arg-CDs / ZIF-8 prepared in step (3) are added, mixed evenly, and then ultrasonically removed to remove air bubbles. After ultraviolet irradiation, CaCl2 solution is sprayed to obtain ACZA hydrogel.

[0007] Further, in step (1), arginine and ethylenediamine are fully dissolved in ultrapure water at a molar ratio of (1.5-2):1, transferred to a hydrothermal reactor, reacted at 150-180°C, and reacted for 4-12 h. The reactants are then cooled to room temperature and removed, dialyzed in a 1000 Da dialysis bag, and freeze-dried at a vacuum of 0.05-0.2 mbar and a temperature of -40--60°C to obtain purified arginine carbon dots, i.e., Arg-CDs.

[0008] Furthermore, step (2) specifically includes the following steps: (2.1) Mix DMF and methanol in a ratio of 4:1 to form a DMF-methanol solution, dissolve Zn(NO3)2∙6H2O in the DMF-methanol solution, and prepare a Zn(NO3)2∙6H2O mixed solution with a concentration of 0.001~0.1 g / mL; (2.2) Mix DMF and methanol in a ratio of 4:1 to prepare a DMF-methanol solution, dissolve 2-methylimidazole in the DMF-methanol solution, and prepare a 2-methylimidazole mixture with a concentration of 0.001 to 0.05 g / mL; (2.3) The Zn(NO3)2∙6H2O mixture obtained in step (2.1) and the 2-methylimidazolium mixture obtained in step (2.2) are mixed at a volume ratio of 1:(1~2). After stirring, the mixture is transferred to a centrifuge tube, precipitated, and then centrifuged. After washing with methanol and drying, the zeolite imidazolium ester skeleton material-8, i.e., ZIF-8, is obtained.

[0009] Further, in step (4), sodium alginate is dissolved in ultrapure water to prepare a sodium alginate solution with a concentration of 1% to 2%. The pH of the solution is adjusted to alkaline using NaOH. Under 0°C ice bath conditions, methacrylic anhydride is added dropwise to the sodium alginate solution to obtain a reaction mixture. At the same time, the pH of the reaction mixture is maintained between 8 and 9 using NaOH solution. After the reaction is complete, the resulting solution is dialyzed and freeze-dried to obtain AlgMA.

[0010] Furthermore, step (5) specifically includes the following steps: (5.1) Dissolve CaCl2 in deionized water to obtain a CaCl2 solution with a concentration of 0.1 g / mL. Then, mix the CaCl2 solution with ammonia and polyethylene glycol-200 (PEG-200) in a flask. Stir the mixture with a magnetic stirrer until fully mixed to obtain a CaCl2 / ammonia / polyethylene glycol-200 mixture. Slowly add H2O2 solution to the CaCl2 / ammonia / polyethylene glycol-200 mixture and continue stirring. Adjust the pH to alkaline with NaOH. After the reaction is complete, centrifuge to separate the precipitate and wash it with ethanol several times to obtain CaO2. (5.2) The AlgMA obtained in step (4.1) was resuspended in PBS buffer containing 0.5% photoinitiator I2959 to prepare a precursor solution; (5.3) Add purchased or CaO2 prepared in step (5.1) to the precursor solution prepared in step (5.2), and then add Arg-CDs / ZIF-8 nanoparticles prepared in step (3). After thorough mixing, sonicate, and then spray CaCl2 solution after ultraviolet irradiation to form Arg-CDs / ZIF-8 / CaO2@DC-HG, i.e., ACZA hydrogel.

[0011] Further, in step (3), the Arg-CDs and ZIF-8 are added to DMF-methanol in a mass ratio of 1:(1~2).

[0012] Further, in step (5.1), the volume ratio of the CaCl2 solution, ammonia water and polyethylene glycol-200 (PEG-200) is (1~2):(0.5~2):(8~15).

[0013] Further, in step (5.3), the concentration of CaO2 in the precursor solution is 0.05 to 0.1 mg / mL, and the concentration of Arg-CDs / ZIF-8 in the precursor solution is 0.25 to 1 mg / mL.

[0014] Furthermore, the ultraviolet irradiation conditions are a wavelength of 360–370 nm and a light intensity of 8–15 mW / cm². 2The centrifugation conditions are as follows: centrifugation at a speed of 8000–11000 rpm for 10–20 min.

[0015] This invention prepares arginine carbon dots (Arg-CDs) from arginine (L-Arg) and ethylenediamine via a hydrothermal method. Arg-CDs possess POD-like enzyme activity and NO-releasing properties, exhibiting antibacterial effects and acting as an antibacterial nanoenzyme. These are then combined with a metal-organic framework material (ZIF-8) to obtain Arg-CDs / ZIF-8 nanoparticles. Simultaneously, calcium peroxide (CaO2) nanoparticles are prepared using a chemical precipitation method. Sodium alginate (SA) is modified by methacrylylation (MA) to obtain methacrylated sodium alginate (AlgMA). The Arg-CDs / ZIF-8 and CaO2 nanoparticles are uniformly dispersed in an AlgMA precursor solution, followed by UV curing and CaCl2 crosslinking to obtain an ACZA composite hydrogel dressing with antibacterial and healing-promoting functions and a dual-crosslinked structure. This invention employs a dual-crosslinking mechanism (photo-initiated polymerization and CaCl2 crosslinking). 2+ Ionic crosslinking endows the hydrogel with excellent mechanical properties and swelling characteristics, enabling it to adapt to irregular wound shapes and effectively absorb exudate. The introduction of reinforcing phase nanoparticles (Arg-CDs / ZIF-8) further enhances the mechanical strength of the hydrogel while maintaining its good biocompatibility. In the ACZA hydrogel system, the endogenous H2O2 provided by CaO2 can activate Arg-CDs / ZIF-8, causing it to release ·OH and NO, achieving a synergistic antibacterial effect. The sustained-release properties of the hydrogel not only prolong the duration of antibacterial action but also reduce the potential toxicity of nanoparticles, improving biosafety.

[0016] The ACZA hydrogel prepared by this invention can significantly promote wound healing in a full-thickness skin defect infection model. When used as a drug in wound healing, it exhibits excellent tissue repair capabilities. This system achieves a balance between efficient antibacterial activity, healing promotion, and biocompatibility through a self-activated antibacterial mechanism and a dual-network cross-linking structure, providing a new solution for the treatment of bacterial infected wounds and applying it to the biomedical field. Attached Figure Description

[0017] Figure 1 The Arg-CDs / ZIF-8 nanoparticles, AlgMA, and ACZA hydrogel prepared in Example 1 of this invention were characterized in terms of physicochemical properties and morphology.

[0018] Figure 2 The graph shows the moisture absorption properties of the ACZA hydrogel prepared in Example 1 of this invention.

[0019] Figure 3 The mechanical properties test diagram is shown for the ACZA hydrogel prepared in Example 1 of this invention.

[0020] Figure 4 The self-excited activity properties of the ACZA hydrogel prepared in Example 1 of this invention.

[0021] Figure 5 The in vitro antibacterial properties of the ACZA hydrogel prepared in Example 1 of this invention.

[0022] Figure 6 This diagram illustrates the wound closure process in mice infected with Staphylococcus aureus after treatment with PBS, DC-HG, Arg-CDs / ZIF-8@DC-HG, CaO2@DC-HG, and ACZA hydrogel, respectively.

[0023] Figure 7 This is a schematic diagram showing the tissue morphology of wounds treated with PBS, DC-HG, Arg-CDs / ZIF-8@DC-HG, CaO2@DC-HG, and ACZA hydrogels, respectively, according to the present invention. Detailed Implementation

[0024] The present invention will be further analyzed, explained and compared below through specific embodiments and comparative examples.

[0025] Example 1 (1) Dissolve 5.7 mmol L-Arg and 2.9 mmol ethylenediamine in 10 mL of ultrapure water, transfer to a hydrothermal reactor, react at 180 °C, react for 8 h, cool the reactants to room temperature, remove them, dialyze them in a 1000 Da dialysis bag, and freeze dry to obtain purified arginine carbon dots, i.e., Arg-CDs; (2) Mix DMF and methanol in a ratio of 4:1 to form a DMF-methanol solution. Weigh 0.3 g of Zn(NO3)2·6H2O and dissolve it in 30 mL of DMF-methanol. Dissolve 0.18 g of 2-methylimidazole in 30 mL of DMF-methanol. Transfer the dissolved Zn(NO3)2·6H2O to a 250 mL round-bottom flask, add the dissolved 2-methylimidazole solution, stir for 5 min, and then transfer the mixed solution to a centrifuge tube. After precipitation for 2 h, continue centrifugation, wash twice with methanol, and dry to obtain zeolite imidazole ester framework material-8, i.e., ZIF-8. (3) Add 50 mg of Arg-CDs and 50 mg of ZIF-8 to DMF-methanol and stir for 30 min. Centrifuge at 8000 rpm for 10 min to obtain precipitate. Wash the precipitate with methanol 5 times to obtain Arg-CDs / ZIF-8 nanoparticles. (4) Dissolve 2 g of sodium alginate in 100 mL of ultrapure water to prepare a 2% sodium alginate solution. Adjust the pH of the solution to 8 using NaOH and stir under ice bath conditions. Slowly add 11.2 mL of methacrylic anhydride to the SA solution while maintaining the pH between 8 and 9 with 6 M NaOH to ensure the efficient progress of the methacrylation reaction. After the reaction lasts for 24 h, dialyze and freeze dry to obtain AlgMA. (5) Dissolve 1 g CaCl2 in 10 mL of deionized water. Then, mix the resulting solution with 5 mL of ammonia solution with a concentration between 25% and 28% and 50 mL of PEG-200 in a flask. At room temperature, stir the mixture uniformly for 15 min using a magnetic stirrer. Slowly add 5 mL of 30% H2O2 solution to the mixture and continue stirring for 120 min. Add an appropriate amount of NaOH solution to the mixture to adjust the pH of the reaction system to 11.5. After the reaction is complete, centrifuge the resulting turbid liquid at 11000 rpm for 10 min using a high-speed centrifuge to achieve solid-liquid separation. Wash with ethanol 5 times to obtain CaO2. Resuspend the AlgMA obtained in step (4) in PBS buffer containing 0.5% photoinitiator I2959 to make the concentration of AlgMA in the PBS buffer 2%. Then, add CaO2 to the solution sequentially to make the concentration of CaO2 in the precursor solution 0.1%. Arg-CDs / ZIF-8 nanoparticles were added at a concentration of 1 mg / mL to achieve an Arg-CDs / ZIF-8 concentration of 1 mg / mL. The mixture was thoroughly stirred to ensure uniform dispersion of each component. The mixture was sonicated to remove air bubbles. The mixture was then irradiated under a 365 nm UV light source for 90 s and sprayed with a 0.025 g / L CaCl2 solution for 4 min to solidify it into a double cross-linked ACZA composite hydrogel, i.e., ACZA hydrogel.

[0026] Example 2 The difference between this embodiment and Embodiment 1 is that the mass ratio of Arg-CDs to ZIF-8 is 1:2, and the mixture is centrifuged at 5000 rpm for 10 min.

[0027] Comparative Example 1 2 g of sodium alginate was dissolved in 100 mL of ultrapure water to prepare a 2% sodium alginate solution. The pH of the solution was adjusted to 8 using NaOH, and the solution was stirred under ice bath conditions. 11.2 mL of methacrylic anhydride was slowly added dropwise to the SA solution, while maintaining the pH between 8 and 9 with 6 M NaOH to ensure the efficient methacrylation reaction. After the reaction continued for 24 h, the solution was dialyzed and lyophilized to obtain AlgMA. AlgMA was dissolved in PBS buffer containing 0.5% photoinitiator I2959 and irradiated with ultraviolet light for 90 s to achieve chemical cross-linking between AlgMA molecular chains. On this basis, 1 mL of 0.025 g / L CaCl2 solution was sprayed, which reacted with the carboxyl groups in AlgMA to form a double-crosslinked AlgMA hydrogel, namely DC-HG hydrogel, after 4 min, serving as a control group.

[0028] Test Example 1 Figure 1 The physicochemical and morphological characteristics of Arg-CDs / ZIF-8 nanoparticles, AlgMA, and ACZA hydrogel (Arg-CDs / ZIF-8 / CaO2@DC-HG) were performed, and the Arg-CDs, ZIF-8, and Arg-CDs / ZIF-8 nanoparticles prepared above were characterized by Fourier transform infrared spectroscopy (FTIR).

[0029] Figure 1 A shows the FTIR analysis results for Arg-CDs, ZIF-8, and Arg-CDs / ZIF-8. The FTIR spectrum of Arg-CDs is at 1640 cm⁻¹. −1 A distinct characteristic peak is observed at 1024 cm⁻¹, which can be attributed to the stretching vibration of C=N in the guanidine group. −1 and 794 cm −1 Characteristic vibrational peaks of CN and C-NH-C were observed respectively. Figure 1 B shows the FTIR analysis results of AlgMA and SA, clearly displaying the characteristic absorption peak of ZIF-8, specifically at 3100 cm⁻¹. −1 With 2900 cm −1 The nearby characteristic peaks correspond to the CH stretching vibrations of the imidazole ring and the methyl group, respectively, at 1564 cm⁻¹. −1 The characteristic peak at 1148 cm⁻¹ can be attributed to the stretching vibration of the C=N bond. −1 and 747 cm −1The characteristic peaks at these locations correspond to the stretching vibration of the CN bond and the out-of-plane bending vibration of the CH bond, respectively. The appearance of these characteristic peaks fully demonstrates that 2-methylimidazole successfully constructed the ZIF-8 framework as an organic ligand. In the FTIR spectrum of the Arg-CDs / ZIF-8 composite material, in addition to retaining the characteristic peaks of ZIF-8, a peak at 1630 cm⁻¹ was also observed. −1 A significant red shift of the C=N bond was observed, a phenomenon attributable to the interaction between the guanidine group and ZIF-8, providing direct evidence for the binding between Arg-CDs and ZIF-8. The FTIR spectral analysis results of AlgMA are shown below. Figure 1 As shown in B, compared to SA, AlgMA at 1740 cm −1 A new characteristic absorption peak appeared at this point, which is attributed to the stretching vibration of the C=O bond of the methacryloyl group. Figure 1 C is the SEM image of DC-HG, 1D is a magnified SEM image of DC-HG, 1E is the SEM image of ACZA hydrogel, and 1FACZA hydrogel is a magnified SEM image. To investigate the microstructural characteristics of the hydrogels and the influence of nanoparticle encapsulation on their structure, SEM was used to characterize the hydrogel morphology. The results are as follows: Figure 1 As shown in CF, the double-crosslinked AlgMA hydrogel exhibits a typical continuous-phase porous structure with uniformly distributed and interconnected pores. This porous structure facilitates the transport of nutrients and metabolites, providing a suitable three-dimensional microenvironment for cell growth. When the DC-HG hydrogel is loaded with Arg-CDs / ZIF-8 and CaO2 nanoparticles, the hydrogel still maintains a continuous three-dimensional network structure, but the pore size is significantly reduced. In the ACZA hydrogel composite system, the pore size is further reduced. This is mainly attributed to the uniform distribution of Arg-CDs / ZIF-8 and CaO2 nanoparticles in the hydrogel matrix. The nanoparticles occupy part of the pore space, thus leading to the reduction of the pore size.

[0030] Test Example 2 Liquid absorption performance test of ACZA hydrogel: First, the dried DC-HG and ACZA hydrogel were ground into a uniform powder, and the initial weight was recorded. M d The samples were placed at 25±0.5℃ and 80±2% relative humidity for hygroscopicity testing, and the change in sample mass was recorded every 10 minutes. M y Continue this process until the sample mass reaches equilibrium. To assess the water retention properties of the hydrogel, accurately weigh the initial mass of the dried hydrogel. M a The samples were then immersed in simulated wound exudate to allow them to fully absorb water and swell for 24 hours. After removing the samples, excess surface moisture was absorbed with filter paper, and their wet weight was immediately measured.M z To evaluate the swelling properties of the hydrogels, three freeze-dried DC-HG and ACZA hydrogels were taken from each group, and their initial mass was recorded as follows: M 0. Subsequently, the samples were immersed in simulated body fluid at 37°C (pH = 7.4). The samples were removed at preset time points of 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h. The free water on the surface of the hydrogel was gently absorbed with filter paper, and then the samples were weighed. M 1. In the study of the degradation performance of hydrogels, the initial mass of the dried DC-HG and ACZA hydrogels was accurately weighed. M x The samples were then immersed in PBS buffer at pH 7.4 and placed in an incubator at 37°C and 5% CO2. Samples were removed at preset time points (days 7, 14, and 21), rinsed with deionized water, and then freeze-dried. Their mass was recorded. M t .

[0031] Figure 2 The graph shows the liquid absorption performance test results of the ACZA hydrogel prepared in Example 1. Figure 2 As shown in Figure A, DC-HG and ACZA hydrogels were placed in a sealed container at 80% humidity for 210 min, and the hydrogels were weighed at different time points. The results showed that the hygroscopicity of DC-HG was slightly higher than that of ACZA hydrogel, but the hygroscopicity of both hydrogels tended to stabilize after 120 min, with hygroscopic rates of 66.94 ± 2.57% and 47.15 ± 3.01%, respectively. Figure 2 Experiment B showed that the water retention rates of DC-HG and ACZA hydrogels were 53.71 ± 3.25% and 43.64 ± 1.43%, respectively, indicating that both have good water retention properties and can provide a stable moist environment for wound healing. Figure 2 The swelling properties of DC-HG and ACZA hydrogels were studied. The results showed that the addition of Arg-CDs / ZIF-8 and CaO2 promoted the tight bonding of DC-HG hydrogel. In the first 90 min, the water absorption rate of both DC-HG and ACZA hydrogels was relatively fast, then gradually stabilized, with final swelling rates reaching 315% and 298%, respectively. Furthermore, Figure 2 The biodegradability of the hydrogels was studied. On day 21, the degradation rates of DC-HG hydrogel and ACZA hydrogel were 66.9 ± 1.994% and 60.55 ± 1.59%, respectively, indicating that both have good biodegradability.

[0032] Test Example 3 Figure 3The diagram illustrates the mechanical properties of ACZA hydrogel. First, the dynamic viscoelasticity of the hydrogel was measured. A circular block with a diameter of 8 mm and a height of 3 mm was made and placed on the bottom tray of the rheometer. After adjusting the rotor to just contact the hydrogel surface, the rheometer was started and switched to rotation mode for dynamic viscosity testing. The test parameters were set as follows: T = 20℃, γ0 = 0.01, f: 0.1-10 Hz. Modulus tests were performed in oscillation mode using an oscillating frequency scan, and the storage modulus (G′) and loss modulus (G′′) of the hydrogel were recorded to evaluate its viscoelastic behavior at different frequencies. Static dynamics of the hydrogel were then measured using a rheometer: using the axial scan mode of a HAKKE rheometer, three sets of samples were taken, and the diameter of the hydrogel was accurately measured with a digital vernier caliper. The hydrogel was placed on the 8 mm bottom tray of the rheometer, and the rotor was adjusted until there was no gap between it and the hydrogel. When the normal stress was 0, a static compression test was performed, with the test parameters set as follows: T = 20℃, Δh =80%, t = 120 s, by recording the cross-sectional area of ​​the hydrogel and the normal stress F at each point. n The stress-strain curves were plotted based on the corresponding height change Δh to analyze the mechanical response under static compression conditions. To determine the linear viscoelastic region of the hydrogel, a rheometer was used to perform dynamic strain scanning on the DC-HG and ACZA hydrogel samples. The DC-HG and ACZA hydrogel samples were placed on the bottom tray of the rheometer, and the rotor was adjusted to just contact the sample surface. An oscillation amplitude scan was performed. The test parameters were set as follows: T = 20℃, and the strain amplitude range was from 0.1% to 1000%. By monitoring the curves of G' and G" as a function of strain amplitude, the range of the linear viscoelastic region of the sample was determined.

[0033] Figure 3 As shown in Figure A, frequency scanning test results indicate that the G′ of the ACZA composite system is significantly higher than that of DC-HG in the frequency range of 0.1-10 Hz, suggesting that the introduction of nanozymes effectively enhances the elastic response capability of the material. Figure 3 B represents the stress-strain curves of DC-HG and ACZA hydrogels. Compared to ACZA hydrogel, DC-HG exhibits superior compressive strength, such as... Figure 3 C is a DC-HG hydrogel. Figure 3Figure D shows the stress-strain curve of the ACZA composite hydrogel. Under low strain conditions, G′ of the DC-HG hydrogel is greater than G′′. As the strain increases, G′ of the hydrogel gradually decreases. When the strain exceeds about 40%, G′ and G′′ begin to intersect, indicating that the network structure of the hydrogel begins to be irreversibly destroyed. The complete failure point of both DC-HG and ACZA hydrogels is around 40%, and the stress-strain curves show similar slopes in the nonlinear region before failure. These results consistently indicate that the introduction of Arg-CDs / ZIF-8 nanozymes slightly reduces the compressive strength of the material, but successfully maintains toughness characteristics comparable to the matrix hydrogel, providing necessary mechanical protection for its application in dynamic wound environments.

[0034] Test Example 4 Figure 4 To assess the self-excitation activity of ACZA hydrogel and investigate the ability of CaO2 to generate H2O2, the DTOD standardization method was used. Samples were placed in PBS buffer and incubated at 37°C. Samples were taken at predetermined time points, and the supernatant was mixed with an equal volume of 0.1 M potassium titanate solution. After reacting at room temperature for 10 min, the absorbance was immediately measured at 380 nm using a UV-Vis spectrophotometer. The H2O2 concentration was calculated using a pre-set standard curve to quantitatively evaluate the H2O2 release kinetics of different samples. Alternatively, ACZA hydrogel solutions (1 mg / mL, 5 μL) containing TMB (10 mg / mL, 25 μL) and H2O2 (0.1 M, 100 μL) or CaO2 solutions (0.2 mg / mL, 5 μL) were added to 10 mM PBS solution at pH 4.0. After incubation at 37°C in the dark for 30 min, the absorbance was immediately measured at 652 nm using UV-Vis absorption spectroscopy. The characteristic absorption peak of oxTMB was determined at nm, and the formation of ·OH was quantitatively analyzed by standard curve method.

[0035] Figure 4 Results A showed that there were significant differences in the intensity of the characteristic absorption peak produced by the reaction of potassium titanium oxalate with different concentrations of H2O2, and the linear regression analysis of the standard curve showed a correlation coefficient R. 2 = 0.999, indicating that the two have excellent linear correlation. Figure 4 As shown in Figure B, DTOD itself does not have an absorption peak at 380 nm. However, when 0.2 mg / mL CaO2 is added, an absorption peak is generated at 380 nm. As the CaO2 concentration increases, a significant absorption peak appears when 0.5 mg / mL H2O2 is added. Figure 4C. The ability of different concentrations of CaO2 to generate H2O2 was investigated. As shown in the figure, the total H2O2 concentration generated by CaO2 gradually increased with time. Within 4 hours, 0.2 mg / mL CaO2 could generate approximately 2.0 mM of H2O2. This provides strong evidence for the source of H2O2 inside the hydrogel. Based on this, the ability of this composite hydrogel system to generate H2O2 was further investigated. Figure 4 As shown in Figure D, both CaO2 and CaO2@DC-HG exhibit absorption peaks at 380 nm, proving that H2O2 is generated inside the hydrogel. Finally, the formation of ·OH and NO inside the hydrogel composite material is investigated. Figure 4 E. When DC-HG is loaded with Arg-CDs / ZIF-8, no absorption peak is generated at 652 nm, but with the addition of CaO2, a significant absorption peak is generated, which proves the formation of ·OH in this composite system. Figure 4 The results show that the hydrogel composite system can effectively generate NO, and the amount gradually increases over time.

[0036] Test Example 5 The antibacterial properties of ACZA hydrogel were determined using *Escherichia coli* and *Staphylococcus aureus* as models of Gram-positive and Gram-negative bacteria, respectively. Figure 5 To determine the antibacterial properties of the hydrogel, the following methods were used: E. coli and S.aureus To evaluate the antibacterial activity of ACZA hydrogels, five groups were selected: PBS, DC-HG, Arg-CDs / ZIF-8@DC-HG, CaO2@DC-HG, and ACZA hydrogels.

[0037] like Figure 5 A represents different hydrogel treatment groups. E. coli and S.aureus Optical photographs of bacterial colonies, Figure 5 B represents different hydrogel treatment groups. E. coli Corresponding survival rate Figure 5 C represents different hydrogel treatment groups S.aureus The corresponding survival rate when DC-HG, Arg-CDs / ZIF-8@DC-HG are applied to E. coli and S.aureus At that time, the bacterial survival rate was approximately 80%, indicating that the material itself does not possess antibacterial properties. In contrast, the bacterial survival rate in the CaO2@DC-HG group was approximately 30%, possibly due to the low concentration of H2O2 generated by CaO2 effectively killing the bacteria. The ACZA hydrogel's effect on... E. coli and S.aureusThe bactericidal rates all exceeded 95%, demonstrating the excellent bactericidal effect of the ACZA hydrogel composite system. This is attributed to the release of H2O2 from the CaO2 loaded in ACZA, which reacts with Arg-CDs / ZIF-8 to produce ·OH and NO, achieving synergistic antibacterial action. Figure 5 D shows SEM images of bacterial morphology treated with different hydrogels, observing changes in bacterial structure. In the ACZA hydrogel group, bacteria underwent large-scale rupture, with contents flowing out. The groups were I: PBS; II: DC-HG; III: Arg-CDs / ZIF-8@DC-HG; IV: CaO2@DC-HG; V: ACZA hydrogel.

[0038] Test Example 6 A standard full-thickness skin defect infection model was established using ICR mice. The specific experimental design was as follows: 5 mm diameter circular full-thickness skin defects were symmetrically prepared on both sides of the spine on the back of the mice using a sterile punch, followed by inoculation. S.aureus A bacterial infection model was established using a suspension.

[0039] Figure 6 This image shows the wound closure process in mice infected with Staphylococcus aureus after treatment with PBS, DC-HG, Arg-CDs / ZIF-8@DC-HG, CaO2@DC-HG, and ACZA hydrogel, respectively. To comprehensively evaluate the efficacy of different treatments, successfully modeled mice were randomly divided into 5 groups (PBS, DC-HG, CaO2@DC-HG, Arg-CDs / ZIF-8@DC-HG, and ACZA hydrogel), with 6 mice in each group. Wound images were recorded using a digital camera at predetermined time points. Figure 6 A), Morphological observation results of wound healing ( Figure 6 B) shows that the wound area in all experimental groups gradually decreased over time, but significant differences in efficacy were observed among the treatment groups. The PBS control group exhibited typical delayed healing characteristics, with significantly delayed wound contraction and epithelial regeneration. In contrast, the ACZA treatment group demonstrated the best healing effect, with a significantly faster wound closure rate than the other experimental groups. Statistical data on wound area are shown in [link to data]. Figure 6 C, confirming that the ACZA group had excellent wound healing effects. Notably, throughout the 10-day observation period, the body weight of mice in all experimental groups remained within the normal range. Figure 6 (D) and no obvious toxic reactions or abnormal behaviors were observed. This result not only confirms the in vivo compatibility of the experimental materials, but also provides important safety data support for subsequent clinical translation applications.

[0040] Test Example 7 Histomorphological testing of the wound: Paraffin sections of the wound and its tissue were taken 10 days after surgery and histological analysis was performed according to the instructions of the H&E staining kit and the Masson staining kit.

[0041] Figure 7 Schematic diagrams of the histomorphological appearance of wounds after treatment with PBS, DC-HG, Arg-CDs / ZIF-8@DC-HG, CaO2@DC-HG, and ACZA hydrogel, respectively. Ten days later, mice were sacrificed, and skin and major organs were collected for histological sections. H&E staining results of the wound skin tissue are shown below. Figure 7 As shown, compared with other groups, the skin tissue of mice treated with ACZA showed the most newly formed hair follicles and blood vessels, while the skin structure of the PBS group only contained basic epithelial structures, with fewer hair follicles and blood vessels. Masson staining revealed that the skin tissue of the ACZA group had the best collagen deposition and actin and other substances were restored and generated. All of these fully demonstrate that ACZA can effectively promote wound healing, accelerate collagen deposition, promote hair follicle generation, and show excellent wound healing effect.

[0042] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing, comprising the following steps: (1) Preparation of Arg-CDs: Arginine and ethylenediamine were dissolved in ultrapure water, transferred to a hydrothermal reactor, and reacted fully to obtain Arg-CDs; (2) Preparation of ZIF-8: Weigh Zn(NO3)2·6H2O and dissolve it in DMF-methanol to obtain a Zn(NO3)2·6H2O mixture. Dissolve 2-methylimidazole in DMF-methanol to obtain a 2-methylimidazole mixture. Mix the Zn(NO3)2·6H2O mixture and the 2-methylimidazole mixture. After the mixture has reacted fully, centrifuge to remove the sediment, wash with methanol and dry to obtain ZIF-8. (3) Preparation of Arg-CDs / ZIF-8: The Arg-CDs prepared in step (1) and the ZIF-8 prepared in step (2) are added to DMF-methanol and stirred. The precipitate is obtained by centrifugation and the precipitate is washed with methanol multiple times to obtain Arg-CDs / ZIF-8. (4) Preparation of AlgMA: After adjusting the sodium alginate solution to alkaline, it is reacted with methacrylic anhydride in an ice bath, and then lyophilized by dialyzing to obtain sodium alginate methacrylated, i.e. AlgMA; (5) Preparation of ACZA hydrogel: The AlgMA prepared in step (4) is resuspended in PBS buffer containing photoinitiator, CaO2 and Arg-CDs / ZIF-8 prepared in step (3) are added, mixed evenly, and then ultrasonically removed to remove air bubbles. After ultraviolet irradiation, CaCl2 solution is sprayed to obtain ACZA hydrogel.

2. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 1, characterized in that, In step (1), arginine and ethylenediamine are fully dissolved in ultrapure water at a molar ratio of (1.5-2):1, transferred to a hydrothermal reactor, and reacted at 150-180°C for 4-12 h. After the reaction is cooled to room temperature, the reactants are removed, dialyzed in a 1000 Da dialysis bag, and then freeze-dried at a vacuum of 0.05-0.2 mbar and a temperature of -40--60°C to obtain purified arginine carbon dots, i.e., Arg-CDs.

3. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 1, characterized in that, Step (2) specifically includes the following steps: (2.1) Mix DMF and methanol in a ratio of 4:1 to form a DMF-methanol solution, dissolve Zn(NO3)2∙6H2O in the DMF-methanol solution, and prepare a Zn(NO3)2∙6H2O mixed solution with a concentration of 0.001~0.1 g / mL; (2.2) Mix DMF and methanol in a ratio of 4:1 to prepare a DMF-methanol solution, dissolve 2-methylimidazole in the DMF-methanol solution, and prepare a 2-methylimidazole mixture with a concentration of 0.001 to 0.05 g / mL; (2.3) The Zn(NO3)2∙6H2O mixture obtained in step (2.1) and the 2-methylimidazolium mixture obtained in step (2.2) are mixed at a volume ratio of 1:(1~2). After stirring, the mixture is transferred to a centrifuge tube, precipitated, and then centrifuged. After washing with methanol and drying, the zeolite imidazolium ester skeleton material-8, i.e., ZIF-8, is obtained.

4. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 3, characterized in that, In step (4), sodium alginate is dissolved in ultrapure water to prepare a sodium alginate solution with a concentration of 1% to 2%. The pH of the solution is adjusted to alkaline using NaOH. Under 0°C ice bath conditions, methacrylic anhydride is added dropwise to the sodium alginate solution to obtain a reaction mixture. At the same time, the pH of the reaction mixture is maintained between 8 and 9 using NaOH solution. After the reaction is complete, the resulting solution is dialyzed and freeze-dried to obtain AlgMA.

5. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 1, characterized in that, Step (5) specifically includes the following steps: (5.1) Dissolve CaCl2 in deionized water to obtain a CaCl2 solution with a concentration of 0.1 g / mL. Then, mix the CaCl2 solution with ammonia and polyethylene glycol-200 (PEG-200) in a flask. Stir the mixture with a magnetic stirrer until fully mixed to obtain a CaCl2 / ammonia / polyethylene glycol-200 mixture. Slowly add H2O2 solution to the CaCl2 / ammonia / polyethylene glycol-200 mixture and continue stirring. Adjust the pH to alkaline with NaOH. After the reaction is complete, centrifuge to separate the precipitate and wash it with ethanol several times to obtain CaO2. (5.2) The AlgMA obtained in step (4.1) was resuspended in PBS buffer containing 0.5% photoinitiator I2959 to prepare a precursor solution; (5.3) Add purchased or CaO2 prepared in step (5.1) to the precursor solution prepared in step (5.2), and then add Arg-CDs / ZIF-8 nanoparticles prepared in step (3). After thorough mixing, sonicate, and then spray CaCl2 solution after ultraviolet irradiation to form Arg-CDs / ZIF-8 / CaO2@DC-HG, i.e., ACZA hydrogel.

6. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 5, characterized in that, In step (3), the Arg-CDs and ZIF-8 are added to DMF-methanol in a mass ratio of 1:(1~2).

7. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 6, characterized in that, In step (5.1), the volume ratio of the CaCl2 solution, ammonia water and polyethylene glycol-200 (PEG-200) is (1~2):(0.5~2):(8~15).

8. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 7, characterized in that, In step (5.3), the concentration of CaO2 in the precursor solution is 0.05 to 0.1 mg / mL, and the concentration of Arg-CDs / ZIF-8 in the precursor solution is 0.25 to 1 mg / mL.

9. The method for preparing an antibacterial nanoenzyme-based composite hydrogel dressing according to claim 8, characterized in that, The ultraviolet irradiation conditions are a wavelength of 360–370 nm and a light intensity of 8–15 mW / cm². 2 The centrifugation conditions are as follows: centrifugation at a speed of 8000–11000 rpm for 10–20 min.

10. Use of the hydrogel according to any one of claims 1 to 9 as a medicine in wound healing.

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