Hydrogel based on self-assembly of cinnamaldehyde and coumaric acid, preparation method and application thereof

Abstract

The application provides a hydrogel based on self-assembly of cinnamaldehyde and coumaric acid, a preparation method and application. The preparation method of the hydrogel based on self-assembly of cinnamaldehyde and coumaric acid comprises the following steps: dissolving cinnamaldehyde and coumaric acid in water and performing dispersion treatment, so that the cinnamaldehyde and the coumaric acid form a nanoparticle suspension; mixing the nanoparticle suspension with a chitosan solution, and reacting to form a hydrogel. The hydrogel exhibits excellent antioxidant and antibacterial properties in vitro, and can promote the polarization of macrophages to M2 phenotype and inhibit the inflammatory signaling pathway of NF-kappa B. In a diabetic mouse wound model, the hydrogel significantly promotes wound closure, neovascularization and collagen deposition, and exhibits good ability to promote wound healing. The multifunctional hydrogel provides a new and efficient treatment strategy for the clinical treatment of diabetic chronic wounds.

Description

Technical Field

[0001] This invention relates to the field of gel materials, and more particularly to hydrogels based on the self-assembly of cinnamaldehyde and coumaric acid, their preparation methods, and applications. Background Technology

[0002] Diabetic chronic wounds are characterized by persistent inflammation, high oxidative stress, impaired angiogenesis, and susceptibility to infection, making healing difficult and imposing a heavy burden on patients and society. Traditional dressings such as gauze and cotton pads have limitations in promoting healing, are prone to infection, and can easily adhere to the wound. Summary of the Invention

[0003] The main objective of this invention is to provide a hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid, its preparation method, and its application, in order to solve the technical problems of existing diabetic wound dressings having limited functionality and limited healing effects.

[0004] To achieve the above objectives, the present invention provides a method for preparing a hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid, comprising the following steps: Cinnamaldehyde and coumaric acid were dissolved in water and dispersed to form a nanoparticle suspension. The nanoparticle suspension is mixed with a chitosan solution to form a hydrogel.

[0005] According to an embodiment of this application, the mass ratio of cinnamaldehyde to coumaric acid is 1:2.

[0006] According to an embodiment of this application, the chitosan solution is a chitosan acetate solution.

[0007] According to an embodiment of this application, in the chitosan acetic acid solution, the concentration of chitosan is 5 wt%, and the solvent is a 1% (v / v) aqueous acetic acid solution.

[0008] According to an embodiment of this application, the nanoparticle suspension and the chitosan solution are mixed at a mass ratio of (3:7) to (7:3).

[0009] According to an embodiment of this application, the nanoparticle suspension and the chitosan solution are mixed at a mass ratio of 5:5.

[0010] This application also provides a hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method.

[0011] This application also provides the use of the hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method in the preparation of antibacterial, antioxidant, anti-inflammatory or angiogenic drugs.

[0012] This application also provides the application of the hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method in the preparation of acute wound healing drugs.

[0013] This application also provides the application of the hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method in the preparation of a drug for healing chronic wounds of diabetes.

[0014] The aforementioned hydrogel is formed by the self-assembly of cinnamaldehyde and coumaric acid into nanoparticles through non-covalent interactions, followed by dynamic cross-linking with chitosan through electrostatic interactions and Schiff base reactions. It possesses multiple physical properties, including injectability, sprayability, self-healing, and tissue adhesion. In vitro, this hydrogel exhibits excellent antioxidant and antibacterial properties, promotes macrophage polarization towards the M2 phenotype, and inhibits inflammatory signaling pathways such as NF-κB. In a diabetic mouse wound model, this hydrogel significantly promotes wound closure, angiogenesis, and collagen deposition, demonstrating a strong ability to promote wound healing. This multifunctional hydrogel provides a novel and efficient treatment strategy for the clinical treatment of chronic diabetic wounds. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 Solubility of cinnamaldehyde, vanillic acid, and CCNPS in water; Tyndall effect of CCNPS aqueous solution.

[0017] Figure 2 This is a scanning electron microscope image of nanoparticles.

[0018] Figure 3 The particle size and PDI distribution of CCNPS are shown.

[0019] Figure 4 The potential values ​​of CA, CIN, CCNPS, CS, and CCCS are displayed for FTIR, UV-Vis absorption spectra, and Zeta potential plots.

[0020] Figure 5 Hydrogels formed by CCNPS and CS in different weight ratios.

[0021] Figure 6 This is a transmission electron microscope image of the CCCS hydrogel.

[0022] Figure 7 The rheological properties of CCCS hydrogel.

[0023] Figure 8 The sprayability and injectability of the hydrogel.

[0024] Figure 9 This demonstrates the self-healing properties of hydrogels.

[0025] Figure 10 and Figure 11 This refers to the adhesive properties of hydrogels.

[0026] Figure 12 The viability of HaCaT and HUVECs cells treated with CCNPS for 24 hours.

[0027] Figure 13 Images of live / dead cells stained with appropriate concentrations of hydrogen peroxide and CCNPS (scale bar: 130 μm).

[0028] Figure 14 Blood compatibility of different concentrations (10, 20, 40, 50 μg / mL) of CCNPS and different hydrogels was studied.

[0029] Figure 15 HUVECs cell migration at 0, 12, 24, 36, and 60 hours in the control and CCNPS groups.

[0030] Figure 16 Representative tube formation and cross-well migration images of HUVECs cells from the control and CCNPS groups.

[0031] Figure 17 For CCNPS against ABTS, DPPH, and The clearance rate.

[0032] Figure 18 Differences in mitochondrial membrane potential are shown in RAW 264.7 cell images of intracellular reactive oxygen species (ROS) levels, superoxide anion levels, and JC-1 staining (scale bar: 130 μm).

[0033] Figure 19 The expression levels of inflammatory factors TNF-α, IL-1β, iNOS, and COX-2, as well as the mRNA expression levels of TLR4, NLRP3, and NF-κB.

[0034] Figure 20 The expression levels and relative quantification results of macrophage polarization markers (iNOS, Arg-1, CD86, CD163) and inflammatory cytokines (TNF-α, IL-1β, iNOS, NF-κB protein) were obtained in the control group and at different concentrations of CCNPS (3 μg / mL, 6 μg / mL).

[0035] Figure 21 Representative images of bacterial colonies after treatment with different concentrations of CCNPS.

[0036] Figure 22 The experimental results show the biofilm disruption effect of CCNPS.

[0037] Figure 23 Representative images of bacterial colonies after CCNPS and different hydrogel treatments.

[0038] Figure 24 Experimental results showing different hydrogel biofilm disruption effects.

[0039] Figure 25 The results show the bacterial inhibition zones after different treatments.

[0040] Figure 26 Scanning electron microscopy images of bacterial morphology before and after different hydrogel treatments and CCNPS treatment.

[0041] Figure 27 Quantitative analysis of wound images and wound area at 0, 3, 6, 9 and 11 days for different groups.

[0042] Figure 28 Representative images (scale bar: 100x) of H&E-stained wound tissue from different groups at 0, 3, 6, 9, and 11 days were obtained. Quantitative analysis of inflammatory cell infiltration, epidermal thickness, and histopathological scoring were also performed.

[0043] Figure 29 Representative images of Masson-stained wound tissue (scale bar: 100x) and collagen deposition in the wound area at 0, 3, 6, 9 and 11 days for different groups.

[0044] Figure 30 Quantitative IHC analysis was performed on representative immunohistochemical staining images and immunofluorescence staining images (scale bar: 100x) of different groups on days 3 and 6, as well as on IL-1β and TNF-α in wound tissue. Semi-quantitative analysis was also performed on immunofluorescence staining (TNF-α) of M1 macrophages in different treatment groups.

[0045] Figure 31 Immunohistochemical staining images of angiogenesis markers CD31 and VEGF in different groups on day 11 (scale bar: 100x) and quantitative analysis of VEGF and CD31 in wound tissue of different treatment groups on day 11 by IHC.

[0046] Figure 32The expression of inflammatory proteins in different groups on day 3 was quantitatively analyzed, as well as the expression of macrophage polarization markers CD86 and CD206 and inflammatory factors TNF-α, IL-1β, iNOS and NF-κB proteins in different groups.

[0047] Figure 33 The expression of inflammatory proteins in different groups on day 6 was quantitatively analyzed, as well as the expression of macrophage polarization markers CD86 and CD206 and inflammatory factors TNF-α, IL-1β, iNOS and NF-κB proteins in different groups.

[0048] Figure 34 The expression of angiogenesis markers CD31, VEGF, α-SMA, and NF-κB proteins on day 11 was quantitatively analyzed, as well as the expression of these markers in different groups.

[0049] Figure 35 Quantitative analysis of wound images and wound area for different groups on days 0, 3, 6, 9, 12 and 15.

[0050] Figure 36 Representative images of H&E-stained wound tissue from different groups at days 0, 3, 6, 9, 12, and 15 (scale bar: 100x).

[0051] Figure 37 Representative images of Masson-stained wound tissue from different groups at days 0, 3, 6, 9, 12, and 15 (scale bar: 100x) and collagen deposition in the wound area.

[0052] Figure 38 Immunofluorescence images of wound tissue after different treatments on day 3, showing the distribution and quantitative analysis results of CD86 (green) and CD206 (red), as well as the immunohistochemical staining and quantitative analysis results of iNOS (scale bar: 130 μm).

[0053] Figure 39 Immunofluorescence images of wound tissue after different treatments on day 9, showing the distribution and quantitative analysis results of CD86 (green) and CD206 (red), as well as the immunohistochemical staining and quantitative analysis results of iNOS (scale bar: 130 μm).

[0054] Figure 40 Immunofluorescence images of wound tissues after different treatments on day 15, showing the distribution and quantitative analysis results of VEGF (red), α-SMA (green), and CD31 (red) (scale bar: 130 μm), as well as the immunohistochemical staining and quantitative results of type I and type II collagen (scale bar: 100x).

[0055] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0056] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0057] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0058] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0059] All animal experiments were approved by the Animal Ethics and Welfare Committee of Nanhua University.

[0060] Embodiments of the present invention provide a method for preparing a hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid, comprising the following steps: S100: Cinnamaldehyde and coumaric acid are dissolved in water and dispersed to form a nanoparticle suspension.

[0061] In this step, the water can be ultrapure water, deionized water, etc. The dispersion treatment can be ultrasonic dispersion or high-speed stirring dispersion.

[0062] In some embodiments, ultrasonic dispersion is employed. The cavitation effect and mechanical vibration of ultrasound cause cinnamaldehyde (CIN) and coumaric acid (CA) to be uniformly dispersed in water, forming a stable nanoparticle suspension. Exemplarily, ultrasonic treatment is performed at 100 W power for 30 minutes.

[0063] This hydrogel is formed by the self-assembly of cinnamaldehyde and coumaric acid into nanoparticles (i.e., CCNPs) through non-covalent interactions. Specifically, hydrogen bonds can be formed between the aldehyde group in the cinnamaldehyde molecule and the phenolic hydroxyl group in the coumaric acid molecule. At the same time, the aromatic ring structure of both can be further stabilized through π-π stacking, which makes the nanoparticles have good dispersibility and stability, laying the foundation for the subsequent cross-linking reaction with chitosan solution.

[0064] In some embodiments, the particle size distribution of the nanoparticles in the nanoparticle suspension is 133~531 nanometers.

[0065] S200: The nanoparticle suspension is mixed with a chitosan solution to form a hydrogel.

[0066] In this step, the chitosan molecular chain contains a large number of amino and hydroxyl groups. When the nanoparticle suspension is mixed with the chitosan solution (i.e., CS), the carboxyl groups and other groups on the surface of the nanoparticles can interact electrostatically with the amino groups of chitosan. At the same time, the aldehyde groups of cinnamaldehyde can undergo a Schiff base reaction with the amino groups of chitosan (-CHO+-NH2→-C=N-+H2O). Through this dynamic covalent crosslinking and electrostatic interaction, the system gradually forms a hydrogel with a three-dimensional network structure.

[0067] The mixing process can be carried out by magnetic stirring or gentle oscillation to ensure a uniform mixture.

[0068] Cinnamaldehyde possesses broad-spectrum antibacterial, anti-inflammatory, and angiogenic activities, while coumaric acid also exhibits antioxidant and anti-inflammatory properties. However, both have poor water solubility and low stability, limiting their bioavailability. Chitosan, as a natural cationic polysaccharide, has good antibacterial, hemostatic, and biocompatibility properties and is often used as a hydrogel matrix.

[0069] In this application, cinnamaldehyde and coumaric acid are self-assembled to form nanoparticles, which can effectively improve their water solubility and stability. Then, a hydrogel system is constructed using chitosan as a matrix. This not only achieves the synergistic effect of the two active ingredients, but also endows the hydrogel with excellent biocompatibility and degradability by taking advantage of the properties of chitosan, thus laying a good foundation for the application of this hydrogel in the biomedical field.

[0070] The aforementioned hydrogel is formed by the self-assembly of cinnamaldehyde and coumaric acid into nanoparticles through non-covalent interactions, followed by dynamic cross-linking with chitosan through electrostatic interactions and Schiff base reactions. It possesses multiple physical properties, including injectability, sprayability, self-healing, and tissue adhesion. In vitro, this hydrogel exhibits excellent antioxidant and antibacterial properties, promotes macrophage polarization towards the M2 phenotype, and inhibits inflammatory signaling pathways such as NF-κB. In a diabetic mouse wound model, this hydrogel significantly promotes wound closure, angiogenesis, and collagen deposition, demonstrating a strong ability to promote wound healing. This multifunctional hydrogel provides a novel and efficient treatment strategy for the clinical treatment of chronic diabetic wounds.

[0071] In some embodiments, the mass ratio of cinnamaldehyde to coumaric acid is 1:2.

[0072] In some embodiments, the chitosan solution is a chitosan acetate solution.

[0073] In some embodiments, the chitosan acetate solution contains 5 wt% chitosan and is in 1% (v / v) aqueous acetic acid solution.

[0074] In some embodiments, the nanoparticle suspension and the chitosan solution are mixed at a mass ratio of (3:7) to (7:3).

[0075] By adjusting the ratio of CCNPs to CS, the formulation can be controlled from a sprayable sol to an injectable gel, meeting the needs of different wound morphologies and application scenarios. For example, a 5:5 mass ratio of CCNPs to CS forms an injectable gel, which can be applied to diabetic foot ulcers, deep sinus tracts, and irregular cavities. As another example, a 3:7 mass ratio of CCNPs to CS forms a sprayable sol, which can be applied to large-area superficial abrasions, burns, and postoperative exudative wounds.

[0076] In some embodiments, the nanoparticle suspension and the chitosan solution are mixed at a mass ratio of 5:5.

[0077] This application also provides a hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method.

[0078] This application also provides the use of the hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method in the preparation of antibacterial, antioxidant, anti-inflammatory or angiogenic drugs.

[0079] This application also provides the application of the hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method in the preparation of acute wound healing drugs.

[0080] This application also provides the application of the hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the above preparation method in the preparation of a drug for healing chronic wounds of diabetes.

[0081] Example 1: Preparation of CCNPs 20 μL of cinnamaldehyde (CIN) and 40 mg of coumaric acid (CA) were added to 4 mL of ultrapure water, mixed thoroughly, and then placed in an ultrasonic cleaner. The mixture was ultrasonically treated at 100 W for 30 minutes to obtain a uniform nanoparticle suspension, which is CCNPs. Based on this, by keeping the amount of ultrapure water constant and adjusting the mass of either CIN or CA, and by adjusting the mass ratio of CIN to CA (1:1, 1:2, 2:1, etc.), CCNPs with different particle sizes and stability can be obtained.

[0082] Example 2: Preparation of CCCS hydrogel CCNP suspensions were mixed with 5 wt% chitosan (CS) solution (dissolved in 1% (v / v) aqueous acetic acid solution) at different mass ratios (1:9 to 9:1) and allowed to stand to form hydrogels.

[0083] Test Example 1: Solubility and Tyndall Effect Test of CCNPs: Equal amounts of cinnamaldehyde (CIN), coumaric acid (CA), and CCNP nanoparticles were added to an equal volume of ultrapure water. After shaking and mixing, the solution was allowed to stand and the dissolution status was observed. A laser pointer was used to illuminate the CCNPs aqueous solution to observe whether there was a clear beam path (Tyndall effect) to preliminarily determine the formation of the nanoparticles. Figure 1 As shown, CIN exhibits clear oil-water separation in water, while CA forms a yellow precipitate, both of which are difficult to dissolve. CCNPs, however, form a homogeneous and transparent solution in water, without precipitation or stratification. Upon laser irradiation, the CCNPs solution displays a distinct red beam path, indicating the presence of a large number of nanoscale particles in the solution, confirming the successful self-assembly of CIN and CA into nanoparticles.

[0084] Test Example 2: Microscopic Morphology Observation of CCNPs (SEM): The prepared CCNPs suspension was dropped onto a silicon wafer, dried at room temperature, and then sputter-coated with gold. The morphology, size distribution, and dispersion state of the nanoparticles were observed using a scanning electron microscope (SEM, Thermo Scientific Apreo-2C) at an accelerating voltage of 5.0 kV. Figure 2 As shown, CCNPs exhibit a uniform spherical structure with clear particle boundaries and no obvious aggregation or adhesion. The relatively concentrated particle diameter distribution indicates that CIN and CA formed a stable nano-assembly under ultrasonic assistance, possessing good dispersibility and structural uniformity, providing a structural basis for its subsequent application in hydrogel construction.

[0085] Test Example 3: Particle Size Distribution and PDI Determination of CCNPs: The particle size distribution and polydispersity index (PDI) of CCNPs prepared at different CIN to CA mass ratios (1:1, 1:2, 2:1, etc.) were determined using a dynamic light scattering particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments, UK). Figure 3 As shown, with changes in CIN content, the particle size and PDI of CCNPs initially decrease and then increase. When the mass ratio of CIN to CA is 1:2, the average particle size of CCNPs is approximately 176 nm, and the PDI is ≤0.3, indicating the narrowest particle size distribution and the most stable system. Nanoparticles at this ratio exhibit uniform particle size and good dispersibility, making them suitable for subsequent hydrogel construction and in vivo applications.

[0086] Test Example 4: FTIR spectral analysis of CCNPs and CCCS hydrogels: Fourier transform infrared spectroscopy (FTIR) was used in the range of 4000–400 cm⁻¹. -1CIN, CA, CS, CCNPs, and CCCS hydrogel samples were scanned within the specified range. The samples were mixed with KBr, compressed into pellets, and the changes in functional groups in each sample were analyzed. Figure 4 As shown in Figure A, CIN is at 1721 cm. -1 A C=O stretching vibration peak appears at 1704 cm⁻¹. -1 (C=O) and 3094 cm -1 A characteristic peak appears at (OH). No new characteristic absorption peaks appear in CCNPs, but the OH and CH vibration peaks show a slight red shift, indicating that CIN and CA form nanoparticles through non-covalent interactions such as hydrogen bonding. In CCCS hydrogel, at 3400 cm⁻¹ -1 The NH peak at 3000 cm⁻¹ and 3000 cm⁻¹ -1 The OH peak intensity at the point of intersection is significantly reduced, indicating that the -NH2 in CS and the -CHO in CIN have undergone a Schiff base reaction to form a dynamic cross-linked network.

[0087] Test Example 5: UV-Vis Spectroscopic Analysis of CCNPs and CCCS Hydrogels: The UV absorption spectra of CIN, CA, CCNPs, and CCCS hydrogels were scanned in the range of 200–600 nm using a UV-Vis spectrophotometer (HITACHI U-3900H, Japan), and the positions and intensity changes of the characteristic absorption peaks of each sample were recorded. For example... Figure 4 As shown in Figure B, CIN exhibits a characteristic absorption peak at 218 nm, and CA exhibits a characteristic absorption peak at 270 nm. CCNPs show absorption peaks at 220 nm and 283 nm, respectively, which are slightly redshifted compared to the single component, indicating that CIN and CA form a new conjugated structure or intermolecular interaction during self-assembly, further confirming the successful construction of nanoparticles.

[0088] Test Example 6: Zeta Potential Measurement of CCNPs and CCCS Hydrogels: The Zeta potentials of CA, CIN, CCNPs, CS, and CCCS hydrogels were measured using a Zetasizer Nano ZS90 potentiometer. Figure 4 As shown in Figure C, CCNPs carry a negative charge (-42.04±0.56 mV), while CS carries a positive charge (+44.46±0.096 mV). The resulting CCCS hydrogel is positively charged, indicating a strong electrostatic interaction between CCNPs and CS. Combined with the FTIR results, the formation of the CCCS hydrogel is a dynamic cross-linked network driven by both electrostatic attraction and Schiff base reaction, endowing it with good structural stability and multifunctionality.

[0089] Test Example 7: Test on the ability of different proportions of CCNPs and CS to form hydrogels: CCNPs suspensions were mixed with 5wt% CS solution at different mass ratios (1:9 to 9:1), and the gel formation was observed after standing. The formation of a gel was determined by the inverted test tube method, and the gel state at each ratio was recorded. Figure 5 As shown, when the mass ratio of CCNPs to CS is within the range of 3:7 to 7:3, a stable gel can be formed after mixing; outside this range, a flowing sol state is formed. This indicates that by adjusting the ratio of CCNPs to CS, it is possible to control the process from a sprayable sol to an injectable gel, meeting the needs of different wound morphologies and application scenarios.

[0090] Test Example 8: Microstructure Observation of CCCS Hydrogel (SEM): After freeze-drying the CCCS hydrogel, a cross-section was cut, sputtered with gold, and its internal microstructure was observed using a scanning electron microscope. Pure CCCS hydrogel was used as a control to compare the pore structure, pore size distribution, and nanoparticle loading of the two.

[0091] like Figure 6 As shown, pure CS hydrogel exhibits a relatively loose lamellar structure, while CCCS hydrogel forms a uniform, dense porous network structure with evenly distributed pores and moderate pore sizes. Magnified images clearly show that CCNPs were successfully loaded into the hydrogel framework, indicating that a stable composite structure was formed between CCNPs and CS, providing structural support for subsequent drug release and cell behavior regulation.

[0092] Test Example 9: Rheological Properties of CCCS Hydrogels: A rotational rheometer was used to characterize the rheological properties of CCCS hydrogels with different mass ratios (3:7, 5:5, 7:3). Hydrogel samples were prepared into cylinders with a diameter of 25 mm and placed on the rheometer platform. Frequency scanning (0.1-100 rad / s) was performed at 25°C with a constant strain of 1%. The changes in storage modulus (G') and loss modulus (G") with angular frequency were recorded. Simultaneously, strain cycling tests were conducted, alternating between 1% and 100% strain, to evaluate the self-healing properties of the hydrogels.

[0093] like Figure 7As shown, all hydrogel ratios exhibited G'>G" across the test frequency range, indicating that they are primarily elastic and possess a stable gel network structure. Among them, the 5:5 ratio CCCS hydrogel showed a significantly higher G' value than other ratios, indicating the highest crosslinking density and the most compact network structure at this ratio. In four consecutive strain cycle tests, the 5:5 ratio hydrogel rapidly recovered to its initial G' value after each high strain release, exhibiting the largest recovery amplitude, demonstrating its excellent self-healing properties. This characteristic is crucial for maintaining the long-term effectiveness of wound dressings; therefore, a 5:5 ratio CCCS hydrogel was used in subsequent experiments. The CCNPs used in subsequent test examples were all CCNPs with a CIN to CA mass ratio of 1:2.

[0094] Test Example 10: Sprayability Test of CCCS Hydrogel: The CCCS hydrogel was transferred to a medical spray bottle, and the hydrogel was evenly sprayed onto the surface of a transparent glass plate by manually pressing the nozzle. The distribution, film-forming properties, and adhesion of the hydrogel on the glass surface were observed, and the smoothness of the spraying process was recorded.

[0095] like Figure 8 As shown in Figure A, CCCS hydrogel can be uniformly sprayed onto a glass surface using a spray bottle, forming a continuous, transparent film without particle aggregation or flow interruption. The sprayed hydrogel quickly adheres to the surface and maintains a stable morphology, indicating its excellent flowability and film-forming ability. This characteristic allows CCCS hydrogel to adapt to irregular wound surfaces, achieving uniform coverage and providing effective protection for difficult-to-heal wounds.

[0096] Test Example 11: Injectability Test of CCCS Hydrogel: CCCS hydrogel was drawn into a standard 1mL medical syringe (needle-free). The hydrogel was manually extruded below the water surface, and its shape formed in the water was observed. Simultaneously, the letters "HVC" were extruded underwater using the syringe, and the forming process and stability were recorded. like Figure 8 As shown in Figure B, the CCCS hydrogel can be successfully extruded using a syringe, forming continuous and stable lines underwater, and successfully constructing the letter "HVC". The extruded hydrogel maintains its intact shape in water, without significant diffusion or dissolution, indicating its excellent injectability and underwater stability. This injectable property allows the CCCS hydrogel to be delivered to deep or irregular wounds via minimally invasive methods, meeting the treatment needs of complex wounds.

[0097] Test Example 12: Using the cutting and repair method, the hydrogel sample was cut in half and placed at room temperature for 30 minutes. The repair process was recorded by photography.

[0098] like Figure 9As shown, to evaluate the self-healing properties, the molded hydrogel was cut, and the cracks between the hydrogels gradually disappeared until they were completely healed.

[0099] Test Example 13: Plasticity Test of CCCS Hydrogel: A suitable amount of CCCS hydrogel was placed on the thumb joint, and the morphological changes of the hydrogel and its adhesion to the skin were observed at different bending angles (0°, 45°, 80°, 90°, 180°). Figure 10 As shown, the CCCS hydrogel maintains its intact structure at the thumb joint regardless of the angle, without breaking or slipping, and remains firmly attached to the skin surface.

[0100] Test Example 14: Adhesion Performance of CCCS Hydrogel on Different Material Surfaces: CCCS hydrogel was coated onto glass, plastic, metal, rubber, and skin surfaces respectively. After standing for 5 minutes, the adhesion of the hydrogel to the different material surfaces was observed. The retention status of the hydrogel on each material surface was recorded.

[0101] like Figure 11 As shown, CCCS hydrogel exhibits excellent adhesion to all tested material surfaces. This broad-spectrum adhesion property allows CCCS hydrogel to be firmly fixed around the wound, preventing dressing displacement and reducing secondary damage caused by friction.

[0102] Test Example 15: In vitro cytotoxicity test of CCNPs: The CCK-8 assay was used to evaluate the cytotoxicity of CCNPs against human immortalized epidermal cells (HaCaT) and human umbilical vein endothelial cells (HUVEC). Cells in logarithmic growth phase were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours to allow them to adhere. The original culture medium was then discarded, and fresh culture medium containing different concentrations of CCNPs was added, followed by another 24 hours of culture. After culturing, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0103] like Figure 12 As shown, after 24 hours of treatment with CCNPs at concentrations ranging from 0 to 50 μg / mL, the cell viability of HaCaT and HUVEC cells remained above 85%, with no significant difference compared to the control group. This indicates that CCNPs have no significant cytotoxicity to normal skin cells and vascular endothelial cells and possess good in vitro biocompatibility, providing a safety basis for their subsequent application in wound dressings.

[0104] Test Example 16: Protective effect of CCNPs on cells damaged by oxidative stress: Mouse embryonic fibroblasts (3T3) were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 12-well plates. After cell attachment, a blank control group, a positive damage group (H2O2 treatment), and a CCNPS treatment group were set up. The H2O2 group was treated with [a specific ingredient]. H2O2 treatment was performed for 4 hours to induce oxidative damage; the CCNPS group was pretreated with CCNPS-containing medium for 24 hours before H2O2 damage treatment. After treatment, cells were stained according to the instructions of the live / dead cell staining kit (Calcein-AM / PI). The stained cells were observed and photographed under a fluorescence microscope; live cells were stained green by Calcein-AM, and dead cells were stained red by PI. like Figure 13 As shown, the control group cells exhibited dense green fluorescence with almost no red fluorescence, indicating good cell viability. The H2O2 group showed abundant red fluorescence and significantly reduced green fluorescence, with shrunken cell morphology, indicating that H2O2-induced oxidative stress led to massive cell death. The CCNPs pretreatment group showed dense green fluorescence and very little red fluorescence, with normal cell morphology, showing no significant difference from the control group. These results indicate that CCNPs can effectively protect cells from oxidative stress damage, maintain cell viability and normal morphology, and possess good antioxidant cell protection effects.

[0105] Test Example 17: Blood Compatibility Test of CCNPs and CCCS Hydrogel: Fresh blood was collected from mice, red blood cells were separated, and washed with PBS to prepare a 4% (v / v) red blood cell suspension. The experiment was divided into a negative control group (PBS), a positive control group (deionized water), a CCNPs group (different concentrations: 10, 20, 40, 50 μg / mL), and a CCCS hydrogel group. 0.5 mL of the diluted red blood cell suspension was mixed with 0.5 mL of each test sample and incubated at 37°C for 4 hours. After incubation, the supernatant was collected by centrifugation (1500 rpm, 15 minutes) and its absorbance at 540 nm was measured. The hemolysis rate was calculated using the formula: Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%.

[0106] like Figure 14 As shown, the deionized water positive control group exhibited obvious red hemolysis with a significantly elevated OD value; the PBS negative control group showed no hemolysis, and the supernatant was clear. The supernatants of all CCNPs concentration groups and the CCCS hydrogel group were clear, without obvious red color, and the hemolysis rate was less than 3%, meeting the requirements for hemolysis of biomaterials (<5%). The results indicate that both CCNPs and CCCS hydrogel have good blood compatibility and do not cause hemolytic reactions, making them suitable for use in wound dressings that come into contact with blood.

[0107] Test Example 18: CCNPs' ability to promote HUVEC cell migration: HUVEC cells were seeded in 24-well plates and cultured until a monolayer of confluent cells was formed. (Using...) A sterile pipette tip was used to draw vertical lines on a monolayer of cells, creating cell-free "scratch" areas. After gently rinsing away the detached cells with PBS, serum-free culture medium (control group) and serum-containing culture medium (control group) were added respectively. Serum-free culture medium was used. The same scratch site was photographed under an inverted microscope at 0, 12, 24, 36, and 60 hours after scratching. The scratch area at each time point was measured using ImageJ software, and cell migration rate was calculated using the formula: Migration rate (%) = (Initial scratch area - Scratch area at each time point) / Initial scratch area × 100%.

[0108] like Figure 15 As shown, at 0 hours, the scratch widths were the same in both groups. At 12 hours, cells in the CCNPs group migrated towards the scratch area, and the scratch width began to decrease; fewer cells migrated in the control group. At 24 and 36 hours, the scratch area in the CCNPs group was covered by a large number of migrated cells, and the scratch width decreased significantly; the migration rate in the control group was slower. At 60 hours, the scratches in the CCNPs group were almost completely healed, while the control group still had obvious unhealed areas. The results indicate that CCNPs can significantly promote the migration ability of HUVEC cells, accelerate the scratch healing process, and have good cell migration-promoting activity, which is beneficial to re-epithelialization during wound repair.

[0109] Test Example 19: Assay on the ability of CCNPs to promote angiogenesis in HUVEC cells: HUVEC cells were resuspended in serum-free medium at a concentration of 1 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells in Transwell chambers (pore size [number]). In the upper chamber, add culture medium containing 10% FBS (control group) or simultaneously containing... The culture medium was prepared. After 24 hours of culture, unmigrated cells in the upper chamber were carefully wiped away with a cotton swab. Cells that migrated to the lower chamber were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. Five fields of view were randomly selected under a microscope for photographing and counting of the positively stained cells.

[0110] like Figure 16As shown in Figure A, HUVEC cells in the control group formed only a few short tubular structures, with dispersed cells and an incomplete network. Cells in the CCNPs group formed a dense and complete tubular network with clear luminal structures, significantly increased branching points, and tight intercellular connections. These results indicate that CCNPs can significantly promote the angiogenesis capacity of HUVEC cells, promote the formation and networking of tubular structures, and possess good pro-angiogenic activity, which is beneficial for neovascularization during wound healing.

[0111] Test Example 20: Assay for CCNPs' ability to promote HUVEC cell migration: HUVEC cells were resuspended in serum-free medium at a concentration of 1 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells in Transwell chambers (pore size [number]). In the upper chamber, add culture medium containing 10% FBS (control group) or simultaneously containing... The culture medium was prepared. After 24 hours of culture, unmigrated cells in the upper chamber were carefully wiped away with a cotton swab. Cells that migrated to the lower chamber were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. Five fields of view were randomly selected under a microscope for photographing and counting of the positively stained cells.

[0112] like Figure 16 As shown in Figure B, the control group showed only a small number of sparsely distributed, crystal violet-stained migrating cells on the lower surface of the Transwell membrane. In contrast, the CCNPs group exhibited a large number of migrating cells on the lower surface of the membrane, with significantly increased cell density and deeper staining. These results indicate that CCNPs can significantly enhance the transmembrane migration ability of HUVEC cells, consistent with the scratch assay results, further confirming that CCNPs possess excellent cell migration-promoting activity.

[0113] Test Example 21: Free Radical Scavenging Ability Test of CCNPs: DPPH ethanol solution, ABTS working solution, and ·OH reaction system were prepared separately. CCNPs solutions of different concentrations were mixed with the above free radical solutions and reacted at room temperature in the dark for 30 minutes (the ·OH reaction system required incubation at 37°C for 1 hour). Subsequently, the absorbance values ​​of each reaction system at specific wavelengths (DPPH at 517 nm, ABTS at 413 nm, and ·OH system at 660 nm) were measured using a UV-Vis spectrophotometer, and the free radical scavenging rate was calculated.

[0114] like Figure 17As shown, the scavenging abilities of CCNPs for DPPH, ABTS, and ·OH all exhibited a concentration-dependent effect. The scavenging rate gradually increased with increasing CCNP concentration. When the CCNP concentration was 750 μg / mL, the DPPH scavenging rate reached 86.2%, the ABTS scavenging rate reached 92.3%, and the ·OH scavenging rate reached 96.5%. These results indicate that CCNPs possess broad-spectrum and highly efficient free radical scavenging capabilities, effectively removing various types of free radicals, providing direct evidence for their antioxidant properties.

[0115] Test Example 22: CCNPs' ability to clear intracellular ROS: RAW 264.7 macrophages were seeded in 6-well plates. Experimental groups included a control group, an LPS stimulation group, and a CCNPs+LPS group. The CCNPs+LPS group was first treated with... Pre-treat the culture medium for 1 hour, then with The cells were incubated for 24 hours to induce intracellular ROS production. After treatment, the DCFH-DA fluorescent probe was added. Incubate the cells at 37°C in the dark for 30 minutes. Wash the cells three times with serum-free medium to remove any probes that did not enter the cells. Finally, observe and photograph changes in intracellular green fluorescence (representing ROS levels) using a fluorescence microscope. Simultaneously, detect intracellular superoxide anion levels (red fluorescence) using a dihydroethidium (DHE) fluorescent probe in a similar manner.

[0116] like Figure 18 As shown in Figure A, the control group cells exhibited weak green fluorescence, indicating a low basal ROS level. The LPS group cells showed bright green fluorescence with significantly enhanced fluorescence intensity, indicating a sharp increase in intracellular ROS levels after LPS induction. The CCNPs group showed significantly weakened green fluorescence, similar to the control group, indicating that CCNPs pretreatment effectively cleared excess ROS induced by LPS. These results demonstrate that CCNPs possess excellent intracellular ROS scavenging ability and can protect cells from oxidative stress damage. Test Example 23: CCNPs' ability to scavenge intracellular superoxide anions: The ability of CCNPs to scavenge intracellular superoxide anions in RAW 264.7 cells was assessed using a dihydroethidium (DHE) fluorescent probe. Logarithmically growing RAW 264.7 cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 24-well plates and cultured for 24 hours. The experiment was divided into four groups: control group, [other groups] ... The LPS group (treated with 1 μg / mL LPS for 24 hours) and the CCNPs group (pretreated with 6 μg / mL CCNPS for 2 hours, then co-cultured with 1 μg / mL LPS for 24 hours) were used. After treatment, the culture medium was discarded, and serum-free medium containing 5 μM DHE was added. The cells were incubated at 37°C in the dark for 30 minutes. The cells were washed three times with serum-free medium, and the fluorescence was observed and images of red fluorescence were captured using a fluorescence microscope. The fluorescence intensity was directly proportional to the intracellular superoxide anion level.

[0117] like Figure 18 As shown in Figure B, the control group cells exhibited weak red fluorescence, indicating a low basal superoxide anion level. The LPS group cells showed bright red fluorescence with significantly enhanced intensity, indicating a sharp increase in intracellular superoxide anion levels after LPS induction. The red fluorescence in the CCNPs group was significantly weakened, similar to the control group, indicating that CCNP pretreatment effectively scavenged excess superoxide anions induced by LPS. These results demonstrate that CCNPs possess excellent intracellular superoxide anion scavenging capabilities and can alleviate oxidative stress-induced cell damage.

[0118] Test Example 24: Test on the protective effect of CCNPs on mitochondrial membrane potential: Cell treatment method as above. After treatment, follow the instructions of the mitochondrial membrane potential detection kit (JC-1). Load cells with JC-1 probes at 37°C for 20-30 minutes. After washing, observe under a fluorescence microscope. In healthy mitochondria, JC-1 forms a polymer and emits red fluorescence; when the mitochondrial membrane potential decreases, JC-1 exists in monomeric form and emits green fluorescence. The relative ratio of red / green fluorescence can be used to determine the functional status of mitochondria.

[0119] like Figure 18 As shown in Figure C, the control group cells exhibited bright red fluorescence, weak green fluorescence, and a high red / green ratio, indicating normal mitochondrial membrane potential. In the LPS group, red fluorescence was significantly weakened, green fluorescence was enhanced, and the red / green ratio was significantly decreased, indicating a decline in mitochondrial membrane potential and impaired mitochondrial function after LPS induction. In the CCNPs group, red fluorescence recovered, green fluorescence weakened, and the red / green ratio was close to that of the control group, indicating that CCNP pretreatment effectively protected mitochondria and mitigated the LPS-induced decrease in mitochondrial membrane potential. These results demonstrate that CCNPs have a good protective effect on mitochondria and can maintain the integrity of mitochondrial function.

[0120] Test Example 25: CCNPs-Regulated Macrophage Polarization and Inflammatory Factor Expression Assay: To investigate the regulatory role of CCNPs on macrophage polarization and inflammatory responses, the inventors used lipopolysaccharide (LPS) to stimulate RAW 264.7 macrophages to establish an in vitro inflammation model and detected the expression of related proteins using Western blotting. RAW 264.7 cells were seeded in 6-well plates and cultured to a density of approximately 80%. The experiment was divided into three groups: a blank control group (Control), a model group (LPS), and a CCNPS treatment group. The CCNPS treatment group was supplemented with different concentrations of... The culture medium was pretreated for 1 hour, and then both the model group and the treatment group were added. Stimulation lasted 24 hours. After stimulation, total cellular protein was collected, and the expression levels of M1 macrophage markers (iNOS, CD86), M2 macrophage markers (Arg-1, CD163), key inflammatory factors (TNF-α, IL-1β), and inflammatory pathway proteins (NF-κB) were detected by Western blotting. Simultaneously, the mRNA levels of related genes were also detected by qRT-PCR.

[0121] like Figures 19-20 As shown in the Western Blot results, compared with the control group, LPS stimulation significantly upregulated the protein expression of M1 macrophage markers iNOS and CD86, while promoting the release of inflammatory cytokines TNF-α and IL-1β. Furthermore, the expression of p-NF-κB p65, a key protein in the NF-κB signaling pathway, was also significantly increased, indicating activation of the inflammatory signaling pathway. However, CCNPS treatment was able to reverse these LPS-induced changes in a dose-dependent manner. The protein levels of iNOS and CD86 were significantly decreased, while the protein levels of M2 macrophage markers Arg-1 and CD163 were significantly increased. Simultaneously, the protein expression of TNF-α, IL-1β, and p-NF-κB p65 was also significantly inhibited. The mRNA level results (Supplementary Figure 5) also corroborate the findings of the Western Blot, namely that CCNPS can downregulate the transcription of pro-inflammatory cytokines (TNF-α, IL-1β, iNOS, COX-2) and inflammation-related genes (TLR4, NLRP3). These results indicate that CCNPS can effectively inhibit LPS-induced macrophage polarization towards the pro-inflammatory M1 phenotype and promote its conversion to the anti-inflammatory M2 phenotype. Its potential mechanism of action may be related to the inhibition of NF-κB inflammatory signaling pathway activation. CCNPS modulates the inflammatory microenvironment in this way, providing a key cellular and molecular basis for its anti-inflammatory and pro-healing effects in vivo.

[0122] Test Example 26: Evaluation of Antibacterial Activity of Different Concentrations of CCNPS: To evaluate the concentration-dependent antibacterial effect of CCNPS, the inventors used the plate count method to test the antibacterial activity of Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). E. coli and S. aureus bacterial suspensions in the logarithmic growth phase were diluted to approximately 10... 7 CFU / mL. 100 μL of bacterial suspension was mixed thoroughly with different concentrations of CCNPS solution (0.1, 0.3, 0.5, 1.0 mg / mL) and incubated at 37°C in a shaker for 4 hours. After incubation, the bacterial suspension was serially diluted 10-fold, and 20 μL of the diluted suspension was evenly spread onto LB agar plates. The plates were incubated at 37°C for 24 hours. The bacterial group without CCNPS treatment served as the control group.

[0123] like Figure 21 As shown, the control group plates exhibited dense and numerous colonies of E. coli and S. aureus. When the CCNPS concentration was 0.1 and 0.3 mg / mL, the number of colonies of both bacteria was not significantly reduced compared to the control group. However, when the CCNPS concentration increased to 0.5 mg / mL, the number of colonies of both S. aureus and E. coli on the plates was significantly reduced, demonstrating significant antibacterial activity. Notably, when the CCNPS concentration reached 1.0 mg / mL, no colonies of either E. coli or S. aureus were observed on the plates, indicating that this concentration of CCNPS completely inhibited the growth of both bacteria. These results demonstrate that CCNPS has a concentration-dependent antibacterial effect against both E. coli and S. aureus, and exhibits a strong bactericidal effect at a concentration of 1.0 mg / mL.

[0124] Test Example 27: Comparison of Antibacterial Activity of Different Hydrogel Formulations: To compare the antibacterial properties of different hydrogel formulations, the inventors conducted plate colony counting experiments on CS hydrogel, CCNPS, and CCCS hydrogel. E. coli and S. aureus bacterial suspensions in the logarithmic growth phase were diluted to approximately 10... 7 CFU / mL. 100 μL of bacterial suspension was mixed with CS hydrogel, CCNPS, and CCCS hydrogel, respectively, and incubated at 37°C in a shaker for 4 hours. A control group without any added materials was used. After incubation, the bacterial suspension was serially diluted 10-fold, and 20 μL was plated onto LB agar plates and incubated at 37°C for 24 hours. Colonies on each plate were photographed and counted to evaluate the antibacterial effect of each group.

[0125] like Figure 23As shown, the colonies of *E. coli* and *S. aureus* grew densely on the control group plates. The number of colonies on the CS hydrogel-treated group plates was reduced compared to the control group, indicating that CS itself has some antibacterial activity, but its effect is limited. The number of colonies on the CCNPS-treated group plates was significantly reduced, showing strong antibacterial ability. Notably, the colonies of *E. coli* and *S. aureus* on the CCCS hydrogel-treated group plates almost completely disappeared, with only a few colonies remaining, and its antibacterial effect was superior to that of CS or CCNPS groups alone. This result indicates that the CCCS hydrogel, by combining CCNPS and CS, exerts a synergistic antibacterial effect, exhibiting the strongest antibacterial activity.

[0126] Test Example 28: Inhibitory Effect of CCNPS and CCCS Hydrogels on Bacterial Biofilm: Biofilm formation is one of the important reasons why chronic wound infections are difficult to clear. The inventors used the crystal violet staining method to evaluate the inhibitory effect of different concentrations of CCNPS and different hydrogel formulations on the formation of biofilms in E. coli and S. aureus. E. coli and S. aureus bacterial suspensions (10... 8 CFU / mL was inoculated into 96-well plates, and different concentrations of CCNPS (0.1, 0.3, 0.5, 1.0 mg / mL) were added simultaneously. The plates were incubated at 37°C for 48 hours to allow bacteria to form a biofilm. Wells without CCNPS served as the control group. Comparison of different hydrogel formulations: Bacterial culture was inoculated into 96-well plates, and CS hydrogel, CCNPS (1.0 mg / mL), and CCCS hydrogel were added respectively. The plates were also incubated at 37°C for 48 hours. After incubation, the culture medium and airborne bacteria were carefully aspirated from the wells, and the plates were gently washed three times with PBS to remove unattached cells. 100 μL of 0.1% crystal violet solution was added to each well, and staining was performed at room temperature for 15 minutes. The staining solution was discarded, and the plates were washed with PBS until the washings were colorless. 100 μL of 33% glacial acetic acid was added to each well to dissolve the crystal violet, and the absorbance at 570 nm was measured using a microplate reader. The absorbance value reflects the amount of biofilm. Figure 22 , Figure 24 As shown, in the CCNPS concentration-dependent experiment, the crystal violet staining intensity of E. coli and S. aureus biofilms gradually decreased with increasing CCNPS concentration. At concentrations of 0.5 and 1.0 mg / mL, the biofilm formation of both bacteria was significantly reduced compared to the control group and the low-concentration group, indicating that CCNPS can effectively inhibit bacterial biofilm formation.

[0127] In comparative experiments using different hydrogel formulations, the CS hydrogel treatment group showed limited inhibitory effect on biofilms of both bacteria, with relatively deep crystal violet staining. The CCNPS treatment group exhibited a strong inhibitory effect, with a significant reduction in biofilm amount. The CCCS hydrogel treatment group showed the lightest crystal violet staining and the most significant inhibitory effect on E. coli and S. aureus biofilms, outperforming the CS and CCNPS groups alone. This result further confirms that CCCS hydrogel, through synergistic action, can not only kill planktonic bacteria but also effectively inhibit biofilm formation, which is of great significance for clearing refractory infections in chronic wounds.

[0128] Test Example 29: Antibacterial Zone Test of Different Hydrogel Formulations: The antibacterial zone test is a commonly used method for evaluating the diffusion and antibacterial activity of antimicrobial materials. E. coli and S. aureus bacterial suspensions (10... 7 Spread evenly (CFU / mL) onto LB agar plates. Wait for the mixture to settle. After the plate surface dried, holes (approximately 8 mm in diameter) were punched at equal intervals on the plate using Oxford cups. 150 μL of CS hydrogel, CCNPS solution, and CCCS hydrogel were added to the wells respectively. Wells containing sterile PBS served as negative controls. The plates were incubated at 37°C for 24 hours. After incubation, the diameter of the inhibition zone formed around each well was observed and measured; a larger inhibition zone indicated stronger diffusive antibacterial activity of the material.

[0129] like Figure 25 As shown, no obvious inhibition zone was observed around the PBS control wells, indicating no antibacterial activity. A weak inhibition zone appeared around the CS hydrogel wells, indicating that CS has some diffusive antibacterial ability, but the activity is weak. Obvious inhibition zones formed around the CCNPS wells, with inhibition zone diameters of approximately 2.5 cm for E. coli and 1.8 cm for S. aureus, showing good diffusive antibacterial activity. The largest inhibition zone formed around the CCCS hydrogel wells, with inhibition zone diameters of approximately 2.7 cm for E. coli and 2.0 cm for S. aureus, and its antibacterial activity was superior to that of the CS and CCNPS groups alone. This result is consistent with the results of plate colony counting and biofilm inhibition experiments, further confirming the excellent antibacterial performance of CCCS hydrogel, and that its antibacterial components can effectively diffuse into the surrounding environment, inhibiting bacteria over a wider range.

[0130] Test Example 30: Scanning Electron Microscopy Observation of Bacterial Morphological Changes: To visually observe the effects of different hydrogel treatments on bacterial cell structure and morphology, the inventors used scanning electron microscopy (SEM) to observe the morphological changes of treated E. coli and S. aureus. E. coli and S. aureus bacterial suspensions in the logarithmic growth phase were co-incubated with PBS (control group), CS hydrogel, CCNPS, and CCCS hydrogel at 37°C for 4 hours. After incubation, the bacteria were collected by centrifugation and washed three times with PBS. 2.5% glutaraldehyde was added to the bacterial pellet, and the pellet was fixed overnight at 4°C. After fixing, the bacteria were washed with PBS and then dehydrated using a gradient of ethanol (30%, 50%, 70%, 85%, 95%, 100%) for 15 minutes each time. The dehydrated samples were replaced with tert-butanol and freeze-dried. The dried bacterial powder was adhered to a conductive adhesive, sputter-coated with gold, and the morphological changes were observed and photographed under a scanning electron microscope.

[0131] like Figure 26 As shown in the SEM images, the control group (untreated) *S. aureus* exhibited typical spherical shapes with smooth, plump cell surfaces and intact cell walls; *E. coli*, on the other hand, showed typical rod-shaped structures with regular morphology and smooth surfaces. In the CS hydrogel treatment group, the morphology of some *S. aureus* and *E. coli* cells changed, showing slight shrinkage and deformation, but most cells maintained a relatively intact structure. In the CCNPS treatment group, the morphological changes of bacteria were more obvious, with a large number of bacterial cells showing roughened surfaces, obvious indentations, shrinkage, and deformation, and some cell membranes showing damage. The CCCS hydrogel treatment group caused the most severe damage to bacteria, with a large number of bacterial cells completely disintegrating and rupturing in the field of view, leaking their contents, and making the cell morphology difficult to identify. These morphological observations directly demonstrate that CCCS hydrogel can exert a powerful bactericidal effect by disrupting the integrity of bacterial cell membranes, and its effect is significantly better than CS or CCNPS alone. This severe destruction of cell structure is the key reason for bacterial death.

[0132] Test Example 31: Establishment of an Acute Wound Model and Experimental Design: To evaluate the wound-healing effect of CCCS hydrogel under normal physiological conditions, the inventors established an acute wound model of full-thickness skin defects on the back of mice. Male BALB / c mice aged 6-8 weeks were selected and, after one week of acclimatization, their back hair was removed using an electric shaver and thoroughly depilated with depilatory cream. Four circular full-thickness skin defects, reaching the fascia layer, were created symmetrically on both sides of the midline of the mouse's back using a 4 mm diameter skin biopsy puncture device. The mice were randomly divided into four groups: control group (PBS treatment), CCCS hydrogel group, VEGF group (positive control, VEGF solution treatment), and CCCS hydrogel group. Each group received the corresponding treatment and was covered and fixed with sterile dressings. On postoperative days 0, 3, 6, 9, and 11, the back wounds of each group were photographed using a digital camera, maintaining a consistent shooting distance and angle. The wound area at each time point was measured using ImageJ image analysis software. The wound healing rate (wound closure rate) is calculated using the formula: Wound closure rate (%) = (Wound area on day 0 - Wound area on day n) / Wound area on day 0 × 100%. The effect of CCCS hydrogel in promoting acute wound healing was evaluated by comparing the wound area and healing rate of each group at different time points.

[0133] like Figure 27 As shown in Figure A, on postoperative day 0, the initial wound size was basically the same in all groups. With the extension of treatment time, the wound area in each group gradually decreased. Visual observation revealed that on days 3, 6, 9, and 11, the wound closure speed in the CCCS hydrogel treatment group was significantly faster than in the other groups. By day 11, the wound in the CCCS group was almost completely closed, leaving only a tiny mark, while the control group and CS group still had relatively obvious unhealed wounds. The healing performance of the VEGF group was better than that of the control group and CS group, but still not as good as that of the CCCS group.

[0134] Figure 27 Quantitative analysis of Figure B further confirmed the above observations. On postoperative day 11, the residual wound area rates in the control group, CS group, VEGF group, and CCCS group were 12.1±1.9%, 11.9±1.2%, 5.2±0.4%, and 0.8±0.2%, respectively. Statistical analysis showed that the residual wound area rate in the CCCS group was significantly lower than that in the other groups (p<0.05). These results indicate that CCCS hydrogel can significantly accelerate the healing process of acute wounds, and its healing-promoting effect is superior to that of CS hydrogel alone and the classic healing-promoting factor VEGF.

[0135] Test Example 32: H&E Staining for Assessment of Wound Histological Repair: To assess wound healing quality at the histological level, the inventors sacrificed mice in each group on postoperative days 0, 3, 6, 9, and 11, collecting full-thickness skin tissue (including surrounding normal skin) from the wound site. Tissue samples were fixed in 4% paraformaldehyde for 24-48 hours, followed by graded ethanol dehydration, xylene clearing, and paraffin embedding. The embedded tissue blocks were cut into 5μm thick sections and stained with hematoxylin and eosin (H&E). The stained sections were observed and photographed under an optical microscope, focusing on assessing epidermal regeneration, granulation tissue formation, the degree of inflammatory cell infiltration, and the regeneration of skin appendages (such as hair follicles). Simultaneously, a semi-quantitative analysis of the degree of inflammatory cell infiltration and histopathological scores was performed using a standard scoring system.

[0136] like Figure 28 As shown in the H&E stained images (Figure A), on postoperative days 3 and 6, the wound sites in the control group, CS group, and VEGF group exhibited abundant inflammatory cell infiltration, minimal granulation tissue formation, and discontinuous epidermal layers. In contrast, the CCCS group showed significantly reduced inflammatory cell infiltration, abundant granulation tissue formation, and the beginning of epidermal migration and coverage. On postoperative days 9 and 11, the control group and CS group still showed significant inflammatory cells and incompletely closed wounds, indicating incomplete epidermal regeneration. The VEGF group showed improved healing, but a small number of inflammatory cells remained. Notably, the CCCS group achieved complete epithelial regeneration by day 11, with an intact epidermal structure and a thickness approaching that of normal skin. Furthermore, the formation of new hair follicles and other skin appendages was observed in the dermis.

[0137] Figure 28 Quantitative analysis of Figure B showed that the inflammatory cell infiltration score of the CCCS group was significantly lower than that of other groups at all time points (p<0.05), while the histopathological score (reflecting tissue structure and repair quality) was significantly higher than that of other groups (p<0.05). These results indicate that CCCS hydrogel can not only accelerate wound closure, but also significantly improve the quality of wound healing, reduce inflammation, and promote the regeneration of skin appendages.

[0138] Test Example 33: Masson Staining for Collagen Deposition: Collagen deposition and remodeling are crucial for tissue strength and functional recovery in the later stages of wound healing. To assess collagen deposition at different wound sites, the inventors used Masson's trichrome staining method to stain wound tissue sections on days 0, 3, 6, 9, and 11. Masson staining stained collagen fibers blue, muscle fibers and erythrocytes red, and cell nuclei blue-purple. The stained sections were observed and photographed under an optical microscope. ImageJ software was used to quantitatively analyze the collagen deposition areas, calculating the percentage of collagen-positive stained area to the total field of view to assess the relative content of collagen deposition.

[0139] like Figure 29 As shown in the Masson stained images in Figure A, on postoperative day 3, only a small amount of scattered blue collagen fibers were present at the wound site in each group. As the healing process progressed, the amount of collagen deposition gradually increased in each group. However, compared with the control group, CS group, and VEGF group, the CCCS group showed abundant and densely arranged blue collagen fibers at all time points (especially on days 6, 9, and 11).

[0140] Figure 29 Quantitative analysis of Figure B further confirmed that, starting from day 6, the percentage of collagen deposition area in the CCCS group was significantly higher than that in other groups (p<0.05), and this advantage persisted until day 11. This result indicates that CCCS hydrogel can effectively promote collagen synthesis and deposition at the wound site, providing better extracellular matrix support for tissue repair, thereby accelerating wound healing and tissue remodeling.

[0141] Test Example 34: Immunohistochemistry and Immunofluorescence Assessment of Early Inflammatory Response: In order to explore the mechanism by which CCCS hydrogel regulates the early inflammatory response, the inventors used immunohistochemistry (IHC) and immunofluorescence (IF) staining techniques to detect the expression of key inflammatory factors and the polarization state of macrophages in wound tissue sections on the 3rd and 6th day after surgery.

[0142] Immunohistochemical staining: Wound tissue sections from days 3 and 6 were stained with TNF-α and IL-1β using IHC. After staining, the tissues were observed and photographed under a microscope; brownish-yellow or brownish-red granules represented positive expression.

[0143] Immunofluorescence staining: Wound tissue sections from days 3 and 6 were stained with IF for TNF-α (an M1 macrophage-associated inflammatory cytokine), and the cell nuclei were counterstained with DAPI. Simultaneously, the M1 macrophage markers CD86 and M2 macrophage markers were also stained. The macrophage marker CD206 was stained with IF (fluorescence induction). The cells were observed and photographed under a fluorescence microscope, and the expression levels of related proteins were assessed by fluorescence intensity.

[0144] like Figure 30 IHC staining results showed that on postoperative day 3, the wound tissues of the control group, CS group, and VEGF group showed extensive and deep positive staining areas for TNF-α and IL-1β, indicating a strong inflammatory response. In contrast, the positive staining areas for TNF-α and IL-1β in the CCCS group were significantly reduced and the staining was lighter. On postoperative day 6, the expression of inflammatory factors decreased in all groups, but the decrease was most significant in the CCCS group, where positive staining was almost invisible.

[0145] IF staining results also showed that the TNF-α fluorescence intensity in the CCCS group was significantly lower than that in other groups on days 3 and 6.

[0146] Figures 32-33 Western blot results also corroborated the above findings. On days 3 and 6, the expression levels of pro-inflammatory factors (TNF-α, IL-1β, iNOS, COX-2) and NF-κB signaling pathway proteins in the CCCS group were significantly lower than those in the control group, CS group, and VEGF group, accompanied by upregulation of the M2 macrophage marker CD163. These results indicate that CCCS hydrogel can effectively inhibit the expression of pro-inflammatory factors in the early stages of wound healing and regulate the local inflammatory microenvironment by promoting macrophage phenotype polarization from M1 to M2, thus laying the foundation for subsequent angiogenesis and tissue regeneration.

[0147] Test Example 35: Immunohistochemistry and Western Blot Assessment of Angiogenesis: The formation of new blood vessels is crucial for providing oxygen and nutrients to regenerating tissue. To assess the effect of CCCS hydrogel on angiogenesis, the inventors used IHC staining and Western Blot techniques to detect the expression of angiogenesis-related markers in wound tissue on postoperative day 11.

[0148] Immunohistochemical staining: IHC staining of wound tissue sections from day 11 post-surgery for CD31 (a marker of vascular endothelial cells) and VEGF (vascular endothelial growth factor) was performed. After staining, the tissues were observed and photographed under a microscope; brownish-yellow granules represented positive expression. ImageJ software was used for quantitative analysis of the positively stained areas. Western Blot: Total protein was extracted from wound tissue from day 11 post-surgery, and the protein expression levels of CD31, VEGF, and α-SMA (smooth muscle actin, a marker of pericyte and vascular smooth muscle cells) were detected by Western blotting. GAPDH was used as an internal control for semi-quantitative analysis of the protein bands.

[0149] like Figure 31 IHC staining results showed that on postoperative day 11, compared with other groups, the CCCS hydrogel treatment group had a more extensive area of ​​positive staining for CD31 and VEGF in the wound tissue, and the staining intensity was higher. This indicates that the CCCS group formed more neovascularization.

[0150] Figure 34 Western blotting results further confirmed the findings of IHC. The protein expression levels of CD31, VEGF, and α-SMA in the CCCS group were significantly higher than those in the control group, CS group, and VEGF group (p<0.05). The upregulation of α-SMA expression indicates that angiogenesis not only increased in number but was also accompanied by pericyte recruitment and vascular maturation.

[0151] These results indicate that CCCS hydrogel can effectively promote angiogenesis at the wound site, increase vascular density, and provide an adequate blood supply for rapid tissue repair and regeneration. Its pro-angiogenic effect is even superior to that of the classic pro-angiogenic factor VEGF, which may be attributed to the synergistic effect of multiple factors in CCCS hydrogel.

[0152] Test Example 36: Establishment of a Diabetic Wound Model and Experimental Design: To evaluate the wound-healing effect of CCCS hydrogel under pathological conditions, the inventors established a full-thickness skin defect model on the back of diabetic mice. Male BALB / c mice aged 6-8 weeks were selected and, after one week of acclimatization, type 1 diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 60 mg / kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5). Blood glucose was monitored starting on day 3 post-injection; mice with two consecutive blood glucose levels ≥16.7 mmol / L were considered to have successfully developed a diabetic model. After maintaining a hyperglycemic state for two weeks, a circular full-thickness skin defect with a diameter of 4 mm was created on the back of the diabetic mice. The diabetic mice were randomly divided into four groups: model group (PBS treatment), CS hydrogel group, VEGF group (positive control), and CCCS hydrogel group. Appropriate treatments were administered according to the group, and the wounds were covered and fixed with sterile dressings. Wounds were photographed on postoperative days 0, 3, 6, 9, 12, and 15, and the wound area at each time point was measured using ImageJ image analysis software. The wound healing rate (wound closure rate) was calculated using the formula: Wound closure rate (%) = (Wound area on day 0 - Wound area on day n) / Wound area on day 0 × 100%. The effect of CCCS hydrogel on promoting wound healing in diabetic patients was evaluated by comparing the wound area and healing rate of each group at different time points.

[0153] like Figure 35As shown in Figure A, on day 0 post-surgery, the initial wound size of diabetic mice in each group was basically the same. With prolonged treatment, the wound area gradually decreased in all groups, but the healing speed differed significantly. Visual observation revealed that on days 3, 6, 9, 12, and 15, the wound closure speed in the CCCS hydrogel-treated group was significantly faster than that in the model group, the CS group, and the VEGF group. By day 15, the wound in the CCCS group was almost completely closed, leaving only a tiny trace, while the model group still had obvious unhealed wounds.

[0154] Figure 35 Quantitative analysis of Figure B further confirmed the above observations. On postoperative day 15, the wound closure rates of the model group, CS group, VEGF group, and CCCS group were 84.38±1.67%, 86.21±1.54%, 89.45±1.32%, and 99.82±0.09%, respectively. Statistical analysis showed that the wound closure rate of the CCCS group was significantly higher than that of the other groups (p<0.05). These results indicate that under the complex pathological conditions of diabetes, CCCS hydrogel can still significantly accelerate the wound healing process, and its healing-promoting effect is significantly better than that of CS hydrogel alone and the classic healing-promoting factor VEGF.

[0155] Test Example 37: H&E Staining Assessment of Histological Repair of Diabetic Wounds: To assess the histological impact of CCCS hydrogel on the healing quality of diabetic wounds, the inventors sacrificed diabetic mice in each group on postoperative days 0, 3, 6, 9, 12, and 15, collecting full-thickness skin tissue from the wound site. Tissue samples were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm thick), and then stained with hematoxylin and eosin (H&E). The stained sections were observed and photographed under an optical microscope, focusing on assessing epidermal regeneration, granulation tissue formation, the degree of inflammatory cell infiltration, and the integrity of the tissue structure.

[0156] like Figure 36As shown in the H&E staining images, throughout the observation period, the wound site in the model group consistently exhibited significant inflammatory cell infiltration, poor granulation tissue formation, and slow epidermal regeneration. By day 15, the epidermis in the model group remained discontinuous, with disordered tissue structure. The healing outcomes in the CS and VEGF groups were improved compared to the model group, but significant inflammatory cell infiltration and incomplete epithelialization remained. Notably, the CCCS group showed superior histological performance at all time points compared to the other groups. On postoperative days 3 and 6, the inflammatory cell infiltration in the CCCS group was significantly less than in other groups. On postoperative days 9 and 12, the CCCS group showed richer granulation tissue, and the epidermis began to effectively cover the wound. By day 15, the wound in the CCCS group had achieved complete epithelial regeneration, with an intact and uniform epidermal structure and a relatively dense dermal structure, approaching that of normal skin. These results indicate that CCCS hydrogel can significantly improve the histological repair quality of diabetic wounds, effectively inhibit inflammation, and promote granulation tissue formation and epithelial regeneration.

[0157] Test Example 38: Masson Staining for Collagen Deposition in Diabetic Wounds: Diabetic wounds are often accompanied by abnormal collagen metabolism, leading to poor healing. To evaluate the regulatory effect of CCCS hydrogel on collagen deposition in diabetic wounds, the inventors used Masson's trichrome staining method to stain wound tissue sections at 0, 3, 6, 9, 12, and 15 days post-surgery. Masson staining stained collagen fibers blue, muscle fibers and erythrocytes red, and cell nuclei blue-purple. The stained sections were observed and photographed under an optical microscope. ImageJ software was used to quantitatively analyze the collagen deposition area, calculating the percentage of collagen-positive stained area to the entire field of view to assess the relative content of collagen deposition.

[0158] like Figure 37 As shown in the Masson staining images (Figure A), from day 3 to day 9 post-surgery, the blue collagen fibers at the wound site in the model group were sparse and loosely arranged. Collagen deposition in the CS and VEGF groups increased compared to the model group, but was still insufficient. In contrast, the CCCS group showed abundant and densely arranged blue collagen fibers at all time points, especially in the later healing stages (days 12 and 15).

[0159] Figure 37 Quantitative analysis of Figure B further confirmed that, starting from day 6, the percentage of collagen deposition area in the CCCS group was significantly higher than that in the model group, CS group, and VEGF group (p<0.05), and this advantage persisted until day 15. This result indicates that CCCS hydrogel can effectively correct abnormal collagen metabolism in diabetic wounds, promote collagen synthesis and orderly deposition, provide better extracellular matrix support for tissue repair, and thus accelerate wound healing and tissue remodeling.

[0160] Test Example 39: Immunofluorescence and Immunohistochemistry Assessment of Macrophage Polarization and Early Inflammation: To investigate the mechanism by which CCCS hydrogel regulates the inflammatory response in diabetic wounds, the inventors used immunofluorescence (IF) and immunohistochemical (IHC) staining techniques to detect the polarization status and inflammation level of macrophages in wound tissues on postoperative days 3 and 9. Immunofluorescence staining: Wound tissue sections from days 3 and 9 were subjected to double IF staining with the M1 macrophage marker CD86 (green fluorescence) and the M2 macrophage marker CD206 (red fluorescence), and the cell nuclei were counterstained with DAPI (blue). The cells were observed and photographed under a confocal microscope, and the macrophage polarization status was assessed by the fluorescence intensity and co-localization of CD86 and CD206.

[0161] Immunohistochemical staining: Inducible nitric oxide synthase (iNOS, an M1 macrophage-associated enzyme) was stained onto wound tissue sections from days 3 and 9. After staining, the tissues were observed and photographed under a microscope. Brownish-yellow granules represented positive expression, and semi-quantitative analysis was performed.

[0162] like Figures 38-39 IF staining results showed that on postoperative days 3 and 9, the green fluorescence intensity of CD86 (M1 marker) in the wound tissue of the model group was significantly higher than that of other groups, while the red fluorescence intensity of CD206 (M2 marker) was weaker, indicating that M1 macrophages were dominant. The M1 / M2 ratio in the CS group and VEGF group was improved compared with the model group, but a large number of CD86-positive cells were still visible. Notably, the green fluorescence of CD86 was the weakest in the CCCS treatment group, while the red fluorescence of CD206 was the strongest, indicating that CCCS hydrogel could effectively induce macrophage polarization towards the M2 anti-inflammatory phenotype. IHC staining results of iNOS were consistent with IF staining results. The iNOS-positive staining area was extensive and the expression level was high in the model group on days 3 and 9. In contrast, the iNOS-positive staining area was the least extensive and the expression level was the lowest in the CCCS group.

[0163] These results indicate that in the persistent inflammatory microenvironment of diabetic wounds, CCCS hydrogel can effectively inhibit the activation of M1 macrophages and promote their conversion to the M2 anti-inflammatory repair phenotype, thereby continuously regulating the local immune microenvironment in the early and middle stages of wound healing and creating favorable conditions for subsequent angiogenesis and tissue regeneration.

[0164] Test Example 40: Immunofluorescence and Immunohistochemical Assessment of Angiogenesis and Collagen Remodeling: In order to evaluate the effect of CCCS hydrogel on angiogenesis and collagen remodeling in the later stage of diabetic wounds, the inventors used immunofluorescence and immunohistochemical staining techniques to examine the wound tissue on the 15th day after surgery.

[0165] Immunofluorescence staining: Wound tissue sections from day 15 post-surgery were stained with VEGF (red fluorescence), α-SMA (green fluorescence), and CD31 (red fluorescence) using immunofluorescence staining. VEGF is a key pro-angiogenic factor, CD31 is a marker of vascular endothelial cells, and α-SMA is a marker of pericytes and myofibroblasts. By observing the expression and distribution of these markers, the quantity and maturity of new blood vessels were assessed.

[0166] Immunohistochemical staining: Wound tissue sections from day 15 post-surgery were stained with IHC for type I and type III collagen. Type I collagen is the predominant collagen type in mature skin, providing tensile strength; type III collagen is abundant in early granulation tissue and is gradually replaced by type I collagen later. The quality of extracellular matrix remodeling was assessed by observing the expression and ratio of these two types of collagen.

[0167] like Figure 40 IF staining results showed that on postoperative day 15, compared with other groups, the fluorescence intensity of VEGF, CD31, and α-SMA in the wound tissue of the CCCS hydrogel treatment group was significantly enhanced. High expression of VEGF and CD31 indicated that the CCCS group formed more neovascularization. Upregulated expression of α-SMA surrounding CD31-positive vessels indicated not only increased angiogenesis but also recruitment of pericytes, resulting in a more mature and stable vascular structure.

[0168] IHC staining results showed that the model group had a high expression level of type III collagen in the wound tissue, while the expression of type I collagen was relatively insufficient, resulting in an imbalance in the type I / III collagen ratio, which is characteristic of immature scar tissue. The collagen type ratios in the CS and VEGF groups were improved compared to the model group. The CCCS group showed a significant increase in type I collagen and a moderate decrease in type III collagen, with the type I / III collagen ratio closer to that of normal skin, indicating more mature and orderly extracellular matrix remodeling. In summary, CCCS hydrogel can effectively promote the angiogenesis and maturation of functional angiogenesis in the later stages of diabetic wound healing, regulate collagen metabolism and remodeling, and guide the normalization of the extracellular matrix, thus laying a solid structural and functional foundation for achieving high-quality wound healing.

[0169] Therefore, this invention successfully constructed a multifunctional hydrogel dressing integrating anti-inflammatory, antioxidant, antibacterial, angiogenesis-promoting, and immunomodulatory functions through a simple and green self-assembly strategy. This CCCS hydrogel has a simple preparation process, low cost, good biocompatibility, and excellent physical properties such as injectability, sprayability, self-healing, and tissue adhesion, enabling it to flexibly adapt to various complex wounds. Its significant promoting effect on the healing of acute and diabetic wounds demonstrated in animal models lays a solid experimental foundation and theoretical basis for its clinical application as a new generation of chronic wound dressings. This invention provides new research ideas and strategies for developing multifunctional wound repair materials derived from natural products, and has broad clinical application prospects and development value.

[0170] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for preparing a hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid, characterized in that, Includes the following steps: Cinnamaldehyde and coumaric acid were dissolved in water and dispersed to form a nanoparticle suspension. The nanoparticle suspension is mixed with a chitosan solution to form a hydrogel.

2. The preparation method according to claim 1, characterized in that, The mass ratio of cinnamaldehyde to coumaric acid is 1:

2.

3. The preparation method according to claim 1, characterized in that, The chitosan solution is a chitosan acetate solution.

4. The preparation method according to claim 3, characterized in that, In the chitosan acetate solution, the concentration of chitosan is 5 wt%, and the solvent is a 1% (v / v) aqueous acetic acid solution.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The nanoparticle suspension and the chitosan solution are mixed at a mass ratio of (3:7) to (7:3).

6. According to the preparation method of claim 5, the nanoparticle suspension and the chitosan solution are mixed at a mass ratio of 5:

5.

7. A hydrogel based on the self-assembly of cinnamaldehyde and coumaric acid prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the hydrogel as described in claim 6 in the preparation of antibacterial, antioxidant, anti-inflammatory or angiogenic drugs.

9. The use of the hydrogel as described in claim 6 in the preparation of an acute wound healing medicament.

10. The use of the hydrogel as described in claim 6 in the preparation of a medicament for the healing of chronic wounds in diabetic patients.

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