Cchg / aps composite hydrogel, preparation method and application

Abstract

This application relates to a CCHG / APS composite hydrogel, its preparation method, and its application. A method for preparing the CCHG / APS composite hydrogel includes adding a chitosan solution dropwise to a carbomer solution, mixing, then adding an astragalus polysaccharide solution, adjusting the pH to 6.5-7.2, and performing cross-linking to obtain the CCHG / APS composite hydrogel. Firstly, performance characterization of the CCHG / APS hydrogel shows that it exhibits a unique loose and porous structure with good stability. Simultaneously, its rheological properties, swelling properties, and water retention capacity are all within suitable ranges, and it demonstrates excellent antibacterial properties, providing a favorable microenvironment for wound healing. In animal experiments, the CCHG / APS hydrogel exhibited a significant healing-promoting effect, and no obvious in vivo toxicity was observed throughout the experiment, confirming its good biocompatibility.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a CCHG / APS composite hydrogel, its preparation method, and its application. Background Technology

[0002] Diabetic wound healing difficulties have escalated into a global health problem. Analysis of global epidemiological data shows that approximately 1 in 16 diabetic patients worldwide suffers from diabetic foot ulcers (DFU), a rate of about 6.3%. Depending on the depth and size of the ulcer, these ulcers can be life-threatening. Furthermore, due to abnormal inflammatory regulation, these wounds may become chronic and extremely difficult to heal. Normal wound healing is a dynamic, orderly, and complex process, typically divided into inflammatory, proliferative, and remodeling phases. The core reason for the difficulty in healing diabetic wounds is the multi-system dysfunction caused by hyperglycemia, which interferes with each stage of normal healing. Currently, traditional treatment methods mainly include debridement, saline-moistened dressings, and decompression techniques. Traditional treatments are largely based on a "symptomatic treatment" approach, lacking systematic intervention for the multi-mechanism damage to diabetic wounds (ischemia, neuropathy, infection, cellular dysfunction, metabolic disorders), and do not actively promote repair at the molecular or cellular level. Therefore, exploring more effective treatment methods is of great significance. In recent years, among various wound dressings, hydrogel wound dressings have attracted researchers' attention due to their unique properties and have become an ideal choice for wound dressings.

[0003] With the increasing global burden of diabetes, diabetic wound healing disorders have become a thorny problem in clinical treatment. The pathological microenvironment of high glucose leads to stagnation of inflammation, bacterial infection, and impaired vascular remodeling, resulting in wound healing disorders that not only significantly prolong patients' hospital stays but also impose a heavy economic burden. Traditional treatments have limited efficacy due to the lack of systematic intervention.

[0004] Therefore, the development of novel repair materials that are highly effective and adapted to the pathological characteristics of diabetic wounds is of great clinical urgency. Summary of the Invention

[0005] The purpose of this application is to provide a CCHG / APS composite hydrogel, its preparation method, and its application, which shows great application potential in the field of diabetic wound treatment, provides new ideas and directions for diabetic wound treatment, and is expected to become an effective means of treating diabetic wounds.

[0006] In a first aspect, this application provides a method for preparing CCHG / APS composite hydrogel, comprising adding chitosan solution dropwise to carbomer solution and mixing, then adding astragalus polysaccharide solution, adjusting the pH to 6.5-7.2 for cross-linking, and obtaining CCHG / APS composite hydrogel.

[0007] Furthermore, the chitosan solution is prepared using carboxymethyl chitosan.

[0008] Further, the carbomer is swollen to obtain the carbomer solution.

[0009] Furthermore, the swelling methods include room temperature swelling or water bath heating swelling.

[0010] Furthermore, triethanolamine was used to adjust the pH.

[0011] Furthermore, cross-linking is carried out simultaneously with stirring.

[0012] Secondly, this application provides CCHG / APS composite hydrogels obtained by any of the above-mentioned methods for preparing CCHG / APS composite hydrogels.

[0013] Thirdly, this application provides the use of any of the above-mentioned CCHG / APS composite hydrogels prepared by any of the above-mentioned methods in the preparation of dressings for the healing or repair of diabetic wounds.

[0014] The beneficial effects of this application are as follows: Firstly, performance characterization of the CCHG / APS hydrogel revealed that it exhibits a unique loose and porous structure with good stability. Simultaneously, its rheological properties, swelling properties, and water retention capacity are all within suitable ranges, and it demonstrates excellent broad-spectrum antibacterial properties, providing a favorable microenvironment for wound healing. In animal experiments, the CCHG / APS hydrogel showed a significant promoting effect on wound healing. Compared to other groups, the CCHG / APS group had only 5% remaining wound area on day 21 of treatment, with a significantly higher wound healing rate than the control group. Furthermore, no significant in vivo toxicity was observed throughout the experiment, confirming its good biocompatibility.

[0015] The preparation method described in this application retains the bioactivity of each component, fully leverages its synergistic function, and makes the cross-linking system safer. In the preparation method described in this application, Astragalus polysaccharide participates in the construction of the gel network, forming a dense fiber adhesion structure, which has stronger water retention, better adhesion, stronger antibacterial properties, and is less prone to detachment.

[0016] The hydrogel in this application has a good structure and properties and is biosafe. It has shown significant effectiveness in promoting the healing of diabetic wounds and has high potential for clinical application. It is expected to become a new and effective solution for the treatment of diabetic wounds. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this application 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 some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram illustrating the preparation process and application of the hydrogel in this application.

[0019] Figure 2 Characterization of the hydrogels in Example 1. a. Scanning electron microscopy (SEM) images of the CCHG hydrogel and the CCHG / APS hydrogel; comparison of porosity between the CCHG and CCHG / APS hydrogels. b. Stability tests of the hydrogels by centrifugation and heat / cold resistance tests. c. Swelling properties of the hydrogels in PBS buffer. d. Water retention capacity test of the CCHG / APS hydrogel. e. Storage modulus and loss modulus of CCHG / APS. Data are expressed as mean ± standard deviation (n=3).

[0020] Figure 3 The in vitro antibacterial properties of the hydrogel in Example 1 were assessed. a. Changes in the culture medium levels of each group were recorded after incubation at 37°C for 12 h, 24 h, and 36 h. b. Measurement results of inhibition zones against *Escherichia coli* and *Staphylococcus aureus*. Data are expressed as mean ± standard deviation (n=3).

[0021] Figure 4 This section describes the effect of hydrogel treatment on diabetic wounds in mice, as shown in Example 1. a. Representative time-series images of the wound healing process from day 0 to day 21. b. Schematic diagrams of wound contraction at days 0, 3, 7, 14, and 21. c. Quantitative analysis of wound healing rate at different healing times. Data are expressed as mean ± standard deviation (n=3).

[0022] Figure 5 Histomorphological analysis of diabetic wound regeneration. Hematoxylin-eosin (H&E) staining images of wounds treated with different methods on days 3, 7, and 19, scale bar 200 μm.

[0023] Figure 6 This is a histological analysis of the major organs in the body. The images show hematoxylin-eosin (H&E) staining of the major organs, including the heart, spleen, lungs, and kidneys, on day 21, scale bar 200 μm. Detailed Implementation

[0024] The technical solution of this application will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0025] In this application, CCHG is carboxymethyl chitosan hydrogel.

[0026] Astragalus polysaccharide (APS) is one of the main active components of Astragalus membranaceus. Extensive research has demonstrated that APS nanoparticles possess antioxidant and anti-inflammatory activities. Astragalus polysaccharide can act as an antioxidant, stimulating SOD production, inhibiting lipid peroxidation in EA.hy926 cells, delaying inflammatory responses, and downregulating the expression of NF-κBp65, IL-8, and ICAM-1. Carboxymethyl chitosan has a regulatory effect on cell function and also possesses antioxidant, antibacterial, and anti-apoptotic activities.

[0027] Carbomer is a high-molecular-weight polymer crosslinked with acrylic acid and propylene sucrose. It exhibits excellent compatibility with skin and mucous membranes, exhibits extremely low irritation to these surfaces, making it suitable for long-term contact with wounds and enabling sustained drug release. Studies have shown that carbomer 940 hydrogel is a non-cytotoxic biomaterial that can improve tissue perfusion and reduce the area of ​​necrotic tissue in burn wounds. Furthermore, carbomer's pH-responsive gelation, shear-thinning fluidity, and cost advantages make it one of the ideal matrices for hydrogels used in diabetic wounds.

[0028] A method for preparing a CCHG / APS composite hydrogel, comprising: 1. Dissolve chitosan in ultrapure water to prepare a chitosan solution, preferably using carboxymethyl chitosan.

[0029] 2. Carbomer is swollen to obtain a carbomer solution. Swelling methods include room temperature swelling or water bath swelling. For example, carbomer is dissolved in ultrapure water overnight to allow it to swell, or it is swollen in a 60°C water bath for 5 hours.

[0030] 3. Add the chitosan solution dropwise to the carbomer solution and mix. Then add the astragalus polysaccharide solution and adjust the pH to 6.5-7.2 for cross-linking, preferably at pH 6.8. Stir during cross-linking to obtain the CCHG / APS composite hydrogel.

[0031] Carbomer molecules contain a large number of carboxyl groups (-COOH), which dissociate under alkaline conditions into negatively charged carboxylate ions (-COO). - After chitosan is carboxymethylated, a negatively charged carboxyl group (-COO) is introduced into the molecular chain.- ) and partially retained amino groups (-NH2, which carry a positive charge under acidic conditions). When the two are mixed, if the pH of the system is adjusted to neutral or weakly alkaline, the -COO of the carbomer - With carboxymethyl chitosan -NH3 + (Protonation under acidic conditions) forms ionic bonds through electrostatic interactions, while Astragalus polysaccharides simultaneously participate in and construct a three-dimensional gel network, achieving integrated in-situ cross-linking and gel formation, such as... Figure 1 As shown. This cross-linking method relies on precise pH changes for triggering and is completed entirely through electrostatic interactions. The reaction conditions are mild, and no chemical cross-linking agents are used. The resulting gel structure is more stable and highly adaptable to the pathological microenvironment of diabetic wounds. The role of pH adjustment in this application is not only to assist gel formation but also to control the cross-linking strength of the gel and to promote the formation of hydrogen bonds between the numerous hydroxyl groups on Astragalus polysaccharides and carbomer and carboxymethyl chitosan, forming a dense fibrous adhesion structure. This enables controlled and sustained release of Astragalus polysaccharides, significantly improving the hydrogel's water-locking capacity, wound adhesion, and structural stability.

[0032] Given the unique advantages of Astragalus polysaccharide, chitosan, and carbomer, combining the three to prepare CCHG / APS composite hydrogel is expected to integrate the anti-inflammatory and antioxidant properties of Astragalus polysaccharide, the antibacterial properties of carboxymethyl chitosan, and the drug sustained-release properties of carbomer, breaking through the limitations of traditional treatments and further exploring its role in the healing of diabetic wounds, providing new ideas and directions for the clinical treatment of diabetic wounds.

[0033] This application describes a CCHG / APS composite hydrogel that can be used for wound healing or repair in diabetic patients, addressing five core issues: high glucose levels, stagnant inflammation, susceptibility to infection, poor vascular remodeling, and slow healing. Due to its high porosity, the composite hydrogel maintains highly efficient fluid exchange and moisturizing capabilities.

[0034] The materials used in the embodiments of this application are from the following sources: 1. Main experimental animals Healthy male ICR mice, aged 5-6 weeks and weighing approximately 20g, of SPF grade, were purchased from the Comparative Medicine Center of Yangzhou University. They were housed in a standard animal enclosure with 12 hours of light / 12 hours of darkness, maintaining relatively constant temperature and humidity. They were given free access to a high-fat diet (containing lard-fried components) and 5% sucrose solution to induce insulin resistance. All animal experiments were approved by the Institutional Animal Care and Use Committee of Yangzhou University (License No.: 202505008) and strictly followed the "Jiangsu Province Laboratory Animal Welfare and Ethics Guidelines" formulated by the Jiangsu Provincial Laboratory Animal Management Committee.

[0035] 2. Main Reagents and Instruments Carbomer 940 and carboxymethyl chitosan were purchased from Solarbio (China). Astragalus polysaccharide (purity ≥70%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Streptozotocin was purchased from Yuan Ye (China), with a purity ≥98.5% and a molecular weight of 265.22 kDa. Triethanolamine was purchased from Hu Shi (China). Sodium citrate buffer was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. *Escherichia coli* and *Staphylococcus aureus* were provided by the College of Veterinary Medicine, Yangzhou University. HE staining images were recorded using an upright microscope on a large instrument platform at the College of Veterinary Medicine, Yangzhou University. The hydrogel microstructure was photographed and recorded using a SEM300 optical microscope.

[0036] Example 1 The preparation method of the CCHG / APS composite hydrogel in this embodiment includes: 1. In a clean bench, dissolve 0.1g of carboxymethyl chitosan in 1mL of ultrapure water to prepare a 10% carboxymethyl chitosan solution.

[0037] 2. Dissolve 0.2g of Carbomer 940 in 10mL of ultrapure water overnight to allow it to swell, thus obtaining a Carbomer 940 solution.

[0038] 3. Weigh 0.25g of Astragalus polysaccharide and dissolve it in 2mL of ultrapure water. Stir magnetically (500rpm, 20min) until completely dissolved to prepare a 12.5% ​​Astragalus polysaccharide solution.

[0039] 4. Add 1 mL of 10% carboxymethyl chitosan solution dropwise to 9 mL of carbomer 940 solution, stir with a magnetic stirrer for 20 minutes, then slowly add 12.5% ​​astragalus polysaccharide solution. Adjust the pH to 6.8 with triethanolamine to initiate cross-linking, stirring simultaneously at 65°C for 30 minutes to obtain the CCHG / APS composite hydrogel. Fill a sterile syringe, seal with sealing film, and store at 4°C for later use.

[0040] I. The in vitro characterization experiments of the CCHG / APS hydrogel in this embodiment are as follows: 1. Microstructure (SEM) of CCHG / APS hydrogel The morphology, microstructure, and porosity of CCHG / APS injectable hydrogels were investigated using a scanning electron microscope (SEM300). To this end, the hydrogels were placed in centrifuge tubes and frozen at -80°C for 24 hours, then freeze-dried in a freeze dryer for 48 hours. The freeze-dried hydrogels were broken apart, and the cross-sections were sputter-coated with gold. The surface morphology was observed in cross-sectional views under an accelerating current of 5.00 kV.

[0041] 2. CCHG / APS hydrogel rheological testing The storage modulus (G') and loss modulus (G'') of the hydrogel were tested using a rheometer, with the following parameters: temperature 37℃ and frequency 0.1-10Hz.

[0042] 3. CCHG / APS hydrogel stability test The stability of the hydrogel was determined by centrifugation (centrifugation at 3000 rpm for 30 minutes) and heat and cold resistance tests (observation after 6 hours in a water bath at 80℃ and 24 hours in a refrigerator at -20℃).

[0043] 4. Evaluation of the equilibrium swelling rate of CCHG / APS hydrogel The hydrogel was cut into small pieces with regular shapes and uniform mass to ensure accurate measurement of the initial dry weight (W1). The swelling of the hydrogel in PBS solution at each time point was recorded. The swelling rate was calculated as follows: Swelling degree (%) = (W2-W1) / W1 × 100% (where W1 represents the initial dry weight of the gel sample and W2 represents the weight of the gel sample after swelling at each time point). The equilibrium swelling rate was marked with time on the horizontal axis and swelling rate (%) on the vertical axis.

[0044] 5. CCHG / APS hydrogel water retention capacity test A certain volume of gel was placed in a 65°C oven and weighed every hour to test its water retention performance.

[0045] The preparation and characterization results of CCHG / APS hydrogels are as follows: CMP940 and carboxymethyl chitosan were mixed to obtain CCHG hydrogel, and its morphology was characterized by scanning electron microscopy (SEM). Figure 2 a) The CCHG hydrogel exhibits a loose, porous, three-dimensional structure. Astragalus polysaccharide was added to the CCHG solution under stirring at 65°C to obtain a homogeneous CCHG / APS composite solution. The CCHG / APS composite hydrogel was obtained by cooling the solution. SEM images of the CCHG / APS hydrogel are shown below. Figure 2 As shown in Figure a, the results not only exhibit a loose and porous three-dimensional structure but also an increased dense fibrous adhesion network structure. This special structure enhances the adhesion of the hydrogel to the wound surface, solving the problems of easy detachment and short action time of traditional dressings. Electron microscopy (such as scanning electron microscopy, SEM) has higher resolution and can more clearly observe the microscopic pore structure of the gel. By processing the captured images using image analysis software and calculating the ratio of pore area to total area, it was found that the porosity of the CCHG group and the CCHG / APS group gels is similar. Figure 2(a) This indicates that the presence of APS did not affect its ability to maintain wound moisture by regulating the liquid exchange rate. Furthermore, the basic properties of hydrogels play a crucial role in wound healing efficiency. Composite hydrogels should possess certain stability and swelling properties. Experimental results showed that neither the CCHG group nor the CCHG / APS group exhibited stratification or water separation in centrifugation or heat and cold resistance tests. Figure 2 (b) This indicates that the stability of the hydrogel was not compromised after the addition of APS; the swelling of both the CCHG group and the CCHG / APS group gels stabilized after 90 min. Figure 2 c) and the CCHG / APS group showed better swelling properties. Subsequently, the water-retention properties of the hydrogel were tested to determine its ability to provide a moist environment at the wound site. Figure 2 As shown in Figure d, the water-locking capacity of the two gel groups far exceeded that of the drug group and the control group, demonstrating their strong water retention capacity, which is beneficial to promoting wound healing. Furthermore, the storage modulus (G') characterizes the rebound of a material after deformation, usually referring to the elasticity of the hydrogel. The loss modulus (G'') reflects the viscous nature of the material, referring to the amount of energy lost due to viscous deformation during deformation, reflecting the viscosity of the material. The storage modulus and loss modulus of CCHG hydrogel and CCHG / APS hydrogel were determined using a frequency-correlated small-amplitude oscillation shear measurement method (temperature 37℃, frequency 0.1-10Hz). Figure 2 e). In all frequency tests, the storage modulus of the hydrogel was higher than its loss modulus, indicating that the hydrogel remained solid over a wide frequency range. During the frequency scan, as the frequency increased from 0.1 to 10 Hz, the storage modulus G' and loss modulus G'' of the CCHG and CCHG / APS hydrogels remained almost constant, indicating that the hydrogel network structure was relatively stable, the gel strength was good, and it could maintain a good gel state.

[0046] II. The in vitro antibacterial effect evaluation experiment of the CCHG / APS hydrogel in this embodiment is as follows: To test the antibacterial activity of CCHG and CCHG / APS hydrogels at diabetic wound sites, Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were used as representative bacteria. In a clean bench, equal volumes of prepared S. aureus and E. coli suspensions were pipetted and added to solidified LB agar plates. The bacterial suspensions were then evenly spread on the agar plates using a sterile spreader, ensuring uniform bacterial distribution. The plates were left to dry at room temperature for 3-5 minutes to allow the bacterial suspensions to fully adhere to the agar surface. Sterile paper discs were punched into 1cm diameter circles using a sterile punch. These discs were then immersed in NS, CCHG, and CCHG / APS hydrogels using sterile forceps, ensuring complete absorption of the hydrogels. Using sterile forceps, carefully place the fully soaked paper discs onto LB agar plates coated with E. coli and S. aureus, respectively. Repeat this process with three identical paper discs on each plate. To ensure accuracy, maintain a proper distance between the paper discs (generally no less than 24 mm) to prevent overlapping inhibition zones from affecting observation. Incubate each culture medium at 37°C for 12, 24, 36, or 48 hours. To determine the size of inhibition zones at different times, measure the size of each inhibition zone using digital calipers and record the measurements with a camera at 12, 24, 36, or 48 hours after the experiment.

[0047] The results of the in vitro antibacterial effect evaluation experiment of CCHG / APS hydrogel are as follows: The high glucose environment of DFU is conducive to bacterial growth, leading to severe wound infections. Therefore, the intrinsic and durable antibacterial activity of wound dressings is crucial for preventing bacterial adhesion, inflammatory responses, and wound infection. Subsequently, we evaluated the in vitro antibacterial properties of CCHG hydrogel and CCHG / APS hydrogel using Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) as representative bacteria. Figure 3 The bacterial concentration was adjusted to 1×10⁻⁶ using the standard paper disc diffusion method. 6 CFU / mL was evenly spread on a plate, and then filter paper (1 cm in diameter) soaked with the sample was placed on the plate surface. After incubation at 37°C for different times, the inhibition zone was measured. Figure 3a). The results showed that the inhibition zone of the CCHG / APS group gradually expanded over time, while no inhibition zone was observed in the NS group. After 36 hours of incubation, the diameters of the inhibition zones of CCHG / APS against Escherichia coli and Staphylococcus aureus were (17.8±1.0) mm and (19.2±1.3) mm, respectively, significantly larger than those of the CCHG group (11.5±0.9) mm and (13.8±1.1) mm (P<0.01). Analysis of the antibacterial mechanism indicated that APS synergistically inhibited bacterial biofilm formation by enhancing the positive charge adsorption capacity of chitosan, while the viscous properties of the CCHG matrix further prolonged the contact time between the antibacterial components and bacteria, thereby improving the antibacterial efficiency.

[0048] III. The following is an experiment on the effect of CCHG / APS composite hydrogel on diabetic wound healing in this embodiment: 1. Establishment of an animal model of type II diabetes After one week of acclimatization, mice were fasted for 12 hours but allowed free access to water. A single injection of 1% STZ solution (150 mg / kg) into the left lower abdominal cavity induced diabetes. Following the injection, mice were allowed free access to a complete diet fried in artificial lard, and their drinking water was replaced with sucrose water. Food and water intake were monitored daily. One week later, blood was collected from the tail to measure blood glucose. A successful model was defined as a random blood glucose level ≥16.7 mmol / L for three consecutive days, accompanied by polydipsia, polyphagia, and polyuria. Mice that did not successfully develop the model were injected again and re-examined after three days.

[0049] 2. Experimental grouping and wound preparation Thirty-two mice were randomly divided into four groups of eight each. Mice were anesthetized with a standard-diluted acetaminophen, fixed in place, and had their back hair shaved. The skin was disinfected with 75% alcohol, and a 1cm × 1cm circular full-thickness skin wound was created on the back using a sterile biopsy punch. After hemostasis, local treatment was administered according to the groups: NS group, CCHG group, APS group, and CCHG / APS group, with 16 wounds in each group.

[0050] 3. Statistical Analysis Each experiment was repeated three times, and data are expressed as mean ± standard deviation (Mean ± SD). Graphs were plotted using GraphPad Prism 9 software. P < 0.05 was considered statistically significant, and statistically significant differences were expressed as follows: P<0.05, P<0.01, P<0.001.

[0051] The experimental results regarding the effect of CCHG / APS composite hydrogel on wound healing in diabetic patients are as follows: The ability of hydrogels to promote wound healing in diabetic patients was determined in vivo. Figure 4Mice in each group received treatment with NS, CCHG, APS monomer, and CCHG / APS composite hydrogel, respectively. Wound healing images were acquired at days 0, 3, 7, and 21, and the healing rate was calculated. Time series analysis ( Figure 4 a) and wound contraction kinetics curve ( Figure 4 (b) indicates that the wound closure rate in the CCHG / APS group was significantly better than that in other groups. By day 21 of treatment, the wounds in the CCHG / APS group had almost completely healed, while the control groups (NS, CCHG, APS) still had unclosed wounds. Quantitative data showed ( Figure 4 c), the CCHG / APS group showed healing advantage from day 7, with a residual wound rate of only 5.1±1.3% on day 14, significantly lower than the NS group's 16.2±3.5% ( P The value is <0.001, indicating that the hydrogel can effectively shorten the repair cycle of diabetic wounds.

[0052] IV. The pathological observation experiment of the wound in this embodiment is as follows: Surgical scissors were used to completely excise regenerated tissue from the wounds of diabetic mice at different postoperative time points and H&E staining was performed to observe the degree of repair within the wound tissue and the integrity of the regenerated skin.

[0053] The results of H&E staining analysis of skin tissue sections are as follows: To further investigate the effects of different treatment methods on the internal repair process of wounded tissue and the integrity of regenerated skin at the defect site, this application performed H&E staining on the wound regeneration tissue of diabetic mice that underwent surgery at 3, 7, and 19 days postoperatively. Figure 5 The study systematically analyzed skin sections from the wound healing site. Wound sections from day 3 showed tissue necrosis and inflammation, caused by bacterial infection and the migration of inflammatory cells. Over time, all wound treatments showed gradual improvement by day 7, with a significant reduction in the number of inflammatory cells within the tissue. Notably, the CCHG / APS treatment group demonstrated particularly outstanding therapeutic effects among the various treatments, while the control groups treated with saline, CCHG alone, or APS showed relatively poor wound repair. Further observation revealed that the CCHG / APS group exhibited a significant advantage in promoting wound tissue repair and improving the integrity of regenerated skin. At key postoperative time points, this group not only showed a continuous decrease in inflammatory cell infiltration but also exhibited some follicle-like structures and collagen fiber deposition. In the composite gel dressing group, collagen fibers were arranged in a regular wavy pattern, while the epidermis of the NS, CCHG, and APS single-drug groups remained incomplete, with disordered collagen deposition. This result indicates that CCHG / APS hydrogel can significantly accelerate the histological repair of diabetic wounds by modulating the inflammatory microenvironment and promoting extracellular matrix remodeling.

[0054] V. The in vivo safety evaluation experiment of this embodiment is as follows: Twenty-one days post-surgery, following standard anatomical procedures, the mouse's heart, spleen, lungs, kidneys, and other vital internal organs were sequentially removed for HE staining to observe for structural changes and inflammatory cell aggregation.

[0055] The results of in vivo safety experiments are as follows: To verify the biocompatibility of the hydrogel, we collected internal organs such as the heart, spleen, lungs, and kidneys from mice in each treatment group 21 days post-surgery for HE staining. Figure 6 HE staining of organs such as the heart, lungs, spleen, and kidneys showed normal physiological structures, indicating that the CCHG / APS gel dressing has good safety in vivo, which provides a basis for long-term use (DFU healing cycle usually requires 4-8 weeks).

[0056] VI. Discussion Currently, traditional surgical debridement and anti-infection treatments, as well as innovative therapies such as hyperbaric oxygen therapy and negative pressure therapy, have become important means of preventing amputation. However, the cure rate of DFU (diuretic lavage fluid) remains unsatisfactory. Hydrogel dressings, with their strong water absorption capacity, complete biocompatibility, biodegradability, and controlled drug release, have become a research hotspot in the field of wound dressing applications. Furthermore, by adjusting and designing the composition and molecular structure of hydrogels, they can be easily manufactured to integrate various wound repair functions. This application successfully prepared a CCHG / APS composite hydrogel, whose unique structure and properties demonstrate advantages in the healing of diabetic wounds. Its loose, porous structure facilitates cell migration and proliferation, mimicking the function of the extracellular matrix (ECM) of tissues; its good stability ensures the effective action time of the hydrogel at the wound site; its swelling properties help maintain a moist wound environment; and its antibacterial properties effectively prevent wound infection.

[0057] CCHG / APS hydrogel showed significant efficacy in promoting wound healing in diabetic mice. The anti-inflammatory activity of Astragalus polysaccharide, the anti-infective capacity of chitosan, and the sustained-release effect of carbomer 940 synergistically reduced inflammation and accelerated wound healing. H&E staining further confirmed its positive impact on wound tissue repair. In vivo safety experiments showed that CCHG / APS hydrogel had no adverse effects on major internal organs of mice, providing a certain level of safety assurance for its clinical application.

[0058] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing a CCHG / APS composite hydrogel, characterized in that, The process involves adding chitosan solution dropwise to carbomer solution and mixing, then adding astragalus polysaccharide solution and adjusting the pH to 6.5-7.2 to perform cross-linking, resulting in CCHG / APS composite hydrogel.

2. The method for preparing the CCHG / APS composite hydrogel according to claim 1, characterized in that, The chitosan solution was prepared using carboxymethyl chitosan.

3. The method for preparing the CCHG / APS composite hydrogel according to claim 1, characterized in that, The carbomer solution is obtained by swelling the carbomer.

4. The method for preparing the CCHG / APS composite hydrogel according to claim 3, characterized in that, Swelling methods include room temperature swelling or water bath heating swelling.

5. The method for preparing the CCHG / APS composite hydrogel according to claim 1, characterized in that, Use triethanolamine to adjust the pH.

6. The method for preparing the CCHG / APS composite hydrogel according to claim 1, characterized in that, The cross-linking process is carried out simultaneously with stirring.

7. The CCHG / APS composite hydrogel obtained by the preparation method of the CCHG / APS composite hydrogel according to any one of claims 1-6.

8. The CCHG / APS composite hydrogel obtained by the preparation method of any one of claims 1-6 or the CCHG / APS composite hydrogel of claim 7, in the preparation of dressings for the healing or repair of diabetic wounds.

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