Visual hydrogel based on multiple response mechanism and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and specifically provides a visual hydrogel based on multiple response mechanisms and a preparation method and application thereof.The preparation method comprises the following steps: (1) preparing an inclusion compound by using beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin and cyanidin; (2) respectively preparing a polyvinyl alcohol / rhein solution and a mixed solution of quaternary ammonium salt chitosan / hyaluronic acid / κ-carrageenan; (3) initiating dynamic crosslinking by using phenylboronic acid, and constructing a multiple network structure in cooperation with tannic acid, the inclusion compound and genipin, and then curing the hydrogel through gradient temperature control.The hydrogel has pH / temperature / ROS response characteristics, and can use the color change of cyanidin to feed back the wound microenvironment state in real time, and meanwhile, hyaluronic acid / polyvinyl alcohol can construct a 3D microchannel structure, promote the ordered arrangement of collagen, and accelerate wound healing, thereby solving the problems of unstable direct loading of cyanidin and difficulty of a single response mechanism in adapting to the complex microenvironment of a diabetic wound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a visualized hydrogel based on a multiple response mechanism, its preparation method, and its applications. Background Technology

[0002] The treatment of diabetic wounds faces multiple challenges, including susceptibility to bacterial infection, imbalance of the inflammatory microenvironment, excessive exudate, and difficulty in real-time monitoring of the healing process. Traditional hydrogel wound dressings have attracted much attention in the field of wound care due to their excellent moisturizing, breathable, and biocompatible properties. However, existing products generally have the following limitations: (1) Most dressings have a single function, only providing a physical barrier and a moist environment, lacking synergistic effects of antibacterial, anti-inflammatory, or healing-promoting effects; (2) Drug release depends on simple diffusion and cannot respond to changes in the wound microenvironment to achieve on-demand release; (3) They cannot intuitively reflect the wound condition and require frequent replacement or reliance on external detection methods; (4) Some materials are prone to swelling and rupture or have poor adhesion, making them difficult to adapt to the treatment of complex wounds such as diabetic ulcers.

[0003] In recent years, the combination of intelligent responsive materials and visualization technology has become a research hotspot. For example, pH-sensitive pigments can indicate the infection status of wounds through color changes, and natural polymers are widely used to construct multifunctional hydrogel networks due to their antibacterial, responsive, and crosslinkable properties. Patent application 202411567623.7 discloses "a bilayer hydrogel for wound repair and infection monitoring, its preparation method, and its application." This patent application discloses a bilayer hydrogel that utilizes the pH-responsive color-changing properties of mulberry anthocyanins to monitor wound infection. The upper hydrogel is based on a sodium alginate hydrogel network crosslinked with metal cations, while the lower hydrogel is a dynamic hydrogel network based on the Schiff base reaction and contains quaternary ammonium salt groups. However, the visualization material used in this patent is easily degraded, has poor direct loading stability, and cannot accurately indicate changes in the degree of wound infection. Furthermore, the single-response mechanism used in this patent is insufficient to meet the needs of complex wound management, such as in cases of diabetes. Summary of the Invention

[0004] To address the aforementioned issues, this invention combines anthocyanins, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin to form an inclusion complex, thereby improving the stability of anthocyanins and providing a visual basis for the color changes of the hydrogel. The hydrogel prepared by this invention exhibits triple-response characteristics of pH / temperature / ROS, overcoming the shortcomings of single-response mechanisms in adapting to the complex microenvironment of diabetic wounds. Furthermore, it effectively reduces the risk of wound infection through the synergistic antibacterial mechanism of quaternary ammonium chitosan, κ-carrageenan, and rhein. Simultaneously, hyaluronic acid / polyvinyl alcohol can construct a 3D microchannel structure, promoting the orderly arrangement of collagen and accelerating the healing of diabetic wounds.

[0005] Therefore, this invention provides a method for preparing a visualized hydrogel based on a multiple response mechanism, the method comprising the following steps:

[0006] S100: Dissolve β-cyclodextrin and hydroxypropyl-β-cyclodextrin in deionized water, add anthocyanins, and perform ultrasonic treatment, magnetic stirring treatment, filtration treatment, and freeze-drying treatment under light-protected conditions to obtain the inclusion complex; S200: Add polyvinyl alcohol and rhein to deionized water to obtain the first solution; dissolve quaternary ammonium salt chitosan, hyaluronic acid, and κ-carrageenan in deionized water to obtain the second solution; S300: Add phenylboronic acid to the first solution and mix it with the second solution to obtain the first mixture; then add tannic acid, the inclusion complex, and genipin to the first mixture in sequence, and perform post-treatment to obtain the hydrogel.

[0007] In one embodiment of the present invention, step S300 specifically includes: S310, adding phenylboronic acid to a first solution, mixing thoroughly, and then slowly adding it to a second solution to obtain a first mixture; S320, adding tannic acid and its inclusion complex to the first mixture, mixing evenly, adding genipin, and reacting at room temperature in the dark to obtain a second mixture; S330, injecting the second mixture into a mold, allowing it to stand at room temperature, and then refrigerating it to solidify to obtain a hydrogel.

[0008] In one embodiment of the present invention, in step S100, the mass concentration of β-cyclodextrin is 2%, the mass concentration of hydroxypropyl-β-cyclodextrin is 2%, and the mass concentration of anthocyanin is 0.4~0.6%.

[0009] In one embodiment of the present invention, in step S200, the mass concentration of polyvinyl alcohol is 5-10%, the mass concentration of rhein is 0.2-0.5%, the mass concentration of quaternary ammonium chitosan is 3-6%, the mass concentration of hyaluronic acid is 2-5%, and the mass concentration of κ-carrageenan is 2-4%.

[0010] In one embodiment of the present invention, in step S300, the mass concentration of phenylboronic acid is 1-3%, the mass concentration of tannic acid is 1-3%, the mass concentration of the inclusion compound is 0.5%, and the mass concentration of genipin is 0.3-0.5%.

[0011] In one embodiment of the present invention, in step S100, ultrasonic treatment is performed for 20 minutes at a power of 400W, a frequency of 30kHz, and a temperature of 25°C. The magnetic stirring treatment is performed for 4 to 6 hours, and a 0.45μm filter membrane is used for filtration.

[0012] In one embodiment of the present invention, in step S330, the temperature for cold curing is 3~5°C and the time is 5~7h.

[0013] In one embodiment of the present invention, a visualization hydrogel based on a multiple response mechanism is also included, wherein the hydrogel is prepared using any of the above-described preparation methods.

[0014] In one embodiment of the invention, an application of a visualized hydrogel based on a multiple response mechanism is also included, in which the hydrogel is used for the repair of diabetic wounds.

[0015] By adopting the technical solution of the present invention, the following technical effects can be achieved:

[0016] (1) Co-modification with β-cyclodextrin and hydroxypropyl-β-cyclodextrin can form a bilayer inclusion structure. The cavity of β-cyclodextrin can accommodate anthocyanin molecules, while the outer layer modification of hydroxypropyl-β-cyclodextrin can further stabilize the inclusion complex and prevent the degradation and loss of anthocyanins in the external environment. β-cyclodextrin has a cone-shaped structure with internal hydrophobicity and external hydrophilicity, which can form inclusion complexes with anthocyanin molecules, thereby improving the water solubility and stability of anthocyanins. Hydroxypropyl-β-cyclodextrin is an etherified derivative of β-cyclodextrin, which has higher water solubility and lower toxicity, and can further enhance the stability and encapsulation efficiency of the inclusion complex. The cavitation effect of ultrasound can provide mechanical energy and promote the formation of inclusion complexes. Through the oscillation and shearing action of ultrasound, the inclusion complex particles can be broken into smaller particle sizes, which helps the inclusion complex to form a stable dispersion system in the hydrogel. When the pH of the wound infection area increases, anthocyanins undergo intramolecular charge transfer, and the color changes from purple to blue-green, reflecting the pH change of the wound microenvironment in real time and avoiding frequent dressing changes;

[0017] (2) Based on the dynamic covalent bond of phenylboronic acid-ortho-dihydroxy and the dual cross-linking network of κ-carrageenan thermosensitive phase change, combined with the characteristic that the solubility of rhein increases with pH, ​​a triple environmental response of pH / reactive oxygen / temperature is achieved, which can be precisely adapted to the complex pathological environment of diabetic wounds, such as high oxidative stress, high temperature and pH waves caused by infection.

[0018] (3) Rhein / tannic acid inhibits the NF-κB and MAPK inflammatory signaling pathways, reduces the expression of pro-inflammatory factors, and enhances the anti-inflammatory ability of hydrogel;

[0019] (4) Quaternary ammonium chitosan and κ-carrageenan can form an electrostatic trapping network, which can effectively capture and fix negatively charged bacteria, preventing bacterial movement and reproduction on the wound surface. Rhein can alter the permeability of bacterial cell membranes, disrupt their integrity, and lead to leakage of intracellular substances, ultimately causing bacterial death. The synergistic effect of the two can enhance the antibacterial ability of the hydrogel and reduce antibiotic dependence;

[0020] (5) Improve tensile strength by optimizing the network topology through a stepwise cross-linking strategy;

[0021] (6) Hyaluronic acid / PVA constructs a 3D microchannel structure to guide the directional migration of fibroblasts, while activating the TGF-β signaling pathway, promoting the orderly arrangement of collagen, and accelerating the healing of diabetic wounds. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings to be used in the description of the embodiments 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 these drawings without creative effort:

[0023] Figure 1 Scanning electron microscope (SEM) image of the hydrogel prepared in Example 1;

[0024] Figure 2 The color change of the hydrogel prepared in Example 1 under different pH conditions;

[0025] Figure 3 The tensile strength test results are for the hydrogel prepared in Example 1;

[0026] Figure 4 The changes in the cumulative release of rhein from the hydrogel prepared in Example 1 over time under different pH values ​​and different H2O2 concentrations are shown in Figure A, where A represents the cumulative release of rhein over time at pH 7.2, and B represents the cumulative release of rhein over time at pH 8.2.

[0027] Figure 5 The cell compatibility test results are for the hydrogel prepared in Example 1.

[0028] Figure 6 Changes in wounds in diabetic mice under different treatment groups;

[0029] Figure 7 Masson staining results for wound infection experiments in diabetic mice. Detailed Implementation

[0030] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0031] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the following description is provided in conjunction with... Figures 1 to 7 Specific embodiments of the present invention will be described in detail below.

[0033] The hydrogel prepared in this invention possesses multiple response mechanisms, enabling it to respond to external stimuli such as pH, temperature, and ROS. In the microenvironment of diabetic wounds, the pH value typically fluctuates. The hydrogel can adjust its properties according to changes in wound pH to better adapt to the dynamic changes in the wound and improve treatment efficacy. Simultaneously, its responsiveness to temperature and ROS allows it to more flexibly address the complex microenvironment of diabetic wounds, achieving precise treatment and repair.

[0034] Furthermore, the addition of a β-CD / HP-β-CD / anthocyanin inclusion complex gives the hydrogel visualization properties. Anthocyanins can change color according to changes in environmental pH. The inclusion of β-CD / HP-β-CD improves the stability and solubility of anthocyanins, enabling them to more effectively perform their visualization and monitoring functions within the hydrogel.

[0035] Preferably, by rationally proportioning and combining materials with different functions, such as PVA providing good gelling and mechanical properties, rhein possessing anti-inflammatory and other pharmacological effects, quaternary ammonium salt chitosan enhancing antibacterial properties, hyaluronic acid promoting cell proliferation and tissue repair, κ-carrageenan participating in the construction of network structures, phenylboronic acid participating in cross-linking reactions, tannic acid providing antioxidant properties, and β-CD / HP-β-CD / anthocyanin inclusion complexes enabling visualization functions, a synergistic effect of multiple functions is achieved, enabling the hydrogel to play a role from multiple angles in the wound repair process, promoting rapid wound healing and tissue regeneration.

[0036] For example, the mass concentration of each material is precisely controlled and optimized, such as PVA 5~10%, rhein 0.2~0.5%, QC 3~6%, HA 2~5%, κ-carrageenan 2~4%, etc., to ensure that the hydrogel has suitable physical properties and chemical stability, so that it can maintain good shape and performance in practical applications, effectively adhere to the wound and provide a lasting repair effect.

[0037] Preferably, the hydrogel in this embodiment is specifically designed for the repair of diabetic wounds, fully considering the special pathophysiological characteristics of diabetic wounds, such as hyperglycemia, microcirculatory disturbances, and severe inflammatory responses. Through the synergistic effect of multiple components, it can effectively improve the wound microenvironment, promote angiogenesis and tissue repair, and improve the healing speed and quality of diabetic wounds, providing an effective wound treatment method for diabetic patients and reducing patient suffering and the social medical burden.

[0038] Furthermore, in clinical applications, this hydrogel can serve as a novel dressing or drug carrier for treating various types of diabetic wounds, such as diabetic foot ulcers and pressure ulcers. Its excellent biocompatibility, multiple response mechanisms, and visualization capabilities make it safer, more effective, and easier to monitor during use, making it a promising product for diabetic wound repair with high clinical application value and market potential.

[0039] Preferably, the selected materials, such as polyvinyl alcohol (PVA) and chitosan, have good biocompatibility, so that the prepared hydrogel will not cause obvious immune or toxic reactions when applied in vivo, ensuring the safety and reliability of use, and is especially suitable for biomedical fields such as diabetic wound repair.

[0040] Ideally, the reaction conditions used in the preparation process are relatively mild, avoiding the damage to materials and active ingredients caused by extreme conditions such as high temperature and high pressure. This helps maintain the bioactivity and stability of each component, while also contributing to energy conservation and reduced production costs. By precisely controlling parameters such as reaction time, temperature, and stirring speed in each step, as well as strictly controlling the mass concentration of each material, the preparation process exhibits good repeatability, enabling the stable production of hydrogel products with consistent quality and stable performance, meeting the stringent requirements for product quality stability in both clinical applications and industrial production.

[0041]

Example 1

[0042] (1) Dissolve 0.2g β-cyclodextrin and 0.2g hydroxypropyl-β-cyclodextrin in 10mL of deionized water, then add 0.05g anthocyanin, sonicate for 20 minutes at 400W power, 30kHz frequency, and 25℃, stir magnetically for 5 hours in the dark, filter through a 0.45 μm filter membrane, and freeze-dry to obtain β-CD / HP-β-CD / anthocyanin inclusion complex;

[0043] (2) Add 60mg PVA powder and 0.5mg rhein to 1mL of deionized water, heat and stir until completely dissolved, cool to room temperature to obtain PVA / rhein solution; add 50mg QC, 40mg HA and 30mg κ-carrageenan to 1mL of deionized water, heat to fully dissolve to obtain QC / HA / carrageenan solution;

[0044] (3) Add 20 mg of phenylboronic acid to the PVA / rhein solution and mix thoroughly. Then slowly add QC / HA / carrageenan solution to obtain mixture A. Add 20 mg of tannic acid and 5 mg of β-CD / HP-β-CD / anthocyanin inclusion complex to mixture A and stir to mix. Then add 4 mg of genipin and mix thoroughly. React at 37°C in the dark for 24 hours. Pour the mixture into a mold and let it stand at room temperature for 1 hour. Then refrigerate at 4°C for 6 hours to form a hydrogel.

[0045]

Example 2

[0046] (1) Dissolve 0.2g of β-cyclodextrin and 0.2g of hydroxypropyl-β-cyclodextrin in 10mL of deionized water, then add 0.04g of anthocyanin, sonicate for 20 minutes at 400W power, 30kHz frequency, and 25℃, and stir magnetically for 5 hours in the dark. Filter through a 0.45 μm filter membrane and freeze-dry to obtain β-CD / HP-β-CD / anthocyanin inclusion complex.

[0047] (2) Add 50mg PVA powder and 4mg rhein to 1mL of deionized water, heat and stir until completely dissolved, cool to room temperature to obtain PVA / rhein solution; add 30mg QC, 20mg HA and 20mg κ-carrageenan to 1mL of deionized water, heat to fully dissolve to obtain QC / HA / carrageenan solution.

[0048] (3) Add 10 mg of phenylboronic acid to the PVA / rhein solution and mix thoroughly. Then slowly add QC / HA / carrageenan solution to obtain mixture A. Add 10 mg of tannic acid and 5 mg of β-CD / HP-β-CD / anthocyanin inclusion complex to mixture A and stir to mix thoroughly. Then add 3 mg of genipin and mix thoroughly. React at 37°C in the dark for 24 hours. Pour the mixture into a mold and let it stand at room temperature for 1 hour. Then refrigerate at 4°C for 6 hours to form a hydrogel.

[0049]

Example 3

[0050] (1) Dissolve 0.2g of β-cyclodextrin and 0.2g of hydroxypropyl-β-cyclodextrin in 10mL of deionized water, then add 0.06g of anthocyanin, sonicate for 20 minutes at 400W power, 30kHz frequency, and 25℃, and stir magnetically for 5 hours in the dark. Filter through a 0.45 μm filter membrane and freeze-dry to obtain β-CD / HP-β-CD / anthocyanin inclusion complex.

[0051] (2) Add 80mg PVA powder and 3mg rhein to 1mL of deionized water, heat and stir until completely dissolved, cool to room temperature to obtain PVA / rhein solution; add 60mg QC, 50mg HA and 40mg κ-carrageenan to 1mL of deionized water, heat to fully dissolve to obtain QC / HA / carrageenan solution.

[0052] (3) Add 30 mg of phenylboronic acid to the PVA / rhein solution and mix thoroughly. Then slowly add QC / HA / carrageenan solution to obtain mixture A. Add 30 mg of tannic acid and 5 mg of β-CD / HP-β-CD / anthocyanin inclusion complex to mixture A and stir to mix. Then add 5 mg of genipin and mix thoroughly. React at 37°C in the dark for 24 hours. Pour the mixture into a mold and let it stand at room temperature for 1 hour. Then refrigerate at 4°C for 6 hours to form a hydrogel.

[0053]

Example 4

[0054] (1) Dissolve 0.2g of β-cyclodextrin and 0.2g of hydroxypropyl-β-cyclodextrin in 10mL of deionized water, then add 0.06g of anthocyanin, sonicate for 20 minutes at 400W power, 30kHz frequency, and 25℃, and stir magnetically for 5 hours in the dark. Filter through a 0.45 μm filter membrane and freeze-dry to obtain β-CD / HP-β-CD / anthocyanin inclusion complex.

[0055] (2) Add 80mg PVA powder and 2mg rhein to 1mL of deionized water, heat and stir until completely dissolved, cool to room temperature to obtain PVA / rhein solution; add 50mg QC, 50mg HA and 40mg κ-carrageenan to 1mL of deionized water, heat to fully dissolve to obtain QC / HA / carrageenan solution.

[0056] (3) Add 30 mg of phenylboronic acid to the PVA / rhein solution and mix thoroughly. Then slowly add QC / HA / carrageenan solution to obtain mixture A. Add 30 mg of tannic acid and 5 mg of β-CD / HP-β-CD / anthocyanin inclusion complex to mixture A and stir to mix. Then add 5 mg of genipin and mix thoroughly. React at 37°C in the dark for 24 hours. Pour the mixture into a mold and let it stand at room temperature for 1 hour. Then refrigerate at 4°C for 6 hours to form a hydrogel.

[0057]

Example 5

[0058] (1) Dissolve 0.2g of β-cyclodextrin and 0.2g of hydroxypropyl-β-cyclodextrin in 10mL of deionized water, then add 0.06g of anthocyanin, sonicate for 20 minutes at 400W power, 30kHz frequency, and 25℃, and stir magnetically for 5 hours in the dark. Filter through a 0.45 μm filter membrane and freeze-dry to obtain β-CD / HP-β-CD / anthocyanin inclusion complex.

[0059] (2) Add 80mg PVA powder and 2mg rhein to 1mL of deionized water, heat and stir until completely dissolved, cool to room temperature to obtain PVA / rhein solution; add 40mg QC, 50mg HA and 30mg κ-carrageenan to 1mL of deionized water, heat to fully dissolve to obtain QC / HA / carrageenan solution.

[0060] (3) Add 30 mg of phenylboronic acid to the PVA / rhein solution and mix thoroughly. Then slowly add QC / HA / carrageenan solution to obtain mixture A. Add 30 mg of tannic acid and 5 mg of β-CD / HP-β-CD / anthocyanin inclusion complex to mixture A and stir to mix. Then add 5 mg of genipin and mix thoroughly. React at 37°C in the dark for 24 hours. Pour the mixture into a mold and let it stand at room temperature for 1 hour. Then refrigerate at 4°C for 6 hours to form a hydrogel.

[0061] Comparative Example 1

[0062] The preparation process of the hydrogel is basically the same as that in Example 1, except that rhein is not added in step 2 and a PVA solution is obtained, and in step 3, the PVA solution is used instead of the PVA / rhein solution to prepare an unloaded hydrogel.

[0063] Performance Characterization

[0064] (1) The hydrogel sample prepared in Example 1 was freeze-dried and then sputter-coated with gold. Its microstructure was observed by scanning electron microscopy. The SEM image is shown below. Figure 1 As shown in the figure. The results show that the hydrogel has a three-dimensional porous structure with pore sizes ranging from 100 to 200 μm, which is beneficial for exudate absorption and gas exchange.

[0065] (2) The hydrogel samples prepared in Example 1 were immersed in buffer solutions with pH values ​​of 5.0, 6.0, 7.0, and 8.0, respectively, and the color changes were recorded, such as... Figure 2 As shown, the hydrogel sample was purple at pH 5.0 (normal skin), blue-purple at pH 6.0 and blue at pH 7.0, and blue-green at pH 8.0 (infected environment), indicating that the hydrogel can reflect the pH changes of the wound microenvironment in real time.

[0066] (3) The hydrogel sample in Example 1 was processed into strips (5 cm long × 1 cm wide × 0.4 cm thick). Stress-strain tests were performed at 25°C using a universal testing machine with a loading rate of 10 mm / min to examine the tensile properties of the hydrogel.

[0067] See results Figure 3 The hydrogel can withstand a tensile strain of up to 186.39%, at which point the stress can reach 67.6 kPa, indicating that it has good tensile strength.

[0068] (4) The hydrogel samples prepared in Example 1 were placed under different pH buffers (pH 7.2 and 8.2) and different H2O2 concentrations (0.1 mM, 1 mM, 10 mM), and the cumulative release of rhein was quantitatively detected by HPLC.

[0069] See results Figure 4 It can be seen that the cumulative release of rhein is affected by pH and H2O2 concentration. When the pH increases and / or the H2O2 concentration increases, the cumulative release of rhein increases, suggesting that this pH / ROS dual-response effect enables rhein to be released precisely in the specific environment of the inflammatory site.

[0070] (5) Using Escherichia coli and Staphylococcus aureus as test strains, the antibacterial activity of the hydrogel samples in Example 1 and the comparative example was determined by plate count method. 0.5 g of hydrogel and 20 mL of bacterial suspension (bacterial density 1×10⁻⁶) were taken. 7 After mixing with CFU / mL, the mixture was placed on a constant temperature shaker and incubated at 37°C for 30 minutes. The bacterial suspension was then serially diluted and spread onto solid LB medium, and incubated at 37°C for 12 hours in an electric thermostatic incubator. The inhibition rate was calculated based on the number of colonies on the plates. Inhibition rate = (number of colonies in blank control group - number of colonies in experimental group) / number of colonies in blank control group * 100%.

[0071] The results are shown in Table 1. It can be seen that the hydrogel samples in the examples and comparative examples all have good antibacterial effects, and the antibacterial rates of the hydrogel samples in the three examples against Escherichia coli and Staphylococcus aureus are significantly higher than those of the comparative examples.

[0072] Table 1. In vitro antibacterial rate of hydrogels

[0073] Escherichia coli inhibition rate (%) Staphylococcus aureus inhibition rate (%) Comparative Example 1 78.2 72.9 Example 1 99.4 97.9 Example 2 94.8 92.6 Example 3 90.3 83.5 Example 4 83.6 79.4 Example 5 80.9 76.2

[0074] (6) The cell compatibility of the hydrogel prepared in Example 1 was evaluated using L929 mouse fibroblasts. L929 mouse fibroblasts were prepared at a ratio of 1 × 10⁶ cells per well. 5Add the culture medium to each well of a 96-well plate and incubate for 24 hours. The control group received 100 μL of complete culture medium to replace the original medium, while the experimental groups received 100 μL of hydrogel extract to replace the original medium. Incubate at 37°C, 5% CO2 for 24, 48, and 72 hours. At each time point, aspirate the culture medium from the wells and add 10 μL of MTT solution (final concentration 0.5 mg / mL) to each well, continuing incubation for another 4 hours. After incubation, carefully aspirate the culture supernatant from the wells and add 100 μL of DMSO to each well, gently shaking for 10 minutes to fully dissolve the formazan crystals. Detect the absorbance (OD) using a microplate reader. 570 Cell viability is calculated using the following formula: Cell viability = (OD) / (OD200) 570 Experimental group / OD 570 (Control group) * 100%.

[0075] The results are as follows Figure 5 As shown, after co-incubation of the hydrogel with L929 mouse fibroblasts, the cell survival rate remained above 90%, indicating that it had no obvious toxic side effects.

[0076] (7) To investigate the effect of the hydrogel prepared in Example 1 on infected wounds in diabetic mice. Eight male KM mice were randomly divided into two groups (control group and hydrogel group). Type I diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (usually 50 mg / kg / day for 5 consecutive days). Fasting blood glucose was monitored 72 hours after injection and throughout the experiment to confirm the successful establishment of the diabetic model (blood glucose ≥11.1 mmol / L). Mice were anesthetized with isoflurane, their back hair was shaved, and their skin was disinfected. Two circular full-thickness skin wounds were created symmetrically on the back using a sterile biopsy puncturist (6 mm in diameter). The control group had their wounds covered with PBS, while the hydrogel group had their wounds covered with hydrogel. The wounds were cleaned and retreated daily. Masson staining was performed on the healed tissues of the rats after the wounds had completely healed.

[0077] Figure 6 Images show the wound healing effects in diabetic mice on days 0, 3, 6, and 12 after hydrogel treatment. It can be seen that, compared to the control group, the hydrogel group exhibited a wound-healing-promoting effect, not only accelerating the healing process but also positively impacting the repair of surrounding tissues. Masson staining results are shown below. Figure 7 As shown, on day 12 of the experiment, the control group had relatively low collagen fiber content and a sparse overall structure, lacking a tight arrangement. In contrast, the hydrogel group showed a significant improvement in collagen fiber formation, with not only a higher quantity but also a more orderly arrangement, demonstrating the significant advantage of this treatment in promoting collagen fiber formation and tissue repair.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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 the present invention.

Claims

1. A method for preparing a visualized hydrogel based on a multiple response mechanism, characterized in that, The preparation method includes the following steps: S100. β-Cyclodextrin and hydroxypropyl-β-Cyclodextrin are dissolved in deionized water, anthocyanins are added, and the mixture is subjected to ultrasonic treatment, magnetic stirring, filtration, and freeze-drying under light-protected conditions to obtain the inclusion complex. S200. Polyvinyl alcohol and rhein are added to deionized water to obtain the first solution; quaternary ammonium salt chitosan, hyaluronic acid, and κ-carrageenan are dissolved in deionized water to obtain the second solution. S300. After adding phenylboronic acid to the first solution, it is mixed with the second solution to obtain a first mixture; then tannic acid, the inclusion complex, and genipin are added to the first mixture in sequence, and after post-treatment, the hydrogel is obtained.

2. The preparation method according to claim 1, characterized in that, Step S300 specifically includes: S310. Add the phenylboronic acid to the first solution, mix thoroughly, and then slowly add it to the second solution to obtain the first mixture. S320. Add the tannic acid and the inclusion complex to the first mixture, mix evenly, add genipin, and react at room temperature in the dark to obtain the second mixture. S330. The second mixture is injected into the mold, left to stand at room temperature, and then refrigerated to solidify, thus obtaining the hydrogel.

3. The preparation method according to claim 1, characterized in that, In step S100, the mass concentration of the β-cyclodextrin is 2%, the mass concentration of the hydroxypropyl-β-cyclodextrin is 2%, and the mass concentration of the anthocyanin is 0.4~0.6%.

4. The preparation method according to claim 1, characterized in that, In step S200, the mass concentration of polyvinyl alcohol is 5-10%, the mass concentration of rhein is 0.2-0.5%, the mass concentration of quaternary ammonium chitosan is 3-6%, the mass concentration of hyaluronic acid is 2-5%, and the mass concentration of κ-carrageenan is 2-4%.

5. The preparation method according to claim 1, characterized in that, In step S300, the mass concentration of the phenylboronic acid is 1-3%, the mass concentration of the tannic acid is 1-3%, the mass concentration of the inclusion complex is 0.5%, and the mass concentration of the genipin is 0.3-0.5%.

6. The preparation method according to claim 1, characterized in that, In step S100, the ultrasonic treatment lasts for 20 minutes at a power of 400W, a frequency of 30kHz, and a temperature of 25℃. The magnetic stirring treatment lasts for 4 to 6 hours. The filtration treatment uses a 0.45μm filter membrane.

7. The preparation method according to claim 2, characterized in that, In step S330, the temperature for cold curing is 3~5℃ and the time is 5~7h.

8. A visualized hydrogel based on a multiple response mechanism, characterized in that, The hydrogel is obtained by the preparation method described in any one of claims 1-7.

9. An application of a visualized hydrogel based on a multiple response mechanism, characterized in that, The hydrogel as described in claim 8 is applied to a diabetic wound repair product.

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