Preparation and application of strong tissue adhesion multifunctional injectable hydrogel responding to wound microenvironment

Abstract

The invention provides a preparation method of strong tissue adhesion multifunctional injectable hydrogel responding to a wound microenvironment and application of the strong tissue adhesion multifunctional injectable hydrogel in the aspect of delivering antibacterial natural products. The injectable hydrogel capable of filling wounds in situ is prepared by mixing arginine modified gelatin with antibacterial and tissue repair promoting functions with dopamine modified carboxymethyl chitosan which provides hemostasis and strong tissue adhesion by adopting an enzymatic and ionic crosslinking strategy and carrying out Schiff base reaction on arginine amino groups and a carboxymethyl chitosan carboxylic acid structure. The prepared hydrogel has dynamic reversibility, can be reconstructed after being broken by external force, and can precisely and controllably release a bacteriostatic natural product according to the pH change of a wound microenvironment. The hydrogel is good in biocompatibility, simple in preparation process, green and low in cost, and has a wide application prospect in wound infection treatment, especially in the field of delivery of antibacterial natural products.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and specifically to the preparation of a strong tissue-adhesive multifunctional injectable hydrogel responsive to the wound microenvironment and its use for the repair and treatment of bacterial-infected wounds. Background Art

[0002] The skin is the body's first line of defense and is vulnerable to trauma. Improper wound treatment may lead to serious consequences. Simple physical coverage is difficult to meet clinical needs. Hydrogels have attracted much attention in the field of wound dressings due to their hydrophilicity and porous structure. Natural polymers, with excellent biocompatibility, have become ideal raw materials for constructing hydrogels. Chitosan (CS) has the properties of biodegradation, hemostasis, antibacterial, etc., but its low solubility in water limits its application. Carboxymethyl chitosan (CMCS) modified by carboxymethylation has good water solubility, which greatly expands the application scope of CS. Gelatin (Gel), as a natural protein product, has excellent degradability in the physiological environment and is an effective raw material for preparing hydrogels. Chinese Patent CN118203693 A uses CMCS and Gel crosslinked with glutaraldehyde to prepare an aerogel with antibacterial and hemostatic effects. However, wound healing is a complex process, including four consecutive stages: hemostasis, inflammation, proliferation, and remodeling. Therefore, it is extremely crucial to develop wound dressings with antibacterial, hemostatic, and tissue repair-promoting functions.

[0003] As an effective means, chemical modification can modify hydrogels, thereby expanding their application scope and improving their performance. Arginine (Arg) carries a positive charge due to the guanidine group in its molecular structure, which endows Arg with certain antibacterial ability. In addition, Arg is an essential amino acid in the wound healing process. It is speculated that a hydrogel based on Arg-Gel can not only play an antibacterial role during wound infection but also potentially promote wound healing. In addition, traditional hydrogels have weak adhesion on the surface of moist tissues. For wounds in high-intensity exercise areas, existing hydrogel dressings are difficult to firmly fix on the repair site, with poor adhesion, which limits the application of hydrogels in the field of wound healing. Dopamine (DA) is structurally inspired by mussel adhesive proteins, and its catechol group can non-covalently bind to proteins on the surface of wound tissues. Therefore, grafting DA in CMCS (DA-CMCS) may be an effective strategy to ensure the stable adhesion of hydrogels to skin tissues to meet the complex and variable wound treatment requirements.

[0004] The present invention for the first time prepared Arg-Gel with bacteriostatic and tissue repair promoting effects and DA-CMCS with hemostatic, strong tissue adhesion and mechanical strength increasing functions, and innovatively proposed a novel method for preparing an "injectable hydrogel" with multiple wound healing enhancing functions by mixing Arg-Gel and DA-CMCS. Its injectability benefits from the Schiff base reaction between the amino group in Arg and the carboxylic acid structure in CMCS. It is worth noting that compared with the traditional "preformed hydrogel" which is difficult to adapt to wounds with complex shapes, the injectable hydrogel in this study can flexibly fill irregular spaces in situ according to the specific shape and depth of the wound, closely adhere to the wound tissue, and create more favorable conditions for wound healing. At the same time, the imine bond formed by the Schiff base reaction not only endows the hydrogel with injectability, but also due to its dynamic reversible characteristics, after the hydrogel is broken under external stress (such as deformation caused by patient movement), it can self-reconstruct under mild physiological conditions, restore its original structure and function, and effectively improve the stability and durability of the hydrogel in a complex dynamic environment.

[0005] In addition, the imine bond is highly sensitive to the environmental pH value and exhibits significant pH-responsive characteristics. The unique structure and properties of this hydrogel make it an ideal carrier for delivering bacteriostatic natural products. Although many bacteriostatic natural products have good antibacterial effects, they often have problems such as poor solubility and low bioavailability, which limit their application in wound treatment. The hydrogel prepared in the present invention has a hydrophilic and porous structure, can effectively encapsulate bacteriostatic natural products, and increase their dispersibility and solubility in the system. During the wound infection stage, the change in the pH value of the local microenvironment can be used as a trigger signal to promote the reversible cleavage and recombination of the imine bond, thereby realizing the precise and controllable release of the bacteriostatic compound by the hydrogel. This intelligent response mechanism with pH stimulation as the "release switch" endows the hydrogel with the function of releasing active ingredients on demand, greatly enhancing its targeting and adaptability during the wound treatment process, and providing strong technical support for realizing personalized and precise wound healing treatment strategies.

[0006] When developing injectable hydrogels, to achieve injectability, their water content is usually relatively high, resulting in weak intermolecular forces and loose network structures, and often facing the problem of low mechanical strength. To address the above challenges, the multi-crosslinking strategy can enhance the network structure stability and improve the mechanical strength without affecting the original advantages of the hydrogel. Among them, the enzymatic crosslinking and ionic crosslinking methods are green, efficient and have good biocompatibility, and have great application potential. Specifically, using horseradish peroxidase (HRP) / H₂O₂ to catalyze the crosslinking of catechol groups in DA can conveniently regulate the gelation rate and crosslinking density. Introducing it into the Schiff base crosslinking system can enhance the mechanical and adhesive strength of mussel-inspired hydrogels and also regulate the gelation time. In addition, in the hydrogel system, Ca 2+ can form a coordination bond with the carboxyl group of CMCS, increasing the crosslinking density and enhancing the mechanical strength of the hydrogel, enabling it to better withstand external forces at the wound site. Meanwhile, Ca 2 + as a key factor in blood coagulation can promote the interaction of coagulation factors and enhance the hemostatic effect of the hydrogel. In the present invention, Arg-Gel and DA-CMCS are innovatively combined to prepare an injectable hydrogel with multiple functions. On this basis, a multi-crosslinking technology is also innovatively introduced to further optimize the performance of the hydrogel. The prepared hydrogel not only has unique functions but also has good biocompatibility. The preparation process is simple, green, without complex equipment and cumbersome processes, greatly reducing the production difficulty and cost, and is easy to promote and use. Summary of the Invention

[0007] Aiming at the problems of existing wound dressings such as single function, difficulty in meeting the diverse needs in the complex process of wound healing, low viscosity, inability to flexibly adapt to the shape of the wound, low mechanical strength, and inability to self-heal, the present invention provides a preparation method of a super-strong tissue-adhesive multifunctional injectable hydrogel that responds to the wound microenvironment. At the same time, aiming at the problems of low solubility and low bioavailability of natural products, the present invention effectively encapsulates natural products by utilizing the unique structure and properties of the hydrogel. Through the reasonable design of the hydrogel, the bioavailability of natural products is improved.

[0008] The technical solution of the present invention is as follows:

[0009] A preparation method of a strong tissue-adhesive multifunctional injectable hydrogel that responds to the wound microenvironment, the specific steps are as follows:

[0010] (1) Take an appropriate amount of phosphate buffer solution (PBS) with pH = 4.5 - 8.4 and place it in a beaker, and add arginine-modified gelatin (Arg-Gel) with a mass fraction of 2.5% - 9.0%, 0.1% - 0.6% horseradish peroxidase (HRP), and 0.1% - 1.0% CaCl 2 ;

[0011] (2) Take an appropriate amount of PBS with pH = 3.5 - 8.5 and place it in a beaker, and add dopamine-modified carboxymethyl chitosan (DA-CMCS) with a mass fraction of 10% - 25% and 0.1% - 0.5% hydrogen peroxide (H 2 O 2 );

[0012] (3) Mix the solutions in steps (1) and (2) with equal volume, and stir at a speed of 150 - 850 rpm for 0.5 - 6 h until a hydrogel is formed.

[0013] Further, the preparation method of the Arg-Gel is as follows:

[0014] Take 5 g of Gel and place it in 100 mL of deionized water. Heat it to dissolve at 50 °C, and add N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) according to a molar ratio of 1.2 - 5.2:1. Stir magnetically for 30 min. Add Arg according to a mass ratio of Gel to Arg of 2 - 10:4, adjust the pH to 2.5 - 8.5, stir at a speed of 100 - 500 rpm at room temperature for 24 h, dialyze for 48 h, and freeze-dry for 36 h to obtain Arg-Gel.

[0015] Furthermore, the preparation method of the DA-CMCS is as follows:

[0016] Take an appropriate amount of CMCS, NHS, and EDC and dissolve them in deionized water according to a molar ratio of 1.2 - 6.5:1:1. Stir magnetically for 30 min; add DA according to a mass ratio of DA to CMCS of 2 - 10:5, adjust the pH to 3 - 9, stir at a speed of 200 - 650 rpm at room temperature for 24 h, dialyze for 48 h, and freeze-dry for 36 h to obtain DA-CMCS.

[0017] Furthermore, it is characterized in that during the preparation of the modified Gel and hydrogel, other amino acids with similar activities (such as lysine, histidine, etc.) are selected, and according to the same mass ratio relationship and similar reaction conditions as in the Arg modification process, replace Arg to modify Gel, and the prepared hydrogel, its preparation method and the functional characteristics it possesses all fall within the protection scope of the present invention.

[0018] Furthermore, it is characterized in that during the preparation of the modified Gel and hydrogel, amino group-containing active components are selected, such as but not limited to amino sugars (such as glucosamine), amino alcohols (such as ethanolamine), and polypeptide fragments with amino side chains, etc. According to a mechanism similar to that of Arg, they participate in the modification of Gel and the preparation of hydrogel in appropriate proportions and reaction conditions, all within the protection scope of the present invention.

[0019] Furthermore, it is characterized in that during the preparation of the modified CMCS and hydrogel, components inspired by mussels (such as mussel adhesive protein) and components containing catechol structures (such as tannic acid) are selected. If they are in accordance with a mechanism similar to that of DA and similar mass ratios and reaction conditions, the resulting similarly functional modified CMCS and hydrogel are all within the protection scope of the present invention.

[0020] Furthermore, it is characterized in that for the preparation of the hydrogel by ionic crosslinking reaction, in addition to Ca 2 +, metal ions with carboxyl crosslinking functions such as iron ions, magnesium ions, zinc ions, etc. are all within the protection scope of the present invention.

[0021] Further, it is characterized in that the hydrogel can respond to the pH change of the wound and intelligently deliver antibacterial natural small molecule compounds, including but not limited to honokiol.

[0022] Further, it is characterized in that the pH change range that the hydrogel can respond to the wound is 4.0 - 6.0.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention innovatively combines natural - sourced Arg - Gel and DA - CMCS to construct a wound - repair platform with multi - functional synergy, enabling the hydrogel to have the functions of hemostasis, antibacterial, promoting repair and strong adhesion, comprehensively and efficiently meeting the requirements of each stage of wound healing, and overcoming the drawback of single - function of traditional dressings.

[0025] 2. The present invention innovatively integrates Schiff - base, enzyme - catalyzed and ionic cross - linking technologies, and the three work together to endow the hydrogel with unique injectability, enabling it to closely fit the wound, precisely control the gel - forming speed and tightness, form a stable network structure, and enhance the overall toughness.

[0026] 3. The present invention proposes a novel antibacterial natural product delivery strategy. Relying on the special structure and properties of the hydrogel, it solves the problems of poor applicability and low utilization rate of natural products at the wound site, and can achieve intelligent release based on the pH - responsive mechanism, greatly improving the treatment efficiency.

[0027] 4. The hydrogel prepared by the present invention has good biocompatibility, can be safely metabolized in vivo, and the preparation process is simple and green, without complex equipment and cumbersome processes, greatly reducing the production difficulty and cost, and is suitable for popularization from the laboratory to actual production. Brief Description of the Drawings

[0028] Figure 1 Shows the injectability, plasticity and self - healing property of the Arg - Gel / DA - CMCS hydrogel. (A: Injectability, B: Plasticity, C: Self - healing property)

[0029] Figure 2 Shows the adhesion and mechanical properties of the Arg - Gel / DA - CMCS hydrogel (A: Adhesion, B: Tensile property).

[0030] Figure 3 Shows the pH - responsive release property of the Arg - Gel / DA - CMCS hydrogel.

[0031] Figure 4 Shows the antibacterial activity of the Arg - Gel / DA - CMCS hydrogel (A: Normal saline group, B: Arg - Gel / DA - CMCS hydrogel group).

[0032] Figure 5 Hemostatic effect of Arg-Gel / DA-CMCS hydrogel on rat tail amputation model (A: normal saline group, B: Arg-Gel / DA-CMCS hydrogel group).

[0033] Figure 6 Wound healing promoting effect of Arg-Gel / DA-CMCS hydrogel (A: normal saline group, B: Arg-Gel / DA-CMCS hydrogel group). Specific implementation manners

[0034] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0035] Example 1

[0036] Preparation of Arg-Gel

[0037] Take 5 g of Gel and place it in 100 mL of deionized water. Heat it to dissolve at 50 °C, add 0.85 g of NHS and 1.35 g of EDC respectively, and stir magnetically for 30 min. Add 4 g of Arg, adjust the pH to 5.5, stir at a speed of 120 rpm at room temperature for 24 h, dialyze for 48 h, and freeze-dry for 36 h to obtain Arg-Gel.

[0038] Example 2

[0039] Preparation of DA-CMCS

[0040] Take 1 g of CMCS, 1.55 g of NHS and 3.89 g of EDC and dissolve them in deionized water respectively, and stir magnetically for 30 min; add 2.31 g of DA, adjust the pH to 7.0, stir at a speed of 300 rpm for 24 h, dialyze for 48 h, and freeze-dry for 36 h to obtain DA-CMCS.

[0041] Example 3

[0042] Preparation of Arg-Gel / DA-CMCS hydrogel

[0043] (1) Take 10 mL of PBS with pH = 7.2 and place it in a beaker, add 2.5% DA-CMCS, 0.1% HRP and 0.5% H 2 O 2 ;

[0044] (2) Take 10 mL of PBS with pH = 7.2 and place it in a beaker, add 20% Arg-Gel and 1% CaCl 2 ;

[0045] (3) Mix the solutions in steps (1) and (2), and stir at a speed of 360 rpm for 1 h until a hydrogel is formed.

[0046] Example 4

[0047] Physical property analysis of Arg-Gel / DA-CMCS hydrogel

[0048] 1. Evaluation of injectability, plasticity and self-healing property

[0049] To comprehensively investigate the 1. injectability, plasticity and self-healing property of the Arg-Gel / DA-CMCS hydrogel prepared in Example 3. First, take 2 mL of the Arg-Gel / DA-CMCS hydrogel and load it into a 5 mL syringe. Then, push the syringe manually to extrude the hydrogel through the needle, and the word "NEAU" is successfully extruded on the plane, which strongly proves from a macroscopic perspective that the hydrogel has good injectability.

[0050] Next, place the Arg-Gel / DA-CMCS hydrogel in various molds with different shapes. After the hydrogel is formed, it can be clearly seen that it can perfectly fit the shape of the mold, which proves that the hydrogel has excellent plasticity.

[0051] In addition, corresponding tests were also carried out on the self-healing property of the Arg-Gel / DA-CMCS hydrogel. The hydrogel was deliberately broken, and then the two broken parts were spliced together. After standing for 10 min, it was found that the hydrogel achieved a firm bond at the splicing site. When a certain external force was applied again, the splicing site did not break again, which fully proves its self-healing property (see Figure 1 ). The above three properties come from the imine bond formed between Arg-Gel and DA-CMCS through the Schiff base reaction.

[0052] 2. Evaluation of adhesion and mechanical properties

[0053] To evaluate the adhesion property of the Arg-Gel / DA-CMCS hydrogel prepared in Example 3, an adhesion property evaluation experiment of organic material (skin) and inorganic material (plastic) was selected. From Figure 2 The shown results can clearly show that the Arg-Gel / DA-CMCS hydrogel exhibits strong adhesion force, and it can firmly adhere a plastic centrifuge tube with gradually increasing mass to the skin. This is due to the catechol group contained in DA, which enables the Arg-Gel / DA-Gel hydrogel to firmly adhere to various organic and inorganic substrates.

[0054] To further explore the mechanical tensile properties of the hydrogel, an experiment simulating the actual use scenario was conducted. The Arg-Gel / DA-CMCS hydrogel was attached to the index finger joint, and then the index finger was slowly bent to different bending angles. During this process, it was observed that the hydrogel could be stretched accordingly as the finger bent, and no fracture occurred in the hydrogel regardless of the degree of finger bending. This experimental result fully demonstrates that the Arg-Gel / DA-CMCS hydrogel has excellent mechanical tensile properties and can effectively resist the damage that may be caused by movement and stretching during actual use. The source of this excellent performance is mainly the enzyme-catalyzed reaction of HRP / HO, and Ca 2 + can form coordination bonds with the carboxyl groups of CMCS, thereby increasing the cross-linking density of the hydrogel.

[0055] 3. Evaluation of the pH-responsive release characteristics of the Arg-Gel / DA-CMCS hydrogel

[0056] To evaluate the pH-responsive release characteristics of the material prepared in Example 3 for the antibacterial natural product, the following experimental operations were carried out: 1 mg of honokiol was accurately weighed and dissolved in 10 mL of PBS containing 10%. After complete dissolution, a stock solution of honokiol with a concentration of 100 μg / mL was obtained. Then, by the two-fold dilution method, a standard curve of honokiol concentration vs. absorbance was plotted at a wavelength of 288 nm.

[0057] Subsequently, 10 mL of PBS solution with pH = 7 was taken and placed in a clean beaker. 3% Arg-Gel, 0.2% HRP, and 0.2% H O were added to it. Another 10 mL of PBS solution with pH = 7 was taken and placed in another beaker, and then 25% DA Gel, 0.5% CaCl, and 0.5% honokiol were added. The above solutions were mixed and continuously stirred at a speed of 360 rpm for 10 min until the Arg-Gel / DA-CMCS hydrogel loaded with honokiol was formed.

[0058] Finally, the prepared hydrogel was placed in PBS solutions containing 10% ethanol, and the solutions were set at three different pH values, namely pH = 4, pH = 7, and pH = 10. Measurements were taken every 3 h for 48 h continuously. Each time a measurement was taken, 2 mL of PBS was taken out from the solution containing the hydrogel, and the release amount of honokiol in it was determined by UV spectroscopy. After the measurement was completed, to keep the total volume of the solution constant, the same volume of fresh PBS was added.

[0059] From Figure 3From the experimental results, it can be seen that the honokiol-loaded Arg-Gel / DA-CMCS hydrogel shows slow-release characteristics in three different pH environments. At the same time, the release amount is the highest in the acidic environment (pH = 4) and the lowest in the neutral environment (pH = 7). This result indicates that the hydrogel has the intelligent release characteristics of responding to the wound environment, can rapidly release honokiol in the acidic environment at the initial stage of wound infection, thereby effectively inhibiting bacterial activity and slowing down the development of inflammation; while during the wound recovery period, as the environmental pH gradually tends to neutral, the drug release amount decreases accordingly. This characteristic is closely related to the imine bond formed between Arg-Gel and DA-CMCS through the Schiff base reaction.

[0060] Example 5

[0061] Bioactivity evaluation of Arg-Gel / DA-CMCS hydrogel

[0062] 1. Antibacterial activity evaluation

[0063] To evaluate the antibacterial activity of the hydrogel in Example 3, the single colony antibacterial method was used to conduct the experiment. First, 100 μL of a bacterial suspension of methicillin-resistant Staphylococcus aureus (MRSA) with a concentration of 10 6 CFU / mL was evenly inoculated on a TSB agar plate. After ultraviolet sterilization treatment of normal saline and the hydrogel in Example 3, 200 μL of each was taken and evenly coated on the TSB agar plate inoculated with bacteria. The plate was placed in a shaker and co-cultured for 24 h under the conditions of a rotation speed of 100 r / min and a temperature of 37 °C. After the culture was completed, a colony counter was used to count the single colonies on the plate to evaluate the antibacterial effect of the hydrogel.

[0064] From Figure 4 the results, it can be clearly seen that among the experimental groups, the number of bacteria in the normal saline group is relatively large. The Arg-Gel / DA-CMCS hydrogel group can significantly inhibit bacterial proliferation. This antibacterial effect is closely related to the antibacterial activities of CMCS and Arg itself.

[0065] 2. Hemostatic activity evaluation

[0066] To evaluate the hemostatic activity of the hydrogel in Example 3, a rat tail truncation model was constructed. SD male rats were selected, and after comprehensive disinfection, a part of the rat's tail was truncated. To ensure appropriate blood loss in the rats, after truncating the tail, the rats were suspended naturally for 15 s. Subsequently, 100 μL of normal saline and Arg-Gel / DA-CMCS hydrogel were quickly applied to the wound on the rat's tail, and the rats were placed on filter paper. At the same time, a group of rats without applying the hydrogel was set as the blank control group. The experimental results show that ( Figure 5), the bleeding area on the filter paper of the rat group smeared with the hydrogel was significantly smaller than that of the blank control group. This fully demonstrates that the Arg-Gel / DA-CMCS hydrogel has good hemostatic activity, and this result mainly depends on the hemostatic activity of CMCS and Ca 2 +.

[0067] 3. Evaluation of wound healing promoting activity

[0068] To evaluate the activity of the hydrogel in Example 3 in promoting wound healing, Kunming mice (male, 6 - 8 weeks old) with full-thickness skin defect models were selected for the experiment. Twelve Kunming mice were evenly divided into two groups, namely the normal saline group and the Arg-Gel / DA-CMCS hydrogel group. Before the experiment started, the backs of the mice were depilated and disinfected. Subsequently, circular wounds with a diameter of 10 mm were created on the backs of the mice. After the modeling was completed, 100 μL of Arg-Gel / DA-CMCS hydrogel was applied to the wounds in the hydrogel group, and the same volume of normal saline was smeared in the normal saline group. On the 0th, 5th, 9th, and 12th days of the experiment, images of the mice's wounds were collected. The entire experimental process was strictly carried out in accordance with the relevant guiding principles of the "Guide for the Care and Use of Laboratory Animals" of the National Research Council, and the animal experiment protocol has also been approved by the Ethics Committee of Northeast Agricultural University (Approval No.: NEAUEC20240379).

[0069] Through the experimental records, the change of the wound area over time can be visually presented in Figure 6 . The results show that after 5 days of treatment, the wound area in the hydrogel group decreased significantly. By the 9th day, the wounds in the hydrogel group had basically healed. Particularly prominent is that the wounds in the Arg-Gel / DA-CMCS hydrogel group had completely healed on the 12th day. In sharp contrast, the wounds in the normal saline group had not healed. This significant advantage is mainly attributed to the Arg component in the hydrogel.

[0070] The present invention presents a preparation scheme for a strong tissue adhesion multifunctional injectable hydrogel that responds to the wound microenvironment, and also elaborates on the application ideas and methods in the field of antibacterial natural product delivery. The specific ways and means to implement this technical solution are rich and diverse, and the above content is only the preferred implementation manner of this invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and optimizations can still be made, and these improvements and optimizations should also be included in the protection scope of the present invention. In addition, each component not described in detail in this embodiment can be realized by means of the existing technology.

Claims

1. A preparation method and application of a multifunctional injectable hydrogel with strong tissue adhesion that responds to wound microenvironment, characterized in that: The specific steps are as follows: (1) Take an appropriate amount of phosphate buffered saline (PBS) with a pH of 4.5 to 8.4 and place it in a beaker, and add 2.5% to 9.0% arginine-modified gelatin (Arg-Gel), 0.1% to 0.6% horseradish peroxidase (HRP), and 0.1% to 1.0% CaCl2 in proportion; (2) Take an appropriate amount of PBS with a pH of 3.5 to 8.5 and place it in a beaker, and add 10% to 25% dopamine-modified carboxymethyl chitosan (DA-CMCS) and 0.1% to 0.5% hydrogen peroxide (H2O2) in proportion; (3) taking equal volumes of the solutions of step (1) and (2) and mixing them, stirring at 150-850 rpm at room temperature for 0.5-6 h until a hydrogel is formed and the antibacterial natural product is delivered.

2. According to claim 1, it is characterized in that: The preparation method of the Arg-Gel is as follows: Take 5g of Gel and place it in 100mL of deionized water, heat it at 50℃ until dissolved, add N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) at a molar ratio of 1.2 to 5.2:1, and stir it magnetically for 30 minutes. Add Arg according to the mass ratio of Gel to Arg of 2 to 10:4, adjust the pH to 2.5 to 8.5, stir at room temperature at 100 to 500 rpm for 24 hours, dialyze for 48 hours, and freeze-dry for 36 hours to obtain Arg-Gel.

3. According to claim 1, it is characterized in that: The preparation method of the DA-CMCS is as follows: Take appropriate amounts of CMCS, NHS and EDC in a molar ratio of 1.2-6.5:1:1 and dissolve them in deionized water, and stir them magnetically for 30 minutes. Add DA according to the mass ratio of DA to CMCS of 2-10:5, adjust the pH to 3-9, stir at room temperature at a speed of 200-650 rpm for 24 hours, dialyze for 48 hours, and freeze-dry for 36 hours to obtain DA-CMCS.

4. According to claim 1, it is characterized in that: In the process of preparing modified Gel and hydrogel, other amino acids (such as lysine, histidine, etc.) with similar activity are selected to replace Arg to modify Gel according to the same mass ratio and similar reaction conditions as in the Arg modification process to obtain the hydrogel. Its preparation method and functional properties all fall within the scope of protection of the present invention.

5. According to claim 1, it is characterized in that: In the process of preparing modified Gel and hydrogel, active ingredients containing amino structures, such as but not limited to amino sugars (such as glucosamine), amino alcohols (such as ethanolamine) and polypeptide fragments with amino side chains, etc., are selected to participate in Gel modification and hydrogel preparation in appropriate proportions and reaction conditions according to a mechanism of action similar to Arg, all of which are within the scope of protection of the present invention.

6. According to claim 1, it is characterized in that: In the process of preparing modified CMCS and hydrogel, components with mussel inspiration (such as mussel mucin) and components containing catechol structure (such as tannic acid) are selected. If the mechanism is similar to DA and the mass ratio and reaction conditions are similar, the modified CMCS and hydrogel with similar functions produced are within the protection scope of the present invention.

7. According to claim 1, it is characterized in that: The ionic crosslinking reaction prepares the hydrogel, and the Ca 2 In addition, metal ions having the function of cross-linking with carboxyl groups, such as iron ions, magnesium ions, zinc ions, etc., are all within the protection scope of the present invention.

8. According to claim 1, it is characterized in that: The hydrogel can respond to changes in wound pH and intelligently deliver antibacterial natural small molecule compounds, including but not limited to magnolol.

9. According to claim 8, it is characterized in that: The hydrogel can respond to changes in wound pH within a range of 4.0 to 6.0.

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