A hydrogel based on double dynamic bond crosslinking and a preparation method thereof

Abstract

The application relates to the technical field of biomaterials, and discloses a preparation method of a hydrogel based on double-dynamic-key crosslinking, which comprises the following steps: adding a modification agent into purified natural paramysin, dissolving in a buffer solution for reaction, and then removing unreacted modification agent to obtain a functionalized paramysin solution; dropwise adding a polyphenol small molecule solution into the functionalized paramysin solution for mixing and stirring to obtain the hydrogel based on double-dynamic-key crosslinking. The hydrogel prepared by the method is constructed by functionalized paramysin and polyphenol small molecules through dynamic covalent bonds and non-covalent bonds, has excellent mechanical properties, self-healing ability, environmental responsiveness and biocompatibility, the preparation method is mild and simple, is suitable for multiple fields of biomedicine, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically, to a hydrogel based on dual dynamic bond crosslinking and its preparation method. Background Technology

[0002] Hydrogels are highly hydrated soft materials with a three-dimensional network structure. Their physicochemical properties are similar to those of the natural extracellular matrix, thus showing great application potential in tissue engineering, drug delivery, regenerative medicine, and biosensing. Ideal hydrogel biomaterials should possess good mechanical properties, injectability and self-healing ability, excellent biocompatibility, and intelligent responsiveness to external physiological or pathological microenvironments.

[0003] To balance mechanical properties and dynamic characteristics, dynamic covalent bonds have been introduced into the design of hydrogels. Dynamic covalent bonds possess the strength of regular covalent bonds while also being reversibly broken and reformed under specific stimuli, thus enabling the gel to simultaneously achieve good mechanical properties and stimulus responsiveness. However, networks constructed solely based on a single dynamic covalent bond exhibit relatively simple response modes, limited flexibility in controlling gelation kinetics and final mechanical properties, and often still require complex chemical modifications to biomolecules or the use of artificially synthesized crosslinking molecules.

[0004] Most existing technical solutions focus on a single cross-linking mode or fail to fully utilize the potential of natural proteins and polyphenols to synergistically construct multi-level dynamic networks. Therefore, developing a novel hydrogel preparation method based on natural materials and achieving synergistic cross-linking of multiple dynamic bonds through ingenious molecular design is urgently needed and has broad application prospects for promoting the development of high-performance intelligent biomaterials. Summary of the Invention

[0005] In view of this, the present invention proposes a hydrogel based on dual dynamic bond crosslinking and its preparation method, aiming to solve the problem that the single crosslinking mechanism in the current hydrogel technology cannot simultaneously satisfy the contradiction of high strength, rapid self-healing, injection molding and multi-mode environmental responsiveness.

[0006] This invention proposes a method for preparing hydrogels based on dual dynamic bond crosslinking, characterized by comprising the following steps: (1) Add the modifier to the purified natural paramyosin, dissolve it in buffer solution for reaction, and then remove the unreacted modifier to obtain a functionalized paramyosin solution; (2) The polyphenol small molecule solution is added dropwise to the functionalized paramyosin solution and mixed, and stirred to obtain the hydrogel based on dual dynamic bond crosslinking.

[0007] More preferably, in step (1), the modifier includes at least one of Traut's reagent, sodium periodate, and 3-aminophenylboronic acid active ester.

[0008] More preferably, in step (2), the polyphenol molecule is a natural polyphenol or its derivative having a catechol or pyrogallol structure.

[0009] More preferably, in step (1), the temperature at which the modifier reacts with natural paramyosin is 4-25°C and the reaction time is 0.5-4 hours.

[0010] More preferably, in step (1), the concentration of the modifier is 0.1mM-10mM, and the molar ratio of the modifier to natural paramyosin is 2:1-20:1.

[0011] More preferably, in step (1), the buffer solution is selected from Tris-HCl, HEPES or phosphate buffer, and the pH value of the buffer solution is 7.0-8.5.

[0012] More preferably, in step (2), the final concentration of the polyphenol small molecules is 1-20 mM.

[0013] More preferably, step (2) is carried out under an inert atmosphere, and the volume ratio of the added polyphenol small molecule solution to the volume of the functionalized paramyosin solution is 1:20-1:5.

[0014] More preferably, the mixing in step (2) is completed at a first temperature to obtain a pregel; then, the pregel is incubated at a second temperature for 10-60 minutes; wherein the first temperature is 10-25°C and the second temperature is 25-37°C.

[0015] On the other hand, the present invention also provides a hydrogel based on dual dynamic bond crosslinking prepared according to the above method, characterized in that it is composed of functionalized paramyosin and polyphenol small molecules through dynamic covalent and non-covalent synergistic crosslinking.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention enhances performance through the synergistic effect of double bonds. Dynamic covalent bonds provide structural stability and reversibility, while non-covalent bonds impart rapid response and self-healing capabilities. The two work together to achieve a balance between high strength and high toughness. Furthermore, this application is based on natural proteins and polyphenols, eliminating the need for toxic cross-linking agents and meeting the safety requirements for biomedical materials.

[0017] The gel prepared by this invention exhibits dual pH / redox response, can be injection molded, and is suitable for minimally invasive implantation and in-situ gelation. Furthermore, the preparation process of this invention is simple and mild: it can be completed at room temperature and pressure, requires no complex equipment, and is suitable for large-scale production. Attached Figure Description

[0018] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 The flowchart illustrates a hydrogel based on dual dynamic bond crosslinking and its preparation method provided by the present invention. Detailed Implementation

[0019] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] See Figure 1 This invention proposes a method for preparing hydrogels based on dual dynamic bond crosslinking, characterized by comprising the following steps: (1) Add the modifier to the purified natural paramyosin, dissolve it in buffer solution for reaction, and then remove the unreacted modifier to obtain a functionalized paramyosin solution; (2) The polyphenol small molecule solution is added dropwise to the functionalized paramyosin solution and mixed, and stirred to obtain the hydrogel based on dual dynamic bond crosslinking.

[0021] Step (1) The modifier is added to the purified natural paramyosin, dissolved in buffer solution for reaction, and then the unreacted modifier is removed to obtain a functionalized paramyosin solution.

[0022] Specifically, this invention first adds a modifier to purified natural paramyosin. The modifier is preferably at least one selected from Traut's reagent, sodium periodate, and 3-aminophenylboronic acid active ester. The concentration of the modifier is preferably 0.1 mM-10 mM, and the molar ratio of the modifier to natural paramyosin is preferably 2:1-20:1. The paramyosin with the added modifier is dissolved in a buffer solution for a thorough reaction. The reaction temperature between the modifier and natural paramyosin is preferably 4-25°C, and the reaction time is preferably 0.5-4 hours. The buffer solution is preferably selected from Tris-HCl, HEPES, or phosphate buffer, and the pH value of the buffer solution is preferably 7.0-8.5. After the reaction is complete, the unreacted modifier is removed to obtain a functionalized paramyosin solution.

[0023] It is understood that the natural paramyosin can only be cross-linked through physical processes (non-covalent bonds). This step chemically modifies the natural paramyosin by adding a modifier, precisely introducing specific functional groups that can undergo reversible chemical reactions with polyphenols onto its molecule. The introduced functional groups determine the type of dynamic covalent bonds that will be formed subsequently (disulfide bonds, imine bonds, or borate ester bonds), thereby directly endowing the hydrogel with specific environmental responsiveness (such as responses to pH, reducing agents, and glucose). Simultaneously, these dynamic covalent bonds are the chemical guarantee for the hydrogel to obtain excellent mechanical strength and stable structure, compensating for the mechanical defects of purely physical gels. The reaction is carried out in a mild buffer system to maximize the preservation of the natural conformation and bioactivity of paramyosin, ensuring the biocompatibility of the final material. Subsequently, unreacted modifiers are removed, and the functionalized product is purified to avoid impurities interfering with subsequent precise cross-linking with polyphenols, ensuring a controllable gelation process and uniform and reliable results.

[0024] It is understood that the Traut's reagent is specifically designed to introduce thiol groups into the paramyosin, providing a foundation for constructing a redox-responsive disulfide bond dynamic network. Sodium periodate is used to oxidize and generate aldehyde groups, constructing a pH-responsive imine bond dynamic network. The 3-aminophenylboronic acid active ester is used to introduce phenylboronic acid groups, thereby constructing a sugar / pH dual-responsive borate ester bond dynamic network. The dynamic covalent bonds introduced by these components form synchronously and synergistically with the inherent dynamic non-covalent bonds between polyphenolic proteins in subsequent reactions, ultimately solving the key technical problem that a single type of hydrogel cannot simultaneously achieve strength, self-healing, injectability, and precise responsiveness. This invention provides multiple chemical pathways for constructing different responsive gels, allowing users to select and customize gels according to their final application needs (such as anti-tumor, anti-inflammatory, and diabetes treatment).

[0025] Understandably, the reaction temperature of the modifier with natural paramyosin is limited to 4-25°C. Within this temperature range, the chemical reaction rate can be significantly increased while most natural paramyosin remains stable. This range avoids side reactions such as partial denaturation, aggregation, or accelerated hydrolysis that may occur at high temperatures. Furthermore, this temperature range ensures the precision of the modification and the uniformity of the product, maximally suppressing the thermal motion and aggregation of proteins, maintaining their natural folded conformation and biological activity, and providing a stable reaction template for the modification reaction.

[0026] Understandably, paramyosin has a limited number of modification sites and significant steric hindrance, making the modification reaction a reversible equilibrium reaction. Maintaining a molar excess of at least 2 times effectively shifts the reaction equilibrium to the right, ensuring a sufficient proportion of protein molecules are successfully modified. An upper limit of 20 times prevents over-modification; excessively high molar ratios can lead to too many modifying groups attaching to individual protein molecules, altering their charge, hydrophobicity, and other physicochemical properties, potentially causing protein denaturation or aggregation. Furthermore, the modification reaction loses site selectivity, attacking more non-target residues and affecting the protein's inherent function and the specificity of subsequent polyphenol cross-linking.

[0027] It is understood that the Tris-HCl, HEPES, or phosphate buffer provides an ionicly stable and pH-constant solution environment for modification reactions (such as sulfhydrylation, oxidation, and amidation), ensuring that the reactions proceed at a predictable rate and direction. The selected buffer components do not inhibit subsequent co-crosslinking reactions with polyphenols, nor do they introduce biotoxic properties into the final hydrogel. In this invention, the aforementioned dynamic covalent bonds prefer to form in a neutral to weakly alkaline pH range, and the paramyosin is most stable within this pH range, making it less susceptible to acid- or base-catalyzed degradation.

[0028] Step (2) The polyphenol small molecule solution is added dropwise to the functionalized paramyosin solution and mixed, and stirred to obtain the hydrogel based on dual dynamic bond crosslinking.

[0029] Specifically, in this invention, a polyphenol small molecule solution is added dropwise to the functionalized paramyosin solution prepared in step (1). Preferably, step (2) is carried out under an inert atmosphere. The polyphenol small molecule is preferably a natural polyphenol or its derivative having a catechol or pyrogallol structure. The final concentration of the polyphenol small molecule is preferably 1-20 mM. The volume ratio of the added polyphenol small molecule solution to the volume of the functionalized paramyosin solution is 1:20-1:5. After thorough stirring, the hydrogel based on dual dynamic bond crosslinking is finally obtained. More preferably, step (2) is carried out under an inert atmosphere. The volume ratio of the added polyphenol small molecule solution to the volume of the functionalized paramyosin solution is 1:20-1:5. Preferably, step (2) is first completed at a first temperature. After obtaining the pregel, the pregel is incubated at a second temperature for 10-60 minutes. The first temperature is preferably 10-25°C, and the second temperature is preferably 25-37°C.

[0030] Understandably, polyphenols, derived from natural plants, possess antioxidant and anti-inflammatory bioactivities, perfectly matching the needs of biomedical applications. Their degradation products are non-toxic and can be metabolized by organisms. When the polyphenol small molecule solution is dropwise into the functionalized paramyosin solution, the abundant phenolic hydroxyl groups and aromatic rings on the polyphenol small molecules instantaneously and spontaneously bind to various residues on the protein surface through hydrogen bonds, hydrophobic interactions, and π-π stacking forces, forming a transient physical cross-linked network in a very short time. This endows the system with preliminary viscoelasticity and shape retention capabilities, forming the basis for the injectability of the hydrogel. Simultaneously, the specific structures on the polyphenol small molecules and the pre-introduced functional groups on the functionalized paramyosin begin to undergo reversible chemical reactions in the close and ordered environment provided by the physical network, forming dynamic covalent bonds. These chemical bonds slowly and firmly reinforce and lock the key nodes of the physical network. The resulting hydrogel, based on dual dynamic cross-linking, utilizes the environmental responsiveness of dynamic covalent bonds to achieve targeted, timed, and controlled drug release at specific lesion sites (such as the microacidic environment of tumors or the high redox environment of inflammation), improving efficacy and reducing side effects. Furthermore, the hydrogel allows for minimally invasive injection into irregular tissue defects, gelling in situ and perfectly conforming to the wound surface. Even if mechanical damage occurs after implantation, its dynamic bonds can spontaneously rebuild, restoring structural integrity and maintaining scaffold function long-term. Its network can adaptively remodel in response to cellular activities (such as secreted enzymes or metabolites), guiding cell growth and tissue regeneration.

[0031] It is understandable that small polyphenol molecules with catechol or pyrogallol structures can undergo specific, reversible chemical reactions with the functionalized paramyosin (containing phenylboronic acid groups, aldehyde groups, etc.), ensuring the cross-linking of double dynamic bonds. If the polyphenol lacks this structure, it cannot participate in the formation of the crucial dynamic covalent bonds.

[0032] Understandably, at the relatively low initial temperature of 10-25°C, molecular thermal motion is more moderate. This temperature range favors the rapid and preferential formation of dynamic non-covalent bonds. These physical effects are unfavorable to entropy change, while the relatively low temperature is conducive to stability. Simultaneously, the lower temperature significantly inhibits the chemical reaction rate of dynamic covalent bonds (such as imine bonds and borate ester bonds). Therefore, after mixing, a relatively weak but complete network dominated by physical effects, namely the pregel, is formed. It possesses the rudiments of injectability and rapid self-healing. Increasing the temperature to the physiological temperature range greatly promotes molecular thermal motion and chemical reactivity. Incubation at the second temperature of 25-37°C for 10-60 minutes provides the necessary activation energy for the formation of dynamic covalent bonds, enabling them to proceed efficiently. Dynamic covalent bonds selectively reinforce key connection points within the framework of the physical network, thereby significantly improving the overall network strength and stability. Through stepwise temperature control, this invention achieves pre-shaping followed by strengthening, allowing the operator sufficient time to inject, fill, or shape the pregel without affecting the final gelation. The subsequent heating and incubation locked in and reinforced this shape, ultimately yielding a final product with significantly improved mechanical properties compared to one-step gelation.

[0033] On the other hand, the present invention also provides a hydrogel based on dual dynamic bond crosslinking prepared according to the above method, characterized in that it is composed of functionalized paramyosin and polyphenol small molecules through dynamic covalent and non-covalent synergistic crosslinking.

[0034] This invention proposes a novel strategy for synergistic crosslinking using dual dynamic bonds. Specifically, by chemically modifying natural paramyosin, specific functional groups are introduced, enabling it to simultaneously undergo dynamic covalent bonding (such as borate ester bonds and imine bonds) and non-covalent bonding (hydrogen bonds, hydrophobic interactions, etc.) crosslinking with small polyphenol molecules containing catechol / triphenol structures under mild conditions. These two bonds synergistically construct a three-dimensional network. The resulting hydrogel exhibits both high strength and rapid self-healing capabilities, allowing for injection molding. Its dynamic covalent bonds endow the material with multiple environmental responsiveness, including pH, glucose, and redox reactions, making it suitable for intelligent drug delivery. The entire preparation process is mild, simple, and uses naturally sourced raw materials with excellent biocompatibility, showing broad application prospects in tissue engineering and biosensing.

[0035] Example 1 Hydrogels based on thiol-disulfide bonds / polyphenol hydrogen bonds with dual dynamic cross-linking 1. Raw materials and reagents Natural paramyosin: extracted and purified from oyster adductor muscle; Modifier: Traut's reagent (2-iminothione hydrochloride); Polyphenol small molecule: epigallocatechin gallate; Buffer: 50 mM Tris-HCl buffer (pH 8.0); Others: Dialysis bag (molecular weight cutoff 10 kDa), nitrogen.

[0036] 2. Preparation method (1) Preparation of functionalized paramyosin solution: 10 mg of purified native paramyosin was dissolved in 5 mL of pH 8.0 Tris-HCl buffer to obtain a protein solution with a concentration of 2 mg / mL. Traut's reagent was added to this solution to a final concentration of 1 mM (modifier to protein molar ratio approximately 10:1). The reaction was carried out at 25°C with gentle stirring for 2 hours. After the reaction, the mixture was transferred to a dialysis bag and dialyzed against a large volume of the same buffer at 4°C for 24 hours (changing the buffer every 4-6 hours) to completely remove unreacted modifier. The dialysis solution was the thiolized paramyosin solution, and its thiol content was determined to be approximately 12 μmol / g protein using Ellman's reagent method.

[0037] (2) Preparation and maturation of hydrogels: EGCG was dissolved in Tris-HCl buffer at pH 7.4 to prepare a 20 mM stock solution, which was sterilized by filtration through a 0.22 μm filter before use. In a nitrogen-filled glove box, 2 mL of the functionalized paramyosin solution was transferred to a vial. With magnetic stirring (500 rpm), 0.1 mL of the EGCG stock solution (final concentration 1 mM, volume ratio 1:20) was added dropwise at a rate of 0.5 mL / min using a microinjection pump. After the addition was complete, stirring was continued at 20 °C (first temperature) for 5 minutes, during which the system transitioned from a solution to a non-flowing, homogeneous pre-gel. The vial containing the pre-gel was then transferred to a 37 °C (second temperature) incubator and incubated for 30 minutes to obtain the final hydrogel (denoted as Gel-1).

[0038] 3. Performance Characterization and Results Gelation time: from the start of dropwise addition until the system loses its fluidity, approximately 3-5 minutes.

[0039] Self-healing properties: Cut a cylindrical gel block in half with a blade, then place the cut surfaces together and let it stand at room temperature. Macroscopically, the two halves of the gel can re-bond themselves within 10 minutes and can withstand their own weight.

[0040] Injectability: Using a 1mL syringe with a 25G needle, the pre-gel or final gel can be smoothly dispensed and retains its shape after injection.

[0041] Rheological properties: Using a rotational rheometer, the storage modulus (G') of Gel-1 is approximately 850 Pa, and the loss modulus (G'') is approximately 120 Pa.

[0042] Redox responsiveness: When small pieces of Gel-1 were immersed in a buffer solution containing 10 mM dithiothreitol, the gel completely dissolved within 4 hours, confirming that its disulfide dynamic covalent bonds are sensitive to the reducing environment.

[0043] Example 2 Double dynamic bond hydrogel based on synergistic aldehyde-imine bond / polyphenol hydrophobic interaction 1. Raw materials and reagents Natural paramyosin: extracted and purified from oyster adductor muscle; Modifier: sodium periodate; Polyphenol molecule: tannic acid; Buffer: 20 mM HEPES buffer (pH 7.4).

[0044] 2. Preparation method (1) Preparation of functionalized paramyosin solution: Dissolve 20 mg of natural paramyosin in 4 mL of HEPES buffer (pH 7.4) (5 mg / mL). Add sodium periodate solid to a final concentration of 5 mM (molar ratio approximately 15:1). React slowly with stirring for 1 hour at 4°C in the dark. Immediately pass the reaction solution through a pre-cooled PD-10 desalting column, elute with the same buffer, and collect the protein fraction to obtain an aldehyde-modified paramyosin solution.

[0045] (3) Preparation of hydrogels: Prepare a 40 mM tannic acid aqueous solution (pH 7.0). In air at room temperature, take 2.5 mL of aldehyde-modified paramyosin solution and add 0.5 mL of tannic acid solution dropwise with stirring (final concentration approximately 6.7 mM, volume ratio 1:5). After the addition is complete, allow to stand at 25°C for 15 minutes to form a brownish-yellow hydrogel (denoted as Gel-2).

[0046] 3. Performance Characterization and Results Gelation time: approximately 8-12 minutes.

[0047] Self-healing properties: After being cut, macroscopic healing is achieved within 30 minutes in a pH 7.4 buffer solution.

[0048] pH responsiveness: When Gel-2 was placed in a pH 5.0 buffer, significant softening was observed within 2 hours, with a modulus decrease of approximately 70%; after being transferred back to a pH 7.4 buffer, the modulus partially recovered. This demonstrates that its imine bonds are sensitive to acidic environments.

[0049] Mechanical strength: Gel-2 has a G' of up to about 2200 Pa, indicating high strength.

[0050] Example 3 Double dynamic bond hydrogel based on phenylboronic acid-boronic acid ester bond / polyphenol π-π stacking synergy 1. Raw materials and reagents Natural paramyosin: extracted and purified from oyster adductor muscle; Modifier: 3-aminophenylboronic acid-N-hydroxysuccinimide ester; Polyphenol small molecule: epigallocatechin gallate; Buffer: 0.1M phosphate buffer (PBS, pH 7.8).

[0051] 2. Preparation method (1) Preparation of functionalized paramyosin solution: Dissolve 3-aminophenylboronic acid-N-hydroxysuccinimide ester in a small amount of DMSO to prepare a 50 mM stock solution. Dissolve 15 mg of natural paramyosin in 3 mL of PBS (5 mg / mL). Add the above-mentioned modifier stock solution to a final concentration of 0.5 mM (molar ratio approximately 5:1). React at 15 °C for 3 hours. Dialyze the reaction solution at 4 °C (to PBS, pH 7.8) for 48 hours to obtain a phenylboronicized paramyosin solution.

[0052] (2) Preparation and programmed temperature control of hydrogels: The EGCG stock solution was prepared as in Example 1. Under nitrogen protection, 3 mL of the functionalized protein solution was taken and stirred in a 15°C water bath. 0.15 mL of EGCG stock solution (final concentration 1 mM, volume ratio 1:20) was slowly added dropwise. After the addition was complete, a pre-gel was obtained at 15°C. The system was then transferred to a 37°C water bath and incubated for 60 minutes to obtain the final hydrogel (denoted as Gel-3).

[0053] 3. Performance Characterization and Results Gelation and curing: The pregel forms at 15°C and can be injected; its strength increases significantly after incubation at 37°C.

[0054] Glucose responsiveness: When Gel-3 was immersed in PBS containing 100 mM glucose, its weight swelling rate was about 150% higher than that in ordinary PBS after 12 hours, indicating that the borate ester bonds were competitively broken and the network swelled.

[0055] Preliminary biocompatibility test: When Gel-3 extract was co-cultured with L929 fibroblasts, the CCK-8 assay showed that the relative cell proliferation rate was >90%, indicating that the extract had no obvious cytotoxicity.

[0056] The performance characterization and results of each embodiment are summarized and compared, resulting in Table 1.

[0057] Table 1 Comparison of Performance Characterization and Results of Examples 1-3 As shown in Table 1, the hydrogels obtained in Examples 1-3 all exhibited excellent comprehensive properties resulting from the dual dynamic crosslinking. All the obtained gels possessed good injectability and could achieve macroscopic self-healing within 10-30 minutes, demonstrating the rapid reconstruction characteristics of the dynamic non-covalent network. By changing the crosslinking system and introducing programmed temperature control steps (as in Examples 1 and 3), the storage modulus (G') of the hydrogels could be effectively controlled within a wide range of approximately 850 Pa to 2200 Pa, meeting the differentiated strength requirements of materials in different application scenarios.

[0058] Each gel exhibited a unique environmental response determined by its specific dynamic covalent bonds: the gel of Example 1 dissolved in response to a reducing agent; the gel of Example 2 underwent a reversible modulus change in response to acidic pH; and the gel of Example 3 swelled significantly in response to glucose concentration. This fully demonstrates the ability of this invention to flexibly customize the material's responsive behavior. Preliminary cytotoxicity tests of Example 3 showed a cell proliferation rate exceeding 90%, indicating that the hydrogel constructed based on this natural protein-polyphenol system possesses potentially excellent biocompatibility.

[0059] It is evident that the hydrogel prepared by the method described in this invention successfully achieves an organic combination of high strength, rapid self-healing, convenient injection, and multi-environment responsiveness, effectively solving the common problem that single cross-linked hydrogels cannot simultaneously achieve multiple properties.

[0060] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a hydrogel based on dual dynamic bond crosslinking, characterized in that, Includes the following steps: (1) Add the modifier to the purified natural paramyosin, dissolve it in buffer solution for reaction, and then remove the unreacted modifier to obtain a functionalized paramyosin solution; (2) The polyphenol small molecule solution is added dropwise to the functionalized paramyosin solution and mixed, and stirred to obtain the hydrogel based on dual dynamic bond crosslinking.

2. The method according to claim 1, characterized in that, In step (1), the modifier includes at least one of Traut's reagent, sodium periodate, and 3-aminophenylboronic acid active ester.

3. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, In step (2), the polyphenol molecule is a natural polyphenol or its derivative having a catechol or pyrogallol structure.

4. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, In step (1), the temperature at which the modifier reacts with natural paramyosin is 4-25°C and the reaction time is 0.5-4 hours.

5. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, In step (1), the concentration of the modifier is 0.1 mM-10 mM, and the molar ratio of the modifier to natural paramyosin is 2:1-20:

1.

6. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, In step (1), the buffer solution is selected from Tris-HCl, HEPES or phosphate buffer, and the pH value of the buffer solution is 7.0-8.

5.

7. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, In step (2), the final concentration of the polyphenol small molecules is 1-20 mM.

8. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, Step (2) is carried out under an inert atmosphere, and the volume ratio of the added polyphenol small molecule solution to the functionalized paramyosin solution is 1:20-1:

5.

9. The method for preparing a hydrogel based on dual dynamic bond crosslinking according to claim 1, characterized in that, The mixing in step (2) is completed at a first temperature to obtain a pregel; then the pregel is incubated at a second temperature for 10-60 minutes; wherein the first temperature is 10-25°C and the second temperature is 25-37°C.

10. A hydrogel based on dual dynamic bond crosslinking prepared by any one of claims 1-9, characterized in that... It is composed of functionalized paramyosin and polyphenol small molecules through dynamic covalent and non-covalent synergistic cross-linking.

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