Method for constructing thiolated gamma-polyglutamic acid gelatin double network hydrogel

Abstract

The present application provides a thiolated gamma-polyglutamic acid / gelatin double network hydrogel and a preparation method thereof, the double network hydrogel comprises a first network and a second network; the first network is a gelatin network, the first network is crosslinked by transglutaminase TG enzyme; the second network is prepared by crosslinking thiolated gamma-polyglutamic acid with a second network crosslinking agent. The present application overcomes the shortcomings of the existing single network or conventional double network hydrogel in mechanical properties, biological functions and preparation flexibility, and provides a series of double network hydrogels based on thiolated gamma-polyglutamic acid / gelatin / TG enzyme and a simple and universal construction method. The method can flexibly control the network structure and performance of the hydrogel by using different click chemistry reaction paths through the same core precursor.

Description

Technical Field

[0001] This invention belongs to the field of dual-network hydrogel technology, and particularly relates to various methods for constructing thiolized γ-polyglutamic acid / gelatin dual-network hydrogels. Background Technology

[0002] Hydrogels, as polymeric materials with a three-dimensional network structure, exhibit great potential in biomedical engineering fields such as tissue engineering, controlled drug release, and wound dressings due to their high water content, good biocompatibility, and physical properties similar to the extracellular matrix. However, the mechanical properties of single-network hydrogels are often limited, making it difficult to meet the mechanical requirements of repairing certain load-bearing tissues (such as cartilage and skeletal muscle). Therefore, dual-network hydrogels have emerged, typically composed of two interpenetrating and complementary polymer networks that synergistically enhance the hydrogel's mechanical strength, toughness, and fatigue resistance.

[0003] γ-Polyglutamic acid (PGA) is a natural anionic polyamino acid produced by microbial fermentation. It possesses excellent biocompatibility, biodegradability, non-immunogenicity, and abundant side-chain carboxyl groups. These carboxyl groups facilitate functional modifications (such as the introduction of thiol groups). Gelatin, a partially hydrolyzed product of collagen, retains its cell recognition sites (such as the arginine-glycine-aspartic acid sequence), exhibiting good cell adhesion and cell proliferation-promoting abilities. Transglutaminase (TG) can specifically catalyze the formation of isopeptide bonds between the γ-amide group of glutamine residues and the ε-amino group of lysine residues in gelatin molecules, thereby constructing a stable and biocompatible enzyme cross-linking network. Therefore, using a TG-crosslinked gelatin network as the first network holds promise for providing an excellent biological interface for cells.

[0004] However, first-network hydrogels composed solely of gelatin typically exhibit weak mechanical properties and insufficient stability at body temperature. Current research has focused on gelatin hydrogels cross-linked with TG enzymes, hydrogels constructed using thiol click chemistry, and γ-PGA / hyaluronic acid-based hydrogels. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies, particularly the limitations of single-network or conventional dual-network hydrogels in terms of mechanical properties, biological functions, and fabrication flexibility. This invention provides a series of dual-network hydrogels based on thiolated γ-polyglutamic acid / gelatin / TG enzymes, along with their simple and universal construction methods. This method allows for flexible control of the hydrogel's network structure and properties using the same core precursor and different click chemistry reaction pathways.

[0006] This invention systematically explores the use of the same core precursor (γ-PGA-SH) through three different thiol click chemical pathways (thiol-aldehyde, thiol-alkynyl, thiol-epoxy) to construct a second network with different functionalized crosslinking agents, and integrates it with the same gelatin / TG enzyme first network to obtain a series of dual-network hydrogels with tunable performance. This invention provides greater flexibility and universality for material design and performance optimization, and is reported for the first time.

[0007] To achieve the above objectives, the present invention provides a thiolated γ-polyglutamic acid / gelatin dual-network hydrogel, wherein the dual-network hydrogel comprises a first network and a second network; the first network is a gelatin network, which is cross-linked by transglutaminase (TG enzyme); the second network is prepared by thiolated γ-polyglutamic acid and a second network cross-linking agent, wherein the second network cross-linking agent comprises any one of aldehyde-based hyaluronic acid HA-CHO, alkyne-bonded hyaluronic acid HA-alk, or polyethylene glycol diglycidyl ether (PEGDE).

[0008] The first network utilizes transglutaminase (TG) to crosslink the ε-NH2 group of gelatin with the glutamine group; the second network is prepared by using thiolated γ-polyglutamic acid as the core and the thiol group of thiolated γ-polyglutamic acid with a second network crosslinking agent. The second network crosslinking agent includes any one of aldehyde-based hyaluronic acid HA-CHO, alkyne-bonded hyaluronic acid HA-alk, or polyethylene glycol diglycidyl ether (PEGDE).

[0009] The present invention provides a modular construction platform for thiolized γ-polyglutamic acid / gelatin dual-network hydrogels, which utilizes a core precursor (γ-PGA-SH) and multiple click chemistry pathways. By simply changing the second network crosslinking agent and reaction type, the chemical microenvironment, crosslinking density, swelling behavior, degradation rate, and mechanical properties of the hydrogel can be systematically controlled, achieving customized design of "one method, multiple materials".

[0010] Preferably, the thiolated γ-polyglutamic acid is synthesized by grafting β-mercaptoethylamine onto the side chain carboxyl group of γ-polyglutamic acid.

[0011] In any of the above-mentioned preferred embodiments, the thiolated γ-polyglutamic acid is prepared using γ-polyglutamic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and β-mercaptoethylamine.

[0012] In any of the above-mentioned preferred embodiments, γ-polyglutamic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are sequentially added to a MES buffer solution at pH=5.0 and stirred; under light-protected nitrogen atmosphere, β-mercaptoethylamine is added to the mixture and stirred continuously; the thiolated γ-polyglutamic acid is obtained by dialyzing and drying.

[0013] Preferably, the alkyne-bonded hyaluronic acid HA-alk is synthesized by grafting propargylamine onto the side chain carboxyl group of hyaluronic acid.

[0014] In any of the above-mentioned preferred embodiments, the alkyne-bonded hyaluronic acid HA-alk is prepared using hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and propargylamine.

[0015] In any of the above preferred embodiments, hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are sequentially added to a MES buffer solution with pH=5.0 and stirred. The pH of the system is adjusted, and propargylamine is added to the mixture while stirring continuously. After the reaction is completed, the mixture is dialyzed and dried to obtain the alkyne-bonded hyaluronic acid HA-alk.

[0016] In any of the above preferred embodiments, the aldehyde-modified hyaluronic acid HA-CHO is obtained by oxidizing hyaluronic acid with sodium periodate.

[0017] In any of the above preferred embodiments, the C2 and C3 C-couplings of the main chain sugar ring of hyaluronic acid are oxidized to aldehyde groups by sodium periodate to obtain the alkyne functionalized polymer HA-CHO.

[0018] The dual-network hydrogel provided by this invention has a first network of gelatin network, which is cross-linked with glutamine groups of gelatin using TG enzyme; the second network is based on thiolized γ-polyglutamic acid (γ-PGA-SH) as the core, and is prepared by thiol-aldehyde Michael addition with aldehyde-based hyaluronic acid (HA-CHO), thiol-triple bond click reaction with alkyne-based hyaluronic acid (HA-alk), and thiol-epoxy bond click reaction with polyethylene glycol diglycidyl ether (PEGDE). In addition to the CS bond generated as the first cross-linking site, the second network cross-linking sites of the three types of dual-network hydrogels also include disulfide bonds formed between γ-PGA-SH thiol groups as the second cross-linking sites.

[0019] The present invention also provides a method for preparing thiolated γ-polyglutamic acid / gelatin dual-network hydrogel.

[0020] Preferably, the dual-network hydrogel is prepared using a one-pot method.

[0021] In any of the above-mentioned preferred embodiments, γ-PGA-SH, HA-CHO / HA-alk / PEGDE, gelatin, and transglutaminase (TG enzyme) are dissolved together in PBS buffer at pH 8.5 to form a homogeneous mixture. This mixture is then injected into a custom mold and reacted at 37°C to form a double-network hydrogel.

[0022] The preferred embodiment of the above is the method for preparing the dual-network hydrogel.

[0023] Includes the following steps:

[0024] Step 1: The thiolated γ-polyglutamic acid was synthesized by grafting β-mercaptoethylamine onto the side chain carboxyl group of γ-polyglutamic acid;

[0025] Step 2: The alkyne-bonded hyaluronic acid HA-alk is synthesized by grafting propargylamine onto the side chain carboxyl group of hyaluronic acid;

[0026] Step 3: Oxidize hyaluronic acid with sodium periodate to obtain the aldehyde-modified hyaluronic acid HA-CHO;

[0027] Step 4: Dissolve gelatin in PBS, add transglutaminase (TG) at 60°C and stir to obtain a gelatin / TG enzyme solution;

[0028] Step 5: Dissolve thiolated γ-polyglutamic acid in PBS at 37°C to obtain γ-PGA-SH solution;

[0029] Step 6: Dissolve aldehyde-modified hyaluronic acid HA-CHO, alkyne-modified hyaluronic acid HA-alk, or polyethylene glycol diglycidyl ether PEGDE in PBS and dissolve at 37°C to obtain HA-CHO solution, HA-alk solution, or PEGDE solution, respectively; the HA-alk solution also contains the photoinitiator LAP.

[0030] The dual-network hydrogel is obtained by following any one of steps 7, 8 or 9;

[0031] Step 7: Mix the γ-PGA-SH solution with the gelatin / TG enzyme solution, add the HA-CHO solution, pour the mixture into a custom mold, and let it stand at 37°C for 1 hour to obtain the double network hydrogel γ-PGA-HA-Gel.

[0032] Step 8: Mix the γ-PGA-SH solution with the gelatin / TG enzyme solution, add the HA-alk solution, and let it stand for 1 hour under 37℃ and 365 nm ultraviolet light to obtain the double network hydrogel HA-γ-PGA-Gel;

[0033] Step 9: Mix the γ-PGA-SH solution with the gelatin / TG enzyme solution, add the PEGDE solution, pour the mixture into a custom mold, and let it stand at 37°C for 1 hour to obtain the double-network hydrogel γ-PGA-PEG-Gel.

[0034] In any of the above preferred embodiments, in step 1, γ-polyglutamic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are sequentially added to a MES buffer solution with pH=5.0 and stirred; under light-protected nitrogen protection, β-mercaptoethylamine is added to this mixture and stirred continuously; the thiolated γ-polyglutamic acid is obtained by dialyzing and drying.

[0035] In any of the above-mentioned preferred embodiments, in step 1, the molecular weight cutoff for dialysis is 100 kDa.

[0036] In any of the above-mentioned preferred embodiments, in step 1, an HCl solution is used as the dialysis medium.

[0037] In any of the above-mentioned preferred embodiments, in step 1, a dilute hydrochloric acid solution with pH=4.5 is used as the dialysis medium.

[0038] In any of the above-mentioned preferred embodiments, in step 1, the drying method is freeze drying.

[0039] Preferably, in any of the above steps, step 1 involves synthesizing the thiol-functionalized polymer γ-PGA-SH by grafting β-mercaptoethylamine onto the side chain carboxyl group of γ-polyglutamic acid: γ-polyglutamic acid (γ-PGA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-hydroxysuccinimide (NHS) are sequentially added to a MES buffer solution at pH 5.0 and stirred for 2 hours. Under a light-protected nitrogen atmosphere, β-mercaptoethylamine (Cys) is added to this mixture, and the mixture is stirred continuously at a certain temperature for 24 hours. After the reaction is complete, the product is dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 100 kDa and HCl solution as the dialysis medium to remove unreacted small molecules. Finally, the γ-PGA-SH sample is obtained by freeze-drying.

[0040] Preferably, in step 1, the molecular weight of γ-polyglutamic acid is 200-300 kDa.

[0041] In any of the above-mentioned preferred embodiments, in step 1, the concentration of the MES solution of γ-polyglutamic acid is 1-10 mg / mL.

[0042] Preferably, in step 1, the reaction system temperature is 25-35℃.

[0043] Preferably, in step 1, the molar ratio of γ-polyglutamic acid, EDC, and NHS is γ-PGA : EDC : NHS = 1 : 1.5 : 1.5 - 1 : 3 : 3, that is, the molar ratio of γ-PGA : EDC : NHS is 1 : (1.5-3) : (1.5-3). Preferably, when the molar amount of γ-PGA is 1 part, the molar amount of EDC is 1.5, 2, or 3 parts or a range thereof, and the molar amount of NHS is 1.5, 2, or 3 parts or a range thereof.

[0044] Preferably, in step 1, the molar ratio of γ-polyglutamic acid to CyS is γ-PGA : CyS = 1 : 1.5 - 1 : 3. More preferably, it is 1:1.5, 1:2, 1:2.5, 1:3 and a range thereof.

[0045] Preferably, in step 1, the pH of the dialysis medium is 4-4.5.

[0046] In any of the above-mentioned preferred embodiments, in step 2, hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are sequentially added to a MES buffer solution with pH=5.0 and stirred to adjust the pH of the system. Propyleneamine is then added to this mixture and the mixture is stirred continuously. After the reaction is completed, the mixture is dialyzed and dried to obtain the alkyne-bonded hyaluronic acid HA-alk.

[0047] In any of the above-mentioned preferred embodiments, in step 2, the pH of the system is adjusted to 8.5-12.5 using NaOH. More preferably, the pH is 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or a range thereof.

[0048] In any of the above-mentioned preferred embodiments, in step 2, after adding propargylamine, the mixture is continuously stirred at room temperature.

[0049] In any of the above-mentioned preferred embodiments, in step 2, the molecular weight cutoff for dialysis is 100 kDa.

[0050] In any of the above-mentioned preferred embodiments, in step 2, deionized water is used as the dialysis medium.

[0051] In any of the above-mentioned preferred embodiments, in step 2, the drying method is freeze drying.

[0052] In any of the above-mentioned preferred embodiments, in step 2, the alkyne-functionalized polymer HA-alk is synthesized by grafting propargylamine onto the side chain carboxyl group of hyaluronic acid: Hyaluronic acid (HA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-hydroxysuccinimide (NHS) are sequentially added to a MES buffer solution at pH=5.0 and stirred for 2 hours. The pH of the system is adjusted with NaOH, and propargylamine (PA) is added to this mixture, and the mixture is stirred continuously at room temperature for 24 hours. After the reaction is complete, the product is dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 100 kDa and deionized water as the dialysis medium to remove unreacted small molecules. Finally, the HA-alk sample is obtained by freeze-drying.

[0053] Preferably, in step 2, the molecular weight of the hyaluronic acid is 100-200 kDa.

[0054] Preferably, in step 2, the concentration of the MES solution containing hyaluronic acid is 10-15 mg / mL. More preferably, it is 10, 13, or 15 mg / mL or a range thereof.

[0055] Preferably, in step 2, the molar ratio of hyaluronic acid, EDC, and NHS is HA : EDC : NHS = 1 : 2 : 2 - 1 : 4 : 4, that is, the molar ratio of HA : EDC : NHS is 1 : (2-4): (2-4). Preferably, when the molar amount of HA is 1 part, the molar amount of EDC is 2, 3, or 4 parts or a range thereof, and the molar amount of NHS is 2, 3, or 4 parts or a range thereof.

[0056] Preferably, in step 2, the molar ratio of hyaluronic acid to PA is HA : PA = 1 : 2 - 1 : 4. More preferably, it is 1:2, 1:3, 1:4, or a range thereof.

[0057] Preferably, in step 3, the molar ratio of hyaluronic acid to sodium periodate is HA : sodium periodate = 1 : 3 - 1 : 6. More preferably, it is 1:3, 1:4, 1:5, 1:6 and a range thereof.

[0058] In step 3, the C2 and C3 C-couplings of the main chain sugar ring of hyaluronic acid are oxidized to aldehyde groups by sodium periodate to obtain the alkyne functionalized polymer HA-CHO.

[0059] Preferably, in step 4, gelatin is dissolved in preheated PBS buffer at pH 8.5; transglutaminase is added; and the reaction is carried out in a water bath.

[0060] In any of the above-mentioned preferred embodiments, the preheating temperature in step 4 is 60°C.

[0061] Preferably, in step 4, the gelatin PBS solution contains a gelatin concentration of 10% (w / v).

[0062] In any of the above-mentioned preferred embodiments, in step 4, the concentration of transglutaminase in the final mixture is 20 U / mL.

[0063] In any of the above-mentioned preferred embodiments, the water bath reaction temperature in step 4 is 60°C.

[0064] In any of the above-mentioned preferred embodiments, in step 4, the reaction is carried out in a water bath for 45 minutes.

[0065] In any of the above-mentioned preferred embodiments, in step 4, after the water bath reaction, the mixture is kept warm in a water bath at 37°C until it is ready for use.

[0066] Preferably, in step 4, the gelatin is dissolved in preheated (60°C) pH 8.5 PBS buffer to prepare a 10% (w / v) solution. After complete dissolution, transglutaminase is added to bring the enzyme concentration in the final mixture to 20 U / mL. The mixture is then reacted in a 60°C water bath for 45 minutes and incubated in a 37°C water bath until ready for use.

[0067] In any of the above-mentioned preferred embodiments, in step 5, γ-PGA-SH is dissolved in PBS buffer at pH=8.5 to prepare a 10% (w / v) solution, which is then dissolved at 37°C.

[0068] In any of the above-mentioned preferred embodiments, in step 6, HA-CHO is dissolved in PBS buffer at pH=8.5 to prepare a 10% (w / v) solution, which is then dissolved at 37°C.

[0069] Preferably, in step 7, at 37°C, a 10% (w / v) gelatin / 20 U / mL transglutaminase mixture, a 10% (w / v) γ-PGA-SH solution, and a 10% (w / v) HA-CHO solution are rapidly mixed at a volume ratio of 1:1:1 and then shaken. More preferably, the mixture is shaken with a vortex mixer for 5 seconds to ensure thorough homogenization, and then immediately injected into a custom-made cylindrical mold (preferably with a diameter of 10 mm and a height of 5 mm). Even more preferably, the mold is placed in a 37°C incubator and allowed to react for 10 minutes to obtain a γ-PGA-HA-Gel dual-network hydrogel.

[0070] In any of the above preferred embodiments, in step 6, HA-alk is dissolved in PBS buffer at pH = 8.5 to prepare a 10% (w / v) solution, dissolved at 37°C, and 0.01 g of photoinitiator LAP is added.

[0071] Preferably, in step 8, mixing and gelation are performed by rapidly mixing a 10% (w / v) gelatin / 20 U / mL transglutaminase mixture, a 10% (w / v) γ-PGA-SH solution, and a 10% (w / v) HA-alk solution at a volume ratio of 1:1:1 at 37°C. More preferably, the mixture is injected into a mold and allowed to react for 15 minutes in a 37°C water bath under 365 nm light irradiation to obtain an HA-γ-PGA-Gel dual-network hydrogel.

[0072] Preferably, in step 6, polyethylene glycol diglycidyl ether is dissolved in PBS buffer at pH 8.5 to prepare a 10% (w / v) solution. Preferably, the molar ratio of the epoxy group of PEGDE to the thiol group of γ-PGA-SH is controlled at 2:1.

[0073] Preferably, in step 9, a 10% (w / v) gelatin / 20 U / mL transglutaminase mixed solution, a 10% (w / v) γ-PGA-SH solution, and a 10% (w / v) polyethylene glycol diglycidyl ether solution are rapidly mixed at 37°C in a volume ratio of 1:1:1, and then vortexed to mix. More preferably, the mixture is injected into a mold and allowed to stand in a 37°C incubator for 40 minutes to obtain a γ-PGA-PEG-Gel dual-network hydrogel.

[0074] The present invention provides a thiolized γ-polyglutamic acid / gelatin dual-network hydrogel prepared according to any one of the above preparation methods.

[0075] The technical solution adopted in this invention is as follows:

[0076] This invention provides a series of methods for constructing thiolized γ-polyglutamic acid / gelatin dual-network hydrogels. Each dual-network hydrogel consists of two interpenetrating networks:

[0077] The first network is a gelatin network, which utilizes transglutaminase to catalyze the cross-linking reaction between the glutamine groups on the gelatin molecules and the ε-amino groups, forming a bio-cross-linked network with good biocompatibility and cell adhesion.

[0078] The second network, centered on thiolized γ-polyglutamic acid, is constructed through efficient click chemistry reactions of its side chain thiol groups with three different functionalized crosslinking agents. Specifically, it involves three pathways:

[0079] Thiol-aldehyde Michael addition pathway: The thiol group of γ-PGA-SH reacts with the aldehyde group of aldehyde-modified hyaluronic acid to form a thioether bond.

[0080] The thiol-alkynyl click reaction pathway: The thiol group of γ-PGA-SH reacts with the alkynyl bond of hyaluronic acid to form a stable CS bond.

[0081] The click reaction pathway of thiol-epoxy bond: The thiol group of γ-PGA-SH reacts with the epoxy group of polyethylene glycol diglycidyl ether to form a crosslink.

[0082] Furthermore, during the formation of the second network, the unreacted thiol groups on the γ-PGA-SH chain can spontaneously form dynamic disulfide bonds under mild oxidative conditions (such as in a buffer solution at pH=8.5), serving as the second crosslinking site, thereby constructing a dual crosslinked second network that combines covalent bond stability and dynamic bond reversibility.

[0083] The dual-network hydrogel was prepared using a one-pot method, comprising the following steps:

[0084] (1) Synthesize functionalized polymer precursors, including thiolized γ-polyglutamic acid (γ-PGA-SH), alkyne-bonded hyaluronic acid (HA-alk) and aldehyde-bonded hyaluronic acid (HA-CHO).

[0085] (2) Preparation of precursor solution: Dissolve gelatin, transglutaminase, γ-PGA-SH and one of the three cross-linking agents (HA-CHO, HA-alk, PEGDE) together in phosphate buffer solution at pH=8.5 to form a homogeneous mixture.

[0086] (3) Gelation: The above mixture is placed in a mold and reacted at 37°C, so that the enzyme crosslinking of the first network and the click chemical crosslinking of the second network occur synchronously or sequentially, and finally a complete three-dimensional double network hydrogel structure is formed.

[0087] Compared with the prior art, the beneficial effects of the present invention are:

[0088] Structural Synergy: This invention constructs a gelatin first network through enzymatic cross-linking and a γ-polyglutamic acid second network through thiol click chemistry. The two networks interpenetrate each other, forming a dual-network structure. This structural design is expected to synergistically leverage the rigid support of the gelatin network and the flexibility of the γ-polyglutamic acid network, providing a structural basis for improving the overall mechanical properties of hydrogels.

[0089] Advantages of Biological Origin: The gelatin, γ-polyglutamic acid, and hyaluronic acid used in this invention are all naturally derived biomolecules with good biocompatibility, degradability, and low immunogenicity potential, laying the foundation for their application in the biomedical field. Gelatin molecules retain cell recognition sites derived from collagen (such as the arginine-glycine-aspartic acid sequence), possessing the potential to provide support for cell adhesion and facilitating the further development of bioactive scaffolds that regulate cell behavior.

[0090] Dynamic structural characteristics: The disulfide bonds introduced in the second network (spontaneously formed by unreacted thiol groups on thiolated γ-polyglutamic acid under mild conditions) have dynamic reversible properties, which theoretically can provide a potential stress dissipation mechanism for hydrogels and are expected to improve the adaptability of materials under cyclic loading environments.

[0091] Flexibility and universality of the construction strategy: This invention proposes a modular construction platform using thiolized γ-polyglutamic acid as the core precursor and combining multiple click chemistry reaction pathways. By selecting different second network crosslinking agents (aldehyde-based hyaluronic acid, alkyne-based hyaluronic acid, or polyethylene glycol diglycidyl ether), the crosslinking density and chemical microenvironment of the hydrogel can be controlled, exhibiting good preparation flexibility.

[0092] Simplicity and mildness of the preparation process: This invention uses a one-pot method for preparation, with mild reaction conditions (37°C, near physiological pH), simple operation, high reaction efficiency, and compatibility with the encapsulation conditions of bioactive substances, which facilitates further development of functional materials for tissue repair or drug delivery. Attached Figure Description

[0093] Figure 1 These are morphological images of the single-network hydrogel and the double-network hydrogel of the present invention.

[0094] Figure 2 This is the proton nuclear magnetic resonance spectrum used in the structural characterization of the preferred embodiment 5 of the present invention.

[0095] Figure 3 This is the infrared spectrum used in the structural characterization of the preferred embodiment 5 of the present invention.

[0096] Figure 4 This is a scanning electron microscope image of the structure characterization in the preferred embodiment 6 of the present invention.

[0097] Figure 5 This is the scanning electron microscope mapping spectrum used in the structural characterization of the preferred embodiment 6 of the present invention. Detailed Implementation

[0098] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0099] Figure 1 The figures shown are morphological images of the single-network hydrogel and the double-network hydrogel of the present invention. Figure 1 Image A is a digital photograph of a single-network hydrogel (gelatin / TG enzyme cross-linked network only), which appears transparent. Figure 1 Images B, C, and D in the image show digital photographs of the dual-network hydrogels γ-PGA-HA-Gel, HA-γ-PGA-Gel, and γ-PGA-PEG-Gel, respectively. Compared to the single-network hydrogels, all three dual-network hydrogels exhibit a uniform pale yellow to milky white appearance, a more uniform texture, and good structural integrity after molding, indicating that the introduction of the second network effectively improves the formability and macroscopic uniformity of the hydrogels.

[0100] Figure 2 The image shows the nuclear magnetic resonance hydrogen spectrum (NMR spectrum) for structural characterization in Example 5. 1 H NMR spectrum, Figure 2 In this context, A represents the compounds γ-PGA, Cys, and γ-PGA-SH. 1 ¹H NMR; B represents compounds HA, PA, and HA-alk. 1 ¹H NMR; C represents compounds HA and HA-CHO. 1 H NMR.

[0101] Figure 3 The infrared (Fourier Transform Infrared Spectroscopy, FT-IR) and Raman (Raman) spectra used in the structural characterization of the preferred embodiment 5 of the present invention are shown. Figure 3 In the diagram, A represents the FT-IR of compounds γ-PGA, Cys, and γ-PGA-SH; B represents the Raman IR of compounds HA, PA, and HA-alk; and C represents the FT-IR of compounds HA and HA-CHO.

[0102] Figure 4 This is a scanning electron microscope (SEM) image of the structure characterization in the preferred embodiment 6 of the present invention.

[0103] Figure 5 This is the scanning electron microscope mapping spectrum used in the structural characterization of the preferred embodiment 6 of the present invention.

[0104] The MES buffer solution of the present invention is prepared by conventional methods of the prior art, namely by dissolving the compound MES in deionized water; the preferred pH of the MES buffer solution is 5; the preferred concentration of the MES buffer solution is 0.1-0.5M, and more preferably 0.1, 0.2, 0.3, 0.4, 0.5M and the range thereof.

[0105] The PBS buffer of the present invention is prepared by conventional methods of the prior art; the preferred pH of the PBS buffer is 8.5; the preferred concentration of the PBS buffer is 0.05-0.1M, and more preferably 0.05 or 0.1M.

[0106] Unless otherwise specified, the reagents and instruments used in this invention are all conventional commercial products in this technical field.

[0107] Example 1

[0108] Example 1 provides the synthesis of functionalized polymers.

[0109] 1.1 Synthesis of thiolated γ-polyglutamic acid.

[0110] Weigh 1 g of γ-polyglutamic acid (250 kDa) and dissolve it in 200 mL of MES buffer (pH 5.0) to prepare a solution with a concentration of 5 mg / mL. Under stirring, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide sequentially, with a molar ratio of γ-PGA, EDC, and NHS of 1:1.5:1.5. Activate the carboxyl groups by stirring at 30°C for 2 hours. Subsequently, under light-protected and nitrogen-protected conditions, add a MES solution of β-mercaptoethylamine dropwise to the activated solution, with a molar ratio of γ-PGA to Cys of 1:2. Maintain the reaction temperature at 30°C and continue stirring in the dark for 24 hours. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 100 kDa. Dialysis was performed using dilute hydrochloric acid solution at pH 4.5 as the dialysis medium at 4°C for 3 days, with the dialysis medium changed 3 times daily to thoroughly remove unreacted EDC, NHS, Cys, and byproducts. Finally, the solution in the dialysis bag was freeze-dried to obtain a white, spongy γ-PGA-SH solid. The thiol content of the product was determined to be approximately 1.8 mmol / g using the Ellman reagent method.

[0111] 1.2 Synthesis of Alkyne-bonded Hyaluronic Acid

[0112] 1 g of sodium hyaluronate with a molecular weight of 150 kDa was weighed and dissolved in 100 mL of MES buffer (pH 5.0) to prepare a solution with a concentration of 10 mg / mL. EDC and NHS were added with stirring, wherein the molar ratio of HA, EDC, and NHS was 1:3:3. The mixture was activated at room temperature with stirring for 2 hours. The pH of the mixture was slowly adjusted to 9.5 using 1 M NaOH solution. Then, PA was added, wherein the molar ratio of HA to PA was 1:3. The reaction was continued with stirring at room temperature for 24 hours. After the reaction was complete, the product solution was placed in a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed against deionized water for 3 days (4℃), changing the water 3 times a day. After freeze-drying, a white solid, HA-alk, was obtained. The presence of characteristic peaks for alkyne protons at δ 2.8–3.0 ppm by ¹H NMR spectroscopy (D₂O) confirmed successful alkyne grafting, with a calculated grafting rate of approximately 25%.

[0113] 1.3 Synthesis of Aldehyde-Tylated Hyaluronic Acid

[0114] 1 g of sodium hyaluronate with a molecular weight of 150 kDa was weighed and dissolved in 100 mL of deionized water to prepare a solution with a concentration of 10 mg / mL. Solid sodium periodate was added under light-protected conditions, with a molar ratio of HA to NaIO4 of 1:4. The reaction was stirred at room temperature in the dark for 6 hours. After the reaction was complete, excess ethylene glycol (approximately 0.1 mL) was added to quench the remaining sodium periodate, and stirring was continued for 30 minutes. The reaction solution was transferred to a dialysis bag (MWCO 100 kDa) and dialyzed against deionized water for 3 days (4℃). After freeze-drying, a white solid, HA-CHO, was obtained. The aldehyde content was determined to be approximately 2.1 mmol / g by hydroxylamine hydrochloride titration.

[0115] Example 2

[0116] In Example 2, a dual-network hydrogel was constructed via thiol-aldehyde Michael addition. This example yielded a γ-PGA-HA-Gel dual-network hydrogel.

[0117] Solution preparation:

[0118] (1) Solution A (gelatin / TG enzyme solution): Dissolve gelatin in preheated (60℃) pH=8.5 PBS buffer to prepare a 10% (w / v) solution. After it is completely dissolved, add transglutaminase to make the enzyme concentration in the final mixture 20 U / mL. React in a 60℃ water bath for 45 minutes, then incubate in a 37℃ water bath until ready for use.

[0119] (2) Solution B (γ-PGA-SH solution): The γ-PGA-SH synthesized in Example 1 was dissolved in PBS buffer at pH=8.5 to prepare a 10% (w / v) solution, which was then dissolved at 37°C.

[0120] (3) Solution C (HA-CHO solution): The HA-CHO synthesized in Example 1 was dissolved in PBS buffer at pH=8.5 to prepare a 10% (w / v) solution, which was then dissolved at 37°C.

[0121] Mixing and gelation: At 37°C, rapidly mix solutions A, B, and C at a volume ratio of 1:1:1 and vortex for 5 seconds to ensure thorough homogenization. Immediately pour the mixture into a custom-made cylindrical mold (10 mm in diameter and 5 mm in height).

[0122] Molding: Place the mold in a 37℃ constant temperature oven and let it stand for 10 minutes. Observation shows that the mixture loses its fluidity within 10-15 minutes, forming an elastic, pale yellow, opaque hydrogel, namely γ-PGA-HA-Gel double network hydrogel.

[0123] Example 3

[0124] Example 3 constructs a dual-network hydrogel via a thiol-alkyne click reaction. In this example, an HA-γ-PGA-Gel dual-network hydrogel was prepared.

[0125] Solution preparation:

[0126] (1) Solution A (gelatin / TG enzyme solution) was prepared in the same way as in Example 2.

[0127] (2) Solution B (γ-PGA-SH solution) was prepared in the same way as in Example 2.

[0128] (3) Solution D (HA-alk solution): The HA-alk synthesized in Example 1 was dissolved in PBS buffer at pH = 8.5 to prepare a 10% (w / v) solution. The solution was dissolved at 37°C and 0.01 g of photoinitiator LAP was added.

[0129] Mixing and gelation: At 37°C, rapidly mix solutions A, B and D in a volume ratio of 1:1:1.

[0130] Molding: The mixture was injected into a mold and allowed to react for 15 minutes in a 37°C water bath under 365 nm light. This resulted in a uniform, pale yellow hydrogel, namely the HA-γ-PGA-Gel dual-network hydrogel.

[0131] Example 4

[0132] Example 4 describes the construction of a dual-network hydrogel via a thiol-epoxy bond click reaction. In this example, a γ-PGA-PEG-Gel dual-network hydrogel was prepared.

[0133] Solution preparation:

[0134] (1) Solution A (gelatin / TG enzyme solution) was prepared in the same way as in Example 2.

[0135] (2) Solution B (γ-PGA-SH solution) was prepared in the same way as in Example 2.

[0136] (3) Solution E (PEGDE solution): Polyethylene glycol diglycidyl ether was dissolved in PBS buffer at pH 8.5 to prepare a 10% (w / v) solution. The molar ratio of the epoxy group of PEGDE to the thiol group of γ-PGA-SH was controlled at 2:1.

[0137] Mixing and gelation: At 37°C, rapidly mix solutions A, B and E in a volume ratio of 1:1:1 and vortex to mix.

[0138] Molding: The mixture is injected into a mold and allowed to react in a 37°C constant temperature oven for 40 minutes. A transparent or translucent elastic hydrogel is formed, namely a γ-PGA-PEG-Gel dual-network hydrogel.

[0139] Example 5

[0140] Example 5 provides structural characterization of the precursor polymer.

[0141] The three polymers obtained in Example 1 were characterized by the following structures:

[0142] Hydrogen NMR spectra were recorded using a Bruker AVANCE III (400 MHz) instrument, with tetramethylsilane (TMS) as an internal standard and heavy water as the solvent. In HA-CHO... 1 H NMR ( Figure 2 A new peak was observed at 4.9–5.0 ppm in γ-PGA-SH, corresponding to a hemiacetal proton formed by an aldehyde group and an adjacent hydroxyl group. This result indicates that the proximal hydroxyl group in HA was successfully oxidized to an aldehyde group. 1 H NMR spectrum ( Figure 2 A set of peaks at 1.5–2.5 ppm and 4.2 ppm from the intermediate methylene (g / f) and methylene (e) of the γ-PGA backbone were also found in A). Similarly, characteristic peaks at 3.0 ppm (SHCH2-, c) and 3.8 ppm (NH2CHCO-, d) from the Cys fragment were also found in γ-PGA-SH. 1The presence of 1H NMR indicates that the thiol was successfully introduced onto the carboxyl group of γ-PGA. (In ( Figure 2 New peaks were observed at 4.1 and 3.1 ppm in (B), corresponding to the methylene and acetylic hydrogen of propargylamine, respectively, indicating that the compound has been successfully prepared, and its grafting rate was further calculated to be 25%.

[0143] Fourier transform infrared (FTIR) spectroscopy was performed using a Nicolet iS 50 spectrometer from Thermo Fisher Scientific, USA. Raman spectroscopy was performed using a Horiba LabRAMHR Evolution spectrometer from Japan. The FTIR spectrum of HA-CHO was obtained at 1730 cm⁻¹. -1 A weak C-O bending vibration peak was observed at this point, confirming the presence of -CHO. Furthermore, in the FT-IR spectrum of γ-PGA-SH, the γ-PGA backbone showed a peak at 3300 cm⁻¹. -1 (O-H stretching vibration), 1656 cm -1 (Amide I band) and 1552 cm -1 The characteristic absorption peak of (amide II band) is clearly distinguishable, and at 2565 cm⁻¹ -1 The peak at [value missing] further confirms the successful introduction of the thiol group. Combined NMR and FT-IR characterization results show that γ-PGA-SH and HA-CHO have been successfully prepared. The Raman spectrum of HA-alk at 2116 cm⁻¹ [value missing] further confirms the successful introduction of the thiol group. -1 The presence of a strong, broad peak at the propargylamine alkyne bond indicates that the polymer HA-alk has been successfully prepared. The results are as follows: Figure 3 As shown.

[0144] Example 6

[0145] Example 6 provides structural characterization of three hydrogels.

[0146] Three hydrogel samples were subjected to 24-hour vacuum freeze-drying, followed by gold sputtering of their cross-sections. The morphology of the samples was then characterized using a scanning electron microscope (SU3800). Figure 4 As shown: SEM images of three single-network and three double-network hydrogels reveal uniform porous and interconnected internal structures in all hydrogels. Figure 5 As shown: SEM mapping images of three types of dual-network hydrogels show the elemental distribution of the main elements C, O, N and S in the hydrogel system, and all elements are uniformly distributed.

[0147] This invention successfully constructed a series of dual-network hydrogels based on thiolated γ-polyglutamic acid / gelatin through an innovative strategy of "enzyme crosslinking + diversified click chemistry".

[0148] This invention has the following significant advantages:

[0149] Structural design advantages: This invention provides a structural basis for improving the mechanical properties of hydrogels by constructing a dual-network structure in which an enzyme-crosslinked gelatin network and a thiol-click chemically crosslinked γ-polyglutamic acid network interpenetrate each other, and has the potential to meet the requirements of complex biomechanical environments.

[0150] Advantages in Raw Material Sources: All raw materials used in this invention (γ-polyglutamic acid, gelatin, and hyaluronic acid) are naturally derived biomolecules with good biocompatibility and biodegradability potential. The gelatin network retains cell recognition sites derived from collagen (such as the arginine-glycine-aspartic acid sequence), providing a structural basis for its application in cell growth and tissue regeneration.

[0151] Dynamic structural characteristics: The disulfide bonds introduced in the second network (spontaneously formed by unreacted thiol groups on thiolated γ-polyglutamic acid under mild conditions) have dynamic reversible characteristics, which can theoretically provide a potential stress dissipation mechanism for materials, and are beneficial to the further development of functionally adaptive biomaterials.

[0152] Flexibility of the construction strategy: This invention provides a modular construction platform that uses thiolated γ-polyglutamic acid as the core precursor and combines multiple click chemistry reaction pathways. By selecting different second network crosslinking agents (aldehyde-based hyaluronic acid, alkyne-bonded hyaluronic acid, or polyethylene glycol diglycidyl ether), the crosslinking density and chemical microenvironment of the hydrogel can be controlled, resulting in good preparation flexibility.

[0153] Simplicity of preparation process: The present invention adopts a "one-pot method" for preparation, with mild reaction conditions (37°C, close to physiological pH), simple operation, high reaction efficiency, and compatibility with the encapsulation conditions of bioactive substances, providing convenience for the preparation of functional bioactive scaffolds.

[0154] Therefore, the dual-network hydrogel provided by this invention has potential applications in tissue engineering scaffolds (such as cartilage, skin, and corneal repair), injectable filler materials, smart drug controlled-release carriers, and advanced wound dressings.

[0155] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent modifications, substitutions, or variations made by those skilled in the art within the technical principles and core concepts disclosed in the present invention, utilizing the content of the present invention's specification and claims, should be included within the scope of protection of the present invention.

[0156] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thiolized γ-polyglutamic acid / gelatin dual-network hydrogel, characterized in that, The dual-network hydrogel comprises a first network and a second network; the first network is a gelatin network, obtained by cross-linking gelatin with transglutaminase (TG); the second network is prepared by thiolated γ-polyglutamic acid and a second network cross-linking agent, the second network cross-linking agent comprising any one of aldehyde-based hyaluronic acid (HA-CHO), alkyne-based hyaluronic acid (HA-alk), or polyethylene glycol diglycidyl ether (PEGDE); the thiolated γ-polyglutamic acid is synthesized by grafting β-mercaptoethylamine onto the side chain carboxyl group of γ-polyglutamic acid; in the second network, the disulfide bond formed between the thiol groups of the thiolated γ-polyglutamic acid serves as the second cross-linking site.

2. The dual-network hydrogel as described in claim 1, characterized in that, The alkyne-bonded hyaluronic acid HA-alk was synthesized by grafting propargylamine onto the side chain carboxyl group of hyaluronic acid.

3. The dual-network hydrogel as described in claim 1, characterized in that, The aldehyde-modified hyaluronic acid HA-CHO was obtained by oxidizing hyaluronic acid with sodium periodate.

4. The method for preparing the dual-network hydrogel according to any one of claims 1 to 3, characterized in that, The dual-network hydrogel is prepared using a one-pot method, comprising the following steps: Step 1: The thiolated γ-polyglutamic acid was synthesized by grafting β-mercaptoethylamine onto the side chain carboxyl group of γ-polyglutamic acid; Step 2: The alkyne-bonded hyaluronic acid HA-alk is synthesized by grafting propargylamine onto the side chain carboxyl group of hyaluronic acid; Step 3: Oxidize hyaluronic acid with sodium periodate to obtain the aldehyde-modified hyaluronic acid HA-CHO; Step 4: Dissolve gelatin in PBS, add transglutaminase (TG) at 60°C and stir to obtain a gelatin / TG enzyme solution; Step 5: Dissolve thiolated γ-polyglutamic acid in PBS at 37°C to obtain γ-PGA-SH solution; Step 6: Dissolve aldehyde-modified hyaluronic acid HA-CHO, alkyne-modified hyaluronic acid HA-alk, or polyethylene glycol diglycidyl ether PEGDE in PBS and dissolve at 37°C to obtain HA-CHO solution, HA-alk solution, or PEGDE solution, respectively; the HA-alk solution also contains the photoinitiator LAP. The dual-network hydrogel is obtained by following any one of steps 7, 8 or 9; Step 7: Mix the γ-PGA-SH solution with the gelatin / TG enzyme solution, add the HA-CHO solution, pour the mixture into a custom mold, and let it stand at 37°C for 1 hour to obtain the double network hydrogel γ-PGA-HA-Gel. Step 8: Mix the γ-PGA-SH solution with the gelatin / TG enzyme solution, add the HA-alk solution, and let it stand for 1 hour under 37℃ and 365 nm ultraviolet light to obtain the double network hydrogel HA-γ-PGA-Gel; Step 9: Mix the γ-PGA-SH solution with the gelatin / TG enzyme solution, add the PEGDE solution, pour the mixture into a custom mold, and let it stand at 37°C for 1 hour to obtain the double-network hydrogel γ-PGA-PEG-Gel.

5. The preparation method according to claim 4, characterized in that, In step 1, γ-polyglutamic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added sequentially to a MES buffer solution at pH 5.0 and stirred. Under light-protected nitrogen atmosphere, β-mercaptoethylamine was added to the mixture and stirred continuously. The thiolated γ-polyglutamic acid was obtained by dialyzing and drying.

6. The preparation method according to claim 5, characterized in that, In step 1, the molecular weight cutoff for dialysis is 100 kDa.

7. The preparation method according to claim 6, characterized in that, In step 1, the molar ratio of γ-polyglutamic acid to β-mercaptoethylamine, γ-PGA : CyS, is 1 : 1.5 - 1 :

3.

8. The preparation method according to claim 4, characterized in that, In step 2, hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are added sequentially to a MES buffer solution with pH=5.0 and stirred to adjust the pH of the system. Propyleneamine is then added to this mixture and stirring is continued. After the reaction was completed, the mixture was dialyzed and dried to obtain the alkyne-bonded hyaluronic acid HA-alk.

9. The preparation method according to claim 8, characterized in that, In step 2, the pH of the system is adjusted to 8.5-12.5 using NaOH.

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