Viscoelasticity-adjustable acylhydrazone bond hydrogel as well as preparation method and application thereof

Abstract

The invention discloses an acylhydrazone bond hydrogel with adjustable viscoelasticity as well as a preparation method and application thereof, and belongs to the technical field of high polymer materials. A skeleton network of the acylhydrazone bond hydrogel is formed by crosslinking multi-arm benzaldehyde polyethylene glycol, multi-arm alkyl hydroformylation polyethylene glycol and hydrazino-modified multi-arm polyethylene glycol or gelatin macromolecules through acylhydrazone bonds. According to the invention, the adjustable viscoelasticity and rigidity of the hydrogel can be changed, and the hydrogel can be endowed with more cell adhesion sites and a cell microenvironment more suitable for the growth of various cells. The hydrogel can be widely applied to three-dimensional culture, amplification and differentiation of different cells in vitro.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer materials, and particularly relates to a hydrazone bond hydrogel with adjustable viscoelasticity, a preparation method thereof, and an application thereof. Background Art

[0002] A polymer hydrogel is a highly hydrophilic three-dimensional network composite material. It has advantages such as a biomimetic three-dimensional pore structure and good biocompatibility, and can be used to simulate the three-dimensional pore structure of the extracellular matrix. During the three-dimensional culture of cells in vitro, in order to achieve complex cell functions such as extracellular matrix deposition, movement, and diffusion, it is necessary to design a crosslinking degradation mechanism in the material to covalently crosslink and synthesize a hydrogel. Its well-defined and easily adjustable biochemical and biophysical properties are particularly suitable for applications in regenerative medicine fields such as three-dimensional cell scaffolds and tissue engineering.

[0003] Although it has been clear that the extracellular matrix ECM and tissues have viscoelastic characteristics, for a long time, research on how to utilize this property, especially considering the time-dependence of the ECM mechanism in the design of hydrogel materials to effectively regulate cell behavior, has been relatively lagging.

[0004] To solve this problem, researchers have begun to explore regulating the viscoelasticity of hydrogels to simulate the stress relaxation or creep behavior of the extracellular matrix, and then investigating the specific effects of these changes on cell behavior (such as cell diffusion, proliferation, and mesenchymal stem cell differentiation, etc.). Using viscoelastic hydrazone bond hydrogels for cell culture can improve our understanding of the interaction between cells and the matrix during development, homeostasis, wound healing, and disease processes, and guide the design of regenerative medical biomaterials. Therefore, it is necessary to propose a hydrazone bond hydrogel with adjustable viscoelasticity to solve the key problems in the prior art, and it also opens up a new path for the development of future biomaterials. Summary of the Invention

[0005] The purpose of the present invention is to provide a hydrazone bond hydrogel with adjustable viscoelasticity, a preparation method thereof, and an application thereof, so as to solve the problem of adjusting the viscoelasticity of existing hydrogels, optimize the performance of the hydrogel, and further promote the normal exertion of cell functions.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses gelatin macromolecules or multi-arm polyethylene glycol with hydrazino groups at the ends to react with aldehyde groups, and prepares a hydrazone bond hydrogel cell system with adjustable viscoelasticity and rigidity through the formation of dynamic or stable hydrazone bonds. Among them, the amino groups and hydrazides on the gelatin macromolecules modified with hydrazino groups can react with the aldehyde groups on the multi-arm alkylated polyethylene glycol through Schiff base reaction to form dynamic hydrazone bonds respectively. The reversible cross-linking mode of the dynamic hydrazone bonds endows the hydrogel system with excellent self-healing performance and injectability; the amino groups and hydrazides on the gelatin macromolecules modified with hydrazino groups can react with the aldehyde groups on the multi-arm benzaldehyde polyethylene glycol through Schiff base reaction to form stable covalent bonds respectively. By regulating the reaction ratio of multi-arm benzaldehyde polyethylene glycol and multi-arm alkylated polyethylene glycol, a series of hydrogels with different viscoelasticities can be formed. The hydrogel has low synthesis cost, simple process, safety, mild reaction conditions, and the obtained hydrogel has good application prospects in biomedical fields such as three-dimensional cell culture, stem cell transplantation, tissue engineering, and drug sustained release. Using this hydrazone bond hydrogel with adjustable viscoelasticity for cell culture can not only deepen our understanding of the interaction mechanism between cells and the matrix in development, homeostasis, wound healing, and disease processes, but also provide a powerful tool and guidance for the design and optimization of biomaterials in the field of regenerative medicine.

[0007] Furthermore, adding collagen or fibronectin hydrogel to the system for preparing the hydrogel of the present invention to form an interpenetrating network hydrogel can not only change the stiffness of the hydrazone bond hydrogel of the present invention, but also endow the hydrogel with more cell adhesion sites and a cell microenvironment more suitable for the growth of various cells. Brief Description of the Drawings

[0008] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0009] Figure 1 Schematic diagram of the relaxation curve of the hydrogel prepared from multi-arm alkylated polyethylene glycol with different ratios in Example 1; Figure 2 Schematic diagram of the relaxation time of the hydrogel prepared from multi-arm alkylated polyethylene glycol with different ratios in Example 1; Figure 3 Oscillation frequency scan diagram of the hydrogel prepared from multi-arm alkylated polyethylene glycol with different ratios in Example 1; Figure 4 Schematic diagram of the stiffness of the hydrogel prepared from multi-arm alkylated polyethylene glycol with different ratios in Example 1; Figure 5 Schematic diagram of the gelation time of hydrogels prepared from multi-armed alkylated polyethylene glycols with different ratios in Example 1. Detailed implementation manners

[0010] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Generally, the components of the present invention described and shown in the accompanying drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0011] The hydrazone conversion reaction has been used to assemble enzyme substrates and form bioconjugates in in vitro biological systems. The reaction rate between the nucleophile of the hydrazide group and the electrophile of the aldehyde group, as well as the stability of the formed hydrazone bond, largely depends on the precise chemical structures of the nucleophile and electrophile. At a pH value of 7.0, its half-life can range from a few minutes to several months, depending on the exact functionality of the groups. Therefore, hydrazone bond hydrogels exhibit covalent adaptation behavior under physiological conditions, characterized by a modulus and stress relaxation that vary with frequency. There is data indicating that the reaction of methylhydrazine (25 μM) with butyraldehyde (25 μM) is very rapid and reaches equilibrium in less than one hour in a buffer solution with a pH value of 7.4. In contrast, the reaction of methylhydrazine (25 μM) and p-nitrobenzaldehyde (25 μM) takes several days to reach equilibrium. Therefore, at a constant molar concentration, by changing the molar ratio of alkyl aldehyde and benzaldehyde reacting with the hydrazide group, the stress relaxation of the hydrogel network can be adjusted.

[0012] Collagen and fibronectin are both important components of the extracellular matrix. Among them, collagen is the most abundant and widely distributed functional protein in mammals. Collagen solution can spontaneously form a hydrogel by adjusting its pH and temperature without the need for a cross-linking agent, and it has excellent biocompatibility. Fibronectinogen and thrombin can form a fibronectin hydrogel by mixing in a certain proportion, and its biological properties are similar to those of mechanical and soft tissues. At present, a large number of studies have shown that collagen and fibronectin hydrogels can be used for in vitro culture of stem cells, embryoid bodies, tumor cells, etc., and good results have been obtained. Therefore, the use of this hydrazone bond hydrogel and interpenetrating network hydrogel can greatly adjust the biochemical and biophysical microenvironment during the three-dimensional cell culture process, while still being able to capture the modulus and adaptability of native tissues. This hydrogel can well explore a series of stress relaxation properties in the native extracellular matrix, and can determine the dynamic changes in the local biophysical environment and its impact on cell function.

[0013] The present invention provides a hydrazone bond hydrogel with adjustable viscoelasticity. The backbone network of the hydrazone bond hydrogel is composed of multi-arm benzaldehyde-modified polyethylene glycol, multi-arm alkyl aldehyde-modified polyethylene glycol, and hydrazine group-modified multi-arm polyethylene glycol or gelatin macromolecules crosslinked by hydrazone bonds;

[0014] The hydrazone bond hydrogel with adjustable viscoelasticity provided by the present invention can also be compounded with collagen, or fibronectin and thrombin in the backbone network of the hydrazone bond hydrogel to form a hydrazone bond hydrogel with adjustable viscoelasticity and stiffness.

[0015] The present invention provides a preparation method of a hydrazone bond hydrogel with adjustable viscoelasticity, which includes the following steps: Step 1: Preparation of multi-arm benzaldehyde-modified polyethylene glycol.

[0016] First, add an appropriate amount of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then, successively add an appropriate amount of multi-arm polyethylene glycol, 4-formylbenzoic acid (p-CBA), and N,N'-diisopropylcarbodiimide (DIC). The molar ratio of multi-arm polyethylene glycol, p-CBA, and DIC is 1∶1∶1 to 1∶50∶50. This reaction is carried out at room temperature for 12 to 24 hours; after the reaction is completed, a first product is obtained. The first product is precipitated in ether to obtain a first solution, and then the first solution is dialyzed in deionized water using a dialysis bag, changing water no less than three times a day. After dialysis for 2 days, it is freeze-dried to obtain multi-arm benzaldehyde-modified polyethylene glycol; Step 2: Preparation of multi-arm alkyl aldehyde-modified polyethylene glycol.

[0017] First, add an appropriate amount of dichloromethane into a 250 mL round-bottom flask. Under the protection of nitrogen, subsequently, add an appropriate amount of multi-arm polyethylene glycol, 2,2,6,6-tetramethylpiperidine-N-oxide (TEMPO), and diacetoxyiodobenzene (DAIB) in sequence. The molar ratio of multi-arm polyethylene glycol, TEMPO, and DAIB is 10∶1∶1~1∶10∶100, and this reaction is carried out at room temperature for 12 to 24 hours; after the reaction is completed, a second product is obtained. The second product is precipitated in diethyl ether to obtain a second solution, and then the second solution is dialyzed in deionized water using a dialysis bag, changing water no less than three times a day. After dialysis for 2 days, it is freeze-dried to obtain multi-arm alkylated polyethylene glycol; Step 3: Preparation of multi-arm hydrazino polyethylene glycol.

[0018] First, add an appropriate amount of dichloromethane into a 250 mL round-bottom flask. Under the protection of nitrogen, subsequently, add an appropriate amount of multi-arm polyethylene glycol amine, tri-Boc-hydrazinoacetic acid, and EDC·HCl in sequence. The molar ratio of multi-arm polyethylene glycol amine, tri-Boc-hydrazinoacetic acid, and EDC·HCl is 10∶1∶1~1∶10∶10, and this reaction is carried out at room temperature for 12 to 24 hours; after the reaction is completed, a third product is obtained. The third product is precipitated in diethyl ether to obtain a fourth product, and the fourth product is vacuum-dried to obtain a fifth product. Then, the fifth product is added to 50:50 DCM:TFA for 2 to 3 hours to obtain a third solution, and then the third solution is dialyzed in deionized water using a dialysis bag, changing water no less than three times a day. After dialysis for 2 days, it is freeze-dried to obtain multi-arm hydrazino polyethylene glycol; Step 4: Preparation of gelatin macromolecules modified with hydrazine groups.

[0019] First, add 40 mL of PBS with a pH of 5.5 into a 100 mL round-bottom flask, and then add a certain amount of type A porcine skin gelatin and stir to dissolve it fully to obtain a fourth solution; add a certain proportion of adipic dihydrazide (ADH) to the fourth solution and stir evenly; then dissolve 1-hydroxybenzotriazole hydrate (HOBT) in DMSO and add it to the reaction flask and stir evenly; finally, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ((EDC·HCl)). The molar ratio of ADH, HOBT, and EDC·HCl is 10∶1∶1~1∶10∶10, and the reaction is carried out at room temperature for 12 to 24 hours to obtain a sixth product. Adjust the pH of the sixth product to 5.0 - 5.3 with NaOH to obtain a seventh product; stir the seventh product at 50 °C for 24 h to obtain a fifth solution, and dialyze the fifth solution in deionized water using a dialysis bag, changing water once a day. After dialysis for one week, it is freeze-dried to obtain gelatin macromolecules modified with hydrazine groups.

[0020] Step 5: Dissolve the products of Step 1, 2, 3 or 4, multi-armed benzaldehyde-polyethylene glycol, multi-armed alkyl aldehyde-polyethylene glycol, and multi-armed hydrazine-polyethylene glycol or gelatin macromolecules modified with hydrazine groups in PBS respectively, neutralize to pH 7.0, and the mass fraction is 1% - 40%; then take different volumes of gelatin macromolecule solution modified with hydrazine groups or multi-armed hydrazine-polyethylene glycol solution, sequentially add multi-armed benzaldehyde-polyethylene glycol solution and multi-armed alkyl aldehyde-polyethylene glycol solution thereto, disperse evenly, and let stand to obtain an acylhydrazone bond hydrogel with adjustable viscoelasticity.

[0021] Step 6: Dissolve the products of Step 1, 2, 3 or 4, multi-armed benzaldehyde-polyethylene glycol, multi-armed alkyl aldehyde-polyethylene glycol, and multi-armed hydrazine-polyethylene glycol or gelatin macromolecules modified with hydrazine groups in PBS respectively, neutralize to pH 7.0, and the mass fraction is 1% - 40%; then take different volumes of gelatin macromolecule solution modified with hydrazine groups or multi-armed hydrazine-polyethylene glycol solution, sequentially add multi-armed benzaldehyde-polyethylene glycol solution, multi-armed alkyl aldehyde-polyethylene glycol solution, and collagen solutions with different concentrations (1 mg / ml - 10 mg / ml) thereto, disperse evenly on ice, and let stand to obtain an acylhydrazone bond hydrogel with adjustable viscoelasticity and rigidity.

[0022] Step 7: Dissolve the products of Step 1, 2, 3 or 4, multi-armed benzaldehyde-polyethylene glycol, multi-armed alkyl aldehyde-polyethylene glycol, and multi-armed hydrazine-polyethylene glycol or gelatin macromolecules modified with hydrazine groups in PBS respectively, neutralize to pH 7.0, and the mass fraction is 1% - 40%; then take different volumes of gelatin macromolecule solution modified with hydrazine groups or multi-armed hydrazine-polyethylene glycol solution, sequentially add multi-armed benzaldehyde-polyethylene glycol solution, multi-armed alkyl aldehyde-polyethylene glycol solution, fibronectin solutions with different concentrations, and thrombin solution thereto, disperse evenly on ice, and let stand to obtain an acylhydrazone bond hydrogel with adjustable viscoelasticity and rigidity.

[0023] The multi-armed polyethylene glycols described in Steps 1 and 2 include polyethylene glycols with 2, 4, 6 or 8 arms, and the molecular weight of the multi-armed polyethylene glycol is 10 - 20 kDa; the multi-armed polyethylene glycol amines described in Step 3 include polyethylene glycol amines with 2, 4, 6 or 8 arms, and the molecular weight of the multi-armed polyethylene glycol amine is 10 - 20 kDa.

[0024] The multi-armed benzaldehyde-polyethylene glycols described in Steps 5, 6 and 7 include one or more of 2, 4, 6 or 8-armed benzaldehyde-polyethylene glycols; the multi-armed alkyl aldehyde-polyethylene glycols include one or more of 2, 4, 6 or 8-armed alkyl aldehyde-polyethylene glycols.

[0025] An acylhydrazone bond hydrogel with adjustable viscoelasticity provided by the present invention is applied in in vitro three-dimensional cell culture. During the application process, this hydrogel has more cell adhesion sites and a cell microenvironment more suitable for the growth of various cells.

[0026] In the following examples, unless otherwise specified, all materials used can be obtained through ordinary channels; the test methods adopted are conventional methods in the art.

[0027] Example 1: Step 1: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then successively add 2 g of 4-arm polyethylene glycol (molecular weight 10 kDa), 0.56 g of 4-formylbenzoic acid (p-CBA), and 0.2 g of N,N'-diisopropylcarbodiimide (DIC), and react at room temperature for 20 hours; after the reaction is completed, precipitate the product in diethyl ether to obtain the product, and then dialyze the solution in deionized water using a dialysis bag, changing the water three times a day. After dialysis for 2 days, freeze-dry to obtain multi-arm benzaldehyde-modified polyethylene glycol; Step 2: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then successively add 2 g of 4-arm polyethylene glycol (molecular weight 10 kDa), 12.5 mg of TEMPO, and 0.77 g of diacetoxyiodobenzene (DAIB), and react at room temperature for 20 hours; after the reaction is completed, precipitate the product in diethyl ether to obtain the product, and then dialyze the solution in deionized water using a dialysis bag, changing the water no less than three times a day. After dialysis for 2 days, freeze-dry to obtain multi-arm alkyl aldehyde-modified polyethylene glycol; Step 3: Preparation of hydrazine-modified gelatin macromolecules. First, add 40 mL of PBS with a pH of 5.5 to a 100 mL round-bottom flask, then add 2 g of type A pigskin gelatin, and stir to dissolve it completely; then add 1 g of adipic dihydrazide and stir evenly; then dissolve 0.5 g of 1-hydroxybenzotriazole hydrate (HOBT) in DMSO and add it to the reaction flask and stir evenly; finally, add 2 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ((EDC·HCl)), and after the reaction is completed, adjust the pH to 5.3 with NaOH; stir the mixture at 50 °C for 24 h, then dialyze the solution in deionized water using a dialysis bag, changing the water once a day. After dialysis for one week, freeze-dry to obtain hydrazine-modified gelatin macromolecules.

[0028] Step 4: Dissolve the products of Steps 1, 2, and 3, multi-armed benzaldehyde-functionalized polyethylene glycol, multi-armed alkyl aldehyde-functionalized polyethylene glycol, and hydrazine-modified gelatin macromolecules separately in PBS, neutralize to pH 7.0, and the mass fraction is 40%; then take different volumes of the hydrazine-modified gelatin macromolecule solution, multi-armed alkyl aldehyde-functionalized polyethylene glycol, and multi-armed benzaldehyde-functionalized polyethylene glycol solution and mix them in this order. Adjust the stress relaxation by changing the crosslinking ratio of multi-armed benzaldehyde-functionalized polyethylene glycol to multi-armed alkyl aldehyde-functionalized polyethylene glycol. The ratio of multi-armed benzaldehyde-functionalized polyethylene glycol to multi-armed alkyl aldehyde-functionalized polyethylene glycol in the aldehyde-functionalized polyethylene glycol component is adjusted to 100:0, 90:10, 70:30, 50:50, 25:75, or 0:100. The final concentration of polyethylene glycol is 1%, and the ratio of hydrazine to aldehyde is 1:1. Let it stand to obtain hydrogels with different viscoelasticities.

[0029] As Figures 1-5 shown, inject the hydrogel solutions with different ratios of multi-armed alkyl aldehyde-functionalized polyethylene glycol into the mold. After gelation, take them out and measure the stiffness and relaxation time of the hydrogel. It can be seen that the gelation time and stiffness of the hydrogels with different ratios are basically the same. As the ratio of benzaldehyde increases, the change in the relaxation curve gradually decreases, and the relaxation time increases, confirming that this kind of hydrogel has the same stiffness but different viscoelasticities.

[0030] Example 2: Step 1: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then sequentially add 2 g of 8-arm polyethylene glycol (molecular weight 20 kDa), 0.56 g of 4-formylbenzoic acid (p-CBA), and 0.2 g of N,N'-diisopropylcarbodiimide (DIC), and react overnight at room temperature; after the reaction is completed, precipitate the product in diethyl ether to obtain the product, and then dialyze the solution in deionized water using a dialysis bag, changing the water three times a day. After dialysis for 2 days, freeze-dry to obtain multi-armed benzaldehyde-functionalized polyethylene glycol; Step 2: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then sequentially add 2 g of 8-arm polyethylene glycol (molecular weight 20 kDa), 12.5 mg of TEMPO, and 0.77 g of diacetoxyiodobenzene (DAIB), and react overnight at room temperature; after the reaction is completed, precipitate the product in diethyl ether to obtain the product, and then dialyze the solution in deionized water using a dialysis bag, changing the water three times a day. After dialysis for 2 days, freeze-dry to obtain multi-armed alkyl aldehyde-functionalized polyethylene glycol; Freeze-dry to obtain multi-armed alkyl aldehyde-functionalized polyethylene glycol; Step 3: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then successively add 2 g of 8-arm polyethylene glycol amine (molecular weight 10 - 20 kDa), 1.5 tris-Boc-hydroxyacetic acid, and 1 g of EDC-hydrochloride. The reaction is carried out overnight at room temperature. After the reaction is completed, the product is precipitated in diethyl ether to obtain the product, which is dried under vacuum. Then the obtained product is added to 50:50 DCM:TFA for 2 hours, and then the solution is dialyzed in deionized water using a dialysis bag, changing the water three times a day. After dialysis for 2 days, it is freeze-dried to obtain multi-arm hydrazide-based polyethylene glycol. Step 4: Dissolve the products of Steps 1, 2, and 3, multi-arm benzaldehyde-functionalized polyethylene glycol, multi-arm alkyl aldehyde-functionalized polyethylene glycol, and polyethylene glycol with hydrazide at the end, respectively, in PBS and neutralize to pH 7.0 with a mass fraction of 15%. Then take different volumes of the polyethylene glycol solution with hydrazide at the end, the multi-arm alkyl aldehyde-functionalized polyethylene glycol solution, and the multi-arm benzaldehyde-functionalized polyethylene glycol solution and mix them in this order. By changing the cross-linking ratio of multi-arm benzaldehyde-functionalized polyethylene glycol to multi-arm alkyl aldehyde-functionalized polyethylene glycol, the stress relaxation is adjusted. The ratios of multi-arm benzaldehyde-functionalized polyethylene glycol and multi-arm alkyl aldehyde-functionalized polyethylene glycol in the aldehyde-functionalized polyethylene glycol component are adjusted to 100:0, 70:30, or 0:100. The final polyethylene glycol concentration is 5%, and the ratio of hydrazide to aldehyde is 1:1. Let it stand to obtain hydrogels with different viscoelasticities.

[0031] Example 3: Step 1: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then successively add 2 g of 6-arm polyethylene glycol (molecular weight 20 kDa), 0.56 g of 4-formylbenzoic acid (p-CBA), and 0.2 g of N,N'-diisopropylcarbodiimide (DIC). The reaction is carried out at room temperature for 20 hours. After the reaction is completed, the product is precipitated in diethyl ether to obtain the product, and then the solution is dialyzed in deionized water using a dialysis bag, changing the water three times a day. After dialysis for 2 days, it is freeze-dried to obtain multi-arm benzaldehyde-functionalized polyethylene glycol. Step 2: First, add 50 mL of dichloromethane to a 250 mL round-bottom flask. Under the protection of nitrogen, then successively add 2 g of 6-arm polyethylene glycol (molecular weight 20 kDa), 12.5 mg of TEMPO, and 0.77 g of diacetoxyiodobenzene (DAIB). The reaction is carried out at room temperature for 20 hours. After the reaction is completed, the product is precipitated in diethyl ether to obtain the product, and then the solution is dialyzed in deionized water using a dialysis bag, changing the water three times a day. After dialysis for 2 days, it is freeze-dried to obtain multi-arm alkyl aldehyde-functionalized polyethylene glycol. Step 3: First, add 40 mL of PBS with a pH of 5.5 into a 100 mL round-bottom flask, then add 2 g of type A porcine skin gelatin and stir to dissolve it completely. Next, add 5 g of adipic dihydrazide and stir evenly. Subsequently, dissolve 1 g of 1-hydroxybenzotriazole hydrate (HOBT) in DMSO and add it to the reaction flask and stir evenly. Finally, add 0.5 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ((EDC·HCl)). After the reaction, adjust the pH to 5.3 with NaOH. Stir the mixture at 50 °C for 24 h, then dialyze the solution in deionized water using a dialysis bag, changing the water once a day. After dialysis for one week, freeze-dry to obtain hydrazide-modified gelatin macromolecules.

[0032] Step 4: Dissolve the products of Steps 1, 2, and 3, multi-armed benzaldehyde-polyethylene glycol, multi-armed alkyl aldehyde-polyethylene glycol, and hydrazide-modified gelatin macromolecules in PBS respectively, and neutralize to pH 7.0 with a mass fraction of 15%. Then take different volumes of hydrazide-modified gelatin macromolecule solution, multi-armed alkyl aldehyde-polyethylene glycol solution, multi-armed benzaldehyde-polyethylene glycol solution, 10 mg / mL fibronectin solution, and 1 U / mL thrombin, and mix them in this order. The multi-armed aldehyde-polyethylene glycol, hydrazide-modified gelatin macromolecules, and fibronectin and thrombin are mixed in a ratio of 1:1:1. Adjust the stress relaxation by changing the cross-linking ratio of multi-armed benzaldehyde-polyethylene glycol to multi-armed alkyl aldehyde-polyethylene glycol. The ratio of multi-armed benzaldehyde-polyethylene glycol to multi-armed alkyl aldehyde-polyethylene glycol in the aldehyde-polyethylene glycol component is adjusted to 100:0, 70:30, or 0:100. The final polyethylene glycol concentration is 10%, and the ratio of hydrazide to aldehyde is 1:1. Let it stand to obtain hydrogels with different viscoelasticities.

[0033] Inject the above 3 groups of hydrogel solutions into molds. After gelation, take them out and measure the stiffness and relaxation time of the hydrogels. It can be found that their stiffnesses are basically the same. As the proportion of benzaldehyde increases, the relaxation time increases, confirming that this kind of hydrogel has the same stiffness but different viscoelasticities.

[0034] In the ranges and any values disclosed in the present invention, the endpoints and any values of the ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. In the following text, in principle, the various technical solutions can be combined with each other to obtain new technical solutions, which should also be regarded as specifically disclosed herein.

[0035] 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 of ordinary skill in the art can still modify or equivalently replace the specific implementation manners of the present invention. Any such modifications or equivalent replacements that do not depart from the spirit and scope of the present invention are within the scope of the claims of the present invention pending approval.

Claims

1. An acylhydrazone bond hydrogel with adjustable viscoelasticity, characterized in that: The skeleton network of the acylhydrazone bond hydrogel is composed of multi-arm benzaldehyde-modified polyethylene glycol, multi-arm alkylaldehyde-modified polyethylene glycol and hydrazine-modified multi-arm polyethylene glycol or gelatin macromolecules cross-linked by acylhydrazone bonds.

2. A method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity, characterized in that: The preparation method is used to prepare the acylhydrazone bond hydrogel with adjustable viscoelasticity and rigidity as claimed in claim 1, and the preparation method comprises: Multi-arm benzaldehyde-polyethylene glycol, multi-arm alkylaldehyde-polyethylene glycol, multi-arm hydrazine-polyethylene glycol and hydrazine-modified gelatin macromolecule are added into phosphate buffer solution to form multi-arm benzaldehyde-polyethylene glycol solution, multi-arm alkylaldehyde-polyethylene glycol solution, multi-arm hydrazine-polyethylene glycol solution and hydrazine-modified gelatin macromolecule solution; A multi-arm hydrazine-polyethylene glycol solution or a hydrazine-modified gelatin macromolecular solution is taken, and a multi-arm benzaldehyde-polyethylene glycol solution and a multi-arm alkylaldehyde-polyethylene glycol solution are added thereto in sequence, and the mixture is dispersed evenly and allowed to stand to obtain an acylhydrazone bond hydrogel with adjustable viscoelasticity.

3. The method for preparing a viscoelastic acylhydrazone bond hydrogel according to claim 2, characterized in that: The preparation method further comprises: Take a multi-arm hydrazine-polyethylene glycol solution or a hydrazine-modified gelatin macromolecular solution, add a multi-arm benzaldehyde-polyethylene glycol solution and a multi-arm alkylaldehyde-polyethylene glycol solution thereto in sequence, then add a collagen solution, or a fibronectin solution and a thrombin solution, disperse them evenly, and let them stand to obtain an acylhydrazone bond hydrogel with adjustable viscoelasticity and stiffness.

4. The method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity according to claim 2, characterized in that: The multi-arm benzaldehyde polyethylene glycol includes one or more of 2-, 4-, 6- or 8-arm benzaldehyde polyethylene glycol; the multi-arm alkylaldehyde polyethylene glycol includes one or more of 2-, 4-, 6- or 8-arm alkylaldehyde polyethylene glycol; The mass fractions of the multi-arm benzaldehyde-polyethylene glycol solution, the multi-arm alkylaldehyde-polyethylene glycol solution, the multi-arm hydrazine-polyethylene glycol solution and the hydrazine-modified gelatin macromolecular solution are all 1% to 40%.

5. The method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity according to claim 2, characterized in that: The preparation of the multi-arm benzaldehyde polyethylene glycol comprises: In a nitrogen atmosphere, multi-arm polyethylene glycol, 4-formylbenzoic acid and N,N'-diisopropylcarbodiimide are sequentially added to dichloromethane, and the mixture is reacted at room temperature for 12 to 24 hours to obtain a first product, and the first product is precipitated in ether to obtain a first solution, and the first solution is dialyzed with deionized water, and after the dialysis is completed, the solution is freeze-dried to obtain a multi-arm benzaldehyde polyethylene glycol; The molar ratio of the multi-arm polyethylene glycol, 4-formylbenzoic acid and N,N'-diisopropylcarbodiimide is 1:1:1-1:50:

50.

6. The method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity according to claim 2, characterized in that: The preparation of the multi-arm alkyl hydroformylation polyethylene glycol comprises: In a nitrogen atmosphere, multi-arm polyethylene glycol, 2,2,6,6-tetramethylpiperidinyl oxide and diacetoxy iodobenzene are sequentially added to dichloromethane, and the mixture is reacted at room temperature for 12 to 24 hours to obtain a second product, and the second product is precipitated in ether to obtain a second solution, and the second solution is dialyzed in deionized water, and after the dialysis is completed, the solution is freeze-dried to obtain a multi-arm alkyl aldehyde polyethylene glycol; The molar ratio of the multi-arm polyethylene glycol, 2,2,6,6-tetramethylpiperidinyl oxide and diacetoxy iodobenzene is 10:1:1-1:10:

100.

7. The method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity according to claim 2, characterized in that: The preparation of the multi-arm hydrazine polyethylene glycol comprises: Under a nitrogen atmosphere, multi-arm polyethylene glycol amine, tri-Boc-tibial acetic acid and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are sequentially added to dichloromethane, and the mixture is reacted at room temperature for 12 to 24 hours to obtain a third product, and the third product is precipitated in diethyl ether to obtain a fourth product, and the fourth product is vacuum dried to obtain a fifth product, and the fifth product is added to 50:50 DCM:TFA for 2 to 3 hours to obtain a third solution, and the third solution is dialyzed in deionized water, and freeze-dried after the dialysis is completed to obtain a multi-arm hydrazine polyethylene glycol; The molar ratio of the multi-arm polyethylene glycol amine, tri-Boc-tibial acetic acid and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 10:1:1-1:10:

10.

8. The method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity according to any one of claims 5 to 7, characterized in that: The multi-arm polyethylene glycol includes polyethylene glycol with 2, 4, 6 or 8 arms, and the molecular weight of the multi-arm polyethylene glycol is 10-20 kDa; the multi-arm polyethylene glycol amine includes polyethylene glycol amine with 2, 4, 6 or 8 arms, and the molecular weight of the multi-arm polyethylene glycol amine is 10-20 kDa.

9. The method for preparing an acylhydrazone bond hydrogel with adjustable viscoelasticity according to claim 2, characterized in that: The preparation of the hydrazine-modified gelatin macromolecule comprises: Under a nitrogen atmosphere, type A pig skin gelatin is added to a phosphate buffer solution and dispersed uniformly to obtain a fourth solution, adipic acid dihydrazide, a DMSO solution of 1-hydroxybenzotriazole hydrate, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are sequentially added to the fourth solution, and the mixture is reacted at room temperature for 12 to 24 hours to obtain a sixth product, and the pH of the sixth product is adjusted to 5.0 to 5.3 to obtain a seventh product, and the seventh product is stirred at 50° C. for 24 hours to obtain a fifth solution, and the fifth solution is dialyzed in deionized water, and freeze-dried after the dialysis is completed to obtain a hydrazine-modified gelatin macromolecule; The molar ratio of adipic acid dihydrazide, 1-hydroxybenzotriazole hydrate and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 10:1:1-1:10:

10.

10. Use of the acylhydrazone bond hydrogel with adjustable viscoelasticity according to claim 1 in three-dimensional cell culture in vitro.

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