Crosslinked copolymers, hydrogels, methods of making and using the same, and products

Abstract

The present application relates to the technical field of high molecular hydrogel, and provides an antioxidant self-repairing hydrogel capable of promoting wound healing, which comprises a copolymer crosslinked by a borate ester linking group and water, and a method for preparing the hydrogel, which comprises the following steps: 1) performing first dissolution on a copolymer Q1 to obtain an aqueous solution A; 2) performing second dissolution on a copolymer Q2 to obtain an aqueous solution B; and 3) mixing the aqueous solution A and the aqueous solution B, and performing reaction at room temperature to obtain the hydrogel. The hydrogel of the present application has low cytotoxicity, good self-repairing ability and antioxidant capacity, and can have long-acting antioxidant function when applied to wound healing.

Description

Technical Field

[0001] This invention relates to the field of polymer hydrogels, specifically to a crosslinked copolymer, a hydrogel, its preparation method and application, and products. Background Technology

[0002] Wound healing can be divided into four consecutive stages: hemostasis, inflammation, cell proliferation, and tissue remodeling. Inflammation is the second stage of wound healing; appropriate inflammation can promote wound repair. However, excessive inflammation releases large amounts of reactive oxygen species, leading to high levels of oxidative stress, which causes oxidative damage to cells and biomolecules (such as DNA and proteins), hindering wound repair. Self-healing hydrogels are a novel type of smart material with excellent hydrophilicity, biocompatibility, injectability, and self-healing properties, showing significant advantages in wound dressings. Therefore, developing self-healing hydrogels with antioxidant properties is essential for the development of novel wound dressings.

[0003] Introducing exogenous antioxidants into hydrogel systems is an effective method for preparing antioxidant hydrogels. However, this approach suffers from problems such as antioxidant burst release, uneven distribution, and poor solubility, limiting the long-term antioxidant function of the hydrogel. Therefore, functional modification of the gelling components to develop self-healing hydrogels with intrinsic antioxidant properties is of great significance in the development of dressings that can promote wound healing. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of uneven distribution of antioxidants, poor solubility, and unstable antioxidant properties in existing hydrogels, and to provide a crosslinked copolymer, hydrogel, preparation method, application, and product thereof.

[0005] To achieve the above objectives, a first aspect of the present invention provides a crosslinked polymer, wherein the crosslinked copolymer comprises a macromolecular chain P1, a macromolecular chain P2, and a borate ester linking group connecting the macromolecular chains P1 and P2; wherein the macromolecular chain P1 contains structural unit A of formula (1), structural unit B of formula (2), and structural unit C of formula (3).

[0006]

[0007] Wherein, R1 is methyl, ethyl, isopropyl, methoxy, hydroxyl or hydrogen atom, R2 and R3 are each independently selected from methoxy, hydroxyl or hydrogen atom, and R4 and R5 are each independently selected from hydrogen atom, dimethyl, C1-C3 alkyl or phenyl.

[0008] The macromolecular chain P2 contains structural unit D as shown in formula (4) and structural unit E as shown in formula (5).

[0009]

[0010] Wherein, R6 is a methyl group and z is a positive integer from 18 to 90, or R6 is a hydrogen atom and z is a positive integer from 43 to 113; * indicates the position where the macromolecular chains P1 and P2 are connected to the borate ester linking group, wherein the * in structural unit D is directly connected to the boron element in the borate ester.

[0011] A second aspect of the present invention provides a hydrogel comprising a copolymer and water filling the copolymer, wherein the copolymer is the aforementioned crosslinked copolymer.

[0012] A third aspect of this invention provides a method for preparing a hydrogel, the method comprising:

[0013] 1) The copolymer Q1 is first dissolved to obtain an aqueous solution A;

[0014] 2) The copolymer Q2 is dissolved a second time to obtain aqueous solution B;

[0015] 3) Mix the aqueous solution A and aqueous solution B, and react at room temperature to obtain the hydrogel;

[0016] The copolymer Q1 contains structural unit A as shown in formula (1), structural unit B as shown in formula (2), and structural unit F as shown in formula (6).

[0017]

[0018] Wherein, R1 is methyl, ethyl, isopropyl, methoxy, hydroxyl or hydrogen atom, R2 and R3 are each independently selected from methoxy, hydroxyl or hydrogen atom, and R4 and R5 are each independently selected from hydrogen atom, dimethyl, C1-C3 alkyl or phenyl.

[0019] The copolymer Q2 contains structural unit H as shown in formula (7) and structural unit E as shown in formula (5).

[0020]

[0021] Where R6 is a methyl group and z is a positive integer from 18 to 90, or R6 is a hydrogen atom and z is a positive integer from 43 to 113.

[0022] A fourth aspect of the present invention provides a hydrogel prepared by the method of the present invention.

[0023] The fifth aspect of the present invention provides the application of the above-mentioned crosslinked copolymer or hydrogel.

[0024] The application is in at least one of the following aspects:

[0025] 1) In the preparation of cell / drug carriers;

[0026] 2) In the preparation of products that promote wound healing;

[0027] 3) In terms of dressing preparation.

[0028] A sixth aspect of the present invention provides a product comprising the crosslinked copolymer or hydrogel of the present invention.

[0029] The product is at least one of the following:

[0030] 1) Cell / drug carrier;

[0031] 2) Products that promote wound healing;

[0032] 3) Dressings.

[0033] Through the above technical solution, this invention incorporates antioxidant dihydropyridine structures into the structures of copolymers Q1 and Q2 via the Hantzsch reaction, and adds boric acid groups to copolymer Q2. In an aqueous solution, both copolymers undergo a partial crosslinking reaction through the hydroxyl groups in the main chain of copolymer Q1 and the boric acid groups on the side groups of the main chain of copolymer Q2 to generate borate ester linkage groups, resulting in a hydrogel with a partially crosslinked structure. Alternating application of different strains (1% and 400%) to the hydrogel causes its morphology to change between near-solid and near-liquid states, indicating that the borate ester bonds in the crosslinked copolymer backbone are dynamic, giving the hydrogel excellent self-healing capabilities. Immersing the hydrogel in a solution containing ABTS... +· In PBS buffer containing (2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonate diammonium salt free radical), the solution color rapidly lightens; when the absorbance of the above solution at 734 nm was detected by microplate reader at different time points, it was found that the concentration of free radicals decreased rapidly, indicating that the hydrogel has good antioxidant capacity.

[0034] In addition to its self-healing and antioxidant properties, the hydrogel provided by this invention possesses a certain degree of softness, allowing it to adapt to and conform to wounds, thus promoting wound healing. Experiments have shown that wounds on the backs of mice treated with the hydrogel of this invention almost completely closed by day 10, while the same wounds not treated with the hydrogel of this invention still showed obvious signs of healing by day 10.

[0035] The hydrogel provided by this invention has a simple preparation method, mild gelation conditions, and safe and readily available raw materials, and has good application value. Attached Figure Description

[0036] Figure 1 It is copolymer Q1-1 in Example 1 1 H-NMR spectrum;

[0037] Figure 2 It is copolymer Q1-2 in Example 2 1 H-NMR spectrum;

[0038] Figure 3 It is copolymer Q2 in Example 3 1 H-NMR spectrum;

[0039] Figure 4 It is the hydrogel material Gel-8 / 3 prepared in Example 4;

[0040] Figure 5 The rheological properties of the hydrogel materials prepared using different contents of copolymer Q2 in Examples 4, 6 and 7 are the test results; Gel-4 / 3, Gel-8 / 3 and Gel-12 / 3 represent hydrogel materials prepared by mixing equal volumes of aqueous solution A with a mass concentration of 3% copolymer Q1 with aqueous solution B with a mass concentration of 4%, 8% and 12% copolymer Q2, respectively.

[0041] Figure 6 a is the process by which the Gel-8 / 3 hydrogel material of Example 4 is broken and then reformed into a complete pentagram-shaped hydrogel.

[0042] Figure 6 b is the rheological property test result of the Gel-8 / 3 hydrogel material in Example 4; the test conditions were alternating between 1% strain and 400% strain, with the frequency fixed at 1.0 Hz;

[0043] Figure 7 It is ABTS with the addition of different copolymers. +· The absorbance test results of the solution, where PVA is polyvinyl alcohol;

[0044] Figure 8 a is ABTS soaked in different hydrogel materials. +· Color change graph of the solution;

[0045] Figure 8 b is ABTS soaked in different hydrogel materials. +· The absorbance test results of the solution at 734 nm;

[0046] Figure 9 This is a characterization diagram of the survival rate of mouse fibroblasts (L929) loaded with the Gel-8 / 3 hydrogel material in Example 4;

[0047] Figure 10 These are photographs showing the healing of mouse wounds 1-10 days with and without the Gel-8 / 3 hydrogel material from Example 4. Detailed Implementation

[0048] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0049] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0050] A first aspect of the present invention provides a crosslinked copolymer, wherein the crosslinked copolymer comprises a macromolecular chain P1, a macromolecular chain P2, and a borate ester linking group connecting the macromolecular chains P1 and P2; wherein the macromolecular chain P1 contains structural unit A of formula (1), structural unit B of formula (2), and structural unit C of formula (3).

[0051]

[0052] Wherein, R1 is methyl, ethyl, isopropyl, methoxy, hydroxyl or hydrogen atom, R2 and R3 are each independently selected from methoxy, hydroxyl or hydrogen atom, and R4 and R5 are each independently selected from hydrogen atom, dimethyl, C1-C3 alkyl or phenyl.

[0053] The macromolecular chain P2 contains structural unit D as shown in formula (4) and structural unit E as shown in formula (5).

[0054]

[0055] Wherein, R6 is a methyl group and z is a positive integer from 18 to 90, or R6 is a hydrogen atom and z is a positive integer from 43 to 113; * indicates the position where the macromolecular chains P1 and P2 are connected to the borate ester linking group, wherein the * in structural unit D is directly connected to the boron element in the borate ester.

[0056] Both macromolecular chains P1 and P2 contain a dihydropyridine ring structure with antioxidant properties. The borate ester linking group is the crosslinking point in the crosslinked copolymer. Because the borate ester bond in the borate ester linking group is a dynamic chemical bond, and the dihydropyridine ring structure has antioxidant function, the crosslinked copolymer has self-healing and antioxidant capabilities in aqueous solution.

[0057] In this invention, the crosslinked copolymer can be obtained by preparing a hydrogel containing the crosslinked copolymer and then freeze-drying it. The freeze-drying temperature is -60°C to -20°C; the vacuum degree is 0.1-35 Pa; and the time is 12-72 h, preferably 24 h. The method for preparing the hydrogel can be the method for preparing the hydrogel provided in the third aspect of this invention.

[0058] According to the present invention, preferably, in formula (5), R6 is methyl and z is a positive integer of 18-20, 31-33, or 88-90, or R6 is a hydrogen atom and z is a positive integer of 43-45 or 111-113. In order to obtain higher antioxidant capacity and more effectively promote wound healing, the crosslinked copolymer preferably has R1 as a hydroxyl group or a hydrogen atom, R2 as a methoxy group or a hydrogen atom, R3 as a hydrogen atom, R4 as a methyl group, R5 as a methyl group, and R6 as a methyl group, and z is a positive integer of 18-20.

[0059] According to the crosslinked copolymer of the present invention, preferably, in the macromolecular chain P1, the molar ratio of structural unit A: structural unit B: structural unit C is 1-10:1-12:78-98; and in the macromolecular chain P2, the molar ratio of structural unit D: structural unit E is 1:1-5.

[0060] According to the present invention, in order to obtain suitable adhesive strength in aqueous solution and to better apply the crosslinked copolymer to the biomedical field, preferably, in the macromolecular chain P2, the molar ratio of structural unit D to structural unit E is 1:2; and the molar ratio of macromolecular chain P1 to macromolecular chain P2 is 1-50:1.

[0061] In the crosslinked copolymer provided by this invention, the borate ester linking groups forming the crosslinking points can be formed by a crosslinking reaction between some hydroxyl groups from the main chain of copolymer Q1 and some borate groups from the side chains of copolymer Q2. The borate ester linking groups give the crosslinked copolymer a crosslinked structure. Simultaneously, unreacted hydroxyl groups from the main chain of copolymer Q1 and unreacted borate groups from the side chains of copolymer Q2 are also present in the crosslinked copolymer. Preferably, the degree of crosslinking of the crosslinked copolymer is 0.17-0.25. The degree of crosslinking can be determined by infrared spectroscopy, and can be defined as the proportion of the amount of borate ester groups in the crosslinked copolymer to the total amount of borate ester groups and unreacted borate groups in the crosslinked polymer. Furthermore, the degree of crosslinking of the crosslinked copolymer in the hydrogel material of this invention can also be measured using the same method.

[0062] In this invention, the crosslinked copolymer can be in the form of a hydrogel, which utilizes the dihydropyridine ring in its own backbone to react with the excess reactive oxygen species released by inflammation, thereby preventing excessive inflammation from causing oxidative damage to cells and biological macromolecules (such as DNA and proteins) and promoting wound healing.

[0063] In this invention, the composition and structure of the crosslinked copolymer can be determined by nuclear magnetic resonance hydrogen spectroscopy, Fourier transform infrared spectroscopy, or by the amount of each material fed during the preparation process.

[0064] A second aspect of the present invention provides a hydrogel comprising a copolymer and water filling the copolymer, wherein the copolymer is the aforementioned crosslinked copolymer.

[0065] In some embodiments of the present invention, preferably, based on the total amount of the hydrogel, the copolymer in the hydrogel has a mass percentage of 3-11%, and the water has a mass percentage of 89-97%.

[0066] In some embodiments of the present invention, preferably, under conditions of room temperature, frequency 1.0 Hz, and strain 1%, the storage modulus (G') of the hydrogel is 50-1500 Pa, and the loss modulus (G″) is 1-150 Pa. The hydrogel material has a good ability to store elastic deformation energy, possesses a certain degree of softness (glue strength), and can adaptively conform to wounds, which is beneficial for promoting wound healing. Experiments have shown that wounds on the backs of mice treated with the hydrogel of the present invention almost completely closed on day 10, while the same wounds not treated with the hydrogel of the present invention still showed obvious signs of healing on day 10.

[0067] A third aspect of this invention provides a method for preparing a hydrogel, the method comprising:

[0068] 1) The copolymer Q1 is first dissolved to obtain an aqueous solution A;

[0069] 2) The copolymer Q2 is dissolved a second time to obtain aqueous solution B;

[0070] 3) Mix the aqueous solution A and aqueous solution B, and react at room temperature to obtain the hydrogel;

[0071] The copolymer Q1 contains structural unit A as shown in formula (1), structural unit B as shown in formula (2), and structural unit F as shown in formula (6);

[0072]

[0073] Wherein, R1 is methyl, ethyl, isopropyl, methoxy, hydroxyl or hydrogen atom, R2 and R3 are each independently selected from methoxy, hydroxyl or hydrogen atom, and R4 and R5 are each independently selected from hydrogen atom, dimethyl, C1-C3 alkyl or phenyl.

[0074] The copolymer Q2 contains structural unit H as shown in formula (7) and structural unit E as shown in formula (5).

[0075]

[0076] Where R6 is a methyl group and z is a positive integer from 18 to 90, or R6 is a hydrogen atom and z is a positive integer from 43 to 113.

[0077] According to the preparation method of the present invention, in order to obtain a hydrogel with higher antioxidant capacity and more effectively promote wound healing, preferably, in copolymer Q1, the molar ratio of structural unit A: structural unit B: structural unit F is 1-10:1-12:78-98; and in copolymer Q2, the molar ratio of structural unit H: structural unit E is 1:1-5.

[0078] According to the preparation method of the present invention, in order to obtain a hydrogel with suitable adhesive strength and self-healing ability, and to better apply it in the biomedical field, preferably, in step 1), the mass fraction of aqueous solution A is 2-10%; in step 2), the mass fraction of aqueous solution B is 4-12%. In step 3), the mixing volume ratio of aqueous solution A and aqueous solution B is 0.5-1.5:1. In the present invention, the mass fraction of aqueous solution A refers to the concentration of copolymer Q1 in aqueous solution A, and the mass fraction of aqueous solution B refers to the concentration of copolymer Q2 in aqueous solution B.

[0079] According to a more preferred embodiment of the present invention, in step 1), the mass fraction of the aqueous solution A is 3%; in step 2), the mass fraction of the aqueous solution B is 4%, 8%, and 12%. In step 3), the mixing volume ratio of the aqueous solution A and the aqueous solution B is 1:1.

[0080] In this invention, the copolymer Q1 can be obtained by modifying polyvinyl alcohol and can be prepared according to the following method:

[0081] (1) Dissolve polyvinyl alcohol in an anhydrous organic solvent, add diketene, and carry out the first step reaction to obtain a polyvinyl alcohol derivative containing β-diketone.

[0082] (2) Under the presence of a catalyst, benzene ring-containing aldehydes, ammonia source compounds, 1,3-diketone compounds and the β-diketone-containing polyvinyl alcohol derivatives are subjected to the Hantzsch reaction.

[0083] (3) Pour the product obtained in step (2) into an organic solvent and collect the precipitate, which is the copolymer Q1.

[0084] Preferably, in step (1), the degree of polymerization of polyvinyl alcohol is 500-1700 and the degree of hydrolysis is 88-99%. The polyvinyl alcohol is commercially available, for example, the polyvinyl alcohol is selected from at least one of PVA1788, PVA1799, and PVA0588.

[0085] Preferably, in step (1), the anhydrous organic solvent is selected from at least one of anhydrous N-methylpyrrolidone (NMP), anhydrous dimethyl sulfoxide (DMSO), and anhydrous N,N-dimethylformamide (DMF).

[0086] Preferably, in step (1), the molar ratio of hydroxyl groups to diketene in polyvinyl alcohol is 1:0.1-0.15; the reaction temperature of the first step is 25-35℃, and the reaction time of the first step is 1-2 hours.

[0087] Preferably, in step (2), the catalyst is selected from amino acids or other weak acids, more preferably glycine, proline or phenylboronic acid.

[0088] Preferably, the benzene ring-containing aldehyde compound is selected from vanillin or benzaldehyde.

[0089] Preferably, the ammonia source compound is selected from at least one of ammonium acetate, ammonium carbonate, and ammonia water.

[0090] Preferably, the 1,3-dione compound is selected from 1,3-cyclohexanedione and its derivatives, more preferably 5,5-dimethyl-1,3-cyclohexanedione.

[0091] Preferably, in step (2), the molar ratio of the benzene-containing aldehyde compound, the ammonia source compound, and the 1,3-diketone compound is 1:1-2:1, more preferably 1:1.2-1.6:1; the molar ratio of the benzene-containing aldehyde compound to diketene is 1-2:1, more preferably 1.1-1.5:1; the molar ratio of the catalyst to the benzene-containing aldehyde compound is 0.05-0.2:1, more preferably 0.1-0.15:1; the Hantzsch reaction temperature is 70-80℃, and the Hantzsch reaction time is 2-4 hours.

[0092] Preferably, the organic solvent in step (3) is at least one of methanol, ethanol, acetone and acetonitrile.

[0093] Preferably, the number-average molecular weight of the copolymer Q1 is 20,000-80,000 g / mol.

[0094] According to a more preferred embodiment of the present invention, the number average molecular weight of the copolymer Q1 is 20,000-31,000 g / mol.

[0095] According to a more preferred embodiment of the present invention, the synthesis route of Q1 can be one of the following two methods, respectively obtaining copolymer Q1-1 of Example 1 and copolymer Q1-2 of Example 2:

[0096]

[0097] The chemical formulas in the above synthetic route diagrams are schematic representations of the corresponding compounds, and there are no special limitations on m and n (the same applies below).

[0098] In this invention, the copolymer Q2 can be prepared according to the following method:

[0099] Under anaerobic conditions, commercially available 4-(3-((2-(methacryloyloxy)ethoxy)carbonyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-4-yl)phenyl)boronic acid (M2) was subjected to free radical polymerization with polyethylene glycol monomethyl ether methacrylate (PEGMA) to obtain the polymer Q2.

[0100] Preferably, the free radical polymerization reaction is carried out in the presence of an initiator.

[0101] Preferably, the initiator may be selected from at least one of azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (ABVN).

[0102] In order to make the final hydrogel have suitable adhesive strength and self-healing ability, and better apply it to the biomedical field, the preferred molar ratio of monomer M2 to PEGMA is 1:1-5, preferably 1:2.

[0103] Preferably, the number-average molecular weight of the copolymer Q2 is 10,000-600,000 g / mol;

[0104] According to a more preferred embodiment of the present invention, the copolymer Q2 has a number-average molecular weight of 533,000 g / mol.

[0105] Preferably, the conditions for the free radical polymerization include: a reaction temperature of 60-80°C, a reaction time of 6-20 hours, an anaerobic environment, and an oil bath.

[0106] Preferably, a purification step is performed after the free radical polymerization reaction.

[0107] More preferably, the purification involves precipitating the solution after the free radical polymerization reaction in diethyl ether.

[0108] In this invention, the polyethylene glycol monomethyl ether methacrylate (PEGMA) is commercially available, for example from Sigma-Aldrich, with a number average molecular weight of 950 g / mol, and contains 100 ppm MEHQ and 200 ppm BHT as polymerization inhibitors.

[0109] According to a more preferred embodiment of the present invention, the synthetic route of Q2 is as follows:

[0110]

[0111] The chemical formulas in the above synthetic route diagrams are schematic representations of the corresponding compounds, and the z in them is not specifically limited (the same applies below).

[0112] A fourth aspect of the present invention provides a hydrogel prepared by the method of the present invention.

[0113] The fifth aspect of the present invention provides the application of the above-mentioned crosslinked copolymer or hydrogel.

[0114] The applications of the crosslinked copolymers or hydrogels provided by this invention include at least one of the following:

[0115] 1) In the preparation of cell / drug carriers;

[0116] 2) In the preparation of products that promote wound healing;

[0117] 3) In terms of dressing preparation.

[0118] The hydrogel described in this invention was applied to the wound on the back of mice, and a Tegaderm transparent dressing was applied to prevent wound contraction; the control group had the Tegaderm transparent dressing applied directly to their wounds. Preferably, the dressing was changed every two days, and the changes in the wound were recorded by photograph. The wounds of the experimental group mice treated with the hydrogel described in this invention were almost completely closed by day 10, while the untreated control group still showed obvious wounds on day 10.

[0119] A sixth aspect of the present invention provides a product comprising the crosslinked copolymer or hydrogel of the present invention.

[0120] Preferably, the product is at least one of the following:

[0121] 1) Cell / drug carrier;

[0122] 2) Products that promote wound healing;

[0123] 3) Dressings.

[0124] The following examples and comparative examples are used to illustrate the technical solutions of the present invention. All raw materials and reagents used are commercially available, and room temperature refers to 15-35°C.

[0125] Polyvinyl alcohol:

[0126] Polyvinyl alcohol 0588, degree of alcoholysis 88%;

[0127] Polyvinyl alcohol 1788, degree of alcoholysis 88%;

[0128] Polyvinyl alcohol 1799, degree of alcoholysis 99%;

[0129] All were purchased from Anhui Wanwei Group Co., Ltd.

[0130] 4-(3-((2-(methacryloyloxy)ethoxy)carbonyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-4-yl)phenyl)boronic acid: purity: 99%; purchased from Bailingwei.

[0131] Polyethylene glycol monomethyl ether methacrylate (PEGMA)

[0132] It has a number average molecular weight of 950 g / mol and contains 100 ppm MEHQ and 200 ppm BHT as polymerization inhibitors.

[0133] Purchased from Sigma-Aldrich.

[0134] The degree of crosslinking was determined by infrared spectroscopy.

[0135] Number-average molecular weight was determined by gel permeation chromatography.

[0136] In the following examples and comparative examples, the composition of the structural units in the obtained polymers was determined by nuclear magnetic resonance hydrogen spectroscopy and X-ray photoelectron spectroscopy.

[0137] Example 1: Preparation of copolymer Q1-1

[0138] The synthetic route for copolymer Q1-1 is as follows:

[0139]

[0140] Polyvinyl alcohol (0588) (1.1 g, hydroxyl molar amount of about 22 mmol) was dissolved in anhydrous N-methylpyrrolidone (10 mL) to form a clear and transparent solution. Diketene (184.9 mg, 2.2 mmol) was added, and the first step reaction was carried out at 25 °C for 1 hour to obtain a polyvinyl alcohol derivative containing β-diketone.

[0141] Vanillin (368.2 mg, 2.42 mmol), 5,5-dimethyl-1,3-cyclohexanedione (339.2 mg, 2.42 mmol), ammonium acetate (279.8 mg, 3.63 mmol), and glycine (18.2 mg, 0.24 mmol) were added to a polyvinyl alcohol derivative containing β-diketone. The reaction mixture was heated to 80 °C and subjected to a Hantzsch reaction for 3.5 hours. After the reaction was completed, the product was precipitated in acetone solution to obtain 1.6 g of copolymer Q1-1.

[0142] The obtained copolymer Q1-1 was subjected to 1H NMR spectroscopy, such as... Figure 1 As shown 1 The H-NMR spectrum shows that a characteristic peak of the benzene ring appears at a chemical shift of 6.42-6.67 ppm, and a nitrogen and hydrogen characteristic peak of the dihydropyridine ring appears at a chemical shift of around 8.92 ppm, indicating that the obtained copolymer Q1-1 is a copolymer containing a 1,4-dihydropyridine structure.

[0143] The number-average molecular weight of copolymer Q1-1 is 31000 g / mol;

[0144] In copolymer Q1-1, the molar ratio of structural unit A: structural unit B: structural unit F is 7:12:81.

[0145] Example 2: Preparation of copolymer Q1-2

[0146] The synthetic route for copolymer Q1-2 is as follows:

[0147]

[0148] The method of Example 1 was followed, except that vanillin was replaced with benzaldehyde. Copolymer Q1-2 was obtained.

[0149] The obtained copolymer Q1-2 was subjected to 1H NMR spectroscopy, such as... Figure 2 As shown 1 The H-NMR spectrum shows that a characteristic peak of the benzene ring appears at a chemical shift of 6.95-7.16 ppm, and a nitrogen and hydrogen characteristic peak of the dihydropyridine ring appears at a chemical shift of around 8.97 ppm, indicating that the obtained copolymer Q1-2 is a copolymer containing a 1,4-dihydropyridine structure.

[0150] The number-average molecular weight of copolymer Q1-2 is 27000 g / mol;

[0151] In copolymer Q1-1, the molar ratio of structural unit A: structural unit B: structural unit F is 7:12:81.

[0152] Example 3: Preparation of copolymer Q2

[0153]

[0154] 4-(3-((2-(methacryloyloxy)ethoxy)carbonyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-4-yl)phenyl)boronic acid (M2) (2.99 g, 6.4 mmol), PEGMA (12.16 g, 12.8 mmol), and ABVN (47.69 mg, 0.192 mmol) were dissolved in 15 mL of DMF. After purging the system with nitrogen, the reaction was carried out in an oil bath at 65 °C for 12 hours. The polymerization reaction was quenched in an ice-water bath. The reaction solution was precipitated in diethyl ether to obtain copolymer Q2.

[0155] The copolymer Q2 was subjected to 1H NMR spectroscopy, such as... Figure 3 As shown 1 The 1H-NMR spectrum shows a characteristic peak of the terminal methyl hydrogen of PEGMA at a chemical shift of approximately 3.28 ppm, a characteristic peak of the methylene hydrogen on the dihydropyridine ring at a chemical shift of 4.79 ppm, a characteristic peak of the benzene ring at chemical shifts of 6.90-7.87 ppm, and a characteristic peak of the nitrogen and hydrogen atoms of the dihydropyridine ring at a chemical shift of approximately 8.97 ppm. This indicates that the obtained copolymer Q2 is a copolymer containing a 1,4-dihydropyridine structure.

[0156] The number-average molecular weight of copolymer Q2 is 533,000 g / mol.

[0157] In copolymer Q2, the molar ratio of structural unit H to structural unit E is 1:2.

[0158] Example 4: Preparation of hydrogel

[0159] 1) Preparation of aqueous solution A

[0160] Copolymer Q1 was dissolved in phosphate buffer (PBS, pH 7.4), heated to 60°C and stirred to obtain aqueous solution A with a mass concentration of 3%.

[0161] 2) Preparation of aqueous solution B

[0162] Copolymer Q2 was dissolved in PBS solution and stirred until completely dissolved to obtain aqueous solution B with a mass concentration of 8%.

[0163] 3) Preparation of hydrogels

[0164] Equal volumes of aqueous solutions A and B were thoroughly mixed and allowed to stand at room temperature for approximately 30 seconds to obtain a hydrogel, named Gel-8 / 3. Figure 4 As shown.

[0165] In Gel-8 / 3, the copolymer accounts for 6% by mass and water accounts for 94% by mass.

[0166] In Gel-8 / 3, the degree of crosslinking of the copolymer is 0.18.

[0167] Example 5

[0168] Following the method of Example 4, copolymer Q1-1 in aqueous solution A was replaced with pure PVA (0588) to prepare a hydrogel, named Gel-PVA-8 / 3.

[0169] Example 6

[0170] Following the method of Example 4, the mass concentration of copolymer Q2 in aqueous solution B was adjusted to 4% to prepare a hydrogel, which was named Gel-4 / 3.

[0171] In Gel-4 / 3, the copolymer accounts for 4% by mass and water accounts for 96% by mass.

[0172] In Gel-4 / 3, the degree of crosslinking of the copolymer is 0.17.

[0173] Example 7

[0174] Following the method of Example 4, the mass concentration of copolymer Q2 in aqueous solution B was adjusted to 12% to prepare a hydrogel, which was named Gel-12 / 3.

[0175] In Gel-12 / 3, the copolymer has a mass percentage of 7.5%, and the water has a mass percentage of 92.5%.

[0176] In Gel-12 / 3, the degree of crosslinking of the copolymer is 0.24.

[0177] Example 8: Preparation of cross-linked polymers

[0178] The hydrogel Gel-8 / 3 obtained in Example 4 was freeze-dried;

[0179] The freeze-drying temperature was -56℃; the vacuum degree was 34Pa; and the time was 24h.

[0180] The degree of crosslinking of the crosslinked polymer is 0.18.

[0181] Example 6: Hydrogel Rheological Properties Testing

[0182] Hydrogels Gel-4 / 3, Gel-8 / 3, and Gel-12 / 3 were respectively prepared into disc-shaped pieces (approximately 1 mL, 20 mm in diameter) and placed on the rheometer test stage. The storage modulus (G') and loss modulus (G″) were recorded under frequency scanning (strain = 1%). The results are as follows: Figure 5As shown, the strength of the hydrogel increases with the increase of the Q2 content of the copolymer, indicating that the strength of the hydrogel is adjustable.

[0183] Example 7: Hydrogel self-healing performance test

[0184] 1) Qualitative characterization: Gel-8 / 3 was placed in a syringe and extruded into a pentagonal mold through a 22G needle. After standing at room temperature for 1 hour, the broken hydrogel formed a complete pentagonal hydrogel that could be easily removed. The results are as follows: Figure 6 As shown in a.

[0185] 2) Quantitative characterization: Using a rheometer, different strains (1%, 400%) were alternately applied to Gel-8 / 3 at a fixed frequency (1.0 Hz), and the values ​​of G' and G″ were measured. The results are as follows: Figure 6 As shown in b, it can be seen that at 1% strain, G' > G″, indicating that the hydrogel maintains its three-dimensional network structure. At 400% strain, G' < G″, indicating that most of the network structure of the hydrogel has been destroyed, exhibiting shear thinning characteristics. When the strain returns to 1%, G' > G″, indicating that the network structure of the hydrogel recovers rapidly. This process is repeatable, demonstrating that the hydrogel prepared by this invention has good self-healing properties.

[0186] Example 8: Antioxidant Capacity Test

[0187] 1) Antioxidant capacity test of copolymer

[0188] ABTS +· The solution was diluted with PBS buffer to an absorbance of approximately 0.7. 8 mM solutions of copolymer Q1-1 and copolymer Q2 were prepared separately with PBS buffer. The copolymer solutions were then mixed with ABTS. +· The solutions were mixed at a volume ratio of 1:19, and the absorbance change of the mixed solution at 734 nm was detected using a microplate reader. PVA and Q1-2 solutions were obtained using the same method, and their absorbance changes at 734 nm were detected as control groups. The results are as follows: Figure 7 As shown, the free radical scavenging rates follow the following order: Q1-1 > Q1-2 > Q2 > PVA, indicating that the dihydropyridine ring itself has antioxidant properties. Introducing the antioxidant functional unit vanillin can further improve the antioxidant properties of the copolymer.

[0189] 2) Antioxidant capacity test of hydrogel

[0190] ABTS +· The solution was diluted with PBS buffer to an absorbance of approximately 0.7. 1 mL of diluted ABTS was added to the glass vial. +·The solution was prepared by immersing approximately 250 μL of Gel-8 / 3 in the solution, taking photos at intervals, and observing the color changes. Gel-PVA-8 / 3 was treated in the same way as a control group. The results are as follows: Figure 8 As shown in Figure a, the color of the solution in the glass bottle soaked in Gel-8 / 3 disappeared faster than that of the solution soaked in Gel-PVA-8 / 3. At different time points, 200 μL of solution was taken from each of the two glass bottles, and the absorbance at 734 nm was measured using a microplate reader. The results are shown below. Figure 8 As shown in b, it can be seen that Gel-8 / 3 scavenge oxygen free radicals faster, indicating that the dihydropyridine ring in the graft copolymer can still maintain its antioxidant capacity after gelation.

[0191] Example 9: Three-dimensional cell culture using hydrogels

[0192] Mouse fibroblasts (L929) were dispersed in solution B (copolymer Q2 solid content 8%) from Example 4 to obtain a cell suspension. This suspension was mixed in equal volumes with aqueous solution A (copolymer Q1-1 solid content 3%) from Example 4 in a culture dish, and the dish was gently shaken to form a uniform gel. The cell-loaded Gel-8 / 3 was incubated at 37°C and 5% CO2 for 24 hours, followed by double staining (FDA / PI). Cell viability was characterized by the percentage of FDA-stained cells (green cells in the figure) to the total cell count. After being placed in the dark for 10 minutes, the cells were observed using a laser confocal microscope, and the results are as follows. Figure 9 As shown, after 24 hours of culture, the cell survival rate is close to 100%. This indicates that the hydrogel provided by the present invention has low cytotoxicity and is suitable for three-dimensional cell carriers.

[0193] Example 10: In vivo study of hydrogel promoting wound healing

[0194] Female BALB / c mice weighing approximately 20g were anesthetized with isoflurane. The hair on the mice's backs was shaved, and depilatory cream was applied to the shaved areas. After 3-5 minutes, excess cream was rinsed off with water. A circular skin lesion with a diameter of 6mm was created in the shaved area on the mice's backs. In the experimental group, 600μL of Gel-8 / 3 was applied to the wound, followed by a Tegaderm transparent dressing to prevent wound contraction. In the control group, a Tegaderm transparent dressing was applied directly to the wound. The dressings were changed every two days, and wound changes were recorded by photography. Results are as follows: Figure 10 As shown, the wounds of mice in the experimental group treated with Gel-8 / 3 were almost completely closed on day 10, while obvious wounds could still be observed in the untreated control group on day 10, indicating that Gel-8 / 3 can effectively promote wound healing.

[0195] As can be seen from the above examples and comparative examples, the hydrogel prepared by this invention has excellent antioxidant capacity and low cytotoxicity, and can effectively promote wound healing. Furthermore, by adjusting the ratio of the raw material copolymers Q1 and Q2, the strength of the prepared hydrogel material can be adjusted to obtain a hydrogel material with a certain degree of softness, thus enabling better application in the biomedical field.

[0196] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various specific technical features in any suitable manner. However, these simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A crosslinked copolymer, characterized in that, The crosslinked copolymer comprises macromolecular chains P1 and P2, and borate ester linking groups connecting macromolecular chains P1 and P2; wherein, macromolecular chain P1 contains structural unit A as shown in formula (1), structural unit B as shown in formula (2), and structural unit C as shown in formula (3). Equation (1) Equation (2), Equation (3), Wherein, R1 is methyl, ethyl, isopropyl, methoxy, hydroxyl or hydrogen atom, R2 and R3 are each independently selected from methoxy, hydroxyl or hydrogen atom, and R4 and R5 are each independently selected from hydrogen atom, dimethyl, C1-C3 alkyl or phenyl. The macromolecular chain P2 contains structural unit D as shown in formula (4) and structural unit E as shown in formula (5). Equation (4) Equation (5), Where R6 is a methyl group and z is a positive integer from 18 to 90, or R6 is a hydrogen atom and z is a positive integer from 43 to 113; This indicates the positions where macromolecular chains P1 and P2 are connected to the borate ester linking groups, wherein structural unit D contains... The boron element in the borate ester is directly connected.

2. The crosslinked copolymer according to claim 1, wherein, In formula (1), R1 is a hydroxyl group or a hydrogen atom, R2 is a methoxy group or a hydrogen atom, R3 is a hydrogen atom, R4 is a methyl group, and R5 is a methyl group.

3. The crosslinked copolymer according to claim 1, wherein, In equation (1), R6 is a methyl group and z is a positive integer between 18 and 20.

4. The crosslinked copolymer according to any one of claims 1-3, wherein, In the macromolecular chain P1, the molar ratio of structural unit A: structural unit B: structural unit C is 1-10:1-12:78-98. In the macromolecular chain P2, the molar ratio of structural unit D to structural unit E is 1:1-5; The molar ratio of macromolecular chain P1 to macromolecular chain P2 is 1-50:

1.

5. The crosslinked copolymer according to claim 1, wherein, The degree of crosslinking of the crosslinked copolymer is 0.17-0.

25.

6. A hydrogel comprising a copolymer and water filled in the copolymer, characterized in that, The copolymer is a crosslinked copolymer as described in any one of claims 1-5.

7. The hydrogel according to claim 6, wherein, Based on the total amount of the hydrogel, the copolymer in the hydrogel has a mass percentage of 3-11%, and the water has a mass percentage of 89-97%.

8. The hydrogel according to claim 6 or 7, wherein, Under the conditions of room temperature, frequency of 1.0 Hz and strain of 1%, the storage modulus (G') of the hydrogel is 50-1500 Pa and the loss modulus (G'') is 1-150 Pa.

9. A method for preparing a hydrogel, the method comprising: 1) The copolymer Q1 is first dissolved to obtain an aqueous solution A; 2) The copolymer Q2 is dissolved a second time to obtain aqueous solution B; 3) Mix the aqueous solution A and aqueous solution B, and react at room temperature to obtain the hydrogel; Wherein, the copolymer Q1 contains structural unit A shown in formula (1), structural unit B shown in formula (2), and structural unit F shown in formula (6). Equation (1) Equation (2), Equation (6), Wherein, R1 is methyl, ethyl, isopropyl, methoxy, hydroxyl or hydrogen atom, R2 and R3 are each independently selected from methoxy, hydroxyl or hydrogen atom, and R4 and R5 are each independently selected from hydrogen atom, dimethyl, C1-C3 alkyl or phenyl. The copolymer Q2 contains structural unit H as shown in formula (7) and structural unit E as shown in formula (5). Equation (7), Equation (5), Where R6 is a methyl group and z is a positive integer from 18 to 90, or R6 is a hydrogen atom and z is a positive integer from 43 to 113.

10. The method according to claim 9, wherein, In the copolymer Q1, the molar ratio of structural unit A: structural unit B: structural unit F is 1-10:1-12:78-98.

11. The method according to claim 9, wherein, In the copolymer Q2, the molar ratio of structural unit H to structural unit E is 1:1-5.

12. The method according to any one of claims 9-11, wherein, In step 1), the mass concentration of the aqueous solution A is 2-10%; In step 2), the mass concentration of the aqueous solution B is 4-12%.

13. The method according to claim 9, wherein, In step 3), the volume ratio of the aqueous solution A and the aqueous solution B is 0.5-1.5:

1.

14. A hydrogel prepared by the method of any one of claims 9-13.

15. The use of a crosslinked copolymer according to any one of claims 1-5, or a hydrogel according to any one of claims 6-8 and 14, in at least one of the following aspects: 1) In the preparation of cell / drug carriers; 2) In the preparation of products that promote wound healing; 3) In terms of dressing preparation.

16. A hydrogel product comprising the crosslinked copolymer of any one of claims 1-5 and the hydrogel of any one of claims 6-8 and 14.

17. The hydrogel product according to claim 16, characterized in that, The product is at least one of the following: 1) Cell / drug carrier; 2) Products that promote wound healing; 3) Dressings.

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