Protein-based phenylboronic acid ester dynamic crosslinking hydrogel material as well as preparation method and application thereof

Abstract

The invention relates to a protein-based phenylboronic acid ester dynamic crosslinking hydrogel material as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving a component A, namely a phenylboronic acid modified protein polymer derivative, in a biocompatible medium to obtain a solution A; dissolving a component B, namely a macromolecular derivative containing a vicinal diol group, into a biocompatible medium to obtain a solution B; and mixing the solution A and the solution B with a certain concentration for a certain time to obtain the protein-based phenylboronic acid ester dynamic crosslinking hydrogel material. The boric acid ester dynamic cross-linking bond belongs to reversible chemical cross-linking reaction, and the prepared protein-based hydrogel material has enough mechanical strength and cheap operability. The protein-based hydrogel material disclosed by the invention can provide the effects of cell adhesion, proliferation and differentiation promotion, tissue regeneration and the like, can load and slowly release beneficial components for promoting tissue repair, is an ideal tissue engineering scaffold material, and can be applied to defect regeneration and repair of soft tissues, cartilages, bones and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and relates to a hydrogel material, in particular to a protein-based phenylborate dynamic cross-linked hydrogel material and its preparation method and application. Background Art

[0002] Hydrogels are three-dimensional network materials formed by cross-linking hydrophilic polymers and are widely used in the fields of tissue engineering, drug delivery, three-dimensional cell culture, and bioprinting. Generally, the backbone materials of hydrogels can be divided into natural polymer materials and synthetic polymer materials. Although synthetic polymer-based hydrogel materials have good mechanical strength and stability, they generally have poor biocompatibility, are difficult to biodegradable, and the uncross-linked small molecule monomers are biotoxic. Natural polymer-based hydrogel materials are considered ideal tissue engineering scaffold materials due to their excellent biocompatibility and biodegradability. Among them, protein-based hydrogel materials have the effects of cell adhesion, proliferation, differentiation, and promoting tissue regeneration, such as collagen, silk fibroin, elastin, recombinant protein, gelatin, polypeptide, etc. However, at present, protein-based hydrogel materials lack an ideal cross-linking method to provide sufficient mechanical strength and take into account cheap operability. For example, the mechanical strength of hydrogels prepared by physical cross-linking methods is generally poor; small molecule aldehyde chemical cross-linking agents represented by glutaraldehyde have obvious improvement in mechanical strength, but have certain biotoxicity; photoinitiated polymerization cross-linking represented by methacrylate modification belongs to covalent bond cross-linking and requires a secondary light irradiation operation step. Therefore, it is urgent to establish a general cross-linking method to prepare protein-based hydrogel materials. Summary of the Invention

[0003] In order to overcome the deficiencies of the above-mentioned cross-linking methods of protein-based hydrogel materials, the present invention provides a protein-based phenylborate dynamic cross-linked hydrogel material and its preparation method and application.

[0004] The object of the present invention can be achieved by the following technical solutions:

[0005] The first object of the present invention is to provide a phenylboric acid-modified protein polymer derivative, and the structure of the phenylboric acid-modified protein polymer derivative is shown in Formula I:

[0006]

[0007] wherein, n≥2, that is, the average number of phenylboric acid groups (i.e., the structure in the brackets in Formula I) on a single P1 polymer chain is greater than or equal to 2;

[0008] P1 is a protein or a protein modifier or a protein degradation product, and the protein is selected from collagen, silk fibroin, elastin, recombinant protein, etc.; the protein degradation product includes gelatin or polypeptide, etc.;

[0009] R1, R2, R3, R4, and R5 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl group, mercapto group, amino group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, ester group, amide group, aryl group, alkyl group, modified alkyl group, etc.

[0010] In one embodiment of the present invention, the alkyl group is a saturated or unsaturated aliphatic straight-chain or branched-chain alkyl group having 1 to 20 carbon atoms.

[0011] The modified alkyl group is such that any carbon atom of the alkyl group is substituted by one selected from -F, -Cl, -Br, -I, -OH, -SH, -NH2, -NO2, -CN, -CHO, -COOH, ester group, amide group, aryl group, etc. The modified alkyl group has 1 to 20 carbon atoms, and its carbon-carbon single bonds can be arbitrarily replaced by carbon-carbon double bonds or carbon-carbon triple bonds.

[0012] In one embodiment of the present invention, preferably, in the structure shown in Formula I, at least two of R1, R2, R3, R4, and R5 are connected to each other to form a saturated or unsaturated alicyclic or heteroalicyclic ring with carbon atoms, or form an aromatic ring or heteroaromatic ring.

[0013] The alicyclic ring is a saturated or unsaturated monocyclic or polycyclic alicyclic ring having 3 to 10 members, and the heteroalicyclic ring is a saturated or unsaturated monocyclic or polycyclic heteroalicyclic ring having 3 to 10 members and containing at least one heteroatom selected from O, S, and N in the ring.

[0014] The aromatic ring is a 5- to 10-membered aromatic monocyclic ring or aromatic fused bicyclic ring, and the heteroaromatic ring is a 5- to 10-membered aromatic monocyclic ring or aromatic fused bicyclic ring containing at least one heteroatom selected from O, S, and N in the ring.

[0015] In one embodiment of the present invention, further, the preferred structures of the alicyclic or heteroalicyclic rings include:

[0016] etc.

[0017] In one embodiment of the present invention, further, the preferred structures of the aromatic or heteroaromatic rings include:

[0018] etc.

[0019] In one embodiment of the present invention, preferably, in the structure shown in Formula I, P1 can be connected to any one or more of the groups of R1, R2, R3, R4, and R5; or is connected to the saturated or unsaturated alicyclic or heteroalicyclic ring formed between R1, R2, R3, R4, and R5, or the aromatic or heteroaromatic ring formed.

[0020] The linking bond is selected from the linking bond P1-O- obtained from hydroxyl groups; or the linking bond P1-S- obtained from mercapto groups; or the linking bond P1-NH- obtained from amino groups; or the linking bond P1- obtained from alkyl groups; or the linking bond P1-COO- obtained from ester bonds; or the linking bond P1-CONH- obtained from amide bonds. One end of the linking bond is connected to P1, and the other end is connected to the benzene ring of the molecule shown in Formula I.

[0021] In one embodiment of the present invention, further preferably, Formula I is selected from the structures of the following components A-1 to A-9:

[0022]

[0023] Among components A-1 to A-9, n≥2, Col is collagen; GL is gelatin; Sil is silk fibroin; Ela is elastin; PGA is polyglutamic acid.

[0024] The second object of the present invention is to provide a preparation method of the phenylboronic acid-modified protein-based polymer derivative.

[0025] The first feasible preparation method of the phenylboronic acid-modified protein-based polymer derivative: Dissolve the carboxyl-containing phenylboronic acid compound (PB-COOH, PB is phenylboronic acid) in deionized water, add the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and the activator N-hydroxysuccinimide (NHS), stir and react at room temperature for 1 h, dropwise add the amino-containing water-soluble protein-based polymer solution, continue to stir and react at 40 °C for 24 h, add the reaction solution into a dialysis bag and dialyze with deionized water for 2-3 d, and then freeze-dry to obtain the phenylboronic acid-modified protein-based polymer derivative.

[0026] The second feasible preparation method of the phenylboronic acid-modified protein-based polymer derivative: Dissolve the carboxyl-containing protein-based polymer in 0.01 mol / L 2-(N-morpholino)ethanesulfonic acid MES buffer solution (pH = 5.2), and add the amino-containing phenylboronic acid compound (PB-NH2, PB is phenylboronic acid), stir until completely dissolved, dissolve 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride DMTMM in the MES buffer solution, dropwise add it to the above reaction solution, react at 40 °C for 24 h, add the reaction solution into a dialysis bag and dialyze with deionized water for 2-3 d, and then freeze-dry to obtain the phenylboronic acid-modified protein-based polymer derivative.

[0027] In the first feasible embodiment and the second feasible embodiment, the amino-containing water-soluble protein-based polymer and the carboxyl-containing protein-based polymer are selected from gelatin, polypeptide, collagen, silk fibroin, elastin, recombinant protein, etc.

[0028] The third object of the present invention is to provide a method for preparing a protein-based phenylborate dynamic cross-linked hydrogel material, which is prepared from the phenylboric acid-modified protein-based polymer derivative described in the first object of the present invention as a raw material.

[0029] The method for preparing the protein-based phenylborate dynamic cross-linked hydrogel material of the present invention comprises the following steps:

[0030] Dissolve component A - phenylboric acid-modified protein-based polymer derivative in a biocompatible medium to obtain solution A;

[0031] Dissolve component B - polymer derivative containing a vicinal diol group in a biocompatible medium to obtain solution B;

[0032] Mix a certain concentration of solution A and solution B for a certain period of time to obtain the protein-based phenylborate dynamic cross-linked hydrogel material.

[0033] The component B - polymer derivative containing a vicinal diol group has a structure shown in formula II:

[0034]

[0035] Wherein, n≥2, that is, the average number of vicinal diol groups (i.e., the structure in brackets in formula II) on a single P2 polymer chain is greater than or equal to 2. In formula II, R1 and R2 are independently selected from hydrogen, chlorine, hydroxyl, amino, carboxyl, amide, aryl, alkyl, modified alkyl, etc.

[0036] In the present invention, the polymer derivative containing a vicinal diol group includes natural polysaccharide substances or their modified products or degraded products, or synthetic polymers containing vicinal diol groups, or vicinal diol-modified protein substances and their modified products or degraded products.

[0037] The natural polysaccharide substances include hyaluronic acid, cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, chitosan, etc. Further preferably, they are hyaluronic acid, cellulose, alginic acid, dextran, etc.

[0038] The synthetic polymer containing a vicinal diol group is preferably polyvinyl alcohol and its modified products.

[0039] Method for preparing vicinal diol-modified protein substances: Dissolve D-(+)-glucono-δ-lactone and triethylamine in a small amount of DMSO, dropwise add the water-soluble polymer solution of protein containing amino groups, continue to stir and react at room temperature for 48 h, add the reaction solution to a dialysis bag and dialyze with deionized water for 2 - 3 d, and then freeze-dry to obtain the vicinal diol-modified protein substances.

[0040] The protein substances include gelatin, polypeptide, collagen, fibroin, elastin, recombinant protein, etc.

[0041] In the preparation method of the hydrogel according to the third object of the present invention, the biocompatible medium is selected from distilled water, physiological saline, buffer solution or cell culture solution. Different media can be selected according to different applications.

[0042] In the preparation method of the hydrogel according to the third object of the present invention, in the hydrogel precursor solution formed by uniformly mixing solution A and solution B, the certain concentration is 0.1% w / v - 60% w / v, preferably 2% w / v - 20% w / v; the molar ratio of phenylboronic acid group to vicinal diol group is 1:0.01 - 100, preferably 1:0.1 - 10.

[0043] In the preparation method of the hydrogel according to the third object of the present invention, the mixing for a certain time is 0.001 - 24 h, preferably 0.1 - 120 min, and further preferably 1 - 30 min.

[0044] In one embodiment of the present invention, in the preparation process of the protein-based phenylboronic acid ester dynamically crosslinked hydrogel material, in addition to adding component A and component B, component C can also be added. Component C is a beneficial component for tissue regeneration and repair, including drugs, bioactive factors or cells.

[0045] In the present invention, the drugs include lidocaine, insulin, local anesthetics, antibiotics, anti-inflammatory drugs, anticancer drugs, etc.; the bioactive factors include BMP-2 - BMP-9 (bone morphogenetic protein), EGF (epidermal growth factor), TGFα, TGFβ (transforming growth factor), FGF (fibroblast growth factor), IGF-I, IGF-II (insulin-like growth factor), NGF (nerve growth factor), etc.

[0046] In the present invention, the drugs and bioactive factors can be loaded on the drug carrier by using drug sustained-release technology. The drug carrier can be selected from microgels, PLGA microspheres, mesoporous silica nanospheres, metal-organic framework nanospheres, etc.

[0047] In the present invention, the cells include osteoblasts, chondrocytes, endothelial cells, myoblasts, fibroblasts, nerve cells, mesenchymal stem cells, embryonic stem cells, etc.

[0048] In the preparation process of the protein-based phenylboronic acid ester dynamically crosslinked hydrogel material of the present invention, component A and component B are necessary components, and component C can be selectively added according to the actual application needs.

[0049] The protein-based phenylboronic acid ester dynamically crosslinked hydrogel material provided by the third object of the present invention has the following crosslinking characteristics and material properties:

[0050] (1) The phenylborate dynamic cross - link bonds belong to reversible chemical cross - linking reactions, making the protein - based hydrogel in a state between cross - linking and de - cross - linking. The material has semi - fluid properties, endowing the hydrogel material with characteristics such as plasticity, shear - thinning, self - healing, and adhesiveness.

[0051] (2) The hydrogel matrix material is mainly composed of protein components and has good biocompatibility, which can provide functions such as cell adhesion, promoting cell proliferation and differentiation, and tissue regeneration. The dynamic cross - link bonds can provide controllable biodegradable characteristics for the protein - based hydrogel.

[0052] The fourth object of the present invention is to provide a product prepared by the preparation method of the protein - based phenylborate dynamic cross - linked hydrogel material, that is, the protein - based phenylborate dynamic cross - linked hydrogel material.

[0053] The present invention provides a hydrogel prepared by the above - mentioned method, which can be called a protein - based phenylborate dynamic cross - linked hydrogel.

[0054] The fifth object of the present invention is to provide the application of the product prepared by the preparation method of the protein - based phenylborate dynamic cross - linked hydrogel material.

[0055] The present invention provides the application of the protein - based phenylborate dynamic cross - linked hydrogel material in the preparation of skin wound repair materials.

[0056] The present invention provides the application of the protein - based phenylborate dynamic cross - linked hydrogel material in the preparation of urethral wound repair materials.

[0057] The present invention provides the application of the protein - based phenylborate dynamic cross - linked hydrogel material in the preparation of articular cartilage defect regeneration and repair materials.

[0058] The present invention provides the application of the protein - based phenylborate dynamic cross - linked hydrogel material in the preparation of bone defect regeneration and repair materials.

[0059] The present invention provides the application of the protein - based phenylborate dynamic cross - linked hydrogel material in the preparation of a substrate material for three - dimensional cell culture.

[0060] The present invention provides the application of the protein - based phenylborate dynamic cross - linked hydrogel material as a 3D printing bio - ink.

[0061] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0062] (1) The present invention discloses a new method for preparing protein - based hydrogel materials by a phenylborate dynamic cross - linking method. Compared with the traditional physical cross - linking method lacking mechanical strength and the chemical covalent cross - linking method lacking operability, the phenylborate dynamic cross - link bonds belong to reversible chemical cross - linking reactions, and the prepared protein - based hydrogel material takes into account sufficient mechanical strength (such as Figure 1as shown) and low cost operability (such as Figure 2 , 3 as shown).

[0063] (2) The protein-based hydrogel material disclosed by the present invention can provide functions such as cell adhesion, promoting proliferation and differentiation, and tissue regeneration. At the same time, it can load and slowly release beneficial components for promoting tissue repair, and is an ideal tissue engineering scaffold material, which can be applied to the regeneration and repair of soft tissue, cartilage, bone and other defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is the crosslinking mechanism and rheological test diagram of p-FPB-GL / PVA hydrogel.

[0065] Figure 2 is the intuitive diagram of the injectability of p-FPB-GL / PVA hydrogel.

[0066] Figure 3 is the intuitive diagram of the self-healing property of p-FPB-GL / PVA hydrogel.

[0067] Figure 4 is the macroscopic view and scanning electron microscope diagram of p-FPB-GL / PVA hydrogel.

[0068] Figure 5 is the intuitive diagram of the tissue adhesion of p-FPB-GL / PVA hydrogel.

[0069] Figure 6 is the phalloidin staining diagram of cells inside p-FPB-GL / PVA hydrogel.

[0070] Figure 7 is the skin defect repair diagram of p-FPB-GL / PVA hydrogel. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0071] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0072] The present invention will be further described below with reference to the drawings and embodiments. However, these embodiments are only descriptions of the best implementation modes of the present invention and do not limit the scope of the present invention in any way. Any other changes and modifications made by those skilled in the art without departing from the spirit and protection scope of the present invention are still included within the protection scope of the present invention.

[0073] Example 1: Synthesis of p-phenylboronic acid modified collagen polymer derivative (p-PB-Col)

[0074] Synthesis of p-PB-Col: Dissolve p-phenylboronic acid compound (p-PB, 50 mg) in deionized water, add condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and activator N-hydroxysuccinimide (NHS, 0.1 g). After stirring the reaction at room temperature for 1 h, gradually add the aqueous solution of collagen (Collagen, derived from rat tail, 1 g) dropwise. Continue to stir the reaction at 40 °C for 24 h, then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d. Freeze-dry to obtain the p-PB-Col polymer derivative (0.85 g, yield 85%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid groups can be calculated to be 23%.

[0075]

[0076] Example 2: Synthesis of p-fluorophenylboronic acid modified gelatin polymer derivative (p-FPB-GL)

[0077] Synthesis of p-FPB-GL: Dissolve p-fluorophenylboronic acid compound (p-FPB, 50 mg) in deionized water, add condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and activator N-hydroxysuccinimide (NHS, 0.1 g). After stirring the reaction at room temperature for 1 h, gradually add the aqueous solution of gelatin (Gelatin, derived from porcine skin, sigma V900863, 1 g) dropwise. Continue to stir the reaction at 40 °C for 24 h, then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d. Freeze-dry to obtain the p-FPB-GL polymer derivative (0.88 g, yield 88%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid groups can be calculated to be 47%.

[0078]

[0079] Example 3: Synthesis of m-fluorochlorophenylboronic acid modified silk fibroin polymer derivative (m-FCPB-Sil)

[0080] Synthesis of m-FCPB-Sil: Dissolve m-fluorochlorobenzeneboronic acid compound (m-FCPB-Sil, 50 mg) in deionized water, add condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and activator N-hydroxysuccinimide (NHS, 0.1 g). After stirring the reaction at room temperature for 1 h, slowly add the aqueous solution of silk fibroin (derived from silk, 1 g) dropwise. Continue to stir the reaction at 40 °C for 24 h, then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze against deionized water for 2 - 3 days. Freeze-dry to obtain the m-FCPB-Sil polymer derivative (0.78 g, yield 78%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of the boronic acid group can be calculated to be 34%.

[0081]

[0082] Example 4: Synthesis of p-methoxyphenylboronic acid modified elastin polymer derivative (p-OPB-Ela)

[0083] Synthesis of p-OPB-Ela: Dissolve p-methoxyphenylboronic acid compound (p-OPB-Ela, 50 mg) in deionized water, add condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and activator N-hydroxysuccinimide (NHS, 0.1 g). After stirring the reaction at room temperature for 1 h, slowly add the aqueous solution of elastin (derived from pig aortic arch, 1 g) dropwise. Continue to stir the reaction at 40 °C for 24 h, then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze against deionized water for 2 - 3 days. Freeze-dry to obtain the p-OPB-Ela polymer derivative (0.71 g, yield 71%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of the boronic acid group can be calculated to be 27%.

[0084]

[0085] Example 5: Synthesis of p-methylaminophenylboronic acid modified polyglutamic acid polymer derivative (p-NPB-PGA)

[0086] Synthesis of p-NPB-PGA: Dissolve polyglutamic acid polymer (PGA, sigma P4886, 1 g in 10 mL deionized water) in 0.01 mol / L 2-(N-morpholino)ethanesulfonic acid MES buffer solution (pH = 5.2), and add p-methylaminophenylboronic acid compound (p-NPB-PGA, 50 mg). Stir until completely dissolved. Dissolve 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 0.1 g) in MES buffer solution and add it dropwise to the above reaction solution. After reacting at 40 °C for 24 h, add the reaction solution to a dialysis bag (MWCO 3500) and dialyze it against deionized water for 2 - 3 d. Then freeze-dry to obtain the p-NPB-PGA polymer derivative (0.86 g, yield 86%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid group can be calculated to be 38%.

[0087]

[0088] Example 6: Synthesis of diol-modified gelatin polymer derivative (diol-GL)

[0089] Synthesis of diol-GL: Dissolve D-(+)-glucono-δ-lactone (0.5 g) and triethylamine (0.2 mL) in 5 mL DMSO, and add it dropwise to the aqueous solution of gelatin (Gelatin, derived from pig skin, sigma V900863, 1 g in 10 mL deionized water) polymer. After continuing to stir and react at 40 °C for 48 h, add the reaction solution to a dialysis bag and dialyze it against deionized water for 2 - 3 d. Then freeze-dry to obtain the diol-GL polymer derivative (0.82 g, yield 82%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of vicinal diol group can be calculated to be 23%.

[0090]

[0091] Example 7: Synthesis of diol-modified polylysine polymer derivative (diol-PLL)

[0092] Synthesis of diol-PLL: Dissolve D-(+)-glucono-δ-lactone (0.5 g) and triethylamine (0.2 mL) in 5 mL DMSO, and add it dropwise to the aqueous solution of polylysine (PLL, sigma P9404, 1 g in 10 mL deionized water) polymer. After continuing to stir and react at 40 °C for 48 h, add the reaction solution to a dialysis bag and dialyze it against deionized water for 2 - 3 d. Then freeze-dry to obtain the diol-PLL polymer derivative (0.87 g, yield 87%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of vicinal diol group can be calculated to be 34%.

[0093]

[0094] Example 8: Preparation of p-FPB-GL / PVA gelatin-polyvinyl alcohol dynamic cross-linked hydrogel

[0095] According to the method of the present invention, component A-phenylboronic acid modified gelatin derivative p-FPB-GL (prepared in Example 2, 0.1g) was selected and dissolved in 1mL D-PBS (pH=7.4), and component B-polyvinyl alcohol (PVA, sigma360627, 20mg) was selected and dissolved in 1mL D-PBS (pH=7.4). The two components were mixed evenly in equal proportions and placed for 5 minutes to obtain p-FPB-GL / PVA gelatin-polyvinyl alcohol dynamic cross-linked hydrogel, which exhibits characteristics such as plasticity, shear thinning, self-healing, and adhesion. Figure 1 As shown in Figure 2, dynamic stress rheological tests show that the hydrogel has good recovery ability under the alternating effects of high stress (γ = 500%) and low stress (γ = 1%), mainly due to the unique plasticity of the borate dynamic cross-linking bonds. Figure 2 As shown in the figure, the p-FPB-GL / PVA hydrogel material can be easily pushed out from a syringe with a 21G needle, presenting a continuous and viscous fluid state, mainly because the shear thinning property imparted by the borate dynamic cross-linking bond enables the hydrogel material to be injected. Figure 3 As shown in the figure, two different p-FPB-GL / PVA borate dynamic cross-linked hydrogel materials can be well fused into a whole, mainly due to the self-healing properties of borate dynamic cross-linking bonds. Figure 4 As shown in Figure 2, the p-FPB-GL / PVA borate dynamic cross-linked hydrogel material exhibits a loose porous structure after freeze-drying. Figure 5 As shown, the p-FPB-GL / PVA borate dynamic cross-linked hydrogel material was placed on pig skin. The phenylboronic acid structure on the hydrogel was able to form hydrogen bonds with active hydrogen on the skin and firmly adhere to the pig skin, demonstrating excellent tissue adhesion.

[0096] Example 9: Preparation of p-PB-Col / diol-GL collagen-gelatin dynamic cross-linked hydrogel

[0097] According to the method of the present invention, component A, phenylboronic acid-modified collagen derivative p-PB-Col (0.1 g prepared in Example 1) is dissolved in 1 mL of D-PBS (pH = 7.4), and component B, vicinal diol-modified gelatin derivative (diol-GL, gelatin sourced from porcine skin, sigmaV900863, 0.1 g) is dissolved in 1 mL of D-PBS (pH = 7.4). The two components are mixed evenly in equal proportions. After standing for 5 minutes, the p-PB-Col / diol-GL collagen-gelatin dynamic cross-linked hydrogel can be obtained. According to the test and analysis method of Example 8, the p-PB-Col / diol-GL hydrogel material exhibits characteristics such as plasticity, shear thinning, self-healing, and adhesiveness.

[0098] Example 10: Preparation of m-FCPB-Sil / HA silk fibroin-hyaluronic acid dynamic cross-linked hydrogel

[0099] According to the method of the present invention, component A, phenylboronic acid-modified silk fibroin derivative m-FCPB-Sil (0.1 g) is dissolved in 1 mL of D-PBS (pH = 7.4), and component B, hyaluronic acid (HA, sourced from biologic fermentation, Freda, molecular weight 340 kDa, 50 mg) is dissolved in 1 mL of D-PBS (pH = 7.4). The two components are mixed evenly in equal proportions. After standing for 5 minutes, the m-FCPB-Sil / HA silk fibroin-hyaluronic acid dynamic cross-linked hydrogel can be obtained. According to the test and analysis method of Example 8, the m-FCPB-Sil / HA hydrogel material exhibits characteristics such as plasticity, shear thinning, self-healing, and adhesiveness.

[0100] Example 11: Preparation of p-OPB-Ela / CS elastin-chondroitin sulfate dynamic cross-linked hydrogel

[0101] According to the method of the present invention, component A, phenylboronic acid-modified elastin derivative p-OPB-Ela (0.1 g) is dissolved in 1 mL of D-PBS (pH = 7.4), and component B, chondroitin sulfate (CS, sourced from bovine cartilage, sigmaC6737, 50 mg) is dissolved in 1 mL of D-PBS (pH = 7.4). The two components are mixed evenly in equal proportions. After standing for 5 minutes, the p-OPB-Ela / CS elastin-chondroitin sulfate dynamic cross-linked hydrogel can be obtained, which exhibits characteristics such as plasticity, shear thinning, self-healing, and adhesiveness.

[0102] Example 12: Preparation of p-NPB-PGA / diol-PLL polyglutamic acid-polylysine dynamic cross-linked hydrogel

[0103] According to the method of the present invention, component A - phenylboronic acid - modified polyglutamic acid derivative p - NPB - PGA (0.1 g) is dissolved in 1 mL of D - PBS (pH = 7.4), and component B - vicinal diol - modified polylysine derivative (diol - PLL, sigma P9404, 0.1 g) is dissolved in 1 mL of D - PBS (pH = 7.4). The two components are mixed evenly in equal proportion. After standing for 5 min, the p - NPB - PGA / diol - PLL polyglutamic acid - polylysine dynamic cross - linked hydrogel can be obtained, which exhibits characteristics such as plasticity, shear thinning, self - healing, and adhesiveness.

[0104] Example XIII: Preparation of phenylborate dynamic cross - linked hydrogel materials

[0105] According to the method of the present invention, the operation is carried out at 37 °C, and polymer aqueous solutions with different concentrations are respectively prepared, with the composition A:B = 1:1 (w / w), as shown in Table 1.

[0106] Table 1

[0107]

[0108] The corresponding concentration of the hydrogel precursor solution of the above - mentioned different components is respectively prepared, mixed evenly in equal proportion, and left standing for 5 min, then hydrogel materials with different chemical compositions can be obtained. Different hydrogel materials have different biological effects, and the composition of the hydrogel materials can be selected according to different applications.

[0109] In addition, beneficial component C for tissue regeneration and repair can be added to the above - mentioned hydrogel, including drugs, bioactive factors or cells. Among them, drugs include lidocaine, insulin, local anesthetics, antibiotics, anti - inflammatory drugs, anti - cancer drugs, etc.; bioactive factors include BMP - 2 to BMP - 9 (bone morphogenetic protein), EGF (epidermal growth factor), TGFα, TGFβ (transforming growth factor), FGF (fibroblast growth factor), IGF - Ⅰ, IGF - Ⅱ (insulin - like growth factor), NGF (nerve growth factor), etc.; cells include osteoblasts, chondrocytes, endothelial cells, myoblasts, fibroblasts, nerve cells, mesenchymal stem cells, embryonic stem cells, etc.

[0110] Example XIV: Rheological test of phenylborate dynamic cross - linked hydrogel

[0111] Rheological analysis is carried out using a HAAKE MARS rheometer, and rheological tests are carried out on a test platform at 37 °C. Figure 1Figure 0 shows the dynamic test curve of the p-FPB-GL / PVA phenylborate dynamic cross-linked hydrogel in Example 8. Under the applied force with alternating strain γ = 1% and 500%, the hydrogel material shows good elasticity and recovery ability, indicating that the hydrogel material has self-healing properties, which is due to the fact that the hydrogel is formed by cross-linking through dynamic phenylborate chemical bonds.

[0112] Example 15: Three-dimensional cell culture experiment of phenylborate dynamic cross-linked hydrogel

[0113] In this experiment, taking the three-dimensional cell culture experiment of the p-FPB-GL / PVA phenylborate dynamic cross-linked hydrogel in Example 8 as an example, a hydrogel precursor solution with a certain concentration was prepared using cell culture medium DMEM (p-FPB-GL: 10% w / v; PVA: 2% w / v; 1:1 mixture). The chondrocytes were digested with trypsin and centrifuged to prepare a cell suspension. Then the cell suspension was thoroughly mixed with the above hydrogel precursor solution and added to a confocal culture dish. After standing for 5 min, it was completely gelled. Then, 1 mL of DMEM containing 10% FBS was added to each well and continued to be cultured for a certain period of time (1 d, 4 d, 7 d) in an environment of 37 °C and 5% CO2. As Figure 6 shown, the phalloidin staining results show that chondrocytes are more conducive to cell spreading, proliferation, differentiation, etc. in the three-dimensional culture matrix of the dynamic cross-linked hydrogel.

[0114] Example 16: Application of phenylborate dynamic cross-linked hydrogel in skin defect repair

[0115] In this experiment, taking the application of the p-PB-Col / diol-GL phenylborate dynamic cross-linked hydrogel in skin defect repair in Example 9 as an example, a hydrogel precursor solution with a certain concentration was prepared (p-FPB-GL: 10% w / v; PVA: 2% w / v; 1:1 mixture). The experiment used a zebra pig model, and a 3 cm × 3 cm square defect model was created on the pig skin, which was divided into 3 groups: ① blank group; ② hydrogel group (p-PB-Col / diol-GL). The hydrogel precursor solution was filled and covered the skin defect site, and after standing for 5 min until it was completely gelled, the wound was dressed every 3 - 5 days, and the wound healing and tissue regeneration were observed one month after the operation. As Figure 7 shown, the moist healing conditions provided by the hydrogel material are conducive to promoting tissue regeneration and successfully realizing the regenerative repair of skin defects.

[0116] Example 17: Application of phenylborate dynamic cross-linked hydrogel in articular cartilage defect repair

[0117] In this experiment, taking the application of the p-OPB-Ela / CS phenylborate dynamic cross-linked hydrogel in the repair of articular cartilage defects in Example XI as an example, a hydrogel precursor solution with a certain concentration was prepared (p-OPB-Ela: 10% w / v; CS: 6% w / v; mixed in a ratio of 1:1). The experiment used a New Zealand rabbit model, and a cylindrical defect model with a diameter of 5 mm was created on the trochlear part of the rabbit joint. The rabbits were divided into 3 groups: ① blank group; ② hydrogel group (p-OPB-Ela / CS); ③ hydrogel + growth factor group (p-OPB-Ela / CS + TGFβ), where TGFβ with a concentration of 10 ng / mL was added during the preparation of the hydrogel precursor solution. The hydrogel precursor solution was filled and covered the articular defect site, and left for 5 min until it was completely gelled. The regeneration and repair of the articular defect were observed at 1 month and 3 months after the operation. The experimental results confirmed that the hydrogel material could stimulate the homing of endogenous stem cells to promote joint regeneration, and the added TGFβ growth factor could accelerate the regeneration and repair speed, successfully achieving the regeneration and repair of articular cartilage defects.

[0118] Example XVIII: Application of Phenylborate Dynamic Cross-Linked Hydrogel in the Repair of Alveolar Bone Defects

[0119] In this experiment, taking the application of the p-NPB-PGA / diol-PLL phenylborate dynamic cross-linked hydrogel in the repair of alveolar bone defects in Example XII as an example, a hydrogel precursor solution with a certain concentration was prepared (p-NPB-PGA: 10% w / v; diol-PLL: 10% w / v; mixed in a ratio of 1:1). The experiment used a New Zealand rabbit model, and a cylindrical defect model with a diameter of 3 mm was created on the alveolar bone part of the rabbit. The rabbits were divided into 3 groups: ① blank group; ② hydrogel group (p-NPB-PGA / diol-PLL); ③ hydrogel + growth factor group (p-NPB-PGA / diol-PLL + BMP-2), where BMP-2 with a concentration of 10 ng / mL was added during the preparation of the hydrogel precursor solution. The hydrogel precursor solution was filled and covered the alveolar bone defect site, and left for 5 min until it was completely gelled. The regeneration and repair of the alveolar bone defect were observed at 1 month and 3 months after the operation. The experimental results confirmed that the hydrogel material could stimulate the homing of endogenous stem cells to promote alveolar bone regeneration, and the added BMP-2 growth factor could accelerate the regeneration and repair speed of bone defects, successfully achieving the regeneration and repair of alveolar bone defects.

[0120] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention as disclosed should fall within the protection scope of the present invention.

Claims

1. A protein-based polymer derivative modified with phenylboronic acid, characterized in that, The structure of the phenylboronic acid-modified protein-based polymer derivative is shown in Formula I: wherein n ≥ 2; P1 is a protein or a protein modifier or a protein degradation product, and the protein is selected from collagen, fibroin, elastin, recombinant protein; the protein degradation product includes gelatin or polypeptide; R1, R2, R3, R4, R5 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl group, mercapto group, amino group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, ester group, amide group, aryl group, alkyl group, modified alkyl group.

2. A phenylboronic acid-modified protein-based polymer derivative according to claim 1, characterized in that, The alkyl group is a saturated or unsaturated aliphatic straight-chain or branched-chain alkyl group having 1-20 carbon atoms; The modified alkyl group is an alkyl group in which any carbon atom is substituted by one selected from -F, -Cl, -Br, -I, -OH, -SH, -NH2, -NO2, -CN, -CHO, -COOH, ester group, amide group, aryl group; The modified alkyl group has 1-20 carbon atoms, and its carbon-carbon single bond can be arbitrarily replaced by a carbon-carbon double bond or a carbon-carbon triple bond; in the structure shown in Formula I, at least two of R1, R2, R3, R4, R5 are connected to each other to form a saturated or unsaturated alicyclic or heteroalicyclic ring with carbon atoms, or form an aromatic ring or heteroaromatic ring; The alicyclic ring is a saturated or unsaturated 3- to 10-membered monocyclic or polycyclic alicyclic ring, and the heteroalicyclic ring is a saturated or unsaturated 3- to 10-membered monocyclic or polycyclic heteroalicyclic ring containing at least one heteroatom selected from O, S, N on the ring; The aromatic ring is a 5- to 10-membered aromatic monocyclic or aromatic fused bicyclic ring, and the heteroaromatic ring is a 5- to 10-membered aromatic monocyclic or aromatic fused bicyclic ring containing at least one heteroatom selected from O, S, N on the ring.

3. A phenylboronic acid-modified protein-based polymer derivative according to claim 1, characterized in that,... In the structure shown in Formula I, P1 is connected to any one or more groups of R1, R2, R3, R4, R5; or is connected to a saturated or unsaturated alicyclic or heteroalicyclic ring formed between R1, R2, R3, R4, R5, or forms an aromatic ring or heteroaromatic ring; The linking bond is selected from the linking bond P1-O- obtained from hydroxyl groups; or the linking bond P1-S- obtained from mercapto groups; or the linking bond P1-NH- obtained from amino groups; or the linking bond P1- obtained from alkyl groups; or the linking bond P1-COO- obtained from ester bonds; or the linking bond P1-CONH- obtained from amide bonds, and one end of this linking bond is connected to P1, and the other end is connected to the benzene ring of the molecule shown in Formula I.

4. A phenylboronic acid-modified protein-based polymer derivative according to claim 1, characterized in that, The Formula I is selected from the structures of the following components A-1 to A-9: In components A-1 to A-9, n ≥ 2, Col is collagen; GL is gelatin; Sil is fibroin; Ela is elastin; PGA is polyglutamic acid.

5. The preparation method of the phenylboronic acid-modified protein-based polymer derivative according to any one of claims 1-4, characterized in that, Dissolve the carboxyl-containing phenylboronic acid compound in deionized water, add the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the activating agent N-hydroxysuccinimide, stir and react at room temperature, dropwise add the amino-containing water-soluble protein-based polymer solution, continue to stir and react, add the reaction solution to a dialysis bag for dialysis, and freeze-dry to obtain the phenylboronic acid-modified protein-based polymer derivative, or, Dissolve the carboxyl-containing protein polymer in 2-(N-morpholino)ethanesulfonic acid MES buffer solution, add the amino-containing phenylboronic acid compound, and stir until completely dissolved. Dissolve 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride DMTMM in MES buffer solution, and add it dropwise to the above reaction solution. React, add the reaction solution to a dialysis bag for dialysis, and freeze-dry to obtain the phenylboronic acid-modified protein polymer derivative.

6. A preparation method of a protein-based phenylborate dynamic cross-linked hydrogel material, characterized in that, It includes the following steps: Dissolve component A - the phenylboronic acid-modified protein polymer derivative described in any one of claims 1-4 in a biocompatible medium to obtain solution A; Dissolve component B - the polymer derivative containing a vicinal diol group in a biocompatible medium to obtain solution B; Mix the solution A and solution B with a certain concentration for a certain time to obtain the protein-based phenylborate dynamic cross-linked hydrogel material; The component B - the polymer derivative containing a vicinal diol group has a structure shown in formula II: Wherein, n≥2. In formula II, R1 and R2 are independently selected from hydrogen, chlorine, hydroxyl group, amino group, carboxyl group, amide group, aryl group, alkyl group, and modified alkyl group.

7. The preparation method of a protein-based phenylborate dynamic cross-linked hydrogel material according to claim 6, characterized in that, The biocompatible medium is selected from distilled water, physiological saline, buffer solution, or cell culture medium solution; In the hydrogel precursor solution formed by mixing solution A and solution B evenly, the certain concentration is 0.1% w / v - 60% w / v, and the molar ratio of the phenylboronic acid group to the vicinal diol group is 1:0.01 - 100; The mixing for a certain time is 0.001 - 24 h.

8. The preparation method of a protein-based phenylborate dynamic cross-linked hydrogel material according to claim 7, characterized in that, During the preparation of the protein-based phenylborate dynamic cross-linked hydrogel material, in addition to adding component A and component B, component C is also added. The component C is a beneficial component for tissue regeneration and repair, including drugs, bioactive factors, or cells; The drugs include lidocaine, insulin, local anesthetics, antibiotics, anti-inflammatory drugs, and anticancer drugs; the bioactive factors include BMP-2 to BMP-9, EGF, TGFα, TGFβ, FGF, IGF-I, IGF-II, NGF; The cells include osteoblasts, chondrocytes, endothelial cells, myoblasts, fibroblasts, nerve cells, mesenchymal stem cells, and embryonic stem cells.

9. The protein-based phenylborate dynamic cross-linked hydrogel material prepared by the method described in any one of claims 6-8.

10. Use of the protein-based phenylborate dynamic cross-linked hydrogel material according to claim 9, characterized in that, Select one of the following applications: The application of the protein-based phenylborate dynamic cross-linked hydrogel material for preparing a skin wound injury repair material; The application of the protein-based phenylborate dynamic cross-linked hydrogel material for preparing a urethral wound injury repair material; The application of the protein-based phenylborate dynamic cross-linked hydrogel material for preparing an articular cartilage defect regeneration and repair material; The application of the protein-based phenylborate dynamic cross-linked hydrogel material for preparing a bone defect regeneration and repair material; The application of the protein-based phenylborate dynamic cross-linked hydrogel material for preparing a substrate material for three-dimensional cell culture; The application of the protein-based phenylborate dynamic cross-linked hydrogel material as a 3D printing bioink.

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