Chemically modified silk proteins and their use for cell and organoid culture

Abstract

The present invention relates to chemically modified silk proteins and their use for the culture of cells and organoids. The chemically modified silk protein is a product obtained by sequentially carboxylating silk protein and modifying the silk protein with organic amine with a phenol group. The invention also provides the hydrogel of the chemically modified silk protein. The hydrogel system disclosed by the invention has similar multilevel structures and mechanical properties to an extracellular matrix, has the characteristics of definite components, controllable physical and chemical properties, low cytotoxicity, good biocompatibility, biodegradability and the like, and can support the growth and differentiation of cells and organoids; the matrigel is suitable for cell and organoid culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tissue engineering and biomaterials, relates to the field of high polymer materials, hydrogel materials and biomaterials, and in particular to a chemically modified silk fibroin, a preparation method thereof and a use for cell and organoid culture. BACKGROUND

[0002] Matrigel, which is widely used for cell and organoid culture at present, is a basement membrane matrix extracted from mouse sarcoma, which has problems of tumor origin, complex and unclear composition, large batch difference, low yield and high price, etc., which limits its wide application in drug development and regenerative medicine. In recent years, the development of hydrogel systems with clear composition, good repeatability, customizable component performance and the ability to simulate extracellular matrix (ECM) for three-dimensional culture of cells and organoids has become a hot spot in the field of biomedical research, and has a broad application and market prospect in the future.

[0003] Silk fibroin (simplified as silk fibroin) is a macromolecular protein derived from natural mulberry silk, which has excellent biocompatibility, mechanical properties and biodegradability. At present, silk fibroin-based materials have been widely used in many fields such as tissue engineering and biomedical engineering, and related medical devices have been approved for use in clinical practice.

[0004] Based on the many advantages of silk fibroin, it has great potential to develop silk fibroin-based hydrogel systems for three-dimensional culture of cells and organoids. In the past few years, various methods have been developed for preparing silk fibroin-based hydrogels, which can be divided into physical crosslinking and chemical crosslinking. Physical crosslinking is to promote silk fibroin to self-assemble by applying external stimuli such as pH change, electric field, shear force, surfactant and organic solvent, etc., to promote silk fibroin to self-assemble, to change from random coil structure to β-sheet structure, and finally to form hydrogel. Chemical crosslinking of silk fibroin usually requires the introduction of chemical crosslinking agents to form a crosslinked network. At present, whether based on physical crosslinking or chemical crosslinking, the silk fibroin-based hydrogels developed cannot simultaneously have the properties of rapid and mild gelation, mechanical properties matching cells, good cell compatibility, stable gelation, etc., and inevitably bring certain cytotoxicity. Not suitable for three-dimensional cell culture.

[0005] Therefore, it is a current research hotspot to develop a new type of silk fibroin-based hydrogel material to meet the needs of cell and organoid culture. SUMMARY

[0006] OBJECTIVE

[0007] One technical objective of the present application is to provide a chemically modified silk fibroin and a preparation method thereof.

[0008] Another technical purpose of the present invention is to provide a series of methods for forming hydrogels from the above-mentioned chemically modified silk proteins and the chemically modified silk protein hydrogels prepared by these methods.

[0009] Another technical purpose of the present invention is to provide the use of the above-mentioned chemically modified silk protein hydrogel material in biomedical materials, especially in cell or organoid culture. SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention provides a method for preparing organic amine-modified carboxylated silk protein, the method comprising: subjecting the carboxylated silk protein to an amidation reaction with an organic amine to obtain the organic amine-modified carboxylated silk protein, wherein:

[0012] The carboxylated silk protein is a silk protein with the following side chain structure:

[0013]

[0014] Wherein, R represents a C2-C6 alkylene group, Indicates the connection to the side chain from here;

[0015] The organic amine carries -NH2 and phenol groups, and undergoes an amidation reaction with the carboxyl group of the carboxylated silk protein through -NH2.

[0016] In a specific embodiment, the carboxylated silk protein is a silk protein whose serine side chain contains the above structure.

[0017] Serine, also known as β-hydroxyalanine, has a structure of In the carboxylated silk protein, the structure Attached to the hydroxyl group on the serine side chain.

[0018] In the carboxylated silk protein, the structure The modification rate of serine is between 20-90%. The molar percentage of serine relative to the total amount of serine.

[0019] The source of the carboxylated silk protein is not particularly limited and can be a commercially available product or can be synthesized according to prior art methods, for example, with reference to the method disclosed in CN115433369A (the entire disclosure of which is incorporated herein by reference). For example, the carboxylated silk protein can be prepared by reacting silk protein with a dianhydride (e.g., succinic anhydride).

[0020] In an embodiment, R represents ethylene or propylene.

[0021] In an embodiment, the organic amine can be represented by the following structure:

[0022]

[0023] wherein R1 represents a C1-C6 alkylene group.

[0024] In a particular embodiment, R1 represents a methylene, ethylene, propylene or butylene group.

[0025] In a particular embodiment, in the organic amine, R1 is attached to the para or meta position, in particular the para position, of the hydroxyl group.

[0026] In a particular embodiment, the organic amine is tyramine, i.e. p-hydroxyphenylethylamine, having the structure

[0027] In a particular embodiment, the organic amine can be used as a free amine or as a salt, such as a hydrochloride salt.

[0028] In a particular embodiment, the molar ratio of carboxylated silk fibroin to organic amine can be from 0.5 to 10:1, such as 1 :2, 1 : 1, 2: 1, 5: 1, 10: 1, in terms of the molar ratio of the carboxyl groups contained in the carboxylated silk fibroin to the amino groups contained in the organic amine; preferably 1 : 1.

[0029] In the specific embodiment, the amidation reaction can be performed in the presence of a condensing agent. The condensing agent can be, for example, an acyl chloride-based condensing agent such as thionyl chloride, phosgene, phosphorus trichloride, phosphorus oxychloride, phosphorus pentachloride; an acid anhydride-based condensing agent such as ethyl chloroformate, isobutyl chloroformate, N,N-carbonyldiimidazole (CDI), dimethyl triflate (CBMIT), methane sulfonyl chloride (MsCl), p-toluenesulfonyl chloride (TsCl), p-nitrobenzenesulfonyl chloride (NsCl), Boc anhydride; a carbodiimide-based condensing agent such as dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC); an onium salt-based condensing agent including a carbonium salt-based condensing agent such as 2-(7-oxabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate quaternary ammonium salt (TNTU), and a phosphonium salt-based condensing agent such as benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxyl)tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP); an organophosphorus-based condensing agent such as diphenylphosphoryl chloride (DPP-Cl), diethyl cyanophosphonate (DECP), diphenyl phosphorazide (DPPA), thiodimethylphosphoryl azide (MPTA), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (BOP-Cl), but the present application is not limited thereto.

[0030] In the case of using a carbodiimide-based condensing agent, a condensation activator is generally required to be added, and the condensation activator commonly used includes 4-N,N-dimethylpyridine (DMAP), 4-pyrrolidinylpyridine (4-PPy), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azobenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (NHPI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), pentafluorophenol (PFPOH), and the like, but the present application is not limited thereto.

[0031] In the specific embodiment, the method is performed as follows, but is not limited thereto,

[0032] S1: preparing a carboxylated silk fibroin solution;

[0033] S2: adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxylated silk protein solution obtained from S1 to activate the carboxylated silk protein;

[0034] S3: adding tyramine hydrochloride to the activated carboxylated silk protein in S2 to react, obtaining tyramine-modified carboxylated silk protein.

[0035] Figure 1 An exemplary schematic diagram showing synthesis of tyramine-modified carboxylated silk protein according to one embodiment, in which silk protein is first reacted with succinic anhydride to obtain carboxylated silk protein, and then reacted with tyramine in the presence of EDC / NHS to obtain amine-modified carboxylated silk protein, but the present application is not limited thereto.

[0036] In a specific embodiment, the carboxylated silk protein solution is prepared by dissolving carboxylated silk protein powder in an aqueous solution of 2-(N-morpholino)ethanesulfonic acid, the concentration of carboxylated silk protein in the carboxylated silk protein solution being 1-100 mg / mL, and the concentration of 2-(N-morpholino)ethanesulfonic acid being 5-100 mM.

[0037] In a specific embodiment, in S2, the amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide added is 172.5-1725 mg, preferably 345 mg, and the amount of N-hydroxysuccinimide added is 103.5-1035 mg, preferably 207 mg, per 1 g of carboxylated silk protein.

[0038] In a specific embodiment, in S3, the amount of tyramine hydrochloride added is 31.4-314 mg per 1 g of carboxylated silk protein. In some embodiments, the reaction temperature is 18-25°C, the reaction time is 1-24 hours, preferably 2 hours, the pH of the reaction system is 6, and preferably, the reaction is carried out with shaking at a rotation speed of 25-100 rpm.

[0039] In a specific embodiment, after step S3, dialysis, lyophilization, and powdering operations can also be included to prepare powdered tyramine-modified carboxylated silk protein.

[0040] In another aspect, the present application provides an organic amine-modified carboxylated silk protein containing

[0041] wherein R represents C2-C6 alkylene, preferably ethylene or propylene;

[0042] R1 represents C1-C6 alkylene, preferably methylene, ethylene, propylene, or butylene;

[0043] denotes the attachment to the side chain from this position.

[0044] In particular embodiments, R1is attached to the para or meta position of the hydroxyl group, in particular the para position.

[0045] In particular embodiments, In particular embodiments, wherein R is defined as above.

[0046] In particular embodiments, the total content of phenol groups in the organic amine-modified carboxylated silk protein is 4.6-10 mol%, for example 7.1 mol%, based on the total number of moles of amino acid units in the silk protein.

[0047] In particular embodiments, the organic amine-modified carboxylated silk protein can be obtained by the method for preparing an organic amine-modified carboxylated silk protein according to the present application.

[0048] In another aspect, the present application provides a method for preparing an organic amine-modified carboxylated silk protein hydrogel, the method comprising:

[0049] S1-1: dissolving the above organic amine-modified carboxylated silk protein in water to obtain an organic amine-modified carboxylated silk protein aqueous solution;

[0050] S1-2: dividing the solution obtained in S1-1 into two equal parts, one part is added with hydrogen peroxide to prepare solution A, and the other part is added with horseradish peroxidase (HRP) to prepare solution B;

[0051] S1-3: mixing the A solution and the B solution in equal volumes to obtain an organic amine-modified carboxylated silk protein hydrogel.

[0052] In particular embodiments, in S1-1, the concentration of the organic amine-modified carboxylated silk protein in the organic amine-modified carboxylated silk protein solution is 25-50 mg / mL.

[0053] In particular embodiments, in S1-2, the concentration of hydrogen peroxide in solution A is 0.01-0.16 wt%, for example 0.06 wt%, and the concentration of horseradish peroxidase in solution B is 1-20 U / mL. It should be noted that the concentration of hydrogen peroxide will affect the mechanical properties of the hydrogel formed, for example when the concentration of hydrogen peroxide in the finally prepared hydrogel is 0.03 wt%, the hydrogel shows excellent mechanical properties.

[0054] In particular embodiments, in S1-3, the mixing temperature of the A solution and the B solution is 20-55°C, for example at 37°C.

[0055] During the hydrogel formation process, HRP catalyzes H2O2 to generate oxygen free radicals, which produces active phenol groups in the organic amine-modified carboxylated silk protein. The phenolic hydroxyl group ortho position of the tyrosine residue or grafted tyramine is coupled with the phenolic hydroxyl group ortho position of another tyrosine residue or grafted tyramine to form dityrosine.

[0056] In another aspect, the present invention provides an organic amine-modified carboxylated silk protein hydrogel prepared by the above method.

[0057] In another aspect, the present invention provides a method for preparing a carboxylated silk protein hydrogel, the method comprising:

[0058] The carboxylated silk protein aqueous solution is allowed to stand at room temperature or under heating conditions to prepare the carboxylated silk protein hydrogel.

[0059] In a specific embodiment, the concentration of carboxylated silk protein in the aqueous solution of carboxylated silk protein is 1-100 mg / mL.

[0060] In a specific embodiment, the solution is preferably placed at a temperature of 20-80° C. for a period of 0.5-240 hours.

[0061] In another aspect, the present invention provides a carboxylated silk protein hydrogel prepared by the above method.

[0062] In another aspect, the present invention provides a matrix gel for cell or organoid culture, which comprises at least the above-mentioned organic amine-modified carboxylated silk protein hydrogel or the above-mentioned carboxylated silk protein hydrogel.

[0063] In a specific embodiment, the hydrogel further comprises natural materials known in the art for promoting interaction between cells and hydrogels, improving cell adhesion, and promoting cell differentiation. The types of these natural materials are not limited, and examples thereof include laminin, collagen, fibronectin, gelatin, serum protein, lactoprotein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate, and carboxymethyl cellulose.

[0064] On the other hand, the present invention provides uses of the organic amine-modified carboxylated silk protein, the organic amine-modified carboxylated silk protein hydrogel, or the carboxylated silk protein hydrogel in the preparation of biomedical materials.

[0065] In a specific embodiment, the biomedical material can be used for organoid culture, cell delivery, biological 3D printing, tissue filling, tissue regeneration, drug sustained release and the like.

[0066] In another aspect, the present invention provides a kit for cell and organoid culture, comprising:

[0067] the carboxylated silk protein modified by the organic amine described above; a hydrogen peroxide solution and a horseradish peroxidase; and optionally, a natural material for promoting the interaction between cells and the material, improving cell adhesion or promoting cell differentiation; or

[0068] the carboxylated silk protein described above; and optionally, a natural material for promoting the interaction between cells and the material, improving cell adhesion or promoting cell differentiation.

[0069] In specific embodiments, the natural material can be selected from the group consisting of laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate, carboxymethyl cellulose.

[0070] In specific embodiments, the concentration of the hydrogen peroxide solution is 1 wt%.

[0071] In specific embodiments, the kit can further comprise a culture medium for cell and organoid culture. For example, the culture medium can be a DMEM medium used in three-dimensional culture of cells, or an EB formation medium, an induction medium, an expansion medium and a maturation medium used in culture of organoids. For the EB formation medium, the induction medium, the expansion medium and the maturation medium, for example, reference can be made to STEMdiff TM A brain-like organ kit.

[0072] In another aspect, the present application provides a method for three-dimensional culture of cells, the method comprising:

[0073] S2-1: dissolve the carboxylated silk protein modified by the organic amine described above in a DMEM medium, divide the solution into two parts, one part is added with hydrogen peroxide to obtain solution A', and the other part is added with horseradish peroxidase to obtain solution B';

[0074] S2-2: digest, collect, centrifuge and resuspend the adherent cells to prepare a cell suspension;

[0075] S2-3: add the cell suspension to solution B' of step S2-1, then mix solutions A' and B' in equal volumes, and incubate in a 37°C incubator for 1 minute;

[0076] S2-4: after the gel of S2-3 is completely formed into a hydrogel, add a culture medium to cover the hydrogel, and place in a 37°C incubator for three-dimensional culture of cells.

[0077] In another aspect, the present application provides a method for three-dimensional culture of organoids, the method being one of the following methods:

[0078] Method (i), using the carboxylated silk protein modified by the organic amine described above as a Matrigel for organoid culture, the method comprising:

[0079] The carboxylated silk fibroin modified by the above organic amine is dissolved in the expansion culture medium, blended with the natural material, and the solution is divided into two parts, one part is added with hydrogen peroxide to obtain solution A", and the other part is added with horseradish peroxidase to obtain solution B";

[0080] Equal volumes of solution A" and B" are simultaneously added dropwise on human induced pluripotent stem cell induced culture to form embryoid bodies (EBs), incubated in a 37°C incubator for 1-20 minutes to obtain EBs encapsulated by the hydrogel, and then added with the expansion culture medium and placed in a 37°C shaker for shaking culture; starting from the 11th day, the culture medium is replaced with an induced brain-like organ maturation culture medium for subsequent culture.

[0081] Method (ii), using the carboxylated silk fibroin hydrogel described above as Matrigel for organoid culture, the method comprising:

[0082] The carboxylated silk fibroin is dissolved in the expansion culture medium, blended with the natural material, and placed in a 37°C incubator for incubation to form a carboxylated silk fibroin hydrogel;

[0083] The carboxylated silk fibroin hydrogel is added dropwise on human induced pluripotent stem cell induced culture to form embryoid bodies (EBs), incubated in a 37°C incubator for 60-180 minutes to obtain EBs encapsulated by the hydrogel, and then added with the expansion culture medium and placed in a 37°C shaker for shaking culture; starting from the 11th day, the culture medium is replaced with an induced brain-like organ maturation culture medium for subsequent culture.

[0084] In specific embodiments, in the above cell three-dimensional culture method or the above organoid three-dimensional culture method, the natural material is as described above. The amount of the natural material added can be determined by a person skilled in the art according to his common sense or routine operation, and is not limited.

[0085] In specific embodiments, in the above cell three-dimensional culture method or the above organoid three-dimensional culture method, the selection of the culture medium depends on the cultured cells, and a person skilled in the art can reasonably select it according to his common knowledge, for example, using DMEM culture medium in cell three-dimensional culture, and using STEMdiff TM A brain-like organ kit, the kit comprising an EB formation culture medium, an induction culture medium, an expansion culture medium and a maturation culture medium, specifically, during the organoid culture process, using the EB formation culture medium for 0-5 days, using the induction culture medium for 5-7 days, using the expansion culture medium for 7-10 days, and using the maturation culture medium for 10-40 days or more, for details of the use of each culture medium, please refer to STEMdiff TM Instructions for using the brain-like organ kit.

[0086] In specific embodiments, in the above-mentioned cell three-dimensional culture method or the above-mentioned organoid three-dimensional culture method, the type of cells for culture includes but is not limited to mouse fibroblasts, human umbilical vein vascular endothelial cells, human induced pluripotent stem cells, and human embryonic stem cells, and the final concentration of the cells in the hydrogel is 3 x 10 5 -1 x 10 6

[0087] In specific embodiments, in the above-mentioned cell culture method or the above-mentioned organoid culture method, the concentration of the organic amine-modified carboxylated silk protein in the culture medium is 25-50 mg / mL; and the concentration of the carboxylated silk protein in the culture medium is 1-100 mg / mL.

[0088] In specific embodiments, the natural material is selected from one or all of laminin, collagen, and fibronectin, and preferably, the concentration of the natural material in the culture medium is: laminin 2-200 μg / mL; collagen 10-1000 μg / mL; fibronectin 4-400 μg / mL; and hyaluronic acid 0.02-2 mg / mL.

[0089] In specific embodiments, the concentration of horseradish peroxidase in the finally formed organic amine-modified carboxylated silk protein hydrogel is 5-10 U / mL; and the concentration of hydrogen peroxide in the finally formed organic amine-modified carboxylated silk protein hydrogel is 0.005wt%-0.03wt%.

[0090] In specific embodiments, human induced pluripotent stem cells are inoculated in an ultra-low adsorption 96-well plate at 9000 cells / well, and after 7 days of induction culture, embryoid bodies (EBs) are formed, and the diameter of the formed embryoid bodies is 400-600 μm.

[0091] Advantages

[0092] The present application further uses an organic amine containing a phenol group (e.g., tyramine) to modify the carboxylated silk protein, thereby increasing the phenol group in the silk protein and accelerating the formation of the silk protein hydrogel.

[0093] Further, according to the present application, the hydrogel system obtained from the carboxylated silk protein or the tyramine-modified carboxylated silk protein has a multi-level structure and mechanical properties similar to those of the extracellular matrix, and has the characteristics of clear composition, controllable physical and chemical properties, low cytotoxicity, good biocompatibility, and biodegradability, and can support the growth and differentiation of cells and organoids. At the same time, the chemical modification silk protein hydrogel has a simple preparation process, can be prepared in large quantities, and the cost is controllable, which helps to realize the batch and high-throughput culture of cells and organoids, and solves the pain points of traditional Matrigel, such as tumor origin, complex composition, and large batch differences. ​

[0094] Further, the present application combines the methods of chemical modification and physical complexation of silk fibroin, avoids the use of chemical cross-linking agents, and further improves the biocompatibility of silk fibroin-based hydrogel materials. In addition, by combining chemical modification with active substance compounding, not only the physical and chemical properties of the hydrogel material are precisely adjusted, but also the biological activity of the hydrogel material is improved, and the interaction between cells and the hydrogel material is enhanced, which is suitable for three-dimensional culture of cells and organoids.

[0095] Further, the carboxylated silk fibroin or tyramine-modified carboxylated silk fibroin hydrogel developed by the present application also has injectability, printability and biodegradability, and has broad application prospects in the fields of organoid culture, cell delivery, biological 3D printing, tissue filling, tissue regeneration, drug release and other biomedical fields. BRIEF DESCRIPTION OF DRAWINGS

[0096] Figure 1 : Synthesis diagram of tyramine-modified carboxylated silk fibroin.

[0097] Figure 2 : Nuclear magnetic hydrogen spectrum of tyramine-modified carboxylated silk fibroin obtained in Preparation Example 1.

[0098] Figure 3 : Preparation of tyramine-modified carboxylated silk fibroin hydrogel, and sample photos before and after gelation.

[0099] Figure 4 : Changes in storage modulus of tyramine-modified carboxylated silk fibroin hydrogel during gelation in Example 4.

[0100] Figure 5 : Ultraviolet spectra of tyramine-modified carboxylated silk fibroin hydrogel before and after gelation in Example 4.

[0101] Figure 6 : Changes in ultraviolet absorption at 335 nm during the gelation of tyramine-modified carboxylated silk fibroin hydrogel in Example 4.

[0102] Figure 7 : Storage modulus of tyramine-modified carboxylated silk fibroin hydrogel (prepared in Examples 1-5, respectively) cross-linked under different hydrogen peroxide concentrations. Note: The hydrogen peroxide concentration in the figure is the concentration in the hydrogel.

[0103] Figure 8 : Compression modulus of tyramine-modified carboxylated silk fibroin hydrogel (prepared in Examples 1-5, respectively) cross-linked under different hydrogen peroxide concentrations. Note: The hydrogen peroxide concentration in the figure is the concentration in the hydrogel.

[0104] Figure 9Compression modulus of tyramide-modified carboxylated silk fibroin hydrogels prepared in Example 4 at different time points during 28 days incubation at 37 °C.

[0105] Figure 10 Beta-sheet structure content of tyramide-modified carboxylated silk fibroin hydrogels prepared in Example 4 at different time points during 28 days incubation at 37 °C.

[0106] Figure 11 Cell viability of NIH / 3T3 cells cultured on the surface of tyramide-modified carboxylated silk fibroin hydrogels in Example 10 at the first and third day.

[0107] Figure 12 Cell viability of NIH / 3T3 cells cultured inside tyramide-modified carboxylated silk fibroin hydrogels for 48 hours in Example 11. Scale bar, 200 μm.

[0108] Figure 13 Schematic diagram of encapsulating embryoid bodies inside tyramide-modified carboxylated silk fibroin hydrogels / carboxylated silk fibroin hydrogels in Examples 12 and 13.

[0109] Figure 14 Growth of organoids inside tyramide-modified carboxylated silk fibroin hydrogels in Example 12 and Comparative Example. Scale bar, 500 μm.

[0110] Figure 15 Immunofluorescence staining of neural differentiation specific markers of organoids inside tyramide-modified carboxylated silk fibroin hydrogels at the 15th day in Example 12. Scale bar, 200 μm.

[0111] Figure 16 Immunofluorescence staining of neural differentiation specific markers of organoids inside tyramide-modified carboxylated silk fibroin hydrogels at the 40th day in Example 12. Scale bar, 50 μm.

[0112] Figure 17 Growth of organoids inside carboxylated silk fibroin hydrogels in Example 13. Scale bar, 500 μm.

[0113] Figure 18 Immunofluorescence staining of neural differentiation specific markers of organoids inside carboxylated silk fibroin hydrogels at the 15th day in Example 13.

[0114] Figure 19 Immunofluorescence staining of neural differentiation specific markers of organoids inside carboxylated silk fibroin hydrogels at the 40th day in Example 13.

[0115] Note: In the above figures, SA represents carboxylated silk protein; SA-TA represents tyramine-modified carboxylated silk protein; and SF represents silk protein. DETAILED DESCRIPTION

[0116] Reagents and cell lines:

[0117] 2-(N-morpholino)ethanesulfonic acid (Aladdin), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (TCI), N-hydroxysuccinimide (TCI), hydrogen peroxide (Aladdin), horseradish peroxidase (ThermoFisher), mouse fibroblast NIH / 3T3 cell line (Beina Biotechnology), human induced pluripotent stem cells, hiPSCs (Frontier Innovation Center, School of Basic Medical Sciences, Fudan University), Alamar Blue kit (Thermo Fisher), Calcein AM / PI live / dead staining kit (Lambo Lead), Gibco TM DMEM high glucose medium (Thermo Fisher), STEMdiff TM Brain organoid kit (which includes EB formation medium, EB induction medium, expansion medium, and maturation medium), Matrigel.

[0118] Terminology: In the present application, organic amine refers to an organic amine containing a phenol group, a representative example of which is tyramine.

[0119] Preparation Example 1: Preparation of tyramine-modified carboxylated silk protein

[0120] 500 mg of carboxylated silk protein powder (synthesized according to Example 1 of CN 115433369 A, except that the reaction time was 3 hours) was dissolved in 25 mL of 1×2-(N-morpholine)ethanesulfonic acid solution. After complete dissolution, 103.5 mg of N-hydroxysuccinimide and 172.5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added and mixed by concussion for 30 minutes. 78.5 mg of tyramine hydrochloride was then added and the mixture was allowed to react by concussion for two hours. The solution was then transferred to a dialysis bag and dialyzed against 0.5×PBS for 36 hours, with the PBS solution replaced every 12 hours. The resulting solution was then centrifuged at 9000 rpm and the supernatant was collected. Finally, the tyramine-modified carboxylated silk protein powder was obtained by freeze drying.

[0121] The obtained tyramine-modified carboxylated silk protein was subjected to nuclear magnetic hydrogen spectrum analysis using a 600 MHz liquid nuclear magnetic resonance spectrometer (ultra-low temperature probe) (Bruker AVANCE NEO, Bruker Corporation).

[0122] Method: 10-20 mg of tyramine-modified carboxylated silk fibroin was dissolved in 0.5 mL of 1.0 M DMSO-d6 / LiCl. 1 H NMR nuclear magnetic resonance spectrum width is 0 ppm to 13 ppm, scanning 16 times. The results are shown in Figure 1. Figure 2 .

[0123] Calculation of the modification rate of tyramine: the peaks at 6.79 and 6.96 ppm in the nuclear magnetic hydrogen spectrum correspond to the four hydrogen atoms between the ortho and meta positions of the tyramine phenolic hydroxyl group, and the peak at 1.26 ppm corresponds to the three hydrogen atoms of the methyl group on the alanine of silk fibroin. The modification ratio of tyramine can be calculated by comparing the peak areas.

[0124] wherein the content of the phenolic group in the tyramine-modified carboxylated silk fibroin is calculated by the following formula:

[0125]

[0126] P(phenol) represents the total content of the phenolic group in the tyramine-modified carboxylated silk fibroin;

[0127] P(Ala) represents the molar ratio of alanine in silk fibroin (28.6 mol%);

[0128] S SA-TA-phenolic represents the area integral of the benzene ring hydrogen in the tyramine and tyrosine residues;

[0129] S SA-TA-Ala represents the area integral of the methyl hydrogen in the alanine residues.

[0130] It is calculated that the total content of the phenolic hydroxyl group in the tyramine-modified carboxylated silk fibroin is 7.1 mol%.

[0131] Example 1: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0132] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.01 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed in equal volume at 37°C to form a hydrogel.

[0133] Example 2: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0134] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.02 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0135] Example 3: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0136] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.04 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0137] Example 4: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0138] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.06 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0139] Example 5: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0140] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.08 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0141] Example 6: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0142] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 50 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.04 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0143] Example 7: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0144] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a 50 mg / mL tyramine-modified carboxylated silk fibroin solution. The solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.08 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0145] Example 8: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0146] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a 50 mg / mL tyramine-modified carboxylated silk fibroin solution. The solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.12 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0147] Example 9: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0148] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a 50 mg / mL tyramine-modified carboxylated silk fibroin solution. The solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.16 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0149] Test Example 1:

[0150] The gelation process of tyramine-modified carboxylated silk fibroin in Example 4 was tested by time sweep using an ARES-G2 rotational rheometer. The test conditions were: 1% strain, frequency 1 Hz, sweep time 1500 seconds, and temperature 37°C. The results are shown in Figure 4 . The figure shows the increase in storage modulus of tyramine-modified carboxylated silk fibroin during the transition from solution to gel state.

[0151] Test Example 2:

[0152] The UV spectra of tyramine-modified carboxylated silk fibroin before and after gelation in Example 4 were determined using a UV-visible spectrophotometer. The results are shown in Figure 5 . The figure shows that a new absorption peak appears at 335 nm after crosslinking of tyramine-modified carboxylated silk fibroin.

[0153] Test Example 3:

[0154] The gelation process of tyramine-modified carboxylated silk fibroin in Example 4 was measured by UV-Vis spectrophotometer at 335 nm. The results are shown in Figure 6 Figure. The results show that tyramine-modified carboxylated silk fibroin can reach the platform of cross-linking reaction in 136 s under the conditions of 10 U / mL HRP, 0.03 wt% H2O2, and temperature 37 °C.

[0155] Test Example 4:

[0156] The hydrogels in Examples 1-5 were tested for storage modulus by ARES-G2 rotational rheometer at 1% strain and 10 Hz. The results are shown in Figure 7 (Note: The H2O2 concentration shown in the figure is the concentration in the hydrogel). The results show that for 25 mg / mL tyramine-modified carboxylated silk fibroin, the optimal H2O2 concentration corresponding to the optimal storage modulus is 0.03 wt% under the conditions of 10 U / mL HRP, temperature 37 °C, and H2O2 concentration range 0.005-0.04 wt%.

[0157] Test Example 5:

[0158] The hydrogels in Examples 1-5 were tested for compression modulus by CellScale Univert mechanical tester at 30% strain and compression speed 0.667% s -1 , the results are shown in Figure 8 (Note: The H2O2 concentration shown in the figure is the concentration in the hydrogel). The results show that for 25 mg / mL tyramine-modified carboxylated silk fibroin, the optimal H2O2 concentration corresponding to the optimal compression modulus is 0.03 wt% under the conditions of 10 U / mL HRP, temperature 37 °C, and H2O2 concentration range 0.005-0.04 wt%.

[0159] Test Example 6:

[0160] The hydrogel in Example 4 was stored in PBS and placed in a 37 °C incubator, and the compression modulus was tested on days 0, 7, 14, 21, and 28, respectively. The results are shown in Figure 9 The results show that the mechanical properties of tyramine-modified carboxylated silk fibroin hydrogel can be maintained stable within 28 days.

[0161] Test Example 7:

[0162] The hydrogel in Example 4 was stored in PBS and placed in a 37 °C incubator, and was freeze-dried on days 0, 7, 14, 21, and 28, respectively, and was subjected to infrared spectrum determination. The spectrum was recorded by 64 scans, and the spectrum range was 400 to 4000 cm -1 , and the resolution was 4.0 cm -1 . The data analysis results show that 1620 and 1698 cm-1 peaks at 1645 and 1685 cm -1 peaks at 1645 and 1685 cm

[0163] The results of infrared spectrum peak separation are shown in Figure 10 The results show that the content of β-sheet structure in tyramine-modified carboxylated silk fibroin hydrogel slightly increases but maintains at a low level (below 5%) within 28 days.

[0164] Example 10: Cell surface culture using tyramine-modified carboxylated silk fibroin hydrogel

[0165] The adherent-cultured NIH / 3T3 cells were digested, centrifuged, supernatant removed, and resuspended to obtain a cell suspension. Tyramine-modified carboxylated silk fibroin powder was dissolved in DMEM medium to form a protein solution with a concentration of 25 mg / mL and filtered to remove bacteria. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (concentration 0.06 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. 50 μL of solution A and 50 μL of solution B were added to a 96-well plate, mixed uniformly to form a hydrogel, and placed in an incubator at 37°C for 1 minute. After complete gelation, 5000 cells were added to each well, 100 μL of culture medium was added to cover the surface of the hydrogel, and placed in a 37°C incubator for culture. On the first day and the third day, cell viability was determined by Alamar Blue reagent, and the results are shown in Figure 11 It can be seen from the figure that NIH / 3T3 cells can proliferate on the surface of tyramine-modified carboxylated silk fibroin hydrogel.

[0166] Example 11: Cell encapsulation using tyramine-modified carboxylated silk fibroin hydrogel

[0167] The adherent-cultured NIH / 3T3 cells were digested, centrifuged, supernatant removed, and resuspended to obtain a cell suspension. Tyramine-modified carboxylated silk fibroin powder was dissolved in DMEM medium to form a protein solution with a concentration of 25 mg / mL and filtered to remove bacteria. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (concentration 0.06 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) and added with cell suspension (final concentration 1*10 6 The adherent-cultured NIH / 3T3 cells were digested, centrifuged, supernatant removed, and resuspended to obtain a cell suspension. Tyramine-modified carboxylated silk fibroin powder was dissolved in DMEM medium to form a protein solution with a concentration of 25 mg / mL and filtered to remove bacteria. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (concentration 0.06 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) and added with cell suspension (final concentration 1*10 Figure 12NIH / 3T3 cells were able to grow inside tyramide-modified carboxylated silk fibroin hydrogels with a survival rate of over 90%.

[0168] Example 12: Tyramide-modified carboxylated silk fibroin hydrogels for brain organoid culture

[0169] According to STEMdiff TM According to the brain organoid kit instruction, hiPSCs were cultured in ultra-low attachment plates to form embryoid bodies. On day 9, the embryoid bodies were observed to be 400-600 pm in size, with smooth edges and forming optically translucent edges. A solution of 25 mg / mL tyramide-modified carboxylated silk fibroin powder was prepared by dissolving the powder in expansion medium, and 20 pg / mL laminin, or 100 pg / mL collagen, or 40 pg / mL fibronectin, or a mixture of the three proteins was mixed in the solution, which was then filtered to remove bacteria. The protein solution was divided into two parts, one of which was prepared into solution A (0.04 wt% concentration) by adding hydrogen peroxide, and the other was prepared into solution B by adding horseradish peroxidase solution (10 U / mL). Parafilm was laid flat and pressed gently with tweezers to form small grooves on the parafilm. The parafilm was then ultraviolet sterilized for 60 minutes. The embryoid bodies were transferred to the grooves on the parafilm using a cut-off dropper, and the original induction medium was removed. Then, 10 pL of solution A and 10 pL of solution B were added in sequence, and the embryoid bodies were embedded in the hydrogel after one minute. The hydrogel with the embedded embryoid bodies was placed in a 24-well plate, and 500 pL of expansion medium was added to each well. The plate was placed in a 37°C incubator and cultured for 3 days with shaking. The surface of the embedded embryoid bodies developed sprouts. The original expansion medium was removed, and 500 pL of maturation medium was added to each well. The plate was placed in a 37°C incubator and cultured with shaking, and the medium was changed every 3 days. TM Brain organoid kit instruction)

[0170] The embryoid bodies will continue to grow and eventually develop into brain organoids. The culture process is shown in Figure 13 The growth and development of the organoids are shown in Figure 14 On day 15 and day 40, the brain organoids were fixed, paraffin-embedded, and sectioned, and immunofluorescence staining was performed on specific markers SOX2 (stem cell marker), Nestin (neural precursor cell marker), Tuj-1 (class III beta tubulin), PAX6 (glial cell marker), FOXG1 (forebrain marker), DCX (doublecortin), MAP2 (microtubule-associated protein 2), CTIP (cortical plate neuron marker), GFAP (astrocyte marker), and NeuN (neuronal nuclear marker). The results are shown in Figure 15 (day 15) and Figure 16(day 40). The staining results showed that the organoids appeared neural differentiation cells and specific "rosette" structure at day 15, and more mature neural differentiation cells at day 40.

[0171] Comparative Example: Matrigel-based brain organoid culture

[0172] According to STEMdiff TM According to the brain organoid kit operation manual, the hiPSCs were cultured to form embryoid bodies in ultra-low adsorption plates. On the 9th day, the embryoid bodies were observed to be 400-600 pm in size, with smooth edges and forming optically translucent edges. The embryoid bodies were removed with a cut-off pipette, and the residual medium was aspirated. Matrigel was thawed in advance in the 4-degree refrigerator, and 15 pL of Matrigel was added to the embryoid bodies using a pre-cooled gun head to completely coat them. The Matrigel-coated embryoid bodies were incubated in a 37°C incubator for half an hour, and then the expansion medium was added and cultured in a 37°C incubator for 3 days. The embedded embryoid bodies developed sprouts on the surface. The original expansion medium was aspirated, and 500 pL of maturation medium was added to each well. The culture was placed in a 37°C incubator for shaking culture, and the medium was changed every 3 days. The embryoid bodies would continue to grow and eventually develop into brain organoids. The brain organoids were fixed on the 15th and 40th days, paraffin-embedded, and sectioned for immunofluorescence staining of specific markers SOX2, Nestin, Tuj-1, PAX6, FOXG1, DCX, MAP2, CTIP, GFAP, and NeuN. The results are shown in FIGS. 6A-6D and 7A-7D. Figure 14 、 Figure 15 、 Figure 16 .

[0173] The results of Example 12 and the comparative example showed that, compared with Matrigel (comparative example), the tyramide-modified carboxylated silk fibroin hydrogel could support the growth and development of brain organoids, with a size close to that of Matrigel. The sections of brain organoids in Matrigel and tyramide-modified carboxylated silk fibroin hydrogel at day 15 had a characteristic "rosette" structure, and had SOX2, Nestin, Tuj-1, PAX6, and FOXG1 protein expression; the section staining at day 40 showed that all had FOXG1, DCX, MAP2, CTIP, GFAP, and NeuN protein expression.

[0174] Example 13: Carboxylated silk fibroin hydrogel for brain organoid culture

[0175] According to STEMdiff TMThe brain-like organ kit operation instruction is that the hiPSCs are cultured in an ultra-low adsorption plate to form a blastoid. On the 9th day of observation, the blastoid is 400-600 μm in size, has a smooth edge and forms an optically translucent edge. Carboxylated silk protein (synthesized in Example 1 of CN 115433369 A, except that the silk protein degumming time is 2 hours and the carboxylation reaction time is 30 minutes) is dissolved in an expansion culture medium to form a solution of 20 mg / mL, 40 μg / mL of laminin, or 20 μg / mL of collagen, or 10 μg / mL of fibronectin, or 0.2 mg / mL of hyaluronic acid, or four ingredients are simultaneously added according to the above concentrations, and filtered to remove bacteria. The carboxylated silk protein solution is incubated in a 37°C incubator in advance to form a hydrogel. The sealing film is laid flat, and a small groove is formed on the sealing film by gently pressing it with tweezers. Ultraviolet sterilization is performed for 60 minutes. The blastoid is transferred to the groove on the sealing film using a cut-off dropper, the original induction culture medium is aspirated, 20 μL of carboxylated silk protein hydrogel is added, and the blastoid is embedded in the hydrogel after being placed in a 37°C incubator for 2 hours. The blastoid-embedded hydrogel is placed in a 24-well plate, and 500 μL of expansion culture medium is added to each well. The plate is placed in a 37°C incubator and shaken for 3 days. The original expansion culture medium is aspirated, and 500 μL of maturation culture medium is added to each well. The plate is placed in a 37°C incubator and shaken, and the culture medium is replaced every 3 days. (Note: The culture medium replacement in the brain-like organ culture refers to STEMdiff TM Brain-like organ kit operation instruction)

[0176] The blastoid will continue to grow and eventually develop into a brain-like organ. The culture process is shown in Figure 17 On the 15th day and the 40th day, the brain-like organ is peeled off from the carboxylated silk protein hydrogel, fixed, frozen sectioned, and subjected to immunofluorescence staining for specific markers SOX2, Nestin, Tuj-1, PAX6, DCX, MAP2, CTIP, GFAP, NeuN, and CD31 (a platelet endothelial cell marker). Among them Figure 18 (day 15) shows the results of using Matrigel, carboxylated silk protein hydrogel, and carboxylated silk protein hydrogel with laminin, fibronectin, or hyaluronic acid added at the above concentrations, or collagen, laminin, fibronectin, and hyaluronic acid added to the brain-like organ culture, Figure 19 (day 40) shows the results of using Matrigel and carboxylated silk protein hydrogel for brain-like organ culture. The staining results show that on the 15th day, the brain-like organ appears to be a neural differentiation cell and a specific "rosette" structure, and on the 40th day, the brain-like organ appears to be a more mature neural differentiation cell.

Claims

1. A method for preparing carboxylated silk protein modified with an organic amine, the method comprising: Carboxylated silk protein is subjected to amidation reaction with organic amine to obtain organic amine-modified carboxylated silk protein, wherein The carboxylated silk protein is a silk protein with the following side chain structure: Wherein, R represents a C2-C6 alkylene group, Indicates the connection to the side chain from here; The organic amine carries -NH2 and phenol groups, and undergoes an amidation reaction with the carboxyl group of the carboxylated silk protein through -NH2. Preferably, In the carboxylated silk protein, structure The modification rate of serine ranges from 20-90%. R represents an ethylene group or a propylene group; The organic amine is represented by the following structure: Wherein, R1 represents a C1-C6 alkylene group, Preferably, R1 represents methylene, ethylene, propylene or butylene, Preferably, in the organic amine, R1 is connected to the para or meta position of the hydroxyl group, especially the para position, In particular, the organic amine is tyramine.

2. The method according to claim 1, wherein the method is carried out as follows: S1: Preparation of carboxylated silk protein solution; S2: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxylated silk protein solution obtained in S1 to activate the carboxylated silk protein; S3: Add tyramine hydrochloride to the carboxylated silk protein activated in S2 to react and obtain tyramine-modified carboxylated silk protein. Preferably, the carboxylated silk protein solution is prepared by dissolving carboxylated silk protein powder in an aqueous solution of 2-(N-morpholino)ethanesulfonic acid, wherein the concentration of carboxylated silk protein in the carboxylated silk protein solution is 1-100 mg / mL, and the concentration of 2-(N-morpholino)ethanesulfonic acid is 5-100 mM. Preferably, in S2, relative to 1 g of carboxylated silk protein, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide added is 172.5-1725 mg, preferably 345 mg, and the amount of N-hydroxysuccinimide added is 103.5-1035 mg, preferably 207 mg. Preferably, in S3, relative to 1 g of carboxylated silk protein, the amount of tyramine hydrochloride added is 31.4-314 mg, the reaction temperature is 18-25° C., the reaction time is 1-24 hours, preferably 2 hours, the pH of the reaction system is 6, and the reaction is carried out at a shaking speed of 25-100 rpm.

3. An organic amine modified carboxylated silk protein containing in, R and R1 are as defined in claim 1, Indicates the connection to the side chain from here, Preferably, based on the total molar number of amino acid units in the silk protein, the total content of phenol groups in the organic amine-modified carboxylated silk protein is 4.6 mol%-10 mol%, for example, 7.1 mol%.

4. A method for preparing an organic amine-modified carboxylated silk protein hydrogel, the method comprising: S1-1: dissolving the organic amine-modified carboxylated silk protein according to claim 3 in water to obtain an organic amine-modified carboxylated silk protein aqueous solution; S1-2: The solution obtained in S1-1 was divided into two equal parts. Hydrogen peroxide was added to one part to prepare solution A, and horseradish peroxidase (HRP) was added to the other part to prepare solution B. S1-3: Mix equal volumes of solution A and solution B to obtain organic amine-modified carboxylated silk protein hydrogel. Preferably, in S1-1, the concentration of the organic amine-modified carboxylated silk protein in the organic amine-modified carboxylated silk protein solution is 25-50 mg / mL; In S1-2, the concentration of hydrogen peroxide in solution A is 0.01 wt%-0.16 wt%, for example, 0.06 wt%; the concentration of horseradish peroxidase in solution B is 1-20 U / mL; In S1-3, the mixing temperature of solution A and solution B is 20-55°C, for example, at 37°C.

5. An organic amine-modified carboxylated silk protein hydrogel prepared by the method according to claim 4.

6. A method for preparing a carboxylated silk protein hydrogel, the method comprising: The carboxylated silk protein aqueous solution is allowed to stand at room temperature or under heating conditions to prepare a carboxylated silk protein hydrogel, wherein the carboxylated silk protein is as defined in claim 1.

7. A carboxylated silk protein hydrogel prepared by the method according to claim 6.

8. A matrix gel for cell or organoid culture, comprising at least: the organic amine-modified carboxylated silk protein hydrogel according to claim 5 or the carboxylated silk protein hydrogel according to claim 7, and Optionally, a natural material, the natural material is selected from the group consisting of laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate and carboxymethylcellulose.

9. Use of the organic amine-modified carboxylated silk protein according to claim 3, the organic amine-modified carboxylated silk protein hydrogel according to claim 5, or the carboxylated silk protein hydrogel according to claim 7 in the preparation of biomedical materials. Preferably, the biomedical material is used for organoid culture, cell delivery, biological 3D printing, tissue filling, tissue regeneration, and drug sustained release.

10. A kit for cell and organoid culture, comprising: The organic amine-modified carboxylated silk protein according to claim 3; hydrogen peroxide solution and horseradish peroxidase; and optionally, a natural material for promoting interaction between cells and materials, improving cell adhesion, and promoting cell differentiation; or Carboxylated silk protein as defined in claim 1; and optionally, a natural material for promoting interaction between cells and materials, improving cell adhesion, and promoting cell differentiation, Preferably, the natural material is selected from laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate and carboxymethyl cellulose, More preferably, the natural material is selected from laminin, collagen, and fibronectin.

11. A three-dimensional cell culture method, comprising: S2-1: dissolving the organic amine-modified carboxylated silk protein as claimed in claim 3 in DMEM culture medium, dividing the solution into two parts, adding hydrogen peroxide to one part to obtain solution A', and adding horseradish peroxidase to the other part to obtain solution B'; S2-2: The adherent cells are digested, collected, centrifuged, and resuspended to prepare a cell suspension; S2-3: Add the cell suspension to solution B' from step S2-1, then mix equal volumes of solutions A' and B', and incubate in a 37°C incubator for 1 minute; S2-4: After the gel of S2-3 is completely formed into a hydrogel, culture medium is added to cover the hydrogel and placed in a 37°C incubator for three-dimensional cell culture.

12. A method for culturing a three-dimensional organoid, the method being one of the following methods: Method (i): The organic amine-modified carboxylated silk protein according to claim 3 is dissolved in an expansion medium, mixed with natural materials, and the solution is divided into two parts, hydrogen peroxide is added to one part to obtain solution A', and horseradish peroxidase is added to the other part to obtain solution B'; Equal volumes of solution A and solution B were simultaneously added dropwise to embryoid bodies (EBs) formed by induced culture of human induced pluripotent stem cells, and incubated in a 37°C incubator for 1-20 minutes to obtain hydrogel-encapsulated EBs. Expansion medium was then added and the cells were placed in a 37°C shaker for shaking culture. Starting on the 11th day, the culture medium was replaced with brain organoid maturation-inducing medium for subsequent culture. Method (ii) dissolving the carboxylated silk protein as defined in claim 1 in an expansion medium, mixing the mixture with a natural material, and incubating the mixture in a 37° C. incubator to form a carboxylated silk protein hydrogel; The carboxylated silk protein hydrogel was dropped onto the embryoid body (EB) formed by the induction culture of human induced pluripotent stem cells, and incubated in a 37°C incubator for 60-180 minutes to obtain hydrogel-encapsulated EB. Then, the expansion medium was added and the cells were placed in a 37°C shaker for shaking culture. Starting from the 11th day, the culture medium was replaced with the brain organoid maturation-inducing medium for subsequent culture.

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