A protein adhesion coating, its preparation method, and its application

Abstract

This application relates to the field of polymer materials technology, and particularly to a protein adhesion coating and its preparation method. The protein adhesion coating provided in this application comprises resin I and resin II. Resin I is obtained by reacting maleic anhydride, polyethylene glycol, pyrrole, and hexanediol diacrylate, while resin II is obtained by polymerizing aminosulfonate-modified polyisocyanate with hexanediol. The mass ratio of resin I to resin II is 6-9:7-8. This application utilizes the curing of resin I and resin II to form a protein adhesion coating. Because these two resins contain hydrophobic segments, they can generate hydrophobic interactions and π-π stacking with substrates such as PS culture dishes to enhance adhesion, preventing the substrate from detaching in water. Simultaneously, these two resins contain a large number of amide bonds and zwitterions, which can form hydrogen bonds and electrostatic adsorption with proteins, promoting cell adhesion.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polymer materials, in particular to a protein adhesion coating and a preparation method and application thereof. BACKGROUND

[0002] Modern biomedical science develops rapidly, and stem cell culture becomes very important basic work in many research and application fields. Adherent cell refers to a cell that must have a support surface to adhere to during animal cell culture, and can grow and proliferate on the surface by secreting adhesion factors by itself or in the culture medium. When the cell grows on the support surface, it generally forms two types of cells, i.e. fibroblast-like cells or epithelial-like cells. Adherent cells are divided into: epithelial cell type, fibroblast cell type, wandering cell type and multiple cell type. When observed under a microscope, the adherent cells stretch and extend into shuttle-shaped or irregular triangular or fan-shaped or other shapes at the bottom of the bottle, and the cells do not move when the culture solution is shaken.

[0003] Adhesion and stretching are basic growth characteristics of most in vitro cultured cells, and play an important role in the formation and stability of animal cell morphology. When the cells cannot adhere well, the growth state of the cells is usually not good, which affects the subsequent application. The main reason for cell adhesion is to secrete extracellular matrix and adhesion factors, so as to improve the structure of the culture dish or culture bottle, and then the cells naturally adhere to the culture dish or culture bottle. Cell adhesion molecules are a general term for a large number of molecules that mediate the contact and combination between cells or between cells and extracellular matrix. Extracellular matrix is a large molecule secreted by animal cells into the extracellular matrix, distributed on the cell surface or between cells. Most of them are glycoproteins, so they have strong hydrophilicity. Therefore, whether the cells can adhere not only depends on whether the cells have this ability, but also depends on whether there is a suitable substrate (culture dish or culture bottle). For a long time, polystyrene (PS) material culture dishes or culture bottles have been widely used as cell culture tools. Polystyrene has good light transmission performance, good strength and easy plasticity, and is non-toxic, so it has become the preferred material for disposable cell culture dishes and cell culture plates and other disposable cell culture consumables. However, the surface of polystyrene is hydrophobic, so it needs to be modified to become hydrophilic before it can be used for cell culture.

[0004] TC treatment (Tissue culture treated) is the most common surface hydrophilic modification treatment method of polystyrene culture dishes, usually using plasma surface treatment technology. The hydrophilic surface formed by plasma modification treatment of polystyrene reaches a hydrophilic contact angle of 60°. Patent US11608447B2 discloses a material for cell patterning, specifically discloses using component A with polyethylene glycol segments and component B with trialkoxysilyl as a light-modifiable polymer. After treating the substrate with this polymer, the contact angle of the substrate in the irradiated area of ultraviolet light (360-370nm) is 42.9°-50.5°, and the contact angle of the substrate in the non-irradiated area is 40.7°-46.8°.

[0005] However, for cells that grow under harsh conditions and adhere to the substrate, the hydrophilic contact angle needs to be maintained below 30°, so it is very important to provide a coating that can maintain the hydrophilic contact angle of the substrate within a reasonable range. SUMMARY

[0006] The embodiments of the present application provide a protein adhesion coating to solve the problem of a large hydrophilic contact angle of a substrate after plasma surface treatment or light-modifiable polymer treatment in the related art.

[0007] In a first aspect, the present application provides a protein adhesion coating, comprising resin I and resin II, wherein the resin I is obtained by reacting maleic anhydride, polyethylene glycol, pyrrole and hexanediol diacrylate, and the resin II is obtained by polymerization reaction of amino sulfonate modified polyisocyanate and hexanediol; and the mass ratio of the resin I to the resin II is 6-9:7-8.

[0008] In some embodiments, the structural formula of the resin I is as follows:

[0009]

[0010] In the formula, n is 9-45.

[0011] In some embodiments, the structural formula of the resin II is as follows:

[0012]

[0013] In the formula, X is C1-C16 alkyl.

[0014] In some embodiments, maleic anhydride is first reacted with polyethylene glycol to obtain polyethylene glycol maleic acid monoester, then the polyethylene glycol maleic acid monoester is reacted with pyrrole to obtain pyrrole-polyethylene glycol maleic acid monoester adduct, and then the pyrrole-polyethylene glycol maleic acid monoester adduct is reacted with hexanediol diacrylate to obtain resin I.

[0015] In some embodiments, the molar ratio of maleic anhydride to polyethylene glycol is 1:1-1.3; the molar ratio of polyethylene glycol maleate monoester to pyrrole is 1:1-1.3; and the molar ratio of pyrrole-polyethylene glycol maleate monoester adduct to hexanediol diacrylate is 1:1.5-2.

[0016] In some embodiments, the molar ratio of sulfamate-modified polyisocyanate to hexanediol is 1:1.3-1.7.

[0017] In some embodiments, the protein adhesion coating further comprises a photoinitiator selected from one or more of 2-hydroxy-2-methyl-1-phenylpropanone (1173), 1-hydroxycyclohexyl phenyl ketone (184), and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO). The photoinitiator can absorb energy of a certain wavelength in the ultraviolet light region (250-420 nm) to generate free radicals, cations, and the like, thereby initiating polymerization and crosslinking of the monomers.

[0018] In some embodiments, the protein adhesion coating further comprises a surfactant selected from one or more of nonylphenol polyoxyethylene ether (NPE), octylphenol polyoxyethylene ether (OPE), and polyvinyl alcohol (PVA). The surfactant can reduce the surface tension of the coating and enhance the ability of cells to adhere to the surface of the coating.

[0019] In some embodiments, the protein adhesion coating further comprises a leveling agent selected from an acrylate leveling agent with a molecular weight of 8000-15000. The leveling agent can effectively reduce the surface tension of the coating, improve its leveling and uniformity, and promote the formation of a smooth, flat, and uniform coating film during the drying and film-forming process.

[0020] In some embodiments, the protein adhesion coating further comprises a photocurable diluent comprising one or more of acrylic acid, itaconic acid, and hydroxyethyl acrylate.

[0021] In some embodiments, the protein adhesion coating further comprises a solvent A selected from one or more of ethyl acetate, butyl acetate, isopropyl alcohol, and ethanol.

[0022] In a second aspect, the present application provides a method for preparing a protein adhesion coating, comprising the following steps:

[0023] In mass parts, 3-5 parts of a surfactant, 30-45 parts of resin I, 35-40 parts of resin II, and 10-25 parts of a photocurable diluent are mixed, solvent A is added, and stirring is performed for 0.5-1 h. Then, 3-5 parts of a photoinitiator and 1-2 parts of a leveling agent are added, and stirring is performed to obtain a protein adhesion coating liquid. The protein adhesion coating liquid is coated on a substrate, and a photo-curing treatment is performed to obtain a protein adhesion coating.

[0024] In some embodiments, the resin I is prepared by the following process:

[0025] (1) adding maleic anhydride and polyethylene glycol into solvent B, heating and reacting to obtain polyethylene glycol maleate monoester;

[0026] (2) adding polyethylene glycol maleate monoester and pyrrole into solvent B, heating and reacting to obtain pyrrole-polyethylene glycol maleate monoester adduct;

[0027] (3) adding pyrrole-polyethylene glycol maleate monoester adduct and hexanediol diacrylate into solvent B, heating and reacting, and after the reaction is completed, washing with water and filtering to obtain the photocuring resin.

[0028] In some embodiments, the solvent B is a mixture of one or more of ethyl acetate, propylene glycol methyl ether and butyl acetate.

[0029] In some embodiments, the molar ratio of maleic anhydride, polyethylene glycol and solvent B is 1:1-1.3:15-25.

[0030] In some embodiments, the molar ratio of pyrrole-polyethylene glycol maleate monoester adduct, hexanediol diacrylate and solvent B is 1:1.5-2:30-40.

[0031] In some embodiments, in step (1), the heating and reaction is carried out at a temperature of 40-70℃ for 6-10h; in step (2), the heating and reaction is carried out at a temperature of 60-90℃ for 8-12h; and in step (3), the heating and reaction is carried out at a temperature of 60-80℃ for 6-10h.

[0032] In some embodiments, the resin II is prepared by the following process: adding aminosulfonic acid salt modified polyisocyanate and hexanediol into solvent C, heating and reacting, removing the solvent after the reaction is completed, and washing with water and filtering to obtain the resin II.

[0033] In some embodiments, the molecular weight of the resin II is 2000-3000.

[0034] In some embodiments, the heating temperature during the preparation of the resin II is 80-120℃, and the heating time is 8-16h.

[0035] In some embodiments, the molar ratio of aminosulfonic acid salt modified polyisocyanate, hexanediol and solvent C is 1:1.3-1.7:30-40.

[0036] In some embodiments, the solvent C is any one or more of ethyl acetate, propylene glycol methyl ether and butyl acetate.

[0037] In some embodiments, the solvent A is added in an amount of 0.3-3 times the total mass of the surfactant, the resin I, the resin II, the light-curable diluent, the photoinitiator, and the leveling agent.

[0038] In some embodiments, the light-curing process is pre-baking at 60-80℃ for 2-3 min, followed by UV LED light curing for 30-60 s, and the energy of the UV LED light is 500-1000 mJ / cm 2 .

[0039] In some embodiments, the coating method is any one of spraying, pouring, dripping, scraping, or rolling.

[0040] In some embodiments, the thickness of the protein-adhesion coating is 5-40 μm.

[0041] In a third aspect, the present application also provides the use of the protein-adhesion coating described above, which can be used for cell adhesion culture, specifically, cells are planted on a PS culture dish / culture flask / culture plate containing the protein-adhesion coating for culture, and the cells can be difficult-to-cultivate cells such as mouse embryonic fibroblasts and mouse adipose adult stem cells.

[0042] The resin I provided by the present application contains hydrophobic structures such as carbon chains and bridged rings, and hydrophilic structures such as ethylene glycol segments in the molecular structure. The resin I as a whole exhibits hydrophilicity. Since the resin I has hydrophobic structures, it can be well attached to a hydrophobic PS culture dish. In addition, the side chain of the resin I contains a large number of carboxyl groups, which helps to adhere proteins. The resin I is a light-curable resin, which can be rapidly cross-linked and cured under UV irradiation.

[0043] The resin II provided by the present application contains isocyanurate groups in the carbon chain skeleton, and thus can produce hydrophobic interaction and π-π stacking with a PS culture dish. At the same time, the resin II contains a large number of amide bonds and zwitterions, which can produce hydrogen bonds and electrostatic adsorption with proteins.

[0044] The resin I and the resin II provided by the present application are both linear structures, and the resin I is topologically entangled with the resin II through hydrogen bonds and hydrophobic interaction during light-curing cross-linking, forming a heterogeneous network structure, thereby improving the mechanical properties of the resin.

[0045] The beneficial effects brought by the technical solutions provided by the present application include:

[0046] 1. The protein-adhesion coating formed by the curing of the resin I and the resin II provided by the present application can produce hydrophobic interaction and π-π stacking with a substrate such as a PS culture dish, thereby enhancing the adhesion and preventing the substrate from falling off in water; at the same time, the two resins contain a large number of amide bonds and zwitterions, which can produce hydrogen bonds and electrostatic adsorption with proteins, thereby promoting cell adhesion;

[0047] 2、The water contact angle of the protein adhesion coating provided by the application is between 14-30°, which is beneficial to cell adhesion; when cells are cultured in a PS culture dish containing the protein adhesion coating provided by the application, the cell adhesion rate can reach 89%-92% and the cell growth state is good, while the adhesion rate of cells cultured in a common TC treated culture dish is only 40%-45%, and the adhesion rate of cells cultured in an untreated culture dish is only 15%-20%;

[0048] 3、The protein adhesion coating provided by the application exhibits excellent water resistance and aging resistance; after the culture dish coated with the protein adhesion coating is placed in a culture medium for 72h-120h, the coating has no obvious peeling, peeling or wrinkling phenomenon; after the culture dish coated with the protein adhesion coating is placed in an open environment for 1-2 years, the coating has no obvious peeling, peeling or wrinkling phenomenon before and after aging;

[0049] 4、The adhesion of the protein adhesion coating provided by the application is 0 level, the coating has low cytotoxicity, and the cell survival rate is above 96% after the cells are cultured in the culture dish containing the protein adhesion coating for 48h;

[0050] 5、The protein adhesion coating provided by the application can be quickly cured by simple UV irradiation, which is simple to operate, low in cost and suitable for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0052] Figure 1 The chemical reaction process schematic diagram for preparing resin I in Example 1 of the application is shown in the figure;

[0053] Figure 2 The chemical reaction process schematic diagram for preparing resin II in Example 1 of the application is shown in the figure;

[0054] Figure 3 The water contact angle diagram of the protein adhesion coating prepared in Example 1 is shown in the figure;

[0055] Figure 4 The bovine whey glycoprotein adhesion rate result schematic diagram is shown in the figure;

[0056] Figure 5 The live and dead staining diagram of human renal epithelial cells (293T) is shown in the figure;

[0057] Figure 6 The fibroblast and adult stem cell adhesion rate result schematic diagram is shown in the figure. DETAILED DESCRIPTION

[0058] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0059] The present application provides a protein adhesion coating, comprising the following raw materials in parts by mass: 3-5 parts of a surfactant, 30-45 parts of resin I, 35-40 parts of resin II, 10-25 parts of a photocurable diluent, 3-5 parts of a photoinitiator, and 1-2 parts of a leveling agent.

[0060] The applicant has found through a large number of experiments that if the mass fraction of the photoinitiator is higher than 5 parts, a large number of free radicals will be generated under ultraviolet light, resulting in a low molecular weight of the final polymer network formed after curing of the coating, a brittle coating film, poor adhesion, and an increased cost of the coating; if the mass fraction of the photoinitiator is lower than 1 part, the generation of free radicals under ultraviolet light will be insufficient, resulting in a high content of residual resin and monomers without photopolymerization, causing poor surface drying of the final coating film, stickiness, and unavailability.

[0061] The raw materials of the protein adhesion coating provided by the present application further comprise solvent A, which is selected from one or more of ethyl acetate, butyl acetate, isopropyl alcohol, and ethanol, and the addition amount of solvent A is 0.3-3 times the total mass of the other components. If the addition amount of solvent A is higher than 3 times the total weight of the other components, the coating is too thin, the coating film thickness is too low, and the hardness of the final coating film is too low, resulting in poor wear resistance. If the addition amount of solvent A is lower than 0.3 times the total weight of the other components, the viscosity of the coating is too large, which easily leads to poor leveling of the coating film, a too thick coating film, and incomplete curing.

[0062] The amino sulfonate-modified polyisocyanate used in the following examples is Bayhydur XP2655 from Covestro.

[0063] Example 1

[0064] Preparation of resin I: 1 mol of maleic anhydride and 1 mol of polyethylene glycol 600 are added to a three-necked flask, followed by 15 mol of ethyl acetate, dissolved by stirring, and reacted at 40℃ for 6 h, after which the solvent is removed to obtain a polyethylene glycol maleate monoester; 1 mol of the polyethylene glycol maleate monoester is added to 1 mol of pyrrole and 20 mol of ethyl acetate, and reacted at 60℃ for 8 h, after which the solvent is removed, washed with water, and filtered to obtain a pyrrole-polyethylene glycol maleate monoester adduct; 1 mol of the pyrrole-polyethylene glycol maleate monoester adduct is added to 1.5 mol of hexanediol diacrylate and 30 mol of ethyl acetate, and reacted at 60℃ for 9 h, after which the solvent is removed, washed with water, and filtered to obtain resin I.

[0065] The chemical reaction process for preparing resin I in Example 1 is shown in Figure 1 .

[0066] Preparation of resin II: 1 mol of aminosulfonate-modified polyisocyanate and 1.3 mol of hexanediol are added to a round-bottom flask, followed by 30 mol of ethyl acetate, dissolved by stirring, and reacted at 80℃ for 8 h, after which the solvent is removed, washed with water, and filtered to obtain resin II.

[0067] The chemical reaction process for preparing resin II in Example 1 is shown in Figure 2 .

[0068] Preparation of a protein-adhesion coating: 5 g of NPE, 30 g of resin I, 40 g of resin II, and 21 g of acrylic acid are weighed out at 1 mass part per 1 g, and then added to a solution of butyl acetate, isopropyl alcohol, and ethanol mixed at a volume ratio of 4:3:3 to form a solution, followed by mixing and stirring for 0.5 h; 3 g of a photoinitiator 1173 and 1 g of an acrylate are then added, mixed and stirred for 0.5 h, to obtain a protein-adhesion coating solution; the protein-adhesion coating solution is sprayed onto a PS culture dish, pre-baked at 60℃ for 2 min, and light-cured under a UV LED lamp for 40 s at an energy of 600 mJ / cm 2 , to obtain a protein-adhesion coating with a thickness of 15 μm.

[0069] In the preparation of the protein-adhesion coating, the amount of the mixed solution of butyl acetate, isopropyl alcohol, and ethanol is 0.5 times the total mass of the other raw materials.

[0070] Example 2:

[0071] Preparation of resin I: 1 mol of maleic anhydride and 1.3 mol of polyethylene glycol 600 are added to a three-necked flask, followed by 25 mol of ethyl acetate, stirred and dissolved, and then the solvent is removed after reaction at 70°C for 10 h to obtain polyethylene glycol maleate monoester; 1 mol of polyethylene glycol maleate monoester is added to 1.3 mol of pyrrole and 40 mol of ethyl acetate, and reacted at 90°C for 12 h, and then the solvent is removed after the reaction is completed, and water washing and filtration are performed to obtain pyrrole-polyethylene glycol maleate monoester adduct; 1 mol of pyrrole-polyethylene glycol maleate monoester adduct is added to 2 mol of hexanediol diacrylate and 40 mol of ethyl acetate, and reacted at 80°C for 10 h, and then the solvent is removed after the reaction is completed, and water washing and filtration are performed to obtain resin I.

[0072] Preparation of resin II: 1 mol of aminosulfonate-modified polyisocyanate and 1.5 mol of hexanediol are added to a round-bottom flask, followed by 35 mol of ethyl acetate, stirred and dissolved, and then the solvent is removed after reaction at 100°C for 12 h, and water washing and filtration are performed to obtain resin II.

[0073] Preparation of protein adhesion coating: 4 g of NPE, 45 g of resin I, 35 g of resin II, and 12 g of itaconic acid are weighed according to 1 mass part per 1 g, and then added to a solution of butyl acetate, isopropyl alcohol, and ethanol mixed at a volume ratio of 4:3:3 to form a solution, followed by stirring for 1 h, and then 3 g of photoinitiator 184 and 1 g of acrylate are added, and stirring is performed for 0.5 h to obtain a protein adhesion coating solution; the protein adhesion coating solution is dip-coated on a PS culture dish, pre-baked at 70°C for 2 min, and then light-cured for 30 s under a UV LED lamp with an energy of 500 mJ / cm 2 to obtain a protein adhesion coating with a thickness of 10 μm.

[0074] In the preparation of the protein adhesion coating, the amount of the mixed solution of ethyl acetate, isopropyl alcohol, and ethanol is 1 times the total amount of other raw materials.

[0075] Example 3:

[0076] Preparation of resin I: 1 mol of maleic anhydride and 1.2 mol of polyethylene glycol 600 are added to a three-necked flask, followed by 20 mol of ethyl acetate, stirred and dissolved, and then the solvent is removed after reaction at 50°C for 8 h to obtain polyethylene glycol maleate monoester; 1 mol of polyethylene glycol maleate monoester is added to 1.1 mol of pyrrole and 30 mol of ethyl acetate, and reacted at 70°C for 10 h, and then the solvent is removed after the reaction is completed, and water washing and filtration are performed to obtain pyrrole-polyethylene glycol maleate monoester adduct; 1 mol of pyrrole-polyethylene glycol maleate monoester adduct is added to 1.7 mol of hexanediol diacrylate and 35 mol of ethyl acetate, and reacted at 70°C for 8 h, and then the solvent is removed after the reaction is completed, and water washing and filtration are performed to obtain resin I.

[0077] Preparation of resin II: 1 mol of aminosulfonate-modified polyisocyanate and 1.7 mol of hexanediol were added to a round-bottom flask, followed by 40 mol of ethyl acetate, dissolved by stirring, and the solvent was removed after 16 h of reaction at 120℃. Resin II was obtained after washing with water and filtration.

[0078] Preparation of a protein-adhesion coating: 3 g of OPE, 40 g of resin I, 38 g of resin II, and 13 g of hydroxyethyl acrylate were weighed out as 1 mass part per 1 g, and then mixed and stirred for 1 h after adding a solution of butyl acetate, isopropyl alcohol, and ethanol mixed in a volume ratio of 4:3:3. Then, 4 g of a photoinitiator TPO and 2 g of an acrylate were added, and mixed and stirred for 0.5 h to obtain a protein-adhesion coating liquid. The protein-adhesion coating liquid was flow-coated on a PS culture dish, pre-baked at 70℃ for 2 min, and light-cured for 35 s under a UV LED lamp with an energy of 800 mJ / cm 2 , to obtain a protein-adhesion coating with a thickness of 5 μm.

[0079] In the preparation of the protein-adhesion coating, the amount of the mixed solution of butyl acetate, isopropyl alcohol, and ethanol was 1.5 times the total mass of the other raw materials.

[0080] Example 4:

[0081] Preparation of resin I: 1 mol of maleic anhydride and 1.1 mol of polyethylene glycol 600 were added to a three-necked flask, followed by 18 mol of propylene glycol methyl ether, dissolved by stirring, and the solvent was removed after 9 h of reaction at 60℃. A polyethylene glycol maleate monoester was obtained. To 1 mol of the polyethylene glycol maleate monoester, 1.2 mol of pyrrole and 25 mol of propylene glycol methyl ether were added, and reacted at 80℃ for 11 h. After the reaction was completed, the solvent was removed, and the product was washed with water and filtered to obtain a pyrrole-polyethylene glycol maleate monoester adduct. To 1 mol of the pyrrole-polyethylene glycol maleate monoester adduct, 1.8 mol of hexanediol diacrylate and 38 mol of propylene glycol methyl ether were added, and reacted at 70℃ for 9 h. After the reaction was completed, the solvent was removed, and the product was washed with water and filtered to obtain resin I.

[0082] Preparation of resin II: 1 mol of aminosulfonate-modified polyisocyanate and 1.4 mol of hexanediol were added to a round-bottom flask, followed by 35 mol of butyl acetate, dissolved by stirring, and the solvent was removed after 15 h of reaction at 100℃. Resin II was obtained after washing with water and filtration.

[0083] Preparation of the protein adhesion coating: 5 g of OPE, 35 g of resin I, 40 g of resin II, and 14 g of acrylic acid were weighed out according to 1 mass part per 1 g, and then mixed and stirred in a solution of butyl acetate, isopropyl alcohol, and ethanol mixed in a volume ratio of 4:3:3 for 1 h. Then, 3 g of a photoinitiator 1173 and 2 g of an acrylate were added, and mixed and stirred for 0.5 h to obtain a protein adhesion coating solution. The protein adhesion coating solution was sprayed on a PS culture dish, pre-baked at 70℃ for 3 min, and light-cured under a UV LED lamp for 50 s at an energy of 600 mJ / cm 2 , to obtain a protein adhesion coating with a thickness of 21 μm.

[0084] In the preparation of the protein adhesion coating, the mixed solution of ethyl acetate, isopropyl alcohol, and ethanol was added in an amount of 1.5 times the total mass of the other raw materials.

[0085] Example 5:

[0086] Preparation of resin I: 1 mol of maleic anhydride and 1.1 mol of polyethylene glycol 600 were added to a three-necked flask, followed by the addition of 22 mol of butyl acetate, and then stirred and dissolved. After reaction at 60℃ for 7 h, the solvent was removed to obtain a polyethylene glycol maleate monoester. Then, 1 mol of the polyethylene glycol maleate monoester was added to 1.2 mol of pyrrole and 25 mol of butyl acetate, and then reacted at 80℃ for 11 h. After the reaction was completed, the solvent was removed, and then washed with water and filtered to obtain a pyrrole-polyethylene glycol maleate monoester adduct. Then, 1 mol of the pyrrole-polyethylene glycol maleate monoester adduct was added to 1.9 mol of hexanediol diacrylate and 32 mol of butyl acetate, and then reacted at 70℃ for 9 h. After the reaction was completed, the solvent was removed, and then washed with water and filtered to obtain resin I.

[0087] Preparation of resin II: 1 mol of aminosulfonate-modified polyisocyanate and 1.6 mol of hexanediol were added to a round-bottom flask, followed by the addition of 32 mol of butyl acetate, and then stirred and dissolved. After reaction at 95℃ for 12 h, the solvent was removed, and then washed with water and filtered to obtain resin II.

[0088] Preparation of the protein adhesion coating: 5 g of OPE, 35 g of resin I, 40 g of resin II, and 14 g of acrylic acid were weighed out according to 1 mass part per 1 g, and then mixed and stirred in a solution of butyl acetate, isopropyl alcohol, and ethanol mixed in a volume ratio of 4:3:3 for 1 h. Then, 3 g of a photoinitiator 1173 and 2 g of an acrylate were added, and mixed and stirred for 0.5 h to obtain a protein adhesion coating solution. The protein adhesion coating solution was sprayed on a PS culture dish, pre-baked at 70℃ for 3 min, and light-cured under a UV LED lamp for 50 s at an energy of 600 mJ / cm 2 , to obtain a protein adhesion coating with a thickness of 21 μm.

[0089] In the process of preparing the protein adhesion coating, the amount of the mixed solution of butyl acetate, isopropyl alcohol and ethanol added is 2 times the total mass of other raw materials.

[0090] Example 6:

[0091] Preparation of resin I: 1 mol of maleic anhydride and 1.1 mol of polyethylene glycol 600 were added to a three-necked flask, followed by 20 mol of butyl acetate, stirring and dissolving, and then removing the solvent after 8 h of reaction at 55℃ to obtain a polyethylene glycol maleate monoester; 1 mol of the polyethylene glycol maleate monoester was added to 1.2 mol of pyrrole and 22 mol of butyl acetate, and the reaction was carried out at 85℃ for 10 h, after which the solvent was removed, and the pyrrole-polyethylene glycol maleate monoester adduct was obtained by water washing and filtration; 1 mol of the pyrrole-polyethylene glycol maleate monoester adduct was added to 1.8 mol of hexanediol diacrylate and 33 mol of butyl acetate, and the reaction was carried out at 75℃ for 8 h, after which the solvent was removed, and the resin I was obtained by water washing and filtration.

[0092] Preparation of resin II: 1 mol of aminosulfonate-modified polyisocyanate and 1.5 mol of hexanediol were added to a round-bottom flask, followed by 36 mol of propylene glycol methyl ether, stirring and dissolving, and then removing the solvent after 10 h of reaction at 100℃, and the resin II was obtained by water washing and filtration.

[0093] Preparation of the protein adhesion coating: 3 g of PVA, 42 g of resin I, 37 g of resin II and 12 g of hydroxyethyl acrylate were weighed according to 1 mass part per 1 g, and then butyl acetate, isopropyl alcohol and ethanol were mixed according to a volume ratio of 4:3:3 to form a solution, followed by mixing and stirring for 1 h, and then 5 g of a photoinitiator TPO and 1 g of an acrylate were added, followed by mixing and stirring for 0.5 h to obtain a protein adhesion coating solution; the protein adhesion coating solution was sprayed on a PS culture dish, pre-baked at 80℃ for 2 min, and then cured by a UV LED lamp for 40 s at an energy of 600 mJ / cm 2 , to obtain a protein adhesion coating with a thickness of 40 μm.

[0094] In the process of preparing the protein adhesion coating, the amount of the mixed solution of butyl acetate, isopropyl alcohol and ethanol added is 2.5 times the total mass of other raw materials.

[0095] Performance test:

[0096] The protein adhesion coatings obtained in Examples 1-6 were subjected to performance tests, and the specific test results and test processes are as follows:

[0097] I. Hydrophilicity test

[0098] The water contact angle of the protein adhesion coatings obtained in Examples 1-6 was tested by using a water contact angle tester: the water contact angle was between 14-30°, indicating that the protein adhesion coating has hydrophilicity.Figure 3 This is a water contact angle diagram of the protein adhesion coating prepared in Example 1. Figure 3 The water contact angle in it is 14°.

[0099] II. Water Resistance Test

[0100] Six PS culture dishes coated with the protein adhesion coatings of Examples 1-6 were immersed in culture medium for 72-120 hours. The coatings were tested before and after aging and no obvious peeling, flaking or wrinkling was observed.

[0101] III. Adhesion Test

[0102] The adhesion of the protein adhesive coatings obtained in Examples 1-6 was tested, and the adhesion of the coatings was measured to be grade 0 using the cross-cut adhesion test.

[0103] IV. Aging Test

[0104] Environmental aging tests were conducted on the protein adhesion coatings obtained in Examples 1-6. The samples were placed in an open-air environment for 1-2 years. Before and after aging, the coatings showed no obvious peeling, flaking, or wrinkling.

[0105] V. Glycoprotein Adhesion Test

[0106] Cell culture dishes containing the protein adhesion coating prepared in Example 1 and uncoated PS culture dishes were respectively incubated with fluorescently labeled bovine whey protein. Afterwards, the culture medium was removed, and the cells were washed twice with PBS solution (pH 10) to lyse the cells. The fluorescence intensity was measured using a microplate reader. The results showed that the adhesion rate of bovine whey protein in the coated culture dishes was 88%, while the adhesion rate in the uncoated culture dishes was 21%. This indicates that the protein adhesion coating improved cell adhesion. Figure 4 This is a schematic diagram showing the adhesion rate of bovine whey protein.

[0107] VI. Cytotoxicity Assessment

[0108] Cytotoxicity was detected using the CCK8 assay kit and the Calcein-AM / PI staining method. The cells used were human renal epithelial cells (293T), mouse peritoneal macrophages (RAW264.7), and human non-small cell lung cancer cells (A549).

[0109] Experimental results showed that the cell viability of the above cells was above 96% after culturing on PS culture dishes containing the protein adhesion coating prepared in Example 1 for 48 hours, indicating that the coating has low cytotoxicity. Figure 5 Image showing the live and dead staining of human renal epithelial cells (293T).

[0110] VII. Fibroblast Adhesion Rate Test

[0111] Mouse embryonic fibroblast (MEF) cell suspension was collected by centrifugation at 1500 rpm for 5 minutes, and then washed twice with 30 ml of growth medium. The cell pellet was resuspended in 15 ml of growth medium and added to PS culture dishes containing the protein adhesion coating prepared in Example 1, TC-treated, and untreated cells, respectively. The cells were then cultured in a 37°C cell culture incubator, and the culture medium was replaced with fresh medium after 24 hours. After the cells reached confluence, they were washed with D-PBS, discarded, and then digested with trypsin and passaged at a 1:5 ratio. Cells were counted at each passage, and cell adhesion was observed using a fluorescence microscope. The cell adhesion rate of PS culture dishes containing the protein adhesion coating was 89%, while the cell adhesion rate of TC-treated PS culture dishes was 40%, and the cell adhesion rate of untreated PS culture dishes was 15%. This indicates that the protein adhesion coating improved the cell adhesion rate.

[0112] VIII. Adult stem cell adhesion rate test

[0113] Mouse adipose-derived adult stem cells (ADAS cells) were cultured in PS culture dishes containing the protein adhesion coating prepared in Example 1, TC-treated, and untreated PS culture dishes, respectively. The cell adhesion rate of PS culture dishes with the protein adhesion coating was 92%. The cell adhesion rate of PS culture dishes treated with TC was 45%, and the cell adhesion rate of untreated PS culture dishes was 20%. The results are as follows. Figure 6 As shown.

[0114] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0115] It should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this application, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0116] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A protein adhesion coating, characterized in that, The resin I is obtained by reacting maleic anhydride, polyethylene glycol and pyrrole with hexanediol diacrylate, and the resin II is obtained by polymerization of amino sulfonate modified polyisocyanate and hexanediol; the mass ratio of the resin I and the resin II is 6-9:7-8; the resin I is prepared by first reacting maleic anhydride and polyethylene glycol to obtain polyethylene glycol maleate monoester, then reacting the polyethylene glycol maleate monoester with pyrrole to obtain pyrrole-polyethylene glycol maleate monoester adduct, and then reacting the pyrrole-polyethylene glycol maleate monoester adduct with hexanediol diacrylate.

2. The protein adhesive coating of claim 1, wherein, The molar ratio of maleic anhydride to polyethylene glycol is 1:1-1.3; the molar ratio of polyethylene glycol maleate monoester to pyrrole is 1:1-1.3; and the molar ratio of pyrrole-polyethylene glycol maleate monoester adduct to hexanediol diacrylate is 1:1.5-2.

3. The protein adhesive coating of claim 1, wherein, The molar ratio of amino sulfonate modified polyisocyanate to hexanediol is 1:1.3-1.

7.

4. The protein adhesive coating of claim 1, wherein, The protein adhesion coating further comprises a photoinitiator selected from one or more of 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexyl phenyl ketone, 2,4,6-trimethylbenzoyl-diphenyl phosphine oxide.

5. The protein adhesive coating of claim 1, wherein, The protein adhesion coating further comprises a surfactant selected from one or more of nonylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, polyvinyl alcohol.

6. The protein adhesive coating of claim 1, wherein, The protein adhesion coating further comprises a leveling agent selected from an acrylate leveling agent; the protein adhesion coating further comprises a photocurable diluent comprising one or more of acrylic acid, itaconic acid, hydroxyethyl acrylate; the protein adhesion coating further comprises a solvent A selected from one or more of ethyl acetate, butyl acetate, isopropyl alcohol, ethanol.

7. A method for the preparation of a protein adhesive coating according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: mixing 3-5 parts of a surfactant, 30-45 parts of resin I, 35-40 parts of resin II, 10-25 parts of a photocurable diluent by mass fraction, adding a solvent A, mixing and stirring for 0.5-1 h, then adding 3-5 parts of a photoinitiator and 1-2 parts of a leveling agent, mixing and stirring to obtain a protein adhesion coating liquid, coating the protein adhesion coating liquid on a substrate, and performing light curing treatment to obtain a protein adhesion coating.

8. The method for preparing a protein adhesive coating according to claim 7, characterized in that, The amount of the solvent A added is 0.3-3 times the total mass of the surfactant, the resin I, the resin II, the photocurable diluent, the photoinitiator and the leveling agent.

9. Use of the protein adhesion coating according to any one of claims 1-6 or prepared by the method according to any one of claims 7-8 in cell adhesion culture.

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