A polypeptide or protein elution coating, and a preparation method and application thereof

Abstract

The application discloses a polypeptide or protein elution coating and a preparation method and application thereof, and belongs to the technical field of biological and medical materials. The method comprises the following steps: performing first incubation on a substrate by using a mixed solution to form an adhesive gel coating on the surface of the substrate, wherein the mixed solution is obtained by mixing polyphenol and polypeptide or protein in an acidic solution system; and performing second incubation on the adhesive gel coating by using a polyamine or polythiol alkaline solution, and in-situ chemical cross-linking of the adhesive gel coating is realized through phenolamine chemistry, so that a solid coating with a highly phenolamine network structure wrapping polypeptide or protein is obtained. The method realizes preparation of a functional polypeptide or protein elution coating with adjustable and controllable multi-scene application, the polypeptide or protein elution coating formed through the above method has the characteristics of wide-spectrum modification, is independent of the material quality of the substrate, and has the stability of chemical structure.

Description

Technical Field

[0001] This invention relates to the field of biological and medical materials technology, and more specifically, to a polypeptide or protein elution coating, its preparation method, and its application. Background Technology

[0002] Peptides and proteins, as biologically functional macromolecules, participate in numerous fundamental biological processes within organisms and mediate specific interactions with different macromolecules. They play crucial roles in various applications such as catalysis, biosensing, diagnostics, and therapy. However, due to the sensitivity of proteins to their environment (temperature, pH, redox, etc.) and their specific requirements for spatial conformation, protein assembly often needs to be tailored to specific circumstances. Furthermore, simple and universal strategies for effectively assembling proteins onto substrates have limitations, as the non-covalent interactions between proteins and substrates are usually insufficient to firmly anchor them to the surface. Therefore, exploring simple, multifunctional peptide or protein loading strategies is of great significance.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide or protein elution coating, its preparation method, and its application to solve or improve the above-mentioned technical problems.

[0005] This application can be implemented as follows: In a first aspect, this application provides a method for preparing a polypeptide or protein elution coating, which includes the following steps: incubating a substrate with a mixed solution for the first time to form an adhesive gel coating on the surface of the substrate, wherein the mixed solution is obtained by mixing polyphenols with polypeptides or proteins in an acidic solution system; The adhesive gel coating is placed in an alkaline solution of polyamine or polythiol for a second incubation. In-situ chemical cross-linking of the adhesive gel coating is achieved through phenolamine chemistry, resulting in a solid coating with a highly phenolamine network structure that encapsulates peptides or proteins.

[0006] In an optional embodiment, the mixed solution is obtained by blending polyphenols and polypeptides in any molar ratio in a solvent with pH = 2-7, or by blending polyphenols and proteins in any molar ratio in a solvent with pH = 2-7.

[0007] In an optional embodiment, the molar ratio of polyphenols to polypeptides in the mixed solution is 0.01:1-100:1, or the molar ratio of polyphenols to proteins is 0.01:1-100:1.

[0008] In an optional embodiment, the concentration of polyphenols in the mixed solution is 0.01-100 mg / mL, and the concentration of polypeptides or proteins is 0.01-100 mg / mL.

[0009] In an optional embodiment, the mixed solution contains one polyphenol and two polypeptides; the molar ratio between the two polypeptides is 0.01:1-10:1, and the total concentration of the two polypeptides in the first incubation system is 0.001-10 mmol / mL.

[0010] In an optional embodiment, the mixed solution contains one polyphenol and two proteins; the molar ratio between the two proteins is 0.01:1 to 10:1; and the total concentration of the two proteins in the first incubation system is 0.001 to 10 mmol / mL.

[0011] In an optional embodiment, the molar ratio between the two peptides is 0.5:1 to 5:1, and the total concentration of the two peptides in the first incubation system is 0.01 to 5 mmol / mL.

[0012] In an optional implementation, the molar ratio between the two proteins is 0.5:1 to 5:1, and the total concentration of the two proteins in the first incubation system is 0.01 to 5 mmol / mL.

[0013] In an optional embodiment, the polyphenols include plant polyphenols containing a polyhydroxyphenolic chemical structure.

[0014] In an optional embodiment, the plant polyphenols include at least one of tannic acid, epigallocatechin, epicatechin gallate, epigallocatechin gallate, salvianolic acid, proanthocyanidins, theaflavins, and brown algae polyphenols.

[0015] In an optional embodiment, the polypeptide includes at least one of hirudin and its derivatives, bivalirudin, antimicrobial peptide LL-37, antimicrobial peptide hCAP-18, antimicrobial peptide C, antimicrobial peptide BLP-7, β-defensin, teriparatide, and osteocalcin.

[0016] In an optional embodiment, the protein includes at least one of lactoferrin, soy protein, whey protein, osteocalcin, insulin, collagen, osteothiin, cathepsin S, lysozyme, glucose oxidase, pepsin, horseradish peroxidase, glucose oxidase, pepsin, horseradish peroxidase, hemoglobin, cytochrome C, immunoglobulin G, fibrinogen, trypsin, and insulin.

[0017] In an optional implementation, the temperature for the first incubation is 0-50°C, and the time is 0.25-96 hours.

[0018] In an optional implementation, the temperature for the first incubation is 15-50°C, and the time is 15 minutes or more.

[0019] In an optional implementation, the temperature for the first incubation is 20-40°C, and the time is 0.25-24 hours.

[0020] In an optional embodiment, the concentration of the polyamine or polythiol alkaline solution is 0.01-1000 mg / mL.

[0021] In an optional embodiment, the solution pH of the polyamine or polythiol alkaline solution is 8-10.

[0022] In an optional embodiment, the polyamine or polythiol has the chemical formula Y-Xn-Y, where n≥2, X=CH2 or CH2OCH2, and Y=primary amino or thiol; or, the polyamine or polythiol has the chemical formula X n Y m Where n≥2, X=CH2 or CH2OCH2, Y=primary amino or mercapto, m≥2, Y can appear at any proton substitution position in the repeating structural unit of X.

[0023] In optional embodiments, the polyamine includes at least one of ethylenediamine, propylenediamine, butanediamine, hexamethylenediamine, heptanediamine, octanediamine, m-phenylenediamine, p-phenylenediamine, benzidine, polyethyleneimine, cystamine, and selenocystamine.

[0024] In an optional embodiment, the polythiol includes at least one selected from hexamethylenedimerol, dimercaptomethane, dimercaptobenzene, and trimercaptomethane.

[0025] In an optional embodiment, the solvent in the polyamine or polythiol alkaline solution is an alkaline buffer solution.

[0026] In an optional embodiment, the alkaline buffer solution includes at least one of carbamate buffer, bicarbonate buffer, PBS buffer, Tris-HCl buffer, ethanolamine buffer, sodium hydroxide solution, and potassium hydroxide solution.

[0027] In an optional embodiment, the alkaline buffer solution is a sodium hydroxide solution.

[0028] In an optional embodiment, the concentration of the alkaline buffer solution is 0.0001-40 mol / L.

[0029] In an optional implementation, the second incubation is carried out at a temperature of 0-50°C for a duration of 30 minutes or more.

[0030] In an optional implementation, the second incubation is carried out at a temperature of 25-40°C for 0.5-24 hours.

[0031] Secondly, this application provides a polypeptide or protein elution coating, which is prepared by any of the preparation methods described in the foregoing embodiments.

[0032] Thirdly, this application provides an application of the polypeptide or protein elution coating as described in the foregoing embodiments, which is used for surface modification of a substrate.

[0033] In optional embodiments, the substrate includes at least one of metallic materials, inorganic non-metallic materials, polymeric materials, biomedical micro / nanoparticles, natural biomaterials, and artificially synthesized polypeptide hydrogel materials.

[0034] In an optional embodiment, the metallic material includes at least one of stainless steel, iron and its alloys, magnesium and its alloys, titanium and its alloys, zinc and its alloys, and cobalt-based alloys.

[0035] In optional embodiments, the inorganic non-metallic material includes at least one of titanium dioxide, titanium oxide, titanium nitride, ceramics, silicon, silicon dioxide, silicon carbide, calcium phosphate silicon nitride, calcium phosphate, aluminosilicate, calcium aluminum bioglass, hydroxyapatite, carbon materials, diamond, and diamond-like carbon.

[0036] In optional embodiments, the polymeric material includes at least one selected from polyester, polyethylene, polyvinyl chloride, polyvinyl alcohol, polypropylene, polycarbonate, polyglycolic acid, polymethyl methacrylate, polytetrafluoroethylene, polyoxymethylene, polystyrene, polyvinyl acetate, polylactic acid, polylactic-glycolic acid copolymer, polytrimethylene carbonate, polyhydroxyalkanoates, polybutylene succinate, polyamide, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives, polyvinyl alcohol, polycaprolactone, and polyurethane.

[0037] In an optional embodiment, the biomedical micro / nanoparticles include at least one of iron oxide nanoparticles, silicon dioxide nanoparticles, titanium dioxide nanoparticles, and zinc oxide nanoparticles.

[0038] In an optional embodiment, the natural biomaterial includes at least one of polysaccharides and decellularized tissues and organs of animal origin; wherein the polysaccharides include at least one of plastic starch-based materials, gelatin, collagen, sodium hyaluronate, fibroin, sodium alginate, agarose, silk fibroin, keratin, cellulose, hemicellulose, lignin, chitin and their derivatives; and the decellularized tissues and organs of animal origin include at least one of blood vessels, valves, heart, bone, lungs, ligaments, bladder, mucous membranes and cornea.

[0039] In an optional embodiment, the synthetic peptide hydrogel material includes at least one of poly-L-lysine and poly-L-glutamic acid.

[0040] The beneficial effects of this application include: This application creatively proposes a method for preparing peptide or protein elution coatings. Utilizing the weak interactions between polyphenols and peptides (or proteins) under acidic conditions, such as hydrogen bonding and hydrophobic forces, a uniform hydrogel coating is deposited, maintaining the original physiological functions of the peptides (or proteins). The film-forming process does not require pH adjustment and can rapidly form films on the surfaces of metals, non-metals, and polymers. The corresponding preparation method is simple and easy to implement, showing great promise for industrial application.

[0041] The hydrogel coating material is placed in an alkaline polyamine or thiol solution, and in-situ chemical cross-linking of the gel coating is achieved through phenol amine chemistry. Thus, the gel coating based on hydrogen bonding and hydrophobic force assembly is transformed into a solid coating with a highly phenol amine network structure that encapsulates peptides or proteins.

[0042] The above method is simple and mild, enabling the preparation of functional peptide or protein elution coatings that are adjustable and controllable for multiple applications. The peptide or protein elution coatings formed by the above method have the characteristics of broad-spectrum modification, are independent of the substrate material, and have chemical structural stability. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a statistical chart showing the cumulative release of valerate in the hydrogel coatings and hydrogel conversion coatings prepared in Examples 3 and 6 of Experimental Example 1; Figure 2 The thrombin (TT) time results are shown in the figures for the hydrogel coatings and hydrogel conversion coatings prepared in Examples 3 and 6 of Experimental Example 2. Figure 3 The images show the anticoagulant effects of the hydrogel coatings before and after modification in each group of Experiment Example 3. Figure 4 The images show the antibacterial effects of the hydrogel coatings in each group before and after modification in Experiment Example 4. Figure 5 This is a comparison diagram of Example 3 and Comparative Example 3 before and after the hydrogel coating modification in Experimental Example 5. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0046] The following provides a detailed description of the polypeptide or protein elution coatings provided in this application, their preparation methods, and their applications.

[0047] The inventors propose that the loading of multifunctional peptides or proteins should consider the following aspects: i) effectively depositing peptides or proteins on various surfaces of different sizes, aspect ratios and material types to meet the needs of different application scenarios; ii) maintaining the integrity of the structure and function of the assembled peptides or proteins to ensure that their activity and properties on the substrate are not affected; iii) allowing the introduction of multiple peptide or protein properties to meet the multifunctional requirements of complex applications, including but not limited to the binding ability, catalytic activity and recognition specificity of different peptides or proteins.

[0048] Based on the above considerations, this application creatively proposes a method for preparing peptide or protein elution coatings. Utilizing the weak interactions between polyphenols and peptides (or proteins) under acidic conditions, such as hydrogen bonding and hydrophobic forces, a uniform hydrogel coating is deposited, maintaining the original physiological functions of the peptides (or proteins). The film-forming process does not require pH adjustment and can rapidly form films on the surfaces of metals, non-metals, polymers, and other materials. The corresponding preparation method is simple and easy to implement, showing great promise for industrial application.

[0049] For reference, the method includes the following steps: incubating a substrate with a mixed solution for the first time to form an adhesive gel coating on the substrate surface, wherein the mixed solution is obtained by mixing polyphenols with peptides or proteins in an acidic solution system; and placing the adhesive gel coating in an alkaline solution of polyamine or polythiol for the second time to achieve in-situ chemical cross-linking of the adhesive gel coating through phenolamine chemistry to obtain a solid coating with a highly phenolamine network structure encapsulating peptides or proteins.

[0050] In the process of forming the adhesive gel coating, peptides (or proteins) and polyphenols can form a hydrogel coating through weak interactions such as hydrogen bonds and hydrophobic forces. The hydrogel coating material is placed in an alkaline polyamine or polythiol solution, and in-situ chemical cross-linking of the gel coating is achieved through phenol-amine chemistry. Thus, the gel coating assembled based on hydrogen bonds and hydrophobic forces is transformed into a solid coating with a highly phenol-amine network structure that encapsulates peptides or proteins.

[0051] The above method is simple and mild, and can realize the preparation of functional peptide or protein elution coatings that are adjustable and controllable for multiple application scenarios. The peptide or protein elution coatings formed by the above method can introduce one or more peptides (or proteins) into the coating and maintain the physiological functions of the peptides (or proteins).

[0052] In this application, the mixed solution can be obtained by blending polyphenols and polypeptides in any molar ratio in a solvent with pH = 2-7 ​​(e.g., pH = 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7). Similarly, the mixed solution can be obtained by blending polyphenols and proteins in any molar ratio in a solvent with pH = 2-7 ​​(e.g., pH = 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7).

[0053] In a mixed solution, the molar ratio of polyphenols to polypeptides can range from 0.01:1 to 100:1, such as 0.01:1, 0.1:1, 0.5:1, 1:1, 5:1, 10:1, 20:1, 50:1, 80:1, or 100:1. Similarly, in a mixed solution, the molar ratio of polyphenols to proteins can range from 0.01:1 to 100:1, such as 0.01:1, 0.1:1, 0.5:1, 1:1, 5:1, 10:1, 20:1, 50:1, 80:1, or 100:1.

[0054] In the mixed solution, the concentration of polyphenols can be 0.01-100 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, or 100 mg / mL. The concentration of polypeptides or proteins can also independently be 0.01-100 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, or 100 mg / mL. In some preferred embodiments, the concentration of polyphenols is 0.25-10 mg / mL, and the concentration of polypeptides or proteins is 0.5-20 mg / mL.

[0055] In some embodiments, the mixed solution contains one polyphenol and two polypeptides. The molar ratio between the two polypeptides can be 0.01:1 to 10:1, such as 0.01:1, 0.05:1, 0.1:1, 0.5:1, 1:1, 2:1, 5:1, 8:1, or 10:1. The total concentration of the two polypeptides in the first incubation system can be 0.001 to 10 mmol / mL, such as 0.001 mmol / mL, 0.005 mmol / mL, 0.01 mmol / mL, 0.05 mmol / mL, 0.1 mmol / mL, 0.5 mmol / mL, 1 mmol / mL, 2 mmol / mL, 5 mmol / mL, 8 mmol / mL, or 10 mmol / mL. In some preferred embodiments, the molar ratio between the two peptides is 0.5:1-5:1, such as 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1, and the total concentration of the two peptides in the first incubation system is 0.01-5 mmol / mL.

[0056] In other embodiments, the mixed solution contains one polyphenol and two proteins. The molar ratio between the two proteins can be 0.01:1 to 10:1, such as 0.01:1, 0.05:1, 0.1:1, 0.5:1, 1:1, 2:1, 5:1, 8:1, or 10:1. The total concentration of the two proteins in the first incubation system can be 0.001 to 10 mmol / mL, such as 0.001 mmol / mL, 0.005 mmol / mL, 0.01 mmol / mL, 0.05 mmol / mL, 0.1 mmol / mL, 0.5 mmol / mL, 1 mmol / mL, 2 mmol / mL, 5 mmol / mL, 8 mmol / mL, or 10 mmol / mL. In some preferred embodiments, the molar ratio between the two proteins is 0.5:1-5:1, such as 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1, and the total concentration of the two proteins in the first incubation system is 0.01-5 mmol / mL.

[0057] By controlling the preferred molar ratio and concentration scheme described above, the specific properties of the two peptides (or proteins) can be better utilized, resulting in an optimal hydrogel coating.

[0058] For reference, the polyphenols used in this application include plant polyphenols containing a polyhydroxyphenolic chemical structure, and may include, by way of example but not by way of limitation, at least one of tannic acid (TA), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), salvianolic acid, proanthocyanidins, theaflavins and brown algae polyphenols.

[0059] The polypeptide may, by way of example but not limitation, include at least one of hirudin and its derivatives, bivalirudin, antimicrobial peptide LL-37, antimicrobial peptide hCAP-18, antimicrobial peptide C, antimicrobial peptide BLP-7, β-defensin, teriparatide and osteocalcin.

[0060] Proteins may, by way of example but not by way of limitation, include at least one of lactoferrin, soy protein, whey protein, osteocalcin, insulin, collagen, osteothiin, cathepsin S, lysozyme, glucose oxidase, pepsin, horseradish peroxidase, glucose oxidase, pepsin, horseradish peroxidase, hemoglobin, cytochrome C, immunoglobulin G (IgG), fibrinogen, trypsin and insulin.

[0061] For reference, the temperature for the first incubation can be 0-50℃, such as 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, or 50℃. The duration of the first incubation can be 0.25-96 hours, such as 0.25h, 1h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, 72h, 78h, 84h, 90h, or 96h. In some preferred embodiments, the temperature for the first incubation is 15-50℃, and the time is 15 minutes or more. In some more preferred embodiments, the temperature for the first incubation is 20-40℃, and the time is 0.25-24 hours.

[0062] After the first incubation reaction is complete, the resulting product is washed and dried.

[0063] In this application, the concentration of the polyamine or polythiol alkaline solution can be 0.01-1000 mg / mL, such as 0.01 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 500 mg / mL, 800 mg / mL, or 1000 mg / mL. The concentration of the polyamine or polythiol in the second incubation system can be 0.05-10 mg / mL, such as 0.05 mg / mL, 0.1 mg / mL, 1 mg / mL, 5 mg / mL, or 10 mg / mL.

[0064] The pH value of alkaline solutions of polyamines or polythiols can be 8-10, such as 8, 8.5, 9, 9.5 or 10.

[0065] In some embodiments, the polyamine or polythiol may have the chemical formula Y-Xn-Y, where n≥2, X=CH2 or CH2OCH2, and Y=primary amine or thiol. In other embodiments, the polyamine or polythiol may have the chemical formula X n Y m Where n≥2, X=CH2 or CH2OCH2, Y=primary amino or mercapto, m≥2, Y can appear at any proton substitution position in the repeating structural unit of X.

[0066] For reference, polyamines may include, by way of example but not by way of limitation, at least one of ethylenediamine, propylenediamine, butylenediamine, hexamethylenediamine, heptamethylenediamine, octamethylenediamine, m-phenylenediamine, p-phenylenediamine, benzidine, polyethyleneimine, cystamine, and selenocystamine.

[0067] Polymerols may, by way of example but not by way of limitation, include at least one of hexamethylene dithiol, methane dithiol, benzene dithiol, and trithiol methane.

[0068] In some preferred embodiments, the polyamine or polythiol is selected from hexadiamine, cystamine, selenocystamine, or hexadithiool.

[0069] The solvent in the above-mentioned polyamine or polythiol alkaline solution is an alkaline buffer solution. The alkaline buffer solution may, by way of example but not limitation, include at least one of carbamate buffer, bicarbonate buffer, PBS buffer, Tris-HCl buffer, ethanolamine buffer, sodium hydroxide solution, and potassium hydroxide solution. In some preferred embodiments, the alkaline buffer solution is a sodium hydroxide solution with a concentration of 0.0001-40 mol / L.

[0070] In this application, the temperature for the second incubation can be 0-50℃, such as 0℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, or 50℃. The time for the second incubation can be 30 minutes or more. In some preferred embodiments, the temperature for the second incubation is 25-40℃, and the time is 0.5-24 hours.

[0071] In this application, the temperature of the second incubation reaction can be carried out within a wide range, and the reaction time should preferably be more than 6 hours. If it is less than 6 hours, some types of proteins cannot be uniformly deposited on the material surface.

[0072] After the second incubation, the incubated product is dried under a nitrogen atmosphere. Specifically, it is washed three times with distilled water, 5 minutes each time. This treatment effectively removes any undeposited reactants from the surface. Finally, it is dried with nitrogen to obtain the target coating.

[0073] By crosslinking the above coating with polyamine or polythiol reagents, the controlled release of peptides (low molecular weight proteins) can be achieved.

[0074] Accordingly, this application also provides a polypeptide or protein elution coating, which is prepared by the above-described preparation method.

[0075] This coating features broad-spectrum modification capabilities, is independent of substrate material, is suitable for deposition on various material surfaces, and exhibits strong adhesion and high chemical structural stability.

[0076] In addition, this application also provides an application of the above-mentioned polypeptide or protein elution coating, for example, it can be used to modify the surface of a substrate.

[0077] The aforementioned substrate can be any solid material of any geometry and material known to humankind.

[0078] For reference, the substrate may, by way of example but not by way of limitation, include at least one of metallic materials, inorganic non-metallic materials, polymeric materials, biomedical micro / nanoparticles, natural biomaterials and synthetic peptide hydrogel materials.

[0079] The metallic material may, by way of example but not by way of limitation, include at least one of stainless steel, iron and its alloys, magnesium and its alloys, titanium and its alloys, zinc and its alloys, and cobalt-based alloys.

[0080] Inorganic non-metallic materials may, by way of example but not limitation, include at least one of titanium dioxide, titanium oxide, titanium nitride, ceramics, silicon, silicon dioxide, silicon carbide (SiC), silicon nitride calcium phosphate (Si3N4), calcium phosphate, aluminosilicate (Na2O·Al2O3·SiO2), calcium aluminum (CaO·Al2O3), bioglass (SiO2·CaO·Na2O·P2O5), hydroxyapatite, carbon materials (C), diamond, and diamond-like carbon.

[0081] Exemplary but not limited polymeric materials may include at least one of polyester (PET), polyethylene (PE), polyvinyl chloride (PVC), polyvinyl alcohol (PVALC), polypropylene (PP), polycarbonate (PC), polyglycolic acid (PGA), polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), polyoxymethylene (POM), polystyrene (PS), polyvinyl acetate (PVA), polylactic acid (PLA), polylactic-co-glycolic acid copolymer (PLGA), polytrimethylene carbonate (PTMC), polyhydroxyalkanoates (PHA), polybutylene succinate (PBS), polyamide (PA), epoxy resin, silicone rubber, silicone gel, polyacrylic acid (PAA) and its derivatives, polyethylene glycol and its derivatives, polyvinyl alcohol (PVA), polycaprolactone (PCL), and polyurethane (PU).

[0082] Biomedical micro- and nanoparticles may, by way of example but not by way of limitation, include at least one of iron oxide nanoparticles, (mesoporous) silica nanoparticles (quantum dots), titanium oxide nanoparticles (quantum dots), and zinc oxide nanoparticles (quantum dots).

[0083] Natural biological materials may, by way of example but not limitation, include at least one of polysaccharides and decellularized tissues and organs of animal origin. Polysaccharides include at least one of plastic starch-based materials (PSM), gelatin, collagen, sodium hyaluronate*, fibrous protein, sodium alginate, agarose, silk fibroin, keratin, cellulose, hemicellulose, lignin, chitin, and their derivatives; decellularized tissues and organs of animal origin include at least one of blood vessels, valves, heart, bone, lungs, ligaments, bladder, mucous membranes, and cornea.

[0084] Artificially synthesized peptide hydrogel materials may, by way of example but not by way of limitation, include at least one of poly-L-lysine and poly-L-glutamic acid.

[0085] As mentioned above, the coating provided in this application is a surface coating with multiple biological functions. Its preparation method is not limited to the types of metals, inorganic non-metals or polymer materials, and the process is simple, mild and easy to implement. In addition, the parameter adjustment range during the preparation process is large, which can meet the needs of different materials for medical scenarios. The raw materials are all common substances or extracts, non-toxic, and the raw materials are interdependent and work together.

[0086] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0087] Example 1 (1) Use SDS to scrub the silicone rubber substrate to remove impurities, wash it thoroughly with distilled water, and then use alcohol to ultrasonically clean it three times for 3 minutes each time. Then blow it dry with nitrogen and set it aside for use.

[0088] (2) Tannic acid and bivalirudin were mixed in distilled water at a molar ratio of 5:4 to obtain a mixed solution with a pH of 5. The concentration of tannic acid in the mixed solution was 1 mg / mL, and the concentration of bivalirudin was 1 mg / mL.

[0089] (3) Immerse the stainless steel sheet to be used in the above mixed solution and sonicate for 1 min. Then place the sonicated system in a constant temperature oven at 37°C and let it stand for 24 h for the first incubation reaction.

[0090] (4) After the first incubation reaction, the silicone rubber was removed, rinsed gently with distilled water three times, and the surface water was absorbed by filter paper from the side. The sample was then dried in a fume hood to obtain a hydrogel coating sample that releases bivalirudin.

[0091] Example 2 This embodiment is basically the same as Example 1, except that the functional peptide is replaced with mersacidin antimicrobial peptide to obtain a hydrogel coating sample that releases antimicrobial peptide.

[0092] Example 3 (1) Mirror polish the 316L stainless steel sheet, scrub with SDS to remove impurities, rinse thoroughly with distilled water, and then ultrasonically clean with acetone three times, 3 minutes each time. Dry with nitrogen. Use a dual-band UV lamp containing 185nm and 253.7nm at 20mw / cm². 2 Irradiate the stainless steel sheet with high intensity for 10 minutes, then remove and set aside for use.

[0093] (2) Brown algae polyphenols and bivalirudin were mixed in distilled water at a molar ratio of 10:1 to obtain a mixed solution with a pH of 4.5. In the mixed solution, the concentration of brown algae polyphenols was 2 mg / mL and the concentration of bivalirudin was 1 mg / mL.

[0094] (3) Immerse the stainless steel sheet to be used in the above mixed solution and sonicate for 1 min. Then place the sonicated system in a constant temperature oven at 37°C and let it stand for 24 h for the first incubation reaction.

[0095] (4) After the first incubation reaction, the stainless steel sheet was removed, rinsed gently with distilled water three times, and the surface water was absorbed by filter paper from the side. The sheet was then dried in a fume hood to obtain a hydrogel coating that releases the anticoagulant peptide bivalirudin.

[0096] Example 4 This embodiment is basically the same as Example 3, except that the plant polyphenol compound is proanthocyanidin, and a coating sample with bivalirudin release is obtained.

[0097] Example 5 This embodiment is basically the same as Example 3, except that bivalirudin is replaced with the anti-inflammatory peptide Chemerin15 to obtain a sample with an anti-inflammatory coating.

[0098] Example 6 Hexamethylenediamine was pre-prepared into an alkaline solution with a concentration of 1 mg / mL and a pH of 8 using an HCl solution with a concentration of 10 mg / mL.

[0099] The sample with the anti-coating hydrogel coating obtained in Example 3 was placed in the above-mentioned hexamethylenediamine alkaline solution and placed in a constant temperature oven at 37°C for 24 hours for a second incubation. After the incubation, it was gently rinsed 3 times with distilled water, and the surface water was absorbed from the side with filter paper. Then it was dried in a fume hood to obtain a hydrogel conversion coating with controllable release of bivalirudin.

[0100] Example 7 (1) A 100 nm thick rutile titanium dioxide surface was obtained on a silicon wafer using a measurement-controlled sputtering deposition method. After rinsing with distilled water, a dual-band ultraviolet lamp containing 185 nm and 253.7 nm wavelengths was used at 20 mW / cm². 2 Irradiate the stainless steel sheet with high intensity for 10 minutes, then remove and set aside for use.

[0101] (2) Tannic acid and bivalirudin were mixed in distilled water at a molar ratio of 1:1 to obtain a mixed solution with a pH of 5. In the mixed solution, the concentration of tannic acid was 5 mg / mL and the concentration of bivalirudin was 4 mg / mL.

[0102] (3) Immerse the titanium dioxide sheet to be used in the above mixed solution and sonicate for 1 min. Then place the sonicated system in a constant temperature oven at 37°C for 24 h to carry out the first incubation reaction.

[0103] (4) After the first incubation reaction, remove the titanium dioxide sheet, rinse it gently with RO water 3 times, absorb the water on the surface with filter paper from the side, and dry it in a fume hood to obtain a hydrogel coating that releases bivalirudin.

[0104] Example 8 This embodiment is basically the same as Example 7, except that the concentration ratio of tannic acid aqueous solution to bivalirudin aqueous solution is 1:2, and the concentration of tannic acid in the system is 1 mg / mL.

[0105] Example 9 Selenocystamine was pre-prepared into an alkaline solution with a concentration of 1 mg / mL and a pH of 8 using a NaOH solution with a concentration of 18 mg / mL.

[0106] The sample with the hydrogel coating that releases bivalirudin obtained in Example 7 was placed in the above-mentioned selenocysteine ​​alkaline solution and incubated for 24 hours in a constant temperature oven at 37°C for a second incubation. Then it was gently rinsed 3 times with RO water, the surface water was absorbed from the side with filter paper, and it was dried in a fume hood to obtain a hydrogel conversion coating with controlled release of bivalirudin.

[0107] Example 10 (1) Remove impurities by rubbing and washing with medical polyurethane and SDS, rinse thoroughly with distilled water, and then ultrasonically clean three times with anhydrous ethanol for 3 minutes each time. Dry with nitrogen. Use a dual-band ultraviolet lamp containing 185nm and 253.7nm at 20mw / cm². 2 Irradiate the silicone rubber with the intensity of the radiation for 10 minutes, then remove and set aside for use.

[0108] (2) Brown algae polyphenols and lysozyme were mixed in distilled water at a molar ratio of 2:1 to obtain a mixed solution with a pH of 4.5. In the mixed solution, the concentration of brown algae polyphenols was 10 mg / mL and the concentration of lysozyme was 1 mg / mL.

[0109] (3) Immerse the silicone rubber sheet to be used in the above mixed solution and sonicate for 1 min. Then place the sonicated system in a constant temperature oven at 37°C for 24 h to carry out the first incubation reaction.

[0110] (4) After the first incubation reaction, the silicone rubber sheet was removed, rinsed gently with RO water 3 times, and the filter paper was used to absorb the water on the surface and then air-dried in the air to obtain a hydrogel coating sample that releases lysozyme.

[0111] Example 11 This embodiment is basically the same as Example 10, except that the plant polyphenol compound is epigallocatechin gallate, and a hydrogel coating sample with lysozyme release is obtained.

[0112] Example 12 1,6-hexanedithiol was pre-prepared into an alkaline solution with a concentration of 1 mg / mL and a pH of 8 using a 10 mg / mL NaOH solution.

[0113] The sample with the anticoagulant and antibacterial hydrogel coating obtained in Example 9 was placed in the above-mentioned 1,6-hexanedithiol alkaline solution and incubated at 37°C for 24 hours for a second incubation. It was then gently rinsed three times with RO water, the surface water was blotted dry with filter paper from the side, and then dried in a fume hood. A hydrogel conversion coating with controllable lysozyme release was obtained.

[0114] Example 13 This embodiment is basically the same as that of Example 11, except that 1,6-hexanedithiol is replaced with selenocysteine ​​solution.

[0115] Example 14 This embodiment is basically the same as Example 10, except that bivalirudin is added to the mixed solution at the same time to obtain a hydrogel coating sample that releases lysozyme and bivalirudin.

[0116] (1) Brown algae polyphenols, lysozyme, and bivalirudin were mixed in distilled water at a molar ratio of 2:1:1 to obtain a mixed solution with a pH of 4.5. In the mixed solution, the concentration of brown algae polyphenols was 10 mg / mL, the concentration of lysozyme was 1 mg / mL, and the concentration of bivalirudin was 0.5 mg / mL.

[0117] (2) Immerse the silicone rubber sheet to be used in the above mixed solution and sonicate for 1 min. Then place the sonicated system in a constant temperature oven at 37°C for 24 h to carry out the first incubation reaction.

[0118] (3) After the first incubation reaction, the silicone rubber sheet was removed, rinsed gently with RO water three times, and the filter paper was used to absorb the water on the surface and then air-dried in the air to obtain a hydrogel coating sample that releases lysozyme and bivalirudin.

[0119] Example 15 Selenocystamine was pre-prepared into an alkaline solution with a concentration of 1 mg / mL and a pH of 8 using a NaOH solution with a concentration of 18 mg / mL.

[0120] The sample with the hydrogel coating that releases bivalirudin and valerate obtained in Example 14 was placed in the above-mentioned selenocysteine ​​alkaline solution and incubated for 24 hours in a constant temperature oven at 37°C for a second incubation. Then it was gently rinsed 3 times with RO water, the surface water was absorbed from the side with filter paper, and it was dried in a fume hood to obtain a hydrogel conversion coating with controllable release of bivalirudin and lysozyme.

[0121] Comparative Example 1 The only difference between this comparative example and Example 1 is that bivalirudin is not added.

[0122] The substrate is made of silicone rubber tubing. The concentration of tannic acid is controlled at 1 mg / mL. The reaction is carried out at 37℃ for 12 hours. The substrate is washed three times with distilled water, the surface moisture is blown off with nitrogen, and the substrate is placed in a dry environment at 37℃ for later use.

[0123] Comparative Example 2 The only difference between this comparative example and Example 2 is that mersacidin antimicrobial peptides are not added.

[0124] The substrate used was 316L SS, the concentration of tannic acid was controlled at 1 mg / mL, the reaction was carried out at 37℃ for 12 h, the substrate was washed three times with distilled water, the surface moisture was blown off with nitrogen, and the substrate was placed in a dry environment at 37℃ for later use.

[0125] Comparative Example 3 The only difference between this comparative example and Example 3 is that the pH of the distilled water was adjusted to 9.

[0126] The substrate was made of silicone rubber, and the concentration of brown algae polyphenols was controlled at 2 mg / mL, and the concentration of bivalirudin was 1 mg / mL. The mixture was reacted at 37℃ for 12 hours, washed three times with distilled water, the surface moisture was blown away with nitrogen, and then dried at 37℃.

[0127] Experimental Example 1 The release amounts of bivalirudin (BVLD) corresponding to Examples 3 and 6 were detected using a fluorescent labeling method as follows, and the results are as follows: Figure 1 As shown.

[0128] The specific process is as follows: (1) A molecule of fluorescein (named BVLD-Fluor) was pre-labeled on the BVLD.

[0129] (2) Prepare hydrogel coatings that release BVLD and hydrogel conversion coatings that controllably release BVLD according to the experimental steps given in Examples 3 and 6.

[0130] (3) The hydrogel coating sample from Example 3 was placed in PBS buffer solution, and the extract was collected at 1, 2, 3, and 5 min for later use. Similarly, the hydrogel conversion coating sample from the example was placed in PBS buffer solution, and the extract was collected at 1, 3, 7, 15, and 30 days for later use.

[0131] (4) Since the BVLD-Fluor released during the coating elution test absorbs light at a wavelength of 490nm, the absorption intensity at this wavelength can be measured by a UV-vis spectrophotometer and the amount of BVLD eluted can be calculated based on the standard curve.

[0132] according to Figure 1 The results showed that the coating in Example 3, after immersion in PBS for 3 minutes, exhibited a tendency for BVLD release to reach equilibrium, with a cumulative release of 595.7 ± 5.8 μg. In contrast, the coating in Example 6 required 15 days of PBS immersion to reach release equilibrium, with a cumulative release of 555.7 ± 10.8 μg. This indicates that the preparation of the release-type and controllable-release BVLD coatings designed in Examples 3 and 6 was successful.

[0133] Experimental Example 2 To determine whether the BVLD released from the BVLD-releasing and controlled-release BVLD coatings possesses anticoagulant capabilities, thrombin time (TT) tests were performed on Examples 3 and 6. Specific experiments are as follows: (1) Plasma preparation: Collected blood was mixed with sodium citrate solution at a ratio of 9:1, and blood evaluation was performed within 12 hours. Platelet-lowering plasma (PPP) was obtained by centrifuging whole blood at 3000 rpm for 15 minutes.

[0134] (2) The TT assay was performed using a TT kit (Shanghai Sun Biotechnology Co., Ltd., TT assay kit (liquid type)). Bare and modified sheets (1cm × 1cm) were placed in a 24-well plate, and 2mL of rabbit PPP was added to each well. The plate was incubated at 37°C for 5 minutes. Then, 200μL of the incubated PPP was transferred to a 1.5mL EP tube, the TT reagent was added, and timing was started. The coagulation time (TT) was determined by visual inspection using a hand-shaking method. The endpoint was the formation of coagulation or flocculent material in the plasma solution.

[0135] according to Figure 2 The observations in Example 3 showed that when the sample was contacted with plasma after elution for 5 minutes, the plasma remained non-clotting in the TT (clotting time) test. However, in Example 6, the sample still showed a higher TT value after elution for 30 days, exceeding the control sample's 28.5 ± 0.8 seconds. This indicates the successful preparation of the release BVLD and the controlled-release BVLD coating.

[0136] Experimental Example 3 Anticoagulation experiments were conducted on the surfaces of Example 1, Comparative Example 1, and unmodified silicone rubber using New Zealand white rabbits. The results are as follows: Figure 3 As shown.

[0137] The specific process is as follows: (1) Connect the above sample to the semi-in vivo circulation catheter and assemble them together. Add physiological saline to the catheter in advance to completely remove air bubbles and ensure that no air embolism is formed during circulation. Then clamp both ends with hemostatic forceps for later use. (2) New Zealand white rabbits (2.5-3kg) were anesthetized by intravenous injection of sodium pentobarbital solution (30mg / mL) via the marginal ear vein, at a dose of 1mL / kg. Subsequently, the left carotid artery and right jugular vein of the rabbit were carefully dissected with a scalpel to expose the blood vessels.

[0138] (3) Remove the soaked indwelling needle and insert it into the rabbit's carotid artery and jugular vein. Then connect the assembled sample to the rabbit's carotid artery and vein to allow blood to flow back to the heart, forming a semi-in vivo blood circulation loop. Throughout the experiment, it is necessary to continuously observe the changes in blood color and temperature in the catheter.

[0139] (4) After 2 hours of circulation, the experiment was stopped, the experimental sample was removed, and rinsed with physiological saline. Subsequently, photographs of the sample and the cross-section of the catheter were taken, and the results were analyzed according to... Figure 3 It can be seen that the example has a significant anticoagulant effect, while the comparative example does not.

[0140] Test Example 4 Antimicrobial coating experiments were conducted on the surfaces of Example 2, Comparative Example 2, and unmodified 316L SS. The results are as follows: Figure 4 As shown.

[0141] The detailed steps are as follows: This experiment used Escherichia coli (E. coli), a typical Gram-negative bacterium, and Staphylococcus epidermidis, a typical Gram-positive bacterium. The experiment was conducted according to the ISO 22196-2011 standard. The experimental steps are as follows: (1) Sample and Culture Medium Sterilization: The prepared samples and PE films were sterilized under UV light for 30 min. Centrifuge tubes, pipette tips, glass petri dishes, etc., used in the experiment were sterilized by high temperature and high pressure. The solid culture medium used in the experiment had the following formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar, with distilled water as the solvent. After all reagents were fully dissolved, they were subjected to routine high temperature and high pressure sterilization and then sealed for storage. The liquid culture medium is obtained by removing the agar from the solid culture medium, while the other components remain the same.

[0142] (2) Bacterial activation, purification, and suspension preparation: After obtaining the bacterial strain from the slant culture medium, it was cultured on a solid culture medium using the streak plate method. After 24 hours, single colonies were obtained and placed at 37℃ with shaking for 24 hours to obtain the activated bacterial strain. Preparation of the suspension: First, the culture medium was prepared (the liquid culture medium was diluted in physiological saline by volume ratio to obtain the culture medium; diluting the liquid culture medium 500 times yielded the Escherichia coli culture medium, and diluting it 100 times yielded the Staphylococcus aureus culture medium). 1 mL of the activated bacterial strain was taken and serially diluted with the culture medium to obtain a concentration of 5.0 × 10⁻⁶. 5 ~1.0×10 6CFU / mL bacterial culture (detectable by spectrophotometer at 600nm wavelength).

[0143] (3) Inoculation of samples with bacteria: Place the sterilized samples in a 24-well plate and drop 100 μL of diluted bacterial solution onto the surface of each sample. Use a pipette tip to cover the entire coated surface with the bacterial solution, being careful not to let the bacterial solution drip off the sample surface. Then cover the sample with a clean PE film. Incubate in a 37°C incubator for 24 hours.

[0144] (4) Plate culture: After preparing and sterilizing the solid culture medium, pour it into petri dishes in a clean workbench to cool and solidify. Add 1 mL of physiological saline to each sample and repeatedly pipette to completely remove bacteria from the sample surface and PE membrane. Then, take 100 μL of bacterial suspension and spread it evenly onto the prepared solid culture medium, and incubate at 37°C for 24 h. Finally, take photos for recording.

[0145] from Figure 4 As can be seen, in Example 2, the antimicrobial peptides in the coating have good bactericidal effects against Escherichia coli (Gram-negative bacteria) and Staphylococcus epidermidis (Gram-positive bacteria).

[0146] By comparing the experimental results of the embodiments and comparative examples, it can be found that all kinds of modified materials in the embodiments of this application have corresponding functional performances. Suitable polyphenols or peptides with anti-inflammatory, antibacterial, and antioxidant activities can be selected for assembly according to the application scenario and scope of use, and cross-linked with appropriate cross-linking agents. By controlling the types of polyphenols and peptides, as well as the types of cross-linking agents, the concentration of reagents, and the length of coating deposition time, a functional protein layer that can be adjusted and controlled for multiple application scenarios can be achieved.

[0147] Experimental Example 5 The results of comparing Example 3 and Comparative Example 3 before and after hydrogel coating modification are as follows: Figure 5 As shown, by Figure 5 It can be seen that when the pH of the system is adjusted to 9, the weak interaction forces such as hydrogen bonds will be broken in the alkaline environment, which will cause the gel coating to fail to adhere to the material surface and form a film.

[0148] In summary, this application involves first incubating a substrate with a mixed solution to form an adhesive gel coating on the substrate surface, followed by a second incubation in an alkaline solution of polyamines or polythiols. This process achieves in-situ chemical cross-linking of the adhesive gel coating through phenolamine chemistry, resulting in a solid coating with a highly phenolamine network structure encapsulating peptides or proteins. This method enables the preparation of tunable and controllable functional peptide or protein elution coatings for various applications. The peptide or protein elution coatings formed by this method exhibit broad-spectrum modification characteristics, are independent of the substrate material, and possess chemical structural stability.

[0149] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a polypeptide or protein elution coating, characterized in that, Includes the following steps: The mixed solution is first incubated on the substrate to form an adhesive gel coating on the surface of the substrate. The mixed solution is obtained by mixing polyphenols and peptides in an acidic solution system, or by mixing polyphenols and proteins in an acidic solution system. The adhesive gel coating is placed in a polyamine or polythiol alkaline solution for a second incubation, and in-situ chemical cross-linking of the adhesive gel coating is achieved through phenolamine chemistry to obtain a solid coating with a highly phenolamine network structure that encapsulates peptides or proteins.

2. The preparation method according to claim 1, characterized in that, The mixed solution is obtained by blending the polyphenol and the polypeptide in any molar ratio in a solvent with pH = 2-7, or the mixed solution is obtained by blending the polyphenol and the protein in any molar ratio in a solvent with pH = 2-7.

3. The preparation method according to claim 2, characterized in that, In the mixed solution, the molar ratio of the polyphenol to the polypeptide is 0.01:1-100:1, or the molar ratio of the polyphenol to the protein is 0.01:1-100:

1.

4. The preparation method according to claim 2, characterized in that, In the mixed solution, the concentration of the polyphenol is 0.01-100 mg / mL, and the concentration of the polypeptide or the protein is 0.01-100 mg / mL.

5. The preparation method according to claim 4, characterized in that, The mixed solution contains one polyphenol and two polypeptides; the molar ratio between the two polypeptides is 0.01:1-10:1, and the total concentration of the two polypeptides in the first incubation system is 0.001-10 mmol / mL; Alternatively, the mixed solution contains one polyphenol and two proteins; the molar ratio between the two proteins is 0.01:1-10:1; the total concentration of the two proteins in the first incubation system is 0.001-10 mmol / mL.

6. The preparation method according to claim 5, characterized in that, The molar ratio between the two peptides was 0.5:1-5:1, and the total concentration of the two peptides in the first incubation system was 0.01-5 mmol / mL. Alternatively, the molar ratio between the two proteins is 0.5:1 to 5:1, and the total concentration of the two proteins in the first incubation system is 0.01 to 5 mmol / mL.

7. The preparation method according to any one of claims 1-6, characterized in that, The polyphenols include plant polyphenols containing polyhydroxyphenolic chemical structures; or, the polypeptides include at least one of hirudin and its derivatives, bivalirudin, antimicrobial peptide LL-37, antimicrobial peptide hCAP-18, antimicrobial peptide C, antimicrobial peptide BLP-7, β-defensin, teriparatide and osteocalcin. Alternatively, the protein may include at least one of lactoferrin, soy protein, whey protein, osteocalcin, collagen, osteothiin, cathepsin S, lysozyme, glucose oxidase, pepsin, horseradish peroxidase, hemoglobin, cytochrome C, immunoglobulin G, fibrinogen, trypsin, and insulin.

8. The preparation method according to claim 7, characterized in that, The plant polyphenols include at least one of tannic acid, epigallocatechin, epigallocatechin gallate, epigallocatechin gallate, salvianolic acid, proanthocyanidins, theaflavins, and brown algae polyphenols.

9. The preparation method according to claim 1, characterized in that, The first incubation should be carried out at a temperature of 0-50℃ for 0.25-96 hours.

10. The preparation method according to claim 1, characterized in that, The first incubation should be carried out at a temperature of 15-50℃ for at least 15 minutes.

11. The preparation method according to claim 10, characterized in that, The initial incubation temperature is 20-40℃, and the time is 0.25-24h.

12. The preparation method according to any one of claims 1-6, characterized in that, The concentration of the polyamine or polythiol alkaline solution is 0.01-1000 mg / mL.

13. The preparation method according to claim 12, characterized in that, The pH value of the alkaline solution of the polyamine or polythiol is 8-10.

14. The preparation method according to claim 12, characterized in that, The polyamine or polythiol has the chemical formula Y-Xn-Y, where n≥2, X=CH2 or CH2OCH2, and Y=primary amino or thiol; or, the polyamine or polythiol has the chemical formula X n Y m Where n≥2, X=CH2 or CH2OCH2, Y=primary amino or mercapto, m≥2, Y can appear at any proton substitution position in the repeating structural unit of X.

15. The preparation method according to claim 14, characterized in that, The polyamines include at least one of ethylenediamine, propylenediamine, butanediamine, hexamethylenediamine, heptanediamine, octanediamine, m-phenylenediamine, p-phenylenediamine, benzidine, polyethyleneimine, cystamine, and selenocystamine.

16. The preparation method according to claim 15, characterized in that, The polythiols include at least one of hexamethylenedimerol, dimercaptomethane, dimercaptobenzene, and trimercaptomethane.

17. The preparation method according to claim 12, characterized in that, The solvent in the alkaline solution of the polyamine or polythiol is an alkaline buffer solution.

18. The preparation method according to claim 17, characterized in that, The alkaline buffer solution includes at least one of carbamate buffer, bicarbonate buffer, PBS buffer, Tris-HCl buffer, and ethanolamine buffer.

19. The preparation method according to claim 17, characterized in that, The concentration of the alkaline buffer solution is 0.0001-40 mol / L.

20. The preparation method according to claim 1, characterized in that, The second incubation should be carried out at a temperature of 0-50℃ for at least 30 minutes.

21. The preparation method according to claim 20, characterized in that, The second incubation should be carried out at a temperature of 25-40℃ for 0.5-24 hours.

22. A polypeptide or protein elution coating, characterized in that, It is prepared by the preparation method according to any one of claims 1-21.

23. An application of the polypeptide or protein elution coating as described in claim 22, characterized in that, The polypeptide or protein elution coating is used to modify the surface of the substrate.

24. The application according to claim 23, characterized in that, The substrate includes at least one of the following: metallic materials, inorganic non-metallic materials, polymeric materials, biomedical micro / nanoparticles, natural biomaterials, and artificially synthesized polypeptide hydrogel materials.

25. The application according to claim 24, characterized in that, The metallic material includes at least one of stainless steel, iron and its alloys, magnesium and its alloys, titanium and its alloys, zinc and its alloys, and cobalt-based alloys.

26. The application according to claim 24, characterized in that, The inorganic non-metallic materials include at least one of titanium dioxide, titanium nitride, ceramics, silicon, silicon carbide, aluminosilicates, calcium aluminum bioglass, hydroxyapatite, carbon materials, diamond, and diamond-like carbon.

27. The application according to claim 24, characterized in that, The polymeric materials include at least one of polyester, polyethylene, polyvinyl chloride, polyvinyl alcohol, polypropylene, polycarbonate, polyglycolic acid, polymethyl methacrylate, polytetrafluoroethylene, polyoxymethylene, polystyrene, polyvinyl acetate, polylactic acid, polylactic-glycolic acid copolymer, polytrimethylene carbonate, polyhydroxyalkanoates, polybutylene succinate, polyamide, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives, polycaprolactone, and polyurethane.

28. The application according to claim 24, characterized in that, The biomedical micro / nanoparticles include at least one of iron tetroxide nanoparticles and zinc oxide nanoparticles.

29. The application according to claim 24, characterized in that, The natural biomaterials include at least one of polysaccharides and decellularized tissues and organs of animal origin; wherein the polysaccharides include at least one of plastic starch-based materials, sodium hyaluronate, sodium alginate, agarose, cellulose, hemicellulose, lignin, chitin and their derivatives; and the decellularized tissues and organs of animal origin include at least one of blood vessels, valves, heart, bone, lungs, ligaments, bladder, mucous membranes and cornea.

30. The application according to claim 24, characterized in that, The synthetic polypeptide hydrogel material includes at least one of poly-L-lysine and poly-L-glutamic acid.

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