CN117427229A - Polypeptide or protein eluting coating, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a polypeptide or protein eluting coating, a preparation method and application thereof, and belongs to the technical field of biological and medical materials. The method comprises the following steps: incubating the substrate for the first time with a mixed solution to form an adhesive gel coating on the surface of the substrate, wherein the mixed solution is prepared by mixing polyphenol and polypeptide or protein in an acidic solution system; and (3) placing the adhesive gel coating in polyamine or polythiol alkaline solution for a second incubation, and chemically realizing in-situ chemical crosslinking of the adhesive gel coating through phenolic amine to obtain the solid coating with the polypeptide or protein wrapped by the highly phenolic amine network structure. The method realizes the preparation of the functional polypeptide or protein eluting coating with adjustable and controllable multi-scene application, and the polypeptide or protein eluting coating formed by the method has the characteristic of broad-spectrum modification, does not depend on the material of a base material, and has the stability of a chemical structure.

Description

Polypeptide or protein eluting coating, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological and medical materials, in particular to a polypeptide or protein eluting coating, a preparation method and application thereof.

Background

Polypeptides and proteins are biologically functional macromolecules, are involved in numerous basic biological processes in the organism, and are capable of mediating specific interactions with different macromolecules. They play an important role in a variety of applications, such as catalysis, biosensing, diagnostics and therapeutics. However, protein assembly often requires special tailoring to the particular situation due to the sensitivity of the proteins to the environment (temperature, pH, redox, etc.) and their particular requirements on spatial conformation. Furthermore, simple and versatile strategies for efficiently assembling proteins onto substrates have certain limitations, as the non-covalent interactions between proteins and substrates are often insufficient to firmly immobilize them on a surface. Therefore, it is of great importance to explore simple multifunctional polypeptide or protein loading strategies.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a polypeptide or protein eluting coating, and a preparation method and application thereof, so as to solve or improve the technical problems.

The application can be realized as follows:

in a first aspect, the present application provides a method of preparing a polypeptide or protein eluting coating comprising the steps of: incubating the substrate for the first time with a mixed solution to form an adhesive gel coating on the surface of the substrate, wherein the mixed solution is prepared by mixing polyphenol and polypeptide or protein in an acidic solution system;

And (3) placing the adhesive gel coating in polyamine or polythiol alkaline solution for a second incubation, and chemically realizing in-situ chemical crosslinking of the adhesive gel coating through phenolic amine to obtain the solid coating with the polypeptide or protein wrapped by the highly phenolic amine network structure.

In alternative embodiments, the mixed solution is obtained by blending the polyphenol and the polypeptide in a solvent having a ph=2 to 7 in an arbitrary molar ratio, or the mixed solution is obtained by blending the polyphenol and the protein in a solvent having a ph=2 to 7 in an arbitrary molar ratio.

In alternative embodiments, the molar ratio of polyphenol to polypeptide in the mixed solution is from 0.01:1 to 100:1, or the molar ratio of polyphenol to protein is from 0.01:1 to 100:1.

In an alternative embodiment, the concentration of polyphenols in the mixed solution is between 0.01 and 100mg/mL and the concentration of polypeptides or proteins is between 0.01 and 100mg/mL.

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

In an alternative embodiment, the mixed solution contains one polyphenol and two proteins; the mol ratio of the two proteins is 0.01:1-10:1; the total concentration of the two proteins in the first incubation system is 0.001-10mmol/mL.

In an alternative embodiment, the molar ratio between the two polypeptides is between 0.5:1 and 5:1, and the total concentration of the two polypeptides in the first incubation system is between 0.01 and 5mmol/mL.

In an alternative embodiment, the molar ratio between the two proteins is between 0.5:1 and 5:1, and the total concentration of the two proteins in the first incubation system is between 0.01 and 5mmol/mL.

In an alternative embodiment, the polyphenol comprises a plant polyphenol comprising a polyhydroxyphenol chemical structure.

In an alternative embodiment, the plant polyphenol comprises at least one of tannic acid, epigallocatechin, epicatechin gallate, epigallocatechin gallate, salvianolic acid, procyanidins, theaflavins, and brown algae polyphenol.

In alternative embodiments, the polypeptide comprises at least one of hirudin and its derivatives, bivalirudin, the antibacterial peptide LL-37, the antibacterial peptide hCAP-18, the antibacterial peptide C, the antibacterial peptide BLP-7, beta-descensin, teriparatide and osteocalcin.

In an alternative embodiment, the protein comprises at least one of lactoferrin, soy protein, whey protein, osteocalcin, insulin, collagen, bone sulfur protein, cathepsin S, lysozyme, glucose oxidase, pepsin, horseradish peroxidase, hemoglobin, cytochrome C, immunoglobulin G, fibrinogen, trypsin, and insulin.

In an alternative embodiment, the first incubation is at a temperature of 0-50℃for a period of 0.25-96 hours.

In an alternative embodiment, the first incubation is at a temperature of 15-50℃for a period of more than 15 minutes.

In an alternative embodiment, the first incubation is at a temperature of 20-40℃for a period of 0.25-24h.

In an alternative embodiment, the polyamine or polythiol alkaline solution is present at a concentration of 0.01 to 1000mg/mL.

In an alternative embodiment, the polyamine or polythiol alkaline solution has a solution pH of 8 to 10.

In an alternative embodiment, the polyamine or polythiol has the formula Y-Xn-Y, wherein n.gtoreq.2, X=CH ₂ Or CH (CH) ₂ OCH ₂ Y=primary amino or mercapto; or, the chemical formula of the polyamine or the polythiol is X _n Y _m Wherein n.gtoreq.2, X=CH ₂ Or CH (CH) ₂ OCH ₂ Y=primary amino or mercapto, m.gtoreq.2, Y may occur at any proton substitution position of the X repeating structural unit.

In an alternative embodiment, the polyamine comprises at least one of ethylenediamine, propylenediamine, butylenediamine, hexylenediamine, heptylenediamine, octylenediamine, metaphenylene diamine, p-phenylenediamine, benzidine, polyethyleneimine, cystamine, and selenocysteine.

In an alternative embodiment, the polythiol comprises at least one of hexanedithiol, dimercaptomethane, dimercaptobenzene, and trismercaptomethane.

In an alternative embodiment, the solvent in the polyamine or polythiol alkaline solution is an alkaline buffer solution.

In an alternative embodiment, the alkaline buffer solution comprises at least one of a carbamate buffer, a bicarbonate buffer, a PBS buffer, a Tris-HCl buffer, an ethanolamine buffer, a sodium hydroxide solution, and a potassium hydroxide solution.

In an alternative embodiment, the alkaline buffer solution is a sodium hydroxide solution.

In an alternative embodiment, the concentration of the alkaline buffer solution is 0.0001 to 40mol/L.

In an alternative embodiment, the second incubation is at a temperature of 0-50℃for a period of more than 30 minutes.

In an alternative embodiment, the second incubation is at a temperature of 25-40℃for a period of 0.5-24h.

In a second aspect, the present application provides a polypeptide or protein eluting coating prepared by the method of any one of the preceding embodiments.

In a third aspect, the present application provides the use of a polypeptide or protein eluting coating as in the previous embodiments for surface modification of a substrate.

In alternative embodiments, the substrate comprises at least one of a metallic material, an inorganic nonmetallic material, a polymeric material, biomedical micro-nanoparticles, a natural biological material, and an artificially synthesized polypeptide hydrogel material.

In an alternative embodiment, the metallic material comprises 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.

In alternative embodiments, the inorganic nonmetallic material includes at least one of titanium dioxide, titanium oxide, titanium nitride, ceramic, silicon dioxide, silicon carbide, calcium silicon nitride phosphate, calcium phosphate, aluminosilicate, calcium aluminum-based bioglass, hydroxyapatite, carbon material, diamond, and diamond-like carbon.

In alternative embodiments, the polymeric material comprises at least one of polyester, polyethylene, polyvinyl chloride, polyvinyl alcohol, polypropylene, polycarbonate, carbon copolymer, polyglycolic acid, polymethyl methacrylate, polytetrafluoroethylene, polyoxymethylene, polystyrene, polyvinyl acetate, polylactic acid-glycolic acid copolymer, polytrimethylene carbonate, polyhydroxyalkanoate, polybutylene succinate, polyamide, polydioxanes, epoxy resins, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives, polyvinyl alcohol, polycaprolactone, and polyurethane.

In an alternative embodiment, the biomedical micro-nanoparticles include at least one of ferroferric oxide nanoparticles, silica nanoparticles, titanium oxide nanoparticles, and zinc oxide nanoparticles.

In an alternative embodiment, the natural biological material comprises a polysaccharide and at least one of decellularized tissue and organs of animal origin; wherein the polysaccharide comprises at least one of plastic starch-based material, gelatin, collagen, sodium hyaluronate, fibrin, sodium alginate, agarose, silk protein, keratin, cellulose, hemicellulose, lignin, chitin and derivatives thereof; the decellularized tissue and organs of animal origin include at least one of blood vessels, valves, heart, bone, lung, ligaments, bladder, mucosa, and cornea.

In an alternative embodiment, the synthetic polypeptide hydrogel material comprises at least one of poly-L-lysine and poly-L-glutamic acid.

The beneficial effects of this application include:

the application creatively provides a preparation method of a polypeptide or protein eluting coating, which utilizes polyphenols and polypeptides (or proteins) to deposit and obtain a uniform hydrogel coating through weak interaction such as hydrogen bonds, hydrophobic force and the like under an acidic condition, and can maintain the original physiological functions of the polypeptides (or proteins). The film forming process can quickly form film on the surfaces of metals, non-metals, polymers and other materials without adjusting the pH value, and the corresponding preparation method is simple and easy to implement and has good industrial application prospect.

The hydrogel coating material is placed in an alkaline polyamine or polythiol solution, and in-situ chemical crosslinking of the gel coating is realized through phenolic amine, so that the gel coating assembled based on hydrogen bonds and hydrophobic force is converted into a solid coating with a highly phenolic amine network structure for coating polypeptide or protein.

The method has the advantages of simple process and mild conditions, realizes the preparation of the functional polypeptide or protein eluting coating with adjustable and controllable multi-scene application, has the characteristic of broad-spectrum modification, does not depend on the material of a base material, and has the stability of a chemical structure.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing cumulative release statistics of valirudin in the hydrogel coating and hydrogel conversion coating prepared in example 3 and example 6 of test example 1;

FIG. 2 is a graph showing thrombin (TT) time results of the hydrogel coating and hydrogel conversion coating prepared in example 3 and example 6 of test example 2;

FIG. 3 is a graph showing the anticoagulant effect of each group of hydrogel coatings before and after modification in test example 3;

FIG. 4 is a graph showing the antibacterial effect of each group of hydrogel coatings before and after modification in test example 4;

FIG. 5 is a graph comparing the hydrogel coating of example 3 and comparative example 3 before and after modification in test example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The following provides a specific description of the polypeptide or protein eluting coating, and a preparation method and application thereof.

The inventors propose that the multifunctional polypeptide or protein loading should take the following aspects into account: i) Effectively depositing polypeptide or protein on various surfaces with different sizes, length-width ratios and material types so as to meet the requirements of different application scenes; ii) maintaining structural and functional integrity of the assembled polypeptide or protein, ensuring that its activity and properties on the substrate are not affected; iii) The properties of a variety of polypeptides or proteins are allowed to be introduced to meet multiple functional needs in complex applications, including but not limited to binding capacity, catalytic activity, recognition specificity, and the like of different polypeptides or proteins.

Based on the above consideration, the application creatively provides a preparation method of a polypeptide or protein eluting coating, which utilizes polyphenols and polypeptides (or proteins) to deposit through weak interactions such as hydrogen bonds, hydrophobic forces and the like under acidic conditions to obtain a uniform hydrogel coating, and can maintain the original physiological functions of the polypeptides (or proteins). The film forming process can quickly form film on the surfaces of metals, non-metals, polymers and other materials without adjusting the pH value, and the corresponding preparation method is simple and easy to implement and has good industrial application prospect.

For reference, the method comprises the steps of: incubating the substrate for the first time with a mixed solution to form an adhesive gel coating on the surface of the substrate, wherein the mixed solution is prepared by mixing polyphenol and polypeptide or protein in an acidic solution system; and (3) placing the adhesive gel coating in polyamine or polythiol alkaline solution for a second incubation, and chemically realizing in-situ chemical crosslinking of the adhesive gel coating through phenolic amine to obtain the solid coating with the polypeptide or protein wrapped by the highly phenolic amine network structure.

Wherein, in the process of forming the adhesive gel coating, the polypeptide (or protein) and the polyphenol can form the hydrogel coating through weak interaction forces such as hydrogen bond, hydrophobic force and the like. The hydrogel coating material is placed in an alkaline polyamine or polythiol solution, and in-situ chemical crosslinking of the gel coating is realized through phenolic amine, so that the gel coating assembled based on hydrogen bonds and hydrophobic force is converted into a solid coating with a highly phenolic amine network structure for coating polypeptide or protein.

The method has simple process and mild condition, can realize the preparation of the functional polypeptide or protein eluting coating with adjustable and controllable multi-scene application, and the polypeptide or protein eluting coating formed by the method can introduce one or more polypeptides (or proteins) into the coating and maintain the physiological functions of the polypeptides (or proteins).

In this application, the mixed solution may be obtained by blending the polyphenol and the polypeptide in a solvent having a ph=2 to 7 (e.g., ph=2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, etc.) in an arbitrary molar ratio. Similarly, the mixed solution may be obtained by blending the polyphenol and the protein in a solvent having a ph=2 to 7 (e.g., ph=2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, etc.) in an arbitrary molar ratio.

The molar ratio of polyphenol to polypeptide in the mixed solution may be 0.01:1-100:1, such as 0.01:1, 0.1:1, 0.5:1, 1:1, 5:1, 10:1, 20:1, 50:1, 80:1, 100:1, etc. Similarly, the molar ratio of polyphenol to protein in the mixed solution is 0.01:1-100:1, 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, etc.

The concentration of polyphenols in the mixed solution may be 0.01-100mg/mL, such as 0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 5mg/mL, 10mg/mL, 20mg/mL, 50mg/mL, 80mg/mL, or 100mg/mL. The concentration of the polypeptide or protein may each independently be 0.01-100mg/mL, such as 0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 5mg/mL, 10mg/mL, 20mg/mL, 50mg/mL, 80mg/mL, or 100mg/mL. In some preferred embodiments, the concentration of polyphenols is from 0.25 to 10mg/mL and the concentration of polypeptides or proteins is from 0.5 to 20mg/mL.

In some embodiments, the mixed solution contains one polyphenol and two polypeptides. The molar ratio between the two polypeptides may 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, etc. The total concentration of the two polypeptides in the first incubation system may be 0.001-10mmol/mL, such as 0.001mmol/mL, 0.005mmol/mL, 0.01mmol/mL, 0.05mmol/mL, 0.1mmol/mL, 0.5mmol/mL, 1mmol/mL, 2mmol/mL, 5mmol/mL, 8mmol/mL, 10mmol/mL, etc. In some preferred embodiments, the molar ratio between the two polypeptides is 0.5:1 to 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, etc., the total concentration of the two polypeptides in the first incubation system is 0.01 to 5mmol/mL.

In other embodiments, the mixed solution contains one polyphenol and two proteins. The molar ratio between the two proteins may 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, etc. The total concentration of the two proteins in the first incubation system may be 0.001-10mmol/mL, such as 0.001mmol/mL, 0.005mmol/mL, 0.01mmol/mL, 0.05mmol/mL, 0.1mmol/mL, 0.5mmol/mL, 1mmol/mL, 2mmol/mL, 5mmol/mL, 8mmol/mL, 10mmol/mL, etc. 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, etc., the total concentration of the two proteins in the first incubation system is 0.01-5mmol/mL.

By controlling the preferred molar ratio and concentration scheme described above, the specificity of the two polypeptides (or proteins) can be better exploited and an optimal hydrogel coating can be formed.

As used herein, polyphenols include plant polyphenols comprising a polyhydroxyphenolic chemical structure, and may include, by way of example and without limitation, at least one of Tannins (TA), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), salvianolic acids, procyanidins, theaflavins, and brown algae polyphenols.

The polypeptides may include, by way of example and not limitation, hirudin and its derivatives, bivalirudin, the antibacterial peptide LL-37, the antibacterial peptide hCAP-18, the antibacterial peptide C, the antibacterial peptide BLP-7, beta-descensin, teriparatide and osteocalcin.

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

As a reference, the temperature of the first incubation may be 0-50℃such as 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, or the like. The first incubation time may be 0.25-96h, such as 0.25h, 1h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, 72h, 78h, 84h, 90h, 96h, etc. In some preferred embodiments, the first incubation is at a temperature of 15-50℃for a period of more than 15 minutes. In some preferred embodiments, the first incubation is at a temperature of 20-40℃for a period of 0.25-24 hours.

After the first incubation reaction is completed, the resulting product is washed and dried.

In the present application, the concentration of the polyamine or polythiol basic solution can be 0.01-1000mg/mL, such as 0.01mg/mL, 1mg/mL, 5mg/mL, 10mg/mL, 50mg/mL, 100mg/mL, 200mg/mL, 500mg/mL, 800mg/mL, 1000mg/mL, or the like. The concentration of polyamine or polythiol in the second incubation system can be from 0.05 to 10mg/mL, such as 0.05mg/mL, 0.1mg/mL, 1mg/mL, 5mg/mL, 10mg/mL, etc.

The solution pH of the polyamine or polythiol alkaline solution can be 8 to 10, such as 8, 8.5, 9, 9.5, or 10, etc.

In some embodiments, the polyamine or polythiol can have the formula Y-Xn-Y, wherein n.gtoreq.2, X=CH ₂ Or CH (CH) ₂ OCH ₂ Y=primary amino or mercapto. In other embodiments, the polyamine or polythiol can have the formula X _n Y _m Wherein n.gtoreq.2, X=CH ₂ Or CH (CH) ₂ OCH ₂ Y=primary amino or mercapto, m.gtoreq.2, Y may occur at any proton substitution position of the X repeating structural unit.

By way of reference, the polyamine may include, by way of example and without limitation, at least one of ethylenediamine (ethylenediamine), propylenediamine (Propylene diamine), butylenediamine (butylenediamine), hexylenediamine (Hexamethylene diamine), heptylenediamine (Heptamethylene diamine), octylenediamine (Octamethylene diamine), m-phenylenediamine (m-Phenylene diamine), p-phenylenediamine (p-Phenylene diamine), benzidine (Benzidine), polyethyleneimine (Polyethyleneimine), cystamine, and selenocysteine.

The polythiol may include, by way of example and not limitation, at least one of hexanedithiol (Hexamethylene dithiol), dimercaptomethane (Methane dithiol), dimercaptobenzene (Benzene dithiol), and tristhiomethane (tristhiol Methane).

In some preferred embodiments, the polyamine or polythiol is selected from hexamethylenediamine, cystamine, selenocysteine, or hexanedithiol.

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

In this application, the temperature of the second incubation may be 0-50deg.C, such as 0deg.C, 10deg.C, 15deg.C, 20deg.C, 25deg.C, 30deg.C, 35deg.C, 40deg.C, 45deg.C, 50deg.C, etc. The second incubation time may be 30 minutes or longer. In some preferred embodiments, the second incubation is at a temperature of 25-40℃for a period of 0.5-24 hours.

In the application, the temperature of the second incubation reaction can be carried out in a wider range, the reaction time is preferably more than 6h, and if the temperature is lower than 6h, the partial type of protein cannot be uniformly deposited on the surface of the material.

After the second incubation was completed, the incubated product was dried under nitrogen atmosphere. Specifically, the washing was performed 3 times with distilled water for 5 minutes each. Through the treatment, the reactant which is not deposited on the surface can be effectively removed, and then the target coating can be obtained by blowing the nitrogen.

Controlled release of polypeptides (low molecular weight proteins) can be achieved by crosslinking the coating as described above with a polyamine or polythiol reagent.

Correspondingly, the application also provides a polypeptide or protein eluting coating, which is prepared by the preparation method.

The coating has the characteristic of broad-spectrum modification, is not dependent on the material of a base material, is suitable for being deposited on the surfaces of various materials, and has stronger adhesion capability and higher chemical structure stability.

In addition, the application also provides application of the polypeptide or protein eluting coating, for example, the polypeptide or protein eluting coating can be used for surface modification of a substrate.

The substrate may be a solid material of any geometry and material known to humans.

For reference, the substrate may include at least one of a metallic material, an inorganic nonmetallic material, a polymeric material, biomedical micro-nano particles, a natural biological material, and an artificially synthesized polypeptide-based hydrogel material, by way of example and not limitation.

The metallic material may include, by way of example and not limitation, 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.

Inorganic nonmetallic materials may include, by way of example and not limitation, titanium dioxide, titanium oxide, titanium nitride, ceramics, silicon dioxide, silicon carbide (SiC), calcium phosphate silicon nitride (Si) ₃ N ₄ ) Calcium phosphate, aluminosilicate (Na) ₂ O·Al ₂ O ₃ ·SiO ₂ ) Calcium-aluminum system (CaO. Al) ₂ O ₃ ) Bioglass (SiO) ₂ ·CaO·Na ₂ O·P ₂ O ₅ ) At least one of hydroxyapatite, a carbon material (C), diamond and diamond-like carbon.

The polymeric material may include, by way of example and not limitation, at least one of dacron (PET), polyethylene (PE), polyvinylchloride (PVC), polyvinyl alcohol (PVALC), polypropylene (PP), polycarbonate (PC), carbon copolymer (PDC), polyglycolic acid (PGA), polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), polyoxymethylene (POM), polystyrene (PS), polyvinyl acetate (PVA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polytrimethylene carbonate (PTMC), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polyamide (PA), polydioxanes (PDS), epoxy (Epoxy), silicone rubber, silicone gel, polyacrylic acid (PAA) and derivatives thereof, polyethylene glycol and derivatives thereof, polyvinyl alcohol (PVA), polycaprolactone (PCL), polyurethane (PU).

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

The natural biological material may include, by way of example and not limitation, polysaccharides and at least one of decellularized tissue and organs of animal origin. Wherein the polysaccharide comprises at least one of plastic starch-based material (PSM), gelatin (gelatin), collagen (collagen), sodium hyaluronate (sodium hyaluronate), fibrin (fibrin), sodium alginate, agarose (agaros), silk protein, keratin, cellulose, hemicellulose, lignin, chitin and derivatives thereof; the decellularized tissue and organs of animal origin include at least one of blood vessels, valves, heart, bone, lung, ligaments, bladder, mucosa, and cornea.

The synthetic polypeptide hydrogel material may include, by way of example and not limitation, at least one of poly-L-lysine and poly-L-glutamic acid.

The coating provided by the application is a surface coating with multiple biological functions, the preparation method of the coating has no limitation on the types of metal, inorganic nonmetal or high polymer materials, the process is simple, the condition is mild, the implementation is easy, in addition, the parameter adjustment range in the preparation process is large, and the requirements of different materials on medical scenes can be met; the preparation raw materials are common substances or extracts, are nontoxic, and are interdependent and act together.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

(1) The silicon rubber substrate is rubbed with SDS to remove impurities, fully washed with distilled water, then ultrasonically washed with alcohol for three times for 3 minutes each time, and dried with nitrogen for standby.

(2) And (3) blending tannic acid and bivalirudin in distilled water according to a molar ratio of 5:4 to obtain a mixed solution with pH of 5. In the mixed solution, the concentration of tannic acid is 1mg/mL, and the concentration of bivalirudin is 1mg/mL.

(3) The stainless steel sheet to be used was immersed in the above mixed solution, and sonicated for 1min. And placing the well-sonicated system in an incubator at 37 ℃ for standing for 24 hours to perform a first incubation reaction.

(4) After the first incubation reaction was completed, the silicone rubber was removed, gently rinsed 3 times with distilled water, and dried in a fume hood after the filter paper had been dried from the side to obtain a bivalirudin-releasing hydrogel coating sample.

Example 2

This example is essentially the same as example 1, except that the functional polypeptide is replaced with a mersacidin antimicrobial polypeptide to obtain a hydrogel coating sample with release of the antimicrobial polypeptide.

Example 3

(1) The 316L stainless steel sheet was mirror polished, washed with SDS to remove impurities, thoroughly washed with distilled water, and then ultrasonically washed three times with acetone for 3 minutes each time. And (5) drying with nitrogen. 20mw/cm using a lamp comprising 185nm, 253.7nm dual band ultraviolet ² The stainless steel sheet is irradiated for 10 minutes and taken out for standby.

(2) And (3) blending the brown algae polyphenol and bivalirudin in distilled water according to a molar ratio of 10:1 to obtain a mixed solution with pH of 4.5. In the mixed solution, the concentration of brown algae polyphenol is 2mg/mL, and the concentration of bivalirudin is 1mg/mL.

(3) The stainless steel sheet to be used was immersed in the above mixed solution, and sonicated for 1min. And placing the well-sonicated system in an incubator at 37 ℃ for standing for 24 hours to perform a first incubation reaction.

(4) After the first incubation reaction was completed, the stainless steel sheet was removed, gently rinsed 3 times with distilled water, and dried in a fume hood after the filter paper had laterally absorbed the surface water, to obtain a hydrogel coating with released anticoagulant polypeptide bivalirudin.

Example 4

This example is essentially the same as example 3 except that the plant polyphenol compound is procyanidin, and a coated sample with release bivalirudin is obtained.

Example 5

This example is essentially the same as example 3, except that bivalirudin is exchanged for the anti-inflammatory polypeptide Chemerin15, and a sample with a release anti-inflammatory coating is obtained.

Example 6

Hexamethylenediamine is prepared in advance with an HCl solution having a concentration of 10mg/mL to give an alkaline solution of hexamethylenediamine having a concentration of 1mg/mL and a pH of 8.

The sample with the anticoagulated hydrogel coating obtained in example 3 was placed in the above hexamethylenediamine alkaline solution, allowed to stand in an incubator at 37 ℃ for 24 hours for the second incubation, after the incubation was completed, gently rinsed 3 times with distilled water, and dried in a fume hood after the filter paper had been dried from the side to obtain the hydrogel conversion coating with controlled release bivalirudin.

Example 7

(1) The method of measuring and controlling sputtering deposition coating film is used to obtain the rutile type titanium dioxide surface with the thickness of 100nm on the silicon chip, and after rinsing with distilled water, the silicon chip is irradiated with ultraviolet light with the double wave bands of 185nm and 253.7nm at the power of 20mW/cm ² The stainless steel sheet is irradiated for 10 minutes and taken out for standby.

(2) Tannic acid and bivalirudin are mixed in distilled water according to the mol ratio of 1:1, and a mixed solution with pH of 5 is obtained. In the mixed solution, the concentration of tannic acid is 5mg/mL, and the concentration of bivalirudin is 4mg/mL.

(3) The titania sheet to be used was immersed in the above mixed solution, and sonicated for 1min. And placing the well-sonicated system in an incubator at 37 ℃ for standing for 24 hours to perform a first incubation reaction.

(4) After the first incubation reaction was completed, the titanium dioxide sheet was removed, gently rinsed 3 times with RO water, the filter paper was side-blotted to dry the surface water, and dried in a fume hood to obtain a hydrogel coating with release bivalirudin.

Example 8

This example is substantially the same as example 7 except that the concentration ratio of tannic acid aqueous solution to bivalirudin aqueous solution is 1:2, and the tannic acid concentration of the system is 1mg/mL.

Example 9

Selenocysteine is prepared into selenocysteine alkaline solution with concentration of 1mg/mL and pH value of 8 in advance by NaOH solution with concentration of 18 mg/mL.

The sample with the release bivalirudin hydrogel coating obtained in example 7 was placed in the selenocysteine alkaline solution described above, allowed to stand in an incubator at 37 ℃ for 24 hours for a second incubation, followed by gently rinsing 3 times with RO water, and the filter paper was dried from the side with water, and dried in a fume hood to obtain a hydrogel conversion coating with controlled release bivalirudin.

Example 10

(1) The medical polyurethane and SDS are rubbed to remove impurities, fully washed by distilled water and then ultrasonically washed by absolute ethyl alcohol for three times for 3 minutes each time. And (5) drying with nitrogen. 20mw/cm using a lamp comprising 185nm, 253.7nm dual band ultraviolet ² The silicone rubber was irradiated for 10 minutes and taken out for use.

(2) And (3) blending the brown algae polyphenol and the lysozyme in distilled water according to a molar ratio of 2:1 to obtain a mixed solution with the pH of 4.5. In the mixed solution, the concentration of brown algae polyphenols is 10mg/mL, and the concentration of lysozyme is 1mg/mL.

(3) The silicone rubber sheet to be used was immersed in the above mixed solution, and sonicated for 1min. And placing the well-sonicated system in an incubator at 37 ℃ for standing for 24 hours to perform a first incubation reaction.

(4) After the first incubation reaction was completed, the silicone rubber sheet was removed, gently rinsed 3 times with RO water, and the filter paper was dried in air after sucking the surface water from the side, to obtain a hydrogel coating sample with lysozyme released.

Example 11

This example is substantially the same as example 10 except that the plant polyphenol compound is epigallocatechin gallate, and a hydrogel coated sample with released lysozyme is obtained.

Example 12

1, 6-hexanedithiol was prepared in advance as 1, 6-hexanedithiol alkaline solution at a concentration of 1mg/mL and a pH of 8 with NaOH solution at a concentration of 10 mg/mL.

The sample having the anti-coagulant antibacterial hydrogel coating obtained in example 9 was placed in the above 1, 6-hexanedithiol alkaline solution, left in an incubator at 37℃for 24 hours for the second incubation, then gently rinsed 3 times with RO water, and dried in a fume hood after the filter paper had been dried from the side. The hydrogel conversion coating with the controllable release lysozyme is obtained.

Example 13

This example is essentially the same as example 11, except that 1, 6-hexanedithiol is replaced with a selenocysteine solution.

Example 14

This example is substantially the same as example 10 except that bivalirudin is added simultaneously to the mixed solution to obtain a hydrogel coating sample having released lysozyme and bivalirudin.

(1) And (3) blending the brown algae polyphenol, lysozyme and bivalirudin in distilled water according to a molar ratio of 2:1:1 to obtain a mixed solution with pH of 4.5. In the mixed solution, the concentration of brown algae polyphenol is 10mg/mL, the concentration of lysozyme is 1mg/mL, and the concentration of bivalirudin is 0.5mg/mL.

(2) The silicone rubber sheet to be used was immersed in the above mixed solution, and sonicated for 1min. And placing the well-sonicated system in an incubator at 37 ℃ for standing for 24 hours to perform a first incubation reaction.

(3) After the first incubation reaction is finished, the silicone rubber sheet is taken out, the silicone rubber sheet is lightly washed 3 times by RO water, the filter paper is dried in the air after the water on the surface is sucked from the side, and a hydrogel coating sample with released lysozyme and bivalirudin is obtained.

Example 15

Selenocysteine is prepared into selenocysteine alkaline solution with concentration of 1mg/mL and pH value of 8 in advance by NaOH solution with concentration of 18 mg/mL.

The sample obtained in example 14 with the hydrogel coating releasing bivalirudin and lysozyme was placed in the above selenocysteine alkaline solution, left to stand in an incubator at 37 ℃ for a second incubation, then gently rinsed 3 times with RO water, and the filter paper was dried from the side, drying in a fume hood, yielding a hydrogel conversion coating with controlled release bivalirudin and lysozyme.

Comparative example 1

This comparative example differs from example 1 only in that: bivalirudin is not added.

The substrate is selected from a silicon rubber catheter, the concentration of tannic acid is controlled to be 1mg/mL, distilled water is reacted for 12 hours at 37 ℃ for three times, nitrogen is used for blowing off surface moisture, and the substrate is put into a drying environment at 37 ℃ for standby.

Comparative example 2

This comparative example differs from example 2 only in that: no mersacidin antibacterial polypeptide was added.

316L SS is selected as a base material, the concentration of tannic acid is controlled to be 1mg/mL, distilled water is reacted for 12h at 37 ℃ for three times, surface moisture is blown off by nitrogen, and the base material is put into a drying environment at 37 ℃ for standby.

Comparative example 3

This comparative example differs from example 3 only in that: the pH of the distilled water was adjusted to 9.

The base material is silicon rubber, the concentration of brown algae polyphenols is controlled to be 2mg/mL, and the concentration of bivalirudin is controlled to be 1mg/mL. The mixture was reacted at 37℃for 12 hours, washed three times with distilled water, surface water was purged with nitrogen, and the mixture was dried at 37 ℃.

Test example 1

The following method was used to detect the amount of Bivalirudin (BVLD) released according to example 3 and example 6, and the results are shown in fig. 1.

The specific process is as follows:

(1) A molecule of fluorescein (designated BVLD-Fluor) was previously labeled on BVLD.

(2) The hydrogel coatings for releasing BVLD and the hydrogel conversion coating for controlled release BVLD were prepared according to the experimental procedure given in example 3, example 6, respectively.

(3) The hydrogel coating samples of example 3 were placed in PBS buffer and the leaching solutions were collected at 1, 2, 3, 5min, respectively, for use. The hydrogel conversion coating samples of the examples were similarly placed in PBS buffer and the leaches were collected for 1, 3, 7, 15, 30 days, respectively, for further use.

(4) Since BVLD-Fluor released in the coating elution test has absorption at 490nm wavelength, the absorption intensity at this wavelength was measured by a UV-vis spectrophotometer and the BVLD elution amount was calculated from the standard curve.

According to the results of fig. 1, it was shown that the coating in example 3 was observed that the release of BVLD was balanced after 3 minutes of immersion in PBS and the cumulative release was 595.7 ±5.8 μg. In contrast, the coating of example 6 required 15 days of PBS soak to reach equilibrium release, and the cumulative release was 555.7 + -10.8 μg. This shows that the preparation of the release BVLD and the controlled release BVLD coatings designed in example 3 and example 6 was successful.

Test example 2

To determine whether BVLD released in the released BVLD and controlled release BVLD coatings has anticoagulation capability, thrombin Time (TT) was tested for examples 3 and 6. The specific experiment is as follows:

(1) Plasma preparation: the collected blood was mixed with a sodium citrate solution at a ratio of 9:1, and blood evaluation was performed within 12 hours. Platelet Poor Plasma (PPP) was obtained by centrifugation of whole blood at 3000 rpm for 15 minutes.

(2) The experiment was performed using a TT kit (Shanghai solar Biotechnology Co., ltd., TT assay kit (liquid)). Bare and modified sheets (1 cm. Times.1 cm) were placed in 24-well plates, 2mL rabbit PPP was added to each well, and incubated at 37℃for 5 minutes. 200. Mu.L of the incubated PPP was then transferred to a 1.5mL EP tube, TT reagent was added and timing was started, and clotting time (TT) was determined by hand-shaking visual inspection at the end of plasma solution clotting or floc generation.

According to the observation results of fig. 2, in example 3, the sample after elution for 5 minutes was brought into contact with plasma, which exhibited a non-coagulated state in the TT (clotting time) test. However, in example 6, the samples after 30 days of elution still showed higher TT values, 28.5.+ -. 0.8 seconds higher than the control samples. This indicates successful preparation of the release BVLD and controlled release BVLD coatings.

Test example 3

Anticoagulation experiments were performed on the surfaces of example 1 and comparative example 1 and unmodified silicone rubber, and the experimental subjects were New Zealand white rabbits, and the results are shown in FIG. 3.

The specific process is as follows:

(1) Connecting the sample with a semi-internal circulation catheter, connecting and assembling the sample, adding physiological saline into the semi-internal circulation catheter in advance, completely discharging bubbles, ensuring that air embolism is not formed in the circulation process, and clamping two ends for standby by using hemostats;

(2) New Zealand white rabbits (2.5-3 kg) were anesthetized (30 mg/mL) by injection of pentobarbital sodium solution by ear margin intravenous injection at a dose of 1mL/kg. Subsequently, the left carotid artery and right jugular vein of the rabbits were carefully dissected out using a scalpel, leaving the blood vessels bare.

(3) The needle was removed and inserted by puncture into the carotid artery and jugular vein of the rabbit, and the assembled sample was then connected to the carotid artery and vein of the rabbit, allowing blood to flow back to the heart to form a semi-internal blood circulation circuit. The change in blood color and temperature in the catheter needs to be observed continuously throughout the experiment.

(4) After 2h of circulation the experiment was stopped, the experimental sample was removed and rinsed with physiological saline. The sample photograph and the catheter cross section were then taken, and as can be seen from fig. 3, the examples have a pronounced anticoagulation effect, whereas the comparative examples do not.

Test example 4

The results of the antibacterial plating experiments performed on the surfaces of example 2 and comparative example 2 and unmodified 316L SS are shown in fig. 4.

The detailed steps are as follows: the experiment used E.coli (E.coil), a typical species of gram-negative bacteria, and Staphylococcus epidermidis (S.epideris), a typical species of gram-positive bacteria. And experiments were completed under ISO 22196-2011 standard. The experimental steps are as follows:

(1) Sample and culture medium sterilization: the prepared samples and PE films were sterilized by UV for 30min. And (3) sterilizing the centrifuge tube, the pipette tip, the glass culture dish and the like which are required to be used for experiments at high temperature and high pressure. The solid medium formulation used in the experiment was: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 15g/L of agar, and the solvent is distilled water. After all the reagents are fully dissolved, conventional high-temperature high-pressure sterilization is carried out, and sealing storage is carried out after sterilization. The agar is removed on the basis of the solid culture medium to obtain the liquid culture medium, and the rest components are consistent.

(2) Bacterial activation, purification and bacterial suspension preparation: after the strain was obtained in the slant medium, it was cultured on the solid medium using the partition streak separation method for 24 hours, and after that, a monoclonal colony was obtained, and it was left to stand at 37℃for 24 hours with shaking, to obtain an activated strain. Preparing a bacterial suspension: firstly, a culture solution is prepared (to be The liquid culture medium is diluted in normal saline according to the volume ratio to obtain a culture solution, the liquid culture medium is diluted by 500 times to obtain an escherichia coli culture solution, and the dilution is 100 times to obtain a staphylococcus aureus culture solution. Taking 1mL of activated strain, and gradient diluting with culture solution to obtain strain with concentration of 5.0X10 ⁵ ～1.0×10 ⁶ CFU/mL of bacterial liquid (detectable by spectrophotometry at 600nm wavelength).

(3) Inoculating bacteria on the surface of the sample: placing sterilized samples in 24 pore plates, dripping 100uL of diluted bacterial liquid on the surface of each sample, covering the bacterial liquid on the whole surface of the coating by using a gun head, avoiding dripping the bacterial liquid from the surface of the sample, and covering the samples by using a clean PE film. Put into a incubator at 37 ℃ for incubation for 24 hours.

(4) Plate culture: after preparing the solid culture medium and sterilizing, pouring the solid culture medium into a culture dish in a clean workbench for cooling and solidifying. 1mL of physiological saline was added to each sample, and the mixture was repeatedly blown with a pipette to completely detach the bacteria from the sample surface and PE film. Then 100 mu L of bacterial liquid is sucked and evenly coated on a solid culture medium prepared in advance, and the culture medium is incubated for 24 hours at the constant temperature of 37 ℃. And finally, photographing and recording.

As can be seen from fig. 4, in example 2, the antibacterial peptide in the coating layer has a good bactericidal effect against escherichia coli (gram-negative bacteria) and staphylococcus epidermidis (gram-positive bacteria).

The experimental results of the comparative examples and comparative examples show that various modified materials in the examples of the application have corresponding functional performances. According to application scenes and application ranges, proper polyphenol or polypeptide with anti-inflammatory activity, antibacterial property, oxidation resistance and the like can be selected for assembly, and proper crosslinking agent is used for crosslinking. The adjustable and controllable functional protein layer for multi-scene application is realized by controlling the types of polyphenol and polypeptide, the types of different cross-linking agents, the concentration of reagents and the deposition time of the coating.

Test example 5

As shown in fig. 5, when the pH of the system is adjusted to 9, weak interaction forces such as hydrogen bonds are broken in an alkaline environment, and thus the gel coating cannot adhere to the surface of the material to form a film.

In summary, the present application provides a solid coating having a highly phenolic amine network structure encapsulating polypeptides or proteins by incubating a mixed solution for the first time to a substrate to form an adhesive gel coating on the surface of the substrate, and then incubating the adhesive gel coating in a polyamine or polythiol alkaline solution for the second time to achieve in situ chemical cross-linking of the adhesive gel coating by phenolic amine. The method realizes the preparation of the functional polypeptide or protein eluting coating with adjustable and controllable multi-scene application, and the polypeptide or protein eluting coating formed by the method has the characteristic of broad-spectrum modification, does not depend on the material of a base material, and has the stability of a chemical structure.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for preparing a polypeptide or protein eluting coating, comprising the steps of: incubating a substrate for the first time with a mixed solution to form an adhesive gel coating on the surface of the substrate, wherein the mixed solution is prepared by mixing polyphenol and polypeptide or protein in an acidic solution system;

and (3) placing the adhesive gel coating in polyamine or polythiol alkaline solution for the second incubation, and realizing in-situ chemical crosslinking of the adhesive gel coating through phenolic amine to obtain the solid coating with high phenolic amine network structure for coating polypeptide or protein.

2. The production method according to claim 1, wherein the mixed solution is obtained by blending the polyphenol and the polypeptide in a solvent having a ph=2 to 7 at an arbitrary molar ratio, or the mixed solution is obtained by blending the polyphenol and the protein in a solvent having a ph=2 to 7 at an arbitrary molar ratio;

Preferably, 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;

preferably, the concentration of the polyphenol in the mixed solution is 0.01-100mg/mL, and the concentration of the polypeptide or the protein is 0.01-100mg/mL.

3. The method of claim 2, wherein the mixed solution comprises one polyphenol and two polypeptides; the molar ratio of the two polypeptides is 0.01:1-10:1, and the total concentration of the two polypeptides in the first incubation system is 0.001-10mmol/mL;

or the mixed solution contains one polyphenol and two proteins; the mol ratio of the two proteins is 0.01:1-10:1; the total concentration of the two proteins in the first incubation system is 0.001-10mmol/mL;

preferably, the molar ratio between the two polypeptides is 0.5:1-5:1, the total concentration of the two polypeptides in the first incubation system is 0.01-5mmol/mL;

or, the molar ratio of the two proteins is 0.5:1-5:1, and the total concentration of the two proteins in the first incubation system is 0.01-5mmol/mL.

4. A method of preparation according to any one of claims 1 to 3 wherein the polyphenols comprise plant polyphenols comprising polyhydroxyphenolic chemical structures; preferably, the plant polyphenol comprises at least one of tannic acid, epigallocatechin, epicatechin gallate, epigallocatechin gallate, salvianolic acid, procyanidins, theaflavins, and brown algae polyphenol;

Or, the polypeptide comprises at least one of hirudin and its derivatives, bivalirudin, antibacterial peptide LL-37, antibacterial peptide hCAP-18, antibacterial peptide C, antibacterial peptide BLP-7, beta-descensin, teriparatide and osteocalcin;

or, the protein comprises at least one of lactoferrin, soy protein, whey protein, osteocalcin, insulin, collagen, bone sulfur protein, cathepsin S, lysozyme, glucose oxidase, pepsin, horseradish peroxidase, hemoglobin, cytochrome C, immunoglobulin G, fibrinogen, trypsin, and insulin.

5. The preparation method according to claim 1, wherein the first incubation is performed at a temperature of 0-50 ℃ for a time of 0.25-96 hours;

preferably, the temperature of the first incubation is 15-50 ℃ and the time is more than 15 min;

more preferably, the first incubation is at a temperature of 20-40℃for a period of 0.25-24h.

6. A process according to any one of claims 1 to 3, wherein the polyamine or polythiol alkaline solution has a concentration of 0.01 to 1000mg/mL;

preferably, the polyamine or polythiol alkaline solution has a solution pH of 8 to 10;

Preferably, the polyamine or polythiol has the formula Y-Xn-Y, wherein n.gtoreq.2, X=CH ₂ Or CH (CH) ₂ OCH ₂ Y=primary amino or mercapto; or, the chemical formula of the polyamine or the polythiol is X _n Y _m Wherein n.gtoreq.2, X=CH ₂ Or CH (CH) ₂ OCH ₂ Y=primary amino or mercapto, m is not less than 2, Y may occur at any proton substitution position of the X repeating structural unit;

preferably, the polyamine comprises at least one of ethylenediamine, propylenediamine, butylenediamine, hexylenediamine, heptylenediamine, octylenediamine, metaphenylene diamine, p-phenylenediamine, benzidine, polyethyleneimine, cystamine and selenocyamine;

preferably, the polythiol comprises at least one of hexanedithiol, dimercaptomethane, dimercaptobenzene, and trismercaptomethane.

7. The method according to claim 6, wherein the solvent in the polyamine or polythiol alkaline solution is an alkaline buffer solution;

preferably, the alkaline buffer solution comprises at least one of a carbamate buffer, a bicarbonate buffer, a PBS buffer, a Tris-HCl buffer, an ethanolamine buffer, a sodium hydroxide solution, and a potassium hydroxide solution;

preferably, the alkaline buffer solution is a sodium hydroxide solution;

preferably, the concentration of the alkaline buffer solution is 0.0001-40mol/L.

8. The preparation method according to claim 1, wherein the temperature of the second incubation is 0-50 ℃ for more than 30 min;

preferably, the second incubation is at a temperature of 25-40℃for a period of 0.5-24h.

9. A polypeptide or protein eluting coating prepared by the method of any one of claims 1 to 8.

10. Use of a polypeptide or protein eluting coating according to claim 9, wherein the polypeptide or protein eluting coating is used for surface modification of a substrate;

preferably, the substrate comprises at least one of a metal material, an inorganic nonmetallic material, a high molecular material, biomedical micro-nano particles, a natural biological material and an artificially synthesized polypeptide hydrogel material;

preferably, the metallic material comprises 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;

preferably, the inorganic nonmetallic material comprises at least one of titanium dioxide, titanium oxide, titanium nitride, ceramic, silicon dioxide, silicon carbide, calcium silicon nitride phosphate, calcium phosphate, aluminosilicate, calcium aluminum bioglass, hydroxyapatite, carbon material, diamond and diamond-like carbon;

Preferably, the polymer material comprises at least one of terylene, polyethylene, polyvinyl chloride, polyvinyl alcohol, polypropylene, polycarbonate, carbon copolymer, polyglycolic acid, polymethyl methacrylate, polytetrafluoroethylene, polyoxymethylene, polystyrene, polyvinyl acetate, polylactic acid-glycolic acid copolymer, polytrimethylene carbonate, polyhydroxyalkanoate, polybutylene succinate, polyamide, polydioxane, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives, polyvinyl alcohol, polycaprolactone, and polyurethane;

preferably, the biomedical micro-nano particles comprise at least one of ferroferric oxide nano particles, silicon dioxide nano particles, titanium oxide nano particles and zinc oxide nano particles;

preferably, the natural biological material comprises at least one of a polysaccharide and an animal-derived decellularized tissue and organ; wherein the polysaccharide comprises at least one of a plastic starch-based material, gelatin, collagen, sodium hyaluronate, fibrin, sodium alginate, agarose, silk protein, keratin, cellulose, hemicellulose, lignin, chitin and derivatives thereof; the decellularized tissue and organs of animal origin include at least one of blood vessels, valves, heart, bone, lung, ligaments, bladder, mucosa, and cornea;

Preferably, the synthetic polypeptide hydrogel material comprises at least one of poly-L-lysine and poly-L-glutamic acid.