Metal protein coating and preparation method thereof

Abstract

The invention belongs to the field of biological materials, and particularly relates to a metal protein coating and a preparation method thereof. The preparation method comprises the following steps: mixing a soluble metal salt solution, a soluble protein solution and a buffer solution to obtain a mixed solution; and immersing the pretreated base material in the mixed solution, and carrying out ultraviolet radiation and reaction. According to the preparation method, an ultraviolet radiation induction method is adopted, ultraviolet is utilized to induce metal and protein to form the coating in a self-assembly mode, the method has the advantages of being efficient, high in controllability, simple and easy to operate and the like, expensive equipment and a complex process are not needed in the preparation process, and the production cost is reduced; in addition, the metal protein coating simultaneously has good biocompatibility and secondary functionalization of protein and antibacterial performance of metal ions, so that the coating has good antibacterial performance and biocompatibility, and can be covalently or non-covalently combined with other molecules or materials to realize other specific functions; and the requirements of different applications are met.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a metal protein coating and a preparation method thereof. Background Art

[0002] In the field of biomaterials, the demand for high-performance coatings continues to rise. Taking bovine serum albumin as an example, proteins show many advantages such as good biocompatibility, adjustable physical and chemical properties, easy acquisition and purification, and multiple functional sites. They can provide an environment for cells to attach and grow, promote tissue repair and regeneration, and play an important role in drug delivery systems, tissue engineering, biosensors, and cell culture. Metal ions, due to their outstanding antibacterial and antiviral properties, can effectively inhibit the growth and reproduction of bacteria and viruses, reduce the risk of infection, and are widely used in medical and food packaging fields. Combining metal ions with proteins to form metal protein coatings is expected to combine the advantages of both and bring new breakthroughs to the field of biomaterials. Although a large number of research results on metal protein coatings have been published, most of them are prepared by simple immersion, deposition, etc. The preparation process has poor controllability, and the coating is easily affected by environmental factors such as temperature, humidity, and pH value. The structure and performance are prone to change, and the reliability is poor in multiple environments and long-term use.

[0003] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for preparing a metal protein coating, the preparation method comprising the following steps:

[0005] (1) mixing a soluble metal salt solution, a soluble protein solution and a buffer solution to obtain a mixed solution;

[0006] (2) Immersing the pretreated substrate in the mixed solution, irradiating it with ultraviolet light and reacting it, thereby obtaining a metal protein coating.

[0007] Preferably, in step (1), the metal ion in the metal salt solution is Ag. + , Pt + One or a combination of two;

[0008] And / or, the concentration of the metal ions is 0.001-10 mg / mL.

[0009] Preferably, in step (1), the soluble protein is one or a combination of two or more of serum albumin, lysozyme, fibrinogen, β-lactoglobulin, α-lactalbumin, and catalase;

[0010] And / or, the concentration of the soluble protein is 0.001-10 mg / mL.

[0011] Preferably, in step (1), the buffer solution is one or a combination of two or more of acetic acid-acetate buffer, Tris-HCl buffer, hydrochloric acid solution, sodium hydroxide solution, distilled water, MES buffer, and HEPES buffer;

[0012] And / or, the pH value of the buffer solution is 2-14.

[0013] Preferably, in step (2), the material of the substrate is an inorganic material, a metal material or a polymer material.

[0014] Preferably, the inorganic material is one of titanium oxide and its nanomaterials, carbon materials, silicon, silicon dioxide and its nanomaterials, hydroxyapatite and calcium phosphate, silicon nitride, silicon carbide, aluminosilicate, calcium aluminum system, glass, hydroxyapatite, calcium carbonate, and titanium nitride;

[0015] And / or, the metal material is one of titanium-based, cobalt-based, magnesium-based, zirconium-based, zinc-based, aluminum alloy and stainless steel, as well as pure metals such as niobium, zirconium, titanium, tantalum, and precious metals such as gold, silver, and platinum;

[0016] And / or, the polymer material includes an inert polymer material such as polyethylene, polypropylene, polyacrylate, aromatic polyester, polysulfone, polytetrafluoroethylene (PTFE), silicone rubber, polyurethane, polyetheretherketone, polyvinyl chloride (PVC), polystyrene (PS), polyacrylic acid, polyacrylamide (PAM), polyvinyl alcohol (PVA) and ethylene-vinyl alcohol copolymer, poly-N-vinylpyrrolidone (PNVP), polyvinyl pyrrolidone (PVP), polyacrylonitrile (PAN), polyphthalamide (PA), polyester fiber, polyoxymethylene, polyethylene glycol, polyurethane (PU), latex, butyl rubber, thermoplastic elastomer (TPE), and a biodegradable polymer material such as aliphatic polyester, polyamino acid, polyvinyl alcohol, polylactic acid (PLA), polycaprolactone, polycarbonate, polyorthoesters, polyanhydrides, polyphosphazenes, etc.

[0017] Preferably, in step (2), the pretreatment is cleaning and drying.

[0018] Preferably, in step (2), the wavelength of the ultraviolet irradiation is 254 to 365 nm, and the ultraviolet irradiation time is 0.1 to 10 h;

[0019] And / or, the reaction temperature is 5-60°C.

[0020] Based on the same technical concept, the present invention further provides a metal protein coating obtained by the above preparation method.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention adopts an ultraviolet irradiation induction method which is different from the traditional protein coating preparation method. It uses ultraviolet to induce metal and protein to form a coating in a self-assembly manner. This method has the advantages of high efficiency, strong controllability, and simple and easy operation. The preparation process does not require expensive equipment and complex processes, thereby reducing production costs.

[0023] 2. The metal protein coating prepared by the preparation method of the present invention has weak dependence on the substrate material, and a stable coating can be prepared on commonly used inorganic materials, metal materials and polymer materials to meet the use of multiple scenarios.

[0024] 3. The metal protein coating prepared by the preparation method of the present invention has good biocompatibility, cell compatibility and anticoagulant properties based on protein, secondary functionalization based on silver ions and antibacterial properties of metal ions, so that the coating itself has good antibacterial properties and biocompatibility.

[0025] 4. The metal protein coating prepared by the preparation method of the present invention is rich in active functional groups, can be secondary modified, and can also covalently or non-covalently bind functional molecules with other molecules or materials to achieve other specific functions and meet the needs of different applications.

[0026] 5. The metal protein coating prepared by the preparation method of the present invention has high stability and can maintain its performance in various environments such as acid, alkali, and buffer solution.

[0027] 6. The metal protein coating prepared by the preparation method of the present invention can specifically respond to and decompose in the environment of oxidants such as H2O2, and has application potential in the field of drug delivery and the field of drug controlled release.

[0028] 7. The present invention is based on the ultraviolet-induced photocatalytic effect and can carry out nano-micrometer-scale patterning processing through methods such as photolithography to prepare patterned biomolecular coatings and achieve high-precision integration of required customized functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 are pictures of metalloprotein coatings prepared on different substrates; among them:

[0031] PVC stands for polyvinyl chloride; PC stands for polycarbonate; PU stands for polyurethane; PTFE stands for polytetrafluoroethylene; PS stands for polystyrene; PLGA stands for polylactic acid-glycolic acid copolymer; GLASS stands for glass; and SS stands for stainless steel.

[0032] Figure 2 This is the XPS analysis result of the metal protein coating based on polyvinyl chloride (PVC).

[0033] Figure 3 This is a diagram showing the adhesion of Pseudomonas aeruginosa, vascular endothelial cells and platelets on the metal protein coating.

[0034] Figure 4 These are the quantitative results of amino groups in metal protein coatings prepared under different UV irradiation times and the results of hyaluronic acid grafting.

[0035] Figure 5 These are photos of the metal protein coating after being immersed in acid and alkaline solutions.

[0036] Figure 6 This is the result of the rapid response of the metal protein coating to the H2O2 environment.

[0037] Figure 7 It is a microscopic pattern of metalloproteins. DETAILED DESCRIPTION

[0038] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0039] Example 1

[0040] This embodiment provides a method for preparing a metal protein coating by ultraviolet irradiation induction. The specific preparation method is as follows:

[0041] (1) The silica glass (inorganic material) substrate is cleaned with deionized water before use, and naturally dried for standby use to obtain a pretreated substrate;

[0042] (2) mixing equal volumes of a 0.02 mg / mL silver nitrate solution and a 2 g / mL bovine serum albumin solution, and adjusting the pH value of the system to 4.7 with a 1 M hydrochloric acid solution to obtain a mixed solution;

[0043] (3) immersing the pretreated substrate in the mixed solution, irradiating it for 1.5 h under a UV crosslinker with a wavelength of 365 nm, and the reaction temperature is 30° C. After the reaction is completed, ultrasonic cleaning is performed with deionized water for 3 min, and the substrate is dried naturally to obtain a metal protein coating.

[0044] Example 2

[0045] This embodiment provides a method for preparing a metal protein coating by ultraviolet irradiation induction. The specific preparation method is as follows:

[0046] (1) The polytetrafluoroethylene (polymer material) substrate is cleaned with deionized water before use, and naturally dried for standby use to obtain a pretreated substrate;

[0047] (2) mixing equal volumes of a 2 mg / mL silver nitrate solution and a 2 g / mL bovine serum albumin solution, and adjusting the pH value of the system to 4.7 with a 1 M hydrochloric acid solution to obtain a mixed solution;

[0048] (3) immersing the pretreated substrate in the mixed solution, irradiating it for 1.5 h under a UV crosslinker with a wavelength of 365 nm and a reaction temperature of 35° C. After the reaction is completed, ultrasonic cleaning is performed with deionized water for 3 min, and the substrate is dried naturally to obtain a metal protein coating.

[0049] Example 3

[0050] This embodiment provides a method for preparing a metal protein coating by ultraviolet irradiation induction. The specific preparation method is as follows:

[0051] (1) The stainless steel (metal material) substrate is cleaned with deionized water before use, and naturally dried for standby use to obtain a pretreated substrate;

[0052] (2) mixing equal volumes of a 20 mg / mL silver nitrate solution and a 2 g / mL β-lactoglobulin solution, and adjusting the pH value of the system to 5.2 with a 1 mM Tris-HCl buffer to obtain a mixed solution;

[0053] (3) immersing the pretreated substrate in the mixed solution, irradiating it for 1.5 h under a UV crosslinker with a wavelength of 365 nm and a reaction temperature of 42° C. After the reaction is completed, ultrasonic cleaning is performed with deionized water for 3 min, and the substrate is dried naturally to obtain a metal protein coating.

[0054] Example 4

[0055] This embodiment provides a method for preparing a metal protein coating by ultraviolet irradiation induction. The specific preparation method is as follows:

[0056] (1) The polystyrene (polymer material) substrate is cleaned with deionized water before use, and naturally dried for standby use to obtain a pretreated substrate;

[0057] (2) mixing equal volumes of a 20 mg / mL platinum nitrate solution and a 2 g / mL α-lactalbumin solution, and adjusting the pH value of the system to 4.5 with a 0.1 M acetic acid-acetate buffer to obtain a mixed solution;

[0058] (3) immersing the pretreated substrate in the mixed solution, irradiating it for 1.5 h under a UV crosslinker with a wavelength of 365 nm and a reaction temperature of 42° C. After the reaction is completed, ultrasonic cleaning is performed with deionized water for 3 min, and the substrate is dried naturally to obtain a metal protein coating.

[0059] Verification Example

[0060] Figure 1 Figure 1 shows metal protein coatings prepared on different substrates (0.3 mL of 2 mg / mL albumin solution was mixed with 0.3 mL of 2 mg / mL silver nitrate solution, the pH was adjusted to 4.7 with 1 M hydrochloric acid solution, and then irradiated with ultraviolet light for 1.5 h). Figure 1 It can be concluded that the metal protein coating can be deposited on the surface of various materials, the coating has a smooth morphology and a light yellow-brown color.

[0061] Figure 2 This is an XPS analysis of metal protein coatings based on polyvinyl chloride (PVC). XPS results confirm that the metal protein coating has a high density and blocks the Cl peak of the PVC substrate.

[0062] Figure 3: It is a graph showing the adhesion results of Pseudomonas aeruginosa, vascular endothelial cells and platelets on the metal protein coating. From the contact sterilization test results of the metal protein coating on Pseudomonas aeruginosa, it can be seen that the metal protein based on the present invention has a good sterilization effect on Pseudomonas aeruginosa, proving that the metal protein coating based on the present invention has a better antibacterial property than the protein coating prepared by the traditional adsorption method. After the metal protein coating was cultured with vascular endothelial cells, it can be observed that a large number of vascular endothelial cells adhered to the metal protein coating prepared by the preparation method of the present invention, and after 3 days of culture, the vascular endothelial cells proliferated in large numbers, while the blank sample and the protein coating prepared by the traditional adsorption method had fewer vascular endothelial cells adhered to the surface. This result shows that the metal protein coating based on the present invention has good cell compatibility. The platelet-rich slurry obtained by centrifugation of whole blood was contacted with and adhered to the metal protein coating. It was observed that only a small amount of platelets adhered to the surface of the metal protein coating prepared by the preparation method described in the present invention, and only some of the platelets extended pseudopodia, while a large amount of platelets were observed to adhere to the surface of the blank sample (PVC) and the protein coating prepared by the traditional adsorption method, and the platelets were spread out, and most of the adhered platelets extended pseudopodia. This result shows that the metal protein coating based on the present invention has an anticoagulant effect.

[0063] Figure 4 The figure is the amino quantitative results of the metal protein coating prepared by different ultraviolet irradiation times and the hyaluronic acid (HA) grafting results. The analysis of the amino quantitative results shows that the amino content of the metal protein coating prepared by the preparation method of the present invention is higher than that of the protein layer prepared by the traditional adsorption method, and has the potential for secondary grafting; after grafting the metal protein coating based on the present invention with hyaluronic acid of different concentrations, it is found that the water contact angle becomes smaller and the hydrophilicity increases, which also proves that the metal protein coating based on the present invention has the ability to be modified secondary.

[0064] Figure 5 This is a photo of the metal protein coating after being immersed in acid and alkali solutions (the acid is hydrochloric acid and the alkali is sodium hydroxide solution, and the photo was taken after immersion for 24 hours). Figure 5 It can be seen that the metal protein coating based on the present invention can withstand 24 hours or more in an environment of pH 1-14 and has good acid-base stability.

[0065] Figure 6 This is the result of the metal protein coating's rapid response to H2O2 environment (the metal protein coating based on glass was immersed in 0.03% H2O2 solution, and the degree of metal decomposition was evaluated by measuring the change in the coating's ultraviolet absorption peak). Figure 6 It can be seen that the metal protein coating based on the present invention can quickly respond to and decompose low-concentration H2O2 environments, with the decomposition rate reaching 38% in 1 minute and 50% in 7 minutes.

[0066] Figure 7 This is a micro-pattern of metalloprotein (200×200μm). It shows that micron-scale patterning can be performed by methods such as photolithography. Patterned biomolecular coatings can be prepared to achieve high-precision integration of required customized functions.

[0067] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a metal protein coating, characterized in that: The preparation method comprises the following steps: (1) mixing a soluble metal salt solution, a soluble protein solution and a buffer solution to obtain a mixed solution; (2) Immersing the pretreated substrate in the mixed solution, irradiating it with ultraviolet light and reacting it, thereby obtaining a metal protein coating.

2. The method for preparing the metal protein coating according to claim 1, characterized in that: In step (1), the metal ion in the metal salt solution is Ag. + , Pt + One or a combination of two; And / or, the concentration of the metal ions is 0.001-10 mg / mL.

3. The method for preparing the metal protein coating according to claim 1, characterized in that: In step (1), the soluble protein is one or a combination of two or more of serum albumin, lysozyme, fibrinogen, β-lactoglobulin, α-lactalbumin, and catalase; And / or, the concentration of the soluble protein is 0.001-10 mg / mL.

4. The method for preparing the metal protein coating according to claim 1, characterized in that: In step (1), the buffer solution is one or a combination of two or more of acetic acid-acetate buffer, Tris-HCl buffer, hydrochloric acid solution, sodium hydroxide solution, distilled water, MES buffer, and HEPES buffer; And / or, the pH value of the buffer solution is 2-14.

5. The method for preparing the metal protein coating according to claim 1, characterized in that: In step (2), the material of the substrate is an inorganic material, a metal material or a polymer material.

6. The method for preparing the metal protein coating according to claim 5, characterized in that: The inorganic material is one of titanium oxide, carbon material, silicon, silicon dioxide, hydroxyapatite and calcium phosphate, silicon nitride, silicon carbide, aluminosilicate, calcium aluminum system, glass, hydroxyapatite, calcium carbonate, and titanium nitride; And / or, the metal material is one of titanium-based, cobalt-based, magnesium-based, zirconium-based, zinc-based, aluminum alloy, stainless steel, niobium, zirconium, titanium, tantalum, gold, silver, and platinum; And / or, the polymer material includes an inert polymer material and a biodegradable polymer material; the inert polymer material is one of polyethylene, polypropylene, polyacrylate, aromatic polyester, polysulfone, polytetrafluoroethylene, silicone rubber, polyurethane, polyetheretherketone, polyvinyl chloride, polystyrene, polyacrylic acid, polyacrylamide, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, poly-N-vinylpyrrolidone, polyvinyl pyrrolidone, polyvinyl phthalate, polyacrylonitrile, polyphthalamide, polyester fiber, polyformaldehyde, polyethylene glycol, polyurethane, latex, butyl rubber, and thermoplastic elastomer; the biodegradable polymer material is one of aliphatic polyester, polyamino acid, polyvinyl alcohol, polylactic acid, polycaprolactone, polycarbonate, polyorthoesters, polyanhydrides, and polyphosphazenes.

7. The method for preparing the metal protein coating according to claim 1, characterized in that: In step (2), the pretreatment is cleaning and drying.

8. The method for preparing the metal protein coating according to claim 1, characterized in that: In step (2), the wavelength of the ultraviolet irradiation is 254 to 365 nm, and the ultraviolet irradiation time is 0.1 to 10 h; And / or, the reaction temperature is 5-60°C.

9. The metal protein coating obtained by the preparation method according to any one of claims 1 to 8.

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