CN112972769B - Method for preparing nano-silver-containing antibacterial peptide functional coating on metal surface - Google Patents

Abstract

The invention discloses a method for preparing a nano-silver-containing antibacterial peptide functional coating on a metal surface, which comprises the following steps: 1. adding silver nitrate powder and antibacterial peptide powder into the silk fibroin solution and mixing to obtain a mixed solution A; 2. carrying out ultraviolet irradiation reduction on the mixed solution A to obtain a mixed solution B; 3. calibrating the Tris solution, adding dopamine hydrochloride into the Tris solution, and soaking metal to obtain metal with a polydopamine auxiliary layer on the surface; 4. and (3) soaking the metal with the polydopamine auxiliary layer on the surface in the mixed solution B to form the nano-silver-containing antibacterial peptide functional coating on the metal surface. The invention adopts an ultraviolet irradiation method to reduce silver ions into nano-silver by utilizing silk fibroin, effectively controls the particle size and the morphology of nano-silver particles, forms coordinate bonds between the antibacterial peptide and the nano-silver, promotes the enrichment of the antibacterial peptide on the surface of the nano-silver particles, and presents a nano-scale dispersion state in the antibacterial peptide functional coating containing the nano-silver, thereby realizing long-term and efficient sterilization under the low dosage of the antibacterial agent.

Description

A method for preparing a functional coating containing nano-silver antimicrobial peptides on a metal surface

technical field

The invention belongs to the field of biomedical materials, and in particular relates to a method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface.

Background technique

Medical pure titanium has been widely used in the field of clinical medicine because of its excellent biocompatibility. As early as the 1950s, pure titanium was processed into bone plates, bone nails, femoral heads and other implanted medical devices for orthopedic repair operations. However, as a common postoperative complication, bacterial infection caused by medical devices has become one of the important problems to be solved in the field of medicine in the 21st century. According to reports, the annual incidence of orthopedic implant-related infections in the United States reaches about 4.3%. According to the relevant data in the "Practical Handbook of Nosocomial Infection Prevention and Control" promulgated by the World Health Organization (WHO), more than 14 million people around the world suffer from nosocomial infections every day, and 60% of bacterial infections are related to the use of medical devices. Postoperative infection in orthopedics will directly cause the patient's wound to not heal for a long time, often lead to operation failure, and even lead to complications such as chronic osteomyelitis. society, etc., causing varying degrees of negative impacts. Therefore, constructing functional coatings loaded with antibacterial agents (antibiotics, antimicrobial peptides, nano-silver, etc.) on the surface of medical titanium alloys has great social and economic significance for eliminating or reducing bacterial infectious diseases caused by related medical devices.

At present, antibiotics are still the most widely used traditional antibacterial agents due to their efficient and reliable bactericidal ability, but antibiotics are prone to cause bacterial resistance, leading to more serious infections caused by drug-resistant bacteria. In contrast, nano-silver and antimicrobial peptides are not easy to cause bacterial drug resistance, and can have broad-spectrum antibacterial and long-term effective inhibition of biofilm, so they have great potential to replace antibiotics. Compared with other existing forms, silver nanoparticles have higher antibacterial activity due to the combined effect of the elution of metal ions and the bactericidal properties of nanoparticles. It is generally believed that nano-silver has a complex antibacterial mechanism: it can adhere to the surface of bacteria, thereby changing the permeability of bacterial cell membranes; it can kill bacteria by interacting with thiol-containing proteins; it can bind to bacterial DNA and destroy bacteria. Its function; in particular, nano-silver can release silver ions with bactericidal ability. Antimicrobial peptides have become a research hotspot in biomolecules and biomaterials due to their various biological activities. Antimicrobial peptides have higher antibacterial efficiency when directly targeting infections caused by bacteria and fungi. At present, in biomedical materials, antimicrobial peptides have received more and more attention, mainly as surface-modified antibacterial components or as carried antibacterial drugs for local release.

Silk fibroin is the main body of silk and a natural biological macromolecule without physiological activity. Compared with other natural polymers, silk fibroin protein has obvious advantages. Studies have shown that it has good biocompatibility, non-toxic, non-polluting, non-irritating, and biodegradable. In recent years, due to its good biocompatibility, silk fibroin has been gradually used in biosensing, biomedical materials, soft tissue compatibility materials, tissue engineering and other fields. In particular, silk fibroin has reducing properties, and can be reduced in situ to obtain nano-silver and antimicrobial peptide particles, avoiding the use of toxic reducing agents.

At present, common antibiotic-loaded coatings are likely to cause bacterial resistance, and cannot cope with bacterial infection environments with higher bacterial concentrations or stronger bacterial activity, and high concentrations of antibacterial agents will produce potential biological toxicity. It is a challenge to achieve long-term and high-efficiency antibacterial effect under dosage.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing a nano-silver antimicrobial peptide-containing functional coating on a metal surface in view of the above-mentioned deficiencies in the prior art. The method uses silk fibroin to reduce silver ions to nano-silver by ultraviolet irradiation, effectively controlling the particle size and shape of nano-silver particles, and forming a coordination bond between the antimicrobial peptide and nano-silver to promote the antimicrobial peptide in nano-silver. The surface enrichment of silver particles makes the nano-silver enriched with antimicrobial peptides on the surface present a nano-scale dispersion state in the functional coating containing nano-silver antimicrobial peptides, which is conducive to the realization of long-term and high-efficiency sterilization with low antibacterial agent dosage.

In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is: a method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface, characterized in that the method comprises the following steps:

Step 1, adding silver nitrate powder and antimicrobial peptide powder to the silk fibroin solution and mixing to obtain a mixed solution A containing silk fibroin, silver ions and antimicrobial peptide; the mass concentration of silk fibroin in the mixed solution A is 2 %～5%, the concentration of silver ion is 1mg/mL～20mg/mL, the concentration of antimicrobial peptide is 0.4mg/mL～1mg/mL;

Step 2. Place the mixed solution A obtained in step 1 under an ultraviolet lamp for irradiation and reduction, so that the color of the mixed solution A gradually changes from milky white to dark brown, and a mixed solution B containing silk fibroin, nano-silver and antimicrobial peptides is obtained. ; The time for the irradiation reduction is 0.5h to 2h;

Step 3: Use standard NaOH solution to calibrate the 10mM Tris solution to pH 8.5, then add dopamine hydrochloride to obtain the working solution, and then immerse the metal in the working solution for 12h-24h under dark conditions to obtain an auxiliary layer of polydopamine on the surface Metal;

Step 4, immerse the metal having the polydopamine auxiliary layer on the surface obtained in step 3 into the mixed solution B obtained in step 2 to form a functional coating containing nano-silver antimicrobial peptide on the metal surface.

The present invention first adopts the reduction method of ultraviolet irradiation, so that silk fibroin can reduce silver ions, and by controlling the key process parameters including the concentration of silk fibroin, silver ion concentration, and irradiation reduction time, the particle size and size of nano-silver particles can be effectively controlled. The morphology of silver nanoparticles with good dispersion and uniform particle size is obtained, and the coordination bond is formed between the antimicrobial peptide and the silver nanoparticle, which promotes the enrichment of the antimicrobial peptide on the surface of the silver nanoparticle, so that the silver nanoparticle enriched with the antimicrobial peptide on the surface The protein-based functional coating containing nano-silver antibacterial peptides presents a nano-scale dispersion state, which improves the sterilization efficiency, greatly shortens the preparation time, and realizes long-term high-efficiency sterilization with low antibacterial agent dosage, that is, low antimicrobial peptide and low nano-silver dosage; and then Introduce a polydopamine auxiliary layer on the surface of the metal substrate, and use the method of soaking to make the silk fibroin-based functional coating containing nano-silver (surface-rich antibacterial peptide) be combined on the surface of the metal substrate through the polydopamine auxiliary layer, Forming a nano-silver antimicrobial peptide functional coating on the metal surface solves the problem of poor binding between the nano-silver antimicrobial peptide functional coating and the metal substrate, and improves the binding effect between the nano-silver antimicrobial peptide functional coating and the metal surface. At the same time, since the nano-silver and the antimicrobial peptide in the nano-silver antimicrobial peptide functional coating have high-efficiency bactericidal ability, especially the antimicrobial peptide has a unique bactericidal mechanism, which usually destroys the integrity of the microbial cell membrane, without activating the adaptive immunity. Rapidly kill the invading microorganisms in the absence of bacteria, effectively avoiding the genetic adaptation mechanism of bacteria, so as not to cause drug resistance, and there is a synergistic effect between nano silver and antimicrobial peptides, and long-term controllable release at the implantation site , which endows the nano-silver antimicrobial peptide functional coating with the ability of long-term and efficient sterilization, effectively solves the problem of implant-related bacterial infection, and does not cause bacterial drug resistance, and reduces the concentration of antimicrobial peptides; in addition, due to Silk fibroin has good human affinity, biocompatibility, non-toxicity, and is beneficial to cell adhesion, and the osteoinductivity of low-concentration nano-silver particles provides excellent osteoinductive ability, therefore, the present invention is in metal The nano-silver antibacterial peptide-containing functional coating prepared on the surface of (especially titanium metal) has good biocompatibility and osteoinductivity, good applicability and high application value.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptides on a metal surface is characterized in that the particle size of nano-silver in the mixed solution B described in step 2 is 20nm to 500nm, and the mixed solution B has a β-sheet structure The mass content of silk fibroin is 15%-30%. The preferred particle size ensures the best antibacterial effect of nano-silver; the preferred quality content of silk fibroin with a β-sheet structure improves the stability of the mixed solution B, thereby ensuring the nano-silver antibacterial peptide function formed on the metal surface coating stability.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface is characterized in that the amino acid sequence of the antimicrobial peptide in the mixed solution A described in step 2 is (NH ₂ )-NGIVKAGPAIAAVLGEAAL-CONH ₂ , the antimicrobial peptide The mass purity is greater than 99%. The preferred antimicrobial peptide has specific and high-efficiency bactericidal properties, and is not easy to cause bacterial drug resistance.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface is characterized in that the metal in step 3 is titanium, titanium alloy or stainless steel. The preparation method of the invention has a wide range of applications and is applicable to various common medical metals.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface is characterized in that the concentration of dopamine hydrochloride in the working solution in step 3 is 1 mg/mL-4 mg/mL.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface is characterized in that the metal described in step 3 is first cleaned and pretreated before being immersed in the working solution: the metal is sequentially immersed in acetone, ethanol and deionized water. Wash for 15 minutes. This preferred cleaning process removes impurities and pollutants on the metal surface, and is conducive to the polymerization of dopamine on the metal surface to form a polydopamine auxiliary layer.

The above method for preparing a functional coating containing nano-silver antibacterial peptide on a metal surface is characterized in that the thickness of the polydopamine auxiliary layer in step 3 is 200nm-500nm. The preferred thickness of the polydopamine auxiliary layer ensures the bonding effect between the metal and the functional coating containing the nano-silver antimicrobial peptide.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface is characterized in that the metal having a polydopamine auxiliary layer on the surface described in step 4 is soaked in the mixed solution B obtained in step 2 and then taken out and placed in In a constant temperature drying oven, dry at 60°C for 10min to 30min. The drying process promotes the formation of a stable structure of the silk fibroin, which is beneficial to improving the binding performance of the metal and the nano-silver antibacterial peptide-containing functional coating.

The above-mentioned method for preparing a functional coating containing nano-silver antimicrobial peptide on a metal surface is characterized in that the thickness of the functional coating containing nano-silver antimicrobial peptide in step 4 is 1 μm to 20 μm.

Compared with the prior art, the present invention has the following advantages:

1. The present invention utilizes silk fibroin to reduce silver ions to nano-silver by ultraviolet irradiation, effectively controlling the particle size and shape of nano-silver particles, and forming a coordination bond between the antimicrobial peptide and nano-silver to promote antimicrobial peptide production. The enrichment on the surface of nano-silver particles makes the nano-silver enriched with antimicrobial peptides on the surface present a nano-scale dispersion state in the functional coating containing nano-silver antimicrobial peptides, which is conducive to achieving long-term high-efficiency sterilization with low antibacterial agent dosage.

2. The present invention improves the bonding effect between the nano-silver antimicrobial peptide functional coating and the metal surface by introducing a polydopamine auxiliary layer on the surface of the metal substrate, and solves the problem of poor bonding between the nano-silver antimicrobial peptide functional coating and the metal substrate , which is conducive to exerting its long-term and efficient synergistic bactericidal effect as a metal implant.

3. Both the nano-silver and the antimicrobial peptide in the functional coating containing nano-silver antibacterial peptide formed on the metal surface of the present invention have high-efficiency bactericidal ability, and there is a synergistic effect between the two, and they can be released in a long-term controllable manner at the implanted position, reaching The purpose of reducing the dosage of antimicrobial peptides (far below the critical concentration of its biological toxicity) and long-term high-efficiency sterilization solves the problem of bacterial drug resistance.

4. The nano-silver antimicrobial peptide functional coating prepared on the metal surface of the present invention has good biocompatibility and osteoinductivity, and after being implanted into the organism, it inhibits the resorption process of the original bone and promotes the formation of new bone, realizing orthopedic The purpose of fast and good integration between the implanted device (prosthesis) and the surrounding bone tissue, and effectively prevent the implantation infection caused by bacteria, prolong its service life, especially suitable for the treatment of patients with pathologically induced bone resorption and fractures, with good applicability , high application value.

The technical solutions of the present invention will be described in further detail below with reference to the drawings and embodiments.

Description of drawings

Fig. 1 is a transmission electron microscope image of nano-silver in the mixed solution B of Example 1 of the present invention.

Fig. 2 is a comparison diagram of the antibacterial effect of each coating formed in Example 1 of the present invention and Comparative Examples 1 to 2 and different parts of the titanium sheet.

Detailed ways

Example 1

This embodiment includes the following steps:

Step 1. Boil the silk in 0.02M Na ₂ CO ₃ solution for 40 minutes, rinse it with deionized water after taking it out, and then dissolve it in 9.3M LiBr solution at 60°C for 4 hours to obtain silk fibroin stock solution;

The silk fibroin stock solution is diluted with deionized water into a silk fibroin solution with a mass concentration of 2%, and then 17mg of silver nitrate powder and 4mg of antimicrobial peptide powder are added to 10mL of the silk fibroin solution with a mass concentration of 2% and mixed to obtain a silk fibroin solution containing 2% mass concentration. The mixed solution A of protein, silver ion and antimicrobial peptide and pH is 8.5;

Step 2. Place the mixed solution A obtained in step 1 under a 40W ultraviolet lamp for irradiation and reduction for 0.5h, so that the color of the mixed solution A gradually changes from milky white to dark brown, and obtains silk fibroin, nano-silver and antimicrobial peptides. Mixed solution B; the particle diameter of nano-silver in the mixed solution B is 20nm, and the mass content of silk fibroin with a β-sheet structure in the mixed solution B is 15%; the amino acid sequence of the antimicrobial peptide in the mixed solution A is ( NH ₂ )-NGIVKAGPAIAAVLGEAAL-CONH ₂ , the mass purity of the antimicrobial peptide is 99.99%;

Step 3: Use standard NaOH solution to calibrate the 10mM Tris (trishydroxymethylaminomethane) solution to pH 8.5, then add dopamine hydrochloride to obtain the working solution, immerse the titanium sheet in acetone, ethanol and deionized water for 15 minutes and ultrasonically clean it. Immerse in the working solution again and soak for 12h under light-proof conditions to obtain a titanium sheet with a polydopamine auxiliary layer on the surface; the concentration of dopamine hydrochloride in the working solution is 2mg/mL; the thickness of the polydopamine auxiliary layer is 200nm;

Step 4. Soak the titanium sheet with the polydopamine auxiliary layer on the surface obtained in step 3 in the mixed solution B obtained in step 2, take it out and place it in a constant temperature drying oven, dry it at 60°C for 10 minutes, and place it on the surface of the titanium sheet A functional coating containing nano-silver antimicrobial peptide is formed; the thickness of the functional coating containing nano-silver antimicrobial peptide is 1 μm.

Fig. 1 is the transmission electron microscope figure of nanometer silver in the mixed solution B of the present embodiment, and black round particle among the figure is nano silver particle, and diameter is about 20nm, and the shadow part around black round particle is antimicrobial peptide, can from Fig. 1 It can be seen that antimicrobial peptides are enriched on the surface of silver nanoparticles.

Comparative example 1

This comparative example includes the following steps:

Step 1. Boil the silk in 0.02M Na ₂ CO ₃ solution for 40 minutes, rinse it with deionized water after taking it out, and then dissolve it in 9.3M LiBr solution at 60°C for 4 hours to obtain silk fibroin stock solution;

The silk fibroin stock solution is diluted with deionized water into a silk fibroin solution with a mass concentration of 2%, and then 17mg of silver nitrate powder is added to 10mL of the silk fibroin solution with a mass concentration of 2% and mixed to obtain silk fibroin and silver ions. And the mixed solution A whose pH is 8.5;

Step 2. Place the mixed solution A obtained in step 1 under a 40W ultraviolet lamp for irradiation and reduction for 0.5h, so that the color of the mixed solution A gradually changes from milky white to dark brown, and a mixed solution B containing silk fibroin and nano-silver is obtained. The particle size of nano silver in the mixed solution B is 20nm, and the mass content of silk fibroin with a β-sheet structure in the mixed solution B is 15%; the amino acid sequence of the antimicrobial peptide in the mixed solution A is (NH ₂ ) -NGIVKAGPAIAAVLGEAAL-CONH ₂ ;

Step 3: Use standard NaOH solution to calibrate the 10mM Tris (trishydroxymethylaminomethane) solution to pH 8.5, then add dopamine hydrochloride to obtain the working solution, immerse the titanium sheet in acetone, ethanol and deionized water for 15 minutes and ultrasonically clean it. Immerse in the working solution again and soak for 12h under light-proof conditions to obtain a titanium sheet with a polydopamine auxiliary layer on the surface; the concentration of dopamine hydrochloride in the working solution is 2mg/mL; the thickness of the polydopamine auxiliary layer is 200nm;

Step 4. Soak the titanium sheet with the polydopamine auxiliary layer on the surface obtained in step 3 in the mixed solution B obtained in step 2, take it out and place it in a constant temperature drying oven, dry it at 60°C for 30 minutes, and place it on the surface of the titanium sheet A functional coating containing nano-silver is formed; the thickness of the functional coating containing nano-silver is 1 μm.

Comparative example 2

This comparative example includes the following steps:

Step 1. Boil the silk in 0.02M Na ₂ CO ₃ solution for 40 minutes, rinse it with deionized water after taking it out, and then dissolve it in 9.3M LiBr solution at 60°C for 4 hours to obtain silk fibroin stock solution;

The silk fibroin stock solution is diluted with deionized water into a silk fibroin solution with a mass concentration of 2%, and then 4mg of antimicrobial peptide powder is added to 10mL of the silk fibroin solution with a mass concentration of 2% and mixed to obtain silk fibroin and antimicrobial peptides. And the mixed solution A whose pH is 8.5;

Step 2. Place the mixed solution A obtained in step 1 under a 40W ultraviolet lamp and irradiate it for 0.5h to obtain a mixed solution B containing silk fibroin and antimicrobial peptide; the silk fibroin with a β-sheet structure in the mixed solution B The mass content of the antimicrobial peptide is 15%, the amino acid sequence of the antimicrobial peptide in the mixed solution A is (NH ₂ )-NGIVKAGPAIAAVLGEAAL-CONH ₂ , and the mass purity of the antimicrobial peptide is 99.99%;

Step 3: Use standard NaOH solution to calibrate the 10mM Tris (trishydroxymethylaminomethane) solution to pH 8.5, then add dopamine hydrochloride to obtain the working solution, immerse the titanium sheet in acetone, ethanol and deionized water for 15 minutes and ultrasonically clean it. Immerse in the working solution again and soak for 12h under light-proof conditions to obtain a titanium sheet with a polydopamine auxiliary layer on the surface; the concentration of dopamine hydrochloride in the working solution is 2mg/mL; the thickness of the polydopamine auxiliary layer is 200nm;

Step 4. Soak the titanium sheet with the polydopamine auxiliary layer on the surface obtained in step 3 in the mixed solution B obtained in step 2, take it out and place it in a constant temperature drying oven, dry it at 60°C for 30 minutes, and place it on the surface of the titanium sheet A functional coating containing antimicrobial peptides is formed; the thickness of the functional coatings containing antimicrobial peptides is 1 μm.

The nano-silver antimicrobial peptide-containing functional coating formed on the titanium sheet surface in Example 1, the nano-silver functional coating formed on the titanium sheet surface in Comparative Example 1, and the antimicrobial peptide-containing antimicrobial peptide formed on the titanium sheet surface in Comparative Example 2 The functional coatings were co-cultured with Staphylococcus aureus (1×10 ⁸ CFU) in a 37°C incubator for 24 hours, and then counted by the plate method, and the number of Staphylococcus aureus after co-cultivation was counted, and the titanium sheet and each group The coatings were selected correspondingly to two different parts, namely part 1 and part 2, to observe the antibacterial effect.

Fig. 2 is the contrast figure of the antibacterial effect of each coating and different parts in the titanium sheet that embodiment 1 of the present invention and comparative example 1～comparative example 2 form, and the green dot in the figure represents live bacterium, and red dot represents dead bacterium, from figure 2 It can be seen that compared with the coatings and titanium sheets formed in Comparative Examples 1 to 2, the gold-yellow color of the functional coating containing nano-silver antimicrobial peptide formed on the surface of the titanium sheet in Example 1 of the present invention after co-cultivation The number of staphylococci was the least, indicating that the functional coating containing nano-silver antimicrobial peptides has excellent synergistic antibacterial effect, which is better than the functional coating containing nano-silver and the functional coating containing antimicrobial peptides.

Example 2

This embodiment includes the following steps:

Step 1. Boil the silk in 0.02M Na ₂ CO ₃ solution for 40 minutes, rinse it with deionized water after taking it out, and then dissolve it in 9.3M LiBr solution at 60°C for 4 hours to obtain silk fibroin stock solution;

The silk fibroin stock solution was diluted with deionized water into a silk fibroin solution with a mass concentration of 3%, and then 10 mg of silver nitrate powder and 8 mg of antimicrobial peptide powder were added to 10 mL of a silk fibroin solution with a mass concentration of 3% and mixed to obtain a silk fibroin solution containing 3% mass concentration. Protein, silver ion and antimicrobial peptide and the mixed solution A that pH is 8;

Step 2. Place the mixed solution A obtained in step 1 under a 40W ultraviolet lamp for irradiation and reduction for 1 hour, so that the color of the mixed solution A gradually changes from milky white to dark brown, and a mixed solution containing silk fibroin, nano-silver and antimicrobial peptides is obtained. Solution B; the particle diameter of nano-silver in the mixed solution B is 300nm, and the mass content of silk fibroin with a β-sheet structure in the mixed solution A is 20%; the amino acid sequence of the antimicrobial peptide in the mixed solution B is (NH ₂ )-NGIVKAGPAIAAVLGEAAL-CONH ₂ , the mass purity of the antimicrobial peptide is 99.99%;

Step 3: Use standard NaOH solution to calibrate the 10mM Tris solution to pH 8.5, then add dopamine hydrochloride to obtain the working solution, immerse the titanium alloy in acetone, ethanol and deionized water for 15 minutes, and then immerse in the working solution in the dark Soaked under the conditions for 18 hours to obtain a titanium alloy with a polydopamine auxiliary layer on the surface; the concentration of dopamine hydrochloride in the working solution is 2mg/mL; the thickness of the polydopamine auxiliary layer is 300nm;

Step 4. Immerse the titanium alloy with polydopamine auxiliary layer on the surface obtained in step 3 into the mixed solution B obtained in step 2, take it out and place it in a constant temperature drying oven, dry it at 60°C for 30 minutes, and place it on the surface of the titanium alloy A functional coating containing nano-silver antimicrobial peptide is formed; the thickness of the functional coating containing nano-silver antimicrobial peptide is 15 μm.

Example 3

This embodiment includes the following steps:

Step 1. Boil the silk in 0.02M Na ₂ CO ₃ solution for 40 minutes, rinse it with deionized water after taking it out, and then dissolve it in 9.3M LiBr solution at 60°C for 4 hours to obtain silk fibroin stock solution;

Dilute the silk fibroin stock solution with deionized water into a silk fibroin solution with a mass concentration of 5%, then add 200 mg of silver nitrate powder and 10 mg of antimicrobial peptide powder into 10 mL of a silk fibroin solution with a mass concentration of 5% and mix to obtain a silk fibroin solution containing 5% mass concentration. Protein, silver ion and antimicrobial peptide and the mixed solution A that pH is 10;

Step 2. Place the mixed solution A obtained in step 1 under a 40W ultraviolet lamp for irradiation and reduction for 2 hours, so that the color of the mixed solution A gradually changes from milky white to dark brown, and a mixed solution containing silk fibroin, nano-silver and antimicrobial peptides is obtained. Solution B; the particle diameter of nano-silver in the mixed solution B is 500nm, and the mass content of silk fibroin with a β-sheet structure in the mixed solution B is 30%; the amino acid sequence of the antimicrobial peptide in the mixed solution A is (NH ₂ )-NGIVKAGPAIAAVLGEAAL-CONH ₂ , the mass purity of the antimicrobial peptide is 99.99%;

Step 3: Use standard NaOH solution to calibrate the 10mM Tris solution to pH 8.5, then add dopamine hydrochloride to obtain the working solution, immerse the stainless steel in acetone, ethanol and deionized water for 15 minutes and ultrasonically clean it, then immerse it in the working solution under dark conditions Soak for 24 hours to obtain stainless steel with a polydopamine auxiliary layer on the surface; the concentration of dopamine hydrochloride in the working solution is 2mg/mL; the thickness of the polydopamine auxiliary layer is 500nm;

Step 4. Immerse the stainless steel with a polydopamine auxiliary layer on the surface obtained in step 3 into the mixed solution B obtained in step 2 for soaking, take it out and place it in a constant temperature drying oven, and dry it at 60° C. for 30 minutes to form a polydopamine-containing layer on the stainless steel surface. Nano silver antimicrobial peptide functional coating; the thickness of the nano silver antimicrobial peptide functional coating is 20 μm.

The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any way. All simple modifications, changes and equivalent changes made to the above embodiments according to the technical essence of the invention still belong to the protection scope of the technical solution of the invention.

Claims (6)

1. A method for preparing a nano-silver-containing antibacterial peptide functional coating on a metal surface is characterized by comprising the following steps:

adding silver nitrate powder and antibacterial peptide powder into a silk fibroin solution, and mixing to obtain a mixed solution A containing silk fibroin, silver ions and antibacterial peptide; the mass concentration of the silk fibroin in the mixed solution A is 2-5%, the concentration of silver ions is 1-20 mg/mL, and the concentration of the antibacterial peptide is 0.4-1 mg/mL;

step two, placing the mixed solution A obtained in the step one under an ultraviolet lamp for irradiation reduction, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and obtaining a mixed solution B containing silk fibroin, nano silver and antibacterial peptide; the time for irradiation reduction is 0.5h to 2h; the amino acid sequence of the antibacterial peptide in the mixed solution A is (NH) ₂ )-NGIVKAGPAIAVLGEAAL-CONH ₂ The mass purity of the antibacterial peptide is more than 99 percent;

step three, calibrating a 10mM Tris solution to the pH value of 8.5 by using a standard NaOH solution, then adding dopamine hydrochloride to obtain a working solution, and then soaking the metal in the working solution for 12h to 24h under the condition of keeping out of the sun to obtain the metal with a polydopamine auxiliary layer on the surface; the thickness of the polydopamine auxiliary layer is 200nm to 500nm;

step four, soaking the metal with the polydopamine auxiliary layer on the surface obtained in the step three into the mixed solution B obtained in the step two to form a nano-silver-containing antibacterial peptide functional coating on the surface of the metal; the thickness of the nano-silver-containing antibacterial peptide functional coating is 1-20 mu m.

2. The method for preparing the functional coating containing the nano-silver antibacterial peptide on the metal surface as claimed in claim 1, wherein the particle size of the nano-silver in the mixed solution B in the second step is 20nm to 500nm, and the mass content of the silk fibroin with a beta-pleated structure in the mixed solution B is 15% -30%.

3. The method for preparing the functional coating containing the nano-silver antibacterial peptide on the metal surface according to the claim 1, wherein the metal in the step three is titanium, titanium alloy or stainless steel.

4. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to claim 1, wherein the concentration of dopamine hydrochloride in the working solution in the third step is 1 mg/mL-4 mg/mL.

5. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to the claim 1, characterized in that the metal is subjected to cleaning pretreatment before being immersed in the working solution in the third step: and sequentially immersing the metal into acetone, ethanol and deionized water for ultrasonic cleaning for 15min.

6. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to claim 1, wherein the metal with the poly-dopamine auxiliary layer on the surface is soaked in the mixed solution B obtained in the step two in the step four, then is taken out and placed in a constant temperature drying oven, and is dried at 60 ℃ for 10min to 30min.

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