Antibacterial material as well as preparation method and application thereof

Abstract

The invention provides an antibacterial material as well as a preparation method and application thereof. The antibacterial material comprises a substrate, a polydopamine layer and n antibacterial layers, the surface of the substrate is coated with the polydopamine layer, the surface of the polydopamine layer is coated with the n antibacterial layers, a polyanion material layer is further arranged between the (n-1) th antibacterial layer and the nth antibacterial layer, and n is larger than or equal to 2; wherein the antibacterial layer comprises antibacterial peptide. The antibacterial material disclosed by the invention has a higher antibacterial effect, lower cytotoxicity and better hydrophilicity, and has potential application value in preparation of antibacterial medical instruments and biodegradable antibacterial implants.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial coating preparation, and in particular to an antibacterial material and a preparation method and application thereof. Background Art

[0002] Due to antibiotic resistance and the poor antibacterial properties of traditional implants, the incidence of infections caused by multidrug-resistant (MDR) pathogens in clinical settings continues to increase, exacerbating the difficulty of clinical treatments such as cancer treatment, invasive surgery and transplantation. Medical devices and implant materials with good biosafety, broad-spectrum and stable antibacterial properties have become one of the most urgently needed materials. Making the surface of medical device materials have stable, controlled and sustained-release antibacterial properties can increase treatment options and the probability of cure, reduce hospitalization time and medical expenses, and facilitate clinical applications.

[0003] Antibacterial peptide (AMP), also known as host defense peptide, is a small molecule polypeptide composed of 12-100 amino acid residues, with biological activity, induced in vivo, and a molecular weight of about 2000-7000. Most of these active cationic antimicrobial peptides have thermal stability and broad-spectrum antibacterial properties, and are generally strongly alkaline. AMP has gradually developed into a research hotspot in biomedicine, veterinary medicine, environmental science and other related fields due to its advantages such as broad antimicrobial spectrum, rapid action, good thermal stability, and low resistance to drug resistance, as well as its excellent therapeutic potential in antibacterial, antiviral, antifungal, anti-inflammatory and anti-tumor. Antimicrobial peptides are widely present in organisms and can help the body resist the invasion of exogenous microorganisms. They are an important component of the innate defense system of organisms. However, AMP is extremely sensitive to proteolytic enzymes, which limits the systemic application of AMP. In addition, due to the particularity of the source of antimicrobial peptides, the stability of antimicrobial peptides themselves is poor and they have certain cytotoxicity, which further limits their application. Summary of the invention

[0004] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention provides an antibacterial material and a preparation method and application thereof. The antibacterial material of the present invention is a controllable sustained-release antibacterial coating constructed based on dopamine and layer-by-layer self-assembly technology. The preparation method of the present invention forms a polydopamine film on the surface of a substrate by self-polymerization of dopamine, and the deposited polydopamine film reacts with an antibacterial component to form a uniform antibacterial coating, and then forms a uniform antibacterial coating with controllable coating thickness and stable and sustainable release through layer-by-layer self-assembly preparation technology.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides an antibacterial material, comprising a substrate, a polydopamine layer coated on the surface of the substrate, n antibacterial layers coated on the surface of the polydopamine layer, and a polyanion material layer is further arranged between the n-1th antibacterial layer and the nth antibacterial layer, n≥2; wherein the antibacterial layer comprises antibacterial peptides.

[0007] According to some embodiments of the present invention, the antimicrobial peptide comprises the antimicrobial peptide LL-37. According to some embodiments of the present invention, N is an integer between 2 and 30. According to some embodiments of the present invention, N is an integer between 2 and 10. According to some embodiments of the present invention, N is an integer between 3 and 6.

[0008] According to some embodiments of the present invention, the substrate includes, but is not limited to, metal-based materials, ceramic-based materials, and bio-based materials, etc. In some preferred embodiments, the substrate includes bio-based polymer materials.

[0009] The bio-based polymer materials of the present invention include, but are not limited to: biodegradable materials (e.g., polylactic acid, polycaprolactone, etc.), bio-based composite materials (e.g., cellulose nanocomposites), and biomimetic materials (e.g., artificial bones, artificial skin, etc.). In some embodiments, the substrate includes polylactic acid, polycaprolactone, and biomimetic materials. In some embodiments, the substrate includes polylactic acid.

[0010] According to some embodiments of the present invention, the polyanionic material includes one or more of hyaluronic acid, sodium alginate, polyacrylic acid, sulfonated dextran, sulfonated polyetheretherketone, and sodium polystyrene sulfonate. In some embodiments, the polyanionic material includes hyaluronic acid. Hyaluronic acid (HA), also known as uronic acid, hyaluronic acid, is a high molecular polymer with a molecular formula of (C 14 H 21 NO 11 )n, is a glycosaminoglycan composed of the basic structure of disaccharides (D-glucuronic acid and N-acetylglucosamine), a linear hydrophilic polysaccharide, and one of the main components of the extracellular matrix. HA, with its unique molecular structure and physicochemical properties, as well as its own advantages of viscoelasticity, biocompatibility and non-immunogenicity, shows a variety of important physiological functions in the body, such as being used as a supplement to arthritis joint fluid to lubricate joints, regulating the permeability of blood vessel walls, regulating protein, water and electrolyte diffusion and operation, promoting wound healing, dermal regeneration and enhancement, and surgical auxiliary equipment for ophthalmic surgery. In the present invention, hyaluronic acid can not only assemble the antibacterial components layer by layer on the substrate, but also improve the hydrophilicity of the antibacterial coating.

[0011] The second aspect of the present invention provides a method for preparing the above antibacterial material, comprising the following steps:

[0012] S1. soaking the substrate in a solution containing dopamine to obtain a substrate coated with a polydopamine layer;

[0013] S2. The substrate coated with the polydopamine layer is immersed in a first antibacterial solution to obtain a substrate coated with an antibacterial layer and a polydopamine layer;

[0014] S3. The substrate obtained in step S2 is sequentially immersed in a solution containing the polyanion material and a second antibacterial solution;

[0015] S4. Repeat step S3 multiple times to obtain an antibacterial material coated with multiple antibacterial layers on the substrate;

[0016] Wherein, the antibacterial components in the first antibacterial solution and the second antibacterial solution include antibacterial peptides.

[0017] According to some embodiments of the invention, the antimicrobial component comprises the antimicrobial peptide LL-37.

[0018] According to some embodiments of the present invention, step S4 includes: repeating step S3 for 1 to 30 times (or 2 to 10 times, or 3 to 6 times) to obtain an antibacterial material having a plurality of antibacterial layers coated on the substrate.

[0019] In the present invention, the immersion process of step S1 is mainly the process of dopamine self-polymerization on the substrate. The concentration of dopamine is crucial for its self-polymerization and has a crucial influence on the construction of the base antibacterial coating. Dopamine of different concentrations has different electrical properties and phenolic hydroxyl content, so it will affect the self-polymerization and the construction of the antibacterial coating. According to some embodiments of the present invention, in the dopamine-containing solution, the concentration of dopamine is 0.5 to 10 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL or any value therebetween, preferably 1 to 4 mg / mL.

[0020] According to some embodiments of the present invention, the pH of the dopamine-containing solution in S1 is 8.0-9.0 (ie, the dopamine self-polymerization process is carried out under the condition of pH 8.0-9.0).

[0021] According to some embodiments of the present invention, the concentration of the antibacterial component in the first antibacterial solution and the second antibacterial solution is the same. According to some other embodiments of the present invention, the concentration of the antibacterial component in the first antibacterial solution and the second antibacterial solution is different.

[0022] In the present invention, the concentration of the antibacterial component in the antibacterial solution is crucial to the antibacterial performance of the antibacterial coating. The antibacterial ability, positive charge amount and functional group amount of antibacterial components of different concentrations are all different, which will affect the construction of the layer-by-layer self-assembled coating and the antibacterial performance of the coating. According to some embodiments of the present invention, in the first antibacterial solution, the concentration of the antibacterial component is 0.25 to 50 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL or any value therebetween. According to some embodiments of the present invention, the concentration of the antibacterial component in the second antibacterial solution is 0.25-50 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL or any value therebetween.

[0023] According to some embodiments of the present invention, the antimicrobial component includes the antimicrobial peptide LL-37. According to some embodiments of the present invention, the concentration of the antimicrobial peptide LL-37 in the first antimicrobial solution and the second antimicrobial solution is independently 0.25 to 5 mg / mL, for example, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL or any value therebetween, preferably 1 to 5 mg / mL.

[0024] According to some embodiments of the present invention, the dopamine-containing solution further contains Tris-HCl buffer.

[0025] According to some embodiments of the present invention, the first antibacterial solution further contains Tris-HCl buffer.

[0026] According to some embodiments of the present invention, the pH of the Tris-HCl buffer is 8.0-9.0, such as 8.0, 8.2, 8.5, 8.8, 9.0 or any value therebetween.

[0027] In some embodiments, in step S1, the dopamine-containing solution is: a dopamine solution prepared with 5-15 mM Tris-HCl buffer (pH=8.0-9.0), and the concentration of dopamine is 1-5 mg / mL.

[0028] In the present invention, the reaction temperature and reaction time are crucial to the self-polymerization of dopamine. According to some embodiments of the present invention, in step S1, the immersion is carried out at 25 to 50°C, for example, at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any value therebetween. In some embodiments, in step S1, the immersion is carried out in a constant temperature oscillator. In some embodiments, the oscillation speed of the constant temperature oscillator is 80 to 150 r / min, and the oscillation time is 4 to 24 h.

[0029] According to some embodiments of the present invention, step S1 further comprises the steps of washing and drying the soaked substrate.

[0030] According to some embodiments of the present invention, the first antimicrobial solution in step S2 is an antimicrobial peptide solution prepared with 5-10 mM Tris-HCl buffer (pH=8.0-9.0), and the concentration of the antimicrobial peptide is 0.25-5 mg / mL.

[0031] According to some embodiments of the present invention, in step S2, the soaking conditions are: temperature 25-40° C., time 4-24 h.

[0032] According to some embodiments of the present invention, in step S2, the reaction of polydopamine and antimicrobial peptide is carried out at a pH of 8.0 to 9.0.

[0033] According to some embodiments of the present invention, step S2 further comprises the steps of washing and drying the soaked substrate.

[0034] According to some embodiments of the present invention, the polyanionic material comprises one or more of hyaluronic acid, sodium alginate, polyacrylic acid, sulfonated dextran, sulfonated polyetheretherketone, and sodium polystyrene sulfonate. In some embodiments, the polyanionic material comprises hyaluronic acid.

[0035] In the present invention, the concentration of the polyanionic material is crucial to the construction of the multilayer coating. The amount of negative charge and the amount of functional groups carried by polyanionic materials of different concentrations are different, so they will affect the construction of the antibacterial coating. According to some embodiments of the present invention, in the solution containing the polyanionic material, the concentration of the polyanionic material is 0.05 to 10 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL or any value therebetween, preferably 0.5 to 5 mg / mL, more preferably 0.5 to 1.5 mg / mL.

[0036] According to some embodiments of the present invention, the solvent of the solution containing the polyanion material is water.

[0037] According to some embodiments of the present invention, the solvent of the second antibacterial solution is water.

[0038] In some embodiments, the solution containing polyanion material in step S3 is: a hyaluronic acid solution prepared with water at a concentration of 0.5-10 mg / mL. In some embodiments, the second antibacterial solution in step S3 is: an antimicrobial peptide solution prepared with water at a concentration of 0.25-5 mg / mL.

[0039] According to some embodiments of the present invention, in step S3, the substrate is immersed in the solution containing the polyanion material for 1 to 60 minutes, preferably 1 to 30 minutes, and more preferably 5 to 10 minutes.

[0040] According to some embodiments of the present invention, in step S3, the substrate is immersed in the second antibacterial solution for 1 to 60 minutes, preferably 1 to 30 minutes, and more preferably 5 to 10 minutes.

[0041] According to some embodiments of the present invention, step S3 further comprises the steps of washing and drying the soaked substrate.

[0042] In a third aspect, the present invention provides an antibacterial medical product, which includes the above-mentioned antibacterial material or the antibacterial material obtained by the above-mentioned preparation method.

[0043] The medical supplies of the present invention include but are not limited to: medical devices, medical materials and implants.

[0044] In some embodiments, the medical supplies include one or more of ophthalmic implants, orthopedic implants, fracture fixation materials, dental implants, surgical fillers, dressings, tissue engineering scaffold materials, tongue depressors, medical thermometers, blood glucose meters, medical robots, surgical tools, and neural prostheses.

[0045] In a fourth aspect, the present invention provides the use of the above-mentioned antibacterial material or the antibacterial material obtained by the above-mentioned preparation method in the preparation of antibacterial medical products.

[0046] The medical supplies described in the present invention include but are not limited to: medical devices, medical materials and implants.

[0047] In some embodiments, the medical supplies include one or more of ophthalmic implants, orthopedic implants, fracture fixation materials, dental implants, surgical fillers, dressings, tissue engineering scaffold materials, tongue depressors, medical thermometers, blood glucose meters, medical robots, surgical tools, and neural prostheses.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The present invention constructs a layer of polydopamine film on the surface of a substrate through the self-polymerization of dopamine. The deposited polydopamine film reacts with antibacterial components such as antimicrobial peptides to form a uniform antibacterial coating. The substrate coated with the antibacterial coating can be used directly, or a layer-by-layer self-assembly preparation technology can be used to construct a basic coating liquid based on polyanions. Antibacterial components such as antimicrobial peptides with a net positive charge are coated on the surface of the substrate through electrostatic forces and hydrogen bonds, thereby obtaining a stable, sustainable and uniform antibacterial material with controllable number of antibacterial layers and thickness.

[0050] 2. The antibacterial material prepared by the present invention can achieve an antibacterial effect close to or even better than that of a pure antibacterial peptide solution when the antibacterial peptide concentration is lower than that of a pure antibacterial peptide solution, and the cytotoxicity of the antibacterial peptide is significantly reduced due to the combination with the substrate. The characterization results show that the controlled sustained-release antibacterial coating prepared by the present application has good biocompatibility.

[0051] 3. The antibacterial material of the present invention can be used to prepare medical devices and implants such as tissue engineering scaffold materials, fracture internal fixation materials, ophthalmic implant materials, etc. with stable and sustainable antibacterial properties, which to a certain extent improves the problems of existing antibacterial materials that cannot continuously and stably release antibacterial properties, have poor biocompatibility, and have resistance to antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The water contact angle test results of the antibacterial coating materials prepared in Examples 4 and 5 of the present application are shown.

[0053] Figure 2 The scanning electron microscopy results of the surface morphology of the antibacterial coating materials prepared in Examples 4 and 5 of the present application are shown.

[0054] Figure 3 The antibacterial effect of the antibacterial coating materials prepared in Examples 4 and 5 of the present application after co-culture with Staphylococcus aureus is shown.

[0055] Figure 4 The scanning electron microscopy results of the antibacterial coating materials prepared in Examples 4 and 5 of the present application after co-culture with Staphylococcus aureus are shown.

[0056] Figure 5 The in vitro cytotoxicity test results of the antibacterial coating materials prepared in Examples 4 and 5 of the present application are shown. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0058] The reagents used in the following experiments of the present invention, unless otherwise specified, are all conventional commercial products or reagents prepared by conventional methods, and the methods used in the experiments, unless otherwise specified, are all conventional experimental methods. The instruments used in the experiments, unless otherwise specified, can all be obtained through commercial channels.

[0059] The substrates used in the following examples are all polylactic acid (PLA) sheets, which are cut to size according to experimental needs; the average molecular weight is 100,000; and they are purchased from Yunnan Juhe Biotechnology Co., Ltd.

[0060] The antimicrobial peptide LL-37 used in the following examples was purchased from Hangzhou Sinoda Pharmaceutical Technology Co., Ltd., CAS: 154947-66-7; dopamine CAS: 51-61-6; ceftriaxone sodium CAS: 64485-93-4.

[0061] Example 1 Preparation of dopamine and antimicrobial peptide (LL-37) composite antibacterial coating

[0062] 1. Polylactic acid substrate: Take the polylactic acid material and trim it into the required shape according to the corresponding mold, then polish it with SiC sandpaper of different particle sizes, ultrasonically clean it in deionized water for 15 to 30 minutes, then repeatedly clean it with deionized water, and set it aside after drying.

[0063] 2. Dissolve dopamine and antimicrobial peptide (LL-37) in 10 mM Tris-HCl buffer (pH = 8.5) respectively, so that the final concentration of dopamine solution is 4 mg / mL, and the final concentration of antimicrobial peptide (LL-37) solution is 1 mg / mL. Then add the above-mentioned polylactic acid substrate to the dopamine solution, place it in a dual-function air bath constant temperature oscillator, and oscillate overnight at 30°C and 115 r / min.

[0064] 3. Take out the substrate coated with the polydopamine film in step 2, wash it, dry it, immerse it in the antimicrobial peptide (LL-37) solution, gently stir it overnight, then take out the substrate, wash off the antimicrobial peptide (LL-37) molecules not connected to the surface of the substrate with deionized water, and place the substrate on a sterile table to dry, thereby obtaining a polylactic acid composite material coated with a layer of polydopamine and a layer of antimicrobial peptide LL-37, recorded as PLA-pDA-AMP-1.

[0065] Example 2 Preparation of dopamine and antibiotic (cefotaxime sodium) composite antibacterial coating

[0066] 1. Polylactic acid substrate: Take the polylactic acid material and trim it into the required shape according to the corresponding mold, then polish it with SiC sandpaper of different particle sizes, ultrasonically clean it in deionized water for 15 to 30 minutes, then repeatedly clean it with deionized water, and set it aside after drying.

[0067] 2. Dopamine and cefotaxime sodium (CS) were dissolved in 10 mM Tris-HCl buffer (pH = 8.5) respectively, so that the final concentration of the dopamine solution was 4 mg / mL, and the final concentration of the cefotaxime sodium solution was 50 mg / mL. Then the above-mentioned polylactic acid substrate was added to the dopamine solution, placed in a dual-function air bath constant temperature oscillator, and oscillated overnight at 30°C and 115 r / min.

[0068] 3. Take out the substrate coated with the polydopamine film in step 2, wash it, dry it, immerse it in a sodium ceftriaxone solution, gently stir it overnight, then take out the substrate, wash off the sodium ceftriaxone not connected to the surface of the substrate with deionized water, place the substrate on a sterile table and dry it for use, and obtain a polylactic acid composite material coated with a layer of polydopamine and a layer of sodium ceftriaxone, PLA-pDA-CS-1.

[0069] Example 3 Preparation of multilayer controlled sustained-release antibacterial coating of dopamine, antimicrobial peptide (LL-37) and hyaluronic acid

[0070] 1. Polylactic acid substrate: Take the polylactic acid material and trim it into the required shape according to the corresponding mold, then polish it with SiC sandpaper of different particle sizes, ultrasonically clean it in deionized water for 15 to 30 minutes, then repeatedly clean it with deionized water, and set it aside after drying.

[0071] 2. Dopamine and antimicrobial peptide (LL-37) were dissolved in 10 mM Tris-HCl buffer (pH = 8.5) respectively, so that the final concentration of the dopamine solution was 2 mg / mL, and the final concentration of the antimicrobial peptide (LL-37) solution was 1.25 mg / mL. Then, the above-mentioned polylactic acid substrate was added to the dopamine solution, placed in a dual-function air bath constant temperature oscillator, and oscillated overnight at 30°C and 100 r / min.

[0072] 3. Take out the substrate coated with the polydopamine film in step 2, wash it, dry it, immerse it in the antimicrobial peptide (LL-37) solution, gently stir it overnight, then take out the substrate, wash off the antimicrobial peptide (LL-37) molecules not connected to the surface of the substrate with deionized water, and place the substrate on a sterile table to dry it for later use.

[0073] 4. Dissolve the antimicrobial peptide (LL-37) and hyaluronic acid in deionized water, respectively, so that the final concentration of the antimicrobial peptide (LL-37) solution is 1 mg / mL, and the final concentration of the hyaluronic acid solution is 0.8 mg / mL. Then immerse the substrate in step 3 into the hyaluronic acid solution, let it stand for 8 minutes, and then take out the substrate.

[0074] 5. Immerse the substrate taken out in step 4 into the LL-37 solution, let it stand for 10 minutes, and then take out the substrate.

[0075] 6. After repeating steps 4 and 5 for a total of 8 times, the substrate is taken out, and the unattached LL-37 molecules on the surface of the substrate are washed off with deionized water. The substrate is placed on a sterile table to dry, and a multi-layer controlled sustained-release antibacterial coating can be coated on the polylactic acid substrate.

[0076] Example 4 Preparation of multilayer controlled sustained-release antibacterial coating of dopamine, antimicrobial peptide (LL-37) and hyaluronic acid

[0077] 1. Polylactic acid substrate: Take the polylactic acid material and trim it into the required shape according to the corresponding mold, then polish it with SiC sandpaper of different particle sizes, ultrasonically clean it in deionized water for 15 to 30 minutes, then repeatedly clean it with deionized water, and set it aside after drying.

[0078] 2. Dopamine and antimicrobial peptide (LL-37) were dissolved in 10 mM Tris-HCl buffer (pH = 8.5) respectively, so that the final concentration of the dopamine solution was 2 mg / mL, and the final concentration of the antimicrobial peptide (LL-37) solution was 1 mg / mL. Then the above-mentioned polylactic acid substrate was added to the dopamine solution, placed in a dual-function air bath constant temperature oscillator, and oscillated overnight at 30°C and 110 r / min to obtain a substrate coated with a polydopamine film, which was recorded as PLA-pDA.

[0079] 3. Take out the substrate coated with the polydopamine film in step 2, wash it, dry it, immerse it in the antimicrobial peptide (LL-37) solution, gently stir it overnight, then take out the substrate, wash off the unattached antimicrobial peptide (LL-37) molecules on the surface of the substrate with deionized water, place the substrate on a sterile table to dry it for use, and then obtain the PLA-pDA-AMP material.

[0080] 4. Dissolve the antimicrobial peptide (LL-37) and hyaluronic acid in deionized water, respectively, so that the final concentration of the antimicrobial peptide (LL-37) solution is 1 mg / mL, and the final concentration of the hyaluronic acid solution is 0.8 mg / mL. Then immerse the substrate in step 3 into the hyaluronic acid solution, let it stand for 8 minutes, and then take out the substrate.

[0081] 5. Immerse the substrate taken out in step 4 into the LL-37 solution, let it stand for 10 minutes, and then take out the substrate.

[0082] 6. Repeat steps 4 and 5 for 3 times, then take out the substrate, wash off the unattached LL-37 molecules on the substrate surface with deionized water, and place the substrate on a sterile table to dry, thus preparing a multi-layer controlled sustained-release antibacterial coating on the polylactic acid substrate, which is recorded as LBL-3 layer;

[0083] After repeating steps 4 and 5 for a total of 5 times, the substrate was taken out, the unattached LL-37 molecules on the surface of the substrate were washed off with deionized water, and the substrate was placed on a sterile bench to dry, thus preparing a multi-layer controlled sustained-release antibacterial coating on the polylactic acid substrate, which was recorded as LBL-5 layer.

[0084] Example 5 Preparation of Dopamine and Ceftriaxone Sodium Controlled-Release Antibacterial Coating

[0085] 1. Polylactic acid substrate: Take the polylactic acid material and trim it into the required shape according to the corresponding mold, then polish it with SiC sandpaper of different particle sizes, ultrasonically clean it in deionized water for 15 to 30 minutes, then repeatedly clean it with deionized water, and set it aside after drying.

[0086] 2. Dopamine and cefotaxime sodium (CS) were dissolved in 10 mM Tris-HCl buffer (pH = 8.5) respectively, so that the final concentration of the dopamine solution was 2 mg / mL, and the final concentration of the cefotaxime sodium solution was 50 mg / mL. Then the above-mentioned polylactic acid substrate was added to the dopamine solution, placed in a dual-function air bath constant temperature oscillator, and oscillated overnight at 30°C and 110 r / min.

[0087] 3. Take out the substrate coated with the polydopamine film in step 2, wash it, dry it, immerse it in cefotaxime sodium, gently stir it overnight, then take out the substrate, wash off the cefotaxime sodium not connected to the surface of the substrate with deionized water, place the substrate on a sterile table to dry it for use, and obtain the PLA-pDA-CS material.

[0088] Experiment 1: Water contact angle test

[0089] The polylactic acid substrate used in Example 4, the PLA-pDA obtained in step 2 of Example 4, the PLA-pDA-AMP obtained in step 3 of Example 4, the LBL-3 layer and the LBL-5 layer obtained in step 6 of Example 4, and the PLA-pDA-CS obtained in Example 5 were tested for water contact angle using a contact angle meter (JY-PHa, Chengde Jinhe Instrument Manufacturing Co., Ltd.) as follows:

[0090] At room temperature, 5 μL of ultrapure water was used as a wetting agent, and three points were selected on both sides of the sample material to be tested to measure its water contact angle. Each material was measured in parallel 3 times, and then the average value was taken.

[0091] The results are as follows Figure 1 As shown. The results show that after the antimicrobial peptide LL-37 and the antibiotic CS were coated on the surface of polylactic acid, the hydrophilicity of polylactic acid was significantly improved, and the water contact angle of the antimicrobial material coated with the antimicrobial peptide LL-37 was smaller than that of the antimicrobial material coated with the antibiotic CS. By comparing the contact angles of the PLA-pDA-AMP obtained in step 3 of Example 4, the LBL-3 layer and the LBL-5 layer obtained in step 6 of Example 4, it is shown that the use of hyaluronic acid as the intermediate of self-assembly has a smaller water contact angle and better hydrophilic performance as the number of assembled layers increases.

[0092] Experiment 2: Material morphology characterization

[0093] Field emission scanning electron microscopy (FE-SEM) was used to characterize the morphology of the polylactic acid substrate used in Example 4, the PLA-pDA obtained in step 2 of Example 4, the PLA-pDA-AMP obtained in step 3 of Example 4, the LBL-3 layer and LBL-5 layer obtained in step 6 of Example 4, and the PLA-pDA-CS material obtained in Example 5. Before observation under the scanning electron microscope, all samples were coated with gold for 1 minute.

[0094] The results are as follows Figure 2 The results show that after the antimicrobial peptide LL-37 and antibiotic CS were coated on the surface of polylactic acid, the surface morphology of polylactic acid changed significantly.

[0095] In addition, from Figure 2 It can be seen that dopamine and antimicrobial peptides can be polymerized in the form of nanoparticles. A large number of nanoparticles aggregate on the surface of polylactic acid, first constructing a polydopamine film and then forming an antibacterial coating with antimicrobial peptides. With the coating of dopamine, antimicrobial peptides and hyaluronic acid on the surface of polylactic acid, the surface morphology and roughness of polylactic acid also changed significantly.

[0096] Experiment 3: Antibacterial experiment

[0097] 1. The antibacterial properties of the polylactic acid substrate used in Example 4, the PLA-pDA obtained in step 2 of Example 4, the PLA-pDA-AMP obtained in step 3 of Example 4, the LBL-3 layer and LBL-5 layer materials obtained in step 6 of Example 4, and the pure AMP solution (LL-37 solution with a concentration of 1 mg / mL) were tested as follows:

[0098] The sample material to be tested was placed in a 96-well plate and 200 μL of 2×10 5CFU / mL of Staphylococcus aureus liquid was then placed in a 37°C constant temperature incubator for co-culture for 24h, 48h, and 72h, and finally the light absorption value of the bacterial liquid at 600nm was detected by an enzyme marker.

[0099] The experimental results are as follows Figure 3 And as shown in Table 1.

[0100] Table 1

[0101]

[0102] It can be seen that after 72 h of co-culture, the self-assembled PLA-pDA-AMP, LBL-3 layer and LBL-5 layer materials still have good antibacterial effects, and the antibacterial effect becomes better with the increase of the number of self-assembled layers.

[0103] In particular, compared with pure AMP solution, the PLA-pDA-AMP, LBL-3 layer and LBL-5 layer materials obtained by coating or self-assembly of the present invention, although having a lower concentration of antimicrobial peptides, can achieve an antibacterial effect comparable to or even higher than that of pure AMP solution.

[0104] 2. The antibacterial properties of the polylactic acid substrate used in Example 4, the PLA-pDA obtained in step 2 of Example 4, the PLA-pDA-AMP obtained in step 3 of Example 4, the LBL-3 layer and the LBL-5 layer obtained in step 6 of Example 4, and the PLA-pDA-CS material prepared in Example 5 were tested as follows:

[0105] Take the sample material to be tested and mix it with 200 μL of 2×10 5 After the Staphylococcus aureus liquid with CFU / mL was co-cultured in a 37°C constant temperature incubator for 24 hours, the solid material was taken into a centrifuge tube, a certain amount of 2.5% glutaraldehyde solution was added for fixation, and after being placed in a refrigerator overnight, it was dehydrated and dried with graded ethanol solutions. Then, FE-SEM was used to analyze the bacterial attachment and diffusion morphology on the surface of the above solid material after Au sputtering.

[0106] The results are as follows Figure 4 The results show that bacteria grow on the surface of the sample without antimicrobial peptide modification, but no bacteria grow on the surface of polylactic acid coated with antimicrobial peptide and antibiotic. Figure 2 The results show that the antibacterial coating has a stable antibacterial effect.

[0107] Experiment 4: In vitro cytotoxicity assay

[0108] The polylactic acid substrate used in Example 4, the PLA-pDA obtained in step 2 of Example 4, the PLA-pDA-AMP obtained in step 3 of Example 4, the LBL-3 layer and the LBL-5 layer obtained in step 6 of Example 4, and the pure AMP solution (LL-37 solution with a concentration of 1 mg / mL) were tested for in vitro cytotoxicity. The specific method is as follows:

[0109] MG-63 cells (Kunming Cell Bank, Chinese Academy of Sciences) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C, 5% CO2 constant temperature incubator, and the medium was changed every 3 days. Cell viability was determined using AlamarBlue cell viability reagent. Each sample material to be tested was placed in a 96-well plate, and the cell suspension was added. The cells were cultured on the surface or the DMSO control group (positive control) surface at a density of 1×10 5 / well. For each group of 6 samples, 20uL CCK-8 reagent was added to each well after 24h, and the well was incubated in a 37℃, 5% CO2 incubator for 2h. The supernatant was aspirated and transferred to a new 96-well plate. The OD value was detected by a microplate reader at 450nm spectrophotometry. The test was performed on 1 day, 3 days, 6 days and 7 days respectively, and the results were expressed as the average of two independent experiments.

[0110] The experimental results are as follows Figure 5 As shown in Table 2, coating or self-assembling the antimicrobial peptide on the surface of polylactic acid is more conducive to cell growth. Compared with the pure AMP solution group, the PLA-pDA-AMP, LBL-3 layer and LBL-5 layer materials coated or self-assembled by the present invention have higher cell survival rates, indicating lower cytotoxicity.

[0111] Table 2

[0112]

[0113] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An antibacterial material comprising a substrate, a polydopamine layer coated on the surface of the substrate, n antibacterial layers coated on the surface of the polydopamine layer, and a polyanion material layer is further arranged between the n-1th antibacterial layer and the nth antibacterial layer, n≥2; wherein, The antimicrobial layer includes antimicrobial peptides.

2. The antibacterial material according to claim 1, characterized in that: 2≤N≤30; preferably, 2≤N≤10, more preferably, 3≤N≤6.

3. The antibacterial material according to claim 1 or 2, characterized in that: The antimicrobial peptide includes the antimicrobial peptide LL-37; and / or, the substrate includes one or more of a metal-based material, a ceramic-based material and a bio-based material, preferably a bio-based polymer material, more preferably polylactic acid, polycaprolactone and a biomimetic material; and / or, The polyanion material includes one or more of hyaluronic acid, sodium alginate, polyacrylic acid, sulfonated dextran, sulfonated polyetheretherketone, and sodium polystyrene sulfonate, preferably hyaluronic acid.

4. A method for preparing the antibacterial material according to any one of claims 1 to 3, comprising the following steps: S1. soaking the substrate in a solution containing dopamine to obtain a substrate coated with a polydopamine layer; S2. The substrate coated with the polydopamine layer is immersed in a first antibacterial solution to obtain a substrate coated with an antibacterial layer and a polydopamine layer; S3. The substrate obtained in step S2 is sequentially immersed in a solution containing the polyanion material and a second antibacterial solution; S4. Repeat step S3 multiple times to obtain an antibacterial material coated with multiple antibacterial layers on the substrate; in, The antimicrobial components in the first antimicrobial solution and the second antimicrobial solution include antimicrobial peptides.

5. The preparation method according to claim 4, characterized in that: In the dopamine-containing solution, the concentration of dopamine is 0.5 to 10 mg / mL, preferably 1 to 4 mg / mL; and / or, The concentration of the antibacterial component in the first antibacterial solution and the second antibacterial solution is the same or different and is independently 0.25 to 50 mg / mL, preferably 0.25 to 5 mg / mL; and / or, The dopamine-containing solution and the first antibacterial solution further contain Tris-HCl buffer, and the pH of the Tris-HCl buffer is preferably 8.0-9.

0.

6. The preparation method according to claim 4 or 5, characterized in that: In the solution containing the polyanion material, the concentration of the polyanion material is 0.05 to 10 mg / mL, preferably 0.5 to 5 mg / mL, more preferably 0.5 to 1.5 mg / mL; and / or, The solvent of the solution containing the polyanion material and the second antibacterial solution is water.

7. The preparation method according to any one of claims 4 to 6, characterized in that: In step S1, the immersion is carried out at 25 to 50° C.; preferably in a constant temperature oscillator, the oscillation speed is preferably 80 to 150 r / min, and the oscillation time is preferably 4 to 24 h; and / or, In step S2, the soaking conditions are: temperature 25-40° C., time 4-24 h; and / or, In step S3, the substrate is immersed in the solution containing the polyanion material for 1 to 60 minutes, preferably 5 to 10 minutes, and immersed in the second antibacterial solution for 1 to 60 minutes, preferably 5 to 10 minutes.

8. An antibacterial medical product, comprising the antibacterial material according to any one of claims 1 to 3 or the antibacterial material obtained by the preparation method according to any one of claims 4 to 7.

9. The antibacterial medical product according to claim 8, characterized in that: The medical supplies include one or more of medical devices, medical materials and implants; preferably include one or more of ophthalmic implants, orthopedic implants, fracture fixation materials, dental implants, surgical fillers, dressings, tissue engineering scaffold materials, tongue depressors, medical thermometers, blood glucose meters, medical robots, surgical tools and neural prostheses.

10. Use of the antibacterial material according to any one of claims 1 to 3 or the antibacterial material obtained by the preparation method according to any one of claims 4 to 7 in the preparation of antibacterial medical products; Preferably, the medical supplies include one or more of medical devices, medical materials and implants; preferably include one or more of ophthalmic implants, orthopedic implants, fracture fixation materials, dental implants, surgical fillers, dressings, tissue engineering scaffold materials, tongue depressors, medical thermometers, blood glucose meters, medical robots, surgical tools and neural prostheses.

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