Antibacterial coating of stable antibacterial peptide as well as preparation method and application of antibacterial coating

Abstract

The invention provides a stable antibacterial peptide antibacterial coating and a preparation method and application thereof, and the method comprises the following steps: pretreating the surface of an orthopedic external fixation frame matrix, suspending in a simulated physiological solution containing polycarboxylate, treating at 60-100 DEG C for 1-24 hours, naturally cooling to room temperature, taking out a substrate material, cleaning, and drying to obtain the stable antibacterial peptide antibacterial coating. And suspending in a solution containing the antibacterial peptide for adsorption treatment, and then rinsing and airing to obtain the antibacterial coating of the stable antibacterial peptide. The antibacterial peptide can be effectively stabilized and prevented from being degraded too early, so that the antibacterial peptide is slowly released on the orthopedic external fixing frame to play a long-acting antibacterial effect. The method is easy to implement, the coating can provide long-acting antibacterial protection, the anti-infection capacity of the orthopedic external fixing frame is improved, the antibacterial duration of the orthopedic external fixing frame is prolonged, the postoperative infection risk is reduced, postoperative recovery of a patient is promoted, the biocompatibility and the antibacterial capacity of the orthopedic external fixing frame are remarkably improved, and a new direction is provided for improvement of orthopedic fixing instruments.

Description

Technical Field

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

[0002] Orthopedic postoperative infection is one of the main reasons for poor postoperative recovery and even surgical failure. Although traditional orthopedic external fixators can provide stable fixation and have certain biocompatibility, they play a key role in treating fractures and promoting bone healing, but the material itself lacks an effective anti-infection mechanism. Since the external fixator is in contact with the skin and the outside air for a long time, it is easy to cause infection, which affects the patient's prognosis and becomes a major challenge in clinical treatment. Under the existing technical background, implant infection is a serious clinical complication, mainly due to the formation of biofilms by bacteria on the surface of the implant, making the infection difficult to treat. At present, although there are release-type and contact-type antibacterial coatings, single-function antibacterial coatings can no longer meet clinical needs. Therefore, it is necessary to develop coatings that are both antibacterial and biocompatible and can reduce the risk of bacterial resistance. The development of orthopedic external fixators with anti-infection function is of great significance to improving the success rate of surgery and the quality of life of patients.

[0003] Antimicrobial peptides are a class of antimicrobial agents with broad-spectrum antimicrobial activity, pathogen specificity and low drug resistance potential. They have broad application prospects in anti-infection treatment, especially in combating drug-resistant bacteria. However, direct application of antimicrobial peptides has problems such as poor stability, easy inactivation, and insufficient antimicrobial effective amount. Therefore, increasing the effective loading amount of antimicrobial peptides on the surface of medical devices and increasing the stability of antimicrobial peptides are feasible methods to achieve long-term antimicrobial effects on medical devices.

[0004] Looking at the clinical application of medical devices in recent years, although a large number of medical implants have produced positive effects, they cannot solve the problems of implant infection and biocompatibility in clinical practice. Therefore, the use of external fixators based on conventional orthopedic implants and the use of antibiotics to alleviate bone implant infection problems cannot fundamentally solve the problem of orthopedic external fixator infection, and the loaded antimicrobial peptides have the challenge of easy premature degradation.

[0005] Therefore, it is necessary to develop a new type of external fixator material that is loaded with antimicrobial peptides and has long-lasting antibacterial properties. Summary of the invention

[0006] The purpose of the present invention is to provide an antibacterial coating of a stable antimicrobial peptide and a preparation method and application thereof, so as to realize a high specific surface area coating of a trauma external fixator that can stabilize the antimicrobial peptide and exert a lasting antibacterial effect.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a method for preparing an antimicrobial coating of a stable antimicrobial peptide is provided, the method comprising:

[0009] Pre-treating the surface of an orthopedic external fixator matrix to obtain a matrix material having a nano-distributed structure on the surface;

[0010] The matrix material with the nano-distributed structure on the surface is suspended in a simulated physiological fluid containing polycarboxylate, treated at 60°C-100°C for 1h-24h, then naturally cooled to room temperature, and the base material is taken out for cleaning and drying to obtain a bottom-top double-layer nested hybrid coating structure material;

[0011] The bottom-top double-layer nested mixed coating structure material is suspended in a solution containing antimicrobial peptides for adsorption treatment, and then rinsed and dried to obtain an antimicrobial coating with stable antimicrobial peptides.

[0012] Furthermore, the orthopedic external fixator matrix is ​​selected from one of aluminum alloy, titanium alloy or stainless steel.

[0013] Furthermore, the preprocessing includes:

[0014] The matrix of the orthopedic external fixator is polished and ground, and after being rinsed, a nano-distributed structure layer is formed on the surface by using one of an electrochemical process, an acid etching process, an alkali thermal chemical process, a micro-arc oxidation process and an anodic oxidation process to obtain a base material with a nano-distributed structure on the surface.

[0015] Furthermore, the formula of the simulated body fluid is:

[0016] Ultrapure water as solvent, containing the following mixed solution of inorganic metal ions: (HPO4) 2- 1.0-3.0mmol / L, (SO4) 2- 0.1-1mmol / L, Cl - 140.5-150.5mmol / L, (HCO3) - 4.1-5.1mmol / L, Ca 2+ 2.1-5.5mmol / L, Na + 140-150mmol / L, Mg 2+ 1.1-2.5mmol / L, K + 4.0-6.0mmol / L;

[0017] And polycarboxylates 10μmol / L-10mmol / L.

[0018] Furthermore, the polycarboxylate includes at least one of polyacrylic acid and polyaspartic acid.

[0019] Furthermore, the conditions of the adsorption treatment include: 2°C-6°C for 1h-24h.

[0020] In the second aspect of the present invention, an antimicrobial coating of a stable antimicrobial peptide prepared by the method is provided.

[0021] In the third aspect of the present invention, there is provided the use of the antibacterial coating of the stable antibacterial peptide in the preparation of medical antibacterial metal equipment.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] 1. The antibacterial coating of the stabilized antimicrobial peptide prepared by the present invention has a surface of a mineralized calcium phosphate structure with a high specific surface area. The high specific surface area structure stabilizes the antimicrobial peptide, prevents the antimicrobial peptide from premature degradation, ensures its antimicrobial activity, and achieves a long-lasting antibacterial effect.

[0024] 2. The high specific surface area surface constructed by the biomimetic mineralization technology of the present invention effectively improves the stability and sustained release of antimicrobial peptides. The preparation method is simple and low-cost. The prepared anti-infection coated orthopedic external fixator has good biocompatibility and antibacterial effect, significantly improves the anti-infection ability of the orthopedic external fixator, reduces the risk of postoperative infection, significantly improves the safety and success rate of orthopedic surgery, and promotes the postoperative recovery of patients.

[0025] 3. The anti-infection coated orthopedic external fixator is clinically used in orthopedic surgery, which can effectively reduce the risk of postoperative infection, promote wound healing, and improve the success rate of surgery. Through comparative experiments, the postoperative infection rate of patients using the anti-infection coated orthopedic external fixator was significantly reduced, and the recovery speed was accelerated. It shows that the high specific surface area coating containing the stable antimicrobial peptides described in the present invention that exerts a lasting antibacterial effect can be used for orthopedic external fixators, orthopedic connection plates, medical wheelchairs, etc. It provides a new anti-infection treatment method for the anti-infection treatment of orthopedic external fixation devices, which has important clinical application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0027] Figure 1 This is the scanning electron microscope (SEM) morphology of the aluminum alloy substrate surface after sandblasting and polishing.

[0028] Figure 2This is the SEM morphology of the aluminum alloy substrate pre-treated surface after alkali heat treatment.

[0029] Figure 3 This is the SEM morphology of the high specific surface area nanofibrous structure coating generated after the pre-treated surface of the aluminum alloy substrate was treated with bionic mineralization.

[0030] Figure 4 This is the release curve of the antimicrobial peptide HHC36 from the high specific surface area coating substrate loaded with antimicrobial peptide in Tris buffer solution in Example 1.

[0031] Figure 5 The following are the macroscopic images of the femur of the rats with aluminum alloy high specific surface area coating substrates implanted two weeks after surgery. a. Control group: aluminum alloy high specific surface area coating substrates without antimicrobial peptides, severe infection, necrosis and abscesses; b. aluminum alloy high specific surface area coating substrates with antimicrobial peptide HHC36, no signs of infection.

[0032] Figure 6 This is the release curve of the antimicrobial peptide HHC36 loaded on the substrate in Tris buffer after alkaline heat treatment in Comparative Example 1.

[0033] Figure 7 This is the release curve of the antimicrobial peptide HHC36 from the high specific surface area coating substrate loaded with antimicrobial peptide in Tris buffer solution in Example 2.

[0034] Figure 8 This is the release curve of the antimicrobial peptide HHC36 loaded on the substrate in Tris buffer after anodization treatment in Comparative Example 2.

[0035] Fig. 9 The antibacterial rates of S. aureus cultured on the surface of the samples obtained in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 at 1h, 6h, 12h and 24h.

[0036] Fig.10 The antibacterial rates of E. coli cultured on the surface of the samples obtained in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 at 1h, 6h, 12h and 24h.

[0037] Fig.11 The present invention provides a flowchart of a method for preparing an antibacterial coating of a stable antibacterial peptide according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0039] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0040] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0041] The general idea of ​​the present invention is as follows:

[0042] According to a typical embodiment of the present invention, a method for preparing an antibacterial coating of a stable antimicrobial peptide is provided, the method comprising:

[0043] Step S1, pre-treating the surface of the orthopedic external fixator matrix to obtain a matrix material having a nano-distributed structure on the surface;

[0044] In the step S1,

[0045] The orthopedic external fixator matrix is ​​selected from one of aluminum alloy, titanium alloy or stainless steel.

[0046] The preprocessing comprises:

[0047] The matrix of the orthopedic external fixator is polished and ground, and after being rinsed, a nano-distributed structure layer is formed on the surface by using one of an electrochemical process, an acid etching process, an alkali thermal chemical process, a micro-arc oxidation process and an anodic oxidation process to obtain a base material with a nano-distributed structure on the surface.

[0048] The specific operations of the electrochemical process, acid etching process and alkaline thermal chemical process are different from those of the micro-arc oxidation and anodizing methods.

[0049] As a specific implementation method, the specific method of constructing the nano-distributed structure layer by electrochemical process, acid etching process, and alkaline thermal chemical process is as follows:

[0050] The surface of the aluminum alloy orthopedic external fixator substrate is subjected to gradient sandblasting, polishing, grinding, and washing, and then the substrate is suspended in an acid etching solution or an alkaline etching solution at 50° C. to 80° C. for 1 to 40 hours, and then taken out for cleaning and drying to obtain a substrate material with a nano-distributed structure on the surface;

[0051] The alkaline etching solution is one of sodium hydroxide, potassium hydroxide or sodium silicate solution, or a mixed solution of two or more alkali metal hydroxide solutions or alkali metal silicate solutions in any proportion, with a concentration of 0.5-50 mol / L;

[0052] As a specific implementation, the specific method of constructing the nano-distributed structure layer on the surface of the trauma external fixator matrix by micro-arc oxidation and anodic oxidation is:

[0053] The surface of the aluminum alloy orthopedic external fixator substrate is subjected to gradient sandblasting, polishing, grinding, and rinsing, and then the substrate is suspended in an electrolyte, the aluminum alloy substrate is used as an anode, and a pure titanium plate is used as a cathode, the distance between the two electrodes is 10-50cm, the reaction voltage is 10-200V, the frequency is 150-500Hz, the duty cycle is 5%-40%, the temperature is 10-40℃, and the reaction time is 1-60min.

[0054] The electrolytic cell solution is composed of: 5-30ml / L sulfuric acid, 20-50ml / L phosphoric acid; 2-30g / L monocalcium phosphate, 1-20g / L calcium glycerophosphate, 2-30g / L calcium acetate, 2-30g / L disodium ethylenediaminetetraacetic acid, and the solvent is deionized water. The electrolyte is a mixed solution of sulfuric acid and phosphoric acid solution in a certain proportion or a mixed solution of monocalcium phosphate, calcium glycerophosphate, calcium acetate, and disodium ethylenediaminetetraacetic acid in a certain proportion. After the reaction is completed, the substrate is taken out, rinsed, and dried with nitrogen to obtain a substrate material with a nano-distributed structure on the surface.

[0055] Step S2, suspending the matrix material with the nano-distributed structure on the surface in a simulated physiological fluid containing polycarboxylate, treating at 60°C-100°C for 1h-24h, then naturally cooling to room temperature, taking out the base material, washing and drying, so as to obtain a bottom-top double-layer nested hybrid coating structure material;

[0056] In step S2, the treatment is carried out at 60°C-100°C for 1h-24h because a coating with a high specific surface area can be formed under this condition range, and the bottom and top layers of the coating are nested with each other, having a high bonding strength. If the temperature is less than 60°C, the coating formation time is too long, the coating bonding strength is low, and the efficiency is low. If the temperature is greater than 100°C, the coating is prone to form a large block, flaky, or layered structure, with a low specific surface area, a low stabilizing effect on antimicrobial peptides, and a short antimicrobial peptide loading amount and antibacterial time of the coating.

[0057] The pH range of the reaction was 7.0-7.8.

[0058] In step S2, the formula of the simulated body fluid is (see Table 1 for details):

[0059] Ultrapure water as solvent, containing the following mixed solution of inorganic metal ions: (HPO4) 2- 1.0-3.0mmol / L, (SO4) 2- 0.1-1mmol / L, Cl - 140.5-150.5mmol / L, (HCO3) -4.1-5.1mmol / L, Ca 2+ 2.1-5.5mmol / L, Na + 140-150mmol / L, Mg 2+ 1.1-2.5mmol / L, K + 4.0-6.0mmol / L;

[0060] And polycarboxylates 10μmol / L-10mmol / L.

[0061] Table 1 1L simulated body fluid formula

[0062]

[0063] The polycarboxylate includes at least one of polyacrylic acid and polyaspartic acid.

[0064] The adsorption treatment conditions include: 2°C-6°C for 1h-24h.

[0065] The solution containing antimicrobial peptides is obtained by dissolving antimicrobial peptides in ethanol solution, and the antimicrobial peptides include at least one of antimicrobial peptides HHC36 (amino acid sequence KRWWKWWRR), SMAP-29 (amino acid sequence H2N-RGLRRLGRKIAHGVKKYGPTVLRIIRIAG-OH), and LL-37 (amino acid sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES). The antimicrobial peptide solution can be HHC36 or any other effective antimicrobial peptide substance. It is ensured to have antimicrobial properties and good biocompatibility.

[0066] Step S3, suspending the bottom-top double-layer nested mixed coating structure material in a solution containing antimicrobial peptides for adsorption treatment, and then rinsing and drying, so as to obtain a mixed coating structure loaded with a large amount of HHC36 antimicrobial peptides.

[0067] In step S3, the adsorption treatment conditions include: 2°C-6°C for 1h-24h.

[0068] According to another typical embodiment of the present invention, an antimicrobial coating of a stable antimicrobial peptide prepared by the method is provided.

[0069] A high specific surface area coating that stabilizes antimicrobial peptides and exerts a lasting antimicrobial effect, the coating being a nested mixed coating structure of a bottom and top double layer, the bottom layer being the surface of an orthopedic external fixator matrix, which is a nano-distributed structural layer, and the top layer being a mineralized calcium phosphate structural layer; the two layers are embedded in each other and diffusely distributed, and a variety of multifunctional inorganic metal ions essential to human physiology are gradiently distributed between the two layers; a large amount of antimicrobial peptides are loaded in the mineralized calcium phosphate structural layer to form a sustained antimicrobial coating surface.

[0070] The matrix of the orthopedic external fixator is: aluminum alloy; titanium alloy; stainless steel;

[0071] The nano-distributed structure layer is a nano-pore structure layer, a nano-grid structure layer or a nano-sheet array structure layer;

[0072] The mineralized calcium phosphate structure layer has a high specific surface area structure, which is a nanofiber structure layer, a nanosheet array structure layer, and an irregular nanoarray structure layer. Its main components are hydroxyapatite and white apatite, with hydroxyapatite accounting for 2wt%-99wt% and white apatite accounting for 2wt%-55wt%, but not limited to these two components. Its main constituent elements are composed of Ca, O, P, C, and N, but not limited to these elements.

[0073] The surface of the antibacterial coating is a mineralized calcium phosphate structure with a high specific surface area, in which a large amount of antibacterial peptides are loaded, the loading amount is increased, and the antibacterial peptides are slowly released to achieve a long-lasting antibacterial effect.

[0074] The surface of the antibacterial coating is a mineralized calcium phosphate structure with a high specific surface area. Its high specific surface area structure stabilizes the antibacterial peptide, prevents premature degradation, ensures its antibacterial activity, and achieves a long-lasting antibacterial effect.

[0075] The present application will be described in detail below with reference to embodiments and experimental data.

[0076] Example 1 Preparation of a high specific surface area coating for trauma external fixator with stable antimicrobial peptide HHC36 and long-lasting antimicrobial effect by alkaline heat treatment combined with biomimetic mineralization

[0077] (1) Surface preparation of aluminum alloy substrate: The aluminum alloy sample was cut into square blocks of 20 mm × 20 mm × 2 mm, and polished with gradient roughness sandpaper. A degreasing agent (such as ethanol or acetone solution) was used to remove grease and impurities on the surface of the aluminum alloy, and then ultrasonically cleaned in an ultrasonic cleaning machine with ultrapure water for 10 minutes. The SEM image of the surface structure of the aluminum alloy is shown in Figure 2. Figure 1 shown.

[0078] (2) Alkali etching treatment: Prepare 4 mol / L sodium hydroxide solution, suspend the square sample in the sodium hydroxide solution, and immerse it at a constant temperature of 50°C for 1 hour. The natural oxide layer on the surface of the aluminum alloy is removed by alkaline etching, and a nanofiber structure is uniformly formed on the surface. The SEM image of the obtained nanofiber array structure is shown in Figure 2 shown.

[0079] (3) Cleaning: Use deionized water for ultrasonic cleaning three times, 5 minutes each time, to remove residual alkali solution.

[0080] (4) Prepare simulated mineralized body fluid: according to Na +142mmol / L, (HPO4) 2- 2.0mmol / L, Ca 2+ 2.5mmol / L, (SO4) 2- 0.2mmol / L, Cl - 145mmol / L, (HCO3) - 4.2mmol / L, Mg 2+ 1.5mmol / L, K + 5.0mmol / L, polyaspartic acid 0.01mmol / L, the solvent is ultrapure water, and a simulated mineralized body fluid containing a small amount of polyaspartic acid is prepared.

[0081] (5) Biomimetic mineralization to form a calcium phosphate nano-network coating structure with a high specific surface area: The square substrate treated in step (3) is suspended in the simulated mineralization solution of step (4), and the pH value is adjusted to 7.4 and the temperature is adjusted to 90°C to promote the deposition of calcium phosphate coatings such as hydroxyapatite on the aluminum alloy substrate to form a biomimetic mineralized coating. The thickness and number of layers of the mineralized coating can be adjusted by adjusting the time. The SEM image of the obtained nanofiber structure of calcium phosphate with a high specific surface area is shown in FIG. Figure 3 shown.

[0082] (6) Post-mineralization treatment: After mineralization, the samples were washed with deionized water to remove the mineral particles not attached to the surface and dried at room temperature.

[0083] (7) Preparation of antimicrobial peptide HHC36 solution: dissolve an appropriate amount of antimicrobial peptide HHC36 in an appropriate amount of ethanol solution.

[0084] (8) The aluminum alloy sample after biomimetic mineralization was immersed in an antimicrobial peptide solution, and the antimicrobial peptide HHC36 was fixed in the pores of the calcium phosphate nanofiber coating with a high specific surface area through physical adsorption and covalent bonding.

[0085] (9) Cleaning and drying after HHC36 loading: Thoroughly clean to remove unbound antimicrobial peptides, air dry, and sterilize by irradiation.

[0086] (10) Performance evaluation: Evaluate the antibacterial performance, durability, biocompatibility, coating hydrophilicity, osteogenic performance, etc. of the treated sample surface. Antibacterial performance test: Evaluate the antibacterial effect of the coating through inhibition zone test, minimum inhibitory concentration (MIC) determination, bacterial colony count, etc. Biocompatibility evaluation: Perform cytotoxicity test, cell adhesion and proliferation test to evaluate the biocompatibility of the coating.

[0087] Example 2 Preparation of a high specific surface area coating for trauma external fixator with stable antimicrobial peptide HHC36 and long-lasting antimicrobial effect by anodic oxidation combined with biomimetic mineralization

[0088] (1) Surface preparation of aluminum alloy substrate: The aluminum alloy sample was cut into square blocks of 20 mm × 20 mm × 2 mm and polished with gradient roughness sandpaper. A degreasing agent (such as ethanol and acetone solution) was used to remove grease and impurities on the surface of the aluminum alloy, and then ultrasonically cleaned in an ultrasonic cleaning machine with ultrapure water for 10 minutes.

[0089] (2) Anodizing treatment: The cleaned aluminum alloy substrate is placed at the two electrodes of an electrolytic cell for anodizing. The distance between the two electrodes is 20 cm, the voltage is 20 V, the electrolyte is composed of deionized water, ethylene glycol and propylene glycol, and the volume ratio of the three is 2:1:1. The electrolyte solute is 5wt% hydrofluoric acid, 2wt% sulfuric acid, and 0.75wt% sodium dodecyl sulfate; the reaction time is 2h.

[0090] (3) Cleaning: Use deionized water for ultrasonic cleaning three times, 5 minutes each time, to remove residual alkali solution.

[0091] (4) Prepare simulated mineralized body fluid: according to Na + 142mmol / L, (HPO4) 2- 2.0mmol / L, Ca 2+ 2.5mmol / L, (SO4) 2- 0.2mmol / L, Cl - 145mmol / L, (HCO3) - 4.2mmol / L, Mg 2+ 1.5mmol / L, K + 5.0mmol / L, polyaspartic acid 0.01mmol / L, the solvent is ultrapure water, and a simulated mineralized body fluid containing a small amount of polyaspartic acid is prepared.

[0092] (5) Biomimetic mineralization to form a calcium phosphate nano-network coating structure with a high specific surface area: The square substrate treated in step (3) is suspended in the simulated mineralization solution of step (4), and the pH value is adjusted to 7.4 and the temperature is adjusted to 90° C. to promote the deposition of calcium phosphate coatings such as hydroxyapatite on the aluminum alloy substrate to form a biomimetic mineralized coating. The thickness and number of layers of the mineralized coating can be adjusted by adjusting the time.

[0093] (6) Post-mineralization treatment: After mineralization, the samples were washed with deionized water to remove the mineral particles not attached to the surface and dried at room temperature.

[0094] (7) Preparation of antimicrobial peptide HHC36 solution: dissolve an appropriate amount of antimicrobial peptide HHC36 in an appropriate amount of ethanol solution.

[0095] (8) The aluminum alloy sample after biomimetic mineralization was immersed in an antimicrobial peptide solution, and the antimicrobial peptide HHC36 was fixed in the pores of the calcium phosphate nanofiber coating with a high specific surface area through physical adsorption and covalent bonding.

[0096] (9) Cleaning and drying after HHC36 loading: Thoroughly clean to remove unbound antimicrobial peptides, air dry, and sterilize by irradiation.

[0097] (10) Performance evaluation: Evaluate the antibacterial performance, durability, biocompatibility, coating hydrophilicity, osteogenic performance, etc. of the treated sample surface. Antibacterial performance test: Evaluate the antibacterial effect of the coating through inhibition zone test, minimum inhibitory concentration (MIC) determination, bacterial colony count, etc. Biocompatibility evaluation: Perform cytotoxicity test, cell adhesion and proliferation test to evaluate the biocompatibility of the coating.

[0098] Example 3 and Example 4

[0099] In Example 3 and Example 4, the antimicrobial peptide HHC36 in Example 1 is replaced by SMAP-29 and LL-37, respectively. In Example 3 and Example 4, the release time of the antimicrobial peptide can be significantly improved, and the release is sustained and slow, so as to achieve long-term antibacterial effect.

[0100] Comparative Example 1: Alkaline heat treatment of aluminum alloy substrate loaded with antibacterial short peptide HHC36 and comparative experiment with Example 1

[0101] (1) Surface preparation of aluminum alloy substrate: The aluminum alloy sample was cut into square blocks of 20 mm × 20 mm × 2 mm, and polished with gradient roughness sandpaper. A degreasing agent (such as ethanol or acetone solution) was used to remove grease and impurities on the surface of the aluminum alloy, and then ultrasonically cleaned in an ultrasonic cleaning machine with ultrapure water for 10 minutes. The SEM image of the surface structure of the aluminum alloy is shown in Figure 2. Figure 1 shown.

[0102] (2) Alkali etching treatment: Prepare 4 mol / L sodium hydroxide solution, suspend the square sample in the sodium hydroxide solution, and immerse it at a constant temperature of 50°C for 1 hour. The natural oxide layer on the surface of the aluminum alloy is removed by alkaline etching, and a nanofiber structure is uniformly formed on the surface. The SEM image of the obtained nanofiber array structure is shown in Figure 2 shown.

[0103] (3) Cleaning: Use deionized water for ultrasonic cleaning three times, 5 minutes each time, to remove residual alkali solution.

[0104] (4) Preparation of antimicrobial peptide HHC36 solution: dissolve an appropriate amount of antimicrobial peptide HHC36 in an appropriate amount of ethanol solution.

[0105] (5) The aluminum alloy sample after alkaline heat treatment was immersed in the antimicrobial peptide solution, and the antimicrobial peptide HHC36 was fixed in the micro-nanostructure through physical adsorption and covalent bonding.

[0106] (6) Cleaning and drying after HHC36 loading: Thoroughly clean to remove unbound antimicrobial peptides, air dry, and sterilize by irradiation.

[0107] (7) Performance comparison evaluation: Evaluate the antibacterial performance, durability, biocompatibility, coating hydrophilicity, osteogenic performance, etc. of the sample surfaces treated by Example 1 and Comparative Example 1. Antibacterial performance test: Evaluate the antibacterial effect of the coating by methods such as inhibition zone test, minimum inhibitory concentration (MIC) determination, and bacterial colony count. Biocompatibility evaluation: Perform cytotoxicity test, cell adhesion and proliferation experiments to evaluate the biocompatibility of the coating.

[0108] (8) The comparison results are as follows:

[0109] Antimicrobial peptide HHC36 release effect: Figure 4 As shown, the antimicrobial peptide loaded on the substrate in Example 1 can be continuously released for up to 180 hours, while the antimicrobial peptide on the substrate in Comparative Example 1 is released explosively and is almost completely released after 48 hours. It can be seen that the high specific surface area calcium phosphate coating in Example 1 of the present invention can significantly improve the release time of the antimicrobial peptide, continuously and slowly release it, and achieve long-term antibacterial effect.

[0110] Comparison of antibacterial effects: Fig. 9 , Fig.10 As shown, the two bacteria, S. aureus and E. coli, were cultured on the surfaces of the samples of Example 1 and Comparative Example 1 for 1 hour, and the antibacterial effects of the two sample surfaces were comparable. When the culture time was extended to 6 hours, 12 hours, and 24 hours, the sample of Example 1 showed stronger antibacterial properties, almost killing the cultured E. coli on the surface at 12 hours, and almost killing the cultured S. aureus on the surface at 24 hours. However, the antibacterial performance of the sample of Comparative Example 1 against the two bacteria did not show a significant increase at 6 hours, 12 hours, and 24 hours, and the antibacterial efficacy was relatively weak. Comparative Example 2: Comparative experiment of Example 2 by loading the antimicrobial peptide HHC36 on an anodized aluminum alloy substrate

[0111] (1) Surface preparation of aluminum alloy substrate: The aluminum alloy sample was cut into square blocks of 20 mm × 20 mm × 2 mm and polished with gradient roughness sandpaper. A degreasing agent (such as ethanol and acetone solution) was used to remove grease and impurities on the surface of the aluminum alloy, and then ultrasonically cleaned in an ultrasonic cleaning machine with ultrapure water for 10 minutes.

[0112] (2) Anodizing treatment: The cleaned aluminum alloy substrate is placed at the two electrodes of an electrolytic cell for anodizing. The distance between the two electrodes is 20 cm, the voltage is 20 V, the electrolyte is composed of deionized water, ethylene glycol and propylene glycol, and the volume ratio of the three is 2:1:1. The electrolyte solute is 5wt% hydrofluoric acid, 2wt% sulfuric acid, and 0.75wt% sodium dodecyl sulfate; the reaction time is 2h.

[0113] (3) Cleaning: Use deionized water for ultrasonic cleaning three times, 5 minutes each time, to remove residual alkali solution.

[0114] (4) Preparation of antimicrobial peptide HHC36 solution: dissolve an appropriate amount of antimicrobial peptide HHC36 in an appropriate amount of ethanol solution.

[0115] (5) The aluminum alloy sample after anodizing treatment was immersed in the antimicrobial peptide solution, and the antimicrobial peptide HHC36 was fixed in the nanofiber structure through physical adsorption and covalent bonding.

[0116] (6) Cleaning and drying after HHC36 loading: Thoroughly clean to remove unbound antimicrobial peptides, air dry, and sterilize by irradiation.

[0117] (7) Performance comparison evaluation: Evaluate the antibacterial performance, durability, biocompatibility, coating hydrophilicity, osteogenic performance, etc. of the sample surfaces treated by Example 1 and Comparative Example 1. Antibacterial performance test: Evaluate the antibacterial effect of the coating by methods such as inhibition zone test, minimum inhibitory concentration (MIC) determination, and bacterial colony count. Biocompatibility evaluation: Perform cytotoxicity test, cell adhesion and proliferation experiments to evaluate the biocompatibility of the coating.

[0118] (8) The comparison results are as follows:

[0119] Antimicrobial peptide HHC36 release effect: Figure 7 As shown, the antimicrobial peptide loaded on the substrate in Example 2 can be continuously released for up to 180 hours, while the antimicrobial peptide on the substrate in Comparative Example 2 is released explosively and is almost completely released after 24 hours. It can be seen that the high specific surface area calcium phosphate coating in Example 2 of the present invention can significantly improve the release time of the antimicrobial peptide, continuously and slowly release it, and achieve long-term antibacterial effect.

[0120] Comparison of antibacterial effects: Fig. 9 , Fig.10As shown, the antibacterial effects of the two samples of Example 2 and Comparative Example 2 were comparable when the two bacteria, S. aureus and E. coli, were cultured on the surface for 1 hour. When the culture time was extended to 6 hours, 12 hours, and 24 hours, the sample of Example 1 showed stronger antibacterial properties, almost killing the cultured E. coli on the surface at 12 hours, and almost killing the cultured S. aureus on the surface at 24 hours. However, the antibacterial performance of the sample of Comparative Example 1 against the two bacteria did not increase significantly at 6 hours, 12 hours, and 24 hours, and the antibacterial efficacy was relatively weak.

[0121] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0122] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0123] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an antibacterial coating of a stable antibacterial peptide, characterized in that: The method comprises: Pre-treating the surface of an orthopedic external fixator matrix to obtain a matrix material having a nano-distributed structure on the surface; The matrix material with the nano-distributed structure on the surface is suspended in a simulated physiological fluid containing polycarboxylate, treated at 60°C-100°C for 1h-24h, then naturally cooled to room temperature, and the base material is taken out for cleaning and drying to obtain a bottom-top double-layer nested hybrid coating structure material; The bottom-top double-layer nested mixed coating structure material is suspended in a solution containing antimicrobial peptides for adsorption treatment, and then rinsed and dried to obtain an antimicrobial coating with stable antimicrobial peptides.

2. The method for preparing an antibacterial coating of a stable antimicrobial peptide according to claim 1, characterized in that: The orthopedic external fixator matrix is ​​selected from one of aluminum alloy, titanium alloy or stainless steel.

3. The method for preparing an antibacterial coating of a stable antimicrobial peptide according to claim 1, characterized in that: The preprocessing comprises: The matrix of the orthopedic external fixator is polished and ground, and after being rinsed, a nano-distributed structure layer is formed on the surface by using one of an electrochemical process, an acid etching process, an alkali thermal chemical process, a micro-arc oxidation process and an anodic oxidation process to obtain a base material with a nano-distributed structure on the surface.

4. The method for preparing an antibacterial coating of a stable antimicrobial peptide according to claim 1, characterized in that: The formula of the simulated body fluid is: Ultrapure water as solvent, containing the following mixed solution of inorganic metal ions: (HPO4) 2- 1.0-3.0mmol / L, (SO4) 2- 0.1-1mmol / L, Cl - 140.5-150.5mmol / L, (HCO3) - 4.1-5.1mmol / L, Ca 2+ 2.1-5.5mmol / L, Na + 140-150mmol / L, Mg 2+ 1.1-2.5mmol / L, K + 4.0-6.0mmol / L; And polycarboxylates 10μmol / L-10mmol / L.

5. The method for preparing an antibacterial coating of a stable antimicrobial peptide according to claim 4, characterized in that: The polycarboxylate includes at least one of polyacrylic acid and polyaspartic acid.

6. The method for preparing an antibacterial coating of a stable antimicrobial peptide according to claim 4, characterized in that: The adsorption treatment conditions include: 2°C-6°C for 1h-24h.

7. The method for preparing an antibacterial coating of a stable antimicrobial peptide according to claim 4, characterized in that: The solution containing antimicrobial peptides is obtained by dissolving antimicrobial peptides in ethanol solution, and the antimicrobial peptides include at least one of antimicrobial peptides HHC36, SMAP-29 and LL-37.

8. An antibacterial coating of a stable antibacterial peptide prepared by the method according to any one of claims 1 to 7.

9. Use of the antibacterial coating of the stable antibacterial peptide according to claim 8 in the preparation of medical antibacterial metal equipment.

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