CN115957385A - Preparation method and application of multifunctional coating of metal polyphenol network coupled antibacterial peptide - Google Patents
Preparation method and application of multifunctional coating of metal polyphenol network coupled antibacterial peptide Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Materials For Medical Uses (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a preparation method and application of a multifunctional coating of metal polyphenol network coupled antibacterial peptide. The multifunctional titanium implant coating of the metal polyphenol network coupling antibacterial peptide is constructed by compounding the titanium dioxide nano Tip (TNS), the Metal Phenol Network (MPN) and the antibacterial peptide (AMP), the combination of physical puncture/photo-thermal/chemical antibacterial capabilities is realized, the osteoconductivity of the coating is enhanced by promoting the deposition of hydroxyapatite, and the coating has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of medical chemistry and materials, and particularly relates to a preparation method and application of a multifunctional coating of metal polyphenol network coupling antibacterial peptide.
Background
With the accelerated aging of the population in China, the amount of medical devices implanted into hard tissues represented by bone/oral implants has been continuously increased in recent years. Titanium (Ti) and its alloys have been widely used in hard tissue implant materials, accounting for 70-80% of orthopedic implants, due to excellent mechanical properties, good corrosion resistance and biocompatibility. However, the inherent biological inertness of titanium material makes it deficient in antibacterial properties and osteoconductivity, resulting in an increased risk of bacterial infection and aseptic loosening, and ultimately, failure of the procedure. Therefore, the titanium implant has good antibacterial performance and excellent bone conduction performance through surface modification, and has good clinical application prospect. Currently, the most widely used clinical antibacterial means for orthopedic implant infection is still antibiotic therapy, but the abuse of antibiotics induces drug resistance of a large number of bacteria, and even causes the situation that the infection is not treatable by drugs. Therefore, there is an increasing interest in finding antibacterial methods that are less likely to induce drug resistance and have good biocompatibility. Photothermal therapy can induce local generation of high temperature by non-invasive light source irradiation, destroy the structure of biological membrane to inactivate its active matrix (such as nucleic acid and protein), and is not easy to generate drug resistance; however, since the photothermal therapy alone requires the introduction of high power laser density and long irradiation time, it inevitably causes side effects on surrounding healthy tissues.
In view of the above, the present research team has considered that it is necessary to explore a method capable of solving the problem of antibacterial property of the implant body and the implant interior.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a preparation method and application of a multifunctional coating of metal polyphenol network coupling antibacterial peptide.
A preparation method of a multifunctional coating of metal polyphenol network coupled antibacterial peptide is characterized by comprising the following steps:
1) Preparing a titanium dioxide nano tip array (TNS array) on the surface of a titanium material in situ by adopting a hydrothermal method to obtain the titanium material with the titanium dioxide nano tip array on the surface; preparing a nano-tip structure which can physically penetrate bacteria and convert light into heat on a titanium material by a high-temperature high-pressure NaOH hydrothermal process, and annealing to obtain the titanium material with a titanium dioxide nano-tip array (TNS array) on the surface;
2) Assembling metal polyphenol nano-film on titanium material surface with titanium dioxide nano-tip array
2.1 Placing the titanium material obtained in the step 1) into a reaction container, and then adding a plant polyphenol solution and a metal ion solution;
2.2 Adjusting the pH of the mixed solution of step 2.1) to physiological pH values (such as: 7.4 );
2.3 In order to make the reaction and assembly more sufficient, the metal polyphenol nano-film formed by the plant polyphenol and the metal ions is quickly assembled on the titanium dioxide nano-tip array by swirling the mixed solution adjusted in the step 2.2), so as to obtain the titanium material TNS-MPN of the titanium dioxide nano-tip array with the metal polyphenol nano-film;
3) Multifunctional coating for preparing metal polyphenol network coupling antibacterial peptide
3.1 Immersing the titanium material obtained in the step 2) in a Tris-HCl solution dissolved with broad-spectrum antibacterial peptide AMP, and placing the titanium material in a shaking table for reaction;
3.2 After the reaction is finished, the titanium material is cleaned and dried, and a multifunctional coating TNS-MPN-AMP of the metal polyphenol network coupling antibacterial peptide is obtained on the surface of the titanium material, wherein the coating has an antibacterial and bone-promoting function brought by physical puncture/photothermal/chemical ternary cooperation.
Further, the step 1) specifically comprises:
1.1 Placing the titanium material at the bottom of a stainless steel autoclave reactor lined with polytetrafluoroethylene, adding NaOH solution with the molar concentration of 0.5-1.5M (preferably 1M), and sealing the autoclave reactor;
1.2 Placing the sealed autoclave reactor in an electric furnace at the temperature of 150-300 ℃ for reaction for 3-5h;
1.3 And) after the reaction is finished, placing the titanium material in a muffle furnace at 400-600 ℃ for annealing for 1-3h to obtain the titanium sheet with the titanium dioxide nano tip array on the surface.
Further, in the step 2), the plant polyphenol in the plant polyphenol solution is catechol, pyrogallic acid, gallic acid or tannic acid;
the metal ion solution is FeCl 3 Solution, znSO 4 Solution, cuSO 4 Solutions or AlCl 3 A solution;
in the step 3), the broad-spectrum antibacterial peptide is antibacterial peptide LL-37, magainin, nisin or epsilon-polylysine;
when the plant polyphenol is catechol, the components are added according to the following parts by weight:
4-7 parts of catechol, 2-5 parts of metal ions and 100-300 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution;
when the plant polyphenol is pyrogallic acid, the components are added according to the following parts by weight:
1-4 parts of pyrogallic acid, 2-5 parts of metal ions and 100-300 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution;
when the plant polyphenol is gallic acid, the components are added according to the following parts by weight:
1-4 parts of gallic acid, 2-5 parts of metal ions and 100-300 parts of AMP dissolved in Tris-HCl solution;
when the plant polyphenol is tannin, the components are added according to the following parts by weight:
2 to 5 portions of tannic acid, 2 to 5 portions of metal ions and 100 to 300 portions of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution.
In order to ensure that the volume of the solution for immersing the titanium material is proper, a plant polyphenol solution, a metal ion solution and a Tris-HCl solution of broad-spectrum antibacterial peptide can be prepared according to the following standards, and then the corresponding volumes are measured according to the parts by weight:
when the plant polyphenol is catechol, the concentration is as follows:
the concentration of the catechol in the plant polyphenol solution is 0.4-0.7 mg mL -1 The concentration of the metal ions in the metal ion solution is 0.2-0.5 mg mL -1 DissolvingThe concentration of the broad-spectrum antibacterial peptide in Tris-HCl solution is 0.01-0.03 g mL -1 ；
When the plant polyphenol is pyrogalloc acid, the concentrations are as follows:
the concentration of pyrogallic acid in the plant polyphenol solution is 0.1-0.4 mg mL -1 The concentration of the metal ions in the metal ion solution is 0.2-0.5 mg mL -1 The concentration of the broad-spectrum antibacterial peptide dissolved in the Tris-HCl solution is 0.01-0.03 g mL -1 ；
When the plant polyphenol is gallic acid, the concentration is as follows:
the concentration of the gallic acid in the plant polyphenol solution is 0.1-0.4 mg mL -1 The concentration of the metal ions in the metal ion solution is 0.2-0.5 mg mL -1 The concentration of the broad-spectrum antibacterial peptide dissolved in the Tris-HCl solution is 0.01-0.03 g mL -1 ；
When the plant polyphenol is tannic acid, the concentrations are as follows:
the concentration of the tannic acid in the plant polyphenol solution is 0.2-0.5 mg mL -1 The concentration of the metal ions in the metal ion solution is 0.2-0.5 mg mL -1 The concentration of the broad-spectrum antibacterial peptide dissolved in the Tris-HCl solution is 0.01-0.03 g mL -1 。
Further, in step 1.3), in order to safely and efficiently realize the annealing of the TNS, the temperature rise rate is set to 10 ℃/min;
in the step 2.2), adjusting the pH value of the mixed solution in the step 2.1) by using a NaOH solution;
in the step 2.3), the vortex time is 30-90s, and 60s is preferred in order to improve the reaction efficiency and ensure full reaction and complete assembly;
in the step 3.1), the reaction is carried out for 6 to 12 hours at room temperature.
Further, before the step 1), the method also comprises the pretreatment of the titanium material, specifically:
cutting a pure titanium material into a required size, sequentially polishing the pure titanium material to mirror gloss by using SiC sandpaper with the granularity of #400, #800, # 1200- #2000, flushing abrasive dust by using deionized water, sequentially carrying out ultrasonic cleaning by using acetone, ethanol and water, removing oil stains on the surface, and drying for later use.
Meanwhile, the invention also provides a multifunctional coating of the metal polyphenol network coupling antibacterial peptide prepared by the method and application of the multifunctional coating in antibacterial promotion of bone modification of titanium materials.
In addition, based on the application, the invention also provides a titanium modified material which is characterized in that the multifunctional coating of the metal polyphenol network coupling antibacterial peptide is prepared on the surface of the titanium material according to the method. And the application of the titanium modified material in preparing the antibacterial osteogenesis-promoting titanium implant and the titanium implant.
The conception and the principle of the invention are as follows:
the research team of the invention considers that pure titanium has biological inertia and no antibacterial and osteoinductive capabilities, and intends to construct various micro/nano structures on the titanium surface, such as: nanotubes, nanopipettes, nanopores, nanophylles, etc., to enhance the bioactivity of the titanium implant. The titanium dioxide nano tip structure can simulate natural extracellular matrix, promote fluid flow, promote molecule and cell transportation, stimulate osteogenesis signals, promote better cell adhesion, migration, proliferation and differentiation, physically destroy a bacterial membrane and leak cell contents to kill bacteria, and more importantly, the titanium dioxide nano tip structure can generate good photothermal conversion efficiency under the assistance of Near Infrared (NIR) light irradiation by improving the light capture performance and the anti-reflection efficiency of a titanium surface, so that a research team thinks that the titanium dioxide nano tip structure constructed on the titanium surface can be used for treating implant infection by using non-invasive photothermal therapy aiming at an infected part. In addition, a metal polyphenol network is modified on the titanium surface, and is coupled on the coating surface through Michael addition/Schiff base reaction between polyphenol and antibacterial peptide (AMP), so that antibacterial capacity is endowed to the titanium implant and bone bioactivity is promoted.
In conclusion, the research team realizes the reduction of the power density and the shortening of the irradiation time of the photothermal therapy by constructing a multi-way synergistic with the photothermal therapy, and simultaneously endows the titanium implant material with good antibacterial property to promote the bone performance and good biocompatibility.
The invention has the advantages that:
1. the method of the invention does not need to introduce a photo-thermal agent, and builds the micro/nano structure with the titanium dioxide nano tip shape on the surface of the titanium material in situ. The structure improves the light capture performance and the anti-reflection efficiency of the surface of the titanium material, so that the titanium material can generate good photo-thermal conversion efficiency under the assistance of Near Infrared (NIR) light irradiation, and is suitable for photo-thermal therapy treatment.
2. The method forms a layer of metal polyphenol network MPN through the complexing action of the plant polyphenol and the metal ions, the network has good adhesion, can quickly form a film on TNS, and has low cost, good biological safety and long reaction time.
3. Compared with pure titanium, the TNS-MPN-AMP coating prepared by the invention introduces abundant bioactive groups such as hydroxyl, carboxyl, amino and the like, can attract phosphate and carbonate in body fluid to the surface of the coating more, promotes the nucleation of hydroxyapatite (HAp) in vivo, and further promotes osseointegration.
4. According to the invention, the physical puncture/photo-thermal/chemical ternary synergistic antibacterial effect is integrated on the surface of the titanium implant, so that a high-efficiency sterilization effect is realized, wherein the in-vitro antibacterial rate exceeds 99.99%, and the in-vivo antibacterial rate exceeds 95%. Meanwhile, compared with a single photothermal therapy, the power laser density of the used light source can be reduced, the irradiation time can be shortened, and the side effect on the surrounding healthy tissues can be effectively inhibited during treatment.
5. The preparation method of the coating is simple, efficient, low in cost and high in biological safety.
Drawings
FIG. 1 is a schematic diagram showing the preparation process of TNS-MPN-AMP obtained in example 1 according to the present invention;
FIG. 2 is an FE-SEM photograph of Ti used in example 1 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained thereby;
FIG. 3 is a diagram of WCA of Ti used in example 2 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained therefrom;
FIG. 4 is a graph showing temperature change curves of Ti used in example 3 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained therefrom;
FIG. 5 is a photograph of a bactericidal coating of gram-negative bacteria E.coli and gram-positive bacteria S.aureus with Ti used in example 1 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained therefrom;
FIG. 6 is an FE-SEM image of the morphology of osteoblasts MC3T3-E1 on the surface of TNS-MPN-AMP obtained in example 1 of the present invention;
FIG. 7 shows the deposition of HAp on the surface of the TNS-MPN-AMP obtained from Ti used in example 6 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the following figures and specific examples:
example 1
A preparation method of a titanium modified material, a schematic preparation flow diagram of which is shown in figure 1, comprises the following steps:
1) Cutting pure titanium material into 1 × 1cm 2 The size of the SiC abrasive paper is equal to that of the SiC abrasive paper (or other required sizes), siC abrasive paper with the granularity of #400, #800, #1200 to #2000 is sequentially polished to mirror surface gloss, the abrasive dust is washed by deionized water, then ultrasonic cleaning is sequentially carried out by acetone, ethanol and water, oil stain on the surface is removed, and the SiC abrasive paper is dried for standby.
2) Placing the pretreated titanium material at the bottom of a polytetrafluoroethylene-lined stainless steel autoclave reactor with the volume of 30mL, and adding 10mL of NaOH solution with the molar concentration of 1.5M; then placing the sealed autoclave reactor in an electric furnace at 300 ℃ for reaction for 3h;
3) After the reaction is finished, taking out the titanium material, placing the titanium material in a muffle furnace, and annealing for 3 hours at 400 ℃ at the heating rate of 10 ℃/min to obtain the titanium material with the surface provided with the titanium dioxide nano tip array, and marking the titanium material as a TNS sample;
4) A50 mL centrifuge tube was taken, the TNS sample was placed in the bottom of the tube, and 10mL tannic acid (0.4 mg mL) was added -1 ) Solution and 10mL ZnSO 4 (0.2mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1M, and vortexing for 60s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) Will resist bacteriaPeptide LL-37 was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.02g mL -1 The concentration of (d); TNS-MPN samples were immersed in 10mL of the above solution and placed on a shaker for reaction at room temperature for 12 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
The morphological structure of the surface of the titanium sheet is characterized by using FE-SEM. The sample prepared in this example was first attached to a conductive tape, and then imaged by gold spraying for 50s, and the result is shown in fig. 2. As can be seen from FIG. 2, the TNS, TNS-MPN and TNS-MPN-AMP sample surfaces all exhibit typical nanotip structures.
Example 2
The difference from example 1 is that:
2) Placing the pretreated titanium material at the bottom of a polytetrafluoroethylene-lined stainless steel autoclave reactor with the volume of 30mL, and adding 10mL of NaOH solution with the molar concentration of 1M; then placing the sealed autoclave reactor in an electric furnace at 150 ℃ for reaction for 5h;
3) After the reaction is finished, taking out the titanium material, placing the titanium material in a muffle furnace, and annealing for 1 hour at 600 ℃ at the heating rate of 10 ℃/min to obtain the titanium material with the surface provided with the titanium dioxide nano tip array, and marking the titanium material as a TNS sample;
4) A50 mL centrifuge tube was placed with TNS at the bottom of the tube, and then 10mL tannic acid (0.4 mg mL) was added -1 ) Solution and 10mL CuSO 4 (0.2mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 0.5M, and vortexing for 60s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) Antimicrobial peptide LL-37 was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.02g mL -1 The concentration of (c); TNS-MPN samples were immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 6 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
The course of the contact angle change of the coating was characterized by means of a contact angle measuring instrument. And (3) slowly sucking deionized water into the water-phase needle tube by using a sitting drop method, transferring the liquid drop at the port of the needle tube to the surface of the sample to be measured, determining the base line of the liquid drop, finishing the transfer of the liquid drop, and reading and recording the measured contact angle. Figure 3 the results show: the contact angle of the prepared nano-tip structure is obviously reduced compared with that of pure titanium, and meanwhile, the contact angle of the surface of the material is further increased along with the assembly and deposition of MPN and AMP, but the hydrophilicity of the surface of the material is still maintained.
Example 3
The difference from example 1 is that:
2) Placing the pretreated titanium material at the bottom of a polytetrafluoroethylene-lined stainless steel autoclave reactor with the volume of 30mL, and adding 10mL of NaOH solution with the molar concentration of 0.5M; then placing the sealed autoclave reactor in an electric furnace at 220 ℃ for reaction for 4h;
3) After the reaction is finished, taking out the titanium material, placing the titanium material in a muffle furnace, and annealing for 2 hours at 550 ℃ at the heating rate of 10 ℃/min to obtain the titanium material with the surface provided with the titanium dioxide nano tip array, and marking the titanium material as a TNS sample;
4) A50 mL centrifuge tube was taken, the TNS sample was placed in the bottom of the tube, and 10mL catechol (0.4 mg mL) was added -1 ) Solution and 10mL AlCl 3 (0.2mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using NaOH solution with the molar concentration of 1.5M, and vortexing for 60s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) Antimicrobial peptide LL-37 was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.02g mL -1 The concentration of (d); a sample of TNS-MPN was immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 12 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
The photo-thermal temperature rise curve of the material under the irradiation of near infrared light is recorded by using a near infrared laser with wavelength of 808nm and a thermal infrared imager. Figure 4 results show that: compared with pure titanium, the prepared nano-tip structure has good photothermal effect, the temperature of Ti is increased from 25.0 ℃ to 39.4 ℃, the temperature of TNS is obviously increased from 25.0 ℃ to 49.7 ℃, and meanwhile, as can be seen from the figure, the material still maintains good photothermal conversion efficiency along with the assembly and deposition of MPN and AMP.
Example 4
The difference from example 1 is that:
4) A50 mL centrifuge tube was taken, the TNS sample was placed in the bottom of the tube, and 10mL tannic acid (0.4 mg mL) was added -1 ) Solution and 10mL FeCl 3 (0.2mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1M, and vortexing for 60s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) Antimicrobial peptide ε -polylysine was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.02g mL -1 The concentration of (d); TNS-MPN samples were immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 12 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
Example 5
The difference from example 1 is that:
4) A50 mL centrifuge tube was placed with TNS sample at the bottom of the tube, then 10mL pyrogalloc acid (0.4 mg mL) was added -1 ) Solution and 10mL FeCl 3 (0.2mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 0.5M, and vortexing for 60s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) The antimicrobial peptide nisin was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.02g mL -1 The concentration of (c); TNS-MPN samples were immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 12 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
Example 6
The difference from example 1 is that:
4) A50 mL centrifuge tube was placed with TNS at the bottom of the tube, and 10mL gallic acid (0.4 mg mL) was added -1 ) Solution and 10mL ZnSO 4 (0.2mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1M, and vortexing for 60s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) Antimicrobial peptide ε -polylysine was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.02g mL -1 The concentration of (d); a sample of TNS-MPN was immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 8 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
In order to verify the effect of the multifunctional coating prepared by the invention, the invention tests the anti-bacterial performance and the cell compatibility of the TNS-MPN-AMP coating prepared in the embodiment 1, and tests the bone conduction promoting performance of the TNS-MPN-AMP coating prepared in the embodiment 6, wherein the specific experiments are as follows:
A. the bactericidal properties of the material in example 1 were verified by testing:
all prepared samples were sterilized in advance, and individual colonies of gram-negative bacteria e.coli and gram-positive bacteria s.aureus were selected from the bacterial culture plate and cultured in LB medium for 4-6 hours until the bacterial population grew to the middle logarithmic phase (optical density at 600nm reached 0.5). Then, 1X 10 is added 7 CFU mL -1 The bacterial solution of (2) was dropped onto the surface of the sample prepared in example 1 and co-incubated in a bacterial incubator at 37 ℃ for 45min, wherein the TNS-MPN-AMP + NIR group samples were subjected to light irradiation at 808nm for 2min, and then 980 μ L of sterile PBS was added to the wells, the bacteria attached to each sample were ultrasonically separated, and then 100 μ L of the bacterial suspension in each well containing the sample was spread on a solid LB agar plate using an L-type spreader and incubated overnight at 37 ℃ to take a photograph. The results in FIG. 5 show that: ti to Staphylococcus aureus and Staphylococcus aureusThe average antibacterial rate of Enterobacter was 0%, TNS was 24.07% and 45.56%, TNS-MPN was 9.64% and 35.03%, TNS-MPN-AMP was 91.79% and 92.80%, and TNS-MPN-AMP + NIR were over 99.99%. Therefore, the prepared coating is proved to have good ternary synergistic anti-infection effect.
B. The cellular compatibility of the material in example 1 was verified by testing:
MC3T3-E1 cells were cultured in a medium containing alpha-MEM, FBS and diabody, after which TNS-MPN-AMP was mixed with 8X 10 3 The cells were co-incubated. At 37 ℃ C, 5% CO 2 After culturing for 24h in a humid environment, fixing the cells with 4% paraformaldehyde, dehydrating with a gradient ethanol solution, spraying gold, and observing with an FE-SEM. As shown in FIG. 6, MC3T3-E1 cells exhibited a spindle-like morphology and were in a well-adherent state. The coating is proved to have good cell compatibility, thereby being beneficial to subsequent proliferation, migration and differentiation.
C. The bone conduction performance of the material in example 6 was verified by testing:
samples of Ti and TNS-MPN-AMP were immersed in Simulated Body Fluid (SBF) at pH 7.4 and maintained at 37 ℃ for 14 days to induce apatite growth, with new SBF every other day. As shown in the FE-SEM image in FIG. 7, a hydroxyapatite layer was formed on the surface of TNS-MPN-AMP.
Example 7
The difference from example 1 is that:
4) A50 mL centrifuge tube was placed with TNS at the bottom of the tube, followed by 10mL catechol (0.7 mg mL) -1 ) Solution and 10mL ZnSO 4 (0.5mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using NaOH solution with the molar concentration of 1.5M, and vortexing for 90s to obtain a titanium material with a titanium dioxide nano tip array assembled with the metal polyphenol nano film, wherein the titanium material is marked as a TNS-MPN sample;
5) Antimicrobial peptide ε -polylysine was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.01g mL -1 The concentration of (d); a sample of TNS-MPN was immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 8 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a coating with multiple functionsThe titanium material (2) was designated as TNS-MPN-AMP sample.
Example 8
The difference from example 1 is that:
4) A50 mL centrifuge tube was placed with TNS at the bottom of the tube, and 10mL pyrogalloc acid (0.1 mg mL) was added -1 ) Solution and 10mL CuSO 4 (0.3mg mL -1 ) Gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1.5M, and performing vortex operation for 90s to obtain a titanium material with a titanium dioxide nano tip array assembled with a metal polyphenol nano film, and marking the titanium material as a TNS-MPN sample;
5) The antibacterial peptide magainin was dissolved in Tris-HCl solution (pH =8.5, 10 mM) to give 0.03g mL -1 The concentration of (d); TNS-MPN samples were immersed in 10mL of the above solution and placed on a shaker for oxidation reaction at room temperature for 10 hours. Subsequently, the sample was removed, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material with a multifunctional coating, designated as TNS-MPN-AMP sample.
In conclusion, the multifunctional titanium implant coating with the metal polyphenol network coupled with the antibacterial peptide is constructed by compounding the titanium dioxide nano Tip (TNS), the Metal Polyphenol Network (MPN) and the antibacterial peptide (AMP), so that the combination of physical puncture/photo-thermal/chemical antibacterial capacity is realized, the bone conductivity of the coating is enhanced by promoting the deposition of hydroxyapatite, and the multifunctional titanium implant coating has a good application prospect.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications or substitutions can be easily made by those skilled in the art within the technical scope of the present disclosure.
Claims (10)
1. A preparation method of a multifunctional coating of metal polyphenol network coupled antibacterial peptide is characterized by comprising the following steps:
1) Preparing a titanium dioxide nano tip array on the surface of a titanium material in situ by adopting a hydrothermal method to obtain the titanium material with the titanium dioxide nano tip array on the surface;
2) Assembling metal polyphenol nano-film on titanium material surface with titanium dioxide nano-tip array
2.1 Placing the titanium material obtained in the step 1) into a reaction container, and then adding a plant polyphenol solution and a metal ion solution into the reaction container;
2.2 Adjusting the pH of the mixed solution obtained in the step 2.1) to a physiological pH value;
2.3 By vortex, the metal polyphenol nano-film formed by the plant polyphenol and the metal ions is assembled on the titanium dioxide nano-tip array to obtain the titanium material of the titanium dioxide nano-tip array with the metal polyphenol nano-film;
3) Multifunctional coating for preparing metal polyphenol network coupled antibacterial peptide
3.1 Immersing the titanium material obtained in the step 2) in a Tris-HCl solution dissolved with broad-spectrum antibacterial peptide, and placing the titanium material in a shaking table for reaction;
3.2 After the reaction is finished, the titanium material is cleaned and dried, and the multifunctional coating of the metal polyphenol network coupled antibacterial peptide is obtained on the surface of the titanium material.
2. The preparation method according to claim 1, wherein the step 1) is specifically:
1.1 Placing the titanium material at the bottom of a stainless steel autoclave reactor with a polytetrafluoroethylene lining, adding NaOH solution with the molar concentration of 0.5-1.5M, and sealing the autoclave reactor;
1.2 Placing the sealed autoclave reactor in an electric furnace at the temperature of 150-300 ℃ for reaction for 3-5h;
1.3 After the reaction is finished, the titanium material is placed in a muffle furnace at the temperature of 400-600 ℃ for annealing for 1-3h, and the titanium sheet with the titanium dioxide nano tip array on the surface is obtained.
3. The production method according to claim 1 or 2, characterized in that:
in the step 2), the plant polyphenol in the plant polyphenol solution is catechol, pyrogallic acid, gallic acid or tannic acid;
the metal ion solution is FeCl 3 Solution, znSO 4 Solution, cuSO 4 Solutions or AlCl 3 A solution;
in the step 3), the broad-spectrum antibacterial peptide is antibacterial peptide LL-37, magainin, nisin or epsilon-polylysine;
when the plant polyphenol is catechol, the components are added according to the following parts by weight:
4-7 parts of catechol, 2-5 parts of metal ions and 100-300 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution;
when the plant polyphenol is pyrogallic acid, the components are added according to the following parts by weight:
1-4 parts of pyrogallol, 2-5 parts of metal ions and 100-300 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution;
when the plant polyphenol is gallic acid, the components are added according to the following parts by weight:
1-4 parts of gallic acid, 2-5 parts of metal ions and 100-300 parts of AMP dissolved in Tris-HCl solution;
when the plant polyphenol is tannin, the components are added according to the following parts by weight:
2 to 5 portions of tannic acid, 2 to 5 portions of metal ions and 100 to 300 portions of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution.
4. The method according to claim 3, wherein:
in the step 1.3), the heating rate is 10 ℃/min;
in the step 2.2), adjusting the pH value of the mixed solution in the step 2.1) by using a NaOH solution;
in the step 2.3), the vortex time is 30-90s;
in the step 3.1), the reaction is carried out for 6 to 12 hours at room temperature.
5. The preparation method according to claim 4, characterized by further comprising a pretreatment of the titanium material before the step 1), specifically:
cutting a pure titanium material into a required size, sequentially polishing the pure titanium material to mirror gloss by using SiC sandpaper with the granularity of #400, #800, # 1200- #2000, flushing abrasive dust by using deionized water, sequentially carrying out ultrasonic cleaning by using acetone, ethanol and water, removing oil stains on the surface, and drying for later use.
6. A multifunctional coating of metal polyphenol network coupled antibacterial peptide is characterized in that: prepared by the process of any one of claims 1 to 5.
7. The use of the multifunctional coating of metal polyphenol network coupled antimicrobial peptide as claimed in claim 6 for the antimicrobial bone-modifying of titanium materials.
8. A titanium modified material is characterized in that: preparing a multifunctional coating of metal polyphenol network coupled antibacterial peptide on the surface of a titanium material according to the method of any one of claims 1 to 5.
9. Use of a titanium modified material according to claim 8 for the preparation of an antibacterial osteogenic titanium implant.
10. A titanium implant, characterized by: the titanium modified material of claim 8 is selected as the material.
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