CN115957385B - 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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- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 64
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- 238000000576 coating method Methods 0.000 title claims abstract description 42
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- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims abstract description 106
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Classifications
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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 coupled antibacterial peptide is constructed through the combination of the titanium dioxide nanotip (TNS), the Metal Phenol Network (MPN) and the antibacterial peptide (AMP), the combination of physical puncture/photo-thermal/chemical antibacterial capability is realized, the bone conductivity of the coating is enhanced through promoting the deposition of hydroxyapatite, and the multifunctional titanium implant coating has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of medicinal chemistry and materials, and particularly relates to a preparation method and application of a multifunctional coating of metal polyphenol network coupled antibacterial peptide, namely a multifunctional titanium implant coating is prepared on the surface of a titanium dioxide nanometer tip structure through the metal polyphenol network coupled antibacterial peptide.
Background
With the acceleration of population aging in China, the use of hard tissue implantation medical devices represented by bone/oral implants has been continuously increasing in recent years. Titanium (Ti) and its alloys are widely used in hard tissue implant materials due to their excellent mechanical properties, good corrosion resistance and biocompatibility, accounting for 70-80% of orthopedic implants. However, the inherent biological inertia of titanium materials makes it lacking antibacterial properties and osteoconductivity, leading to an increased risk of bacterial infection and aseptic loosening, and ultimately to failure of the procedure. Therefore, the titanium implant has good antibacterial property and excellent bone conduction property through surface modification, and has good clinical application prospect. At present, the most widely used antibacterial means clinically for orthopedic implant infection is still antibiotic treatment, but abuse of antibiotics has induced a large number of bacteria to develop drug resistance, and even can generate a non-drug treatable environment for infection. Therefore, there is increasing interest in finding antibacterial methods that are not easily induced to drug resistance and have good biocompatibility. Phototherapy can induce local high temperature by non-invasive light source irradiation, and destroy the structure of biological membrane to inactivate active matrix (such as nucleic acid and protein), and is not easy to generate drug resistance; however, since phototherapy alone requires the introduction of high power laser density and long irradiation time, it is inevitable to have side effects on surrounding healthy tissues.
For the above reasons, the research team of the present invention has considered it necessary to explore a method to solve the antibacterial problem of the implant body and after implantation in the body.
Disclosure of Invention
The invention aims to solve the defects existing in the prior art and provides a preparation method and application of a multifunctional coating of metal polyphenol network coupled antibacterial peptide.
The preparation method of the multifunctional coating of the metal polyphenol network coupled antibacterial peptide is characterized by comprising the following steps of:
1) Preparing a titanium dioxide nanotip array (TNS array) on the surface of the titanium material in situ by adopting a hydrothermal method to obtain the titanium material with the titanium dioxide nanotip array on the surface; preparing a nano-tip structure capable of physically penetrating bacteria and converting 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 a physiological pH (such as: 7.4 A) is provided;
2.3 In order to make the reaction and assembly more sufficient, the metal polyphenol nano-film formed by plant polyphenol and metal ions is quickly assembled on the titanium dioxide nano-tip array through vortex of the mixed solution regulated in the step 2.2), so as to obtain the titanium TNS-MPN 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 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, cleaning and drying the titanium material, and obtaining a multifunctional coating TNS-MPN-AMP of the metal polyphenol network coupled antibacterial peptide on the surface of the titanium material, wherein the coating has the antibacterial function of promoting bone function brought by the physical puncture/photo-thermal/chemical ternary synergy.
Further, the step 1) specifically comprises:
1.1 Placing titanium material at the bottom of a polytetrafluoroethylene-lined stainless steel autoclave reactor, 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 150-300 ℃ for reaction for 3-5h;
1.3 After the reaction is completed, 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 nanotip array on the surface.
Further, in step 2), the plant polyphenol in the plant polyphenol solution is catechol, pyrogallol, gallic acid or tannic acid;
the metal ion solution is FeCl 3 solution, znSO 4 solution, cuSO 4 solution or AlCl 3 solution;
In the step 3), the broad-spectrum antibacterial peptide is antibacterial peptide LL-37, xenopus, nisin or epsilon-polylysine;
When the plant polyphenol is catechol, the following components are added according to the 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 pyrogallol, the following components are added according to the parts by weight:
jiaosuan to 4 parts of metal ions, 2 to 5 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution, and 100 to 300 parts of broad-spectrum antibacterial peptide;
when the plant polyphenol is gallic acid, the following components are added according to the 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 tannic acid, the following components are added according to the parts by weight:
2 to 5 parts of tannic acid, 2 to 5 parts of metal ions and 100 to 300 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution.
In order to make the volume of the solution for immersing the titanium material appropriate, 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 standard, and then the corresponding volumes are measured according to the parts by weight:
When the plant polyphenol is catechol, the concentrations are as follows:
The concentration of catechol in solution of plant polyphenol is 0.4-0.7 mg mL -1, the concentration of metal ions in solution of metal ions is 0.2-0.5 mg mL -1, and the concentration of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution is 0.01-0.03 g mL -1;
when the plant polyphenol is pyrogallol, the concentrations are as follows:
the concentration of pyrogallol in the plant polyphenol solution is 0.1-0.4 mg mL -1, the concentration of metal ions in the metal ion solution is 0.2-0.5 mg mL -1, and the concentration of broad-spectrum antibacterial peptide dissolved in 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 gallic acid in the plant polyphenol solution is 0.1-0.4 mg mL -1, the concentration of metal ions in the metal ion solution is 0.2-0.5 mg mL -1, and the concentration of 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 concentration is as follows:
The concentration of tannic acid in the plant polyphenol solution is 0.2-0.5 mg mL -1, the concentration of metal ions in the metal ion solution is 0.2-0.5 mg mL -1, and the concentration of 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 TNS, the heating rate is set to 10 ℃/min;
In the step 2.2), naOH solution is utilized to adjust the pH value of the mixed solution in the step 2.1);
In the step 2.3), the vortex duration is 30-90s, and in order to improve the reaction efficiency and ensure full reaction and complete assembly, 60s is preferable;
In step 3.1), the reaction is carried out at room temperature for 6-12h.
Further, the method further comprises the pretreatment of the titanium material before the step 1), specifically:
cutting pure titanium material into required size, sequentially polishing with SiC sand paper with granularity of #400, #800, # 1200- #2000 to specular gloss, flushing abrasive dust with deionized water, sequentially ultrasonic cleaning with acetone, ethanol and water, removing greasy dirt on the surface, and drying for later use.
Meanwhile, the invention also provides a multifunctional coating of the metal polyphenol network coupled antibacterial peptide prepared by the method and application of the multifunctional coating in antibacterial 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 coupled 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 antibacterial bone titanium implant and titanium implant.
The conception and 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 capacity, and intends to construct various micro/nano structures on the surface of titanium, such as: nanotubes, nanotips, nanopores, nanolobes, etc., to enhance the biological activity of the titanium implant. The titanium dioxide nanotip structure not only can simulate natural extracellular matrix, promote fluid flow, promote molecular and cell transportation, excite an osteogenic signal, promote better cell adhesion, migration, proliferation and differentiation, but also can physically destroy bacterial membranes and leak cell contents to kill bacteria, and more importantly, the titanium dioxide nanotip structure can generate good photo-thermal conversion efficiency with the aid of Near Infrared (NIR) light irradiation by improving the photo-capturing performance and anti-reflection efficiency of the titanium surface, so that a research team considers that the construction of the titanium dioxide nanotip structure on the titanium surface can be used for treating implant infection by using non-invasive photo-thermal therapy aiming at an infection site. In addition, the metallic polyphenol network is modified on the surface of titanium, and the metallic polyphenol network is coupled on the surface of the coating through Michael addition/Schiff base reaction between polyphenol and antibacterial peptide (AMP), so that the antibacterial capability and bone bioactivity are endowed to the titanium implant.
In conclusion, the research team of the invention realizes the reduction of the power density and the shortening of the irradiation time of the photothermal therapy by constructing a multi-component mode of the photothermal therapy cooperation, and simultaneously endows the titanium implant material with good antibacterial property, bone promotion performance and good biocompatibility.
The invention has the advantages that:
1. the method does not need to introduce a photo-thermal agent, and builds the micro/nano structure of the titanium dioxide nanotip morphology on the surface of the titanium material in situ. The structure improves the light capturing performance and the anti-reflection efficiency of the titanium surface, so that the titanium surface has good light-heat conversion efficiency under the assistance of Near Infrared (NIR) light irradiation, and is suitable for photothermal therapy treatment.
2. According to the method, a layer of metal polyphenol network MPN is formed through the complexation of plant polyphenol and metal ions, the network has good adhesiveness, can rapidly form a film on TNS, and is low in cost, good in biological safety and short in reaction time.
3. Compared with pure titanium, the TNS-MPN-AMP coating prepared by the invention introduces rich biological active groups such as hydroxyl, carboxyl, amino and the like, can attract more phosphate and carbonate in body fluid to the surface of the coating, promotes in vivo hydroxyapatite (HAp) nucleation, 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 the efficient sterilization effect is realized, wherein the in-vitro antibacterial rate exceeds 99.99%, and the in-vivo antibacterial rate exceeds 95%. Meanwhile, compared with single photothermal therapy, the power excitation density of the light source used can be reduced, the irradiation time is shortened, and side effects on surrounding healthy tissues can be effectively restrained 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 a preparation scheme of TNS-MPN-AMP obtained in example 1 of the present invention;
FIG. 2 is an FE-SEM image of Ti used in example 1 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained;
FIG. 3 is a WCA diagram of Ti, TNS-MPN and TNS-MPN-AMP used in example 2 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained;
FIG. 4 is a graph showing the temperature change of Ti used in example 3 of the present invention, and TNS, TNS-MPN and TNS-MPN-AMP obtained;
FIG. 5 is a photograph of a bactericidal plating of Ti, TNS-MPN and TNS-MPN-AMP obtained for the gram-negative bacteria E.coli and the gram-positive bacteria S.aureus used in example 1 of the present invention;
FIG. 6 is an FE-SEM image of the morphology of MC3T3-E1 osteoblasts on the surface of TNS-MPN-AMP obtained in example 1 of the present invention;
FIG. 7 shows the deposition of the HAp on the surface of the TNS-MPN-AMP obtained in example 6 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the attached drawings and specific examples:
Example 1
A preparation method of a titanium modified material is shown in a preparation flow diagram as shown in figure 1, and comprises the following steps:
1) Cutting pure titanium material into a size of 1X 1cm 2 (other needed sizes can be adopted), sequentially polishing the pure titanium material with SiC sand paper with the granularity of #400, #800, # 1200- #2000 to have mirror luster, washing abrasive dust with deionized water, sequentially carrying out ultrasonic cleaning with acetone, ethanol and water to remove greasy dirt on the surface, and drying for later use.
2) Placing the pretreated titanium material at the bottom of a stainless steel autoclave reactor with a polytetrafluoroethylene lining, wherein 10mL of NaOH solution with the molar concentration of 1.5M is added; then placing the sealed autoclave reactor in an electric furnace at 300 ℃ for reaction for 3 hours;
3) After the reaction is completed, taking out the titanium material, placing the titanium material in a muffle furnace, and annealing for 3 hours at 400 ℃ at a heating rate of 10 ℃/min to obtain the titanium material with the titanium dioxide nanotip array on the surface, and marking the titanium material as a TNS sample;
4) Taking a 50mL centrifuge tube, placing a TNS sample at the bottom of the centrifuge tube, then adding 10mL of tannic acid (0.4 mg mL -1) solution and 10mL of ZnSO 4(0.2mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with a molar concentration of 1M, and swirling for 60 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) Antibacterial peptide LL-37 was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.02g mL -1; TNS-MPN samples were immersed in 10mL of the above solution and placed on a shaker to react at room temperature for 12 hours. Subsequently, the sample was taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
The morphology and structure of the titanium sheet surface were characterized by FE-SEM. The sample prepared in the implementation is firstly stuck on a conductive adhesive tape, then sprayed with metal for 50s to shoot and image, 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 present typical nanotip structures.
Example 2
The difference from example 1 is that:
2) Placing the pretreated titanium material at the bottom of a stainless steel autoclave reactor with a polytetrafluoroethylene lining, wherein 10mL of NaOH solution with the molar concentration of 1M is added; then placing the sealed autoclave reactor in an electric furnace at 150 ℃ for reaction for 5 hours;
3) After the reaction is completed, taking out the titanium material, placing the titanium material in a muffle furnace, annealing for 1 hour at 600 ℃ at a heating rate of 10 ℃/min to obtain the titanium material with the titanium dioxide nanotip array on the surface, and marking the titanium material as a TNS sample;
4) Taking a 50mL centrifuge tube, placing TNS at the bottom of the centrifuge tube, then adding 10mL of tannic acid (0.4 mg mL -1) solution and 10mL of CuSO 4(0.2mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 0.5M, and swirling for 60 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) Antibacterial peptide LL-37 was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.02g mL -1; 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
The course of the contact angle of the coating was characterized using a contact angle meter. And slowly sucking deionized water into the water phase needle tube by adopting a sitting drop method, transferring liquid drops at the port of the needle tube to the surface of a sample to be detected, determining the base line of the liquid drops, completing the transfer of the liquid drops, and reading and recording the measured contact angle. The results of fig. 3 show that: 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 material surface is further increased along with the assembly and deposition of MPN and AMP, but the hydrophilicity of the material surface is still maintained.
Example 3
The difference from example 1 is that:
2) Placing the pretreated titanium material at the bottom of a stainless steel autoclave reactor with a polytetrafluoroethylene lining, wherein 10mL of NaOH solution with the molar concentration of 0.5M is added; then placing the sealed autoclave reactor in an electric furnace at 220 ℃ for reaction for 4 hours;
3) After the reaction is completed, taking out the titanium material, placing the titanium material in a muffle furnace, and annealing for 2 hours at 550 ℃ at a heating rate of 10 ℃/min to obtain the titanium material with the titanium dioxide nanotip array on the surface, and marking the titanium material as a TNS sample;
4) Taking a 50mL centrifuge tube, placing a TNS sample at the bottom of the centrifuge tube, then adding 10mL of catechol (0.4 mg mL -1) solution and 10mL of AlCl 3(0.2mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1.5M, and swirling for 60 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) Antibacterial peptide LL-37 was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.02g mL -1; 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
The photo-thermal heating curve of the material under the irradiation of near infrared light is recorded by using a 808nm near infrared laser and a thermal infrared imager. The results of fig. 4 show: compared with pure titanium, the prepared nano-tip structure has good photo-thermal 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, along with the assembly and deposition of MPN and AMP, the material still maintains good photo-thermal conversion efficiency.
Example 4
The difference from example 1 is that:
4) Taking a 50mL centrifuge tube, placing a TNS sample at the bottom of the centrifuge tube, then adding 10mL of tannic acid (0.4 mg mL -1) solution and 10mL of FeCl 3(0.2mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with a molar concentration of 1M, and swirling for 60 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) Antibacterial peptide epsilon-polylysine was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.02g mL -1; 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
Example 5
The difference from example 1 is that:
4) Taking a 50mL centrifuge tube, placing a TNS sample at the bottom of the centrifuge tube, then adding 10mL of pyrogallol (0.4 mg mL -1) solution and 10mL of FeCl 3(0.2mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 0.5M, and swirling for 60 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) The antibacterial peptide nisin was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.02g mL -1; 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
Example 6
The difference from example 1 is that:
4) Taking a 50mL centrifuge tube, placing TNS at the bottom of the centrifuge tube, then adding 10mL gallic acid (0.4 mg mL -1) solution and 10mL ZnSO 4(0.2mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1M, and swirling for 60 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) Antibacterial peptide epsilon-polylysine was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.02g mL -1; TNS-MPN samples were 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
In order to verify the effect of the multifunctional coating prepared in the invention, the TNS-MPN-AMP coating prepared in the invention has antibacterial property and cell compatibility, and the TNS-MPN-AMP coating prepared in the invention in the example 6 has bone conduction promoting property, and the specific experiment is as follows:
A. the sterilization performance of the material in example 1 was verified by the test:
All prepared samples were sterilized in advance, individual colonies of the gram-negative bacteria E.coli and the gram-positive bacteria S.aureus were selected from the bacterial culture plates and cultured in LB medium for 4-6 hours until the bacterial population grew to mid-log (optical density at 600nm reached 0.5). Then, 1×10 7CFU mL-1 bacteria were dropped onto the surface of the sample prepared in example 1 and incubated in a bacteria incubator at 37 ℃ for 45min, wherein 808nm light was applied to the TNS-MPN-amp+nir group samples for 2min, then 980 μl of sterile PBS was added to the wells, bacteria attached to each sample were ultrasonically separated, and then 100 μl of bacterial suspension in each well containing the sample was spread on a solid LB agar plate with an L-type spreader and incubated overnight at 37 ℃ for photographing. The results of fig. 5 show that: the average antibacterial rate of Ti to staphylococcus aureus and escherichia coli is 0%, TNS is 24.07% and 45.56%, TNS-MPN is 9.64% and 35.03%, TNS-MPN-AMP is 91.79% and 92.80%, and TNS-MPN-AMP+NIR is more than 99.99%. Therefore, the prepared coating has good ternary synergistic anti-infection effect.
B. the cytocompatibility of the materials in example 1 was verified by testing:
MC3T3-E1 cells were cultured with medium containing alpha-MEM, FBS and diabody, after which TNS-MPN-AMP samples were incubated with 8X 10 3 cells. The cells were cultured in a humidified atmosphere of 5% CO 2 at 37℃for 24 hours, then fixed with 4% paraformaldehyde, dehydrated with a gradient ethanol solution, and observed with FE-SEM after metal spraying. As shown in FIG. 6, MC3T3-E1 cells were in spindle-like morphology and in good adhesion. The coating has good cell compatibility, and is beneficial to subsequent proliferation, migration and differentiation.
C. The bone conduction performance of the material of example 6 was verified by testing:
Ti and TNS-MPN-AMP samples were immersed in a Simulated Body Fluid (SBF) at pH 7.4 and maintained at 37 ℃ for 14 days to induce apatite growth, with new SBF being exchanged every other day. As shown in the FE-SEM image of FIG. 7, a hydroxyapatite layer was formed on the surface of TNS-MPN-AMP.
Example 7
The difference from example 1 is that:
4) Taking a 50mL centrifuge tube, placing TNS at the bottom of the centrifuge tube, then adding 10mL of catechol (0.7 mg mL -1) solution and 10mL of ZnSO 4(0.5mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1.5M, and swirling for 90 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) Antibacterial peptide epsilon-polylysine was dissolved in Tris-HCl solution (ph=8.5, 10 mM) to give a concentration of 0.01g mL -1; TNS-MPN samples were 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
Example 8
The difference from example 1 is that:
4) Taking a 50mL centrifuge tube, placing TNS at the bottom of the centrifuge tube, then adding 10mL of pyrogallol (0.1 mg mL -1) solution and 10mL of CuSO 4(0.3mg mL-1 solution, gradually adjusting the pH of the mixed solution to 7.4 by using a NaOH solution with the molar concentration of 1.5M, and swirling for 90 seconds to obtain a titanium material with a titanium dioxide nanotip array assembled with a metal polyphenol nano-film, and marking the titanium material as a TNS-MPN sample;
5) The antibacterial peptide Xenopus laevis was dissolved in Tris-HCl solution (pH=8.5, 10 mM) to give a concentration of 0.03g mL -1; 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 taken out, rinsed with DI water for 1 minute, and dried at room temperature to obtain a titanium material having a multifunctional coating, which was designated as a TNS-MPN-AMP sample.
In conclusion, the multifunctional titanium implant coating of the metal polyphenol network coupled antibacterial peptide is constructed through the combination of the titanium dioxide nanotip (TNS), the Metal Polyphenol Network (MPN) and the antibacterial peptide (AMP), so that the combination of physical puncture/photo-thermal/chemical antibacterial capability is realized, the bone conductivity of the coating is enhanced through promoting the deposition of the hydroxyapatite, and the method has a good application prospect.
While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made without departing from the spirit and scope of the invention.
Claims (10)
1. The preparation method of the multifunctional coating of the metal polyphenol network coupled antibacterial peptide is characterized by comprising the following steps of:
1) Preparing a titanium dioxide nanotip array on the surface of the titanium material in situ by adopting a hydrothermal method to obtain the titanium material with the titanium dioxide nanotip 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) in 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 of step 2.1) to a physiological pH value;
2.3 Assembling a metal polyphenol nano film formed by plant polyphenol and metal ions on the titanium dioxide nano tip array by vortex to obtain a titanium material with the titanium dioxide nano tip array of 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 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 completed, cleaning and drying the titanium material, and obtaining the multifunctional coating of the metal polyphenol network coupled antibacterial peptide on the surface of the titanium material.
2. The preparation method according to claim 1, wherein the step 1) is specifically:
1.1 Placing titanium material at the bottom of a polytetrafluoroethylene-lined stainless steel autoclave reactor, 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 150-300 ℃ for reaction for 3-5h;
1.3 After the reaction is completed, 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 nanotip array on the surface.
3. The preparation method according to claim 1 or 2, characterized in that:
In the step 2), the plant polyphenol in the plant polyphenol solution is catechol, pyrogallol, gallic acid or tannic acid;
the metal ion solution is FeCl 3 solution, znSO 4 solution, cuSO 4 solution or AlCl 3 solution;
In the step 3), the broad-spectrum antibacterial peptide is antibacterial peptide LL-37, xenopus, nisin or epsilon-polylysine;
When the plant polyphenol is catechol, the following components are added according to the 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 pyrogallol, the following components are added according to the parts by weight:
jiaosuan to 4 parts of metal ions, 2 to 5 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution, and 100 to 300 parts of broad-spectrum antibacterial peptide;
when the plant polyphenol is gallic acid, the following components are added according to the 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 tannic acid, the following components are added according to the parts by weight:
2 to 5 parts of tannic acid, 2 to 5 parts of metal ions and 100 to 300 parts of broad-spectrum antibacterial peptide dissolved in Tris-HCl solution.
4. A method of preparation according to claim 3, characterized in that:
In the step 1.3), the heating rate is 10 ℃/min;
In the step 2.2), naOH solution is utilized to adjust the pH value of the mixed solution in the step 2.1);
in the step 2.3), the vortex duration is 30-90s;
In step 3.1), the reaction is carried out at room temperature for 6-12h.
5. The method according to claim 4, further comprising pretreatment of the titanium material prior to step 1), specifically:
cutting pure titanium material into required size, sequentially polishing with SiC sand paper with granularity of #400, #800, # 1200- #2000 to specular gloss, flushing abrasive dust with deionized water, sequentially ultrasonic cleaning with acetone, ethanol and water, removing greasy dirt on the surface, and drying for later use.
6. A multifunctional coating of metal polyphenol network coupled antibacterial peptide is characterized in that: obtained by the method of any one of claims 1 to 5.
7. The use of the multifunctional coating of metal polyphenol network coupled antibacterial peptide of claim 6 for antibacterial promotion of bone modification of titanium materials.
8. A titanium-modified material, characterized in that: a multifunctional coating of metal polyphenol network coupled antibacterial peptide is prepared on the surface of a titanium material according to the method of any one of claims 1-5.
9. Use of the titanium-modified material of claim 8 for the preparation of an antimicrobial osteo-titanium implant.
10. A titanium implant, characterized by: the titanium modified material of claim 8 is used as the material.
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