A photothermal-activated carbon monoxide-releasing antibacterial coating on a titanium substrate, its preparation method and application

Abstract

The present invention discloses a photothermal-activated carbon monoxide-releasing antibacterial coating on a titanium substrate, its preparation method and application, including: 1) performing surface activation treatment on a pure titanium sheet under oxygen; 2) preparing a levodopa Tris-HCL solution, and then adding an acetone solution of pentaerythritol tetra(3-mercaptopropionate) thereto, and mixing evenly to obtain a reaction solution; 3) suspending and immersing the titanium sheet treated in step 1) in the reaction solution prepared in step 2), after magnetic stirring treatment, immersing the titanium sheet in an acetone solution of dodecacarbonyltriiron, performing deposition treatment in a nitrogen atmosphere in the dark, and then performing cleaning and drying treatments to obtain a photothermal-activated carbon monoxide-releasing antibacterial coating on the titanium substrate. The antibacterial coating prepared by the present invention can respond to near-infrared laser to release carbon monoxide from the implant abutment, so as to reduce bacterial adhesion and plaque formation, promote the formation of a good biological seal at the soft tissue bonding site, thereby reducing the occurrence of peri-implantitis and lowering the implant failure rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of dental implant materials, and particularly relates to a photothermally activated carbon monoxide-releasing antibacterial coating on the surface of a titanium substrate, and a preparation method and application thereof. Background Art

[0002] Oral implantation has become the preferred treatment method for tooth loss repair. However, implant treatment may also fail due to some complications, such as peri-implant mucositis, peri-implantitis and other peri-implant infections, which seriously threaten the quality of life of patients. Implants need to serve in the oral cavity for a long time, and the existence of complex oral microbiota poses a huge challenge to implants. Once bacteria start to colonize on the surface of implants, it will form a biofilm that is difficult to remove.

[0003] In recent years, researchers in multiple fields have cooperated with each other to construct antibacterial coatings on the surface of titanium implants to endow the implant surface with antibacterial properties. There is currently no unified classification standard for antibacterial coatings on the surface of titanium implants. According to different antibacterial mechanisms of the coatings, they can be divided into: antibacterial adhesion coatings, contact sterilization coatings, release sterilization coatings, etc. Anti-adhesion coatings and contact sterilization coatings belong to passive coatings and cannot actively kill bacteria in the surrounding environment of implants. Therefore, the antibacterial effect still needs to be improved. Release sterilization coatings can actively release sterilizing substances to kill bacteria on the surface and around implants. However, such coatings often lack controllability and face the problem of burst release of sterilizing drugs, and cannot achieve controllable drug delivery antibacterial function.

[0004] In addition, compared with natural teeth, the connective tissue healing around implants is similar to scar tissue, which has fewer blood vessels and cells, lacks structures such as hemidesmosomes that produce adhesion with natural teeth, and only has a small amount of collagen fibers parallel to the long axis of the implant, resulting in a weak ability to resist bacterial invasion after the formation of soft tissue closure, and then complications such as inflammation occur; especially when implanting under adverse microecological environment conditions such as periodontal disease, maxillofacial defect and alveolar socket infection, it is easier to produce peri-implant infection, and once it occurs, it is difficult to reverse, affecting the success rate of implants. Summary of the Invention

[0005] In order to overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a photothermally activated carbon monoxide-releasing antibacterial coating on the surface of a titanium substrate, and a preparation method and application thereof, so as to solve the technical problem that the coating on the surface of the existing implant abutment has poor ability to resist bacterial invasion and is prone to inflammation, thus affecting the success rate of implants.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A preparation method of a photothermally activated carbon monoxide-releasing antibacterial coating on the surface of a titanium substrate disclosed by the present invention includes the following steps:

[0008] 1) Activate the surface of the pure titanium sheet in an oxygen atmosphere;

[0009] 2) Prepare a levodopa Tris-HCL solution, and then add an acetone solution of pentaerythritol tetraallyl (3-mercaptopropionic acid) to it, and mix well to obtain a reaction solution;

[0010] 3) Suspend and immerse the titanium sheet treated in step 1) in the reaction solution prepared in step 2), perform magnetic stirring for 24 - 48 h, then immerse the titanium sheet in an acetone solution of dodecacarbonyltriiron, and perform light-shielded deposition treatment for 8 - 14 h in a nitrogen atmosphere, and then perform cleaning and drying treatments to obtain a photothermal activation carbon monoxide-releasing antibacterial coating on the surface of the titanium substrate.

[0011] Preferably, in step 1), place the pure titanium sheet in a low-temperature and low-pressure plasma spraying machine, and the working parameters are: oxygen flow rate is 80 mL / min, voltage is 220 V, current is 0.6 A, and treatment time is 300 s.

[0012] Preferably, in step 2), the pH value of the levodopa Tris-HCL solution is 8.0 - 9.0, and the concentration is 4.2 mM.

[0013] Further preferably, the pH value of the levodopa Tris-HCL solution is preferably 8.5.

[0014] Preferably, in step 2), the mass concentration of the acetone solution of pentaerythritol tetraallyl (3-mercaptopropionic acid) is 1 mg / mL, and the volume fraction of pentaerythritol tetraallyl (3-mercaptopropionic acid) in the acetone solution of pentaerythritol tetraallyl (3-mercaptopropionic acid) is 3 - 5%.

[0015] Preferably, the volume fraction of pentaerythritol tetraallyl (3-mercaptopropionic acid) in the acetone solution of pentaerythritol tetraallyl (3-mercaptopropionic acid) is 3.85%.

[0016] Preferably, in step 2), the molar ratio of levodopa to pentaerythritol tetraallyl (3-mercaptopropionic acid) in the reaction solution is 4:1.

[0017] Preferably, in step 3), the mass concentration of the acetone solution of dodecacarbonyltriiron is 4 - 5 mg / mL.

[0018] Further preferably, in step 3), the mass concentration of the acetone solution of dodecacarbonyltriiron is 5 mg / mL.

[0019] Preferably, in step 3), the cleaning is to alternately rinse the surface of the titanium sheet with absolute ethanol and ultrapure water to remove unbound substances, and the drying is to blow dry with nitrogen.

[0020] Preferably, before use, the pure titanium sheet is polished step by step with sandpaper on its surface, and then ultrasonic cleaned successively with acetone, absolute ethanol, and deionized water, and dried for standby after high-pressure sterilization and disinfection.

[0021] The present invention also discloses a photothermal-activated carbon monoxide-releasing antibacterial coating on the surface of a titanium substrate prepared by the above preparation method. This antibacterial coating can play an antibacterial role by synergistically combining the photothermal effect and releasing CO gas molecules in response to near-infrared laser.

[0022] The present invention also discloses the application of the above-mentioned photothermal-activated carbon monoxide-releasing antibacterial coating on the surface of a titanium substrate in the preparation of dental implant abutments.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The preparation method of the photothermal-activated carbon monoxide-releasing antibacterial coating on the surface of a titanium substrate disclosed in the present invention first performs surface activation treatment on the pure titanium sheet in an oxygen atmosphere, so that the titanium surface has abundant hydroxyl groups, which is beneficial for the subsequent deposition of levodopa. In the present invention, levodopa is selected, which has better biocompatibility than commonly used dopamine, and can enhance the hydrophilicity of the titanium surface at the same time. At the same time, the cross-linking agent pentaerythritol tetra(3-mercaptopropionate) used in the present invention has abundant mercapto groups. On the one hand, it can be linked to the benzene ring of levodopa through Michael addition reaction, and on the other hand, it can be coupled with the carbon monoxide donor dodecacarbonyltriiron. The antibacterial coating prepared by the present invention can be photothermally activated to release carbon monoxide on the surface of the part where the abutment is combined with soft tissue, and the implant abutment that can release carbon monoxide in response to near-infrared laser can reduce the adhesion of bacteria and the formation of dental plaque, promote the formation of a good biological seal at the soft tissue combination site, thereby reducing the occurrence of peri-implantitis and reducing the implant failure rate. Therefore, it can effectively solve the technical problem that the surface coating of the existing implant abutment has poor ability to resist bacterial invasion and is prone to cause inflammation.

[0025] Furthermore, the present invention uses a low-temperature and low-pressure plasma spraying machine (working parameters: oxygen flow rate is 80 mL / min, voltage is 220 V, current is 0.6 A, and treatment time is 300 s) to activate the titanium sheet, so that the titanium surface has abundant hydroxyl groups.

[0026] Furthermore, the present invention studied the influence of the deposition time on the coating thickness and found that before 48 h, the thickness increased with time, while at 72 h, the thickness decreased significantly. Therefore, the preferred deposition time of levodopa is 48 h.

[0027] Furthermore, the present invention studied the effect of the feeding ratio of levodopa to crosslinking agent on the hydrophilicity of the coating, and found that the hydrophilicity was excellent when the feeding ratio was 1:1 or 4:1, while the hydrophilicity decreased significantly when the feeding ratio was 8:1 or 16:1. Considering that one molecule of crosslinking agent contains four molecules of thiol groups that can bind, we finally selected the feeding ratio of 4:1.

[0028] Furthermore, the present invention studied the effect of the concentration of iron dodecacarbonyl on biocompatibility, and found that the biocompatibility decreased when the concentration exceeded 5 mg / mL. Therefore, the concentration was selected as 5 mg / mL.

[0029] Furthermore, it can synergistically exert the antibacterial effect by combining the photothermal effect and the release of CO gas molecules in response to near-infrared laser, and can effectively kill Staphylococcus aureus and Porphyromonas gingivalis around the implant, achieving the purpose of preventing peri-implantitis. Description of the Drawings

[0030] Figure 1 SEM images of each group of samples; among them, (a) is Ti; (b) is Ti-DOPA; (c) is Ti-DOPA-CO;

[0031] Figure 2 AFM observation results of each group of samples;

[0032] Figure 3 Hydrophilic angle measurement results of the surfaces of each group of samples; among them, (a) is Ti; (b) is Ti-DOPA; (c) is Ti-DOPA-CO;

[0033] Figure 4 XPS characterization results of the coating;

[0034] Figure 5 Photothermal performance test results of the coating;

[0035] Figure 6 Pictures taken by the thermal imager;

[0036] Figure 7 Photothermal response release test data of the coating;

[0037] Figure 8 Fluorescence intensity data of the near-infrared laser irradiation test;

[0038] Figure 9 Bacterial spotting results of the antibacterial performance of the coating against Staphylococcus aureus

[0039] Figure 10 Antibacterial rate data of the coating against Staphylococcus aureus

[0040] Figure 11 Bacterial spotting results of the antibacterial performance of the coating against Porphyromonas gingivalis

[0041] Figure 12 Data on the antibacterial rate of the coating against Porphyromonas gingivalis

[0042] Figure 13 Results of Live / Dead staining after co - culturing the sample and bacteria;

[0043] Figure 14 SEM photos of the morphology of two kinds of bacteria on different sample surfaces. Detailed implementation manners

[0044] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0045] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above - mentioned drawings are used to distinguish similar objects, and do not necessarily need to describe a specific order or sequence. It should be understood that such used data can be interchanged under appropriate circumstances so that the embodiments of the present invention described here can be implemented in an order different from those illustrated or described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non - exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0046] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0047] 1. Preparation of the titanium surface coating

[0048] a) Select commercially pure titanium sheets, polish their surfaces step - by - step with 400 - 1200 - mesh silicon carbide sandpaper, ultrasonically clean them with acetone, absolute ethanol, and deionized water for 10 minutes in sequence, and perform high - pressure sterilization and disinfection before use, and set aside after drying.

[0049] b) Place the pure titanium sheets in a low - temperature and low - pressure plasma spraying machine to perform surface activation in an oxygen atmosphere. The working parameters are: oxygen flow rate is 80 mL / min, voltage is 220 V, current is 0.6 A, and the treatment time is 300 s.

[0050] c) Prepare a 4.2 mM levodopa Tris-HCl solution (pH = 8.5, 10 mM), stir magnetically until completely dissolved, add an acetone solution of 1 mg / mL pentaerythritol tetra(3-mercaptopropionate) (PETMP) such that the final volume fraction of PETMP is 3.85%, and the molar ratio of levodopa to PETMP is 4:1. Suspend and immerse the titanium sheet in the above reaction solution and stir magnetically for 48 h. Subsequently, immerse the titanium sheet in an acetone solution of 5 mg / mL dodecacarbonyltriiron and deposit it for 12 h in a nitrogen atmosphere protected from light. Rinse the titanium surface 3 times alternately with absolute ethanol and Milli-Q to remove unbound substances, then dry it with nitrogen and store it sealed for later use.

[0051] 2. Characterization of the titanium surface coating

[0052] Characterize the morphology of the titanium surface coating using a field emission scanning electron microscope (FESEM). Cut the titanium sheet into a size of 1 * 1 cm, fix it to the SEM sample stage with conductive glue, spray gold (10 mA for 300 s) and then observe and scan. The working voltage is 3.0 kV. The results are shown in Figure 1 , which are the SEM images of each group of samples. It can be seen that the tiny grooves on the surface of pure titanium after silicon carbide polishing disappear after depositing the DOPA coating, forming a uniform coating; when the CO donor coating is deposited, a relatively rough and dense coating can be seen on the titanium surface.

[0053] Further characterize the surface morphology and roughness of the coating using an atomic force microscope (AFM). Use a silicon CONT probe to record the AFM image (resonance frequency: 13 kHz, force parameter: 0.2 N / m). The AFM observation results are shown in Figure 2 , and it can be seen that the polished titanium sheet is relatively smooth with a low roughness, and the Ra value is ~17.07 nm. After depositing DOPA and the CO donor, the roughness of the titanium surface gradually increases, and the Ra values are ~20.5 nm and 27.7 nm respectively.

[0054] Measure the contact angle of the coating surface using the sessile drop mode of a contact angle measuring instrument to characterize the hydrophilic-hydrophobic degree of the coating. The results are shown in Figure 3 , and it can be seen that Figure 3 in (a) shows that the maximum hydrophilic angle of the pure titanium surface is 106.8 ± 2.48°, Figure 3 in (b) shows that when the DOPA coating is deposited, the surface hydrophilic angle decreases to 22.47 ± 3.03°, indicating that the deposition of the DOPA coating significantly increases the surface hydrophilicity. Figure 3Figure (c) shows that after the deposition of the CO donor, the hydrophilic angle of the sample increased to 41.47±6.60°. Although the deposition of the CO donor increased the surface hydrophobicity to some extent, the hydrophilic degree of the surface was still significantly enhanced compared to the pure titanium surface.

[0055] X-ray photoelectron spectroscopy (XPS) was used to characterize the changes in the elements contained before and after the construction of the coating. The results are shown in Figure 4 , it can be seen that in addition to C and O elements, the pure titanium surface mainly contains Ti elements. After the deposition of DOPA, characteristic N elements and S elements characteristic of the crosslinker PETMP were added to the titanium surface, proving that DOPA and the crosslinker were successfully deposited on the titanium surface. After the deposition of the CO donor, characteristic Fe elements appeared on the titanium surface, and at the same time, the Ti element peak decreased significantly, which is because the coating covered the titanium surface, proving the successful preparation of the Ti-DOPA-CO coating.

[0056] 3. Characterization of the in vitro photothermal performance of the coating

[0057] To screen for the appropriate near-infrared laser power, a 808 nm near-infrared laser was used to irradiate the titanium surface coating at different powers (0.4, 0.6, 0.8, 1.0, 1.2 W / cm 2 ), and the temperature change of the coating in PBS was measured. The temperature value was recorded every 50 s, and a thermal imaging map was taken using a photothermal imager every 100 s. The results are shown in Figure 5 . As shown in the photothermal performance test results of the coating, too high temperature will cause damage to the cells around the titanium surface. Therefore, in order to screen for the appropriate photothermal temperature, near-infrared lasers with different powers were used to test the coating surface. The results showed that when the coating surface was irradiated with lasers with different powers (0.4, 0.6, 0.8, 1.0, 1.2 W / cm 2 ) for 10 minutes, the temperatures of the samples in PBS reached 26.8, 32.0, 37.0, 42.5, and 46.3 °C respectively. And the temperature that half of human cells can tolerate is no more than 43 °C. Therefore, 1.0 W / cm 2 was selected as the power of the near-infrared laser used in the subsequent experiments. The pictures taken using the thermal imager every 100 seconds at this power are shown in Figure 6 .

[0058] 4. In vitro photothermal response carbon monoxide release test of the coating

[0059] The in vitro carbon monoxide release of the coating was characterized by the FL-CO-1 fluorescence probe method. First, a fluorescence probe solution system was prepared: a mixed solution of FL-CO-1 and PbCl2 with a concentration of 5 μM each was prepared in a PBS buffer solution (10 mM, pH 7.4, containing 0.5% by volume of DMSO). The experiment was divided into two groups: a non-irradiation group and an irradiation group. The titanium sheet with the coating was placed in a 24-well plate, and 1 mL of the fluorescence probe solution was added. The non-irradiation group was not irradiated, and the irradiation group was irradiated with an 808 nm near-infrared laser (1.0 W / cm 2 ) every 10 minutes, the probe solution was transferred to a quartz cuvette and the fluorescence intensity was measured using a fluorescence spectrometer (λ ex = 485 nm, λ em = 500 - 650 nm) to plot the fluorescence spectrum. To further test the photothermal response carbon monoxide release performance of the coating, the titanium sheet with the coating was also placed in 1 mL of the fluorescence probe solution and irradiated with an 808 nm near-infrared laser for 5 minutes. The fluorescence intensity was measured as above, and then the laser was turned off for 5 minutes and the fluorescence intensity was measured again. This was one cycle, and the measurement was repeated three cycles. The fluorescence intensity at a wavelength of 520 nm was taken as the ordinate and the time as the abscissa to plot the curve.

[0060] The results are shown in Figure 7 . Under the condition of no irradiation, the fluorescence intensity only increased slightly within 0 - 60 minutes. However, when irradiated with the near-infrared laser, the fluorescence intensity of the probe solution system increased significantly. This indicates that under non-irradiated conditions, the coating hardly releases CO molecules. Only when the near-infrared laser irradiates the coating surface and the temperature rises, the thermoresponsive CO donor will release CO molecules. Subsequently, we further tested the photothermal response release performance of the coating. We irradiated the coating with the near-infrared laser for 5 minutes and then turned it off for 5 minutes as one cycle, and tested the response release of the coating under 3 cycles. As Figure 8 shows, within 3 cycles, when irradiated with the near-infrared laser for 5 minutes, the fluorescence intensity of the probe solution system increased significantly. When the laser was turned off for 5 minutes, the fluorescence intensity of the probe solution system did not change significantly, which further proved that the coating has the performance of releasing CO molecules in response to the near-infrared laser.

[0061] 5. In vitro antibacterial experiment

[0062] 5.1 Antibacterial experiment by plate coating method

[0063] The aerobic Staphylococcus aureus (S. aureus, ATCC 25923) and anaerobic Porphyromonas gingivalis (P. gingivalis, ATCC 33277), which are closely related to peri-implant infection, were used as representative bacteria of Gram-positive and Gram-negative bacteria respectively for this part of the experiment. Staphylococcus aureus was cultured using LB medium, and Porphyromonas gingivalis was cultured using BHI medium (containing 10% defatted sheep blood and 1% hemin-vitamin K) under anaerobic conditions. For Staphylococcus aureus, the samples were immersed in 1 mL of a bacterial solution with a concentration of 10 7 in a 24-well plate and co-incubated at 37 °C for 12 h. Subsequently, they were irradiated with an 808 nm near-infrared laser (1.0 W / cm 2 ) for 10 min, while the control group was not irradiated. The samples of each group were taken out and gently rinsed 3 times with sterile PBS to remove non-adherent bacteria. Then the samples were placed in a centrifuge tube containing 500 μL of sterile PBS and sonicated for 5 minutes to resuspend the bacteria adhered to the sample surface in PBS. 100 μL of the bacterial suspension was taken and serially diluted 10-fold in a sterile 96-well plate. 10 μL of the finally diluted suspension was inoculated onto LB agar medium for colony counting, and another 100 μL of the bacterial suspension diluted 1000-fold was evenly spread onto LB agar medium for qualitative observation of the number of surviving colonies. For Porphyromonas gingivalis, the samples were immersed in 1 mL of a bacterial solution with a concentration of 10 7 in a 24-well plate and co-incubated at 37 °C under anaerobic conditions for 48 h. Subsequently, they were irradiated with an 808 nm near-infrared laser (1.0 W / cm 2 ) for 10 min, while the control group was not irradiated. The samples of each group were taken out and gently rinsed 3 times with sterile PBS to remove non-adherent bacteria. Then the samples were placed in a centrifuge tube containing 500 μL of sterile PBS and sonicated for 5 minutes to resuspend the bacteria adhered to the sample surface in PBS. 100 μL of the bacterial suspension was taken and serially diluted 10-fold in a sterile 96-well plate. 10 μL of the finally diluted suspension was inoculated onto LB agar medium for colony counting, and another 100 μL of the bacterial suspension diluted 100-fold was evenly spread onto BHI agar medium for qualitative observation of the number of surviving colonies.

[0064] 5.2 Observation of bacterial morphology

[0065] 1 mL of a bacterial solution with a concentration of 10 6 of Staphylococcus aureus or Porphyromonas gingivalis was co-incubated with the samples of each group at 37 °C for 12 h and 48 h respectively. The irradiated groups were irradiated with an 808 nm near-infrared laser (1.0 W / cm 2)Irradiate for 10 min, and the non-irradiated group is not treated. Subsequently, the bacterial solution is aspirated, gently rinsed 3 times with PBS, and fixed with 4% paraformaldehyde at 4 °C for 2 h. After rinsing 3 times with PBS, gradient ethanol dehydration (20%, 40%, 60%, 80%, 90%, 100%) is carried out. The first three concentrations are each for 15 min, and the last three concentrations are each for 30 min. Finally, the samples are air-dried and fixed to the sample stage with conductive glue, and after sputtering with gold (10 mA, 300 s), FESEM observation is carried out.

[0066] As Figure 9 and Figure 10 shown, under the condition of NIR-, the survival rates of S. aureus in the Ti-DOPA group and the Ti-DOPA-CO group are 85.0 ± 6.6% and 81.7 ± 6.5% respectively, which may be due to the enhanced hydrophilicity of the titanium surface after the deposition of the coating, thus inhibiting the surface adhesion of bacteria. When under the condition of NIR+, the survival rates of S. aureus in the Ti-DOPA group and the Ti-DOPA-CO group are reduced to 79.7 ± 5.9% and 31.3 ± 3.5% respectively, indicating that the temperature increase caused by simple near-infrared laser irradiation has no obvious effect on S. aureus. However, when the temperature increase caused by the coating in response to near-infrared laser releases CO, the survival rate of S. aureus significantly decreases. This result confirms that the Ti-DOPA-CO coating can jointly exert antibacterial efficacy by synergizing the photothermal effect and CO release. As Figure 11 and Figure 12 shown, for P. gingivalis, when NIR- is applied, the survival rates of P. gingivalis in the Ti-DOPA group and the Ti-DOPA-CO group are 85.3 ± 4.2% and 81.3 ± 4.5% respectively. When under the condition of NIR+, the survival rates of P. gingivalis in the two groups of samples are reduced to 77.4 ± 6.0% and 25.5 ± 4.4% respectively, indicating that for anaerobic P. gingivalis, the Ti-DOPA-CO coating can also jointly exert antibacterial efficacy by synergizing the photothermal effect and CO release.

[0067] 5.3 Bacterial Live / Dead staining characterization

[0068] Prepare the Live / Dead reagent: Dissolve the contents of one pipette of component A (containing orange-yellow solid) and one pipette of component B (containing red solid) in a centrifuge tube containing 5 mL of sterile water to obtain a 2X stock solution of the staining reagent mixture. Mix the 2X stock solution sample with an equal volume of the bacterial suspension. The final concentration of each dye is 6 μM SYTO 9 and 30 μM PI. Place the samples of each group flat in a 24-well plate, and add 1 mL of 10 7Bacterial suspension, incubation time and incubation conditions were the same as in 5.1. After reaching the incubation time, the samples were gently rinsed with sterile PBS to remove non - adherent bacteria. Subsequently, the samples were placed in a centrifuge tube containing 500 μL of sterile PBS, sonicated for 5 min to resuspend the bacteria in PBS, and then centrifuged. Add 30 μL for resuspension, add 30 μL of the prepared mixed staining solution under light - avoiding conditions, and incubate in a bacterial incubator for 30 min. Finally, take 10 μL of the bacterial suspension and place it in a 35 - mm confocal culture dish, cover it with a cover glass, and observe and take pictures under a confocal microscope.

[0069] The results are shown in Figure 13 , it can be seen that under the condition of NIR -, the antibacterial ability of the three groups of samples against the two kinds of bacteria is weak, showing a large amount of green fluorescence; while under the condition of NIR +, only very weak antibacterial ability is shown in the Ti and Ti - DOPA groups, indicating that when no CO donor is deposited, the temperature change caused by simple near - infrared laser is not enough to show the killing ability against the two kinds of bacteria; while the antibacterial ability of the Ti - DOPA - CO group is significantly enhanced under the condition of NIR +, and the staining result shows a large amount of red fluorescence, further proving that the Ti - DOPA - CO coating can jointly exert the antibacterial performance against S. aureus and P. gingivalis by synergistic photothermal effect and CO release. As Figure 14 shown, under NIR -, the two kinds of bacteria show a smooth and plump morphology on the surfaces of the three groups of samples. Under NIR +, the morphology of the two kinds of bacteria on the surfaces of the Ti and Ti - DOPA group samples does not change significantly. However, on the surface of the Ti - DOPA - CO group samples, the morphology of the two kinds of bacteria shows obvious shrinkage and cell membrane rupture, which indicates that the Ti - DOPA - CO coating can affect the morphology of bacteria adhering to its surface under the irradiation of near - infrared laser. Therefore, the synergistic photothermal effect and CO release of the Ti - DOPA - CO coating to jointly exert antibacterial efficacy may be caused by damaging the bacterial cell membrane.

[0070] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A method for preparing an antibacterial coating on a titanium substrate surface by photothermal activation of carbon monoxide release, characterized in that: The following steps are involved: 1) Surface activation treatment of pure titanium sheet in oxygen atmosphere; 2) preparing a 4.2 mM levodopa solution in Tris-HCl, then adding a solution of pentaerythritol tetraenyl (3-mercaptopropionic acid) in acetone and mixing to prepare a reaction solution; The mass concentration of the acetone solution of pentaerythritol tetraenyl (3-mercaptopropionic acid) is 1 mg / mL, the volume fraction of pentaerythritol tetraenyl (3-mercaptopropionic acid) in the acetone solution of pentaerythritol tetraenyl (3-mercaptopropionic acid) is 3-5%, and the molar ratio of levodopa to pentaerythritol tetraenyl (3-mercaptopropionic acid) in the reaction solution is 4:1; 3) Suspending and immersing the titanium sheet treated in step 1) in the reaction solution prepared in step 2) and magnetically stirring the titanium sheet for 24 to 48 hours, then immersing the titanium sheet in an acetone solution of triiron dodecacarbonyl with a mass concentration of 4 to 5 mg / mL and depositing the titanium sheet in a nitrogen atmosphere in the dark for 8 to 14 hours. The titanium sheet is then cleaned and dried to obtain a photothermally activated carbon monoxide-releasing antibacterial coating on the titanium substrate surface.

2. The method for preparing a photothermally activated carbon monoxide-releasing antibacterial coating on a titanium substrate according to claim 1, wherein in step 1), a pure titanium sheet is placed in a low-temperature, low-pressure plasma sputtering machine with the following operating parameters: an oxygen flow rate of 80 mL / min, a voltage of 220 V, a current of 0.6 A, and a treatment time of 300 s.

3. The method for preparing a photothermally activated carbon monoxide-releasing antibacterial coating on a titanium substrate according to claim 1, wherein in step 2), the pH value of the levodopa Tris-HCl solution is 8.0-9.

0.

4. The method for preparing a photothermally activated carbon monoxide-releasing antibacterial coating on a titanium substrate according to claim 1, wherein in step 3), the cleaning step is to alternately rinse the titanium sheet surface with anhydrous ethanol and ultrapure water to remove unbound matter, and the drying step is to blow dry with nitrogen.

5. The method for preparing a photothermal activated carbon monoxide-releasing antibacterial coating on a titanium substrate according to any one of claims 1 to 4, characterized in that: Before use, the surface of the pure titanium sheet was polished step by step with sandpaper, and then ultrasonically cleaned with acetone, anhydrous ethanol, and deionized water in sequence, sterilized by high pressure, and dried for use.

6. The antibacterial coating on the titanium substrate surface prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The antibacterial coating can synergize the photothermal effect and release CO gas molecules in response to near-infrared laser to exert antibacterial effect.

7. Use of the photothermally activated carbon monoxide-releasing antibacterial coating on the titanium substrate surface as described in claim 6 in the preparation of a dental implant abutment.

Images