An antibacterial coating and its application

Abstract

The present invention discloses an antibacterial coating and its application, relating to the technical field of surface modification of dental implants, in particular to an antibacterial coating. The preparation method of the antibacterial coating is as follows: in a Tris solution, under normal temperature and aerobic conditions, a mixture of dissolved catecholamine, bacitracin, and metal ions is reacted on a titanium sheet for 24 hours, and then the surface precipitate is ultrasonically cleaned and dried to obtain the antibacterial coating. The antibacterial coating has good antibacterial properties against Staphylococcus aureus, and the bacteriostatic rate reaches 72%. At the same time, the antibacterial coating is non-toxic to the human body and has good biocompatibility.

Description

Technical Field

[0001] The invention relates to the technical field of dental implant surface modification, and in particular to an antibacterial coating used for implant surface modification. Background Art

[0002] At present, in oral clinical practice, dental implants have become one of the effective ways to repair edentulous defects and are accepted and recognized by more and more patients. However, implant-related infection is still one of the most serious complications after implant surgery. In the worst case, it often leads to loosening and falling off of the implant. Some patients need to undergo a secondary operation, which brings psychological trauma and economic burden to patients. Therefore, clarifying the pathogenesis of peri-implant infection and exploring the prevention methods of peri-implant infection are crucial to reducing and preventing the occurrence of peri-implant infection, thereby improving the success rate of implantation and the service life of implants. Peri-implant infection is essentially an inflammatory lesion that destroys the tissue around the implant. It can be divided into early infection and late infection according to the time of occurrence. Early infection occurs before the implant is combined with the bone, that is, in the initial healing period after implant placement. It is mainly due to bacterial invasion and surgical trauma, which may damage or hinder the combination of the implant and the bone; late infection occurs after the implant is combined with the bone. It is mainly related to peri-implantitis. If the infection is not treated in time, it will cause progressive damage to the formed implant-bone interface. Compared with late infection, early postoperative infection of implants is the main cause of treatment failure. Since the oral environment is a multi-bacterial environment, it is extremely difficult to control the amount of local bacteria during surgery. In severe cases, it will lead to bone dissolution around the implant, which in turn causes the implant to loosen or even fall off. Therefore, antibacterial is one of the important factors in maintaining the long-term stability of the implant.

[0003] The microbiome in the oral cavity may have a substantial impact on the biofilm formation of newly implanted implants. In a comparative study of the initial adhesion bacteria around implants and teeth, Mirjam M. et al. found that Staphylococcus aureus accounted for a higher proportion of bacteria that colonized the implant surface in the early stage. Currently, there is increasing evidence that Staphylococcus aureus may be an important pathogen in some cases of implant periodontitis, and many studies have also reported the association between Staphylococcus aureus, Enterobacter and Candida albicans and peri-implant inflammation.

[0004] Titanium and its alloys are considered to be the best implant materials due to their good biocompatibility, mechanical properties, and excellent corrosion resistance. However, titanium-based materials do not have antibacterial properties. In the early stage of implantation, infections around the implant and poor osseointegration result in the loss of the supporting tissues around the implant, which is often the main cause of treatment failure. Compared with natural teeth, implant dentures have their particularities. The implant cuff, that is, the oral soft tissue surrounding the implant where it penetrates the gingiva, and the junctional epithelium here mainly plays a barrier role, forming a good biological seal around the implant. However, compared with natural teeth, the junctional epithelium attachment at the implant cuff is thinner, so its barrier effect is relatively weak. In addition, the blood vessels in the connective tissue around the implant are fewer than those in natural teeth, and the interface between the implant and the bone lacks the periodontal ligament. When bacteria invade, the inflammatory response in this area will be lower, and the immune defense ability will also be relatively weak. Bacteria will aggregate and multiply in this area, leading to peri-implant infection.

[0005] Antibiotic antibacterial coatings mainly antibacterial through the unique antibacterial methods of various antibiotics, with the advantages of good biocompatibility and targeted antibacterial, while the generation of drug resistance is its biggest drawback. Bacitracin is a widely used metal peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis, with powerful bactericidal activity, mainly targeting Gram-positive bacteria. This antibiotic requires a divalent metal ion (such as Zn) to obtain its biological activity. Despite decades of widespread use, bacterial resistance to bacitracin remains rare. A recently published article showed that the emergence of resistance to antibiotic peptides is lower than that to traditional antibiotics. Therefore, bacitracin can be used as a prototype to better understand the structure and function relationship of antibiotic peptides and as a leading drug for the rational design of antibiotic peptides in the future. In addition, the success of the chemical synthesis of bacitracin has also greatly reduced its use cost. In recent years, several metal peptide families have been designed as model systems to explore the structure and function of metalloproteins. Understanding the structure and function relationship of the antibiotic peptide bacitracin also points out a new direction for the rational design of metal peptides as potential models of metalloproteins. Bacitracin can specifically bind to the precursor of peptidoglycan, the main component of the cell wall, and block the synthesis of the cell wall, but has no obvious effect on the cells of eukaryotic animals. Therefore, it can have bactericidal properties without cytotoxicity, and no drug resistance has been found since it was proposed in the 1980s. It is an ideal antibacterial substance.

[0006] The bacitracin molecule contains a cyclododecapeptide, a special thiazoline ring, a cycloheptapeptide structure, and four D - amino acids. These unusual structural features can protect the peptide from protease degradation. The "tail" of bacitracin bends towards the seven - membered ring, bringing the thiazoline ring, Glu, and His closer, forming a potential metal - binding site in solution. Through the amino group on bacitracin, we found a self - polymerizing substance, norepinephrine (NE), that can covalently bind to it. And through the coordination with Zn, the positive - negative point attraction, and by a one - step blending method, it helps bacitracin to be efficiently loaded on the coating surface.

[0007] Through the analysis of the characteristics of the bacitracin molecule, the present invention finds a coating construction method suitable for its loading and exerting its antibacterial function. Utilizing the charge interaction, covalent interaction between NE and bacitracin, and the coordination interaction between NE and Zn ions, a stable coating is formed by a one - step blending method, taking into account both antibacterial properties and cell compatibility, providing a new strategy for the surface modification of implants with similar structures. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a coating suitable for the loading of bacitracin and exerting its antibacterial function.

[0009] To achieve the above - mentioned purpose, the present invention provides an antibacterial coating formed by the polymerization of catecholamine, bacitracin, and metal ions.

[0010] In a preferred embodiment of the present invention, a method for preparing the antibacterial coating is as follows: in a Tris solution, at room temperature and under aerobic conditions, a mixture of dissolved catecholamine, bacitracin, and metal ions is reacted on a titanium sheet for 24 h, and then the surface precipitate is ultrasonically cleaned and dried to obtain the antibacterial coating.

[0011] Preferably, the pH of the Tris solution is 7.5 - 9.5.

[0012] Preferably, the aerobic condition is exposure to air.

[0013] Preferably, the catecholamine is norepinephrine.

[0014] Preferably, the metal ion is ZnCl2.

[0015] Preferably, the concentration of norepinephrine is 1 - 5 mg / ml, the concentration of bacitracin is 0.12 - 1 mg / ml, and the concentration of ZnCl2 is 0.5 - 2 mg / ml.

[0016] More preferably, the concentration of norepinephrine is 3 mg / ml, the concentration of bacitracin is 0.25 mg / ml, and the concentration of ZnCl2 is 1 mg / ml.

[0017] In another preferred embodiment of the present invention, the antibacterial coating is applied to the surface modification of interventional medical devices.

[0018] Preferably, the interventional medical device is a dental implant.

[0019] The antibacterial coating constructed by the present invention has good antibacterial properties against Staphylococcus aureus, and the antibacterial rate reaches 72%. At the same time, the antibacterial coating has no toxicity to the human body and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows the detection results of the antibacterial properties of the antibacterial coatings prepared by the one-step blending method and the multi-step blending method against Staphylococcus aureus, where Group A is the NE antibacterial coating (NE + rod + Zn) prepared by the multi-step grafting method, Group B is the dopamine (Dopa) antibacterial coating (Dopa + rod + Zn) prepared by the multi-step grafting method, Group C is the NE antibacterial coating (NE@rod@Zn) prepared by the one-step blending method, Group D is the dopamine (Dopa) antibacterial coating (Dopa@rod@Zn) prepared by the one-step blending method, and C is the blank control group;

[0021] Figure 2 shows the detection results of the antibacterial properties of the antibacterial coatings prepared by various processes against Staphylococcus aureus;

[0022] Figure 3 shows the detection results of the antibacterial properties of the NE@rod@Zn antibacterial coatings prepared with different concentrations of bacitracin against Staphylococcus aureus;

[0023] Figure 4 shows the cck-8 proliferation detection results of the NE@rod@Zn antibacterial coatings prepared with different concentrations of bacitracin;

[0024] Figure 5 shows the fluorescence staining results of the proliferation of MC3T3-E1 by the NE@rod@Zn antibacterial coatings prepared with different concentrations of bacitracin. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The following introduces multiple preferred embodiments of the present invention with reference to the accompanying drawings of the specification, making its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0026] Example 1

[0027] The present invention first selects the substrate material. Among many catecholamine materials, norepinephrine is selected as the substrate material, and a coating with antibacterial properties can be prepared. In the present invention, dopamine, a common catecholamine material, is used as a comparative material.

[0028] 1. Coating preparation:

[0029] 1) One-step blending method: Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and aerobic conditions, react the dissolved catecholamine (3 mg / ml), bacitracin (0.25 mg / ml), ZnCl2 (1 mg / ml) with titanium sheets for 24 h, then ultrasonically clean the surface precipitate, dry it, and obtain the antibacterial coating.

[0030] 2) Multi-step grafting method: Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and aerobic conditions, first react the dissolved catecholamine (3 mg / ml) on titanium sheets for 24 h, ultrasonically clean it, and dry it; then under the same pH, temperature and oxygen conditions, react bacitracin (0.25 mg / ml) on titanium sheets for 24 h, ultrasonically clean it, and dry it; finally under the same pH, temperature and oxygen conditions, react ZnCl2 (1 mg / ml) on titanium sheets for 24 h, ultrasonically clean it, and dry it to obtain the antibacterial coating.

[0031] 2. In vitro antibacterial performance evaluation

[0032] The turbidimetry method was used to evaluate the anti-Staphylococcus aureus performance of the prepared catecholamine coatings. Group A was the NE antibacterial coating (NE + bac + Zn) prepared by the multi-step grafting method, Group B was the dopamine (Dopa) antibacterial coating (Dopa + bac + Zn) prepared by the multi-step grafting method, Group C was the NE antibacterial coating (NE@bac@Zn) prepared by the one-step blending method, Group D was the dopamine (Dopa) antibacterial coating (Dopa@bac@Zn) prepared by the one-step blending method, and Group C was the blank control group. As Figure 1 shown, only the coating in Group C showed obvious antibacterial performance against Staphylococcus aureus.

[0033] Example 2

[0034] In order to explore the relationship between the coating loading mechanism and antibacterial performance, the present invention prepared antibacterial coatings with various processes.

[0035] 1. Coating preparation:

[0036] 1) Blank control.

[0037] 2) Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and aerobic conditions, first react the dissolved NE (3 mg / ml) on titanium sheets for 24 h, ultrasonically clean it, and dry it to obtain the NE coating.

[0038] 3) Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first mix the dissolved NE (3 mg / ml) and ZnCl2 (1 mg / ml) on a titanium sheet and react for 24 h, then ultrasonically clean and

[0039] dry it to obtain the NE@Zn coating.

[0040] 4) Multi-step grafting method: Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first mix the dissolved NE (3 mg / ml) and ZnCl2 (1 mg / ml) on a titanium sheet and react for 24 h, then ultrasonically clean and dry it; then, under the same pH, temperature and oxygen conditions, react bacitracin (0.25 mg / ml) on the titanium sheet for 24 h, ultrasonically clean and dry it to obtain the NE@Zn + bacitracin antibacterial coating.

[0041] 5) Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first react the dissolved NE (3 mg / ml) on a titanium sheet for 24 h, then ultrasonically clean and dry it; then, under the same pH, temperature and oxygen conditions, react bacitracin (0.25 mg / ml) on the titanium sheet for 24 h, ultrasonically clean and dry it to obtain the NE + bacitracin coating.

[0042] 6) Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first mix the dissolved NE (3 mg / ml) and bacitracin (0.25 mg / ml) on a titanium sheet and react for 24 h, then ultrasonically clean and dry it to obtain the NE@bacitracin coating.

[0043] 7) Multi-step grafting method: Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first mix the dissolved NE (3 mg / ml) and bacitracin (0.25 mg / ml) on a titanium sheet and react for 24 h, then ultrasonically clean and dry it; then, under the same pH, temperature and oxygen conditions, react ZnCl2 (1 mg / ml) on the titanium sheet for 24 h, ultrasonically clean and dry it to obtain the NE@bacitracin + Zn antibacterial coating.

[0044] 8) One-step blending method: The NE@bacitracin@Zn antibacterial coating was prepared as shown in Example 1.

[0045] 9) Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first react the dissolved NE (3 mg / ml) on the titanium sheet for 24 h, ultrasonically clean, and dry. Then, under the same pH, temperature, and oxygen conditions, react ZnCl2 (1 mg / ml) on the titanium sheet for 24 h, ultrasonically clean, and dry to obtain the NE+ rod coating.

[0046] 10) Multi-step grafting method: Prepare the NE+ rod+Zn antibacterial coating as shown in Example 1.

[0047] 11) Multi-step grafting method: Under alkaline conditions, using Tris solution (pH = 8.5, 1.21 mg / ml), at room temperature (25 °C) and in the presence of oxygen, first react the dissolved NE (3 mg / ml) on the titanium sheet for 24 h, ultrasonically clean, and dry. Then, under the same pH, temperature, and oxygen conditions, react ZnCl2 (1 mg / ml) on the titanium sheet for 24 h, ultrasonically clean, and dry. Finally, under the same pH, temperature, and oxygen conditions, react bacitracin (0.25 mg / ml) on the titanium sheet for 24 h, ultrasonically clean, and dry to obtain the NE+Zn+ rod antibacterial coating.

[0048] 2. In vitro antibacterial performance evaluation

[0049] The turbidimetry method was used to evaluate the anti-Staphylococcus aureus performance of the prepared coatings. C is the blank control, NE is the NE coating, 3 is the NE@Zn coating, 4 is the NE@Zn+ rod antibacterial coating, 5 is the NE+ rod coating, 6 is the NE@ rod coating, 7 is the NE@ rod+Zn antibacterial coating, 8 is the NE@ rod@Zn antibacterial coating, 9 is the NE+ rod coating, 10 is the NE+ rod+Zn antibacterial coating, 11 is the NE+Zn+ rod antibacterial coating. As Figure 2 shown, the 8 and 11 coatings showed obvious antibacterial performance against Staphylococcus aureus. We made a conjecture on the above results. NE@Zn+ rod has no antibacterial property, while NE+Zn+ rod has antibacterial property. It may be that the NE@Zn blend wraps Zn, making it difficult to achieve antibacterial effect, while there is a large amount of Zn on the surface of the NE+Zn+ rod coating, thus also producing good antibacterial performance. NE@ rod+Zn and NE+ rod+Zn both have no antibacterial property, which may be due to the fact that without the addition of Zn, it is difficult for bacitracin to be loaded in large amounts on the surface of NE. The one-step blending method does not have the problem of bacitracin and Zn being wrapped and unable to be released, and has good antibacterial performance.

[0050] Example 3

[0051] Since the one-step blending method has the advantages of simple operation and shortened preparation process, the present invention further explores its antibacterial performance for the one-step blending method.

[0052] Prepare NE@rod@Zn antibacterial coatings containing 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml, and 1 mg / ml bacitracin according to the process shown in Example 1. Evaluate the anti-Staphylococcus aureus performance of the prepared coatings using the turbidimetric method. As Figure 3 shown, after the bacitracin concentration increases to 0.25 mg / ml, the antibacterial effect tends to be stable.

[0053] Further consider the biocompatibility of the NE@rod@Zn antibacterial coatings prepared by the one-step blending method. In our previous study, the NE coatings had good biocompatibility. Now, explore the biocompatibility for different concentrations of bacitracin and Zn.

[0054] Prepare NE@rod@Zn antibacterial coatings containing 0.125 mg / ml, 0.25 mg / ml, and 0.5 mg / ml bacitracin according to the process shown in Example 1.

[0055] Use BMSC / MC3T3-E1 as the experimental material to perform cck-8 (cell counting kit-8) detection to quantitatively detect the cell growth quantity, and measure the OD values at 1 / 3 d respectively; at the same time, use fluorescence staining to observe the cell morphology, culture for 1 d / 3 d respectively, and take fluorescence photos. As Figure 4A shown, the biocompatibility of the NE@rod@Zn antibacterial coatings containing 0.125 mg / ml, 0.25 mg / ml, and 0.5 mg / ml bacitracin is all good, and the results of fluorescence staining are consistent with those of cck-8 detection, as Figure 4B shown. Considering economic factors, the bacitracin concentration is selected as 0.25 mg / ml.

[0056] Further explore the influence of Zn concentration on biocompatibility.

[0057] Prepare NE@rod@Zn antibacterial coatings containing 0.25 mg / ml, 0.5 mg / ml, and 1 mg / ml ZnCl2 according to the process shown in Example 1.

[0058] Use BMSC / MC3T3-E1 as the experimental material to perform cck-8 (cell counting kit-8) detection to quantitatively detect the cell growth quantity, and measure the OD values at 1 / 3 d respectively; at the same time, use fluorescence staining to observe the cell morphology, culture for 1 d / 3 d respectively, and take fluorescence photos. As Figure 5A shown, the NE@rod@Zn antibacterial coatings prepared with 0.25 mg / ml and 0.5 mg / ml ZnCl2 show toxicity, while the NE@rod@Zn antibacterial coating with 1 mg / ml ZnCl2 has good biocompatibility. The results of fluorescence staining are consistent with those of cck-8 detection, as Figure 5BAs shown. The preferred concentration of ZnCl2 used in the antibacterial coating of the present invention is 1 mg / ml.

[0059] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. An antibacterial coating, and its preparation method is as follows: in a Tris solution, under normal temperature and aerobic conditions, a mixture of dissolved catecholamine, bacitracin, and metal ions is reacted on a titanium sheet for 24 h, and then the surface precipitate is ultrasonically cleaned and dried to obtain the antibacterial coating; The catecholamine is norepinephrine, and the metal ion is ZnCl2.

2. The antibacterial coating according to claim 1, wherein The pH of the Tris solution is 7.5 - 9.

5.

3. The antibacterial coating according to claim 1, wherein The aerobic condition is exposure to air.

4. The antibacterial coating according to claim 1, wherein The concentration of norepinephrine is 1 - 5 mg / ml, the concentration of bacitracin is 0.12 - 1 mg / ml, and the concentration of ZnCl2 is 0.5 - 2 mg / ml.

5. The antibacterial coating according to claim 4, wherein The concentration of norepinephrine is 3 mg / ml, the concentration of bacitracin is 0.25 mg / ml, and the concentration of ZnCl2 is 1 mg / ml.

6. The application of the antibacterial coating according to any one of claims 1 - 5 in the surface modification of an interventional medical device.

7. The application according to claim 6, characterized in that, The interventional medical device is a dental implant.

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