Long-acting antibacterial surface modified titanium metal material as well as preparation method and application thereof

Abstract

The invention discloses a long-acting antibacterial surface modified titanium metal material as well as a preparation method and application thereof. The titanium dioxide nanotube array titanium is constructed on the surface of the titanium implant, and the PLGA coating with the optimized layer number is deposited, so that precise slow-release drug loading of the antibacterial polypeptide is realized. The coating not only overcomes the defects of uncontrollable initial release and limited lasting time of a traditional single-layer coating, but also remarkably prolongs the antibacterial validity period, can provide stable antibacterial protection as long as several days for the surrounding environment of a postoperative implant, and reduces the infection incidence rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a long-acting antibacterial surface-modified titanium metal material, a preparation method thereof, and an application thereof. Background Art

[0002] Medical implants (such as artificial joints, bone plates, dental implants, etc.) are widely used in clinics, but postoperative infection is still one of the main complications. Once bacteria colonize on the surface of the implant and form a biofilm, it will lead to serious infections, and it may be necessary to remove the implant and perform long-term antibiotic treatment. To reduce the risk of infection, various antibacterial surface modification technologies have been studied, including loading antibiotics, antibacterial polypeptides on the surface of the implant or coating materials with bactericidal effects such as silver. Among them, antibacterial polypeptides, as a new type of antibacterial agent, have attracted much attention due to their broad-spectrum high efficiency and low tendency to induce drug resistance. For example, the antibacterial polypeptide HHC36 (KRWWKWWRR) consists of 9 amino acids (Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg), is a cationic antibacterial polypeptide, and has a strong killing effect on pathogenic bacteria such as Staphylococcus aureus. The present invention selects this antibacterial peptide as a model drug, which is representative. However, whether using antibiotics or antibacterial polypeptides for coating, existing antibacterial implant materials often have problems of too fast drug release or short duration: the initial release amount is large and is released completely within a short time after surgery, and it is impossible to maintain an effective concentration for a longer time, so that the implant loses its antibacterial effect in the subsequent period and the infection risk increases. On the contrary, if the initial release amount is reduced to extend the action time, it may not be able to provide enough antibacterial dose in the early stage of implantation, making it difficult to inhibit bacterial colonization in time.

[0003] Poly(lactic-co-glycolic acid) (PLGA) is often used for drug controlled-release coatings due to its biodegradability and slow-release characteristics. However, traditional single-layer PLGA coatings are difficult to achieve precise regulation of drug release in practical applications. Single-layer coatings often release more drugs in the initial stage, and then the release rate drops rapidly, unable to maintain a stable release curve. In addition, the thickness and drug content of single-layer coatings are limited. If the drug loading is increased, it may cause coating cracking or initial burst release. To solve these problems, researchers have begun to explore multi-layer coatings or other means to extend the drug release time, but there is currently a lack of a simple and reliable method that can balance initial release control and long-acting continuous release. Therefore, there is an urgent need to develop a material that can precisely control the release rate and significantly extend the antibacterial effective period, and apply it to implants. Summary of the Invention

[0004] In order to overcome the deficiencies and drawbacks of the prior art, the primary object of the present invention is to provide a surface modification method for a long-acting antibacterial surface-modified titanium metal material. This surface modification method is achieved by constructing a titanium dioxide nanotube structure on the surface of a titanium-based metal material and loading the antibacterial polypeptide HHC36, and then superimposing multiple layers of poly(lactic-co-glycolic acid) (PLGA) coatings. The surface coating of the titanium-based metal material constructed by this surface modification method can achieve the sustained release of drugs and long-acting antibacterial protection, and is suitable for being widely promoted and applied to implants in the fields of orthopedics, dentistry, etc.

[0005] The second object of the present invention is to provide a long-acting antibacterial surface-modified titanium metal material prepared by the above surface modification method.

[0006] The third object of the present invention is to provide an application of a long-acting antibacterial surface-modified titanium metal material in the preparation of antibacterial implants.

[0007] The primary object of the present invention is achieved through the following technical solutions:

[0008] A surface modification method for a long-acting antibacterial surface-modified titanium metal material, comprising the following steps:

[0009] (1) Pretreatment of the titanium substrate: Select pure titanium metal or a titanium alloy as the substrate material, cut and polish it to the required size and surface finish, ultrasonically clean it with acetone, absolute ethanol, and deionized water, perform acid treatment (remove the oxide layer on the surface of the pure titanium metal), and thoroughly clean and dry it with deionized water for later use;

[0010] (2) Preparation of a titanium dioxide nanotube array titanium substrate by anodic oxidation: Use the pretreated titanium substrate as the anode and the untreated titanium sheet as the cathode, perform the first anodic oxidation in the No. 1 electrolyte system and the second anodic oxidation in the No. 2 electrolyte system in sequence. After the two anodic oxidations are completed, a highly ordered titanium dioxide nanotube array is formed on the surface of the titanium substrate. Rinse it with deionized water and dry it to prepare a titanium dioxide nanotube array titanium substrate;

[0011] (3) Loading of the antibacterial polypeptide HHC36: Immerse the alcoholic solution of the antibacterial polypeptide HHC36 into the titanium dioxide nanotube array titanium substrate prepared in step (2), evacuate, repeatedly immerse the titanium dioxide nanotube array titanium substrate in the alcoholic solution of the antibacterial polypeptide HHC36, and let it stand to load the antibacterial polypeptide HHC36 on the titanium dioxide nanotube array titanium substrate to prepare a titanium dioxide nanotube array titanium substrate loaded with the antibacterial polypeptide HHC36;

[0012] (4) Construction of multi-layer PLGA sustained-release coating: Spin coat the PLGA / organic solvent layer by layer on the titanium substrate of the titanium dioxide nanotube array loaded with the antibacterial polypeptide HHC36 in step (3), and repeat the spin coating deposition 1 to 21 times to obtain a PLGA coating with the desired number of layers. Vacuum dry until the surface is uniform and complete, without visible bubbles or cracks to the naked eye, and store it sealed to prepare a titanium substrate of a titanium dioxide nanotube array loaded with the antibacterial polypeptide HHC36 and a multi-layer PLGA sustained-release coating, that is, a long-acting antibacterial surface-modified titanium metal material.

[0013] Preferably, in step (1), the ultrasonic cleaning time is 5 to 10 minutes, the solution used for acid treatment is a 5 to 10% by volume HF solution (prepared by adding deionized water), and the acid treatment time is 0.5 to 1 minute.

[0014] Preferably, the No. 1 electrolyte system in step (2) is prepared by adding solid NH4F to a (NH4)2SO4 solution with a concentration of 1-10 mmol / L, fully dissolving until the concentration of NH4F in the solution is 0.5-3 wt%, and then adding sulfuric acid with a mass fraction of 98%. The volume ratio of the (NH4)2SO4 solution to the sulfuric acid solution is 1:500 to 1000; the No. 2 electrolyte system is prepared by adding sulfuric acid with a mass fraction of 98% to a (NH4)2SO4 solution with a concentration of 1-10 mmol / L, and the volume ratio of the (NH4)2SO4 solution to the sulfuric acid solution is 1:500 to 1000.

[0015] Preferably, in step (2), the constant voltage applied for the first anodic oxidation is 10-40V, and the anodic oxidation treatment time is 15-50 minutes; the constant voltage for the second anodic oxidation is 10-40V, and the anodic oxidation treatment time is 5-10 minutes.

[0016] Preferably, the constant voltage applied for the first anodic oxidation is 22V, and the anodic oxidation treatment time is 30 minutes; the constant voltage applied for the second anodic oxidation is 22V, and the anodic oxidation treatment time is 5 minutes.

[0017] Preferably, in step (2), the pore diameter of the nanotubes in the titanium dioxide nanotube array is about 20-130 nm, and the tube length is 600-1500 nm.

[0018] Preferably, in order to improve the crystallinity and stability of the titanium dioxide nanotube array, the titanium substrate of the titanium dioxide nanotube array can be further heat-treated at 400-500°C for 2-3 h. A dense oxide layer is formed on the surface of the prepared titanium substrate after drying, forming a highly ordered titanium dioxide nanotube array and improving stability.

[0019] Preferably, in step (3), the concentration of the antibacterial polypeptide HHC36 alcohol solution is 1-10 mmol / mL; the number of repeated impregnation times is 10-20 times, the standing temperature is 20-26 °C, and the standing time is 5-20 minutes.

[0020] Preferably, in step (4), the concentration of the PLGA / organic solvent is 10-15% (w / v), and the PLGA / organic solvent is prepared by dissolving PLGA in an anhydrous organic solvent;

[0021] The PLGA (polylactic acid-glycolic acid copolymer) is prepared from lactic acid and glycolic acid in a molar ratio of 50:50, and the molecular weight of the PLGA is 10-20 kDa;

[0022] The anhydrous organic solvent is one of dichloromethane (DCM) or chloroform.

[0023] Preferably, the spin-coating parameters are as follows: the spin-coating speed is 2000-5000 rpm, and the spin-coating time is 20-60 seconds.

[0024] Among them, the reason for limiting the molar ratio of lactic acid and glycolic acid is that different ratios of lactic acid and glycolic acid result in different degradation rates of the prepared PLGA. When the PLGA is prepared from lactic acid and glycolic acid in a molar ratio of 50:50, the degradation rate of the PLGA is faster than that of the other two; when the PLGA is prepared from lactic acid and glycolic acid in a molar ratio of 75:25, the degradation time of the PLGA is as long as 45 days, which is not conducive to early drug release. The PLGA (polylactic acid-glycolic acid copolymer) selected in the present invention is prepared from lactic acid and glycolic acid in a molar ratio of 50:50.

[0025] Preferably, in step (4), after each layer of PLGA is deposited and left standing at room temperature and dried, the PLGA solution is dropped again for spin-coating the next layer.

[0026] Preferably, the standing time is 1-3 minutes.

[0027] Preferably, the vacuum drying temperature is room temperature, and the vacuum drying time is 2 hours.

[0028] The second object of the present invention is achieved by the following technical solution:

[0029] A long-acting antibacterial surface-modified titanium metal material is prepared by the above surface modification method.

[0030] The third object of the present invention is achieved by the following technical solution:

[0031] An application of a long-acting antibacterial surface-modified titanium metal material in the preparation of antibacterial implants.

[0032] Preferably, the antibacterial implant is an orthopedic implant or a dental implant.

[0033] In view of the problems of rapid drug release and short antibacterial duration of implants in the prior art, the present invention provides a long-acting antibacterial surface-modified titanium metal material based on a titanium dioxide nanotube structure loaded with antibacterial polypeptides and a multi-layer PLGA coating, and a surface modification method thereof. By constructing an ordered titanium dioxide nanotube array on the titanium substrate surface and loading the antibacterial polypeptide HHC36, and then spin-coating a 1-21 layer PLGA coating of any number of layers by a specific process, precise regulation of antibacterial drug release is achieved. And an orthopedic implant or a dental implant is prepared by using the long-acting antibacterial surface-modified titanium metal material.

[0034] Among them, the preferred embodiment is to deposit 14 layers of PLGA coating on the titanium dioxide nanotubes. Experiments have found that the coating of this number of layers achieves the best balance between the drug release rate and the antibacterial duration, and can maintain long-term stable drug release.

[0035] Specifically, the present invention provides a long-acting antibacterial surface-modified titanium metal material, including: a titanium or titanium alloy substrate, a titanium dioxide nanotube array formed by anodization thereof, an antibacterial polypeptide HHC36 loaded in the nanotubes, and a multi-layer PLGA sustained-release coating covering the nanotubes. The number of layers of the PLGA coating can be selected according to the required release duration, and can be any number of layers above 1 layer, preferably 14 layers.

[0036] The present invention also provides a preparation method of the above antibacterial coating, and the steps thereof include: anodizing to prepare nanotubes on the surface of the implant, loading the antibacterial polypeptide HHC36 into the nanotubes, and spin-coating a PLGA solution layer by layer to form a multi-layer coating. Through the multi-layer sustained-release coating structure prepared by the above method, the antibacterial polypeptide is effectively encapsulated and gradually released, so as to achieve slow and controllable drug release.

[0037] In addition, the technical solution of the present invention has wide applicability and can be applied to various types of medical implants (such as the surface of artificial joints, bone fixation plates and screws, the surface of dental implants, etc.). Only by preparing a nanotube structure on the surface of the corresponding implant material and implementing a multi-layer PLGA coating process, its long-acting antibacterial performance can be imparted. Further, the antibacterial drug is not limited to the HHC36 antibacterial polypeptide, and other drugs with antibacterial activity (such as antibacterial polypeptides of other sequences, antibiotics, antibacterial small molecules, etc.) can be loaded into the nanotubes in a similar manner and sustained-release is achieved by the multi-layer PLGA coating. The nanotube preparation time, reaction voltage, number of PLGA coating layers, thickness, and drug loading amount in the present invention can be adjusted according to different application requirements, so as to provide a customized antibacterial protection plan for clinical use.

[0038] The technical solution of the present invention has the following advantages and beneficial effects over the prior art:

[0039] (1) The nanotube structure enhances drug loading and stability: The titanium dioxide nanotube array prepared on the titanium surface by anodic oxidation provides a storage space with a high specific surface area for drugs. By optimizing the nanotube preparation parameters (reaction time, reaction voltage, electrolyte ratio), titanium dioxide nanotubes with different diameters and lengths can be prepared. The present invention verifies that by performing anodic oxidation once at 22V for 30 minutes, titanium dioxide nanotubes with regular structures can be obtained and the best drug loading can be achieved. By performing secondary anodic oxidation at 22V for 5 minutes, the nanotube substrate is strengthened and the stability of the nanotubes is improved. The vacuum impregnation drug loading strategy enables the antibacterial polypeptide HHC36 to fully enter the nanotube pores and firmly stay, improving the drug loading capacity and stability. The nanotube structure also provides a mechanical interlocking effect, avoiding the rapid loss of drugs in the initial stage.

[0040] (2) The multi-layer PLGA coating enables precise slow release: The multi-layer PLGA coating is formed by the method of layer-by-layer spin coating, and the drug release curve can be precisely regulated by adjusting the number of coating layers. The more the number of coating layers, the greater the overall coating thickness and the longer the diffusion path required for drugs, thereby reducing the initial release rate and prolonging the release time. However, too many layers will also result in insufficient initial release. The present invention determines through a large number of experiments that 14 layers of PLGA coating can achieve continuous and stable release while suppressing the initial burst release, achieving the best balance between the release rate and the duration.

[0041] (3) Long-acting and highly efficient antibacterial performance: The antibacterial polypeptide HHC36 loaded in the present invention has a broad-spectrum and highly efficient bactericidal effect, and its activity can be maintained for a long time through the slow release of multi-layer PLGA. Experiments prove that the nanotube drug-loaded surface with 14 layers of PLGA coating can maintain a high antibacterial effect for 5 consecutive days in the in vitro antibacterial test of co-culturing with Staphylococcus aureus, which are 100%, 99.99%, 99.99%, 70%, and 58% respectively, and the antibacterial rate always remains at a high level during the whole period. In contrast, the following numbers of layers (such as 1 layer, 4 layers, 7 layers, 21 layers) have significantly shortened antibacterial effective maintenance time in the same test and are difficult to provide a long enough infection protection period.

[0042] (4) Good biocompatibility: The titanium dioxide and PLGA materials used in the present invention are both recognized biocompatible materials and have no toxic or side effects on surrounding tissues. The nanotube structure and its coating will not significantly affect the mechanical properties and bone integration process of the implant, ensuring the safety and effectiveness of the implant while providing antibacterial functions.

[0043] In summary, the present invention realizes the precise slow-release drug loading of antibacterial polypeptides by constructing a nanotube array on the surface of a titanium implant and depositing an optimized number of layers of PLGA coating. This coating not only overcomes the defects of uncontrollable initial release and limited duration of traditional single-layer coatings, but also significantly extends the antibacterial effective period, providing stable antibacterial protection for the environment around the implant for several days after surgery and reducing the incidence of infection. Since the preparation processes (anodic oxidation and spin coating) used in the present invention are simple and feasible, with good repeatability and scalability, they have broad application prospects in the fields of orthopedic implants, dental implants, etc. In particular, the efficient antibacterial protection provided by the present invention for more than 5 days covers the critical period of high risk of infection after implantation (usually within about one week after surgery), so it is expected to significantly reduce the incidence of postoperative infection and improve the success rate of implantation surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of preparing a titanium dioxide nanotube array by anodic oxidation in Examples 1 to 5; wherein the cathode is an untreated titanium sheet, the anode is a titanium sheet after acid etching treatment, and a gray titanium dioxide nanotube structure as shown in the figure is formed after anodic oxidation.

[0045] Figure 2 It is a schematic diagram of the structure of the antibacterial coating in Examples 1 to 5, which shows the structure of the titanium dioxide nanotube array on the surface of the titanium substrate and the PLGA coating deposited layer by layer thereon (schematic cross-sectional view, where the green is the titanium dioxide nanotube layer loaded with AMP, and the blue is the multi-layer PLGA coating).

[0046] Figure 3 It is a top view of the SEM of the titanium dioxide nanotubes prepared by anodic oxidation once at a constant voltage of 22V and a reaction time of 15 minutes in Example 1.

[0047] Figure 4 It is a cross-sectional view of the titanium dioxide nanotubes prepared by anodic oxidation once at a constant voltage of 22V and a reaction time of 15 minutes in Example 1.

[0048] Figure 5 It is a top view of the SEM of the titanium dioxide nanotubes prepared by anodic oxidation once at a constant voltage of 22V and a reaction time of 20 minutes in Example 2.

[0049] Figure 6 It is a cross-sectional view of the titanium dioxide nanotubes prepared by anodic oxidation once at a constant voltage of 22V and a reaction time of 20 minutes in Example 2.

[0050] Figure 7 It is a schematic diagram of the structure of the titanium dioxide nanotube array prepared by the first anodic oxidation and the second anodic oxidation in Example 3, where Figure 7 (A) is a top view of the titanium dioxide nanotubes after the first anodic oxidation,Figure 7 (C) is the cross-sectional view of titanium dioxide nanotubes after the first anodization. Figure 7 (B) is the top view of titanium dioxide nanotubes after the second anodization based on the first anodization. Figure 7 (D) is the cross-sectional view of titanium dioxide nanotubes after the secondary anodization based on the first anodization.

[0051] Figure 8 is the bar graph of the maximum drug loading amount of HHC36 with different numbers of PLGA coating layers in Example 3.

[0052] Figure 9 is the drug release curve graph of HHC36 with different numbers of PLGA coating layers in Example 3.

[0053] Figure 10 is the antibacterial performance characterization of each group of materials against Staphylococcus aureus (S. aureus) in Example 3.

[0054] Figure 11 is the SEM result of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) on the material surface in Example 3.

[0055] Figure 12 is the top view of the SEM of titanium dioxide nanotubes prepared by the first anodization with a constant voltage of 22V and a reaction time of 40 minutes in Example 4.

[0056] Figure 13 is the cross-sectional view of the titanium dioxide nanotubes prepared by the first anodization with a constant voltage of 22V and a reaction time of 40 minutes in Example 4.

[0057] Figure 14 is the top view of the SEM of titanium dioxide nanotubes prepared by the first anodization with a constant voltage of 22V and a reaction time of 50 minutes in Example 5.

[0058] Figure 15 is the cross-sectional view of the titanium dioxide nanotubes prepared by the first anodization with a constant voltage of 22V and a reaction time of 50 minutes in Example 5. Detailed implementation manners

[0059] The present invention will be further described in detail below in conjunction with specific embodiments, but the implementation manners of the present invention are not limited thereto. The materials used in the examples of the present invention can all be obtained by commercial purchase.

[0060] Figure 1Schematic diagram of preparing titanium dioxide nanotube arrays by anodization in Examples 1 to 5; where the cathode is an untreated titanium sheet, the anode is an acid-etched titanium sheet, and a gray titanium dioxide nanotube structure as shown in the figure is formed after anodization.

[0061] Figure 2 Schematic diagram of the structure of the antibacterial coating in Examples 1 to 5, which shows the structure of the titanium dioxide nanotube array on the surface of the titanium substrate and the PLGA coating deposited layer by layer thereon (schematic cross-sectional view, where green - titanium dioxide nanotube layer loaded with AMP, blue - multi-layer PLGA coating).

[0062] Example 1

[0063] A surface modification method for a long-acting antibacterial surface-modified titanium metal material, the steps are as follows,

[0064] (1) Pretreatment of the titanium substrate: Select pure titanium metal as the substrate material, cut and polish it to the required size and surface finish; ultrasonically clean it with acetone, absolute ethanol, and deionized water for 8 minutes, then immerse it in a 10% HF solution for pretreatment for 0.5 minutes to remove the oxide layer on the titanium sheet surface, and then wash it thoroughly with deionized water again and dry it for use;

[0065] (2) Preparation of nanotube arrays by anodization: Use the pretreated titanium substrate as the anode and the untreated titanium sheet as the cathode, and place them in an electrolyte containing fluoride ions for anodization. Preferably, add 1 wt% NH4F to 500 mL of 1 mmol / L (NH4)2SO4 solution, fully dissolve it, and then add 500 μL of sulfuric acid to prepare Electrolyte System No. 1, apply a constant voltage of 22 V at room temperature, and perform anodization for 15 minutes; preferably, add 500 μL of sulfuric acid to 500 mL of 1 mmol / L (NH4)2SO4 to prepare Electrolyte System No. 2, apply a constant voltage of 22 V at room temperature, and perform anodization for 5 minutes. The optimized anodization parameters can form a highly ordered titanium dioxide nanotube array on the titanium surface. The obtained nanotube pore diameter is about 35 - 40 nm, the tube length is about 700 - 800 nm, the tube wall is smooth and perpendicular to the titanium substrate; after the anodization is completed, take out the sample and rinse it with deionized water to remove the residual electrolyte, and then dry it in the air; if it is necessary to improve the crystallinity and stability of the titanium dioxide nanotubes, perform heat treatment on the sample at 450 °C for 2 hours (but this step is optional); after the anodization treatment is completed, the titanium substrate surface presents a regularly arranged nanotube array structure; Figure 3 Top view of the SEM of the titanium dioxide nanotubes prepared by performing anodization once with a constant voltage of 22 V and a reaction time of 15 minutes in this example; Figure 4 Cross-sectional view of the titanium dioxide nanotubes prepared by performing anodization once with a constant voltage of 22 V and a reaction time of 15 minutes in this example;

[0066] (3) Loading of antibacterial polypeptide HHC36: Prepare an aqueous solution of antibacterial polypeptide HHC36 (e.g., with a concentration of 1 mmol / mL and the solvent being absolute ethanol); place the dried nanotube sample flat in a petri dish, and drop 30 - 50 μL of HHC36 solution onto its surface, so that the solution evenly covers and fully penetrates into the pores of the nanotubes. To increase the penetration depth and loading amount of the drug into the nanotubes, the container containing the sample can be placed in a vacuum desiccator, evacuated for 5 minutes to allow the air in the nanotubes to escape, and when the atmospheric pressure is restored, the antibacterial polypeptide solution is quickly sucked into the tubes. Repeat the vacuum impregnation process 15 times, and let it stand for several hours (overnight at 4°C) to allow the antibacterial polypeptide to be fully adsorbed on the inner wall of the nanotubes;

[0067] (4) Construction of multilayer PLGA sustained-release coating:

[0068] 4.1. Preparation of PLGA solution: Select a medical-grade poly(lactic-co-glycolic acid) (PLGA, prepared with a molar ratio of lactic acid to glycolic acid of 50:50 and a molecular weight of 10 - 20 kDa); dissolve PLGA in an anhydrous organic solvent, such as dichloromethane (DCM) or chloroform, to prepare a 15% (w / v) PLGA / organic solvent solution to ensure a moderate viscosity for spin-coating into a film;

[0069] 4.2. Spin-coating process parameters: Use the spin coating method to layer-deposit the PLGA solution on the titanium substrate surface of the titanium dioxide nanotube array that has been loaded with the drug. Fix the dried drug-loaded titanium dioxide nanotube sample on the sample stage of the spin coater, and drop an appropriate amount of PLGA solution (e.g., 0.1 mL) onto the sample surface; set the spin coating program, pre-spin at 1000 rpm for 5 seconds, and then increase to 3000 rpm and rotate for 30 seconds to ensure that the solution spreads evenly under the action of centrifugal force to form a film. After one spin coating, let the sample stand at room temperature for about 1 minute to allow the organic solvent to volatilize and the coating to solidify;

[0070] (5) Construction of multilayer coatings: Repeat the spin coating process in step (4) multiple times to obtain PLGA coatings with the desired number of layers. After each deposition of a layer of PLGA and drying, drop the PLGA solution again for the next spin coating. By controlling the number of spin coatings, coating samples with different numbers of layers can be obtained; in this example, a series of samples with different numbers of layers were prepared for comparison, including a single-layer PLGA coating sample obtained by spin coating only once, a 4-layer coating sample obtained by spin coating 4 times, a sample spin coated 7 times, as well as 14-layer and 21-layer PLGA coating samples obtained by spin coating 14 times and 21 times respectively according to the above method; the film-forming conditions for each layer are kept consistent, so that the differences between different samples only lie in the number of PLGA coating layers;

[0071] (6) Curing and preservation: When the target number of layers is reached, the sample is placed in a vacuum drying oven for further drying at room temperature for 2 hours to ensure that the residual organic solvent in the coating is completely volatilized; the surface of the resulting multilayer PLGA coating should be uniform and complete, without visible bubbles or cracks; finally, the sample is sealed and stored for later use. During the entire multilayer coating construction process, the sample should be kept away from moisture or high temperature to prevent premature degradation of PLGA or drug loss.

[0072] Example 2

[0073] A surface modification method for a long-lasting antibacterial surface-modified titanium metal material, comprising the following steps:

[0074] (1) Pretreatment of titanium substrate: Pure titanium metal was selected as the substrate material, which was cut and polished to the required size and surface finish; ultrasonic cleaning was performed with acetone, anhydrous ethanol, and deionized water for 8 minutes, and then pretreatment was performed with 10% HF solution for 0.5 minutes to remove the oxide layer on the surface of the titanium sheet, and then the sheet was thoroughly cleaned with deionized water again and dried for later use;

[0075] (2) Preparation of nanotube arrays by anodization: The pretreated titanium substrate is used as the anode and the untreated titanium sheet is used as the cathode, and they are placed in an electrolyte containing fluoride ions for anodization. Preferably, 1 wt% NH4F is added to 500 mL of 1 mmol / L (NH4)2SO4 solution, and after sufficient dissolution, 500 μL of sulfuric acid is added to prepare electrolyte system No. 1, and a constant voltage of 22 V is applied at room temperature, and anodization treatment is performed for 20 minutes. Preferably, 500 mL of 1 mmol / L (NH4)2SO4 is prepared, 500 μL of sulfuric acid is added, and electrolyte system No. 2 is prepared, and a constant voltage of 22 V is applied at room temperature, and anodization treatment is performed for 5 minutes. The optimized anodizing parameters can form a highly ordered array of titanium dioxide nanotubes on the titanium surface. The obtained nanotubes have a pore size of about 40-50nm, a tube length of 800-900nm, and a smooth tube wall perpendicular to the titanium substrate. After the anodizing is completed, the sample is taken out and rinsed with deionized water to remove the residual electrolyte, and then dried in the air. If it is necessary to improve the crystallinity and stability of the titanium dioxide nanotubes, the sample is heat treated at 450°C for 2 hours (but this step is optional). After the anodizing treatment is completed, the surface of the titanium substrate presents a regularly arranged nanotube array structure. Figure 5 This is a SEM top view of titanium dioxide nanotubes prepared by anodization at a constant voltage of 22V and a reaction time of 20 minutes in this embodiment; Figure 6 This is a cross-sectional view of titanium dioxide nanotubes prepared by anodization at a constant voltage of 22V and a reaction time of 15 minutes in this embodiment;

[0076] (3) Loading of antibacterial polypeptide HHC36: Prepare an aqueous solution of antibacterial polypeptide HHC36 (for example, with a concentration of 1 mmol / mL and the solvent being absolute ethanol); place the dried nanotube sample flat in a petri dish, and drop 30 - 50 μL of HHC36 solution onto its surface, so that the solution evenly covers and fully penetrates into the pores of the nanotubes. To increase the penetration depth and loading amount of the drug into the nanotubes, the container containing the sample can be placed in a vacuum dryer, evacuated for 5 minutes to allow the air in the nanotubes to escape, and the antibacterial polypeptide solution is quickly sucked into the tubes when the atmospheric pressure is restored. Repeat the vacuum impregnation process 15 times, and let it stand for several hours (overnight at 4°C) to allow the antibacterial polypeptide to be fully adsorbed on the inner wall of the nanotubes;

[0077] (4) Construction of multi-layer PLGA sustained-release coating:

[0078] 4.1. Preparation of PLGA solution: Select a medical-grade poly(lactic-co-glycolic acid) (PLGA, prepared with a molar ratio of lactic acid to glycolic acid of 50:50 and a molecular weight of 10 - 20 kDa); dissolve PLGA in an anhydrous organic solvent, such as dichloromethane (DCM) or chloroform, to prepare a 15% (w / v) PLGA / organic solvent solution to ensure a moderate viscosity for spin-coating into a film;

[0079] 4.2. Spin-coating process parameters: Use the spin coating method to layer-deposit the PLGA solution on the surface of the nanotube titanium that has been loaded with the drug. Fix the dried drug-loaded nanotube sample on the sample stage of the spin coater, and drop an appropriate amount of PLGA solution (for example, 0.1 mL) on the sample surface; set the spin coating program, pre-spin at 1000 rpm for 5 seconds, and then increase to 3000 rpm and rotate for 30 seconds to ensure that the solution spreads evenly under the action of centrifugal force to form a film. After one spin coating, let the sample stand at room temperature for about 1 minute to allow the organic solvent to volatilize and the coating to cure;

[0080] (5) Construction of multi-layer coatings: Repeat the spin coating process in step (4) multiple times to obtain PLGA coatings with the desired number of layers. After each deposition of a layer of PLGA and drying, drop the PLGA solution again for the next spin coating. By controlling the number of spin coatings, coating samples with different numbers of layers can be obtained; in this example, a series of samples with different numbers of layers were prepared for comparison, including a single-layer PLGA coating sample obtained by spin coating only once, a 4-layer coating sample obtained by spin coating 4 times, a sample spin coated 7 times, and 14-layer and 21-layer PLGA coating samples obtained by spin coating 14 times and 21 times respectively according to the above method; the film-forming conditions for each layer are kept consistent, so that the differences between different samples only lie in the number of PLGA coating layers;

[0081] (6) Solidification and storage: When the target number of layers is reached, the sample is placed in a vacuum drying oven and further dried at room temperature for 2 hours to ensure that the residual organic solvents in the coating are completely volatilized; the surface of the obtained multi-layer PLGA coating should be uniform and complete, without visible bubbles or cracks to the naked eye; finally, the sample is sealed and stored for future use. During the entire multi-layer coating construction process, the sample should be prevented from contacting moisture or high temperature to avoid premature degradation of PLGA or drug loss.

[0082] Example 3

[0083] A surface modification method for a long-acting antibacterial surface-modified titanium metal material, the steps are as follows:

[0084] (1) Pretreatment of titanium substrate: Select pure titanium metal as the substrate material, cut and polish it to the required size and surface finish; ultrasonically clean it with acetone, absolute ethanol, and deionized water for 8 minutes, then immerse it in a 10% HF solution for pretreatment for 0.5 minutes to remove the oxide layer on the titanium sheet surface, and then wash it thoroughly with deionized water again and dry it for use;

[0085] (2) Preparation of nanotube arrays by anodic oxidation: The pretreated titanium substrate is used as the anode, and the untreated titanium sheet is used as the cathode, and placed in an electrolyte containing fluoride ions for anodic oxidation. Preferably, 1 wt% NH4F is added to 500 mL of 1 mmol / L (NH4)2SO4 solution, and after fully dissolving, 500 μL of sulfuric acid is added to prepare the No. 1 electrolyte system. A constant voltage of 22 V is applied at room temperature for 30 minutes of anodic oxidation treatment; preferably, 500 μL of sulfuric acid is added to 500 mL of 1 mmol / L (NH4)2SO4 to prepare the No. 2 electrolyte system. A constant voltage of 22 V is applied at room temperature for 5 minutes of anodic oxidation treatment. Through optimized anodic oxidation parameters, a highly ordered titanium dioxide nanotube array can be formed on the titanium surface. The obtained nanotube pore diameter is about 60 - 70 nm, the tube length is about 900 - 1100 nm, and the tube wall is smooth and perpendicular to the titanium substrate; after the anodic oxidation is completed, the sample is taken out and rinsed with deionized water to remove the residual electrolyte, and then dried in the air; if it is necessary to improve the crystallinity and stability of the titanium dioxide nanotubes, the sample is heat-treated at 450 °C for 2 hours (but this step is optional); after the anodic oxidation treatment is completed, the titanium substrate surface presents a regularly arranged nanotube array structure; Figure 7 This is the structural schematic diagram of the preparation of titanium dioxide nanotube arrays by the first anodic oxidation and the second anodic oxidation in this example, where Figure 7 (A) is the top view of the titanium dioxide nanotubes after the first anodic oxidation, Figure 7 (C) is the cross-sectional view of the titanium dioxide nanotubes after the first anodic oxidation, Figure 7 (B) is the top view of the titanium dioxide nanotubes after the second anodic oxidation based on the first anodic oxidation, Figure 7(D) is a cross-sectional view of titanium dioxide nanotubes after secondary anodization on the basis of the first anodization. After secondary anodization, a dense oxide layer about 50 μm thick is formed at the bottom of the titanium dioxide nanotubes, improving the stability of the overall structure.

[0086] (3) Loading of antibacterial polypeptide HHC36: Prepare an aqueous solution of antibacterial polypeptide HHC36 (for example, with a concentration of 1 mmol / mL and the solvent being anhydrous ethanol); place the dried nanotube sample flat in a petri dish, and drop 30 - 50 μL of HHC36 solution onto its surface, so that the solution evenly covers and fully penetrates into the pores of the nanotubes. To increase the penetration depth and loading amount of the drug into the nanotubes, the container containing the sample can be placed in a vacuum dryer, evacuated for 5 minutes to allow the air in the nanotubes to escape. When the atmospheric pressure is restored, the antibacterial polypeptide solution is quickly sucked into the tubes. Repeat the vacuum impregnation process 15 times, and let it stand for several hours (overnight at 4°C) to allow the antibacterial polypeptide to be fully adsorbed on the inner wall of the nanotubes.

[0087] (4) Construction of multi-layer PLGA sustained-release coating:

[0088] 4.1. Preparation of PLGA solution: Select a medical-grade poly(lactic-co-glycolic acid) (PLGA, prepared with a molar ratio of lactic acid to glycolic acid of 50:50 and a molecular weight of 10 - 20 kDa); dissolve PLGA in an anhydrous organic solvent, such as dichloromethane (DCM) or chloroform, to prepare a 15% (w / v) PLGA / organic solvent solution to ensure an appropriate viscosity for spin-coating into a film.

[0089] 4.2. Spin-coating process parameters: Use the spin coating method to layer by layer deposit the PLGA solution on the surface of the nanotube titanium that has been loaded with the drug. Fix the dried drug-loaded nanotube sample on the sample stage of the spin coater, and drop an appropriate amount of PLGA solution (for example, 0.1 mL) onto the surface of the sample; set the spin coating program, pre-spin at 1000 rpm for 5 seconds, and then increase to 3000 rpm and rotate for 30 seconds to ensure that the solution evenly spreads under the action of centrifugal force to form a film. After one spin coating, let the sample stand at room temperature for about 1 minute to allow the organic solvent to volatilize and the coating to solidify.

[0090] (5) Construction of multi-layer coating: Repeat the spin-coating process in step (4) multiple times to obtain a PLGA coating with the desired number of layers. After each deposition of a layer of PLGA and drying, drop the PLGA solution again for spin-coating the next layer. By controlling the number of spin-coating times, coating samples with different numbers of layers can be obtained; in this example, a series of samples with different numbers of layers were prepared for comparison, including a single-layer PLGA coating sample obtained by spin-coating only once, a 4-layer coating sample obtained by spin-coating 4 times, a sample spin-coated 7 times, and 14-layer and 21-layer PLGA coating samples obtained by spin-coating 14 times and 21 times respectively according to the above method; the film-forming conditions for each layer are kept consistent, so that the difference between different samples lies only in the number of PLGA coating layers;

[0091] (6) Curing and storage: When the target number of layers is reached, place the sample in a vacuum drying oven and further dry it at room temperature for 2 hours to ensure that the residual organic solvents in the coating are completely volatilized; the surface of the obtained multi-layer PLGA coating should be uniform and complete, without visible bubbles or cracks to the naked eye; finally, seal and store the sample for future use. During the entire process of constructing the multi-layer coating, the sample should be kept away from moisture or high temperature to prevent premature degradation of PLGA or loss of drugs.

[0092] Example 4

[0093] A surface modification method for a long-acting antibacterial surface-modified titanium metal material, the steps are as follows,

[0094] (1) Pretreatment of titanium substrate: Select pure titanium metal as the substrate material, cut and polish it to the required size and surface finish; ultrasonically clean it with acetone, absolute ethanol, and deionized water for 8 minutes, then immerse it in a 10% HF solution for pretreatment for 0.5 minutes to remove the oxide layer on the titanium sheet surface, and then wash it thoroughly with deionized water again and dry it for use;

[0095] (2) Preparation of nanotube arrays by anodization: Using the pretreated titanium substrate as the anode and the untreated titanium sheet as the cathode, place them in an electrolyte containing fluoride ions for anodization. Preferably, add 1 wt% NH4F to 500 mL of 1 mmol / L (NH4)2SO4 solution, fully dissolve it, then add 500 μL of sulfuric acid to prepare Electrolyte System No. 1. Apply a constant voltage of 22 V at room temperature for 40 minutes of anodization treatment; preferably, add 500 μL of sulfuric acid to 500 mL of 1 mmol / L (NH4)2SO4 to prepare Electrolyte System No. 2. Apply a constant voltage of 22 V at room temperature for 5 minutes of anodization treatment. The optimized anodization parameters can form a highly ordered titanium dioxide nanotube array on the titanium surface. The obtained nanotube pore diameter is about 50 - 65 nm, the tube length is about 1100 - 1200 nm, the tube wall is smooth and perpendicular to the titanium substrate; after the anodization is completed, take out the sample and rinse it with deionized water to remove the residual electrolyte, and then dry it in the air; if it is necessary to improve the crystallinity and stability of the titanium dioxide nanotubes, perform a heat treatment on the sample at 450 °C for 2 hours (this step is optional); after the anodization treatment is completed, the titanium substrate surface presents a regularly arranged nanotube array structure;

[0096] (3) Loading of antibacterial polypeptide HHC36: Prepare an aqueous solution of antibacterial polypeptide HHC36 (for example, with a concentration of 1 mmol / mL and the solvent being anhydrous ethanol); place the dried nanotube sample flat in a petri dish, and drop 30 - 50 μL of HHC36 solution on its surface to make the solution evenly cover and fully penetrate into the nanotube pores. To improve the penetration depth and loading amount of the drug into the nanotubes, the container containing the sample can be placed in a vacuum desiccator, evacuated for 5 minutes to allow the air in the nanotubes to escape, and the antibacterial polypeptide solution is quickly sucked into the tubes when the atmospheric pressure is restored. Repeat the vacuum impregnation process 15 times, and let it stand for several hours (place it overnight at 4 °C) to allow the antibacterial polypeptide to be fully adsorbed on the inner wall of the nanotubes; Figure 12 This is the SEM top view of the titanium dioxide nanotubes prepared by anodization once with a constant voltage of 22 V and a reaction time of 40 minutes in this example; Figure 13 This is the cross-sectional view of the titanium dioxide nanotubes prepared by anodization once with a constant voltage of 22 V and a reaction time of 40 minutes in this example;

[0097] (4) Construction of multilayer PLGA sustained-release coating:

[0098] 4.1. Preparation of PLGA solution: Select a medical-grade poly(lactic-co-glycolic acid) (PLGA, prepared with a molar ratio of lactic acid to glycolic acid of 50:50 and a molecular weight of 10 - 20 kDa); dissolve PLGA in an anhydrous organic solvent, such as dichloromethane (DCM) or chloroform, to prepare a 15% (w / v) PLGA / organic solvent solution to ensure appropriate viscosity for spin-coating into a film;

[0099] 4.2. Spin Coating Process Parameters: The PLGA solution was deposited layer by layer on the surface of nanotube titanium loaded with drugs by spin coating. The dried drug-loaded nanotube sample was fixed on the sample stage of the spin coater, and an appropriate amount of PLGA solution (e.g., 0.1 mL) was dropped on the sample surface; the spin coating program was set to pre-spin coat at 1000 rpm for 5 seconds, and then increased to 3000 rpm and rotated for 30 seconds to ensure that the solution was evenly spread under the action of centrifugal force to form a film. After one spin coating, the sample was left standing at room temperature for about 1 minute to allow the organic solvent to volatilize and the coating to cure;

[0100] (5) Construction of Multilayer Coatings: The spin coating process in step (4) was repeated multiple times to obtain the desired number of layers of PLGA coatings. After each deposition of a layer of PLGA and drying, PLGA solution was dropped again for the next spin coating. By controlling the number of spin coatings, coating samples with different numbers of layers could be obtained; in this example, a series of samples with different numbers of layers were prepared for comparison, including a single-layer PLGA coating sample obtained by spin coating only once, a 4-layer coating sample obtained by spin coating 4 times, a sample spin coated 7 times, and 14-layer and 21-layer PLGA coating samples obtained by spin coating 14 times and 21 times respectively according to the above method; the film-forming conditions for each layer were kept consistent, so that the difference between different samples was only in the number of PLGA coating layers;

[0101] (6) Curing and Storage: When the target number of layers was reached, the sample was placed in a vacuum drying oven and further dried at room temperature for 2 hours to ensure that the residual organic solvent in the coating completely volatilized; the surface of the obtained multilayer PLGA coating should be uniform and complete, without visible bubbles or cracks to the naked eye; finally, the sample was sealed and stored for later use. During the entire process of constructing the multilayer coating, the sample should be avoided from contacting moisture or high temperature to prevent premature degradation of PLGA or drug loss.

[0102] Example 5

[0103] A surface modification method for a long-acting antibacterial surface-modified titanium metal material, the steps are as follows,

[0104] (1) Pretreatment of Titanium Substrate: Select pure titanium metal as the substrate material, cut and polish it to the required size and surface finish; ultrasonically clean it with acetone, absolute ethanol, and deionized water for 8 minutes, then immerse it in 10% HF solution for pretreatment for 0.5 minutes to remove the oxide layer on the titanium sheet surface, and then wash it thoroughly with deionized water again and dry it for use;

[0105] (2) Preparation of nanotube arrays by anodization: The pretreated titanium substrate was used as the anode, and the untreated titanium sheet was used as the cathode. They were placed in an electrolyte containing fluoride ions for anodization. Preferably, 1 wt% NH4F was added to 500 mL of 1 mmol / L (NH4)2SO4 solution, and after complete dissolution, 500 μL of sulfuric acid was added to prepare the No. 1 electrolyte system. A constant voltage of 22 V was applied at room temperature for 50 minutes of anodization treatment; preferably, 500 μL of sulfuric acid was added to 500 mL of 1 mmol / L (NH4)2SO4 to prepare the No. 2 electrolyte system. A constant voltage of 22 V was applied at room temperature for 5 minutes of anodization treatment. The optimized anodization parameters can form a highly ordered titanium dioxide nanotube array on the titanium surface. The obtained nanotubes have a pore diameter of about 45 - 50 nm, a tube length of about 1200 - 1300 nm, and smooth tube walls perpendicular to the titanium substrate; after anodization, the sample was taken out and rinsed with deionized water to remove the residual electrolyte, and then dried in air; if it is necessary to improve the crystallinity and stability of the titanium dioxide nanotubes, the sample was heat-treated at 450 °C for 2 hours (this step is optional); after the anodization treatment, the titanium substrate surface presents a regularly arranged nanotube array structure; Figure 14 The top view SEM of titanium dioxide nanotubes prepared by anodization once with a constant voltage of 22 V and a reaction time of 50 minutes in this example; Figure 15 The cross-sectional view of titanium dioxide nanotubes prepared by anodization once with a constant voltage of 22 V and a reaction time of 50 minutes in this example;

[0106] (3) Loading of antibacterial polypeptide HHC36: Prepare an aqueous solution of antibacterial polypeptide HHC36 (for example, with a concentration of 1 mmol / mL and the solvent being absolute ethanol); place the dried nanotube sample flat in a petri dish, and drop 30 - 50 μL of HHC36 solution onto its surface to evenly cover the solution and fully infiltrate the nanotube pores. To increase the penetration depth and loading amount of the drug into the nanotubes, the container containing the sample can be placed in a vacuum dryer and evacuated for 5 minutes to allow the air in the nanotubes to escape. When the atmospheric pressure is restored, the antibacterial polypeptide solution is quickly sucked into the tubes. Repeat the vacuum impregnation process 15 times and let it stand for several hours (overnight at 4 °C) to allow the antibacterial polypeptide to be fully adsorbed on the inner wall of the nanotubes;

[0107] (4) Construction of multilayer PLGA sustained-release coating:

[0108] 4.1. Preparation of PLGA solution: Select a medical-grade poly(lactic-co-glycolic acid) (PLGA, prepared with a molar ratio of lactic acid to glycolic acid of 50:50 and a molecular weight of 10 - 20 kDa); dissolve PLGA in an anhydrous organic solvent, such as dichloromethane (DCM) or chloroform, to prepare a 15% (w / v) PLGA / organic solvent solution to ensure moderate viscosity for spin-coating into a film;

[0109] 4.2 Spin Coating Process Parameters: The PLGA solution was deposited layer by layer on the surface of nanotube titanium loaded with drugs by spin coating. The dried drug-loaded nanotube sample was fixed on the sample stage of the spin coater, and an appropriate amount of PLGA solution (e.g., 0.1 mL) was dropped onto the sample surface; the spin coating program was set to pre-spin coat at 1000 rpm for 5 seconds, and then increased to 3000 rpm and rotated for 30 seconds to ensure that the solution was evenly spread under the action of centrifugal force to form a film. After one spin coating, the sample was left standing at room temperature for about 1 minute to allow the organic solvent to volatilize and the coating to cure;

[0110] (5) Construction of Multilayer Coatings: The spin coating process in step (4) was repeated multiple times to obtain PLGA coatings with the desired number of layers. After each deposition of a layer of PLGA and drying, the PLGA solution was dropped again for the next spin coating. By controlling the number of spin coatings, coating samples with different numbers of layers could be obtained; in this example, a series of samples with different numbers of layers were prepared for comparison, including single-layer PLGA coating samples obtained by spin coating only once, 4-layer coating samples obtained by spin coating 4 times, samples spin coated 7 times, and 14-layer and 21-layer PLGA coating samples obtained by spin coating 14 times and 21 times respectively according to the above method; the film-forming conditions for each layer were kept consistent, so the difference between different samples was only in the number of PLGA coating layers;

[0111] (6) Curing and Storage: When the target number of layers was reached, the sample was placed in a vacuum drying oven and further dried at room temperature for 2 hours to ensure that the residual organic solvent in the coating was completely volatilized; the surface of the obtained multilayer PLGA coating should be uniform and complete, without visible bubbles or cracks to the naked eye; finally, the sample was sealed and stored for later use. During the entire process of constructing the multilayer coating, the sample should be prevented from contacting moisture or high temperature to prevent premature degradation of PLGA or drug loss.

[0112] Drug Release Performance Test.

[0113] To evaluate the effect of different numbers of PLGA coating layers on the release behavior of the antimicrobial polypeptide HHC36, in vitro drug release experiments were carried out on each group of samples obtained in step (5) of Example 3. Each sample was placed in its respective centrifuge tube, and a certain volume (e.g., 0.2 mL) of phosphate buffer solution (PBS, pH 7.4) was added to simulate the body fluid environment. All samples were placed in a constant temperature shaker at 37 °C at 85 rpm to simulate the slow flow conditions in the body. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 14, 21, 28 days), the leaching solution was aspirated, and an equal volume of fresh PBS buffer solution was quickly replenished before the samples were placed back for continued soaking. The concentration of HHC36 in the leaching solution could be determined by the BCA method. Three parallel specimens were tested for each group of samples, the amount of drug released at each time point was recorded and the average value was calculated, and the cumulative release curve was plotted accordingly (as Figure 8 and Figure 9 shown). Figure 8 is a bar chart of the maximum drug loading of HHC36 drugs with different numbers of PLGA coating layers, including the comparison results of the maximum drug loading of 1 layer, 4 layers, 7 layers, 14 layers and 21 layers of coatings. Figure 9 is a drug release curve of HHC36 drugs with different numbers of PLGA coating layers (cumulative release percentage vs. time), including the comparison results of 1 layer, 4 layers, 7 layers, 14 layers and 21 layers of coatings. It can be seen that the drug release rate of the 14-layer coating is the most stable and continuous.

[0114] The experimental results showed that the samples with different numbers of coating layers exhibited significantly different release characteristics.

[0115] Samples with 14 layers of PLGA coating: This group of samples showed the best sustained release performance. The initial 24-hour release amount accounted for only about 40% of the total amount, and the drug was released continuously at a relatively constant rate. The cumulative release was about 60% at day 5 and gradually approached complete release (about 80% or more) at day 21. The entire release curve was smooth without obvious early spikes, indicating that the 14-layer coating effectively inhibited the initial burst release and achieved long-term stable release.

[0116] The above results indicate that with the increase in the number of PLGA coating layers, the release behavior of HHC36 gradually changes from rapid release to slow and continuous release. However, the more coating layers are not necessarily better: too thick a coating will overly reduce the initial release rate, which may prevent the minimum inhibitory concentration (MIC) of antibacterial drugs from being reached in a timely manner in the early stage. Considering both the initial release control and the long-term release ability, the design of the 14-layer PLGA coating achieved the optimal balance, avoiding the early rapid depletion of drugs and ensuring a continuous effective drug concentration during the critical postoperative period. This is consistent with the expected goal of the present invention.

[0117] The 14-layer PLGA-coated sample exhibited the most persistent antibacterial effect: during 4 consecutive days of repeated bacterial suspension challenges, the antibacterial rate remained at a high level of approximately 99% every day until signs of decline began on the 5th day (the antibacterial rate dropped to approximately 60% on the 5th day). That is to say, this sample could effectively inhibit the reproduction of Staphylococcus aureus within the first 5 days, proving that the concentration of the sustained-release HHC36 met the MIC level during this period. In contrast, samples with 7 or fewer coating layers failed to achieve such a long effective antibacterial period (as Figure 10 and Figure 11 described). Figure 10 This is a comparison graph of the in vitro antibacterial experiment results of the coating of the present invention, showing the change in the antibacterial rate over time (days) when the leachate released from samples with different coating layer numbers was co-cultured with Staphylococcus aureus. The curve shows that the 14-layer PLGA-coated sample maintained a high antibacterial rate throughout the 5 days, significantly superior to the samples with fewer coating layers. Figure 11 This is a comparison graph of the SEM results of the in vitro antibacterial experiment of the coating of the present invention, showing the bactericidal effect diagrams of the antibacterial polypeptide HHC36 and the sample of the antibacterial polypeptide HHC36 loaded on PLGA against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA).

[0118] The antibacterial experiment results further verified the effectiveness of the multi-layer coating design of the present invention: the surface of the drug-loaded nanotubes with 14-layer PLGA coating showed the best antibacterial strength and duration, being able to provide strong antibacterial protection at the initial stage of implantation and maintain high-efficiency antibacterial for at least 5 days or more. This significantly reduced the risk of early infection after implantation. In contrast, too few coating layers would lead to rapid depletion of the drug, and too many coating layers would result in insufficient initial release, neither of which could meet the immediate and long-term antibacterial requirements. The 14-layer PLGA sustained-release coating described in the present invention effectively balanced these two requirements, demonstrating its superiority as a long-acting antibacterial surface modification for medical implants.

[0119] In summary, as can be seen from the preparation and testing of the above Examples 1 to 5, with the increase of the reaction time, the length of the nanotubes increases linearly, the pore diameter of the nanotubes first increases and then decreases, and the nanotubes with different aspect ratios have an impact on the drug loading amount and the long-term cell adhesion behavior. The nanotube multi-layer PLGA-coated antibacterial implant surface provided by the present invention has significant advantages in terms of drug sustained release and antibacterial persistence. This coating structure realizes the fine regulation of the release behavior of the antibacterial polypeptide HHC36, enabling the implant to continuously provide an antibacterial dose during the critical postoperative period to prevent infection. This technology has the characteristics of simple preparation method, reliable effect and wide application range, and has important application value for various implant medical devices that require long-term antibacterial performance. In particular, the high-efficiency antibacterial protection provided by the present invention for more than 5 days covers the critical period of high risk of postoperative infection (usually within about one week after surgery), and is expected to significantly reduce the incidence of postoperative infection and improve the success rate of implant surgery.

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A surface modification method for a long-acting antibacterial surface-modified titanium metal material, characterized in that, It includes the following steps: (1) Pretreatment of titanium substrate: Select pure titanium metal or titanium alloy as the substrate material, cut and polish it to the required size and surface finish, ultrasonically clean it with acetone, absolute ethanol, and deionized water, perform acid treatment, and thoroughly clean and dry it with deionized water for later use; (2) Preparation of titanium dioxide nanotube array titanium substrate by anodic oxidation: Use the pretreated titanium substrate as the anode and the untreated titanium sheet as the cathode. First, perform anodic oxidation in the No. 1 electrolyte system and then perform second anodic oxidation in the No. 2 electrolyte system. After the two anodic oxidations are completed, a highly ordered titanium dioxide nanotube array is formed on the surface of the titanium substrate. Rinse it with deionized water and dry it to obtain the titanium dioxide nanotube array titanium substrate; (3) Loading of antibacterial polypeptide HHC36: Immerse the alcoholic solution of antibacterial polypeptide HHC36 into the titanium dioxide nanotube array titanium substrate prepared in step (2), evacuate, repeatedly immerse the titanium dioxide nanotube array titanium substrate in the alcoholic solution of antibacterial polypeptide HHC36, and let it stand to load antibacterial polypeptide HHC36 on the titanium dioxide nanotube array titanium substrate to obtain the titanium dioxide nanotube array titanium substrate loaded with antibacterial polypeptide HHC36; (4) Construction of multi-layer PLGA sustained-release coating: Use the spin-coating method to deposit PLGA / organic solvent layer by layer on the titanium dioxide nanotube array titanium substrate loaded with antibacterial polypeptide HHC36 in step (3), repeat the spin-coating deposition 1 - 21 times to obtain the PLGA coating with the required number of layers, dry it in vacuum until the surface is uniform and complete, without visible bubbles or cracks to the naked eye, seal and store it to obtain the titanium dioxide nanotube array titanium substrate loaded with antibacterial polypeptide HHC36 and multi-layer PLGA sustained-release coating, that is, the long-acting antibacterial surface-modified titanium metal material.

2. The surface modification method of the long-acting antibacterial surface-modified titanium metal material according to claim 1, characterized in that, In the No. 1 electrolyte system described in step (2), it is prepared by adding solid NH4F to a (NH4)2SO4 solution with a concentration of 1 - 10 mmol / L, fully dissolving it until the concentration of NH4F in the solution is 0.5 - 3 wt%, and then adding sulfuric acid with a mass fraction of 98%. The volume ratio of the (NH4)2SO4 solution to the sulfuric acid solution is 1:500 - 1000; the No. 2 electrolyte system is prepared by adding sulfuric acid with a mass fraction of 98% to a (NH4)2SO4 solution with a concentration of 1 - 10 mmol / L, and the volume ratio of the (NH4)2SO4 solution to the sulfuric acid solution is 1:500 - 1000.

3. The surface modification method of the long-acting antibacterial surface-modified titanium metal material according to claim 1, characterized in that, In step (2), the constant voltage applied for the first anodic oxidation is 10 - 40 v, and the anodic oxidation treatment time is 15 - 50 minutes; the constant voltage for the second anodic oxidation is 10 - 40 v, and the anodic oxidation treatment time is 5 - 10 minutes.

4. The surface modification method of the long-acting antibacterial surface-modified titanium metal material according to claim 1, characterized in that, In step (2), the pore diameter of the nanotubes in the titanium dioxide nanotube array is about 20 - 130 nm, and the tube length is 600 - 1500 nm.

5. The surface modification method of the long-acting antibacterial surface-modified titanium metal material according to claim 1, characterized in that, In step (3), the concentration of the alcoholic solution of antibacterial polypeptide HHC36 is 1 - 10 mmol / mL; the number of repeated immersions is 10 - 20 times, the standing temperature is 20 - 26 °C, and the standing time is 5 - 20 minutes.

6. The surface modification method of the long-acting antibacterial surface-modified titanium metal material according to claim 1, characterized in that, In step (4), the concentration of the PLGA / organic solvent is 10-15% (w / v), and the PLGA / organic solvent is prepared by dissolving PLGA in an anhydrous organic solvent; The PLGA is prepared from lactic acid and glycolic acid in a molar ratio of 50:50, and the molecular weight of the PLGA is 10-20 kDa; The anhydrous organic solvent is one of dichloromethane or chloroform.

7. The surface modification method of the long-acting antibacterial surface-modified titanium metal material according to claim 1, characterized in that, The spin coating parameters are as follows: the spin coating speed is 2000-5000 rpm, and the spin coating time is 20-60 seconds.

8. A long-acting antibacterial surface-modified titanium metal material, characterized in that, Prepared by the surface modification method according to any one of claims 1 to 7.

9. Use of the long-acting antibacterial surface-modified titanium metal material according to claim 8 in the preparation of antibacterial implants.

10. Use of the long-acting antibacterial surface-modified titanium metal material according to claim 9 in the preparation of antibacterial implants, characterized in that, The antibacterial implant is an orthopedic implant or a dental implant.

Images