Multi-stage microporous titanium alloy containing nanometer copper selenide antibacterial peptide and preparation method thereof

Abstract

The application discloses a preparation method of a multi-stage microporous titanium alloy loaded with nanometer copper selenide antibacterial peptides, and comprises the following steps: one, substrate preparation; two, surface modification; three, functional treatment, to obtain the multi-stage microporous titanium alloy loaded with nanometer copper selenide antibacterial peptides; the application also provides a multi-stage microporous titanium alloy loaded with nanometer copper selenide antibacterial peptides. The multi-stage microporous titanium alloy loaded with nanometer copper selenide antibacterial peptides provided by the application has a load design and a photothermal synergistic mechanism, can effectively inhibit the breeding of bacteria, greatly reduces the infection risk of implant surgery, promotes bone integration, and has a multi-scale structure and ion release design, can accelerate bone tissue growth and material fusion, reduce long-term complications, optimize the bone repair microenvironment, opens up a new idea for the treatment of complex conditions, has good stability and safety, ensures long-term stable function in the body, meets strict standards, lays a solid foundation for large-scale clinical application, and is expected to revolutionize the field of bone implant medical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical titanium alloy technology, specifically relating to a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides and its preparation method. Background Technology

[0002] In the biomedical field, the development of bone implant materials has always been a key research direction in medicine and materials science. With the increasing aging population and the growing number of patients with various traumas and bone diseases, the demand for high-performance bone implant materials is becoming increasingly urgent. Traditional bone implant materials, such as stainless steel, cobalt-based alloys, and traditional porous titanium, while meeting basic clinical needs to some extent, have revealed many problems during long-term use, severely limiting their application effectiveness in complex clinical scenarios.

[0003] Traditional stainless steel and cobalt-based alloy implant materials suffer from poor biocompatibility, triggering varying degrees of immune and inflammatory responses in the body. The slow release of metal ions may not only cause allergic reactions in surrounding tissues but also potentially harm normal physiological functions. Furthermore, the elastic modulus of these materials differs significantly from that of human bone tissue, easily leading to stress shielding effects. This can cause abnormal stress on the implant site, resulting in bone resorption and decreased bone strength, ultimately affecting the long-term stability and osseointegration of the implant.

[0004] While traditional porous titanium materials offer some improvements in biocompatibility and osteoconductivity, their insufficient antibacterial properties remain a key factor limiting their clinical application. During implantation surgery, bacteria can easily adhere to the material surface, leading to implant-related infections. Once infection occurs, it can not only cause surgical failure but also trigger a series of serious complications, such as osteomyelitis and sepsis, causing significant suffering and financial burden to patients. Furthermore, traditional porous titanium materials lack effective loading and release mechanisms for drugs or bioactive substances, failing to meet the synergistic therapeutic needs of bone repair and anti-infection under complex physiological conditions.

[0005] In the process of bone repair, promoting osteoblast migration and proliferation, achieving precise release of antimicrobial substances, and enhancing immune regulation are key elements for achieving efficient bone repair. Current bone implant materials struggle to simultaneously meet these complex functional requirements. For example, existing materials cannot effectively control the release rate of antimicrobial agents; insufficient release in the early stages may lead to poor antimicrobial effects, while excessive release in later stages may cause cytotoxicity. Simultaneously, there is a lack of effective means to regulate immune function, making it difficult to fully stimulate the body's own immune defense mechanisms to promote bone repair and fight infection.

[0006] With the continuous advancement of medical technology, patients have increasingly higher performance requirements for bone implant materials. They not only expect materials to possess good biocompatibility and mechanical properties, but also hope that they can play a synergistic role in anti-infection, promoting bone integration, and immune regulation, achieving more precise and efficient treatment results. Therefore, developing a novel bone implant material with multi-scale structural design, intelligent release system, and photothermal synergistic mechanism to solve the aforementioned problems of traditional materials has become an important issue that urgently needs to be addressed in the field of biomedical materials. Summary of the Invention

[0007] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides. This multi-level microporous titanium alloy, with its innovative loading design and photothermal synergistic mechanism, effectively inhibits bacterial growth, significantly reducing the risk of infection during implantation surgery. Its excellent stability and safety ensure long-term stable function in vivo, meeting the performance requirements for bone implant materials.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides, characterized in that the method includes the following steps: Step 1: Substrate preparation: Selective laser melting is used to form a honeycomb structure titanium alloy substrate; Step 2, Surface modification: The titanium alloy substrate formed in Step 1 is subjected to surface etching and micro-arc oxidation in sequence to obtain a surface-modified titanium alloy substrate. Step 3, Functionalization: A copper selenide film is deposited on the surface-modified titanium alloy substrate obtained in Step 2 and loaded with pH-responsive antimicrobial peptides to obtain a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides.

[0009] This invention utilizes selective laser melting to easily provide a honeycomb structure for a titanium alloy matrix, achieving high forming precision and controllable structure. It precisely controls the pore size of the honeycomb structure to 200μm~400μm and the wall thickness to 60μm~100μm, achieving a balance between pore connectivity and mechanical support. This solves the problems of uneven pore size and poor connectivity in traditional porous titanium preparation, and also provides excellent mechanical properties. During the forming process, the powder is completely melted, resulting in a matrix density greater than 99%. Combined with subsequent stress annealing, the residual stress is reduced from 215MPa to 58MPa, and the elastic modulus can match that of human bone (10GPa~30GPa), avoiding the stress shielding effect of traditional materials. Furthermore, it boasts high powder utilization, adaptability to complex shapes, and supports personalized customization. It can be directly formed according to the patient's bone defect morphology without subsequent processing, reducing material waste. Simultaneously, it meets the needs of preparing complex orthopedic implants, such as irregular wound repair devices.

[0010] This invention constructs a micron-level rough surface through surface etching, achieving a surface roughness Sa of 1.2±0.3μm after etching. This increases the specific surface area, providing anchoring points for osteoblast adhesion and migration, thus solving the problem of insufficient cell adhesion caused by the smooth surface of traditional materials. Furthermore, it removes surface impurities and oxide layers. After selective laser melting, the substrate surface still contains oxide scale and residual powder. Surface etching selectively dissolves these impurities while avoiding excessive corrosion of the substrate, thus improving surface activity. In addition, it enhances the bonding strength of subsequent processes. The rough surface forms a physical interlocking structure, increasing the bonding strength between the micro-arc oxide film layer and the substrate by more than 30%, and providing sufficient adsorption sites for subsequent copper selenide film deposition and antimicrobial peptide loading.

[0011] This invention utilizes micro-arc oxidation to form a multifunctional oxide film, creating a dense oxide film of 8μm~10μm thickness containing Si and P elements on the surface of titanium alloys. This increases the hardness to over 500HV and reduces wear resistance by 40%, solving the problem of insufficient corrosion resistance in traditional titanium alloys. Furthermore, it constructs a mesopore control system, forming secondary mesopores of 5nm~50nm in the oxide film. The Cu content is precisely controlled by adjusting the surface zeta potential to -25mV. 2+ The sustained-release rate, with a cumulative release of 15±2ppm over 7 days, avoids cytotoxicity caused by burst ion release. In addition, the optimized bioactive environment, with Si and P elements in the oxide film promoting hydroxyapatite deposition, and a Ca / P ratio close to 1.67 after 7 days of SBF immersion, enhances the material's mineralization capacity and lays the foundation for osseointegration.

[0012] This invention utilizes a copper selenide (CuSe) film to form a photothermal antibacterial core carrier. CuSe exhibits excellent near-infrared photothermal conversion efficiency, heating to 48±2℃ after 10 minutes of 808nm laser irradiation. This localized high temperature directly disrupts bacterial cell membranes, as observed under electron microscopy, leading to bacterial fold rupture. Simultaneously, it enhances photocatalytic activity, promotes reactive oxygen species (ROS) generation, and possesses synergistic ion antibacterial and immunomodulatory effects, with the slow release of Cu... 2+ The minimum inhibitory concentration (MIC) of 8 μg / mL can disrupt bacterial metabolism. Se can promote macrophage polarization towards the M1 type, increase the CD86+ ratio to 65%, enhance the body's innate immunity, reduce the levels of inflammatory factors IL-6 (67%) and TNF-α (48%). In addition, it has long-term stability assurance, with the membrane layer firmly bonded to the substrate and no shedding in the wear resistance test. After 12 months of accelerated aging, the residual rate of antibacterial elements is greater than 85%, ensuring long-term antibacterial effect.

[0013] This invention utilizes pH-responsive antimicrobial peptides for targeted and intelligent release. PLGA microspheres slowly release the peptides under normal physiological conditions (pH=7.4), with a 24-hour release rate of 25%. Degradation is accelerated at the infection site (pH<6.5), with a 24-hour release rate exceeding 90% at pH=6.0. This addresses the problems of insufficient early release and excessive late release in traditional antimicrobial agents, and synergistically enhances the antimicrobial effect. The antimicrobial peptide (MIC=4.1μg / mL) and Cu... 2+ After forming a composite system, the MIC decreased to 1.9 μg / mL, the inhibition zone diameter reached 21.5 mm, and the bactericidal rate against Escherichia coli and Staphylococcus aureus was >99%. In addition, it has high biosafety, the PLGA microspheres are biodegradable, have no residual toxicity, and the cell survival rate after loading with antimicrobial peptides is still greater than 90% (CCK-8 test), which meets the ISO10993 biosafety standard.

[0014] The photothermal synergistic antibacterial mechanism of this invention: Under near-infrared light irradiation at 808 nm, the CuSe film achieves highly efficient antibacterial activity through the following synergistic pathways: Direct photothermal sterilization: CuSe converts light energy into heat energy, raising the surface temperature of the material to 45℃~50℃. This temperature can destroy the lipid bilayer of the bacterial cell membrane. Electron microscopy reveals bacterial wrinkling and rupture, leading to bacterial death; Enhanced ion antibacterial activity: High temperature promotes the release of Cu from the CuSe film. 2+ And Cu under high temperature environment 2+ The efficiency of disrupting bacterial metabolic enzymes was improved, reducing the MIC from 8 μg / mL to 2 μg / mL; ROS generation was promoted, with the photothermal effect activating the photocatalytic performance of CuSe, reacting with H2O2 produced by bacterial metabolism to generate ROS, such as •OH. ROS can oxidize bacterial DNA and proteins, further enhancing the bactericidal effect; the release of antimicrobial peptides was accelerated: the high temperature, combined with the acidic environment of the infection site (pH < 6.5), accelerated the degradation of PLGA microspheres, resulting in the rapid release of antimicrobial peptides, which reacted with Cu... 2+ An ion-peptide complex antibacterial system is formed, and the diameter of the inhibition ring is increased to 21.5 mm.

[0015] The preparation method of the above-mentioned multi-level microporous titanium alloy loaded with nano-copper selenide antibacterial peptides is characterized in that the selective laser melting process in step one is as follows: using Ti-6Al-4V-0.5Mo pre-alloy powder with a particle size of 10μm~50μm and an oxygen mass content of less than 0.15%, forming is carried out in an Ar gas environment with an oxygen content of no more than 50ppm using laser power of 200W~300W, scanning speed of 800mm / s~1200mm / s, and layer thickness of 20μm~50μm. The scanning strategy adopts a 4mm~6mm×4mm~6mm checkerboard fractal filling and a rotation angle of 60°~75°. After forming, stress annealing is performed to obtain a titanium alloy matrix with a honeycomb structure. This invention controls the particle size of the pre-alloyed powder to ensure powder flow rate, adapting to the requirements of selective laser melting for layer-by-layer powder spreading. This prevents the shortcomings of poor flowability due to excessively small particle size and low forming accuracy due to excessively large particle size. By controlling the oxygen content, it avoids the oxidation of titanium alloy to form brittle Ti2O3, ensuring the compositional stability of the titanium alloy matrix, guaranteeing the mechanical properties of the matrix, and achieving a compressive strength retention rate of greater than 89% and biocompatibility. By controlling the laser power, scanning speed, and layer thickness, the power and scanning speed are matched to ensure complete powder melting without splashing, and the layer thickness controls forming efficiency and accuracy. This combination of parameters results in a matrix density of greater than 99%. By controlling the scanning strategy, the laser energy is dispersed, reducing thermal stress concentration, avoiding forming cracks, and improving structural uniformity.

[0016] The above-mentioned method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antibacterial peptides is characterized in that the stress annealing temperature is 720℃~780℃, and the holding time is 1.5h~2.5h; after stress annealing, a segmented cooling method is adopted, specifically furnace cooling to 280℃~320℃ followed by air cooling. This invention effectively eliminates residual stress by controlling the stress annealing parameters to make the temperature range close to the recrystallization temperature of the titanium alloy, avoids excessive temperature differences that could generate new stress through segmented cooling, and simultaneously preserves the integrity of the honeycomb structure.

[0017] The above-mentioned method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is characterized in that the surface etching process in step two is as follows: etching is performed using an etching solution containing HF, HNO3 and H2O, followed by ultrasonic cleaning to form a rough surface.

[0018] The above-mentioned method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antibacterial peptides is characterized in that the volume fraction of HF in the etching solution is 1.5%~3%, the volume fraction of HNO3 is 8%~12%, and the balance is H2O; the etching temperature is 20℃~30℃, the stirring speed is 120r / min~180r / min, and the time is 4min~6min; the ultrasonic cleaning frequency is 35kHz~45kHz, and the time is 8min~12min. This invention controls the composition of the etching solution, where HF acts as the main etchant, selectively dissolving the TiO2 oxide layer and part of the titanium alloy matrix on the surface of the titanium alloy. Through the chemical reactions TiO2+4HF→TiF4↑+2H2O; Ti+4HF→TiF4↑+2H2↑, micron-level pits are formed on the surface, ultimately constructing a rough surface that provides physical anchors for the subsequent micro-arc oxide film layer. HNO3 acts as both an oxidant and a corrosion inhibitor, dually regulating the etching process. The oxidation effect is to remove the Ti produced during etching. 3+ Ion oxidation to Ti 4+ Ions, 3Ti 3+ +NO3 - +4H + →3Ti 4+ +NO↑+2H2O, avoid Ti 3 The surface deposition leads to uneven etching. The corrosion inhibition effect is to form an extremely thin TiO2 oxide passivation film on the titanium alloy surface, which slows down the excessive corrosion of the substrate by HF. If the HNO3 content is less than 8%, it will result in an excessively rough surface Sa>2μm and excessive weight loss of the substrate. If it is higher than 12%, the etching will be insufficient Sa<1μm, and an effective rough surface cannot be formed. By controlling the parameters of ultrasonic cleaning, etching residues can be effectively removed without residual liquid corrosion. The time takes into account both the cleaning effect and the protection of the surface structure.

[0019] The above-mentioned method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is characterized in that the micro-arc oxidation process in step two is as follows: micro-arc oxidation is performed using an electrolyte containing Na2SiO3, KOH and Na3PO4 to obtain an oxide film.

[0020] The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antibacterial peptides is characterized in that the electrolyte contains 6 g / L to 10 g / L of Na2SiO3, 1.5 g / L to 3 g / L of KOH, and 4 g / L to 6 g / L of Na3PO4; the pH value of the electrolyte is 11 to 12; the forward voltage of the micro-arc oxidation is 260 V to 300 V; the frequency of the micro-arc oxidation is 400 Hz to 600 Hz, the duty cycle is 15% to 25%, and the time is 12 min to 18 min; the temperature of the electrolyte during the micro-arc oxidation is controlled below 38 °C. This invention controls the composition of the electrolyte, with Na2SiO3 content ranging from 6 g / L to 10 g / L as a hardness enhancer. During micro-arc discharge, Si elements are incorporated into the oxide film to form TiSiO4 silicon-titanium composite oxide, increasing the film hardness from 300 HV of pure titanium oxide film to over 500 HV, improving wear resistance by 45%, and reducing wear rate. This solves the problem of surface wear caused by friction after traditional titanium alloy implantation. KOH content ranging from 1.5 g / L to 3 g / L acts as a conductivity modifier, controlling the conductivity to 15 mS / cm to 25 mS / cm by adjusting the electrolyte ion concentration. This ensures stable micro-arc discharge, avoids uneven film thickness caused by voltage fluctuations, and prevents insufficient conductivity (too low for KOH content, hindering continuous micro-arc formation) and excessive conductivity (too high for KOH content, leading to localized overheating and film cracking). The Na3PO4 content is also used. With a content of 4g / L~6g / L as a bioactive regulator, phosphorus (P) incorporated into the oxide film can induce hydroxyapatite (HA) deposition in simulated body fluid (SBF). After 7 days, the Ca / P ratio reaches 1.65±0.03, close to the 1.67 of natural bone HA, significantly improving the osteoconductivity of the material and providing a chemical basis for osseointegration. By controlling the pH value, a strongly alkaline environment is provided, which is conducive to micro-arc discharge and the formation of a stable oxide film, preventing uneven discharge and decreased film density caused by excessively high or low pH values. By controlling the forward voltage, frequency, and duty cycle, where the voltage ensures micro-arc formation and the frequency and duty cycle regulate the discharge intensity, the film is prevented from breaking and a uniform and dense oxide film is formed. By controlling the time, the film thickness is controlled to 8μm~10μm to prevent problems such as easy detachment due to excessive thickness and insufficient performance due to excessive thinness. By controlling the temperature of the electrolyte, the decomposition of the electrolyte is prevented, ensuring the stability of the film composition.

[0021] The above-mentioned method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antibacterial peptides is characterized in that the process of depositing the copper selenide film in step three is as follows: Reactive magnetron sputtering is used to deposit the copper selenide film under the conditions of Ar gas and H2Se gas being introduced, with the Ar gas introduction rate being 40 sccm~60 sccm and the H2Se gas introduction rate being 4 sccm~6 sccm, using a Cu target sputtering power of 120W~180W and a Se target sputtering power of 80W~120W, followed by vacuum annealing; the temperature of the titanium alloy substrate during the reactive magnetron sputtering is 280℃~320℃, and the deposition temperature during the reactive magnetron sputtering is... The accumulation rate is 1.5 nm / min to 2.5 nm / min, the vacuum annealing temperature is 380℃ to 420℃, the holding time is 0.8 h to 1.2 h, and the thickness of the copper selenide film layer is 80 nm to 120 nm. The process of loading the pH-responsive antimicrobial peptide is as follows: antimicrobial peptides are loaded onto PLGA microspheres to obtain antimicrobial peptide carrier microspheres, and then the antimicrobial peptide carrier microspheres are loaded through impregnation-vacuum drying. The particle size of the antimicrobial peptide carrier microspheres is 1.5 μm to 2.5 μm, and the drug loading of the antimicrobial peptide carrier microspheres is 8% to 12%. The impregnation time in the impregnation-vacuum drying process is 20 h to 28 h.This invention controls the sputtering power of Cu and Se targets, ensuring a Cu:Se ratio of approximately 1:1 to form a pure CuSe phase and prevent impurities from affecting photothermal performance. By precisely supplying a selenium source through the introduction of H2Se gas, which acts as a reactive selenium source, the selenium reacts with Cu atoms generated by sputtering on the titanium alloy substrate, resulting in a Cu + Se → CuSe phase. This ensures the film is a pure CuSe phase, preventing compositional inhomogeneity due to insufficient selenium source and guaranteeing photothermal performance and ion release stability. Furthermore, by adjusting the H2Se flow rate, the stoichiometry of the film is precisely controlled to maintain a Cu:Se atomic ratio of 1:1. This ratio is crucial for CuSe's excellent near-infrared photothermal conversion efficiency, preventing excessive Se from increasing film brittleness and excessive Cu from decreasing photothermal performance. Additionally, it inhibits film oxidation; the H2Se creates a reducing atmosphere in the sputtering chamber, preventing carbon oxidation. The oxidation of u and se atoms prevents the formation of ineffective components such as CuO and SeO2. Simultaneously, it works with Ar gas to maintain an inert environment within the cavity, ensuring film density and a porosity of less than 2%. Controlling the substrate temperature enhances atomic diffusion and strengthens film adhesion. Controlling the deposition rate ensures a dense, pore-free film. Controlling vacuum annealing parameters eliminates internal stress. During reactive magnetron sputtering, the rapid atomic deposition rate easily generates internal stress, leading to film detachment. Vacuum annealing releases this stress through atomic diffusion, increasing the film-substrate bonding strength by 30% and improving crystallinity. Before annealing, the film is partially amorphous; after annealing, a highly crystalline CuSe phase is formed, increasing photothermal conversion efficiency from 35% to 48%. The heating rate is increased by 20% at the same laser power. Controlling the ion release rate reduces defects such as micropores and dislocations after annealing. 2+ The release is more gradual, with a stable cumulative release of 15±2ppm over 7 days, avoiding burst ion release (>25ppm) caused by defects before annealing, reducing the risk of cytotoxicity, promoting film crystallization, improving photothermal conversion efficiency, and preventing the deficiencies of excessively large crystals due to excessively high temperatures and insufficient crystallization due to excessively low temperatures; the effective heating of 45℃~50℃ is achieved by controlling the thickness of the copper selenide film layer, and the bonding is firm, preventing the deficiencies of poor photothermal effect due to excessive thinness and easy detachment due to excessive thickness; by controlling the parameters of the loaded pH-responsive antimicrobial peptide, including particle size matching the mesoporous structure, drug loading to ensure antimicrobial dosage, and impregnation time to ensure loading rate greater than 85%, long-lasting antimicrobial effect is achieved.

[0022] In addition, the present invention provides a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides, characterized in that the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is prepared by the above-described method.

[0023] The above-mentioned multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is characterized in that the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is used in the preparation of orthopedic implants or wound repair devices.

[0024] Compared with the prior art, the present invention has the following advantages: 1. The multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides provided by this invention has achieved remarkable results as a novel bone implant material preparation scheme with broad application prospects. In terms of antibacterial properties, the innovative loading design and photothermal synergistic mechanism can effectively inhibit bacterial growth, significantly reduce the infection risk of implantation surgery, and provide patients with a safer treatment option. In terms of promoting bone integration, the multi-scale structure and ion sustained-release design can accelerate bone tissue growth and material fusion, help patients recover faster, and reduce long-term complications. Its immunomodulatory function enhances the body's immunity through element regulation and optimizes the bone repair microenvironment, opening up new ideas for the treatment of complex diseases. Its good stability and safety ensure that the material can play a stable role in vivo for a long time, meet strict standards, lay a solid foundation for large-scale clinical application, and is expected to revolutionize the field of bone implantation medicine and benefit more patients.

[0025] 2. Superior Antibacterial Properties: This invention achieves synergistic antibacterial effects through a dual design of antibacterial substance deposition and antibacterial peptide loading. Under near-infrared photothermal action, the local temperature rises to 45℃~50℃, significantly enhancing the antibacterial photocatalytic activity. The released reactive oxygen species can effectively destroy bacterial structures. Simultaneously, the released Cu... 2+ It forms an ion-peptide complex antibacterial agent with antimicrobial peptides, further enhancing the antibacterial effect. Animal experiments showed that after 8 weeks of implantation in the infection model group, the bacterial load decreased by 3.8 logCFU / mL. Compared with traditional materials, it can more effectively inhibit bacterial infection at the implantation site, reduce the risk of postoperative infection, and improve the success rate of surgery.

[0026] 3. Highly efficient osteointegration capability: The multi-scale structure constructed during the matrix preparation process of this invention plays a crucial role. Primary pores provide ideal channels for osteoblast migration, promoting osteoblast adhesion and growth on the material surface and accelerating bone tissue formation. Secondary mesopores precisely control Cu through surface zeta potential (-25mV). 2+ Sustained release provides a suitable microenvironment for osteocyte proliferation and differentiation. Animal experiments show that it increases bone integration rate by 42%, effectively enhances the bonding strength between implant material and bone tissue, shortens bone healing time, and facilitates rapid postoperative recovery for patients.

[0027] 4. Excellent immunomodulatory function: The introduction of Se element in this invention is a major innovation. Se element can promote macrophage polarization, enhance innate immunity, enable the body's own immune defense mechanism to play a better role, and reduce inflammatory response. Experimental data show that the inflammatory factor IL-6 decreased by 67%, effectively reducing the degree of inflammation at the implantation site, creating a good immune microenvironment for bone repair, and reducing the risk of complications caused by inflammation.

[0028] 5. Excellent stability and safety: The material strength retention rate is greater than 92% after 12 months of accelerated aging test by PBS immersion, ensuring stable mechanical properties during long-term use and meeting the load-bearing requirements of bone implant materials. The surface antibacterial agent residue rate is greater than 85%, ensuring long-term antibacterial effect. At the same time, there is no abnormal release of toxic elements, strictly following medical device standards (ISO5832-2, ISO10993), fully guaranteeing biosafety and giving patients greater peace of mind.

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0030] Figure 1 The image shows the in vitro biocompatibility (CCK-8) results of the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides obtained in Example 1 of this invention.

[0031] Figure 2 The image shows the results of an in vitro antibacterial experiment on a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides obtained in Example 1 of this invention.

[0032] Figure 3 This is an electron microscope image of the multi-level microporous titanium alloy carrying nano-copper selenide antimicrobial peptides obtained in Example 1 of the present invention after co-culturing with Staphylococcus aureus for 24 hours.

[0033] Figure 4 The image shows the morphology of the multi-level microporous titanium alloy carrying nano-copper selenide antimicrobial peptides obtained in Example 1 of this invention after co-culturing with Escherichia coli for 24 hours under an electron microscope. Detailed Implementation

[0034] Example 1 This embodiment includes the following steps: Step 1: Matrix Preparation TI-6AL-4V-0.5MO pre-alloying powder conforming to ASTM F3001 standard was selected, with a particle size range of 15μm to 45μm and an oxygen content of less than 0.15%. Before use, the pre-alloying powder was placed under a vacuum degree not exceeding 1×10⁻⁶. -2 In a vacuum drying oven at 80°C for 4 hours, the raw material powder was dried. After drying, the powder flowability was tested using a Hall effect flow meter. The flow rate was required to be no more than 35 s / 50 g to ensure the smooth progress of subsequent molding processes. Using an EOS M290 or equivalent selective laser melting (SLM) device with dried pre-alloyed powder as raw material, Ar gas with a purity of not less than 99.999% and an oxygen content of not more than 50ppm is used as a protective atmosphere to prevent oxidation of the alloy powder during the forming process. The laser power is set to 250W, the scanning speed to 900mm / s, the layer thickness to 30μm, and the scanning strategy to adopt checkerboard fractal filling with a partition size of 5×5mm and a rotation angle of 67°. Through careful design of the SLM scanning path, a honeycomb structure titanium alloy substrate is directly formed. The pore size of this structure is 250μm~350μm and the wall thickness is 70μm~90μm, avoiding the complex process of post-processing interference technology required in traditional processes. The formed honeycomb structure titanium alloy substrate was subjected to stress annealing under Ar gas protection. It was placed in a high-temperature furnace, heated to 750°C and held for 2 hours, and then cooled to 300°C with the furnace. After that, it was taken out and cooled naturally in the air to eliminate the residual stress generated during the forming process and improve the structural stability of the substrate, thus obtaining the annealed formed honeycomb structure titanium alloy substrate. Step 2, Surface Modification: An etching solution was prepared by diluting 2% HF and 10% HNO3 by volume with deionized water. The annealed and formed honeycomb structure titanium alloy substrate obtained in step one was immersed in the etching solution. The etching was carried out at 23℃~27℃ using a magnetic stirrer at a speed of 150 r / min for 5 min. After etching, the substrate was immediately placed in deionized water and then ultrasonically cleaned at 40 kHz for 10 min to remove residual etching solution. Finally, the substrate was dried with nitrogen to obtain the surface etched substrate. Scanning electron microscopy (SEM) showed that the surface etched substrate formed a micron-level rough surface with a surface roughness parameter Sa = 1.2 ± 0.3 μm. An electrolyte containing 8g Na₂SiO₃, 2g KOH, and 5g Na₃PO₄ per liter was prepared, and the pH was adjusted to 11.3–11.7. Using a surface-etched substrate as the anode, micro-arc oxidation was performed at a forward voltage of 280V, a frequency of 500Hz, and a duty cycle of 20% for 15 minutes. During the treatment, the electrolyte temperature was controlled to not exceed 35℃ using a water-cooling circulation system. The resulting surface-modified titanium alloy substrate had an oxide film thickness of 8μm–10μm. The surface zeta potential of the surface-modified titanium alloy substrate was measured to be -25mV in 0.1M KCl solution using a Zeta potentiometer. III. Functionalization: Reactive magnetron sputtering was employed using Cu and Se targets. During sputtering, the Ar gas flow rate was 50 sccm and the H2Se gas flow rate was 5 sccm. The temperature of the surface-modified titanium alloy substrate obtained in step two was controlled at 300℃. The sputtering power of the Cu target was 150W, and the sputtering power of the Se target was 100W. The deposition rate was 2 nm / min, resulting in a copper selenide film with a thickness of 100 nm. After deposition, vacuum annealing was performed under Ar protection at 400℃ for 1 h to obtain the titanium alloy substrate with the deposited copper selenide film. X-ray diffraction (XRD) confirmed that the copper selenide film in the titanium alloy substrate formed a CuSe phase. Loaded antimicrobial peptides: Polylactic acid-glycolic acid copolymer (PLGA) microspheres with a particle size of 2 μm were prepared as antimicrobial peptide carriers using an emulsion solvent evaporation method, with a drug loading of 10%. The microspheres were dispersed in phosphate buffered saline (PBS) at pH 7.4, and a titanium alloy substrate with a deposited copper selenide film was immersed in the solution for 24 h and then vacuum dried to obtain a hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides. The antimicrobial peptide loading rate in the hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides was determined by fluorescent labeling, and the result was 85±5%. In vitro release experiments verified that the microspheres degraded rapidly at pH less than 6.5, and the release rate exceeded 90% after 24 h at pH 6.0, showing good pH response characteristics.

[0035] The hierarchical microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides prepared in this embodiment was subjected to the following performance tests: 1. Matrix molding quality test: Test Name: Cellular Structure Dimensional Test, Residual Stress Test (XRDsin) 2 (ψ method), compressive strength test, fatigue performance test, wear resistance test; Results: The pore size of the honeycomb structure was 298±14 μm, the wall thickness was 81±3 μm, the residual stress before annealing was 215±13 MPa, and the residual stress after annealing decreased to 58±5 MPa; the compressive strength was 380±25 MPa; the elastic modulus was 25±3 GPa; the yield strength was 220±18 MPa, and the yield strength was 1×10⁻⁶ MPa. 6 No crack propagation occurred after the second cycle, and the fracture surface exhibited ductile fracture characteristics. Dimples were visible in the SEM image, and the wear rate was 0.8 × 10⁻⁶. -6 mm 3 / (N•m), the wear rate of pure titanium material is 1.45×10 -6 mm 3 / (N•m) decreased by 45%; Description: The uniform honeycomb structure ensures even distribution of cyclic loads, avoiding localized stress concentration and meeting the dynamic load tolerance requirements of long-term implants such as artificial joints. Combined with the dense oxide film formed by subsequent micro-arc oxidation, it significantly improves surface wear resistance and reduces material loss caused by friction due to joint movement after implantation. Stress annealing effectively eliminates residual stress during molding, preventing matrix cracking. The residual stress is reduced from 215±13MPa to 58±5MPa, effectively preventing matrix cracking during molding and ensuring that the compressive strength meets the load-bearing requirements of orthopedic implants. The compressive strength of human bone is approximately 100MPa~200MPa. The elastic modulus matches that of human bone, 10GPa~30GPa, avoiding stress shielding effects.

[0036] 2. Surface modification property test: Test names: Surface roughness test (SEM), oxide film characteristic test (film thickness, zeta potential, mesopore size), inductively coupled plasma mass spectrometry (ICP-MS) test; Results: After etching, the surface roughness Sa = 1.2 ± 0.3 μm, the micro-arc oxide film thickness was 8 μm ~ 10 μm, the zeta potential was -25 mV, and the mesopore size was 5 nm ~ 50 nm. Cu content was measured within 7 days. 2+ The cumulative release amount was 15±2 ppm; Explanation: A rough surface adapted for osteoblast adhesion is formed. The oxide film is dense and possesses ion-regulating capabilities, laying the foundation for subsequent functionalization treatments. This verifies that the mesopores of the film layer affect Cu through zeta potential. 2+ The regulatory effect of release.

[0037] 3. Functional layer characteristic testing Test names: Composition and thickness test of copper selenide film (XRD, film thickness gauge), antimicrobial peptide loading and release test (fluorescent labeling method, HPLC); Results: The membrane was a pure CuSe phase with a thickness of 100 nm; the antimicrobial peptide loading rate was 85±5%; the release rate after 24 h was >90% at pH=6.0 and 25±4% at pH=7.4. Note: The copper selenide film has pure components and controllable thickness, and the antimicrobial peptides achieve pH-responsive targeted release, avoiding ineffective release and cytotoxicity.

[0038] 4. Photothermal performance test Test Names: Photothermal Heating Test (Infrared Thermal Imager), Photothermal Conversion Efficiency Test; Result: 808nm laser (1W / cm) 2 After irradiation for 10 minutes, the surface temperature rises to 48±2℃; the photothermal conversion efficiency meets the clinical antibacterial requirements.

[0039] Note: It has excellent near-infrared photothermal conversion capability, which can achieve effective local sterilization temperature.

[0040] 5. Antibacterial performance test Test names: In vitro antibacterial test (plate count method), minimum inhibitory concentration (MIC) test, biofilm inhibition test (crystal violet staining method); Results: The bactericidal rate against both Escherichia coli and Staphylococcus aureus was greater than 99%; Cu 2+ The MIC of the complex system with antimicrobial peptides was 1.9 ± 0.2 μg / mL, which is less than that of Cu alone. 2+ The MIC of the antimicrobial peptide alone was 8.3 ± 0.5 μg / mL, and the MIC of the antimicrobial peptide alone was 4.1 ± 0.3 μg / mL; the biofilm formation was reduced by 70% compared with the control group. Explanation: By leveraging the synergistic effects of photothermal, ion, and antimicrobial peptides, highly efficient sterilization and biofilm inhibition are achieved, addressing the shortcomings of traditional materials in terms of antibacterial efficacy.

[0041] 6. Biocompatibility testing Test names: Cell viability test (CCK-8 assay), osteoblast migration test (Transwell assay); Results: Cell viability was greater than 90%, with no significant difference compared to pure titanium; osteoblast (MC3T3-E1) migration rate was increased by 35% compared to pure titanium; Description: It is non-cytotoxic, and its multi-level microporous structure and surface properties promote osteoblast migration, creating conditions for bone integration. The primary pores provide unobstructed migration channels for osteoblasts, solving the problem of difficult cell migration caused by the small pore size of traditional porous titanium. This lays a key foundation for subsequent bone tissue inward growth and improved bone integration rate.

[0042] 7. Osteointegration Performance Testing (In vivo Animal Experiments) Test Name: Bone Volume Fraction (BV / TV) Test (Micro-CT); Results: The BV / TV ratio in the experimental group was 44.7 ± 2.8%, while that in the control group (pure titanium) was 31.5 ± 3.6%. Note: The multi-level structure and ion sustained-release design significantly improve the osseointegration rate and enhance the bonding strength between the implant and bone tissue.

[0043] 8. Immune regulation test (in vivo animal experiment) Test names: Inflammatory factor detection (ELISA), macrophage polarization test (flow cytometry), bacterial load; Results: In the experimental group, IL-6 decreased by 52% and TNF-α decreased by 48% (p<0.05); the proportion of CD86+ macrophages increased to 65%; and the bacterial load in the experimental group decreased by 2.1 logCFU / mL compared with the control group, which was statistically significant (p<0.01). Note: Se promotes macrophage polarization, effectively reduces inflammatory response at the implantation site, and optimizes the bone repair microenvironment, thereby achieving immune regulation. The bacterial load in the experimental group was 2.1 logCFU / mL lower than that in the control group, and the difference was statistically significant (p<0.01).

[0044] 9. Long-term stability test Test names: Accelerated aging test (PBS immersion for 12 months), mechanical property test (ISO5832-2 standard), antimicrobial agent residue test (XPS), biosafety test; Results: Compressive strength retention rate was 89±3%; the surface Cu:Se atomic ratio remained at 1:1 with an error of ±5%; Cu in the immersion solution was detected by ICP-MS. 2+ The release amount is less than 0.1 ppm; Note: The material exhibits stable mechanical properties during long-term use, sufficient residual antibacterial elements, and no abnormal release of toxic ions, indicating that it has good biocompatibility and meets the requirements for long-term clinical implantation.

[0045] 10. Mineralization capacity test Test Name: In Vitro Mineralization Test (SBF Immersion for 7 Days, EDS Detection); Results: Hydroxyapatite was deposited on the material surface, with a Ca / P ratio of 1.65 ± 0.03; Note: It has good in vitro mineralization ability, which is beneficial to subsequent bone tissue growth and fusion.

[0046] 11. pH response Test Name: High Performance Liquid Chromatography (HPLC) Test Results: Under simulated infection microenvironment (pH 6.0), the release of antimicrobial peptides was determined by high performance liquid chromatography (HPLC), and the results showed that the release was not less than 80%. Results: The effectiveness of the pH response mechanism was confirmed.

[0047] All test results focused on the four core functions of antibacterial activity, bone integration, immune regulation, and stability, verifying the inventiveness and practicality of this invention.

[0048] Figure 1 This is a graph showing the in vitro biocompatibility (CCK-8) results of the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides obtained in this embodiment. Figure 1 Blank represents the blank control group, Ti represents the pure titanium control group, and Ti@Se+AMPs represents a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides. Figure 1 As can be seen, the absorbance value of the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is almost the same as that of pure titanium, and the cell survival rate reaches more than 90%, showing good biocompatibility.

[0049] Figure 2 This figure shows the results of the in vitro antibacterial experiment on the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides obtained in this embodiment. Figure 2 Blank represents the blank control group, Ti represents the pure titanium control group, and Ti@Se+AMPs represents a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides. Figure 2 As can be seen, the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides exhibits excellent antibacterial effects, with a bactericidal rate of over 99% against Escherichia coli and Staphylococcus aureus.

[0050] Figure 3 The image shows the morphology of the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides obtained in this embodiment after co-culturing with Staphylococcus aureus for 24 h under an electron microscope. Figure 3 Blank represents the blank control group, Ti represents the pure titanium control group, and Ti@Se+AMPs represents a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides. Figure 3 As can be seen from the data, compared with the control group, the bacterial cell membranes treated with the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides showed obvious wrinkles and even ruptures. This indicates that the multi-level microporous titanium alloy loaded with antimicrobial peptides can destroy bacterial cell membranes, thereby achieving a high-quality antibacterial effect.

[0051] Figure 4 The image shows the morphology of the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides obtained in this embodiment after co-culturing with Escherichia coli for 24 h under an electron microscope. Figure 4 Blank represents the blank control group, Ti represents the pure titanium control group, and Ti@Se+AMPs represents a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides. Figure 4 As can be seen from the data, compared with the control group, the bacterial cell membranes treated with the multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides showed obvious wrinkles and even ruptures. This indicates that the multi-level microporous titanium alloy loaded with antimicrobial peptides can destroy bacterial cell membranes, thereby achieving a high-quality antibacterial effect.

[0052] Example 2 This embodiment includes the following steps: Step 1: Matrix Preparation TI-6AL-4V-0.5MO pre-alloying powder conforming to ASTM F3001 standard was selected, with a particle size range of 20μm~50μm and an oxygen content of less than 0.15%. Before use, the pre-alloying powder was placed under a vacuum degree not exceeding 1×10⁻⁶. -2In a vacuum drying oven at 90°C for 5 hours, the raw material powder was dried. After drying, the powder flowability was tested using a Hall effect flow meter. The flow rate was required to be no more than 38 s / 50 g to ensure the smooth progress of subsequent molding processes. Using an EOS M290 or equivalent selective laser melting (SLM) device with dried pre-alloyed powder as raw material, Ar gas with a purity of not less than 99.999% and an oxygen content of not more than 50ppm is used as a protective atmosphere to prevent oxidation of the alloy powder during the forming process. The laser power is set to 280W, the scanning speed to 800mm / s, the layer thickness to 35μm, and the scanning strategy to adopt checkerboard fractal filling with a partition size of 6mm×6mm and a rotation angle of 75°. Through careful design of the SLM scanning path, a honeycomb structure titanium alloy substrate is directly formed. The pore size of this structure is 300μm~400μm and the wall thickness is 80μm~100μm, avoiding the complex process of post-processing interference technology required in traditional processes. The formed honeycomb structure titanium alloy substrate was subjected to stress annealing under Ar gas protection. It was placed in a high-temperature furnace, heated to 780℃ and held for 2.5 hours. Then it was cooled to 320℃ with the furnace and then taken out and allowed to cool naturally in the air. This eliminated the residual stress generated during the forming process and improved the structural stability of the substrate, resulting in the annealed formed honeycomb structure titanium alloy substrate. Step 2, Surface Modification: An etching solution was prepared by diluting 3% HF and 12% HNO3 by volume with deionized water. The annealed and formed honeycomb structure titanium alloy substrate obtained in step one was immersed in the etching solution and etched for 6 minutes at 180 r / min using a magnetic stirrer at a temperature of 26℃~30℃. After etching, it was immediately placed in deionized water and then ultrasonically cleaned at 45 kHz for 12 minutes to remove residual etching solution from the surface. Finally, it was dried with nitrogen to obtain the surface etched substrate. Scanning electron microscopy (SEM) showed that the surface etched substrate formed a micron-level rough surface. An electrolyte containing 10g Na2SiO3, 3g KOH and 6g Na3PO4 per liter was prepared, and the pH value was adjusted to 11.6~12.0. Using the surface-etched substrate as the anode, micro-arc oxidation was performed under the conditions of a forward voltage of 300V, a frequency of 600Hz and a duty cycle of 25% for 18min. During the treatment, the electrolyte temperature was controlled to not exceed 32℃ through a water cooling circulation system. After treatment, a surface-modified titanium alloy substrate was obtained. III. Functionalization: Reactive magnetron sputtering was employed using Cu and Se targets. During sputtering, the Ar gas flow rate was 60 sccm and the H2Se gas flow rate was 6 sccm. The temperature of the surface-modified titanium alloy substrate obtained in step two was controlled at 320℃. The sputtering power of the Cu target was 180W, and the sputtering power of the Se target was 120W. The deposition rate was 2.5 nm / min, resulting in a copper selenide film with a thickness of 120 nm. After deposition, vacuum annealing was performed under Ar protection at 420℃ for 1.2 h to obtain the titanium alloy substrate with the deposited copper selenide film. X-ray diffraction (XRD) confirmed that the copper selenide film in the titanium alloy substrate formed a CuSe phase. Loaded antimicrobial peptides: Polylactic acid-glycolic acid copolymer (PLGA) microspheres with a particle size of 2.5 μm were prepared as antimicrobial peptide carriers using an emulsion solvent evaporation method, with a drug loading of 12%. The microspheres were dispersed in phosphate buffered saline (PBS) at pH 7.4, and a titanium alloy substrate with a deposited copper selenide film was immersed in the solution for 28 h and then vacuum dried to obtain a hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides. The antimicrobial peptide loading rate in the hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides was determined by fluorescent labeling, and the result was 85±5%. In vitro release experiments verified that the microspheres degraded rapidly at pH less than 6.5, and the release rate exceeded 90% after 24 h at pH 6.0, showing good pH response characteristics.

[0053] Example 3 This embodiment includes the following steps: Step 1: Matrix Preparation TI-6AL-4V-0.5MO pre-alloying powder conforming to ASTM F3001 standard was selected, with a particle size range of 10μm to 40μm and an oxygen content of less than 0.15%. Before use, the pre-alloying powder was placed under a vacuum degree not exceeding 1×10⁻⁶. -2 In a vacuum drying oven at 70°C for 3 hours, the raw material powder was dried. After drying, the powder flowability was tested using a Hall effect flow meter. The flow rate was required to be no more than 32 s / 50 g to ensure the smooth progress of subsequent molding processes. Using an EOS M290 or equivalent selective laser melting (SLM) device with dried pre-alloyed powder as raw material, Ar gas with a purity of not less than 99.999% and an oxygen content of not more than 50ppm is used as a protective atmosphere to prevent oxidation of the alloy powder during the forming process. The laser power is set to 220W, the scanning speed to 1000mm / s, the layer thickness to 25μm, and the scanning strategy to adopt checkerboard fractal filling with a partition size of 4mm×4mm and a rotation angle of 60°. Through careful design of the SLM scanning path, a honeycomb structure titanium alloy substrate is directly formed. The pore size of this structure is 200μm~300μm and the wall thickness is 60μm~80μm, avoiding the complex process of post-processing interference technology required in traditional processes. The formed honeycomb structure titanium alloy substrate was subjected to stress annealing under Ar gas protection. It was placed in a high-temperature furnace, heated to 720°C and held for 1.5 hours. Then it was cooled to 280°C with the furnace and then taken out and allowed to cool naturally in the air. This eliminated the residual stress generated during the forming process and improved the structural stability of the substrate, resulting in the annealed formed honeycomb structure titanium alloy substrate. Step 2, Surface Modification: An etching solution was prepared by diluting 1.5% HF and 8% HNO3 by volume with deionized water. The annealed and formed honeycomb structure titanium alloy substrate obtained in step one was immersed in the etching solution. The etching was carried out at 20℃~24℃ using a magnetic stirrer at a speed of 120r / min for 4 minutes. After etching, the substrate was immediately placed in deionized water and then ultrasonically cleaned at 35kHz for 8 minutes to remove residual etching solution. Finally, the substrate was dried with nitrogen to obtain the surface etched substrate. Scanning electron microscopy (SEM) showed that the surface etched substrate formed a micron-level rough surface. An electrolyte containing 6g Na2SiO3, 1.5g KOH and 4g Na3PO4 per liter was prepared, and the pH value was adjusted to 11.0~11.4. Using the surface-etched substrate as the anode, micro-arc oxidation was performed under the conditions of a forward voltage of 260V, a frequency of 400Hz and a duty cycle of 15% for 12 minutes. During the treatment, the electrolyte temperature was controlled to not exceed 38℃ through a water cooling circulation system. After treatment, a surface-modified titanium alloy substrate was obtained. III. Functionalization: Reactive magnetron sputtering was employed using Cu and Se targets. During sputtering, the Ar gas flow rate was 40 sccm and the H2Se gas flow rate was 4 sccm. The temperature of the surface-modified titanium alloy substrate obtained in step two was controlled at 280℃. The sputtering power of the Cu target was 120W, and the sputtering power of the Se target was 80W. The deposition rate was 1.5 nm / min, ultimately yielding a copper selenide film with a thickness of 80 nm. After deposition, vacuum annealing was performed under Ar protection at 380℃ for 0.8 h to obtain the titanium alloy substrate with the deposited copper selenide film. X-ray diffraction (XRD) confirmed that the copper selenide film in the titanium alloy substrate formed a CuSe phase. Loaded antimicrobial peptides: Polylactic acid-glycolic acid copolymer (PLGA) microspheres with a particle size of 1.5 μm were prepared as antimicrobial peptide carriers using an emulsion solvent evaporation method, with a drug loading of 8%. The microspheres were dispersed in phosphate buffered saline (PBS) at pH 7.4, and a titanium alloy substrate with a deposited copper selenide film was immersed in the solution for 20 h and then vacuum dried to obtain a hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides. The antimicrobial peptide loading rate in the hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides was determined by fluorescent labeling, and the result was 85±5%. In vitro release experiments verified that the microspheres degraded rapidly at pH less than 6.5, and the release rate exceeded 90% after 24 h at pH 6.0, showing good pH response characteristics.

[0054] Example 4 This embodiment includes the following steps: Step 1: Matrix Preparation TI-6AL-4V-0.5MO pre-alloying powder conforming to ASTM F3001 standard was selected, with a particle size range of 18μm to 48μm and an oxygen content of less than 0.15%. Before use, the pre-alloying powder was placed under a vacuum degree not exceeding 1×10⁻⁶. -2 In a vacuum drying oven at 85°C for 4.5 hours, the raw material powder was dried. After drying, the powder flowability was tested using a Hall effect flow meter. The flow rate was required to be no more than 36 s / 50 g to ensure the smooth progress of subsequent molding processes. Using an EOS M290 or equivalent selective laser melting (SLM) device with dried pre-alloyed powder as raw material, Ar gas with a purity of not less than 99.999% and an oxygen content of not more than 50ppm is used as a protective atmosphere to prevent oxidation of the alloy powder during the forming process. The laser power is set to 260W, the scanning speed to 850mm / s, the layer thickness to 32μm, and the scanning strategy to adopt checkerboard fractal filling with a partition size of 5mm×5mm and a rotation angle of 70°. Through careful design of the SLM scanning path, a honeycomb structure titanium alloy substrate is directly formed. The pore size of this structure is 280μm~380μm and the wall thickness is 75μm~95μm, avoiding the complex process of post-processing interference technology required in traditional processes. The formed honeycomb structure titanium alloy substrate was subjected to stress annealing under Ar gas protection. It was placed in a high-temperature furnace, heated to 760℃ and held for 2.2 hours. Then it was cooled to 310℃ in the furnace and then taken out and allowed to cool naturally in the air. This eliminated the residual stress generated during the forming process and improved the structural stability of the substrate, resulting in the annealed formed honeycomb structure titanium alloy substrate. Step 2, Surface Modification: An etching solution was prepared by diluting 2.5% HF and 11% HNO3 by volume with deionized water. The annealed and formed honeycomb structure titanium alloy substrate obtained in step one was immersed in the etching solution and etched for 5.5 minutes at 160 r / min using a magnetic stirrer at a temperature of 24℃~28℃. After etching, the substrate was immediately placed in deionized water and then ultrasonically cleaned at 42 kHz for 11 minutes to remove residual etching solution. Finally, it was dried with nitrogen to obtain the surface etched substrate. Scanning electron microscopy (SEM) showed that the surface of the surface etched substrate formed a micron-level rough surface. An electrolyte containing 9g Na2SiO3, 2.5g KOH and 5.5g Na3PO4 per liter was prepared, and the pH value was adjusted to 11.4~11.8. Using the surface-etched substrate as the anode, micro-arc oxidation was performed under the conditions of a forward voltage of 290V, a frequency of 550Hz and a duty cycle of 22% for 16min. During the treatment, the electrolyte temperature was controlled to not exceed 34℃ through a water cooling circulation system. After treatment, a surface-modified titanium alloy substrate was obtained. III. Functionalization: Reactive magnetron sputtering was employed using Cu and Se targets. During sputtering, the Ar gas flow rate was 55 sccm and the H2Se gas flow rate was 5.5 sccm. The temperature of the surface-modified titanium alloy substrate obtained in step two was controlled at 310℃. The sputtering power of the Cu target was 160W, and the sputtering power of the Se target was 110W. The deposition rate was 2.2 nm / min, ultimately yielding a copper selenide film with a thickness of 110 nm. After deposition, vacuum annealing was performed under Ar protection at a temperature of 410℃ for 1.1 h to obtain the titanium alloy substrate with the deposited copper selenide film. X-ray diffraction (XRD) confirmed that the copper selenide film in the titanium alloy substrate formed a CuSe phase. Loaded antimicrobial peptides: Polylactic acid-glycolic acid copolymer (PLGA) microspheres with a particle size of 2.2 μm were prepared as antimicrobial peptide carriers using an emulsion solvent evaporation method, with a drug loading of 11%. The microspheres were dispersed in phosphate buffered saline (PBS) at pH 7.4, and a titanium alloy substrate with a deposited copper selenide film was immersed in the solution for 26 h and then vacuum dried to obtain a hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides. The antimicrobial peptide loading rate in the hierarchical microporous titanium alloy carrying nano-copper selenide antimicrobial peptides was determined by fluorescent labeling, and the result was 85±5%. In vitro release experiments verified that the microspheres degraded rapidly at pH less than 6.5, and the release rate exceeded 90% after 24 h at pH 6.0, showing good pH response characteristics.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides, characterized in that, The method includes the following steps: Step 1: Substrate preparation: Selective laser melting is used to form a titanium alloy substrate with a honeycomb structure; Step 2, Surface modification: The titanium alloy substrate formed in Step 1 is subjected to surface etching and micro-arc oxidation in sequence to obtain a surface-modified titanium alloy substrate; Step 3, Functionalization: A copper selenide film is deposited on the surface-modified titanium alloy substrate obtained in Step 2 and pH-responsive antimicrobial peptide carrier microspheres are loaded to obtain a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides.

2. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 1, characterized in that, The selective laser melting process described in step one is as follows: Ti-6Al-4V-0.5Mo pre-alloyed powder with a particle size of 10μm~50μm and an oxygen mass content of less than 0.15% is used for forming in an Ar gas environment with an oxygen content of no more than 50ppm. The forming parameters are 200W~300W laser power, 800mm / s~1200mm / s scanning speed, and 20μm~50μm layer thickness. The scanning strategy adopts a 4mm~6mm×4mm~6mm checkerboard fractal filling and a rotation angle of 60°~75°. After forming, stress annealing is performed to obtain a titanium alloy matrix with a honeycomb structure.

3. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 2, characterized in that, The stress annealing temperature is 720℃~780℃, and the holding time is 1.5h~2.5h; after stress annealing, a segmented cooling method is adopted, specifically furnace cooling to 280℃~320℃ followed by air cooling.

4. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 1, characterized in that, The surface etching process described in step two is as follows: etching is performed using an etching solution containing HF, HNO3 and H2O, followed by ultrasonic cleaning to form a rough surface.

5. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 4, characterized in that, The etching solution contains 1.5% to 3% HF by volume, 8% to 12% HNO3 by volume, and the balance is H2O; the etching temperature is 20℃ to 30℃, the stirring speed is 120r / min to 180r / min, and the time is 4min to 6min; the ultrasonic cleaning frequency is 35kHz to 45kHz, and the time is 8min to 12min.

6. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 1, characterized in that, The micro-arc oxidation process described in step two is as follows: micro-arc oxidation is performed using an electrolyte containing Na2SiO3, KOH and Na3PO4 to obtain an oxide film.

7. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 6, characterized in that, The electrolyte contains 6 g / L to 10 g / L of Na2SiO3, 1.5 g / L to 3 g / L of KOH, and 4 g / L to 6 g / L of Na3PO4. The pH of the electrolyte is 11 to 12. The forward voltage of the micro-arc oxidation is 260 V to 300 V. The frequency of the micro-arc oxidation is 400 Hz to 600 Hz, the duty cycle is 15% to 25%, and the time is 12 min to 18 min. The temperature of the electrolyte in the micro-arc oxidation is controlled below 38 °C.

8. The method for preparing a multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 1, characterized in that, The process of depositing the copper selenide film in step three is as follows: Reactive magnetron sputtering is used to deposit the copper selenide film under the conditions of Ar gas and H2Se gas, with the Ar gas flow rate being 40 sccm~60 sccm and the H2Se gas flow rate being 4 sccm~6 sccm. The sputtering power of the Cu target is 120W~180W and the sputtering power of the Se target is 80W~120W, followed by vacuum annealing. The temperature of the titanium alloy substrate during reactive magnetron sputtering is 280℃~320℃, and the deposition rate is 1.5nm / min~2.5nm. The vacuum annealing temperature is 380℃~420℃, the holding time is 0.8h~1.2h, and the thickness of the copper selenide film layer is 80nm~120nm. The process of loading pH-responsive antimicrobial peptide carrier microspheres is as follows: antimicrobial peptides are loaded onto PLGA microspheres to obtain antimicrobial peptide carrier microspheres, and then the antimicrobial peptide carrier microspheres are loaded through impregnation-vacuum drying. The particle size of the antimicrobial peptide carrier microspheres is 1.5μm~2.5μm, and the drug loading of the antimicrobial peptide carrier microspheres is 8%~12%. The impregnation time in the impregnation-vacuum drying process is 20h~28h.

9. A multi-level microporous titanium alloy loaded with nano-copper selenide antibacterial peptides, characterized in that, The multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is prepared by the method described in any one of claims 1 to 8.

10. A multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides according to claim 9, characterized in that, The multi-level microporous titanium alloy loaded with nano-copper selenide antimicrobial peptides is used to prepare orthopedic implants or wound repair devices.

Images