Preparation method of piezoelectric response antibacterial / endothelial cell growth promoting coating material

Abstract

The invention relates to the technical field of novel functional and structural materials, and discloses a preparation method of a piezoelectric response antibacterial / endothelial cell growth promoting coating material, the coating material takes a titanium alloy as a matrix, an alkali heat treatment technology is utilized to form a microstructure with a large number of OH <-> in pores on the surface of the titanium alloy, and a cation exchange method is utilized to prepare the piezoelectric response antibacterial / endothelial cell growth promoting coating material. Zinc ions are fixed to the surface of the titanium alloy, then a zinc-based metal organic framework is generated on the surface of the titanium alloy through an in-situ synthesis method, and finally the HZnO nanometer material which is stably combined and is of a hollow structure is obtained on the surface of the titanium alloy through a high-temperature calcination method. The HZnO nano material has piezoelectric characteristics, can generate micro-current and ROS, has the effects of promoting growth of endothelial cells and resisting bacteria, and further shows excellent anticoagulation and antibacterial effects. In addition, the composite coating has slow physiological degradability, and degradation products are non-toxic. The method is simple in preparation process and has potential application prospects in the field of novel functional and structural materials.

Description

Technical Field

[0001] This invention relates to the field of novel functional and structural materials technology, and in particular to a method for preparing a piezoelectrically responsive antibacterial / endothelial cell growth promoting coating material. Background Technology

[0002] Cardiovascular disease remains a major global health burden, exacerbated by an aging population and unhealthy lifestyles. Implantable devices such as artificial valves, vascular grafts, occluders, and stents have significantly improved treatment outcomes, but adverse biological reactions at the blood-material interface often lead to impaired device performance. Stent implantation, in particular, can cause endothelial abruption and medial tearing, subsequently triggering inflammation and thrombosis, promoting smooth muscle cell proliferation and extracellular matrix deposition, ultimately resulting in neointimal hyperplasia and in-stent restenosis. While drug-eluting stents can alleviate in-stent restenosis through local release of anti-proliferative drugs, they often delay or inhibit vascular reendothelialization, increasing the risk of late thrombosis. Furthermore, drug-eluting stent materials often face issues such as poor coating adhesion and unstable efficacy. The vascular microenvironment is dynamic and influenced by multiple factors, necessitating more precise materials to regulate repair responses. To improve endothelial cell integration, researchers have developed various functional coatings. Traditional chemical or biological modification strategies, due to their reliance on functional components, lack stability and reliability in the complex in vivo microenvironment. In contrast, strategies based on physical signals (including surface topology, mechanical stimulation, and electrical signals) provide stable non-biochemical means for regulating cell behavior.

[0003] Electrical stimulation (ES) can promote endothelial cell proliferation while regulating smooth muscle cell behavior and reducing neointimal formation. Piezoelectric materials can generate charge through mechanical stress, making them a highly promising self-powered stimulation platform; however, the lack of continuous mechanical input in passive systems limits their activation. Among various stimuli, ultrasound is a highly attractive option due to its deep tissue penetration, high spatial precision, and good safety profile. Unlike light or electromagnetic fields, sound waves can penetrate opaque, highly scattering tissues without requiring nanoparticle loading or high-gradient magnetic fields. Furthermore, it can achieve non-invasive drug release through stable / inertial cavitation, acoustic flow, or piezoelectric conversion, thereby reducing off-target exposure and the risk of late-stage thrombosis. Studies have demonstrated that incorporating piezoelectric nanomaterials (such as ZnO, BaTiO3, and BiO) can effectively reduce the risk of late-stage thrombosis. 2-x(e.g.,) Loaded onto the surface of cardiovascular implantable devices, it can generate an internal electric field under external ultrasound stimulation, thereby exerting the effect of electrical stimulation (Zheng T, Feng Y, Wu H, et al. Ultrasound-activated piezoelectric nano-armor for in-situ vascular repair on vascular implants[J]. Biomaterials,2025: 123746). It is worth noting that vascular graft or endovascular stent infection is a serious complication after open or endovascular vascular surgery, with an incidence of approximately 1%–6%, and this incidence is increasing with aging and the rising prevalence of atherosclerosis. Despite its low incidence, the mortality and disability rate of vascular graft and endograft infections exceeds 30%, and even after treatment, long-term sequelae are often left (Chakfé N, Diener H, Lejay A, et al. Editor's Choice-European Society for Vascular Surgery (ESVS) 2020 clinical practice guidelines on the management of vascular graft and endograft infections[J]. Eur. J. Vasc. Endovasc. 2020, 59(3): 339−384). Therefore, developing cardiovascular implantable devices with both antibacterial and endothelial cell proliferation-promoting properties is currently a key goal in the research and development of vascular repair materials.

[0004] Studies have shown that piezoelectric materials, under ultrasonic stimulation, can not only generate an internal electric field, but the electron-hole pairs they produce can also undergo redox reactions with H2O or O2, generating reactive oxygen species (such as... • OH and • O 2–This allows for the achievement of piezoelectric acoustic-dynamic therapy (SDT) effects, exhibiting significant antibacterial properties (Cai L, Sun T, Han F, et al. Degradable and piezoelectric hollow ZnO heterostructures for sonodynamic therapy and pro-death autophagy[J]. J. Am. Chem. Soc. 2024, 146: 34188−34198). Therefore, combining piezoelectric materials with cardiovascular implant materials to prepare functional coatings for medical devices is of great significance for clinical applications. However, the bonding force between nanomaterials and medical metal substrates is usually weak. How to enhance the bonding stability between nano-coatings and metal substrates is currently a key research focus in cardiovascular implant devices.

[0005] This invention employs a two-step synthesis method to prepare stable, porous zinc oxide nanomaterials (HZnO) on the surface of medical titanium alloys. An alkaline thermal treatment method is used to introduce a large amount of OH groups onto the titanium alloy surface. – Then, using ion exchange, Zn is promoted to... 2+ Stable composite with a titanium alloy substrate. Furthermore, a stable loaded HZnO was prepared on the titanium alloy surface via a two-step synthesis method. The hollow structure of HZnO promotes charge separation under ultrasonic conditions, enhancing the piezoelectric effect and SDT performance. Summary of the Invention

[0006] Objective of the Invention: In a first aspect, the present invention provides a piezoelectric responsive antibacterial / endothelial cell growth promoting coating material, wherein the piezoelectric responsive antibacterial / endothelial cell growth promoting coating material uses titanium alloy as the base material, and firstly forms a porous OH group on the surface of the titanium alloy using an alkaline heat treatment method. - The microstructure; then, using cation exchange, zinc ions (Zn) were... 2+ The process involves fixing the coating material onto the surface of a titanium alloy; then, using an in-situ synthesis method, generating a zinc-based metal-organic framework (ZIF-8) on the surface of the titanium alloy; finally, using a high-temperature calcination method, obtaining a stably bonded ZnO nanomaterial (HZnO) with a hollow structure on the surface of the titanium alloy; the piezoelectric responsive antibacterial / endothelial cell growth promoting coating material is denoted as HZnO-Ti coating material.

[0007] In this HZnO-Ti coating material, the diameter of the HZnO nanomaterials ranges from 20 to 400 nm, while the diameter of its hollow structure ranges from 10 to 200 nm. Furthermore, the HZnO-Ti coating material exhibits stable bonding capabilities, remaining stable for at least 30 days in simulated bodily fluid environments. This robust bonding between the HZnO coating and the titanium alloy substrate is a crucial foundation for the effective functioning of medical implant materials. In addition, the HZnO nanomaterials possess piezoelectric properties, generating microcurrents and ROS under ultrasonic triggering, promoting endothelial cell growth and exhibiting antibacterial effects, thus demonstrating excellent anticoagulant and antibacterial properties. Its hollow structure enhances the deformation of the piezoelectric material under ultrasonic stimulation, strengthening the internal electric field and ROS production levels, thereby achieving effective antibacterial and endothelial cell growth-promoting effects.

[0008] Secondly, the present invention provides a method for preparing a piezoelectrically responsive antibacterial / endothelial cell growth promoting coating material as described above, comprising the following steps: (1) The surface-treated titanium alloy substrate is placed in an alkaline treatment solution and reacted under heating conditions for a period of time. After washing and vacuum drying, a surface with a large number of OH groups is obtained. - The microstructured titanium alloy substrate is denoted as OH-Ti; (2) The OH-Ti obtained in step (1) is immersed in Zn(NO3)2·6H2O solution and reacted for a period of time. After washing with deionized water, it is placed in a methanol solution containing Zn(NO3)2·6H2O and 2-methylimidazole and reacted at room temperature for a period of time. After taking it out, it is washed three times with methanol and ethanol and dried under vacuum to obtain a titanium alloy with ZIF-8 material on the surface, which is denoted as ZIF-8-Ti. (3) The ZIF-8-Ti obtained in step (2) is placed in a mixture of methanol, ethanol and ultrapure water, stirred evenly, and then poured into the liner of a polytetrafluoroethylene reactor. The liner of the polytetrafluoroethylene reactor is then placed in a high-pressure reactor, heated in an oven for a period of time, and then taken out. It is washed three times with ultrapure water and ethanol and then vacuum dried to obtain a titanium alloy with a hollow ZnO structure on the surface, namely HZnO-Ti coating material.

[0009] Further, in step (1), the titanium alloy substrate is first sanded with sandpaper, then polished with diamond plaster, and finally ultrasonically cleaned with ethanol and acetone in sequence to obtain a surface-treated titanium alloy substrate; wherein the titanium alloy substrate is Ti-6Al-4V titanium alloy.

[0010] Further, in step (1), the dimensions of the titanium alloy substrate are: length:width:height = 5-10 mm:5-10 mm:1-3 mm; the alkaline treatment solution is one of Ca(OH)2, Ba(OH)2, KOH or NaOH; the volume of the alkaline treatment solution is 200-500 ml, and the concentration is 3-8 mol·L. -1 The reaction conditions are: reaction temperature of 50-100℃ and reaction time of 12-36 h; the vacuum drying temperature is 50-100℃.

[0011] Furthermore, in step (2), the volume of the Zn(NO3)2·6H2O solution used to soak OH-Ti is 20-80 ml, and the concentration is 0.02-0.08 mol·L⁻¹. -1 The methanol solution containing Zn(NO3)2·6H2O and 2-methylimidazole was prepared with a methanol-to-Zn(NO3)2·6H2O and 2-methylimidazole ratio of 30-80 ml : 0.2-0.5 g : 0.2-0.5 g. The reaction time at room temperature was 12-36 h. The vacuum drying temperature was 50-100 ℃.

[0012] Further, in step (3), the volume of the mixture is 5-20 ml, wherein the volume ratio of methanol, ethanol and ultrapure water is 3-10 ml: 3-10 ml: 0.001-0.09 ml; the reaction conditions are: reaction temperature of 150-240 ℃, reaction time of 12-36 h; and the vacuum drying temperature is 50-100 ℃.

[0013] Thirdly, the present invention provides an application of the piezoelectric responsive antibacterial / endothelial cell growth promoting coating material as described above, which can be used as a cardiovascular implant material.

[0014] Beneficial effects: Compared with the prior art, the specific beneficial effects of this invention are as follows: To enhance the bonding stability between nanomaterials and the titanium alloy matrix, this invention utilizes alkaline heat treatment and cation exchange methods to bond Zn... 2+ A stable HZnO coating material with a hollow structure was prepared on the surface of a titanium alloy substrate using in-situ synthesis and high-temperature calcination (HZnO-Ti coating material). By adjusting parameters such as the hollow structure size and coating thickness of the HZnO coating, the piezoelectric properties were optimized to obtain a coating material with the best electrostimulation and SDT effects. This invention features a simple, low-cost, safe, and controllable preparation process, and has potential applications in the field of novel functional and structural materials. Attached Figure Description

[0015] Figure 1 Images of the HZnO-Ti coating material prepared in Example 1 after immersion in PBS for 0 days and 30 days; Figure 2 Scanning electron microscope images of the HZnO-Ti coating material prepared in Example 1 after immersion in PBS for 0 days and 30 days; Figure 3 Scanning electron microscope image of the microstructure of the HZnO-Ti coating material prepared in Example 1; Figure 4 The ROS detection curves of Examples 1-3 and Comparative Examples 1-2 under ultrasonic stimulation are shown. Detailed Implementation

[0016] The present invention will now be described in detail with reference to the embodiments. Example 1

[0017] The HZnO-Ti coating material provided in this embodiment uses titanium alloy (Ti-6Al-4V) as the substrate for the coating material, and firmly bonds HZnO nanomaterials with hollow structures to the surface of the titanium alloy. The preparation method is as follows: (1) Use a size of 10 × 10 × 2 mm 3 A titanium alloy (Ti-6Al-4V) substrate was used. The titanium alloy substrate sample was first sanded, then polished with diamond plaster, and finally ultrasonically cleaned sequentially with ethanol and acetone to obtain a surface-treated titanium alloy substrate sample. The treated titanium alloy substrate was placed in 100 ml of 5 M NaOH solution and reacted at 80 °C for 24 h. It was then washed three times with deionized water to obtain a surface with numerous pores containing OH groups. - Microstructured titanium alloy substrate (OH-Ti); (2) The titanium alloy substrate treated in step (1) was immersed in 50 ml of Zn(NO3)2·6H2O (0.05 M) solution for 30 min. After washing with deionized water, the sample was placed in 50 ml of methanol solution containing 0.37 g Zn(NO3)2·6H2O and 0.41 g 2-methylimidazole and reacted at room temperature for 24 h. The substrate sample was removed, washed three times with methanol and ethanol, and dried under vacuum at 60 °C to obtain a titanium alloy (ZIF-8-Ti) with ZIF-8 material on its surface. (3) The ZIF-8-Ti obtained in step (2), 5 ml of methanol, 5 ml of ethanol, and 0.006 ml of deionized water were mixed and placed in a 25 ml polytetrafluoroethylene (PTFE) reactor liner. The mixture was stirred continuously to ensure uniform mixing. Then, the PTFE reactor liner was placed in an autoclave and heated in an oven at 220°C for 24 h. Finally, the substrate sample was removed, washed three times with ultrapure water and ethanol, and vacuum dried at 60°C to obtain a titanium alloy (HZnO-Ti) with a porous ZnO structure on its surface. Example 2

[0018] The difference from Example 1 is that in step (3), the amount of deionized water used is 0.02 ml. By precisely adjusting the amount of deionized water, the pore structure of ZnO can be adjusted to obtain porous ZnO with a good pore structure. Example 3

[0019] The difference from Example 1 is that in step (3), the amount of deionized water used is 0.05 ml.

[0020] The HZnO-Ti coated material sample prepared in Example 1 was immersed in PBS for 30 days. Figure 1 Photographs of HZnO-Ti samples after soaking in PBS for 0 days and 30 days; Figure 2 Scanning electron microscope images of the HZnO-Ti coating material prepared in Example 1 after immersion in PBS for 0 days and 30 days.

[0021] The microstructure of the HZnO-Ti coating prepared in Example 1 was characterized, and the results are as follows: Figure 3 As shown, the coating material has a nanostructure with a diameter of approximately 40 nm. Comparative Example 1

[0022] The titanium alloy matrix material provided in this comparative example is prepared by the following method: (1) Use a size of 10 × 10 × 2 mm 3 The titanium alloy (Ti-6Al-4V) substrate was first sanded, then polished with diamond plaster, and finally ultrasonically cleaned with ethanol and acetone in sequence to obtain a surface-treated titanium alloy substrate sample. Comparative Example 2

[0023] The solid ZnO-Ti coating material provided in this comparative example is prepared by the following method: (1) Preparation of a surface with a large number of OH groups in the pores - The microstructured titanium alloy substrate (OH-Ti) was prepared using the same method as in Example 1. (2) The titanium alloy substrate treated in step (1) was immersed in 50 ml of Zn(NO3)2·6H2O (0.05 M) solution for 30 min. After washing with deionized water, the sample was placed in 50 ml of methanol containing 0.37 g Zn(NO3)2·6H2O and stirred to mix the sample evenly. The mixture was then placed in a 25 ml polytetrafluoroethylene (PTFE) reactor liner. The PTFE reactor liner was then placed in an autoclave and heated in an oven at 220ºC for 24 h. Finally, the substrate sample was removed, washed three times with ultrapure water and ethanol, and vacuum dried at 60ºC to obtain a titanium alloy (ZnO-Ti) with solid ZnO on the surface.

[0024] The properties of the materials prepared in Examples 1-3 and Comparative Examples 1-2 were tested: 1. Testing of electrochemical performance under ultrasonic stimulation: To test the ultrasonic electrochemical performance of Examples 1-3 and Comparative Examples 1-2, the current intensity of each group of samples under ultrasonic stimulation was measured, as shown in Table 1. The experimental results show that the current intensity of Examples 1, 2, 3, and Comparative Examples 1 and 2 gradually decreases, proving that the synthesis method in Example 1 has the best effect on the ultrasonic stimulation current generation of HZnO-Ti coating materials.

[0025]

[0026] 2. Test of ROS performance generated by ultrasound stimulation: To test the ultrasonic stimulation ROS production capacity of Examples 1-3 and Comparative Examples 1-2, 1,3-diphenylisobenzofuran (DPBF) was used as a ROS scavenger, and the effect of ultrasonic stimulation on the absorbance of DPBF in each group of samples was detected. Figure 4 As shown, the degree of decrease in DPBF absorbance in Examples 1, 2, 3, and Comparative Examples 1 and 2 gradually decreased, indicating that the ROS yield gradually weakened. This demonstrates that the synthesis method in Example 1 was the most effective for ROS production of HZnO-Ti coated materials under ultrasonic stimulation.

[0027] 3. Hemolysis test The hemolysis test was used to verify the blood compatibility of each group of samples. As shown in Table 2, the hemolysis rates of Examples 1, 2, 3, and Comparative Examples 1 and 2 were close to those of the negative control, and the hemolysis rates were low, which proves that all experimental samples have good blood compatibility.

[0028]

[0029] 4. Antibacterial test: For the antibacterial experiment, *Escherichia coli* and *Staphylococcus aureus* were used as the experimental strains. The revived bacteria were placed in liquid culture medium and shaken overnight at 37°C. Samples from Examples 1-3 and Comparative Examples 1-2 were placed in 12-well plates. 500 µL of bacterial suspension was added to the sample surface, and each group of samples was then sonicated for 10 min. Next, 1 mL of PBS solution was added to each group of samples, followed by sonication. 50 μL of bacterial suspension was then plated and incubated overnight at 37°C on a shaker. The number of bacterial colonies was then observed.

[0030]

[0031] The statistical data of plate colonies in the in vitro antibacterial experiment are shown in Table 3 above. The results show that Examples 1-3 all exhibited effective antibacterial effects against Escherichia coli and Staphylococcus aureus, with Example 1 showing the best antibacterial efficiency.

[0032] 5. Endothelial cell generation stimulation experiment To investigate the effects of ultrasound stimulation on endothelial cell growth activity in Examples 1-3 and Comparative Examples 1-2, cell viability was monitored after co-culturing endothelial cells with each group of samples under ultrasound conditions. As shown in Table 4, compared with the blank control group, the cell viability of Examples 1, 2, and 3 was enhanced, with Example 1 exhibiting the highest cell viability, demonstrating that the sample obtained using the experimental method in Example 1 had the best effect on promoting endothelial cell growth. This experimental result is consistent with the experimental data on current intensity and ROS production efficiency, all indicating that Example 1 has the best effect on ultrasound-stimulated ROS production and promoting endothelial cell proliferation.

[0033]

[0034] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A piezoelectrically responsive antibacterial / endothelial cell growth promoting coating material, characterized in that: The piezoelectric responsive antibacterial / endothelial cell growth promoting coating material uses titanium alloy as the base material. First, an alkaline heat treatment method is used to form a porous OH group on the surface of the titanium alloy. - The microstructure was obtained; then zinc ions were fixed to the surface of the titanium alloy using a cation exchange method; zinc-based metal-organic framework ZIF-8 was generated on the surface of the titanium alloy using an in-situ synthesis method; finally, ZnO nanomaterials with a stable bond and hollow structure were obtained on the surface of the titanium alloy using a high-temperature calcination method; the piezoelectrically responsive antibacterial / endothelial cell growth promoting coating material is denoted as HZnO-Ti coating material.

2. The method for preparing the piezoelectrically responsive antibacterial / endothelial cell growth promoting coating material according to claim 1, characterized in that, Includes the following steps: (1) The surface-treated titanium alloy substrate is placed in an alkaline treatment solution and reacted under heating conditions for a period of time. After washing and vacuum drying, a surface with a large number of OH groups is obtained. - The microstructured titanium alloy substrate is denoted as OH-Ti; (2) The OH-Ti obtained in step (1) is immersed in Zn(NO3)2·6H2O solution and reacted for a period of time. After washing with deionized water, it is placed in a methanol solution containing Zn(NO3)2·6H2O and 2-methylimidazole and reacted at room temperature for a period of time. After taking it out, it is washed three times with methanol and ethanol and dried under vacuum to obtain a titanium alloy with ZIF-8 material on the surface, which is denoted as ZIF-8-Ti. (3) The ZIF-8-Ti obtained in step (2) is placed in a mixture of methanol, ethanol and ultrapure water, stirred evenly, and then poured into the liner of a polytetrafluoroethylene reactor. The liner of the polytetrafluoroethylene reactor is then placed in a high-pressure reactor, heated in an oven for a period of time, and then taken out. It is washed three times with ultrapure water and ethanol and then vacuum dried to obtain a titanium alloy with a hollow ZnO structure on the surface, namely HZnO-Ti coating material.

3. The preparation method according to claim 2, characterized in that: In step (1), the titanium alloy substrate is first sanded with sandpaper, then polished with diamond plaster, and finally ultrasonically cleaned with ethanol and acetone in sequence to obtain a surface-treated titanium alloy substrate; wherein the titanium alloy substrate is Ti-6Al-4V titanium alloy.

4. The preparation method according to claim 2, characterized in that: In step (1), the dimensions of the titanium alloy substrate are: length:width:height = 5-10 mm:5-10 mm:1-3 mm; the alkaline treatment solution is one of Ca(OH)2, Ba(OH)2, KOH or NaOH; the volume of the alkaline treatment solution is 200-500 ml, and the concentration is 3-8 mol·L. -1 The reaction conditions are: reaction temperature of 50-100℃ and reaction time of 12-36 h; the vacuum drying temperature is 50-100℃.

5. The preparation method according to claim 2, characterized in that: In step (2), the volume of the Zn(NO3)2·6H2O solution used to soak OH-Ti is 20-80 ml, and the concentration is 0.02-0.08 mol·L⁻¹. -1 The methanol solution containing Zn(NO3)2·6H2O and 2-methylimidazole was prepared with a methanol-to-Zn(NO3)2·6H2O and 2-methylimidazole ratio of 30-80 ml : 0.2-0.5 g : 0.2-0.5 g. The reaction time at room temperature was 12-36 h. The vacuum drying temperature was 50-100 ℃.

6. The preparation method according to claim 2, characterized in that: In step (3), the volume of the mixture is 5-20 ml, wherein the volume ratio of methanol, ethanol and ultrapure water is 3-10 ml: 3-10 ml: 0.001-0.09 ml; the reaction conditions are: reaction temperature of 150-240 ℃, reaction time of 12-36 h; and the vacuum drying temperature is 50-100 ℃.

7. The application of the piezoelectric responsive antibacterial / endothelial cell growth promoting coating material according to claim 1, characterized in that: The piezoelectrically responsive antibacterial / endothelial cell growth promoting coating material can be used as a cardiovascular implant material.

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