Magnesium alloy intravascular stent material with coating capable of catalytically releasing nitric oxide in situ and preparation method of magnesium alloy intravascular stent material

Abstract

The invention discloses a magnesium alloy intravascular stent material with a coating capable of catalyzing and releasing nitric oxide in situ and a preparation method of the magnesium alloy intravascular stent material. The preparation method comprises the following steps: constructing a polylipoic acid-arginine coating with the capability of catalyzing and releasing nitric oxide in situ on the surface of a pretreated magnesium alloy intravascular stent material; the magnesium alloy intravascular stent mainly solves the problems of excessive hyperplasia and endothelialization delay of the current magnesium alloy intravascular stent. The polylipoic acid-arginine coating is tightly adhered to the surface of the pretreated magnesium alloy stent based on hydrogen-bond interaction, arginine fixed by the coating serves as fuel for synthesizing nitric oxide, endothelial cells generate nitric oxide through in-situ catalysis of endothelial nitric oxide synthase, lipoic acid serves as an endothelial nitric oxide synthase enhancer, and the endothelial cells generate nitric oxide through in-situ catalysis of the endothelial nitric oxide synthase. The level of endothelial nitric oxide synthase in endothelial cells is increased, arginine is decomposed into nitric oxide, and sufficient supply of in-situ nitric oxide is ensured. The coating obviously delays the degradation rate of the magnesium alloy stent, promotes reconstruction of natural endothelium and prevents restenosis.

Description

Technical Field

[0001] The invention belongs to the field of surface treatment of magnesium alloy vascular stent materials, and in particular relates to a magnesium alloy vascular stent material with an in-situ catalytic nitric oxide releasing coating and a preparation method thereof. Background Art

[0002] Cardiovascular diseases (CVDs) are currently the leading cause of death and chronic disability. Currently, drug therapy, surgical treatment and intravascular intervention are the most commonly used methods for the clinical treatment of cardiovascular diseases. In cases where drug treatment is ineffective or arterial stenosis recurs, stent angioplasty becomes an important treatment method. Currently, the materials of stents used in clinical practice are mainly stainless steel, nickel-titanium alloy and cobalt-chromium alloy. Since they are all made of non-degradable materials, as permanent foreign bodies in the body, they will lead to the risk of vascular inflammation and recurrence of atherosclerosis. Therefore, their long-term safety and efficacy are still questioned. Guided by the concept of "intervention without implantation", biodegradable vascular stents have gradually become the focus and hot spot of attention. Compared with non-degradable stents, biodegradable stents have unique abilities to gradually degrade and eventually disappear after vascular remodeling, effectively avoiding a series of immune rejection problems caused by long-term foreign body stimulation of permanent implants.

[0003] Magnesium alloys are considered to be excellent candidate materials for biodegradable implants due to their appropriate mechanical properties, good biocompatibility and inherent bioactivity. Currently, magnesium alloy drug-eluting stents can slow down the degradation rate of magnesium stents and have anti-proliferative effects. In 2023, Germany's Biotronik's third-generation magnesium-based drug-eluting coronary stent DREAMS 3G completed a new round of research, and the results showed that within 12 months after implantation, 99.3% of the stent beams could be completely absorbed, and regardless of the characteristics of the lesion, the stent could maintain consistent excellent performance. However, drug-eluting magnesium alloy stents still face some difficult clinical problems. The anti-proliferative drugs released by the stent coating will hinder the growth of endothelial cells while effectively promoting smooth muscle cell proliferation, leading to thrombosis and new atherosclerosis caused by delayed endothelialization.

[0004] In response to the problems faced by magnesium alloy vascular stents, researchers have brainstormed and developed a series of cardiovascular stent coatings that mimic the functions of endothelial cells. The healthy endothelium continuously catalyzes L-arginine (arginine for short) to produce nitric oxide (NO) through endothelial nitric oxide synthase (eNOS), thereby achieving multiple functions such as inhibiting coagulation, inhibiting excessive proliferation of smooth muscle cells (SMCs), and promoting self-repair of damaged endothelial cells (ECs). Currently, introducing catalysts on the surface of materials to catalyze endogenous NO donors is currently considered a relatively stable strategy. Endogenous nitric oxide donors can be oxidized by some traditional metal ions (such as Fe 2+ 、Zn 2+, Cu 2+ ) catalytically decomposes into nitric oxide gas, in which Cu 2+ The catalytic efficiency of is higher than that of other metal ions. Gao et al. constructed a copper / dopamine / hexamethylenediamine (Cu(II) / DA / HD) coating on the stent surface by a one-step molecular / ion self-assembly method, and then grafted the anticoagulant molecule bivalirudin (BVLD) onto the surface. 2+ It provides the catalytic activity of glutathione peroxidase, which can be used for the sustained and controllable production of nitric oxide gas; the HD molecule provides abundant amine groups, providing a large number of sites for the surface grafting of the anticoagulant molecule bivalirudin. Both in vivo and in vitro results show that the nitric oxide gas produced by the synergistic catalysis of bivalirudin improves the stent's ability to resist thrombosis, restenosis, and promote re-endothelialization [Biomaterials, 2020, 248: 119981]. However, the design of nitric oxide coatings that catalyze the production of nitric oxide from endogenous nitric oxide donors often does not take into account the low local concentration and short half-life of endogenous nitric oxide donors in the actual pathophysiological microenvironment, so they may not support sufficient nitric oxide catalysts in vivo. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a magnesium alloy vascular stent material with an in-situ catalytic release of nitric oxide coating and a preparation method thereof based on the pathway of nitric oxide synthesis by endothelial cells, mainly to solve the problems of excessive proliferation and delayed endothelialization faced by current magnesium alloy vascular stents.

[0006] Based on the above purpose, the present invention adopts the following technical solution: A method for preparing a magnesium alloy vascular stent material with an in-situ catalytic nitric oxide releasing coating comprises the following steps: (1) Take a magnesium alloy vascular stent material, use sandpaper to grind it until the surface is flat and smooth, and then clean it, dry it, and set it aside; (2) immersing the magnesium alloy vascular stent material of step (1) in a fluoridation treatment solution, reacting at room temperature for 8 to 48 hours, cleaning, and drying to obtain a magnesium alloy vascular stent material with a magnesium fluoride layer on the surface; (3) After the lipoic acid powder is heated and melted into liquid, add arginine, continue to heat to 130-150°C, stir until the arginine is completely dissolved, and set aside; (4) The polylipoic acid-arginine liquid prepared in step (3) is applied to the surface of the magnesium alloy vascular stent material prepared in step (2) and cooled to obtain a magnesium alloy vascular stent material with a coating that releases nitric oxide in situ.

[0007] Preferably, the magnesium alloy vascular stent material in step (1) is ZE21B, WE43, AZ31 or AZ91.

[0008] Preferably, the fluorination treatment liquid in step (2) is at least one of hydrofluoric acid (35-40 wt%), sodium fluoride aqueous solution (0.1 M-0.3 M) and potassium fluoride aqueous solution (0.1-0.3 M).

[0009] Preferably, the molar ratio of lipoic acid to arginine in step (3) is (5-1):1. The melting temperature of the lipoic acid powder in step (3) is 110-130°C.

[0010] Preferably, the polylipoic acid-arginine coating in step (4) is prepared by dip coating or spin coating.

[0011] Preferably, the drying in steps (1) to (4) refers to drying at 40°C to 60°C for 5 to 30 minutes.

[0012] The magnesium alloy vascular stent material with a coating layer for in-situ catalytic release of nitric oxide prepared by the preparation method.

[0013] The thickness of the magnesium fluoride layer is 0.8 μm~3.0 μm, and the thickness of the polylipoic acid-arginine coating is 11.0 μm~16.0 μm.

[0014] Application of the magnesium alloy vascular stent material with the coating for in-situ catalytic release of nitric oxide in the preparation of in vivo implants.

[0015] The technical effect directly brought about by the above technical scheme is that the reaction conditions are mild, the operation is simple and safe, and it is easier to be accepted by researchers. The developed polylipoic acid-arginine coating is tightly adhered to the surface of the pretreated magnesium alloy stent based on hydrogen bonding. The polylipoic acid-arginine coating is chemically bonded through salt bridge hydrogen bonds. The arginine fixed in the coating acts as a fuel for synthesizing nitric oxide. Endothelial cells produce nitric oxide by in situ catalysis of endothelial nitric oxide synthase, and lipoic acid acts as an endothelial nitric oxide synthase enhancer, increasing the level of endothelial nitric oxide synthase in endothelial cells and decomposing arginine into nitric oxide, ensuring an adequate supply of in situ nitric oxide. The coating system significantly slows down the degradation rate of the magnesium alloy stent, promotes the reconstruction of the natural endothelium, and prevents restenosis. The method of the present invention is highly operable and effective, and provides a new method for the surface coating of magnesium alloy vascular stent materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the preparation of the magnesium fluoride / polylipoic acid-arginine coating prepared in Example 1; Figure 2 The SEM images of the magnesium alloy in the first step of Example 1, the magnesium fluoride coating prepared in the second step, and the polylipoic acid-arginine coating prepared in the third step; Figure 3The EDS images of the magnesium fluoride coating prepared in the second step and the polylipoic acid-arginine coating prepared in the third step of Example 1 are shown; Figure 4 The potentiodynamic polarization curves of the magnesium alloy in the first step of Example 1, the magnesium fluoride coating prepared in the second step, and the polylipoic acid-arginine coating prepared in the third step; Figure 5 The endothelial cell CCK8 test of the blank group of Example 1, the magnesium alloy in the first step, the magnesium fluoride layer prepared in the second step, and the polylipoic acid-arginine coating prepared in the third step showed statistically significant differences (**p<0.01, *p<0.05); Figure 6 The NO release of the blank group of Example 1, the magnesium alloy in the first step, the magnesium fluoride coating prepared in the second step, and the polylipoic acid-arginine coating prepared in the third step were statistically significantly different (**p<0.01, *p<0.05); Figure 7 The endothelial cell nitric oxide synthase of the blank group of Example 1, the magnesium alloy in the first step, the magnesium fluoride coating prepared in the second step, and the polylipoic acid-arginine coating prepared in the third step were statistically significantly different (**p<0.01, *p<0.05); Note: Using independent samples t - test The significance level was determined, and the differences were considered significant at different levels when **p<0.01 and *p<0.05, and *p<0.05 was considered statistically significant. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below in conjunction with examples, but the protection scope of the present invention is not limited thereto.

[0018] The magnesium alloy vascular stent material used in the present invention can be purchased through the Materials Research Center of Zhengzhou University or commercial channels.

[0019] Embodiment 1: A method for preparing a magnesium alloy vascular stent material with an in-situ catalytic nitric oxide releasing coating, such as Figure 1 As shown, the following steps are included: The material of the magnesium alloy vascular stent is Mg-2.0Zn-0.5Y-0.5Nd magnesium alloy (brand name ZE21B, the specific preparation process can refer to patent ZL 201110043303.8).

[0020] Step 1: Pretreatment of magnesium alloy The magnesium alloy was cut into cylinders with a diameter of 10 mm and a thickness of 3 mm, and polished to be smooth using 100 mesh, 400 mesh, 800 mesh, and 1000 mesh sandpapers in sequence. The cylinders were then rinsed with deionized water and anhydrous ethanol in sequence, dried at 50 °C for 5 min, and set aside.

[0021] Step 2: Surface fluorination of magnesium alloy The pretreated ZE21B magnesium alloy sheet was placed in a 24-well plate (with a diameter of about 1.5 cm, a well depth of about 2 cm, and could hold about 3.3 mL of solution); Add 2 mL of 40 wt% hydrofluoric acid solution to the wells containing the magnesium alloy sheets, and place the 24-well plate in a fume hood at room temperature for 12 h; The fluorinated ZE21B magnesium alloy sheet was taken out, rinsed with deionized water and anhydrous ethanol in sequence for three cycles to remove the residual reaction solution, dried at 50°C for 5 min, and set aside.

[0022] Step 3 Preparation of polylipoic acid-arginine coating 10 g of lipoic acid was placed in a 100 mL beaker, heated in an oil bath to 120°C, and melted to a light yellow transparent fluid, and then arginine was added, with the molar ratio of arginine to lipoic acid being 1:2.5, and the temperature was continued to rise to 140°C to obtain a polylipoic acid-arginine fluid; The polylipoic acid-arginine coating was prepared on the surface of the fluorinated magnesium alloy sample by a dip coating method (temperature maintained at 140°C for 2 s) and then cooled.

[0023] The prepared magnesium alloy sample modified with the magnesium fluoride / polylipoic acid-arginine coating is placed in a sample bag and sealed for later use.

[0024] The sample of the ZE21B magnesium alloy surface modified with magnesium fluoride / polylipoic acid-arginine coating prepared in Example 1 was subjected to scanning electron microscopy (SEM) observation, energy spectrum (EDS) analysis, potentiodynamic polarization curve analysis, endothelial cell NO test, endothelial cell nitric oxide synthase test and endothelial cell CCK8 test. Figure 1 The experimental results are shown in Figures 2 to 7 shown.

[0025] Figure 1 This is a formation mechanism diagram of the magnesium fluoride / polythioic acid-arginine coating prepared in Example 1. Lipoic acid forms polythioic acid through disulfide self-polymerization, and the added arginine and polythioic acid build high entropy penalty salt bridge hydrogen bonds to stabilize the polythioic acid polymer network. The carboxyl group of polythioic acid can form dense hydrogen bonds with the magnesium fluoride coating, and then adhere to the fluorinated magnesium alloy surface.

[0026] Figure 2The SEM images of the samples prepared in Example 1 were gold-sprayed before the morphology was observed. Figure 2 It can be seen that there are scratches on the surface of the magnesium alloy substrate, which is caused by the grinding process. After fluorination treatment, the surface shows a relatively smooth morphology, and a dense and uniform fluoride coating can be observed. After coating with polylipoic acid-arginine coating, the surface morphology of the magnesium alloy vascular stent material is uniform and dense. In addition, the thickness of the magnesium fluoride coating and the polylipoic acid-arginine coating in the polylipoic acid-arginine sample are 2.38±0.27 μm and 13.32±1.93 μm, respectively.

[0027] Figure 3 The EDS data of each sample prepared in Example 1 is shown in Figure 1. The elemental composition of the magnesium fluoride coating sample is mainly Mg (43.35 wt%) and F (55.74 wt%), corresponding to the magnesium fluoride coating. After preparing the polylipoic acid-arginine coating, the main elements are C (53.50 wt%), S (27.72 wt%), N (11.24 wt%) and O (7.38 wt%). The C, S and O elements mainly come from lipoic acid, while the N element comes from arginine.

[0028] The corrosion resistance of magnesium fluoride / polylipoic acid-arginine coating samples was investigated using an electrochemical workstation. The specific test steps are as follows: the electrochemical workstation is the Interface 1000 model produced by Gamry Electrochemical Company of the United States, the electrolyte is Hanks' solution (pH 7.4, 0.14 g / L CaCl2, 0.1 g / L MgCl2·6H2O, 0.1 g / L MgSO4·7H2O, 0.4 g / L KCl, 0.06 g / L KH2PO4, 0.35 g / L NaHCO3, 8.0 g / L NaCl, 0.154g / L Na2HPO4·12 H2O, 1.0g / L glucose), and the temperature is 37 °C. All electrochemical measurements were performed in a traditional three-electrode system, in which a platinum sheet was used as the counter electrode, a saturated calomel electrode was used as the reference electrode, and an exposed area of ​​0.78 cm 2 The samples were used as working electrodes. Before the electrochemical test, the samples were immersed in Hanks' solution for 10 min to allow the open circuit potential (OCP) to reach a stable state. The scan rate of the potentiodynamic polarization curve was 1 mV·s -1 The corrosion potential and corrosion current density of the sample can be obtained by Tafel extrapolation. Generally speaking, the lower the corrosion current density, the slower the degradation rate. Figure 4 The potentiodynamic polarization curves of each sample are shown. The corrosion current density of ZE21B magnesium alloy is 1.10 × 10 -5 A / cm2 After fluoridation treatment, the corrosion current density of the magnesium fluoride sample is 1.85×10 -7 A / cm 2 , indicating that the degradation rate of the magnesium alloy matrix was significantly reduced. After modification with polylipoic acid-arginine, the corrosion current density of the coating sample dropped to 4.62×10 -8 A / cm 2 , further slowing down the degradation rate of the magnesium alloy matrix.

[0029] Human umbilical vein endothelial cells were cultured at 6×10 3 The cells were seeded at a concentration of 100 μL / well on the bottom of a 24-well plate, and 400 μL of endothelial cell culture medium was added to each well and cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 h. At the same time, the extracts of each sample group were prepared: the samples were transferred to a culture dish, irradiated with ultraviolet light (UV) on the front and back for 60 min, and the sample surface area / culture medium volume ratio (1.25 cm 2 / mL) to prepare sample extracts, referring to ISO 10993-12:2004. After 24 h of pre-culture, the old culture medium was removed, and 200 μL of culture medium and sample extract were added to each well at a ratio of 1:1, and 20 μL of fetal bovine serum and 2 μL of penicillin-streptomycin solution were added. This step was repeated every 24 h to replace fresh culture medium. After that, the culture medium was removed, and each well was washed with phosphate buffered saline (PBS), and 100 μL of culture medium containing 10% CCK-8 (Beijing Solebold Technology Co., Ltd., China) was added to each well and cultured in a cell culture incubator for 2 h. Subsequently, the absorbance value of each sample at 450 nm was detected by a microplate reader (BIO-RAD680, Bio-Rad, USA). In addition, at 24 h and 72 h of culture, the old culture medium removed from the cells was used to detect the content of NO and endothelial nitric oxide synthase. The specific steps were as follows: (1) The NO generation was evaluated using a NO detection kit (Biyuntian Biotechnology Co., Ltd., China). The released NO was easily oxidized to NO2 -, and quickly form a pink diazo compound with Griss reagent. The test process was strictly carried out in accordance with the manual of the NO detection kit, and the NO detection wavelength was 540nm. (2) Use the ELISA kit (Elerite Biotechnology Co., Ltd.) to test the endothelial cell nitric oxide synthase content. Add 100 μL of the sample group culture medium to the ELISA plate, incubate at 37℃ for 90 min, shake off the liquid in the well, add 100 μL of biotinylated antibody working solution to the well, incubate at 37℃ for 1 h, shake off the liquid in the well, wash the plate 3 times, pat dry on clean absorbent paper, add 100 μL of HRP enzyme conjugate working solution to each well, incubate at 37℃ for 30 min, shake off the liquid in the well, wash the plate 5 times, add 90 μL of substrate color development solution to each well, incubate at 37℃ in the dark for 15 min, add 50 μL of stop solution to each well, stop the reaction, and immediately measure the absorbance of each well at a wavelength of 450 nm using an ELISA reader. For details, refer to the steps of the ELISA kit.

[0030] like Figure 5 The results of the endothelial cell proliferation test for all samples are shown. Higher absorbance values ​​indicate more endothelial cell proliferation. After 24 h of culture, the absorbance values ​​of the blank group, ZE21B magnesium alloy, and magnesium fluoride coating samples were between 0.48 and 0.50, while the absorbance value of the polylipoic acid-arginine coating group was 0.53 ± 0.014, indicating that the polylipoic acid-arginine coating promoted the initial adhesion and proliferation of endothelial cells. After 72 h of culture, the absorbance values ​​of all sample groups increased significantly. It is worth noting that the absorbance values ​​of the ZE21B magnesium alloy (1.24 ± 0.01) and magnesium fluoride coating groups (1.17 ± 0.021) were lower than those of the blank control group (1.25 ± 0.018), indicating that it was unfavorable for the growth of endothelial cells. In contrast, the absorbance value of the polylipoic acid-arginine coating group was 1.30±0.02, which exceeded that of the blank control group, ZE21B magnesium alloy group and magnesium fluoride coating group, indicating that it has the ability to promote endothelial cell proliferation, which is attributed to the polylipoic acid-arginine coating system significantly promoting the release of NO, thereby promoting the self-repair of damaged endothelial cells.

[0031] As Figure 6The results of the NO release test showed that during the entire culture period, the NO release of the polylipoic acid-arginine coating group was significantly higher than that of the other groups: 1 d blank group (0.50±0.005), ZE21B magnesium alloy (0.52±0.006), magnesium fluoride coating (0.54±0.01), polylipoic acid-arginine coating (0.58±0.015); 3 d blank group (0.54±0.01), ZE21B magnesium alloy (0.54±0.015), magnesium fluoride coating (0.57±0.005), polylipoic acid-arginine coating (0.63±0.015), indicating that the LBL coating can stimulate EC to release more NO, indicating that it is superior to other sample groups. The above results are due to the fact that the lipoic acid and arginine contained in the polylipoic acid-arginine coating can act as enhancers of endothelial nitric oxide synthase and fuels for the synthesis of NO, respectively, thereby promoting the release of NO.

[0032] In order to verify the above conclusion, the content of endothelial nitric oxide synthase was quantitatively detected. Figure 7 The data show the content of endothelial nitric oxide synthase in all sample groups. The content of endothelial nitric oxide synthase in the polylipoic acid-arginine coating group was as high as 397.25±8.94 pg / mL (24h) and 4577.68±357.63 pg / mL (72h) during the culture period, which was much higher than that in other groups, indicating that lipoic acid significantly increased the activity of endothelial nitric oxide synthase, released more NO, and then promoted the proliferation and growth of endothelial cells. The above description is only a partial implementation mode of the present invention. For ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the principle of the present invention. These improvements and substitutions should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a magnesium alloy vascular stent material with an in-situ catalytic release of nitric oxide coating, characterized in that: The following steps are involved: (1) Take a magnesium alloy vascular stent material, use sandpaper to grind it until the surface is flat and smooth, and then clean it, dry it, and set it aside; (2) immersing the magnesium alloy vascular stent material of step (1) in a fluoridation treatment solution, reacting at room temperature for 8 to 48 hours, cleaning, and drying to obtain a magnesium alloy vascular stent material with a magnesium fluoride layer on the surface; (3) After the lipoic acid powder is heated and melted into a liquid, add arginine, continue to heat to 130-150°C, stir until the arginine is completely dissolved, and set aside; (4) The polylipoic acid-arginine liquid prepared in step (3) is applied to the surface of the magnesium alloy vascular stent material prepared in step (2) and cooled to obtain a magnesium alloy vascular stent material with a coating that releases nitric oxide in situ.

2. The preparation method according to claim 1, characterized in that The magnesium alloy vascular stent material described in step (1) is ZE21B, WE43, AZ31 or AZ91.

3. The preparation method according to claim 1, characterized in that: The fluorination treatment solution in step (2) is at least one of a 35-40 wt % hydrofluoric acid solution, a 0.1 M-0.3 M sodium fluoride aqueous solution, and a 0.1-0.3 M potassium fluoride aqueous solution.

4. The preparation method according to claim 1, characterized in that: The molar ratio of lipoic acid to arginine in step (3) is (5-1):

1.

5. The method for preparing a polylipoic acid-arginine coating according to claim 1, wherein: The melting temperature of the lipoic acid powder in step (3) is 110-130°C.

6. The method for preparing a polylipoic acid-arginine coating according to claim 1, wherein: The polylipoic acid-arginine coating described in step (4) is prepared by dip coating or spin coating.

7. A magnesium alloy vascular stent material with a coating for in-situ catalytic release of nitric oxide, prepared by the preparation method according to any one of claims 1 to 6.

8. The magnesium alloy vascular stent material with an in-situ catalytic release of nitric oxide coating according to claim 7, characterized in that: The thickness of the magnesium fluoride layer is 0.8 μm~3.0 μm, and the thickness of the polylipoic acid-arginine coating is 11.0 μm~16.0 μm.

9. Use of the magnesium alloy vascular stent material with an in-situ catalytic nitric oxide releasing coating as claimed in claim 7 or 8 in the preparation of an in vivo implant.

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