A cardiovascular stent material surface Cu-MOF / sulfonated hyaluronic acid hybrid coating and a preparation method thereof

Abstract

The application discloses a cardiovascular stent material surface Cu-MOF / sulfonated hyaluronic acid hybrid coating and a preparation method thereof, and belongs to the field of cardiovascular stent material surface treatment. The main purpose of the application is to solve the problem of endothelialization delay of the current cardiovascular stent, and the application has the function of promoting the tissue repair of the damaged blood vessel part. The method mainly includes three steps: preparation of Cu-MOF powder, synthesis of sulfonated hyaluronic acid material and preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating. The cardiovascular stent material surface Cu-MOF / sulfonated hyaluronic acid hybrid coating obtained by the method is uniform and dense, has strong adhesion, can significantly promote endothelial cell proliferation and migration, inhibit smooth muscle cell proliferation, and inhibit platelet adhesion and degeneration. The application has strong operability, remarkable effect and provides a new method for surface modification of cardiovascular stent material.

Description

Technical Field

[0001] This invention belongs to the field of surface treatment of cardiovascular stent materials, specifically relating to a Cu-MOF / sulfonated hyaluronic acid hybrid coating on the surface of cardiovascular stent materials and its preparation method. Background Technology

[0002] In recent years, improved living standards and changes in dietary structure have led to a significant increase in the incidence of cardiovascular and cerebrovascular diseases. These diseases are characterized by high morbidity, high disability rates, and high mortality rates, posing a serious threat to the health and lives of middle-aged and elderly individuals. Magnesium alloy vascular stents, due to their suitable mechanical properties, good biocompatibility, and biodegradability, have significant advantages in the treatment of cardiovascular and cerebrovascular diseases caused by embolism. However, delayed endothelialization after magnesium alloy stent implantation can easily lead to dangerous events such as late thrombosis and in-stent restenosis, which is a significant factor limiting the clinical translation of magnesium alloy vascular stents. Therefore, surface modification of magnesium alloy stents to accelerate endothelialization and promote the repair of damaged vascular tissue is of great significance for the clinical application of magnesium alloy vascular stents.

[0003] Metal-organic frameworks (MOFs) are porous materials formed by the coordination of metal ions and organic ligands. Their high porosity and high specific surface area are beneficial for the storage and release of biological signaling molecules and cell adhesion. Copper ions (Cu(II)) can catalyze the production of nitric oxide (NO) molecules from endogenous nitrosothiol donors. NO has multiple biological functions, including inhibiting platelet adhesion and smooth muscle cell proliferation, scavenging free radicals, preventing atherosclerosis, and promoting the repair of damaged intima. Cu-MOFs, with Cu(II) as the central metal atom, promote the recovery of damaged vascular tissue by catalyzing the release of NO from the donor.

[0004] Hyaluronic acid (HA) is a major structural component of the brain's extracellular matrix. It can neutralize the large amount of reactive oxygen species in the brain caused by stroke, promote the expression of brain-derived neurotrophic factor and nerve growth factor in astrocytes, and participate in tissue repair processes such as cell differentiation, neuronal proliferation, cell migration, and angiogenesis. However, hyaluronic acid is easily decomposed by hyaluronidase in the human body, which is not conducive to the factor regulation of hyaluronic acid at sites of vascular damage. Yu et al. [ACS. Appl. Mater. Interfaces 12 (2020) 46827–46836] found that sulfonated hyaluronic acid, prepared by adjusting the sulfur content to sulfonate hyaluronic acid, has significantly improved resistance to hyaluronidase degradation and has better effects in promoting endothelial cell growth, regulating macrophages to the M2 phenotype, and maintaining or transforming smooth muscle cells to the contractile phenotype.

[0005] Furthermore, the inventors combined Cu-MOF with sulfonated hyaluronic acid and successfully prepared it on the surface of cardiovascular stent materials. Utilizing NO generated in vivo and released sulfonated hyaluronic acid molecules, they achieved rapid in-situ endothelialization after stent implantation and tissue recovery at the site of vascular injury. The Cu-MOF / sulfonated hyaluronic acid functional coating has significant clinical implications in enhancing the biological properties of magnesium alloy vascular stents, achieving rapid endothelialization, and promoting the self-repair of damaged tissues. Summary of the Invention

[0006] To address the issue of delayed endothelialization in current clinical applications of magnesium alloy cardiovascular stents, this invention provides a multifunctional Cu-MOF / sulfonated hyaluronic acid hybrid coating. This coating is low-cost to prepare, and produces a uniform, dense coating with strong adhesion. It promotes endothelial cell adhesion, proliferation, and migration for rapid endothelialization, inhibits smooth muscle cell proliferation, exhibits anti-inflammatory and antithrombotic effects, and significantly reduces the late-stage in-stent restenosis rate.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a Cu-MOF / sulfonated hyaluronic acid hybrid coating on the surface of a cardiovascular stent material includes the following steps: (1) Take the cardiovascular stent material, sand it with sandpaper until the surface is smooth, rinse it with deionized water and anhydrous ethanol in turn, dry it and set it aside; (2) Preparation of Cu-MOF powder: Copper(II) nitrate trihydrate was dissolved in deionized water to obtain a 2 mmol / L to 20 mmol / L copper(II) nitrate solution, and 2,3,6,7,10,11-hexahydroxytrimethylene was dissolved in dimethyl sulfoxide to obtain a 2 mmol / L to 20 mmol / L 2,3,6,7,10,11-hexahydroxytrimethylene solution. The 2,3,6,7,10,11-hexahydroxytrimethylene solution was added to the copper(II) nitrate trihydrate solution, stirred, and the pH was adjusted to 7 to 8 using Tris-HCl solution. After stirring and centrifugation, the precipitate was collected and washed multiple times with methanol and ultrapure water, and dried to obtain Cu-MOF powder. (3) Synthesis of sulfonated hyaluronic acid material: Prepare a 5 g / L to 30 g / L sodium hyaluronate aqueous solution, add cation exchange resin to the sodium hyaluronate aqueous solution, continue stirring for 12 h to 48 h, let stand in the dark, filter, take the supernatant, adjust the pH of the solution to 8 to 9 with tetrabutylammonium hydroxide solution, heat to a viscous state, freeze dry to obtain quaternary ammonium salt hyaluronic acid, dissolve the quaternary ammonium salt hyaluronic acid and sulfur trioxide pyridine complex in N,N-dimethylformamide respectively, stir under ice bath conditions, add the sulfur trioxide pyridine complex solution dropwise to the quaternary ammonium salt hyaluronic acid solution, continue stirring in an ice bath, adjust the pH of the solution to 8 to 9 with sodium hydroxide solution, wash with acetone, dialyze in a dialysis bag, dry to a viscous state, freeze dry to obtain sulfonated hyaluronic acid sample; the concentration of ion exchange resin in sodium hyaluronate aqueous solution is 100 to 150 g / L; (4) Preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating: Sulfonated hyaluronic acid was dissolved in ultrapure water to prepare a sulfonated hyaluronic acid solution with a concentration of 0.5-5.0 mg / mL. Cu-MOF powder was dissolved in the sulfonated hyaluronic acid solution to make the concentration of Cu-MOF powder in the solution 1-10 μg / mL. The solution was stirred and dissolved to obtain a Cu-MOF / sulfonated hyaluronic acid mixed solution. N-hydroxysuccinimide solution with a concentration of 0.1-0.5 mg / mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution with a concentration of 0.5-2.5 mg / mL were added to the Cu-MOF / sulfonated hyaluronic acid mixed solution to obtain a Cu-MOF / sulfonated hyaluronic acid coating solution. This solution was prepared on the surface of the cardiovascular stent material and dried to obtain a Cu-MOF / sulfonated hyaluronic acid coating.

[0008] Preferably, the cardiovascular stent material in step (1) is a magnesium alloy, zinc alloy, iron alloy, 316L stainless steel, nickel-titanium alloy, or cobalt-chromium alloy.

[0009] Preferably, in step (2), the molar ratio of 2,3,6,7,10,11-hexahydroxytriphenylene and copper nitrate is (1-3):1.

[0010] Preferably, in step (3), the mass ratio of the quaternary ammonium hyaluronic acid and the sulfur trioxide pyridine complex is 1:(10-15); the molecular weight cutoff of the dialysis bag is 3000Da-5000Da, and dialysis is performed in ultrapure water.

[0011] Preferably, the surface of the cardiovascular stent material in step (4) is coated with a magnesium fluoride / polydopamine coating, and a Cu-MOF / sulfonated hyaluronic acid coating is prepared on the magnesium fluoride / polydopamine coating on the surface of the cardiovascular stent material. The preparation of the magnesium fluoride / polydopamine coating is a prior art technique.

[0012] Preferably, the ratio of N-hydroxysuccinimide solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and Cu-MOF / sulfonated hyaluronic acid mixed solution in step (4) is 1:1:(4-18); Preferably, in step (4), the Cu-MOF / sulfonated hyaluronic acid hybrid coating is prepared by dip coating, spray coating, or spin coating.

[0013] The cardiovascular stent material prepared by the above method has a Cu-MOF / sulfonated hyaluronic acid hybrid coating on its surface.

[0014] The direct technical benefits of the above-mentioned solution are: lower cost; and a dense, uniform Cu-MOF / sulfonated hyaluronic acid hybrid coating on the surface of the obtained cardiovascular stent material with strong adhesion, which can significantly promote endothelial cell proliferation and migration, inhibit smooth muscle cell proliferation, and inhibit platelet adhesion and degeneration. The method of this invention is highly operable and effective, providing a new approach for the surface modification of cardiovascular stent materials. Attached Figure Description

[0015] Figure 1 The image shows the XRD pattern of the Cu-MOF powder prepared in Example 1. Figure 2 (a) is a SEM image of the Cu-MOF powder prepared in Example 1; (b) is a SEM image of the sulfonated hyaluronic acid material prepared in Example 1; (c, d) are SEM images of the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1. Figure 3 The FT-IR spectrum of the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1 is shown below. Figure 4 XPS spectrum of Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1; Figure 5 Nanoscale scratch pattern of the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1; Figure 6 The potentiodynamic polarization curve of the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1 is shown. Figure 7 The NO release curve of the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1 is shown. Figure 8 The results of endothelial cell activity tests on the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1 are shown. Figure 9Morphological diagram of smooth muscle cells in the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1; Figure 10 This is an adhesion diagram of macrophages to the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1; Figure 11 The results of whole blood experiments on the Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared in Example 1 are shown. Note: The experimental groups with added exogenous glutathione and S-nitroso-N-acetylpenicillamine were designated as (+) donor groups, the experimental groups without added glutathione and S-nitroso-N-acetylpenicillamine were designated as (-) donor groups, the sample groups with a magnesium fluoride / polydopamine / sulfonated hyaluronic acid composite coating on the surface of the cardiovascular stent material were designated as sulfonated hyaluronic acid, and the sample groups with a magnesium fluoride / polydopamine / Cu-MOF / sulfonated hyaluronic acid composite coating on the surface of the cardiovascular stent material were designated as Cu-MOF / sulfonated hyaluronic acid. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to examples, but the scope of protection of the present invention is not limited thereto.

[0017] The cardiovascular stent materials used in this invention can all be obtained through the Materials Research Center of Zhengzhou University or through commercial channels.

[0018] Example 1: A method for preparing a Cu-MOF / sulfonated hyaluronic acid hybrid coating on the surface of a magnesium alloy cardiovascular stent material includes the following steps: The magnesium alloy cardiovascular stent is made of Mg-2.0Zn-0.5Y-0.5Nd magnesium alloy (grade ZE21B; for the specific preparation process, please refer to patent CN201110043303.8). The preparation method of the Cu-MOF / sulfonated hyaluronic acid hybrid coating is as follows: Step 1: Pretreatment of magnesium alloys Cut the magnesium alloy into cylinders with a diameter of 9 mm and a thickness of 3 mm. Grind the surface with sandpaper of 200 grit, 400 grit, 600 grit, 800 grit and 1000 grit in sequence and polish until the surface of the material is smooth and flat. Rinse with deionized water and anhydrous ethanol in sequence and dry for later use.

[0019] Step 2: Preparation of magnesium fluoride coating The polished ZE21B magnesium alloy sheet was placed in a 24-well plate (approximately 1.5 cm in diameter and 2 cm in depth). 2 mL of 40 wt% hydrofluoric acid solution was added to each well containing the magnesium alloy matrix. The 24-well plate was then placed in a fume hood and left at room temperature for 48 h. Remove the magnesium alloy sheet with magnesium fluoride coating, rinse it sequentially with deionized water and anhydrous ethanol to remove residual hydrofluoric acid, and dry it for later use.

[0020] Step 3: Preparation of polydopamine coating A 10 mM Tris solution was prepared, and dilute hydrochloric acid (HCl) solution was added dropwise to adjust the pH to 8.5 to obtain a Tris-HCl solution. Then, dopamine hydrochloride was dissolved in the above pH 8.5 Tris-HCl solution to prepare a 2 mg / mL polydopamine / Tris-HCl solution. Magnesium fluoride samples were placed in 24-well plates, and 1.5 mL of polydopamine / Tris-HCl solution was added to each well. The plates were then fixed on a constant temperature shaker and deposited at 37 °C for 6 h. Remove the sample coated with magnesium fluoride / polydopamine, rinse it sequentially with deionized water and anhydrous ethanol, and dry it for later use.

[0021] Step 4: Preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating (1) Preparation of Cu-MOF powder Copper nitrate trihydrate (II) was dissolved in deionized water, and 2,3,6,7,10,11-hexahydroxytriphenylene (CAS:4877-80-9) was dissolved in dimethyl sulfoxide, both to a concentration of 10 mM. The 2,3,6,7,10,11-hexahydroxytriphenylene solution was slowly added dropwise to the copper(II) nitrate trihydrate solution at a volume ratio of 2:1, and the mixture was stirred until the temperature dropped to room temperature. The pH of the mixed solution was adjusted to 7.4 using Tris-HCl solution (20 mM, pH=8), and stirred at room temperature for 1 h. The obtained solution was centrifuged at high speed (10,000 rpm, 1 h), the precipitate was collected and washed 5 times with methanol and ultrapure water, and then dried under vacuum at 80 °C. The resulting Cu-MOF powder was rapidly ground and stored under vacuum for later use.

[0022] (2) Synthesis of sulfonated hyaluronic acid materials Prepare a 10 g / L sodium hyaluronate aqueous solution and stir until the powder is completely dissolved. Add cation exchange resin [polymer of divinylbenzene and sulfonated (styrene and vinyl ethylbenzene) (CAS: 69011-20-7)] to the sodium hyaluronate aqueous solution until the concentration is 100 g / L, and continue stirring at room temperature for 24 h. After standing in the dark for 1 hour, the supernatant was filtered until the solution was clear and transparent. Then, 25 wt% tetrabutylammonium hydroxide solution was slowly added dropwise to adjust the pH of the solution to 8.0. The solution was dried at 65 °C until it became viscous. After freezing it into a solid in a refrigerator, it was freeze-dried at -60 °C in a freeze dryer to obtain white flocculent quaternary ammonium hyaluronic acid. Quaternary ammonium hyaluronic acid and sulfur trioxide pyridine complex were dissolved in N,N-dimethylformamide solvent, with a mass ratio of quaternary ammonium hyaluronic acid to sulfur trioxide pyridine complex of 1:12. Under ice bath conditions, the solutions were stirred until the temperature dropped to 0 °C. The sulfur trioxide pyridine complex solution was then added dropwise to the quaternary ammonium hyaluronic acid solution, and the mixture was stirred in an ice bath for 2 hours. After adjusting the pH of the quaternary ammonium hyaluronic acid solution to 8.5 by adding 1 mol / L sodium hydroxide solution, the solution was washed 5 times with acetone and then transferred to a dialysis bag (molecular weight cutoff: 3500 Da) for dialyzing in ultrapure water for 7 days. Remove the liquid from the dialysis bag, dry it at 60 ℃ until it becomes viscous, freeze it into a solid in a refrigerator, and then freeze-dry it at -60 ℃ in a freeze dryer to obtain the sulfonated hyaluronic acid sample. Store it away from light for later use.

[0023] (3) Preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating Sulfonated hyaluronic acid was dissolved in ultrapure water at a concentration of 2.0 mg / mL and stirred thoroughly. Cu-MOF powder was dissolved in sulfonated hyaluronic acid solution with a Cu-MOF concentration of 5 μg / mL and stirred until fully dissolved to prepare a Cu-MOF / sulfonated hyaluronic acid mixed solution. A Cu-MOF / sulfonated hyaluronic acid coating solution was prepared by thoroughly mixing an aqueous solution of N-hydroxysuccinimide with a concentration of 0.24 mg / mL and an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a concentration of 1.0 mg / mL with a Cu-MOF / sulfonated hyaluronic acid mixed solution at a volume ratio of 1:1:9. 1 mL of Cu-MOF / sulfonated hyaluronic acid coating solution was added to the surface of the magnesium fluoride / polydopamine sample, and deposition was carried out at 37℃ and 60 rpm for 6 h. After cleaning and drying, a magnesium alloy sample coated with magnesium fluoride / polydopamine / Cu-MOF / sulfonated hyaluronic acid was obtained, which was named Cu-MOF / sulfonated hyaluronic acid hybrid coating sample.

[0024] As a comparison, 1 mL of sulfonated hyaluronic acid coating solution was added to the surface of the ZE21B sample with magnesium fluoride / polydopamine coating obtained in the third step, and deposited at 37 ℃ and 60 rpm for 6 h. After cleaning and drying, a magnesium alloy sample coated with magnesium fluoride / polydopamine / sulfonated hyaluronic acid was obtained and named sulfonated hyaluronic acid sample.

[0025] The preparation process of the sulfonated hyaluronic acid coating solution is as follows: sulfonated hyaluronic acid is dissolved in ultrapure water at a concentration of 2.0 mg / mL and stirred thoroughly; an aqueous solution of N-hydroxysuccinimide with a concentration of 0.24 mg / mL and an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a concentration of 1.0 mg / mL are thoroughly mixed with the sulfonated hyaluronic acid solution at a volume ratio of 1:1:9 to obtain the sulfonated hyaluronic acid coating solution.

[0026] The samples prepared in Example 1 with a Cu-MOF / sulfonated hyaluronic acid hybrid coating were observed by SEM, detected by XRD, analyzed by FT-IR, XPS, and subjected to bonding force testing, potentiodynamic polarization curve analysis, NO release assay, endothelial cell proliferation assay, smooth muscle cell morphology analysis, macrophage adhesion analysis, and whole blood assay. The experimental results are as follows: Figures 1-11 .

[0027] Phase analysis of Cu-MOF powder was performed using a CT computed tomography (CT) X-ray diffraction (XRD) system. The development of Cu-MOF crystals can be observed through XRD patterns. Figure 1 As shown, the XRD pattern of Cu-MOF powder shows that its crystals are well-developed, with diffraction peaks at 5.1°, 9.5°, 12.6°, 16.5° and 27.8°, indicating the successful synthesis of the copper-based framework.

[0028] The surface morphology of Cu-MOF samples, sulfonated hyaluronic acid coated samples, and Cu-MOF / sulfonated hyaluronic acid hybrid coated samples was observed using dual-beam scanning electron microscopy. Before observation, Cu-MOF samples were dissolved in ultrapure water, and a small amount of the liquid was dropped onto a glass slide, dried, and then sputter-coated with gold. The remaining samples were directly sputter-coated with gold. SEM images are shown below. Figure 2 As shown in (a), the crystal morphology of Cu-MOF is micro-nano-sized particles. Figure 2 As shown in (c,d), the Cu-MOF / sulfonated hyaluronic acid hybrid coating exhibits good uniformity.

[0029] Cu-MOF powder was prepared into potassium bromide tablets, and the tablets were scanned using a Fourier transform infrared spectroscopy (FTIR) instrument with a wavenumber range of 500-4000 cm⁻¹. −1 .like Figure 3As shown, the Cu-MOF / sulfonated hyaluronic acid hybrid coating sample at 2960 cm⁻¹ -1 A small, prominent shoulder appeared near the wavenumber, at 2930 and 2857 cm. -1 Two small, raised shoulders appeared near the wavenumber, indicating that the sample may contain methyl (-CH3) and methylene (-CH2-) groups, which may originate from sulfonated hyaluronic acid. (1730, 1458, and 1373 cm⁻¹) -1 The presence of absorption peaks near the wavenumber indicates the presence of amide bonds (-CONH-) and CN in the sample. The presence of these chemical groups proves that the amino group in polydopamine and the carboxyl group in sulfonated hyaluronic acid underwent a dehydration condensation reaction during self-assembly, generating amide bonds. At 1242 cm⁻¹ -1 The presence of SO2 functional groups, characteristic of sulfonated hyaluronic acid, near the wavenumber indicates the successful fabrication of a Cu-MOF / sulfonated hyaluronic acid hybrid coating on a magnesium alloy surface.

[0030] This application used X-ray photoelectron spectroscopy to detect the chemical elemental composition of the Cu-MOF sample surface, using a 150 W Al Kα monochromatic X-ray source and an internal chamber pressure <5 × 10⁻⁶ W. -9 The obtained spectrum from Torr needs to be baseline-calibrated by setting the C 1s peak to 284.8 eV before further processing. The XPS spectrum is shown below. Figure 4 As shown, a distinct peak appeared on the F 1s orbital, confirming the successful preparation of the magnesium fluoride coating. The presence of the N 1s peak confirms the successful coating of the polydopamine coating. The Cu-MOF / sulfonated hyaluronic acid coating sample showed a smaller peak on the S 2p orbital, confirming the successful coating of the sulfonated hyaluronic acid coating. Simultaneously, the presence of Cu element also confirms the successful assembly of Cu-MOF.

[0031] like Figure 5 As shown, a nanoindenter was used in quasi-static dotting mode with a diamond probe. The scratch distance was set to 200 μm, and the scratch rate was 2.5 mN / s. The experimental results show that the adhesion strength of the Cu-MOF / sulfonated hyaluronic acid coating is 20-25 mN, indicating that the coating has strong adhesion.

[0032] The electrochemical corrosion behavior of Cu-MOF / sulfonated hyaluronic acid coating samples was studied using an electrochemical workstation (RST 5000). A platinum electrode and a saturated hot wire electrode were used as the electrode and reference electrode, respectively. The sample thickness (0.68 cm) was [not specified in the original text]. 2 The sample was immersed in Hanks' solution and allowed to stand for 1200 s, followed by potentiometric polarization at a scan rate of 1 mV / s. Figure 6As shown, the corrosion current density of the ZE21B alloy is 8.86 × 10⁻⁶. -5 A / cm 2 The corrosion current density of the Cu-MOF / sulfonated hyaluronic acid coating sample was 9.65 × 10⁻⁶. -7 A / cm 2 The corrosion current density decreased significantly, the corrosion potential increased significantly, and the corrosion resistance of magnesium alloys was enhanced.

[0033] Samples were placed in 24-well plates. 1 mL of a phosphate-buffered saline (PBS) solution containing 130 μM S-nitroso-N-acetylpenicillamine and 60 μM glutathione was added to the (+) donor group, and 1 mL of PBS solution (pH 7.4) was added to the (-) donor group. After incubation at 37 °C in the dark for 1 h, 2 h, and 3 h, 50 μL of the supernatant was added to a 96-well plate, followed by 50 μL of Griess I solution and 50 μL of Griess II solution. After thorough mixing, the absorbance was measured at 540 nm using a microplate reader. Figure 7 As shown, compared to the ZE21B alloy, the Cu-MOF / sulfonated hyaluronic acid coating group showed a higher concentration of NO production after the addition of glutathione and S-nitroso-N-acetylpenicillamine, indicating that Cu(II) has a catalytic effect on the decomposition of NO donors.

[0034] Endothelial cells were 1×10 4 Cells were seeded at a concentration of [cells / mL] at the bottom of 48-well adhesive plates, with 400 μL of endothelial cell culture medium added to each well. The plates were then incubated at 37 ℃ with 5% CO2 for 24 h. Afterward, the waste liquid was removed, and 200 μL of culture medium and extraction buffer were added to each well at a 1:1 ratio, along with 20 μL of fetal bovine serum and 2 μL of penicillin-streptomycin solution. This process was repeated every 24 h, replacing the medium with fresh medium. The control group used complete culture medium, with the same replacement conditions as the other groups. In the (+) donor group, glutathione and S-nitroso-N-acetylpenicillamine were added every 6 h to a final concentration of 10 μM. After culturing in an incubator for 24 h and 72 h respectively, the cells were removed and waste liquid was discarded. 440 μL of a mixture of culture medium and CCK-8 at a ratio of 9:1 was added, and the cells were incubated in a constant temperature incubator for 3 h. Then, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader. The relative viability of the endothelial cells was calculated. Figure 8As shown, compared with the ZE21B sample group and the sulfonated hyaluronic acid coated sample group, the Cu-MOF / sulfonated hyaluronic acid coated sample group exhibited higher cell activity, with a further increase in cell activity from 24 h to 72 h. Samples with added glutathione and S-nitroso-N-acetylpenicillamine showed even higher cell activity. The experimental results indicate that the Cu-MOF / sulfonated hyaluronic acid coating plays a beneficial role in the activity of endothelial cells.

[0035] Smooth muscle cells were collected at a rate of 1×10 4 Cells / mL were seeded at the bottom of 48-well adhesive plates containing 8 mm cell spreaders, and the cell culture procedure was the same as described above. For the (+) donor group, 10 μM glutathione and S-nitroso-N-acetylpenicillamine solution were added every 6 h. After incubation for 24 h and 72 h, the samples were removed, rinsed three times, and fixed with 100 μL of 4% paraformaldehyde fixative for 2 h. After rinsing, permeabilization was performed for 5 min, and finally, bovine serum albumin blocking solution was used for 30 min. 80 μL of phalloidin-FITC working solution (200 nmol / L) was added to the surface of each sample, and staining was performed in the dark for 50 min. Then, 80 μL of DAPI solution (10 μg / ml) was added, and staining was performed in the dark for 5 min. After rinsing three times with immunostaining washing solution, the samples were observed under a laser confocal microscope. Figure 9 As shown, compared with the ZE21B group, the Cu-MOF / sulfonated hyaluronic acid coated sample group had fewer smooth muscle cells and a smaller cell spreading area. Furthermore, the samples with added glutathione and S-nitroso-N-acetylpenicillamine exhibited even fewer cells and a smaller cell spreading area. The experimental results indicate that Cu(II) inhibits smooth muscle cell proliferation by catalyzing the decomposition of NO donors.

[0036] Macrophages were harvested at a rate of 2.5 × 10⁻⁶. 4 Cells / mL were seeded at a concentration of [number] cells / mL at the bottom of an adhesive 48-well plate containing an 8 mm cell spreader. Cell culture and staining were performed following the same procedures as described above. Observation was then performed under a laser confocal microscope. Figure 10 As shown, compared with the ZE21B sample group and the sulfonated hyaluronic acid coating sample group, the Cu-MOF / sulfonated hyaluronic acid coating sample group had the fewest macrophage adhesions and the smallest increase in cell proliferation from 24 h to 72 h. The sample groups with added glutathione and S-nitroso-N-acetylpenicillamine showed the most significant effect in inhibiting macrophage proliferation.

[0037] The sample was placed in a 24-well plate, and 100 μL of fresh human blood was added to its surface. The plate was then placed in a shaker (37 ℃, 100 rpm, 1 h). The sample was slowly rinsed with physiological saline, and 4% paraformaldehyde fixative was added to the surface for fixation. After 20 min, the sample was rinsed several times with physiological saline, followed by gradient dehydration with ethanol solution, and then dried for later use. After gold sputtering, the morphology was observed using SEM. Figure 11 As shown, compared to the ZE21B sample surface which has a large amount of fibrin network, the samples coated with sulfonated hyaluronic acid coating and Cu-MOF / sulfonated hyaluronic acid hybrid coating have significantly fewer blood cells adhering to their surfaces, and more platelets appear as inactive round shapes. This demonstrates that the sulfonated hyaluronic acid coating and Cu-MOF / sulfonated hyaluronic acid hybrid coating have better anticoagulant properties, with the Cu-MOF / sulfonated hyaluronic acid hybrid coating exhibiting the most significant anticoagulant properties.

[0038] Example 2: A method for preparing a Cu-MOF / sulfonated hyaluronic acid hybrid coating on the surface of a 316L stainless steel cardiovascular stent material includes the following steps: The material selected for cardiovascular and cerebrovascular stents is medical-grade 316L stainless steel, and the preparation method is as follows: The first step is the pretreatment of 316L stainless steel. Cut 316L stainless steel into 10 × 10 × 1 mm square pieces. Polish them with 200#, 400#, 600#, 800# and 1000# sandpaper in sequence until the surface is smooth and flat. Rinse with deionized water and anhydrous ethanol in sequence, and dry for later use. Step 2: Preparation of polydopamine coating A 10 mM Tris solution was prepared, and dilute hydrochloric acid (HCl) solution was added dropwise to adjust the pH to 8.5 to obtain a Tris-HCl solution. Then, dopamine hydrochloride was dissolved in the above pH 8.5 Tris-HCl solution to prepare a 2 mg / mL polydopamine / Tris-HCl solution. The polished 316L stainless steel sample was placed in a 24-well plate, and 2 mL of polydopamine / Tris-HCl solution was added to each well. The plate was then fixed on a constant temperature shaker and deposited at 37 °C for 8 h. Remove the sample, rinse it sequentially with deionized water and anhydrous ethanol, and dry it for later use.

[0039] Step 3: Preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating (1) Preparation of Cu-MOF powder (2) Copper nitrate trihydrate (II) was dissolved in deionized water and 2,3,6,7,10,11-hexahydroxytriphenylene (CAS:4877-80-9) was dissolved in dimethyl sulfoxide, respectively, and the concentration of each was prepared to be 10 mM. The 2,3,6,7,10,11-hexahydroxytriphenylene solution was slowly added dropwise to the copper(II) nitrate trihydrate solution at a volume ratio of 2:1, and the mixture was stirred until the temperature dropped to room temperature. The pH of the mixed solution was adjusted to 7.5 using Tris-HCl solution (20 mM, pH=8), and stirred at room temperature for 1 h. The obtained solution was centrifuged at high speed (10,000 rpm, 1 h), the precipitate was collected and washed 5 times with methanol and ultrapure water, and then dried under vacuum at 80 °C. The resulting Cu-MOF powder was rapidly ground and stored under vacuum for later use.

[0040] (2) Synthesis of sulfonated hyaluronic acid materials Prepare a sodium hyaluronate aqueous solution with a concentration of 15 g / L, stir until the powder is completely dissolved, add cation exchange resin [polymer of divinylbenzene and sulfonated (styrene and vinyl ethylbenzene) (CAS: 69011-20-7)] to the sodium hyaluronate aqueous solution to a concentration of 100 g / L, and continue stirring at room temperature for 24 h; After standing in the dark for 1 hour, the supernatant was filtered until the solution was clear and transparent. Then, 25 wt% tetrabutylammonium hydroxide solution was slowly added dropwise to adjust the pH of the solution to 8.5. The solution was dried at 65 °C until it became viscous. After freezing it into a solid in a refrigerator, it was freeze-dried at -60 °C in a freeze dryer to obtain white flocculent quaternary ammonium hyaluronic acid. Quaternary ammonium hyaluronic acid and sulfur trioxide pyridine complex were dissolved in N,N-dimethylformamide solvent, with a mass ratio of quaternary ammonium hyaluronic acid to sulfur trioxide pyridine complex of 1:12. Under ice bath conditions, the solutions were stirred until the temperature dropped to 0 °C. The sulfur trioxide pyridine complex solution was then added dropwise to the quaternary ammonium hyaluronic acid solution, and the mixture was stirred in an ice bath for 2 hours. After adjusting the pH of the quaternary ammonium hyaluronic acid solution to 8.5 with 1 mol / L sodium hydroxide solution, the solution was washed five times with acetone and then transferred to a dialysis bag (molecular weight cutoff: 3500 Da) for dialysis for 7 days. Remove the liquid from the dialysis bag, freeze it into a solid in a refrigerator, and then freeze-dry it at -60 ℃ in a freeze dryer to obtain a sulfonated hyaluronic acid sample. Store it away from light for later use.

[0041] (3) Preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating Sulfonated hyaluronic acid was dissolved in ultrapure water at a concentration of 1.5 mg / mL and stirred thoroughly. Cu-MOF powder was dissolved in sulfonated hyaluronic acid solution with a Cu-MOF concentration of 7 μg / mL and stirred until fully dissolved to prepare a Cu-MOF / sulfonated hyaluronic acid mixed solution. A Cu-MOF / sulfonated hyaluronic acid coating solution was prepared by thoroughly mixing an aqueous solution of N-hydroxysuccinimide with a concentration of 0.24 mg / mL and an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a concentration of 1.0 mg / mL with a Cu-MOF / sulfonated hyaluronic acid mixed solution at a volume ratio of 1:1:12. 1.5 mL of Cu-MOF / sulfonated hyaluronic acid solution was added to the surface of the polydopamine-coated sample, and deposition was carried out at 37 °C and 60 rpm for 8 h. After washing and drying, a 316L stainless steel sample coated with polydopamine / Cu-MOF / sulfonated hyaluronic acid was obtained.

[0042] The above description is only a partial embodiment of the present invention. For those skilled in the art, several improvements and substitutions can be made without departing from the principle of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a Cu-MOF / sulfonated hyaluronic acid hybrid coating on the surface of a cardiovascular stent material, characterized in that, Includes the following steps: (1) Take the cardiovascular stent material, sand it with sandpaper until the surface is smooth, rinse it with deionized water and anhydrous ethanol in turn, dry it and set it aside; (2) Preparation of Cu-MOF powder: Copper(II) nitrate trihydrate was dissolved in deionized water to obtain a 2 mmol / L to 20 mmol / L copper(II) nitrate solution, and 2,3,6,7,10,11-hexahydroxytrimethylene was dissolved in dimethyl sulfoxide to obtain a 2 mmol / L to 20 mmol / L 2,3,6,7,10,11-hexahydroxytrimethylene solution. The 2,3,6,7,10,11-hexahydroxytrimethylene solution was added to the copper(II) nitrate trihydrate solution, stirred, and the pH was adjusted to 7 to 8 using Tris-HCl solution. The mixture was stirred, centrifuged, the precipitate was collected, and washed multiple times with methanol and ultrapure water. The precipitate was dried to obtain Cu-MOF powder. The molar ratio of 2,3,6,7,10,11-hexahydroxytrimethylene to copper nitrate was (1 to 3):

1. (3) Synthesis of sulfonated hyaluronic acid material: Prepare a 5 g / L to 30 g / L sodium hyaluronate aqueous solution, add cation exchange resin to the sodium hyaluronate aqueous solution, continue stirring for 12 h to 48 h, let stand in the dark, filter, take the supernatant, adjust the pH of the solution to 8 to 9 with tetrabutylammonium hydroxide solution, heat to a viscous state, freeze dry to obtain quaternary ammonium salt hyaluronic acid, dissolve the quaternary ammonium salt hyaluronic acid and sulfur trioxide pyridine complex in N,N-dimethylformamide respectively, stir under ice bath conditions, add the sulfur trioxide pyridine complex solution dropwise to the quaternary ammonium salt hyaluronic acid solution, continue stirring in an ice bath, adjust the pH of the solution to 8 to 9 with sodium hydroxide solution, wash with acetone, dialyze in a dialysis bag, dry to a viscous state, freeze dry to obtain sulfonated hyaluronic acid sample; the concentration of ion exchange resin in sodium hyaluronate aqueous solution is 100 to 150 g / L; (4) Preparation of Cu-MOF / sulfonated hyaluronic acid hybrid coating: Sulfonated hyaluronic acid was dissolved in ultrapure water to prepare a sulfonated hyaluronic acid solution with a concentration of 0.5-5.0 mg / mL. Cu-MOF powder was dissolved in the sulfonated hyaluronic acid solution to make the concentration of Cu-MOF powder in the solution 1-10 μg / mL. The solution was stirred and dissolved to obtain a Cu-MOF / sulfonated hyaluronic acid mixed solution. N-hydroxysuccinimide solution with a concentration of 0.1-0.5 mg / mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution with a concentration of 0.5-2.5 mg / mL were added to the Cu-MOF / sulfonated hyaluronic acid mixed solution to obtain a Cu-MOF / sulfonated hyaluronic acid coating solution. This solution was prepared on the surface of the cardiovascular stent material and dried to obtain a Cu-MOF / sulfonated hyaluronic acid coating.

2. The preparation method according to claim 1, characterized in that, The cardiovascular stent material in step (1) is a magnesium alloy, zinc alloy, iron alloy, 316L stainless steel, nickel-titanium alloy, or cobalt-chromium alloy.

3. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the quaternary ammonium salt hyaluronic acid to the sulfur trioxide pyridine complex is 1:(10-15); the molecular weight cutoff of the dialysis bag is 3000Da-5000Da, and dialysis is performed in ultrapure water.

4. The preparation method according to claim 1, characterized in that, In step (4), the surface of the cardiovascular stent material is coated with magnesium fluoride / polydopamine, and a Cu-MOF / sulfonated hyaluronic acid coating is prepared on the surface of the cardiovascular stent material coated with magnesium fluoride / polydopamine.

5. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of N-hydroxysuccinimide solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and Cu-MOF / sulfonated hyaluronic acid mixed solution is 1:1:(4-18).

6. The preparation method according to claim 1, characterized in that, In step (4), the Cu-MOF / sulfonated hyaluronic acid hybrid coating is prepared by dip coating, spray coating, or spin coating.

7. The cardiovascular stent material with a surface of Cu-MOF / sulfonated hyaluronic acid hybrid coating prepared by the preparation method according to any one of claims 1 to 6.

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