A coating with surface double-gas release function, magnesium alloy, preparation method and application

Abstract

The application discloses a coating with surface double-gas release function, a magnesium alloy, a preparation method and application, and the coating is a 2-methacryloyloxyethyl phosphorylcholine-diallyl disulfide-acrylamide ternary copolymer with a carbon monoxide release molecular side chain. The ternary copolymer coating with the carbon monoxide release molecular side chain is covalently combined on the surface of the magnesium alloy through a thermal initiation polymerization mode, which can not only significantly improve the corrosion resistance of the magnesium alloy, but also in-situ release H2S and CO under the stimulation of a physiological environment, so that the corrosion resistance and physiological regulation capacity of the magnesium alloy are simultaneously improved, the blood compatibility and the oxidation resistance of the magnesium alloy material are synergistically improved, and the growth of vascular endothelial cells is selectively promoted, so that the vascular endothelial in-situ repair and regeneration are induced.

Description

Technical Field

[0001] This invention relates to a coating, a magnesium alloy, a preparation method, and an application, and more particularly to a coating, a magnesium alloy, a preparation method, and an application with surface dual gas release function. Background Technology

[0002] Cardiovascular disease (CVD) has become the leading cause of death worldwide. Coronary atherosclerotic heart disease (CAD) is one of the main types of stenotic cardiovascular disease. Metallic vascular stents can dilate narrowed arteries, thereby restoring blood flow and maintaining patency, improving patients' quality of life and saving lives. They are widely used in the clinical treatment of stenotic cardiovascular disease, and developing ideal coronary stents has always been the ultimate goal of interventional cardiology. Although traditional non-degradable metallic stents (316L, Co-Cr, NiTi) have significantly improved the treatment effect of CAD, currently used metallic stents (including drug-eluting stents) remain permanently in the body after implantation. Factors such as metal ion leakage, the inhibitory effect of anti-proliferative drugs on vascular endothelial regeneration, and the poor biocompatibility between metallic materials and drug carrier polymer materials inevitably induce clinical complications such as inflammation, excessive intimal proliferation, late thrombosis, and late in-stent restenosis. Furthermore, patients need to take antiplatelet drugs for life, which places a great psychological and economic burden on them. Therefore, the development of absorbable vascular stents has become a key issue that urgently needs to be addressed in the current treatment of cardiovascular diseases.

[0003] Biodegradable vascular stents, once implanted in the human body, can completely degrade after the damaged blood vessels have been repaired. Furthermore, their degradation products can be absorbed or metabolized by the body, preventing permanent retention and avoiding the clinical side effects caused by long-term retention of non-degradable metal stents. This offers significant advantages and application potential in cardiovascular interventional therapy. Magnesium alloys possess excellent mechanical properties and good biodegradability. Their degradation products are excreted through metabolism or absorbed by the body, completely overcoming the clinical complications caused by the permanent retention of traditional metal stents. Therefore, they are one of the most promising materials in the field of absorbable cardiovascular stents. However, research on magnesium alloy vascular stent materials still faces many challenges, such as premature loss of mechanical strength due to excessively rapid corrosion rates and uneven corrosion degradation, and complications such as thrombosis, delayed endothelialization, and inflammatory reactions due to poor biocompatibility. Surface modification can simultaneously improve the corrosion resistance and biocompatibility of magnesium alloys and is currently the main method for improving the performance and function of magnesium alloy vascular stent materials. However, existing surface modification methods still face many key challenges: First, magnesium alloys have poor corrosion resistance, and the bioactive substances loaded on the surface of most existing strategies are prone to rapid inactivation, lack specificity, and have low in vivo stability, making it difficult to exert a stable effect in the long term. Furthermore, they are costly and difficult to prepare on a large scale, further limiting their application. Second, the surface modified layers or coatings constructed by existing strategies have weak anticoagulant properties and cannot effectively selectively induce and promote the growth of vascular endothelial cells. As a result, the endothelialization performance after implantation is poor, which can easily lead to clinical complications such as late thrombosis and late restenosis.

[0004] Studies have shown that gaseous signaling molecules within physiological concentration ranges have unique advantages in regulating human physiological responses and promoting endothelialization of material surfaces. Hydrogen sulfide (H2S), as an important endogenous gaseous signaling molecule, can effectively dilate blood vessels, inhibit platelet adhesion and aggregation, and significantly improve the blood compatibility of materials. Simultaneously, it can inhibit excessive proliferation and migration of vascular smooth muscle cells, alleviate implant-induced intimal hyperplasia and restenosis, and also exert anti-inflammatory, antioxidant, and endothelial protective effects, reducing local oxidative stress and inflammatory responses, and improving the implantation microenvironment. Carbon monoxide (CO) is also an endogenous gaseous signaling molecule. Within physiological concentration ranges, CO can not only effectively reduce the risk of thrombosis but also selectively promote the adhesion and growth of endothelial cells and induce endothelial progenitor cells to differentiate into endothelial cells, thereby inhibiting restenosis. Therefore, constructing a bioactive coating on the surface of magnesium alloys that can simultaneously release H2S and CO can leverage their synergistic effect to significantly improve the biocompatibility of implanted materials. CN115089773A discloses a biomimetic coating for vascular stents, a vascular stent, and a method for preparing the same. The coating can self-crosslink and adhere to a polymer matrix on the stent surface, adaptively generating corresponding signaling factors such as CO and H2S. However, it only attaches polymeric macromolecules and adaptive small molecules to the matrix surface via photo-initiated polymerization, and the bonding strength between the coating and the matrix, as well as between coating components, needs improvement. Simultaneously, CO-releasing molecules are mainly physically embedded in the polymer, resulting in poor controllable release performance, burst release behavior, and difficulty in stably and continuously releasing CO molecules. Furthermore, while the coating enhances its anticoagulant properties through hydrophilic polymers, its resistance to biofouling is insufficient. Additionally, this technology primarily generates the polymer coating through photocrosslinking, requiring specific equipment, and the polymerization conditions are difficult to control. Summary of the Invention

[0005] Objectives of the Invention: The objective of this invention is to provide a coating with high bonding strength that can release H2S and CO in situ under physiological environmental stimulation. The second objective of this invention is to provide a magnesium alloy containing the coating. The third objective of this invention is to provide a method for preparing the magnesium alloy. The fourth objective of this invention is to provide applications of the magnesium alloy.

[0006] Technical solution: The coating with surface dual gas release function of the present invention is a terpolymer of 2-methacryloyloxyethyl phosphorylcholine-diallyl disulfide-acrylamide (MPC-DATS-AM) with carbon monoxide releasing molecular side chains.

[0007] Among them, 2-methacryloyloxyethylphosphorylcholine (MPC), as an amphoteric antifouling monomer, can mimic the cell membrane structure and significantly inhibit the adsorption of proteins and platelets. Immobilizing it on the magnesium alloy surface can significantly improve the anticoagulant properties of the material. Diallyl disulfide (DATS), containing a disulfide bond structure, can stably cleave and continuously release H2S under physiological conditions, exhibiting high biocompatibility. Acrylamide (AM), with its strong hydrophilicity, can significantly improve the film-forming properties and coating adhesion of the copolymer, and provides amino side chains for grafting carbon monoxide-releasing molecules. In the presence of a thermal initiator, MPC, DATS, and AM can form a uniform and dense ternary copolymer while simultaneously forming a covalent bond with the functionalized magnesium alloy matrix, significantly improving the bonding strength between the matrix and the coating.

[0008] The molar ratio of 2-methacryloyloxyethyl phosphorylcholine, diallyl disulfide, and acrylamide in the terpolymer is 10~30:30~50:30~50. If the molar ratio of MPC is <10, it will lead to insufficient zwitterionic components, resulting in a significant decrease in the coating's anti-protein adsorption, anticoagulation, and anti-biofouling properties, making it prone to inducing thrombosis after implantation. If the molar ratio of MPC is >30, the proportions of DATS and AM decrease. The reduced DATS content leads to insufficient H2S release, and the reduced AM content leads to insufficient grafting of carbon monoxide molecules, resulting in insufficient CO release. At the same time, the coating's hydrophilicity and film-forming properties deteriorate, making the coating prone to cracking. If the molar ratio of DATS is <30, the three... The low disulfide bond content in the terpolymer coating results in insufficient H2S release under physiological conditions, leading to a lack of vasodilatory, anti-inflammatory, and intimal hyperplasia-inhibiting effects. If the molar ratio of DATS is greater than 50, the coating's mechanical strength and corrosion resistance decrease, and the degradation rate becomes too fast. If the molar ratio of AM is less than 30, the number of amino groups in the terpolymer coating is insufficient, significantly reducing the grafting rate of carbon monoxide-releasing molecules and drastically decreasing CO release capacity. If the molar ratio of AM is greater than 50, the coating absorbs water and swells severely, easily peeling and flaking off under long-term immersion, resulting in decreased overall stability.

[0009] The carbon monoxide-releasing molecule is CORM-401, which has good solubility in aqueous solution and can be decomposed by reactive oxygen species or cysteine ​​in a physiological environment to induce the release of CO. CORM-401 has a carboxyl group, which can form a covalent bond with the amino group of AM through a condensation reaction, and is firmly bound to the side chain of the MPC-DATS-AM terpolymer to form a stable H2S / CO dual-gas release functional coating.

[0010] The magnesium alloy with surface dual gas release function described in this invention has the aforementioned coating on its surface. The coating has a thickness of 5~50μm on the magnesium alloy surface and its structure consists of MPC, DATS and AM arranged in parallel on the magnesium alloy surface. The ends of AM are connected to carbon monoxide releasing molecules (CORM401). The DATS polymer chain segment formed by thermal polymerization can release H2S gas, while the CORM401 grafted to the ends of AM can release CO gas.

[0011] The method for preparing a magnesium alloy with surface dual-gas release function according to the present invention includes the following steps: immersing a surface-allylated magnesium alloy substrate in a monomer mixture of 2-methacryloyloxyethyl phosphorylcholine (MPC), diallyl disulfide (DATS), acrylamide (AM), and a thermal initiator to initiate an in-situ thermal polymerization reaction to obtain a magnesium alloy substrate with a ternary copolymer coating; after the reaction, grafting carbon monoxide-releasing molecules onto the side chains of the ternary copolymer to obtain a magnesium alloy with surface dual-gas release function.

[0012] Preferably, the in-situ thermal polymerization reaction is carried out at a temperature of 60-80°C for 4-8 hours under an inert gas atmosphere. The in-situ thermal polymerization reaction causes MPC, DATS, and AM to form a uniform and dense ternary polymer on the magnesium alloy surface, which then bonds to the magnesium alloy surface, improving the bonding strength between the coating and the substrate surface.

[0013] Preferably, the thermal initiator is one of ammonium persulfate, potassium persulfate, and azobisisobutyronitrile, and its addition amount is 0.5% to 2% of the total mass of MPC, DATS, and AM; MPC, DATS, AM, and the thermal initiator are prepared as a monomer mixture using a polar solvent; the polar solvent is at least one of deionized water, ethanol, and dimethylformamide.

[0014] The surface allylation treatment involves immersing a surface-alkali-thermally treated magnesium alloy substrate in a solution containing allyl functionalizing agents to initiate a surface self-assembly reaction. The surface alkali-thermally treated magnesium alloy surface contains -OH groups, enabling the functionalizing agents to form covalent bonds with the -OH matrix and adhere to the substrate surface. The allylation treatment introduces double bonds into the magnesium alloy surface, allowing MPC, DATS, and AM to react with these double bonds to form covalent bonds and polymerize on the magnesium alloy surface, thereby enhancing the polymerization strength between the coating and the substrate.

[0015] Preferably, the surface alkaline heat treatment involves immersing the polished and cleaned magnesium alloy substrate in a strong alkaline solution at 60-80°C for 12-24 hours; the strong alkaline solution is a sodium hydroxide solution with a concentration of 1-3 mol / L.

[0016] The allyl functionalizing agent is allyltrimethoxysilane, with a mass fraction of 0.5% to 2% in the solution, which is an anhydrous ethanol solution. Allyltrimethoxysilane forms a Mg-O-Si covalent bond with the -OH groups on the magnesium alloy surface, with the allyl groups having double bonds facing outwards.

[0017] Preferably, the surface self-assembly reaction is carried out at room temperature for 8-12 hours.

[0018] The grafting process involves activating carbon monoxide-releasing molecules with an activating solution, then immersing a magnesium alloy substrate with a terpolymer coating in the activated carbon monoxide-releasing molecule solution to initiate a grafting reaction.

[0019] The activation solution is a mixed activation solution (EDC / NHS) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), with a molar ratio of EDC to NHS of 3:1. The activation solution is used to activate the carboxyl group (-COOH) in the carbon monoxide release molecule into a highly reactive intermediate, allowing for direct grafting with the amino group in AM.

[0020] Preferably, the activation involves mixing carbon monoxide-releasing molecules with an activation solution, with the activation temperature at room temperature and the activation time at 30-60 minutes; the mass concentration of the carbon monoxide-releasing molecules in the solution is 3%-8%, and the solution is an aqueous solution.

[0021] Preferably, the grafting reaction is carried out at a temperature of 25~40℃ for 6~12h, and the reaction is conducted in the dark.

[0022] The applications of the magnesium alloy with surface dual gas release function described in this invention include the preparation of medical magnesium alloy orthopedic implants, vascular stents, bone repair materials, medical magnesium alloy wound repair modification materials, or anti-inflammatory and corrosion-resistant medical magnesium alloy functional devices.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) By thermally initiated polymerization, a ternary copolymer coating with carbon monoxide-releasing molecular side chains is constructed on the surface of magnesium alloy. This not only significantly improves the corrosion resistance of magnesium alloy, but also releases H2S and CO in situ under the stimulation of the physiological environment, thereby simultaneously improving the corrosion resistance and physiological regulation of magnesium alloy, synergistically improving the blood compatibility and antioxidant properties of magnesium alloy material, selectively promoting the growth of vascular endothelial cells, thereby inducing in situ repair and regeneration of vascular endothelium; (2) The dual-gas (H2S / CO) release coating containing MPC segments endows magnesium alloy with excellent hydrophilicity and anti-biofouling properties, prevents non-specific adhesion of plasma proteins and blood cells, improves the anticoagulant properties of the material, and inhibits thrombus formation; (3) The coating preparation method is simple and reproducible. The thickness, composition and gas release of the surface coating can be adjusted as needed, thus facilitating its promotion and application. At the same time, the surface modification method of the present invention can also be used for the surface modification of other biomaterials and implantable medical devices, and has a wide range of application value. Attached Figure Description

[0024] Figure 1 The results are the water contact angle measurements of the magnesium alloy surfaces prepared in the embodiments and comparative examples of this invention.

[0025] Figure 2 The H2S release curves of the magnesium alloy surfaces prepared in the embodiments and comparative examples of the present invention are shown.

[0026] Figure 3 The CO release curves of the magnesium alloy surfaces prepared in the embodiments and comparative examples of this invention are shown.

[0027] Figure 4 The weight loss corrosion rate of the magnesium alloys prepared in the embodiments and comparative examples of this invention;

[0028] Figure 5 The hemolysis rate of the magnesium alloys prepared in the embodiments and comparative examples of this invention;

[0029] Figure 6 The partial thromboplastin time (APTT) of the magnesium alloys prepared in the embodiments and comparative examples of the present invention is given.

[0030] Figure 7 The effect of partial thromboplastin time on the magnesium alloys prepared in the embodiments and comparative examples of the present invention;

[0031] Figure 8 The endothelial cell adhesion on the surface of the magnesium alloy prepared in the embodiments and comparative examples of the present invention;

[0032] Figure 9 This is a schematic diagram of the magnesium alloy surface coating structure prepared according to an embodiment of the present invention. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the embodiments and comparative examples. Unless otherwise stated, all reagents used are commercially available and are used directly without purification.

[0034] Example 1

[0035] (1) Alkali heat treatment of magnesium alloy + silane modification: After polishing, cleaning and drying, the magnesium alloy was immersed in 2 mol / L NaOH solution, alkali heat treatment at 70℃ for 18h and then taken out. After rinsing with deionized water and drying, the magnesium alloy substrate with surface alkali heat treatment was placed in a 1% mass fraction allyltrimethoxysilane-anhydrous ethanol solution and self-assembled at room temperature for 10h. After self-assembly, it was cleaned with ethanol and dried to obtain the magnesium alloy with surface allylation treatment.

[0036] (2) Preparation of monomer solution: Weigh MPC, DATS and AM as reaction monomers in a molar ratio of 10:30:30. The total amount of monomers is 0.08 mol. Dissolve them in deionized water to prepare a monomer mixture. Add 1% of the total mass of ammonium persulfate as a thermal initiator and stir until completely dissolved.

[0037] (3) In-situ thermal polymerization: The magnesium alloy with surface allylation treatment is completely immersed in the monomer mixture and polymerized at 70°C for 6 hours; after removal, it is cleaned with deionized water and ethanol and dried at room temperature to obtain a magnesium alloy matrix with a terpolymer coating.

[0038] (4) CORM401 covalent grafting: Prepare a 5% CORM401 aqueous solution, add EDC / NHS mixed activation solution (molar ratio 3:1), and activate for 45 min; immerse the magnesium alloy substrate with the terpolymer coating into the activated CORM401 solution, react at 30℃ in the dark for 9 h, remove the magnesium alloy substrate after the reaction, clean to remove free CORM401, and dry to obtain a magnesium alloy with a dual gas release function coating.

[0039] Example 2

[0040] (1) Alkali heat treatment of magnesium alloy + silane modification: After polishing, cleaning and drying, the magnesium alloy was immersed in 2 mol / L NaOH solution, alkali heat treatment at 70℃ for 18h and then taken out. After rinsing with deionized water and drying, the magnesium alloy substrate with surface alkali heat treatment was placed in a 1% mass fraction allyltrimethoxysilane-anhydrous ethanol solution and self-assembled at room temperature for 10h. After self-assembly, it was cleaned with ethanol and dried to obtain the magnesium alloy with surface allylation treatment.

[0041] (2) Preparation of monomer solution: Weigh MPC, DATS and AM as reaction monomers in a molar ratio of 20:40:40. The total amount of monomers is 0.08 mol. Dissolve them in deionized water to prepare a monomer mixture. Add 1.2% of potassium persulfate as a thermal initiator and stir until completely dissolved.

[0042] (3) In-situ thermal polymerization: The magnesium alloy with surface allylation treatment is completely immersed in the monomer mixture and polymerized at 70°C for 6 hours; after removal, it is cleaned with deionized water and ethanol and dried at room temperature to obtain a magnesium alloy matrix with a terpolymer coating.

[0043] (4) CORM401 covalent grafting: Prepare a 5% CORM401 aqueous solution, add EDC / NHS mixed activation solution (molar ratio 3:1), and activate for 45 min; immerse the magnesium alloy substrate with the terpolymer coating into the activated CORM401 solution, react at 30℃ in the dark for 9 h, remove the magnesium alloy substrate after the reaction, clean to remove free CORM401, and dry to obtain a magnesium alloy with a dual gas release function coating.

[0044] Example 3

[0045] (1) Alkali heat treatment of magnesium alloy + silane modification: After polishing, cleaning and drying, the magnesium alloy was immersed in 2 mol / L NaOH solution, alkali heat treatment at 70℃ for 18h and then taken out. After rinsing with deionized water and drying, the magnesium alloy substrate with surface alkali heat treatment was placed in a 1% mass fraction allyltrimethoxysilane-anhydrous ethanol solution and self-assembled at room temperature for 10h. After self-assembly, it was cleaned with ethanol and dried to obtain the magnesium alloy with surface allylation treatment.

[0046] (2) Preparation of monomer solution: Weigh MPC, DATS and AM as reaction monomers in a molar ratio of 30:50:50. The total amount of monomers is 0.08 mol. Dissolve them in deionized water to prepare a monomer mixture. Add 1.5% of the total mass of azobisisobutyronitrile as a thermal initiator and stir until completely dissolved.

[0047] (3) In-situ thermal polymerization: The magnesium alloy with surface allylation treatment is completely immersed in the monomer mixture and polymerized at 75°C for 7 hours; after removal, it is cleaned with deionized water and ethanol and dried at room temperature to obtain a magnesium alloy matrix with a terpolymer coating.

[0048] (4) CORM401 covalent grafting: Prepare a 6% CORM401 aqueous solution, add EDC / NHS mixed activation solution (molar ratio 3:1), and activate for 50 min; immerse the magnesium alloy substrate with the terpolymer coating into the activated CORM401 solution, react at 35°C in the dark for 10 h, remove the magnesium alloy substrate after the reaction, clean to remove free CORM401, and dry to obtain a magnesium alloy with a dual gas release function coating.

[0049] Comparative Example 1: The polished and cleaned magnesium alloy was subjected to alkaline heat treatment using the method described in Example 2. Then, using the complete process of Example 2, an MPC / DATS / AM copolymer coating was directly copolymerized onto the alkaline-treated magnesium alloy surface. Furthermore, CORM401 was grafted onto the surface using the process of Example 2. The difference from Example 2 is that the magnesium alloy substrate was not subjected to surface allylation treatment. Because the magnesium alloy surface did not undergo self-assembly surface modification after alkaline heat treatment, it lacked allylic groups. The ternary copolymer could not form covalent bonds with the substrate and adhered only through physical adsorption, resulting in poor coating adhesion. Part of the coating peeled off during cleaning. Therefore, allylation modification is a prerequisite for achieving covalent bonding between the copolymer and the magnesium alloy; without this step, only a loosely adhered physically formed coating can be obtained.

[0050] Comparative Example 2: The polished and cleaned magnesium alloy underwent surface pretreatment using the alkaline heat treatment and self-assembly process described in Example 2. A monomer mixture of MPC:DATS = 20:40 (molar ratio, total monomer content 0.08 mol) was prepared. The remaining preparation process and reaction parameters were identical to those in Example 2. The difference from Example 2 was that the in-situ thermal polymerization yielded an MPC-DATS binary copolymer. Since the coating did not contain AM, CORM401 could not be covalently grafted onto it, and therefore the coating did not have CO release capability. After polymerization, a polymer coating that only releases H2S was obtained.

[0051] Comparative Example 3: The polished and cleaned magnesium alloy underwent surface pretreatment using the alkaline heat treatment and self-assembly process described in Example 2. A mixed solution of MPC:AM = 20:40 (molar ratio, total monomer content 0.08 mol) was prepared. The remaining preparation process and reaction parameters were completely consistent with Example 2. The difference from Example 2 was that the in-situ thermal polymerization reaction yielded an MPC-AM binary copolymer. After polymerization, CORM401 was grafted using the process described in Example 2 to obtain a polymer coating that only releases CO.

[0052] The magnesium alloys prepared in the examples and comparative examples were subjected to the following performance tests:

[0053] 1. Surface water contact angle measurement

[0054] Water contact angle measurements were performed using a water contact angle meter (KrüSS GmbH, Germany). During measurement, 10 μL of deionized water was dropped onto the material surface, and the instrument automatically measured the contact angle after 10 seconds. Measurements were conducted at room temperature. Surface water contact angle tests were performed on unmodified magnesium alloy (Mg), alkali-heat-treated magnesium alloy (Mg-OH), allyltrimethoxysilane-modified magnesium alloy (Mg-Si), and the magnesium alloys of Comparative Examples 1-3 and Examples 1-3. Specific test results are as follows: Figure 1 As shown. By Figure 1 It is evident that alkaline heat treatment significantly improves the hydrophilicity of the magnesium alloy surface, but the self-assembled allyltrimethoxysilane makes the surface hydrophobic again. Due to the introduction of highly hydrophilic MPC and AM segments into the magnesium alloy surface by the in-situ thermal polymerization reaction, the hydrophilicity of the comparative and examples is good, with a significantly lower contact angle compared to Mg and Mg-Si. Notably, the water contact angles of Examples 1-3 do not show a significant difference, likely because the surface MPC and AM segments exhibit excellent hydrophilicity.

[0055] 2. H2S gas release experiment

[0056] The hydrogen sulfide release of different samples was determined by methylene blue spectrophotometry (comparative and example samples). The specific procedure was as follows: The sample was placed at the bottom of a 4 mL centrifuge tube pre-filled with 2 mL of phosphate-buffered saline (PBS) at pH 7.4, and 100 μL of glutathione (10 mM) was added. At preset time points (5 min, 10 min, 20 min, 40 min, 60 min), 200 μL of zinc acetate solution, 400 μL of p-aminodimethylaniline sulfuric acid solution, and 400 μL of ferric chloride solution were added sequentially to initiate the colorimetric reaction. After standing for 20 min, the absorbance was measured at 670 nm, and the H2S release curve was plotted based on the standard curve.

[0057] H2S release curves of different samples are as follows Figure 2 As shown, Examples 1-3, due to the introduction of DATS that can release H2S, all exhibited good hydrogen sulfide gas release behavior under the catalysis of glutathione, and there was no burst release phenomenon. Due to the different DATS content on the surface, the amount of H2S released from the surface varied. Among them, Comparative Example 1 had the weak adhesion of the surface coating, and the coating was partially detached during the cleaning process, resulting in the smallest amount of hydrogen sulfide released. Comparative Example 2 had the highest DATS content on the surface, and its release amount was also the largest.

[0058] 3. CO gas release experiment

[0059] CO release was assessed using a myoglobin (Mb) assay. 100 μM myoglobin (Mb) was dissolved in PBS, degassed with N2 for 30 min, and then 20 mg of sodium dithionite (Na2S2O3) was added to completely deplete oxygen, converting it to deoxymyoglobin (DeoMb). 1 mL of the DeoMb solution was then mixed with 3 mg of CORM-401 and 0.5 mg of cysteine ​​to convert all of the DeoMb to carbonylhemoglobin (Mb-CO), and the absorbance change of Mb-CO at 540 nm was recorded. Samples were placed in 3 mL of DeoMb solution, and the absorbance changes at 540 nm were measured for samples with and without 200 μL of cysteine ​​(catalyst, 10 μM), with the catalyst added every 6 hours. The CO release concentration was determined using the following formula:

[0060]

[0061] OD 540 It is the absorbance of the Mb-CO solution at 540 nm, ΔOD 540 The change in absorbance at 540 nm of Mb-CO solution after sample addition with and without cysteine ​​is ΔOD. iso510 This represents the change in absorbance of the solution at its isoabsorption point of 510 nm, ε = 15.4 mM. -1 cm -1 It is the extinction coefficient of Mb-CO.

[0062] CO release curves from different sample surfaces are shown below. Figure 3 As shown, after CORM401 was fixed on the surface, CO was continuously released from the surface for more than 7 days under the catalysis of cysteine. Except for Comparative Example 1, where the surface coating was lost due to poor adhesion, resulting in a significant reduction in CO release, the CO release from the surface of the other samples was mainly related to the amount of CORM401 fixed on the surface, and there was no burst release behavior.

[0063] 4. Weight loss corrosion test

[0064] Immersion tests were used to evaluate the corrosion behavior of the materials. Simulated human body fluid (NaCl 8 g / L, KCl 0.4 g / L, NaHCO3 0.35 g / L, CaCl2 0.14 g / L, Na2HPO4 0.06 g / L, KH2PO4 0.06 g / L, MgSO4·7H2O 0.01 g / L, glucose 1 g / L, pH=7.4) was used for testing, with the temperature controlled at 37±1℃. The ratio of solution volume to exposed sample surface area was consistently maintained at 20 mL / cm². 2After cleaning and drying, the test samples were immersed in simulated human body fluid for 15 days, with the liquid changed every two days. After the corrosion test, the samples were immersed in a chromate cleaning solution (containing 200 g / L chromium trioxide and 2 g / L silver nitrate) at room temperature for 8-10 seconds to remove surface corrosion products. The samples were then rinsed with deionized water, dried with cold air, and finally weighed. To ensure reliable and repeatable experimental results, three parallel samples were used in each experiment. The average corrosion rate was calculated based on the weight difference before and after corrosion.

[0065] The average corrosion rate of different samples over 15 days is as follows: Figure 4 As shown, the unmodified magnesium alloy exhibits poor corrosion resistance, resulting in the highest corrosion rate. Alkali heat treatment (Mg-OH) and allyltrimethoxysilane self-assembly modification (Mg-Si) can improve corrosion resistance to some extent, thus reducing the corrosion rate. After preparing an H2S / CO bioactive polymer coating on the surface, the average corrosion rate of Examples 1-3 further decreased, but there was no significant difference among the three, indicating that all three coatings can significantly improve the corrosion resistance of the magnesium alloy. It is worth noting that the coating obtained in Comparative Example 1 has poor adhesion to the substrate, making it prone to peeling off. Consequently, its effect on improving the corrosion resistance of the magnesium alloy is not as good as that of Examples 1-3. Furthermore, the coatings in Comparative Examples 2 and 3 have lower density than those of the ternary polymer, resulting in inferior corrosion resistance compared to the examples.

[0066] 5. Hemolysis test

[0067] Red blood cells isolated from healthy human whole blood (from the Second People's Hospital of Huai'an City) were centrifuged at 1000 rpm and prepared into a 2% red blood cell suspension with physiological saline. 2 ml of this suspension was added to the sample surface. Simultaneously, 2 ml of physiological saline was prepared as a cathode control; 2 ml of a 2% red blood cell suspension (prepared with distilled water) was prepared as an anode control. After incubation at 37℃ for 3 hours, 1 ml of the incubation solution was transferred to a centrifuge tube and centrifuged at 3000 rpm for 5 minutes. 200 μL of the supernatant was added to a 96-well plate, and the absorbance at 545 nm was measured using a microplate reader. The hemolysis rate was calculated using the following formula:

[0068] Hemolysis(%)=(A-A2) / (A1-A2)×100%

[0069] In the formula: A is the absorbance of the sample, A1 is the absorbance of the anodic control, and A2 is the absorbance of the cathode control.

[0070] Hemolysis rate test results for different samples are as follows Figure 5As shown, due to the poor corrosion resistance of unmodified magnesium alloy Mg, rapid corrosion leads to a hemolysis rate close to 30%. Alkali heat treatment and subsequent self-assembly modification improve the corrosion resistance of the material, thus significantly reducing the hemolysis rate, but it is still higher than 5%, posing a certain risk of hemolysis and failing to meet the relevant national standard requirements (<5%). The hemolysis rates of all samples modified with the polymer coating are less than 5%, and the hemolysis rate of the examples is lower than that of the comparative example, indicating that the polymer coating prepared in this invention can improve the anticoagulant properties of magnesium alloy materials.

[0071] 6. Approximately Adaptive Time (APTT) Test

[0072] Platelet-Potential Plasma (PPP) was obtained by centrifuging fresh anticoagulated whole blood from healthy volunteers (from the Second People's Hospital of Huai'an City). The sample and PPP were incubated together at 37°C for 15 min. Then, 50 μL of the incubated PPP was added to a test cup, followed by 50 μL of activated partial thromboplastin time (APTT) reagent, and incubated for another 3 min at 37°C. Next, 50 μL of 0.025 mol / L calcium chloride solution was added, and the APTT was automatically measured using a Sysmex CA-1500 coagulation analyzer. Three parallel samples were used for each group, and the average result was taken as the final result.

[0073] Figure 6 The coagulation time measurements of different samples showed that the coagulation times of Mg, Mg-OH, and Mg-Si were all shorter than those of blank plasma, indicating that these three substances may induce blood coagulation and thrombus formation. However, the coagulation times of the magnesium alloy samples in Examples 1-3 were significantly prolonged (longer than those of blank plasma), indicating that the coating has the effect of prolonging coagulation time, thus making it less likely to cause thrombus formation and coagulation when in contact with blood. Notably, the coagulation times of Examples 1-3 were significantly greater than those of Comparative Examples 1-3, indicating that the coating prepared in this invention can significantly improve the anticoagulant properties of magnesium alloys. Furthermore, the APTT time of Examples 1-3 under gas release conditions was tested, and the results are as follows... Figure 7 As shown, it can be seen that the release of H2S (Example - H2S) prolongs the clotting time, while the release of CO (Example - H2S / CO) further prolongs the clotting time. On the surface, H2S and CO have a synergistic effect in improving the anticoagulant properties of the material, with Example 2 showing the best effect.

[0074] 7. Endothelial cell adhesion experiment

[0075] The sealed samples were sterilized by ultraviolet irradiation for 12 h, followed by the addition of 0.5 ml of endothelial cell suspension (purchased from the Cell Bank of the Chinese Academy of Sciences) (5 × 10⁶ cells / mL). 4Cells were cultured in 1.5 ml of cell culture medium at 37°C and 5% CO2 for 24 h. After washing with physiological saline, the cells were fixed with 2.5% glutaraldehyde at 4°C for 3 h. After washing again with physiological saline, the cells were stained with 100 μL of rhodamine (10 μg / ml) and 100 μL of 4,6-diamidinyl-2-phenylindole (DAPI, 500 ng / ml) for 20 min and 10 min, respectively. Finally, the morphology of the cells adhering to the sample surface was observed and fluorescent images of the cells were captured using an inverted fluorescence microscope (Carl Zeiss A2 inverted) in the dark. The results are as follows: Figure 8 As shown, in the absence of H2S or CO release, the number of endothelial cells adhering to the surface of Examples 1-3 is less than that of the Mg-OH and Mg-Si surfaces. This is because the MPC block in the MPC-DATS-AM terpolymer coating of the magnesium alloy surface has anti-biofouling properties. Medical magnesium alloy orthopedic implants, vascular stents, bone repair materials, medical magnesium alloy wound repair modification materials, or anti-inflammatory and anti-corrosion medical magnesium alloy functional devices prepared using this magnesium alloy can significantly resist foreign cell contamination. However, since the magnesium alloy of Examples 1-3 has improved corrosion resistance, the number of cells adhering to the surface is significantly greater than that of the unmodified magnesium alloy Mg. Furthermore, when H2S is released from the magnesium alloy surface, the number of cells adhering to the magnesium alloy surface is improved. When H2S / CO is released simultaneously from the magnesium alloy surface, the number of endothelial cells adhering to the surface increases significantly. This means that in the simulated biological environment of H2S / CO catalytic release, CO can synergistically promote the adhesion and growth of endothelial cells with H2S. The magnesium alloys prepared in Examples 1-3 have good endogenous properties for promoting the proliferation and regeneration of endothelial cells, thereby accelerating the endothelialization process on the material surface.

Claims

1. A coating with surface dual-gas release function, characterized in that, The coating is a terpolymer of 2-methacryloyloxyethyl phosphorylcholine-diallyl disulfide-acrylamide with carbon monoxide-releasing molecular side chains.

2. The coating with dual surface gas release function according to claim 1, characterized in that, The molar ratio of 2-methacryloyloxyethyl phosphorylcholine, diallyl disulfide, and acrylamide in the terpolymer is 10~30:30~50:30~50.

3. The coating with dual surface gas release function according to claim 1, characterized in that, The carbon monoxide-releasing molecule is CORM-401.

4. A magnesium alloy with surface dual-gas release function, characterized in that, The magnesium alloy surface has a coating as described in any one of claims 1-3.

5. A method for preparing a magnesium alloy with surface dual-gas release function as described in claim 4, characterized in that, The process includes the following steps: immersing a surface-allylated magnesium alloy substrate in a monomer mixture of 2-methacryloyloxyethyl phosphorylcholine, diallyl disulfide, acrylamide, and a thermal initiator to initiate an in-situ thermal polymerization reaction to obtain a magnesium alloy substrate with a ternary copolymer coating; after the reaction, grafting carbon monoxide-releasing molecules onto the side chains of the ternary copolymer to obtain a magnesium alloy with surface dual-gas release function.

6. The preparation method according to claim 5, characterized in that, The surface allylation treatment involves immersing a surface-alkali-treated magnesium alloy substrate in a solution containing an allyl functionalizing agent to initiate a surface self-assembly reaction.

7. The preparation method according to claim 6, characterized in that, The allyl functionalizing agent is allyltrimethoxysilane.

8. The preparation method according to claim 5, characterized in that, The grafting process involves activating carbon monoxide-releasing molecules with an activating solution, then immersing a magnesium alloy substrate with a terpolymer coating in the activated carbon monoxide-releasing molecule solution to initiate a grafting reaction.

9. The preparation method according to claim 8, characterized in that, The activation solution is a mixed activation solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

10. An application of the magnesium alloy with surface dual gas release function as described in claim 4, the application including the preparation of medical magnesium alloy orthopedic implants, vascular stents, bone repair materials, medical magnesium alloy wound repair modification materials, or anti-inflammatory and corrosion-resistant medical magnesium alloy functional devices.

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