Ultra-smooth anticoagulant functional material as well as preparation method and application thereof

Abstract

The invention provides a super-smooth anticoagulant functional material and a preparation method and application method thereof, and the functional material comprises a base material and a coating formed on the base material through a noradrenaline monomer self-assembly synthesis reaction, the surface of the coating formed by the self-assembly synthesis reaction of the noradrenaline monomer is provided with a sustained-release medicine which can be covalently grafted with phenolic hydroxyl groups or amino groups. Through a noradrenaline monomer self-assembly synthesis reaction, an ultra-smooth polymer coating with the thickness of 1-500nm is formed on the surface of the coronary stent. The surface of the coating has extremely high smoothness, adhesion of biomacromolecules can be effectively reduced, and the risk of thrombosis is reduced; the surface of the coating is rich in functional groups with chemical activity, so that the carrying capacity and the loading rate of the medicine can be greatly improved, and the medicine release time is prolonged; the coating can effectively fix trace NO gas in the blood vessel and enhance the elasticity and relaxation function of the blood vessel; the hydrophilic and chemical active functional groups on the surface of the coating significantly improve the biocompatibility of the stent.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a super-smooth anticoagulant functional material, its preparation method and application. Background Art

[0002] As the main interventional treatment method for treating coronary artery stenosis and blockage, coronary stents have been widely used in clinical practice. Its basic principle is to implant a metal stent into the narrowed blood vessel to provide structural support and maintain the patency of the blood vessel, thereby improving blood flow and reducing the risk of heart attack. Since the 1990s, the technology of coronary stents has been continuously developed. In particular, the introduction of drug-eluting coronary stents (DES) has significantly reduced the incidence of restenosis.

[0003] DES is the most widely used type of coronary stent at present. Its surface is coated with a layer of drug, which is mainly used to inhibit intimal hyperplasia and reduce the risk of restenosis. The most common drug coatings such as Sirolimus or Paclitaxel prevent excessive proliferation of vascular wall cells and restenosis by locally releasing drugs. The structure of drug-eluting stents is usually composed of metal materials such as stainless steel, cobalt-chromium alloy or nickel-titanium alloy. These metals have strong mechanical strength and elasticity and can effectively support blood vessels.

[0004] Although drug-eluting stents have significantly improved the treatment effect, they still have some problems, mainly including the attachment of biological macromolecules, the colonization of microorganisms in blood vessels, and the limitations of drug release. In addition, the long-term use of drug-eluting stents may cause some complications, such as stent thrombosis (ST), intimal hyperplasia, stent splitting and other problems. With the increasing number of coronary stent implantations, these problems have attracted more and more clinical attention.

[0005] Although drug-eluting stents have achieved remarkable results in reducing restenosis, they still face the following several technical problems and limitations:

[0006] 1. Problem of biological macromolecule attachment

[0007] Existing drug-eluting coronary stents have problems such as rough surface and poor hydrophilicity, resulting in easy attachment of biological macromolecules (such as proteins, cells, platelets, etc.) in the blood to the stent surface. This attachment may not only lead to complications such as thrombosis, intimal hyperplasia, and microbial infection, but also accelerate the occurrence of vascular restenosis. The coatings of existing technologies cannot completely and effectively prevent the attachment of these biological macromolecules, thus affecting the treatment effect.

[0008] 2. Low drug release efficiency and insufficient persistence

[0009] Currently, the drug-eluting stents used in clinical applications mainly employ degradable or non-degradable polymers (such as poly-lactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (PVA), etc.) as drug sustained-release carriers. By controlling the drug release rate, these drug-loaded polymers are expected to provide continuous anti-restenosis treatment after stent implantation. However, the existing polymer systems generally have the following problems: on the one hand, the drug release curve often exhibits an "explosive release" phenomenon in the initial stage, resulting in too high local drug concentration and increasing the risk of cytotoxicity; on the other hand, the duration of drug efficacy is limited and it is difficult to cover the time window required for the complete repair of endothelial cells, thus affecting the long-term inhibition effect of restenosis.

[0010] 3. Mechanical stress problems in the intravascular microenvironment

[0011] After the rigid metal material of traditional coronary stents is implanted into blood vessels, it may generate mechanical stress on the vessel wall, resulting in impaired elasticity and structural recovery of the blood vessels and increasing the risk of vessel splitting or microvascular rupture. In addition, the contact between the metal material and endothelial cells of blood vessels may trigger a foreign body reaction, affecting the repair and biocompatibility of vascular endothelium and increasing the risk of complications.

[0012] 4. Biocompatibility problems in the long-term use of stents

[0013] During the long-term use of existing drug-eluting stents, there are still biocompatibility problems such as incomplete fusion between the stent material and vascular tissue and incomplete endothelial repair, which may lead to complications such as in-stent thrombosis, intimal hyperplasia of the stent, and restenosis in patients, and require patients to undergo secondary or even multiple surgeries. Summary of the Invention

[0014] The purpose of the present invention is to provide a super-smooth anti-coagulation functional material, its preparation method and application to solve the technical problems existing in the background technology.

[0015] The technical solution adopted by the present invention is as follows:

[0016] A super-smooth anti-coagulation functional material, the functional material includes a substrate and a coating formed by self-assembly synthesis reaction of norepinephrine monomers on the substrate, and the coating surface formed by the self-assembly synthesis reaction of norepinephrine monomers has a sustained-release drug that can be covalently grafted with phenolic hydroxyl groups or amino groups.

[0017] Through the self-assembly synthesis reaction of norepinephrine monomers, a super-smooth polymer coating with a thickness of 1 - 500 nm is formed on the surface of the coronary stent. The coating surface has extremely high smoothness, which can effectively reduce the attachment of biological macromolecules, and is rich in hydrophilic functional groups on the surface, which can form a dense water film on the stent surface, further reducing the possibility of biological molecule attachment.

[0018] The coating is rich in chemically active functional groups (such as phenolic hydroxyl groups, amino groups, etc.), and these functional groups can interact with drug molecules, providing a higher drug loading rate and loading capacity. The functional groups on the surface of the coating not only increase the affinity between the drug and the stent surface, but also can slowly release the drug after the stent is implanted, thereby improving the efficacy of the drug and reducing the occurrence of restenosis. The drug release behavior is closely related to the therapeutic effect of the stent. If the drug is released too quickly, although it can effectively inhibit the proliferation of smooth muscle cells in the initial stage, the drug concentration will rapidly decrease subsequently, which may lead to vascular restenosis; if the release time is too short, it is impossible to achieve long-term regulation of blood vessels and support tissue repair, and it is also difficult to form a stable re-endothelialization environment. The present invention realizes the slow, controllable, and long-term release of drugs on the surface of the stent through interface structure regulation and material modification means.

[0019] The coating can also effectively fix trace amounts of NO gas in the blood vessel, enhance the elasticity of the blood vessel, and reduce the risks of blood vessel rupture and splitting. The fixation of NO gas can promote the repair of the vascular endothelium, improve the biocompatibility of the blood vessel, and reduce postoperative complications.

[0020] Further preferably, the above-mentioned sustained-release drugs include immunosuppressants and / or blood thinners.

[0021] Further preferably, the sustained-release drugs include one or more of heparin, sirolimus, and paclitaxel.

[0022] Further preferably, the material of the above-mentioned substrate is medical stainless steel, cobalt-chromium alloy, nickel-titanium alloy, or medical polymer material. Using a biocompatible material as the substrate provides the mechanical support of the stent and ensures sufficient patency in the blood vessel.

[0023] Based on the same technical concept, the present invention provides a preparation method of a super-smooth anticoagulant functional material, including the following steps:

[0024] After cleaning and activating the substrate, a self-assembly synthesis reaction of norepinephrine is carried out on the surface of the substrate in an alkaline environment to form a coating, and then a sustained-release drug is covalently grafted on the surface of the coating.

[0025] Further preferably, the above-mentioned activation step includes placing the cleaned substrate in an oxygen plasma processor and treating it at a power of 100 W for more than 10 minutes.

[0026] Use a mixed solution of deionized water, absolute ethanol, and acetone for triple ultrasonic cleaning to remove surface oil stains and impurities; place the cleaned stent in an oxygen plasma processor for treatment to enhance surface hydrophilicity and reactivity.

[0027] Further preferably, the step of forming a coating by the self-assembly synthesis reaction of norepinephrine includes immersing the activated substrate into a solution containing norepinephrine and oscillating the reaction at room temperature for 10 - 12 hours.

[0028] Further preferably, the pH value of the above solution containing norepinephrine is 8 - 9, and the solution contains norepinephrine with a concentration of 0.1 - 10 mg / mL and copper sulfate with a concentration of 0.05 - 0.2 μM.

[0029] By regulating the concentration in the process of self-assembly synthesis of norepinephrine coating and adding a copper sulfate solution with an appropriate concentration, the prepared coating is smoother and flatter. The alkaline environment and 0.05 - 0.2 μM copper sulfate in the solution provide necessary conditions for the self-assembly synthesis reaction. Under this environment, norepinephrine monomers polymerize through oxidation reaction and self-assemble into a polymer coating, which uniformly covers the surface of the coronary stent. The benzene ring structure in the norepinephrine monomer can form stable chemical bonds with the stent surface through chemical reactions, ensuring the firmness of the coating.

[0030] Further preferably, after each coating in the above self-assembly synthesis reaction of norepinephrine to form a coating, it is pre-dried at 30 - 60 °C and repeated multiple times to control the coating thickness within 1 - 500 nm, and the surface roughness Ra / coating thickness D < 10%. By controlling the concentration and number of times in the self-assembly synthesis reaction of norepinephrine, a dense and uniform nano-scale smooth coating is obtained.

[0031] Further preferably, the step of covalently grafting a sustained-release drug on the coating surface includes immersing the substrate with the coating into a solution containing the sustained-release drug and reacting at 30 - 40 °C for 10 - 12 hours.

[0032] Based on the same technical idea, the present invention provides an application of the above ultra-smooth anti-coagulation functional material or the ultra-smooth anti-coagulation functional material prepared by the above preparation method in a coronary stent.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] 1. Effectively reduce the attachment of biological macromolecules and reduce the risk of thrombus formation

[0035] Through the design of the ultra-smooth coating, the present invention significantly reduces the microscopic roughness of the stent surface and reduces the attachment of biological macromolecules (such as proteins, cells, platelets, etc.) in the blood. This effect of reducing attachment can effectively reduce thrombus formation and reduce the incidence of intimal hyperplasia of the stent, thereby reducing the risk of vascular restenosis. The hydrophilicity of the coating surface helps to form a dense water film on the stent surface, further isolating the blood from the stent surface and enhancing the anti-attachment performance.

[0036] 2. Prolong the drug release time and improve the drug loading rate

[0037] The surface of the coating of the present invention is rich in chemically active functional groups such as phenolic hydroxyl groups and amino groups, which can greatly improve the drug loading capacity and loading rate. Compared with traditional drug-coated stents, this technology can not only improve the drug release efficiency, but also prolong the continuous drug release time, ensure that the drug can be released long-term and stably after implantation, and reduce the occurrence of restenosis. At the same time, the coating can adjust the drug release according to the changes in the vascular environment and has better potential for personalized treatment.

[0038] 3. Enhance vascular elasticity and reduce the risk of vascular splitting

[0039] The coating of the present invention can effectively fix trace amounts of NO gas in the blood vessel, enhance the elasticity and diastolic function of the blood vessel. The role of NO gas in the blood vessel helps the relaxation of vascular smooth muscle, improves the hemodynamic environment of the blood vessel, and reduces the risk of vascular splitting and microvascular rupture after surgery. By enhancing the biocompatibility and elasticity of the blood vessel, promoting endothelial repair, and reducing the mechanical stress damage of the blood vessel.

[0040] 4. Prolong the service life of the stent and reduce the need for secondary surgery

[0041] The coating of the present invention has excellent properties such as super-smoothness, durability, and anti-adhesion. Compared with existing drug-coated stents, it has a longer service life. The coating can effectively reduce the risk of re-blockage of the blood vessel, reduce the possibility of restenosis and thrombosis, thereby reducing the need for patients to undergo secondary or even multiple stent implantation surgeries. Patients can thus enjoy a more long-term and safe treatment effect.

[0042] 5. Simple and easy preparation process with wide applicability

[0043] The present invention uses a method of self-assembling and synthesizing a coating with norepinephrine monomer solution in an alkaline and 0.05 - 0.2 uM copper sulfate solution environment. This method is not only simple and easy, but also the reaction conditions can be adjusted, and the thickness, smoothness, and chemical functional characteristics of the coating can be flexibly controlled. This method is applicable to coronary stents made of various materials and does not require complex equipment or technology. It has wide applicability and can achieve large-scale production.

[0044] 6. Improve biocompatibility and reduce the incidence of complications

[0045] The hydrophilicity and chemically active functional groups on the surface of the coating significantly improve the biocompatibility of the stent. By reducing the direct contact between the metal stent material and the vascular endothelium, the occurrence of foreign body reactions and immune reactions is reduced. The optimized stent surface can promote the repair and regeneration of vascular endothelial cells and reduce the incidence of complications such as intimal hyperplasia and thrombosis of the stent. Description of the Drawings

[0046] Figure 1 It is a schematic structural diagram of the super-smooth anticoagulant functional material of the present invention;

[0047] Figure 2 It is an electron micrograph of the PNE coating on the surface of the NiTi stent in the present invention;

[0048] Figure 3 It is an electron micrograph of the surface roughness of the stent in the present invention;

[0049] Figure 4 It is a graph of the change in heparin level in mice of the functional material obtained by the present invention;

[0050] Figure 5 It is a graph of the blood anti-adhesion test results of the functional material obtained by the present invention;

[0051] Figure 6 It is a detection graph of the NO capture ability of the functional material obtained by the present invention. Detailed implementation manners

[0052] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be described and illustrated below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments provided by the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. If there is no special description, all implementation manners and optional implementation manners of the present invention can be combined with each other to form a new technical solution.

[0053] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0054] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0055] Example 1:

[0056] Such as Figure 1 shown, a preparation method of a super-smooth anticoagulant functional material with the following specific steps:

[0057] Step 1: Substrate pretreatment

[0058] Cleaning treatment: Immerse the nickel-titanium alloy peripheral vascular stent in acetone and ultrasonically clean it for 30 minutes to remove surface grease and impurities; then rinse it 3 times with deionized water and dry it with nitrogen for standby.

[0059] Plasma activation: Place the cleaned stent in an oxygen plasma processor and treat it at a power of 100 W for 10 minutes to enhance surface hydrophilicity and reactivity.

[0060] Step 2: Coating of poly-norepinephrine intermediate layer

[0061] Solution preparation: Prepare Tris-HCl buffer (pH 8.5), add norepinephrine monomer and CuSO4 solution, and adjust to a final concentration of 1.5 mg / mL of norepinephrine monomer and 0.1 uM of CuSO4.

[0062] Reaction process: Immerse the pretreated stent in the above solution and oscillate the reaction at room temperature for 12 hours. After the reaction is completed, take out the stent and rinse it 3 times with deionized water to remove the unpolymerized norepinephrine monomer.

[0063] Repeat the above reaction process 3 times, and pre-dry at low temperature (40 °C) after each coating. Finally, obtain a stent with a poly-norepinephrine (PNE) coating with a thickness of 35 nm covering the surface as Figure 2 shown (labeled PNE@NiTi). Measure the surface flatness of the stent. As Figure 3 shown, a is the surface of the bare stent without coating, which is uneven and has a roughness of 380 nm; b is PNE@NiTi covered with a coating, with a smooth and flat surface and a roughness of 1 nm.

[0064] Step 3: Construction of sustained-release drug-PNE coating

[0065] 1. Preparation of sustained-release drug solution: Dissolve sodium heparin (10 mg / mL), EDC (1.2 mg / mL) and NHS (0.25 mg / mL) in MES buffer (pH 5.5) and activate at room temperature for 30 minutes.

[0066] 2. Grafting reaction: Immerse the PNE@NiTi stent in the activated heparin solution and react at 37 °C for 12 hours. Through amino-carboxyl condensation reaction, heparin is covalently fixed on the coating surface (labeled Hep-PNE@NiTi).

[0067] 3. Post-treatment: Take out the stent, rinse it 3 times with PBS buffer to remove the unbound heparin, and dry it in vacuum to obtain a peripheral vascular stent with super-smooth anticoagulant function.

[0068] Effect verification

[0069] Drug release performance: Detect the content of heparin in mouse blood samples every 1 h by HPLC. As Figure 4As shown, it was found that the heparin content in the Hep-PNE@NiTi group was stable within 48 hours. In the Hep@NiTi group without the PNE coating, the heparin content in the mouse blood showed an explosive increase 1 hour after the stent implantation, and no heparin signal could be detected after 40 hours. The experiment showed that Hep-PNE@NiTi significantly prolonged the drug release time of the stent.

[0070] Anticoagulant performance: Through dynamic clotting time tests, it was found that the clotting time of the Hep-PNE@NiTi group was extended to 45 minutes (the bare stent in the control group was 15 minutes), indicating that Hep-PNE@NiTi significantly improved the anticoagulant performance.

[0071] Anti-adhesion test: Simulating the blood environment in the body after stent implantation, the Hep-PNE@NiTi and Hep@NiTi stents were immersed in bovine serum albumin (2 mg / mL) for 4 hours and then rinsed three times with PBS solution. Fluorescence method was used to characterize the proteins attached to the stents. As Figure 5 shown, it was found that the proteins attached to Hep-PNE@NiTi were almost zero, while different amounts of proteins were attached to the bare stent and the Hep-NiTi stent. This indicates that Hep-PNE@NiTi has strong anti-adhesion characteristics.

[0072] NO capture ability detection: As Figure 6 shown, by comparing the Fourier transform infrared spectra of the film before and after NO treatment in the mouse experiment, it was found that implanting into the mouse body could effectively fix trace amounts of NO gas in the organism.

[0073] It should be noted that the present invention is not limited to the above embodiments. The above embodiments are only preferred examples, and embodiments with the same composition and the same function and effect as the technical idea within the technical solution scope of the present invention are all included in the technical scope of the present invention. In addition, within the scope not departing from the gist of the present invention, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways constructed by combining some of the constituent elements in the embodiments should also be regarded as within the scope of the present invention.

Claims

1. A super-smooth anti-coagulation functional material, characterized in that: The functional material includes a substrate and a coating formed by a self-assembly synthesis reaction of norepinephrine monomers on the substrate. The surface of the coating formed by the self-assembly synthesis reaction of norepinephrine monomers has a sustained-release drug capable of covalently grafting with a phenolic hydroxyl group or an amino group.

2. The super-smooth anti-coagulation functional material according to claim 1, characterized in that: The sustained-release drug includes an immunosuppressant and / or a blood thinner.

3. The super-smooth anticoagulant functional material according to claim 1, characterized in that: The material of the substrate is medical stainless steel, cobalt-chromium alloy, nickel-titanium alloy or medical polymer material.

4. A preparation method of a super-smooth anticoagulant functional material, characterized in that: It includes the following steps: After cleaning and activating the substrate, a self-assembly synthesis reaction of norepinephrine is carried out on the surface of the substrate in an alkaline environment to form a coating, and then a sustained-release drug is covalently grafted on the surface of the coating.

5. The preparation method according to claim 4, characterized in that: The activation step includes placing the cleaned substrate in an oxygen plasma processor and treating it at a power of 100 W for more than 10 minutes.

6. The preparation method according to claim 4, characterized in that: The step of forming a coating by the self-assembly synthesis reaction of norepinephrine includes immersing the activated substrate in a solution containing norepinephrine and oscillating and reacting at room temperature for 10 - 12 hours.

7. The preparation method according to claim 6, characterized in that: The pH value of the solution containing norepinephrine is 8 - 9, and the solution contains norepinephrine with a concentration of 0.1 - 10 mg / mL and copper sulfate with a concentration of 0.05 - 0.2 μM.

8. The preparation method according to claim 7, characterized in that: After each coating in the step of forming a coating by the self-assembly synthesis reaction of norepinephrine, it is pre-dried at 30 - 60 °C, and repeated multiple times to control the coating thickness within 1–500 nm, and the surface roughness Ra / coating thickness D < 10%.

9. The preparation method according to any one of claims 4-8, characterized in that: The step of covalently grafting a sustained-release drug on the surface of the coating includes immersing the substrate with the coating in a solution containing the sustained-release drug and reacting at 30 - 40 °C for 10 - 12 hours.

10. Application of the ultra-smooth anticoagulant functional material according to any one of claims 1 - 3 or the ultra-smooth anticoagulant functional material prepared by the preparation method according to any one of claims 4 - 9 in a coronary stent.

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