A functional coating with both anticoagulant and antioxidant functions and a preparation method thereof

Abstract

The application discloses a coating preparation method with anticoagulant and antioxidant functions, which comprises the following steps: step one, amino treatment is performed on the surface of a substrate; step two, double bond modification is performed on the amino surface; step three, an anticoagulant function layer is constructed through in-situ copolymerization; and step four, an antioxidant is grafted through ring-opening reaction of an epoxy group. The application further discloses a functional coating with anticoagulant and antioxidant functions. Through reasonable chemical structure design, the application realizes excellent anticoagulant and antioxidant performances in the same coating system, and effectively overcomes the limitations of the existing single-function coating. Meanwhile, the coating is firmly combined with the substrate through multilayer chemical covalent bonds, has high bonding strength and stability, and is not easy to fall off, so that the long-term implantation or long-term use requirement can be met.

Description

Technical Field

[0001] This invention relates to a bio-coating material, specifically to a functional coating with both anticoagulant and antioxidant functions and its preparation method, belonging to the field of biomaterial surface modification technology. Background Technology

[0002] Blood-contact materials, such as vascular stents, artificial heart valves, extracorporeal circulation tubing, hemodialysis membranes, and various implantable sensors, are key components of the modern medical system. However, when these material surfaces come into direct contact with human blood or tissues, a series of complex biological reactions are triggered. Among these, thrombosis and oxidative stress damage are two core pathological challenges leading to device failure and serious complications. Thrombosis begins with the non-specific adsorption and conformational changes of plasma proteins (such as fibrinogen and von Willebrand factor) on the material surface. This "conditioned" protein membrane then activates platelet adhesion and aggregation, initiating an intrinsic and extrinsic coagulation cascade, ultimately forming a fibrin network that constitutes an occlusive thrombus. Thrombi not only directly obstruct the device lumen, causing functional loss, but their detachment can also trigger fatal embolic events such as myocardial infarction and stroke. Therefore, endowing material surfaces with durable and efficient anticoagulant properties is the primary goal of biocompatibility modification. Meanwhile, the oxidative stress caused by material intervention has received increasing attention in recent years. The interaction between materials and the biological environment can activate cells to produce a large number of reactive oxygen species (ROS); the materials themselves may also catalyze the generation of free radicals. Excessive ROS attack lipids, proteins, and nucleic acids, leading to cell dysfunction, apoptosis, and continuous activation of pro-inflammatory signaling pathways. In the field of cardiovascular implantation, this directly exacerbates endothelial damage and promotes excessive proliferation and migration of smooth muscle cells, which are important causes of in-stent restenosis and late endothelialization. Clinically, thrombosis and oxidative stress are not isolated processes, but rather an intertwined and mutually reinforcing vicious cycle: the inflammatory response during thrombosis exacerbates oxidative stress, while oxidatively damaged endothelium and activated platelets further promote thrombus development. Therefore, an ideal blood-contact material surface must be able to simultaneously address these two major challenges.

[0003] To inhibit thrombus formation, traditional surface modification strategies primarily focus on constructing anticoagulant or antifouling coatings. Heparinized coatings, by immobilizing the natural anticoagulant heparin through covalent or ionic bonds, directly inhibit thrombin activity; however, their bioactivity is easily degraded by heparinase, and there is a risk of inducing heparin-induced thrombocytopenia. Polyethylene glycol (PEG) coatings rely on their high hydration capacity and steric hindrance effect to passively reduce protein adsorption and cell adhesion through an "antifouling" mechanism; however, PEG chains may undergo oxidative cleavage in vivo, resulting in insufficient long-term stability, and their antifouling effect gradually diminishes in complex biofluids. Zwitterionic polymer coatings (such as sulfobetaine-based polymers) exhibit excellent antiprotein and non-specific cell adhesion properties by forming a strong ionic hydration layer, and are considered an important direction for next-generation anticoagulant coatings. However, the above strategies mainly focus on physical barrier or local anticoagulation, and their mechanism of action is essentially passive defense, lacking the ability to actively intervene and neutralize oxidative stress. On the other hand, modification research targeting oxidative stress has mostly focused on introducing antioxidant functions. For example, slow release can be achieved by physically embedding or loading small-molecule antioxidants such as vitamin E and polyphenols onto biodegradable matrices; or by utilizing the catechol structure of biomimetic coatings such as polydopamine to scavenge free radicals. However, these methods often face problems such as limited antioxidant loading, uncontrollable release behavior, short duration of action, or insufficient stability and biosafety of the coating itself. Crucially, these simple antioxidant coatings typically lack excellent anticoagulant properties; some rough surfaces rich in phenolic hydroxyl groups may even conversely promote protein adsorption and platelet activation, thereby increasing the risk of thrombosis.

[0004] This reveals a significant problem in the current field of biomaterial surface engineering: mainstream anticoagulation technologies fail to address oxidative stress, while antioxidant solutions cannot meet anticoagulation requirements. This single-function modification strategy is severely out of sync with the complex and interconnected pathological challenges faced in clinical practice. Developing a technology that organically integrates and synergistically enhances both long-acting active anticoagulation and highly efficient sustained antioxidant functions within a single stable coating system has become crucial for overcoming existing bottlenecks and improving the long-term safety and effectiveness of blood contact materials. An ideal solution not only requires a strong bond between the coating and the substrate and excellent biocompatibility but also necessitates rational chemical design to enable synergistic action between anticoagulation and antioxidant units at the molecular level, rather than simple stacking. Based on this, this invention aims to propose a modular and universally applicable surface functionalization method. This method constructs an anticoagulation polymer brush backbone through in-situ copolymerization and utilizes highly reactive epoxy groups as a "chemical handle" to precisely and stably graft highly efficient free radical scavengers, thereby achieving active and bidirectional regulation of the material surface's biological environment and ultimately achieving a synergistic improvement in anticoagulation and antioxidant properties. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a functional coating that is flexible in process, has a wide range of applications, stable coating, and excellent anticoagulant and antioxidant functions.

[0006] The present invention is implemented as follows:

[0007] A method for preparing a coating with both anticoagulant and antioxidant functions includes the following steps:

[0008] Step 1: Amination treatment of the substrate surface.

[0009] The substrate surface is cleaned and activated, and then an amino functional layer is introduced on the substrate surface to obtain an aminated substrate;

[0010] Step 2: Amino surface double bond modification.

[0011] The aminated substrate was reacted with methacrylic anhydride and then washed to obtain a double-bonded substrate with carbon-carbon double bonds on its surface.

[0012] In this step, methacrylic anhydride undergoes an amidation reaction with the amino groups on the substrate surface, while simultaneously grafting methacryloyl groups with terminal double bonds onto the surface.

[0013] Step 3: In-situ copolymerization construction of the anticoagulant functional layer.

[0014] A polymerization reaction solution containing glycidyl methacrylate and double-bonded anticoagulant monomers is prepared. The double-bonded substrate is immersed in the reaction solution. Under the action of an initiator, the monomers in the solution undergo an in-situ free radical copolymerization reaction with the double bonds on the substrate surface, forming a covalently grafted polymer brush coating on the substrate surface.

[0015] Step 4: Ring-opening grafting of antioxidants with epoxy groups.

[0016] The substrate obtained in step three is immersed in a buffer solution containing amino-TEMPO. Under mild heating conditions, the epoxy groups in the polymer brush on the substrate surface undergo a ring-opening nucleophilic addition reaction with the amino groups on the amino-TEMPO, thereby stably covalently grafting TEMPO radicals onto the coating.

[0017] TEMPO is a highly efficient nitrogen oxide free radical that can continuously and effectively scavenge reactive oxygen free radicals, giving the coating an active antioxidant function.

[0018] A further step is:

[0019] The substrate can be any one or more of the following: metallic materials, polymers, ceramics, or silicon materials.

[0020] A further step is:

[0021] An amino functional layer was introduced onto the substrate surface using a dopamine / polyethyleneimine (PEI) co-deposition method, specifically:

[0022] The substrate is immersed in a Tris-HCl buffer solution containing dopamine hydrochloride and polyethyleneimine, and the reaction is carried out at room temperature with shaking or standing for several hours. An amino-rich polydopamine / polyethyleneimine composite layer is formed on the substrate surface through the self-polymerization and co-deposition of dopamine.

[0023] A further step is:

[0024] An amino functional layer was introduced onto the substrate surface using an alpha-silylating agent (APTES) deposition method, specifically:

[0025] The substrate is immersed in an ethanol solution (1-5% by mass) containing γ-aminopropyltriethoxysilane and reacted by shaking at room temperature or heating under reflux; or, after activating the substrate by plasma treatment, APTES is introduced by vapor deposition to form an amino-containing silanized layer on the substrate surface.

[0026] A further step is:

[0027] The monomer includes glycidyl methacrylate (GMA), and also includes at least one of sulfobetaine methacrylate (SBMA), polyethylene glycol diacrylate (PEGDA), sodium styrene sulfonate (SS), and 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0028] The coating provides resistance to protein adsorption and platelet adhesion using SBMA, PEGDA, SS, or MPC, while also containing active epoxy groups from GMA.

[0029] A further step is:

[0030] The initiator is a thermal initiator such as APS or VAO-44, or a photoinitiator such as I2959 or LAP.

[0031] A further step is:

[0032] The amino-TEMPO is a 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxo radical derivative containing an amino group.

[0033] A further step is:

[0034] The mild heating conditions are 37-60°C.

[0035] The present invention also provides a functional coating with both anticoagulant and antioxidant functions, which is prepared by the preparation method of the functional coating with both anticoagulant and antioxidant functions provided by the present invention.

[0036] Compared with the prior art, the present invention has at least the following outstanding technical effects:

[0037] 1. Functional Integration: Through ingenious chemical design, this invention achieves excellent anticoagulant (through MPC / SBMA / PEGDA / SS) and antioxidant (through covalently grafted TEMPO) properties in a single coating system, overcoming the limitations of single-function coatings.

[0038] 2. High stability: The coating is bonded to the substrate through multi-layered chemical covalent grafting (amylation layer - double bond - polymer brush - antioxidant), which results in high firmness and resistance to detachment, meeting the requirements for long-term implantation or use.

[0039] 3. Wide applicability: The amination method in step one is diverse and can be applied to a variety of substrate materials such as metals (titanium alloys, stainless steel), polymers (polyurethane, polyvinyl chloride), ceramics, and silicon wafers.

[0040] 4. Clearly defined active sites: By using the epoxy groups of GMA as active handles, efficient, quantitative, and site-specific grafting of TEMPO antioxidants can be achieved, ensuring the reliability and consistency of antioxidant activity.

[0041] 5. Good biocompatibility: The selected SBMA and PEGDA are both recognized materials with excellent biocompatibility, and TEMPO is also a widely studied biocompatible antioxidant. No toxic or harmful reagents are used in the entire preparation process. Attached Figure Description

[0042] Figure 1 This is a schematic diagram showing the number and distribution of platelets in the embodiments and comparative examples of the present invention;

[0043] Figure 2 This is a schematic diagram of the ROS levels in endothelial cells in the embodiments and comparative examples of the present invention. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments.

[0045] Example 1

[0046] This embodiment provides a method for preparing a functional coating on a silicon wafer surface that has both anticoagulant and antioxidant functions, specifically as follows:

[0047] 1. Substrate pretreatment: The silicon wafer is ultrasonically cleaned with ethanol and deionized water in sequence, dried with nitrogen, and pretreated with plasma for 5 minutes to increase the hydrophilicity of the silicon wafer surface.

[0048] 2. Immerse the substrate in 10 mM Tris-HCl buffer (pH 8.5) containing 2 mg / mL dopamine hydrochloride and 5 mg / mL polyethyleneimine, and react with shaking at room temperature for 4 hours. After removal, rinse thoroughly with deionized water to obtain the aminated substrate.

[0049] 3. Double Bonding: The aminated substrate was immersed in a sufficient amount of PBS buffer containing 1% (w / w) methacrylic anhydride and reacted at room temperature for 8 hours. After the reaction, it was washed with ethanol and deionized water to obtain the double-bonded substrate.

[0050] 4. In-situ copolymerization: Prepare an aqueous monomer solution containing 0.5 mol / L SBMA and 0.1 mol / L GMA, and add 1 wt% thermal initiator VA0-44. Immerse the double-bonded substrate in a sufficient amount of this solution, purge with nitrogen to remove oxygen, and then react in a 40°C water bath for 6 hours. After the reaction, remove the substrate and rinse thoroughly with hot water and deionized water to remove physically adsorbed homopolymer.

[0051] 5. Grafting Antioxidant: Immerse the above substrate in sufficient phosphate buffer (pH 7.4) containing 50 mM 4-amino-TEMPO and react at 37°C in the dark for 24 hours. After the reaction, thoroughly wash with ethanol and deionized water, and dry to obtain the final product.

[0052] Example 2

[0053] This embodiment provides a method for preparing a functional coating on the surface of a stainless steel stent that has both anticoagulant and antioxidant functions, specifically as follows:

[0054] 1. Substrate pretreatment: The stainless steel sheet is activated by alkaline washing, acid washing, and plasma cleaning.

[0055] 2. Place the activated stainless steel sheet in a 2% (w / w) APTES solution and shake at room temperature for 4 hours to form an APTES layer on the surface, introducing amino groups.

[0056] 3. Double bond formation: Same as step 3 in Example 1.

[0057] 4. In-situ copolymerization: Prepare a monomer ethanol solution containing 0.3 mol / L PEGDA and 0.2 mol / L GMA, and add 0.5 wt% of the photoinitiator Irgacure 2959. Immerse the double-bonded titanium sheet in the solution and irradiate it with 365 nm ultraviolet light for 30 minutes under nitrogen protection. Subsequent cleaning is the same as in Example 1.

[0058] 5. Grafting antioxidant: Same as step 5 in Example 1.

[0059] Example 3

[0060] This embodiment provides a method for preparing a functional coating on the surface of polydimethylsiloxane (PDMS) that has both anticoagulant and antioxidant functions, specifically as follows:

[0061] 1. Substrate pretreatment: PDMS sheets are washed sequentially with ethanol and deionized water, and then dried with inert gas.

[0062] 2. Place the dried PDMS sheet in a plasma vapor deposition apparatus, introduce APTES vapor, and treat it at a power of 50W for 5 minutes to form an APTES layer on the surface and introduce amino groups.

[0063] 3. Double bond formation: Same as step 3 in Example 1.

[0064] 4. In-situ copolymerization: Prepare a monomer ethanol solution containing 0.5 mol / L MPC and 0.2 mol / L GMA, and add 0.5 wt% of photoinitiator Irgacure 2959. Immerse the double-bonded titanium sheet in the solution and irradiate it with 365 nm ultraviolet light for 30 minutes under nitrogen protection. Subsequent cleaning is the same as in Example 1.

[0065] 5. Grafting antioxidant: Same as step 5 in Example 1.

[0066] Example 4

[0067] This embodiment provides a method for preparing an anticoagulant functional coating on a silicon wafer surface, specifically: only steps 1-4 of Example 1 are performed, without performing the TEMPO grafting reaction in step 5.

[0068] Experimental Example 1

[0069] The water contact angles of the sheets in Examples 1, 2, and 3 before and after surface coating modification were measured using a water contact angle meter.

[0070] The experimental method was as follows: Sheets obtained in Examples 1, 2, and 3, before and after modification, were cut to 10 mm x 10 mm. A water contact angle meter was used for testing at room temperature (25 ± 1 ℃). Before testing, the samples were fixed on the instrument stage to ensure a horizontal surface. A 5 μL droplet of deionized water was slowly added to the sample surface using a micro-syringe. After the droplet stabilized for 10 s, the instrument automatically collected the droplet profile and calculated the contact angles on both sides. The average value was taken as the static water contact angle at that point. At least five samples were prepared and tested in parallel for each material, and the average value was taken as the final contact angle result, expressed as "mean ± standard deviation".

[0071] Table 1 Results of water contact angles on different substrates

[0072] Table 1 shows that the water contact angle of all substrates was significantly reduced after coating modification. For silicon wafers, it decreased from 76.8±3.4° to below 5°; for stainless steel, from 59.2±0.33° to 18.04±3.3°; and for PDMS, from 115.9±5.2° to 14.5±3.2°. Among these, the silicon wafer surface reached a superhydrophilic level, while PDMS showed the largest decrease. This indicates that the coating can significantly improve the hydrophilicity of different material surfaces, exhibiting good versatility and laying the foundation for further improvements in anti-protein adsorption, anti-platelet adhesion, and anticoagulation properties.

[0073] Experimental Example 2

[0074] The anticoagulant properties of the sheets before and after surface coating modification in Examples 1, 2, and 3 were evaluated.

[0075] The experimental method was as follows: The modified sheets obtained in Examples 1, 2, and 3 were cut into 10mm diameter discs and placed in 48-well plates for later use. Fresh rabbit whole blood was collected from healthy adult New Zealand white rabbits via the ear vein into centrifuge tubes containing 3.8 wt% sodium citrate anticoagulant (anticoagulant to whole blood volume ratio 1:9). The anticoagulant was centrifuged at 1500 rpm for 10 min at room temperature, and the supernatant platelet-rich plasma (PRP) was collected for later use. 0.5 mL of PRP was added to the surface of each sample to completely cover the sample surface, and the samples were statically incubated at 37 ℃ for 60 min. After incubation, the supernatant was aspirated, and the sample surface was gently rinsed three times with PBS. Subsequently, 0.5 mL of calcein AM working solution with a mass concentration of 1 μg / mL was added to each sample, and the samples were incubated at 37 ℃ in the dark for 30 min to allow the active platelets adhering to the sample surface to be stained with fluorescence. After staining, the samples were slowly rinsed three times with PBS. The platelet adhesion on the sample surface was observed and photographed using a fluorescence microscope, with the excitation wavelength set at 488 nm and the emission wavelength at 515–530 nm. Using the unmodified sheet as a control group, the number and distribution of platelets adhering to the sample surface before and after modification in Examples 1, 2, and 3 were compared to evaluate the anticoagulant properties of the coating.

[0076] The results are attached. Figure 1 As shown, the unmodified substrate surface exhibited a large number of green fluorescent platelets, which were densely distributed and partially aggregated. After coating modification, the number of platelets adhering to the surface of each embodiment was significantly reduced, with only a small number of scattered platelets remaining. Specifically, the modified samples of Examples 1 and 2 showed significantly better anti-platelet adhesion ability than the unmodified substrate, with a reduction in the number of adhering platelets exceeding 95%. The results indicate that the functional coating constructed in this invention can effectively inhibit platelet adhesion and aggregation, exhibiting excellent anticoagulant properties.

[0077] Experimental Example 3

[0078] The performance of Example 1, Example 4 and unmodified silicon wafer in scavenging intracellular reactive oxygen species (ROS) was compared.

[0079] The experimental method was as follows: Silicon wafers from Examples 1 and 4, and unmodified wafers, were sterilized by immersion in 75% ethanol for 3 hours. After washing with sterile PBS, the materials were placed in 48-well plates. Human umbilical vein endothelial cells (purchased from Huatuo Biotechnology Co., Ltd., catalog number HTX3606) were digested and resuspended in normal complete culture medium, and seeded at a concentration of 20,000 cells per well. After culturing at 37°C and 5% CO2 for 24 hours, 1 mM H2O2 was added to the culture medium. The expression of ROS in the endothelial cells grown on the material was qualitatively observed by DCFH-DA staining.

[0080] The results are attached. Figure 2 As shown, DCFH-DA fluorescence staining results revealed strong green fluorescence in endothelial cells of both the unmodified group and the Example 4 group, indicating a high level of intracellular ROS under H2O2 stimulation. In contrast, the green fluorescence in the Example 1 group was significantly weakened, indicating that intracellular ROS was significantly scavenged. These results demonstrate that the coating obtained in Example 1 possesses excellent antioxidant properties, effectively scavenging H2O2-induced reactive oxygen species and mitigating oxidative damage to endothelial cells. In contrast, Example 4, without the grafted antioxidant component, did not exhibit significant ROS scavenging ability, indicating that antioxidant grafting is crucial for imparting antioxidant function to the coating.

[0081] Although the present invention has been described herein with reference to illustrative embodiments, the above embodiments are merely preferred embodiments of the present invention, and the implementation of the present invention is not limited to the above embodiments. It should be understood that those skilled in the art can devise many other modifications and implementations, which will fall within the scope and spirit of the principles disclosed in this application.

Claims

1. A method for preparing a coating with both anticoagulant and antioxidant functions, characterized in that... Includes the following steps: Step 1: Amination treatment of the substrate surface The substrate surface is cleaned and activated, and then an amino functional layer is introduced on the substrate surface to obtain an aminated substrate; Step 2: Amino surface double bond modification The aminated substrate was reacted with methacrylic anhydride and then washed to obtain a double-bonded substrate with carbon-carbon double bonds on its surface. Step 3: In-situ copolymerization construction of the anticoagulant functional layer A polymerization reaction solution containing glycidyl methacrylate and double-bonded anticoagulant monomers is prepared. The double-bonded substrate is immersed in the reaction solution. Under the action of an initiator, the monomers in the solution undergo an in-situ free radical copolymerization reaction with the double bonds on the substrate surface, forming a covalently grafted polymer brush coating on the substrate surface. Step 4: Ring-opening grafting of epoxy groups with antioxidants The substrate obtained in step 3 is immersed in a buffer solution containing amino-TEMPO; under mild heating conditions, the epoxy groups in the polymer brush on the substrate surface undergo a ring-opening nucleophilic addition reaction with the amino group on the amino-TEMPO, thereby stably covalently grafting TEMPO radicals onto the coating.

2. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: The substrate can be any one or more of the following: metallic materials, polymers, ceramics, or silicon materials.

3. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: An amino functional layer was introduced onto the substrate surface using a dopamine / polyethyleneimine co-deposition method, specifically: Dopamine / polyethyleneimine co-deposition method: The substrate is immersed in a Tris-HCl buffer solution containing dopamine hydrochloride and polyethyleneimine, and the reaction is carried out at room temperature by shaking or standing for several hours. Through the self-polymerization and co-deposition of dopamine, an amino-rich polydopamine / polyethyleneimine composite layer is formed on the substrate surface.

4. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: An amino functional layer was introduced onto the substrate surface using a silanizing agent deposition method, specifically: The substrate is immersed in an ethanol solution containing γ-aminopropyltriethoxysilane and reacted by shaking at room temperature or heating under reflux; or, after activating the substrate by plasma treatment, APTES is introduced by vapor deposition to form an amino-containing silanized layer on the substrate surface.

5. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: The monomers include glycidyl methacrylate, and further include at least one of the following: anticoagulant monomer sulfobetaine methacrylate, polyethylene glycol diacrylate, sodium styrene sulfonate, and 2-methacryloyloxyethyl phosphorylcholine.

6. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: The initiator is either the thermal initiator ammonium persulfate or the photoinitiator I2959.

7. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: The amino-TEMPO is a 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxo radical derivative containing an amino group.

8. The method for preparing the coating with both anticoagulant and antioxidant functions according to claim 1, characterized in that: The mild heating conditions are 37-60°C.

9. A functional coating with both anticoagulant and antioxidant functions is prepared by the preparation method of the coating with both anticoagulant and antioxidant functions as described in any one of claims 1 to 8.

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