Synergistic dual-mode anticoagulation and anti-inflammatory bionic central venous catheter coating and preparation method thereof

Abstract

The invention discloses a synergistic dual-mode anticoagulation and anti-inflammatory central venous catheter BC-BCD double-layer bionic hydrogel coating and a preparation method thereof. BSA / CBAA hydrogel formed by copolymerizing bovine serum albumin and carboxyl betaine acrylamide is used as an outer coating of the catheter, dopamine containing catechol groups is introduced as a main chain network, and BSA / CBAA / DA hydrogel is obtained and used as an inner coating of the catheter. The two layers are combined through a two-step impregnation method to obtain the BC-BCD double-layer bionic coating, thrombus can be defended on a catheter interface in an active mode and a passive mode, oxidation factors in a body can be removed, oxidative stress and bacterial infection can be reduced, and the BC-BCD double-layer bionic coating can be tightly attached to a base material. Experimental results show that the prepared bionic hydrogel coating has excellent anticoagulation and anti-inflammatory performance and long-term stable adhesion performance, the main defects of a traditional central venous catheter are remarkably overcome, and the bionic hydrogel coating has a positive effect on clinical prevention of thrombus and infection.

Description

Technical Field

[0001] The present invention belongs to the fields of biocompatible material preparation and biomedical applications. Specifically, it relates to a BC-BCD double-layer bionic hydrogel coating with synergistic dual-mode anticoagulant and anti-inflammatory properties crosslinked by bovine serum albumin, carboxybetaine acrylamide, and dopamine, which is applied to central venous catheters, and a preparation method thereof. Background Art

[0002] Central venous catheters (CVCs) are increasingly widely used clinically, especially in the diagnosis and treatment of heart diseases, cardiovascular diseases, cancers, and other diseases. However, the complication of CVCs-related thrombosis (CRT) cannot be ignored, with an annual mortality rate as high as 12%-25%. Among them, natural hydrophobicity and high roughness are considered to be two main factors leading to such complications. At present, the use of anticoagulants or antibiotics is considered to be the basic method to solve these problems, while the interference with natural blood coagulation and the generation of antibiotic-resistant super bacteria severely limit the wide clinical application of these treatment methods. Therefore, designing a new CVCs coating with ideal anticoagulant performance to solve CRT complications has become an urgent need in clinical and scientific research. The surface modification of CVCs provides a promising method to alleviate CRT and can be used as an alternative to traditional anticoagulants or antibiotics.

[0003] In recent years, scientists have been dedicated to designing hydrogel materials with excellent performance for wide applications in many fields. Zwitterionic polymers with the same number of positive and negative charges in the same molecular chain are expected to prevent the adsorption of proteins, blood cells, and bacteria to prevent thrombosis due to their special hydration ability. For example, Patent CN118557809A discloses a highly stable superhydrophilic and superlubricant coating, which is formed by chemical covalent crosslinking between polyamino quaternized polyethyleneimine polymer and zwitterionic phosphocholine-based PVP hydrophilic macromolecule through the amino group and epoxy functional group. Based on the strong hydration effect induced by the electrostatic force of the zwitterionic component, a firm and stable hydration layer is formed. The cured coating has excellent hydrophilic lubrication function, stability, bactericidal performance, antifouling performance, and blood compatibility. However, this strategy achieves effective anticoagulation by reducing the adsorption of blood components, which is defined as a passive defense mechanism. However, creating such a bio-inert antifouling surface to reduce thrombus formation is not sufficient to fundamentally solve the problem of CRT, especially to achieve the purpose of inhibiting the coagulation process and providing long-term prevention. Bovine serum albumin (BSA) also has a wide range of applications in the biomedical field. It can provide abundant anticoagulant functional groups (such as sulfonic acid and carboxyl groups) to inhibit the expression of certain coagulation factors. Another example is that Patent CN119055839A discloses an anti-inflammatory and repair-promoting coating based on hydrogen sulfide donors, its preparation method and application, which can endow biomedical materials with excellent biological activity. The coating of this invention contains hydrogen sulfide donors, which can spontaneously or under external stimuli release hydrogen sulfide or its precursors, and has significant functions in anticoagulation, vasodilation, scavenging reactive oxygen species, and regulating inflammation. This method of achieving anticoagulation is called an active attack mechanism. However, CRT is usually related to a complex pathological environment because thrombus and inflammation will exacerbate each other. In this case, a single active attack mechanism may not be sufficient to obtain long-lasting and effective anticoagulation. In addition, the reduced catechol form of dopamine (DA) can strongly adhere to any hydrophobic surface by participating in various physical interactions with the substrate. Another example is that Patent CN108815586A discloses a bioantifouling coating with long-term superhydrophilicity. Specifically, after the surface of the substrate material is pretreated and placed in a slightly acidic buffer solution, polyphenol compounds, polyamine compounds, and oxidants are added for reaction, and then obtained by ultrasonic cleaning and nitrogen drying. The coating material prepared by this method has polyphenol compounds and a large number of functional groups such as carboxyl groups, phenolic hydroxyl groups, quinone groups, and amine groups, and has excellent adhesion to the substrate material. However, the preparation steps are relatively cumbersome and not suitable for batch production. Based on the above considerations, we intend to construct a new type of bionic CVCs coating with passive / active anticoagulant performance and improved interfacial affinity, which is expected to solve CRT.

[0004] Based on this, the present invention proposes a brand-new blood vessel endothelium-mimicking bilayer hydrogel coating (BC-BCD), which is composed of carboxybetaine zwitterion (CBAA), BSA, and DA-crosslinked BSA / CBAA and BSA / CBAA / DA hydrogels. The BSA / CBAA (outer layer) and BSA / CBA / DA (inner layer) are coated on the surface of CVCs through a simple two-step dip coating method. Its superhydrophilic property can prevent the catheter from being adhered by biomolecules to achieve passive anticoagulation, and the sulfonic acid groups with heparin-like properties can achieve active anticoagulation by inhibiting the coagulation cascade reaction. The internal DA realizes the scavenging of reactive oxygen species and the super-strong adhesion to the substrate, and the bilayer gradient structure is beneficial to improving the mechanical properties of the hydrogel, and also has a modulus similar to that of vascular endothelium, reducing mechanical damage. Such a hydrogel coating not only has passive and active dual-mode anticoagulant capabilities, but also has excellent anti-inflammatory and antibacterial properties, and can stably adhere for a long time and the preparation process is simple. The test results in the rabbit in vitro arteriovenous reflux model also show that this coating can significantly reduce CVCs complications, thereby maintaining efficient blood delivery. Summary of the Invention

[0005] To solve the above technical problems, the present invention aims to provide a BC-BCD bilayer biomimetic hydrogel coating with synergistic dual-mode anticoagulation and anti-inflammation and its preparation method. By mimicking the anticoagulation of the human blood vessel endothelium and mussel adhesion, multifunctional materials containing different functional groups are introduced to endow this series of biomimetic hydrogel adhesives with good anticoagulant performance, excellent interfacial adhesion effect, and antibacterial activity. It is expected to become a new type of coating for CVCs in clinical applications.

[0006] To achieve the above invention purpose, the technical solutions provided by the present invention are as follows:

[0007] A preparation method of a BC-BCD bilayer biomimetic hydrogel coating for CVCs with synergistic dual-mode anticoagulation and anti-inflammation, comprising the following steps:

[0008] (1) Dissolve N,N-dimethylaminopropyl acrylamide (DMAPAA) and ethyl bromoacetate in acetonitrile, and react at 60 °C for 12 hours to obtain solution S1;

[0009] (2) Pour anhydrous ether into solution S1, continuously stir at room temperature to obtain white precipitate of ethyl-carboxybetaine acrylamide (ethyl-CBAA), vacuum dry for 3 h and then dissolve in ethanol, and stir at room temperature to obtain solution S2;

[0010] (3) Pour the ion exchange resin (IRA-400) soaked in 4% (V / V) NaOH into solution S2, stir and filter to obtain solution S3;

[0011] (4) Pour anhydrous diethyl ether into its supernatant, stir to obtain a white precipitate of carboxybetaine acrylamide (CBAA), and then dissolve it in deionized water to obtain solution S4;

[0012] (5) Dissolve bovine serum albumin (BSA) in deionized water and stir at room temperature to obtain solution S5;

[0013] (6) Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in deionized water to obtain solution S6;

[0014] (7) Dissolve N-hydroxysuccinimide (NHS) in deionized water and stir to obtain solution S7;

[0015] (8) Dissolve N,N'-methylenebis(acrylamide) (MBAA) in deionized water and stir to obtain solution S8;

[0016] (9) Dissolve N,N,N',N'-tetramethylethylenediamine (TEMED) in deionized water and stir to obtain solution S9;

[0017] (10) Dissolve ammonium persulfate (APS) in deionized water and stir at room temperature to obtain solution S10;

[0018] (11) Dissolve dopamine hydrochloride (DA) in deionized water and stir to obtain solution S11;

[0019] (12) Mix solution S4 and solution S5 evenly and stir to obtain solution S12;

[0020] (13) Add solution S8, S9, and S10 to solution S12 in sequence, mix evenly at room temperature to obtain an outer layer BSA / CBAA pre-gel solution; then add solution S6, S7, Tris hydrochloride, and solution S11 to the BSA / CBAA pre-gel solution to obtain an inner layer BSA / CBAA / DA pre-gel solution; To prepare the hydrogel coating, first cut the medical-grade central venous catheter sample into the required size, and clean it with ethanol or deionized water to remove surface impurities. The purified sample is soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the sample is ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature to obtain a BC-BCD double-layer hydrogel coating.

[0021] Preferably, in step (1), the mass fraction of DMAPAA in solution S1 is 1.0 - 99.7 wt%, the mass fraction of ethyl bromoacetate is 0.1 - 99.7 wt%, the mass fraction of acetonitrile is 1.0 - 99.7 wt%, the stirring temperature is 60 °C, and the stirring time is 12 - 14 h.

[0022] Preferably, in the solution S2 in step (2), the mass fraction of anhydrous ether is 0.1 - 99.7 wt%, the stirring temperature is 10 - 40 °C, and the stirring time is 1 - 20 min.

[0023] Preferably, in the solution S3 in step (3), the mass fraction of the ion exchange resin is 0.1 - 99.7 wt%.

[0024] Preferably, in the solution S4 in step (4), the mass fraction of CBAA is 0.1 - 50.0 wt%, the stirring temperature is 10 - 40 °C, and the stirring time is 5 - 10 min.

[0025] Preferably, in the solution S5 in step (5), the mass fraction of BSA is 0.1 - 25.0 wt%, the stirring temperature is 10 - 40 °C, and the stirring time is 1 - 10 min.

[0026] Preferably, the concentration of the EDC solution in step (6) is 0.001 - 0.1 g / mL; the stirring temperature is 5 - 45 °C, and the stirring time is 2 - 150 min.

[0027] Preferably, the concentration of the NHS solution in step (7) is 0.001 - 1 g / mL; the stirring temperature is 5 - 45 °C, and the stirring time is 2 - 150 min.

[0028] Preferably, the concentration of the MBAA solution in step (8) is 0.002 - 1 g / mL; the stirring temperature is 5 - 45 °C, and the stirring time is 2 - 150 min.

[0029] Preferably, the concentration of the TEMED solution in step (9) is 0.002 - 1 g / mL; the stirring temperature is 5 - 45 °C, and the stirring time is 2 - 150 min.

[0030] Preferably, the concentration of the APS solution in step (10) is 0.002 - 1 g / mL; the stirring temperature is 5 - 45 °C, and the stirring time is 2 - 150 min.

[0031] Preferably, the concentration of the DA solution in step (11) is 0.002 - 1 g / mL; the stirring temperature is 5 - 45 °C, and the stirring time is 2 - 150 min.

[0032] Preferably, in the solution S12 in step (12), the mass ratio of the solution S4:S5 is 100:1 - 1:100; the stirring temperature is 5 - 55 °C, and the stirring time is 2 - 150 min.

[0033] Preferably, in the BSA / CBAA pre-gel solution in step (13), the mass ratio of S8:S9:S10:S12 is 10:1000:1:1:1 to 1000:10:1:1:1; in the BSA / CBAA / DA pre-gel solution, the mass ratio of S6:S7:S8:S9:S10:S11:S12 is 10:1000:1:1:1:1:1 to 1000:10:1:1:1:1:1; the stirring temperature is 5-55°C, and the stirring time is 1-150 min.

[0034] Compared with the prior art, the present invention has the following technical advantages:

[0035] (1) A series of BC-BCD bilayer biomimetic hydrogel coatings for CVCs prepared by the present invention have strong interfacial adhesion. A series of BC-BCD bilayer biomimetic hydrogel coatings with synergistic dual-mode anticoagulation and anti-inflammatory effects use the hydrogel formed by free radical polymerization of BSA and CBAA as the main chain network. Further, DA containing catechol groups is introduced, and cross-linking is generated with the main chain network through hydrogen bonding to form a secondary network. This hydrogel coating mimics the adhesion mode of marine mussels and can form effective adhesion on the surface of CVCs. The introduced DA side chain is rich in aldehyde groups, which can form a non-covalent bond cross-linking network with the amino groups on the BSA side chain in the form of Schiff base binding, and can also form Schiff base binding with the amino groups on CVCs, achieving strong adhesion to the substrate. Moreover, based on the strong interfacial adhesion ability and powerful network structure of the hydrogel coating, the ability to dissipate hysteretic energy is maximized, and the series of biomimetic hydrogel coatings for CVCs can achieve long-term stable adhesion effects in vivo.

[0036] (2) A series of BC-BCD bilayer biomimetic hydrogel coatings for CVCs prepared by the present invention have the effect of self-adaptive hydration lubrication. Due to the presence of a large number of hydrophilic groups, a series of BC-BCD bilayer coatings prepared by the present invention can form a dense hydrogen bond network with water molecules in the blood vessel due to the grafting of zwitterions. This strong hydrogen bond site formed with water molecules achieves the effect of super-hydrophilic lubrication, which helps to achieve ultra-low friction during relative movement, reduce shear damage during contact with blood vessels during clinical use, and avoid out-of-control inflammation and thrombus problems caused thereby.

[0037] (3) A series of BC-BCD double-layer bionic hydrogel coatings for CVCs prepared by the present invention have good anticoagulant effects. Due to having a large number of hydrophilic groups, a series of BC-BC hydrogel coatings prepared by the present invention can rapidly absorb the moisture in the blood to form a hydrated layer, and achieve passive anticoagulation by resisting the adhesion of red blood cells, monocytes and bacteria; most of the sulfhydryl groups in BSA in the hydrogel are converted into hydrophilic sulfonic acid groups through hydrogen peroxide sterilization before clinical application, showing the properties of heparin-like to achieve active anticoagulation. In vitro and in vivo experiments have confirmed that a series of bionic hydrogel coatings for CVCs with dual-mode anticoagulation and anti-inflammatory effects prepared by the present invention have excellent and efficient passive, active anticoagulation and anti-inflammatory effects.

[0038] (4) A series of BC-BCD double-layer bionic hydrogel coatings for CVCs prepared by the present invention have high anti-inflammatory performance. While preventing thrombus formation, a series of BC-BCD double-layer bionic hydrogel coatings prepared by the present invention have excellent antioxidant performance due to the catechol groups contained in DA and the disulfide bonds formed by the oxidation of sulfhydryl groups in BSA, can capture reactive oxygen species, and play a role in reducing oxidative stress protection in the initial stage of implantation, thereby contributing to reducing the formation of inflammation.

[0039] (5) A series of BC-BCD double-layer bionic hydrogel coatings for CVCs prepared by the present invention have excellent antibacterial properties. A series of BC-BCD bionic hydrogel coatings prepared by the present invention can adhere to the phosphate groups of cell membrane phospholipids through electrostatic interaction due to the presence of groups with positive and negative charges inside the material, so as to affect the denaturation of microbial proteins and the replication of DNA and inhibit the surrounding bacteria, so they show good antibacterial activity against microorganisms. In addition, a series of BC-BCD bionic hydrogel coatings prepared by the present invention can also affect the colonization of microorganisms on their surface to form biofilms through strong hydration and free radical scavenging ability, thereby achieving significant antibacterial ability.

[0040] (6) A series of BC-BCD double-layer bionic hydrogel coatings for CVCs prepared by the present invention have good tissue compatibility. While achieving strong adhesion and superlubrication, a series of double-layer bionic BC-BC coatings prepared by the present invention have a modulus (10 kPa) matching that of the vascular intima due to their double-layer longitudinal gradient distribution, and greatly improve the comfort of catheter implantation by reducing the interface stress and enhancing the adhesion stability.

[0041] In summary, a series of BC-BCD bilayer bionic hydrogel coatings for CVCs prepared by the present invention have good interfacial adhesion, super hydration lubricity, anticoagulation, antibacterial, anti-inflammatory, and tissue adaptation capabilities. This bionic hydrogel coating combines strong interfacial adhesion, anticoagulation, anti-inflammatory, and antibacterial functions, preventing the interfacial detachment between the ordinary coating and the substrate, reducing mechanical damage caused by high roughness, alleviating the foreign body reaction caused by hydrophobicity, significantly solving the main drawbacks and complications of traditional CVCs, and is expected to cope with the complex physiological environment during in vivo implantation. Description of the Drawings

[0042] Figure 1 Morphology and structure characterization of the hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2. A. Scanning electron microscope images and corresponding pore size distributions of BSA / CBAA and BSA / CBAA / DA hydrogels. B. Average porosity of BSA / CBAA and BSA / CBAA / DA hydrogels. C. FTIR spectra of BSA, BSA / CBAA, and BSA / CBAA / DA. D. Bilayer structure of the BC-BCD bionic hydrogel coating.

[0043] Figure 2 Quantitative analysis diagrams of the mechanical properties of the hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2. A. Tensile stress-strain curves of the bilayer bionic BC-BCD hydrogel coating for CVCs. B. Quantitative analysis of the tensile strength and elongation at break of the bionic BC-BC hydrogel coating. C. Compressive stress-strain curves of the bilayer bionic BC-BCD hydrogel coating. D. Comparison diagram of the tensile Young's modulus between the bilayer bionic BC-BCD hydrogel coating and biological tissues.

[0044] Figure 3 Analysis of the adhesion and lubrication properties of the hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2. A. Adhesion strength of the BC-BCD bilayer bionic hydrogel coating to wood, glass, plastic, rubber, and metal. B. Friction coefficient of the bilayer BC-BCD bionic hydrogel coating. C. Picture of the BC-BCD coating changing from viscous to lubricated state when encountering water. D. Contact angle of the bilayer BC-BCD bionic hydrogel coating.

[0045] Figure 4 Anti-inflammatory and antibacterial activities of the hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2. A. Infrared analysis of BSA / CBAA and BSA / CBAA hydrogels after hydrogen peroxide treatment. B. Free radical scavenging rate of the BC-BCD hydrogel coating. C. Ability of the BC-BCD bionic hydrogel coating to inhibit the formation of biofilms by Escherichia coli on the surface.

[0046] Figure 5Anticoagulant performance characterization of the BC-BCD double-layer bionic hydrogel coating prepared in Example 7. A. Activated partial thromboplastin time test of the BC-BCD bionic hydrogel coating. B. Bicinchoninic acid assay (BCA) test of the BC-BCD bionic hydrogel coating. C. Coagulation time test caused by the double-layer BC-BCD bionic hydrogel coating. D. Coagulation index of the BC-BCD hydrogel.

[0047] Figure 6 In vitro biocompatibility evaluation of the BC-BCD double-layer bionic hydrogel coating prepared in Example 7. A. Live / dead staining images of the BC-BCD bionic hydrogel coating co-cultured with HUVECs cells for 1 and 3 days. B-C. Evaluation of cytotoxicity and OD value at 450 nm of the hydrogel coating co-cultured with HUVECs by the CCK-8 method. D. Hemolysis image of the bionic BC-BCD hydrogel. E-F. Hemolysis rate and hemolysis OD value at 450 nm of the BC-BCD hydrogel.

[0048] Figure 7 Anti-inflammatory performance evaluation of the BC-BCD double-layer bionic hydrogel coating prepared in Example 7 in mice. A. Schematic diagram of implanting a CVC coated with BC-BCD into the back of a mouse. B. H&E staining image of the surrounding tissue after catheter implantation with the BC-BCD bionic hydrogel coating. C. Semi-quantitative analysis of IL-6 in the surrounding tissue after catheter implantation. D. Staining photographs of IL-6 and TNF-α in the surrounding tissue after catheter implantation of the CVCs double-layer BC-BCD anticoagulant hydrogel coating.

[0049] Figure 8 Performance evaluation of the BC-BCD double-layer bionic hydrogel coating prepared in Example 7 after being coated on a catheter in an in vitro arteriovenous circulation model of New Zealand white rabbits. A. Schematic diagram of the rabbit in vitro arteriovenous circulation model. B. Observation of thrombus formation inside the catheter coated with the BC-BCD double-layer bionic hydrogel coating after testing. C. Scanning electron microscope images to observe the attachment of red blood cells and thrombus formation on the surface of the BC-BCD bionic coating. D. Occlusion rate of the catheter coated with the double-layer BC-BCD bionic coating hydrogel coating after testing. E. Quantitative analysis of the number of red blood cells attached to the surface of the double-layer bionic BC-BCD coating hydrogel coating.

[0050] Figure 9The bilayer biomimetic BC-BCD hydrogel coating prepared in Example 7 was coated on the catheter, and blood analysis was performed after the rabbit atrioventricular shunt circulation model. A. Flow chart of blood detection during extracorporeal circulation. B. Measurement of blood flow rate of coated and uncoated catheters before and after circulation. C-D. APTT (0 to 60 minutes) of uncoated and BC-BCD coated catheters during circuit testing. E. Difference in F1+2 between bare catheter and BC-BCD coated catheter. F-G. Evaluation of anti-inflammatory performance of BC-BCD coating by inflammatory indexes IL-10 and TNF-α in blood. Detailed Description of the Invention

[0051] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0052] The present invention discloses a method for preparing a new material of a biomimetic hydrogel coating with anticoagulant and anti-inflammatory effects, comprising the following steps:

[0053] (1) Dissolve N,N-dimethylaminopropyl acrylamide (DMAPAA) and ethyl bromoacetate in acetonitrile, and react at 60 °C for 12 hours to obtain solution S1;

[0054] (2) Pour anhydrous ether into solution S1, stir continuously at room temperature to obtain a white precipitate of ethyl-carboxybetaine acrylamide (ethyl-CBAA), vacuum dry it for 3 h, then dissolve it in ethanol, and stir at room temperature to obtain solution S2;

[0055] (3) Pour the ion exchange resin (IRA-400) soaked in 4% (V / V) NaOH into solution S2, stir and filter to obtain solution S3;

[0056] (4) Pour anhydrous ether into its supernatant, stir to obtain a white precipitate of carboxybetaine acrylamide (CBAA), and then dissolve it in deionized water to obtain solution S4;

[0057] (5) Dissolve bovine serum albumin (BSA) in deionized water, and stir at room temperature to obtain solution S5;

[0058] (6) Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in deionized water, and stir to obtain solution S6;

[0059] (7) Dissolve N-hydroxysuccinimide (NHS) in deionized water, and stir to obtain solution S7;

[0060] (8) Dissolve N,N'-methylenebis(acrylamide) (MBAA) in deionized water and stir to obtain solution S8;

[0061] (9) Dissolve N,N,N',N'-tetramethylethylenediamine (TEMED) in deionized water and stir to obtain solution S9;

[0062] (10) Dissolve ammonium persulfate (APS) in deionized water and stir at room temperature to obtain solution S10;

[0063] (11) Dissolve dopamine hydrochloride (DA) in deionized water and stir to obtain solution S11;

[0064] (12) Mix solution S4 and solution S5 evenly and stir to obtain solution S12;

[0065] (13) Add solution S8, S9, and S10 to solution S12 in sequence, stir and mix evenly at room temperature to obtain the outer layer BSA / CBAA pre-gel solution; then add S6, S7, Tris hydrochloride, and S11 solution to the BSA / CBAA pre-gel solution, stir and mix evenly to obtain the inner layer BSA / CBAA / DA pre-gel solution; To prepare the hydrogel coating, first cut the medical-grade central venous catheter sample into the required size and clean it with ethanol or deionized water to remove surface impurities. The purified sample is soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the sample is ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature to obtain the BC-BCD double-layer hydrogel coating.

[0066] In the present invention, first dissolve N,N'-dimethylaminopropyl acrylamide (DMAPAA) and ethyl bromoacetate in acetonitrile and react at 60 °C for 12 hours to obtain solution S1. More preferably, the mass fraction of DMAPAA is 1.0 - 99.7 wt%, the mass fraction of ethyl bromoacetate is 0.1 - 99.7 wt%, the mass fraction of acetonitrile is 1.0 - 99.7 wt%, the stirring temperature is 60 °C, and the stirring time is 12 - 14 h.

[0067] Pour anhydrous ether into solution S1, stir continuously at room temperature to obtain white precipitate of ethyl-carboxybetaine acrylamide (ethyl-CBAA), dissolve it in ethanol after vacuum drying for 3 h, and stir at room temperature to obtain solution S2. More preferably, the mass fraction of anhydrous ether is 0.1 - 99.7 wt%, the stirring temperature is 10 - 40 °C, and the stirring time is 1 - 20 min.

[0068] Pour the ion exchange resin (IRA-400) soaked in 4% (V / V) NaOH, stir and filter to obtain solution S3. More preferably, the mass fraction of the ion exchange resin is 0.1-99.7 wt%.

[0069] Pour anhydrous ether into its supernatant, stir to obtain white precipitate of carboxybetaine acrylamide (CBAA), and then dissolve it in deionized water to obtain solution S4;

[0070] At room temperature, dissolve bovine serum albumin (BSA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), N,N'-methylenebis(acrylamide) (MBAA), N,N,N',N'-tetramethylethylenediamine (TEMED), ammonium persulfate (APS), and dopamine hydrochloride (DA) in deionized water respectively, and stir at room temperature to obtain solutions S5, S6, S7, S8, S9, S10, and S11; More preferably, the mass fraction of BSA is 0.1-25.0 wt%, the concentration of EDC solution is 0.001-0.1 g / mL, the concentration of NHS is 0.001-1 g / mL, the concentration of MBAA is 0.002-1 g / mL, the concentration of TEMED is 0.002-1 g / mL, the concentration of APS is 0.002-1 g / mL, the concentration of DA is 0.002-1 g / mL, the stirring temperature is 5-45 °C, and the stirring time is 2-150 min.

[0071] Mix solution S4 and solution S5 evenly to obtain solution S12, and the mass ratio of solution S4:S5 is 100:1-1:100; the stirring temperature is 5-55 °C, and the stirring time is 2-150 min.

[0072] At room temperature, add solutions S7, S8, and S9 to solution S12 in sequence, and then add solutions S5, S6, S7, S8, S9, and S10 to solution S12 in sequence, and mix evenly at room temperature in sequence; obtain two kinds of pre-gel solutions of outer layer BSA / CBAA and inner layer BSA / CBAA / DA. Cut the medical grade CVCs samples into required sizes, soak them in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time, ultrasonically clean and dry them at room temperature to obtain BC-BCD double-layer hydrogel coatings. More preferably, the mass ratio of solution S7:S8:S9:S12 is 10:1000:1:1:1-1000:10:1:1:1; the mass ratio of solution S5:S6:S7:S8:S9:S10:S12 is 10:1000:1:1:1:1:1-1000:10:1:1:1:1:1; the stirring temperature is 5-55 °C, and the stirring time is 1-150 min.

[0073] To further understand the present invention, the following describes the preparation method of a new material for wound repair with hemostatic effect provided by the present invention in conjunction with embodiments. The protection scope of the present invention is not limited by the following embodiments.

[0074] Example 1

[0075] Dissolve 4.2 g of N,N-dimethylaminopropyl acrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) in 25.0 mL of acetonitrile, react at 60 °C for 12 h, wash with 150.0 mL of diethyl ether, and vacuum dry for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). At the same time, soak IRA-400 resin in 4% (V / V) NaOH for 4 h and wash with 99% (V / V) ethanol until neutral. To remove ethyl, dissolve the ethyl-CBAA precipitate in ethanol and then mix it with IRA-400 resin. After stirring and filtering, transfer the solution to a round-bottom flask, rotary evaporate at 50 °C at a rotation speed of 60 rpm for 20 min, and finally add 300.0 mL of diethyl ether and stir slowly to obtain a white precipitate, which is dried in a vacuum dryer to obtain a white powder CBAA.

[0076] Mix 3.0 g of BSA and 3.0 g of CBAA and completely dissolve them in 10.0 mL of deionized water. Then, sequentially add 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS during stirring to obtain a BSA / CBAA pre-gel solution. Then, add 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first cut the medical-grade central venous catheter sample into the required size and wash it with ethanol or deionized water to remove surface impurities. The purified sample is soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, ultrasonically clean the sample three times in deionized water, cure it at 60 °C, and dry it at room temperature for subsequent use to obtain a BC-BCD double-layer hydrogel coating.

[0077] Example 2

[0078] Dissolve 4.2 g of N,N-dimethylaminopropylacrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) in 25.0 mL of acetonitrile, react at 60 °C for 12 h, wash with 150.0 mL of ether, and dry in vacuum for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). At the same time, soak IRA-400 resin in 4% (V / V) NaOH for 4 h and wash with 99% (V / V) ethanol until neutral. To remove the ethyl group, dissolve the ethyl-CBAA precipitate in ethanol and then mix it with IRA-400 resin. Then transfer the solution to a round-bottom flask, perform rotary evaporation at 50 °C at a rotation speed of 60 rpm for 20 min, and finally add 300.0 mL of ether and stir slowly to obtain a white precipitate, which is dried in a vacuum dryer to obtain a white powder CBAA.

[0079] Mix 3.0 g of BSA and 4.0 g of CBAA and dissolve them completely in 10.0 mL of deionized water. Then, add 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS successively during stirring to obtain a BSA / CBAA pre-gel solution. After that, add 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first cut the medical-grade central venous catheter sample into the required size and wash it with ethanol or deionized water to remove surface impurities. The purified sample is soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, ultrasonically clean the sample three times in deionized water, cure it at 60 °C, and dry it at room temperature for subsequent use to obtain a BC-BCD double-layer hydrogel coating.

[0080] Example 3

[0081] Dissolve 4.2 g of N,N-dimethylaminopropylacrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) in 25.0 mL of acetonitrile, react at 60 °C for 12 h, wash with 150.0 mL of ether, and dry in vacuum for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). At the same time, soak IRA-400 resin in 4% (V / V) NaOH for 4 h and wash with 99% (V / V) ethanol until neutral. To remove the ethyl group, dissolve the ethyl-CBAA precipitate in ethanol and then mix it with IRA-400 resin. Then transfer the solution to a round-bottom flask, perform rotary evaporation at 50 °C at a rotation speed of 60 rpm for 20 min, and finally add 300.0 mL of ether and stir slowly to obtain a white precipitate, which is dried in a vacuum dryer to obtain a white powder CBAA.

[0082] 3.0 g of BSA and 5.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were added successively during stirring to obtain a BSA / CBAA pre-gel solution. Subsequently, 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA were added to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first, a medical-grade central venous catheter sample was cut into the required size and washed with ethanol or deionized water to remove surface impurities. The purified sample was immersed in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the sample was ultrasonically washed three times in deionized water, cured at 60 °C, and dried at room temperature for subsequent use to obtain a BC-BCD double-layer hydrogel coating.

[0083] Example 4

[0084] 4.2 g of N,N-dimethylaminopropylacrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) were dissolved in 25.0 mL of acetonitrile, reacted at 60 °C for 12 h, washed with 150.0 mL of ether, and dried in vacuo for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). Meanwhile, IRA-400 resin was soaked in 4% (V / V) NaOH for 4 h and washed with 99% (V / V) ethanol until neutral. To remove the ethyl group, the ethyl-CBAA precipitate was dissolved in ethanol and then mixed with IRA-400 resin. Then the solution was transferred to a round-bottom flask, rotary evaporated at 50 °C at a rotation speed of 60 rpm for 20 min, and finally, 300.0 mL of ether was added and stirred slowly to obtain a white precipitate, which was dried in a vacuum dryer to obtain a white powder of CBAA.

[0085] 3.0 g of BSA and 6.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were sequentially added during stirring to obtain a BSA / CBAA pre-gel solution. Subsequently, 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA were added to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first, medical-grade central venous catheter samples were cut into the required size and washed with ethanol or deionized water to remove surface impurities. The purified samples were immersed in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the samples were ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature for subsequent use to obtain a BC-BCD double-layer hydrogel coating.

[0086] Example 5

[0087] 4.2 g of N,N-dimethylaminopropyl acrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) were dissolved in 25.0 mL of acetonitrile and reacted at 60 °C for 12 h. The reaction product was washed with 150.0 mL of diethyl ether and dried under vacuum for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). Meanwhile, IRA-400 resin was soaked in 4% (V / V) NaOH for 4 h and washed with 99% (V / V) ethanol until neutral. To remove the ethyl group, the ethyl-CBAA precipitate was dissolved in ethanol and then mixed with IRA-400 resin. Then the solution was transferred to a round-bottom flask and rotary evaporated at 50 °C at a speed of 60 rpm for 20 min. Finally, 300.0 mL of diethyl ether was slowly added with stirring to obtain a white precipitate, which was dried in a vacuum dryer to obtain a white powder of CBAA.

[0088] 3.0 g of BSA and 7.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were successively added during stirring to obtain a BSA / CBAA pre-gel solution. Subsequently, 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA were added to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first, medical-grade central venous catheter samples were cut into the required sizes and washed with ethanol or deionized water to remove surface impurities. The purified samples were soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the samples were ultrasonically washed three times in deionized water, cured at 60 °C, and dried at room temperature for subsequent use to obtain a BC-BCD double-layer hydrogel coating.

[0089] Example 6

[0090] 4.2 g of N,N-dimethylaminopropylacrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) were dissolved in 25.0 mL of acetonitrile, reacted at 60 °C for 12 h, washed with 150.0 mL of diethyl ether, and dried in vacuo for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). Meanwhile, IRA-400 resin was soaked in 4% (V / V) NaOH for 4 h and washed with 99% (V / V) ethanol until neutral. To remove the ethyl group, the ethyl-CBAA precipitate was dissolved in ethanol and then mixed with IRA-400 resin. Then the solution was transferred to a round-bottom flask, rotary evaporated at 50 °C at a rotation speed of 60 rpm for 20 min, and finally, 300.0 mL of diethyl ether was added and stirred slowly to obtain a white precipitate, which was dried in a vacuum dryer to obtain a white powder of CBAA.

[0091] 3.0 g of BSA and 8.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were sequentially added during stirring to obtain a BSA / CBAA pre-gel solution. Subsequently, 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA were added to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first, medical-grade central venous catheter samples were cut into the required size and washed with ethanol or deionized water to remove surface impurities. The purified samples were soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the samples were ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature for subsequent use to obtain a BC-BCD double-layer hydrogel coating.

[0092] Example 7

[0093] 4.2 g of N,N-dimethylaminopropylacrylamide (25.0 mmol) and 5.8 g of ethyl bromoacetate (37.5 mmol) were dissolved in 25.0 mL of acetonitrile and reacted at 60 °C for 12 h. The reaction product was washed with 150.0 mL of diethyl ether and dried under vacuum for 3 h to obtain a white precipitate of ethyl carboxybetaine acrylamide (ethyl-CBAA). Meanwhile, IRA-400 resin was soaked in 4% (V / V) NaOH for 4 h and washed with 99% (V / V) ethanol until neutral. To remove the ethyl group, the ethyl-CBAA precipitate was dissolved in ethanol and then mixed with IRA-400 resin. Then the solution was transferred to a round-bottom flask and rotary evaporated at 50 °C at a speed of 60 rpm for 20 min. Finally, 300.0 mL of diethyl ether was slowly added with stirring to obtain a white precipitate, which was dried in a vacuum dryer to obtain a white powder of CBAA.

[0094] 3.0 g of BSA and 9.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were added sequentially during stirring to obtain a BSA / CBAA pre-gel solution. Subsequently, 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA were added to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. To prepare the hydrogel coating, first, medical-grade central venous catheter samples were cut into the required sizes and cleaned with ethanol or deionized water to remove surface impurities. The purified samples were soaked in the BSA / CBAA and BSA / CBAA / DA pre-gel solutions for a predetermined time. Finally, the samples were ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature to obtain a BC-BCD double-layer hydrogel coating.

[0095] Comparative Example 1

[0096] 3.0 g of BSA and 9.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were added sequentially during stirring to obtain a BSA / CBAA pre-gel solution. Medical-grade central venous catheter samples were cut into the required sizes and cleaned with ethanol or deionized water to remove surface impurities. The samples were soaked in the BSA / CBAA pre-gel solution for a predetermined time. Finally, the samples were ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature to obtain a BSA / CBAA hydrogel coating.

[0097] Comparative Example 2

[0098] 3.0 g of BSA and 9.0 g of CBAA were mixed and completely dissolved in 10.0 mL of deionized water. Then, 12.0 mg of MBAA, 24.0 mg of TEMED, and 66.0 mg of APS were added sequentially during stirring to obtain a BSA / CBAA pre-gel solution. Subsequently, 50.0 mg of EDC, 50.0 mg of NHS, 1.0 mL of Tris hydrochloride (Tris-HCl), and 30.0 mg of DA were added to the BSA / CBAA pre-gel solution to obtain a BSA / CBAA / DA pre-gel solution. Medical-grade central venous catheter samples were cut into the required sizes and cleaned with ethanol or deionized water to remove surface impurities. The samples were soaked in the BSA / CBAA / DA pre-gel solution for a predetermined time. Finally, the samples were ultrasonically cleaned three times in deionized water, cured at 60 °C, and dried at room temperature to obtain a BSA / CBAA / DA hydrogel coating.

[0099] The morphological and structural characterization of the bionic hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2 is as follows Figure 1 As shown, the introduction of the material is mainly physical cross-linking by intermolecular forces during the cross-linking process with the substrate adhesive, without chemical reactions occurring. The hydrogels prepared in Comparative Example 1 and Comparative Example 2 both have obvious porous network structures, with pore sizes of 10.2 ± 1.8 μm and 5.1 ± 1.0 μm, respectively Figure 1 (A, B). As can be seen from Figure 1 C, the porosity of the hydrogel in Comparative Example 2 was significantly reduced from that of Comparative Example 1 (36.58% ± 0.46%) to (18.46% ± 3.71%). The porous structure of the hydrogel in Comparative Example 1 as the top layer is conducive to the storage of water molecules, thus making the surface lubricated. And the dense vertically oriented porous structure of the hydrogel in the bottom layer of Comparative Example 2 helps the overall hydrogel coating to bear and maintain sufficient mechanical properties.

[0100] The quantitative analysis diagrams of the mechanical properties of the bionic hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2 are as follows Figure 2 As shown. The tensile strength of the composite BC-BCD hydrogel in Example 7 was 21.0 ± 96.2 kPa, and the elongation at break was approximately 199.0% ± 52.4%, showing an elastomer with no obvious stress yield. Consistent with the tensile test results; the compressive strength of the hydrogel in Comparative Example 2 (202.3 ± 0.1 kPa) was higher than that of the sample in Comparative Example 1 (177.7 ± 5.2 kPa). At the same time, the double-layer BC-BCD composite gel in Example 7 also showed high compressive resistance (200.4 ± 9.7 kPa), fully verifying its good elasticity, ductility, and external compressive resistance. It is worth noting that the Young's modulus of the BC-BCD hydrogel coating (10.1 ± 0.9 kPa) falls within the range of the vascular intima (≈10.0 kPa), endowing it with the required biomechanical matching characteristics.

[0101] The adhesion and lubrication performance analysis of the bionic hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2 is as follows Figure 3As shown in the figure. The adhesion strengths of the hydrogel of Comparative Example 2 to wood, glass, PS, rubber, and metal were significantly different, being 67.9 ± 3.4 kPa, 23.7 ± 1.5 kPa, 89.5 ± 2.3 kPa, and 127.2 ± 16.2 kPa, respectively. In addition, after 20 friction sliding cycles, the CoF of the coating of Comparative Example 1 remained in a relatively low range, varying from 0.003 ± 0.04 to 0.108 ± 0.072. The double-layer bionic BC-BCD coating of Example 7 would immediately turn into a lubricated state after encountering water, enabling a glass bottle filled with water to slide down from a 30° inclined plane. Its water contact angle (WCA) value (24.15° ± 1.42°) was also significantly lower than that of different coating materials (>50°). It was proved that the double-layer BC-BCD hydrogel coating prepared in Example 7 not only had a strong adhesion effect with various substrates, enhancing the interfacial stability, but also achieved self-adaptive hydration lubrication by forming strong hydrogen bond sites with water molecules, which helped to reduce the shear damage during contact with blood vessels and prevent biofouling adhesion in clinical applications.

[0102] The anti-inflammatory and antibacterial activities of the hydrogel coatings prepared in Example 7, Comparative Example 1, and Comparative Example 2 are as Figure 4 shown. After being soaked in 1 mM hydrogen peroxide, the number of sulfonic acid groups in the hydrogels of both Comparative Example 1 and Comparative Example 2 increased, and other groups were not affected, ensuring long-term application in vivo. In the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate test, the scavenging rate of Comparative Example 1 was only 22.99% ± 9.30%. In contrast, the ratio of Comparative Example 2 increased significantly to 69.7% ± 3.50%, while that of BC-BCD in Example 7 was as high as 79.42% ± 1.34%. In addition, after culturing bacteria on the surface of the BC-BCD double-layer bionic hydrogel coating proposed in the present invention, compared with the bare surface, the biofilm formation rate decreased from 100% to 60% ± 1.36%, showing significant anti-inflammatory and antibacterial properties.

[0103] The anticoagulant performance characterization of the BC-BCD double-layer bionic hydrogel coating prepared in Example 7 is as Figure 5As shown, the prepared BC-BC coating significantly prolonged the normal blood APTT from 105±5 s to 693±67 s. In the BCA test, after the hydrogel was soaked in the BSA solution, almost no protein adhesion was observed on the coating surface. In addition, the clot formation time induced by normal blood was 13±0.82 min, while the clot formation time in the BC-BCD group was significantly prolonged to more than 80±8.16 min. Similarly, during the BCI test (0-85 minutes), the values in the BC-BCD hydrogel were higher compared with normal blood, indicating that the hydrogel led to a slower coagulation rate of free hemoglobin. All these results suggest that the BC-BCD bilayer biomimetic hydrogel coating of the present invention not only blocks the coagulation cascade by reducing the activities of coagulation factors VIII, IX or XI and preventing their activation, but also can prevent the attachment of proteins and blood components to reduce thrombus formation.

[0104] The in vitro biocompatibility of the bilayer BC-BCD biomimetic hydrogel coating prepared in Example 7 is as Figure 6 shown. After culturing the prepared BC-BCD hydrogel coating with human umbilical vein endothelial cells (HUVECs) for 1 day and 3 days, the fluorescence images of the live / dead staining test of HUVECs showed that the cultured cells survived normally and had a healthy morphology. The results of the Cell Counting Kit-8 (CCK-8) showed that the cell survival rate in each group was higher than 90% on the 1st day and the 3rd day, and the cell viability in the BC-BCD hydrogel group was higher than that in the control group on the 3rd day, indicating that the hydrogel did not damage the growth of HUVECs, but instead promoted cell proliferation. In the hemolysis test, the hemolysis rate of the hydrogel prepared in Example 7 was less than 3%, which is a polymer material with good blood compatibility.

[0105] The in vivo anti-inflammatory performance characterization of the bilayer BC-BCD biomimetic hydrogel coating prepared in Example 7 in mice is as Figure 7 shown. One week after implantation into mice, the prepared BC-BCD coating did not cause abnormal surrounding tissue nodules, and no obvious inflammatory cell infiltration and fibrosis were observed. Moreover, compared with the control group, the level of IL-6 in the surrounding tissue decreased by 70.10%, providing a basis for its in vivo biocompatibility and immunomodulatory ability.

[0106] The application evaluation of the dual-mode anticoagulant BC-BCD bilayer biomimetic hydrogel coating prepared in Example 7 in the rabbit's in vitro arteriovenous circulation is as Figure 8 shown. After the catheter coated with the BC-BCD biomimetic hydrogel coating was tested, almost no thrombus was formed inside, significantly reducing the catheter occlusion rate. SEM also hardly observed blood components attached to the surface. All these indicate that the bilayer biomimetic hydrogel coating provided by the present invention can effectively prevent thrombus formation and is expected to become a new generation of CVCs coating.

[0107] The double-layer BC-BCD bionic hydrogel coating prepared in Example 7 was coated on the catheter. After in vitro arteriovenous circulation, the blood analysis results of the animals were as Figure 9 shown. After circulation, the catheter coated with the BC-BCD hydrogel coating still maintained the flow rate of the normal catheter without circulation. The APTT time was significantly prolonged, and the F1+2 level in the blood showed a downward trend, confirming its effect of actively anticoagulating by cutting off the coagulation cascade. In addition, the inflammatory indexes of IL-10 and TNF-α in the blood did not increase significantly compared with the control group. It shows that the bionic hydrogel coating provided by the present invention can not only effectively anticoagulate, but also play a good role in preventing the infiltration of inflammatory factors and thus anti-inflammatory, and is expected to be applied clinically.

[0108] The above descriptions of the embodiments are only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0109] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a BC-BCD double-layer bionic hydrogel coating for a central venous catheter with synergistic dual-mode anticoagulation and anti-inflammatory properties, characterized in that: The following steps are involved: (1) dissolving N,N-dimethylaminopropyl acrylamide and ethyl bromoacetate in acetonitrile and reacting at 60° C. for 12 hours to obtain solution S1; (2) Anhydrous ether was poured into the S1 solution and stirred continuously at room temperature to obtain a white precipitate of ethyl-carboxybetaine acrylamide, which was then vacuum dried for 3 h and dissolved in ethanol and stirred at room temperature to obtain a solution S2; (3) pouring the ion exchange resin IRA-400 soaked in 4% V / V NaOH into the S2 solution, stirring, filtering, and rotary evaporating to obtain a solution S3; (4) anhydrous ether was poured into the supernatant, and after stirring, a white precipitate of carboxybetaine acrylamide was obtained, which was then dissolved in deionized water to obtain solution S4; (5) dissolving bovine serum albumin in deionized water and stirring at room temperature to obtain solution S5; (6) dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in deionized water to obtain solution S6; (7) dissolving N-hydroxysuccinimide in deionized water and stirring to obtain solution S7; (8) dissolving N,N'-methylenebis(acrylamide) in deionized water and stirring to obtain solution S8; (9) dissolving N,N,N',N'-tetramethylethylenediamine in deionized water and stirring to obtain solution S9; (10) dissolving ammonium persulfate in deionized water and stirring at room temperature to obtain a solution S10; (11) dissolving dopamine hydrochloride in deionized water and stirring to obtain a solution S11; (12) Mix solution S4 and solution S5 evenly, and stir to obtain solution S12; (13) Solutions S8, S9, and S10 are sequentially added to solution S12, and stirred and mixed at room temperature to obtain an outer layer BSA / CBAA pregel solution; then, S6, S7, Tris hydrochloric acid, and S11 solutions are added to the BSA / CBAA pregel solution, and stirred and mixed to obtain an inner layer BSA / CBAA / DA pregel solution; in order to prepare the hydrogel coating, a medical-grade central venous catheter sample is first cut into a desired size and cleaned with ethanol or deionized water to remove surface impurities; the purified sample is immersed in BSA / CBAA and BSA / CBAA / DA pregel solutions for a predetermined time; finally, the sample is ultrasonically cleaned in deionized water three times, cured at 60°C, and dried at room temperature to obtain a BC-BCD double-layer hydrogel coating.

2. The preparation method according to claim 1, characterized in that: In the solution S1 of step (1), the mass fraction of N,N-dimethylaminopropyl acrylamide is 1.0-99.7wt%, the mass fraction of ethyl bromoacetate is 0.1-99.7wt%, the mass fraction of acetonitrile is 1.0-99.7wt%, the stirring temperature is 60°C, and the stirring time is 12-14h.

3. The preparation method according to claim 1, characterized in that: In the solution S2 of step (2), the mass fraction of anhydrous ether is 0.1-99.7wt%, the stirring temperature is 10-40°C, and the stirring time is 1-20min; in the solution S3 of step (3), the mass fraction of ion exchange resin is 0.1-99.7wt%; in the solution S4 of step (4), the mass fraction of carboxybetaine acrylamide is 0.1-50.0wt%, the stirring temperature is 10-40°C, and the stirring time is 5-10min; in the solution S5 of step (5), the mass fraction of bovine serum albumin is 0.1-25.0wt%, the stirring temperature is 10-40°C, and the stirring time is 1-10min.

4. The preparation method according to claim 1, characterized in that: In step (6), the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is 0.001 to 0.1 g / mL; the stirring temperature is 5 to 45° C., and the stirring time is 2 to 150 min. In step (7), the concentration of N-hydroxysuccinimide is 0.001 to 1 g / mL; the stirring temperature is 5 to 45° C., and the stirring time is 2 to 150 min.

5. The preparation method according to claim 1, characterized in that: In step (8), the concentration of N,N'-methylenebis(acrylamide) is 0.002-1 g / mL; the stirring temperature is 5-45°C, and the stirring time is 2-150 min.

6. The preparation method according to claim 1, characterized in that: In step (9), the concentration of N,N,N',N'-tetramethylethylenediamine is 0.002-1 g / mL; the stirring temperature is 5-45° C., and the stirring time is 2-150 min.

7. The preparation method according to claim 1, characterized in that: In step (10), the concentration of ammonium persulfate is 0.002-1 g / mL; the stirring temperature is 5-45° C., and the stirring time is 2-150 min.

8. The preparation method according to claim 1, characterized in that: In step (11), the concentration of dopamine hydrochloride is 0.002-1 g / mL; the stirring temperature is 5-45° C., and the stirring time is 2-150 min.

9. The preparation method according to claim 1, characterized in that: In the solution S12 of step (12), the mass ratio of solution S4:S5 is 100:1 to 1:100; the stirring temperature is 5 to 55° C., and the stirring time is 2 to 150 min.

10. The preparation method according to claim 1, characterized in that In step (13), the mass ratio of S8:S9:S10:S12 in the BSA / CBAA pregel solution is 10:1000:1:1:1 to 1000:10:1:1:1; the mass ratio of S6:S7:S8:S9:S10:S11:S12 in the BSA / CBAA / DA pregel solution is 10:1000:1:1:1:1:1 to 1000:10:1:1:1:1:1; the stirring temperature is 5 to 55°C, and the stirring time is 1 to 150 min.

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