Heparin-like ternary copolymer containing zwitterion, sulfonic acid group and carboxyl side chain, coating, preparation method and application

Abstract

A heparin-like ternary copolymer containing zwitterionic, sulfonic, and carboxyl side chains is prepared by atom transfer radical polymerization (ATRP) from a first monomer containing a sulfonic acid group and an olefinic group, a second monomer containing an oxygen-carbon chain-linked zwitterionic group and an olefinic group, and a third monomer containing an oxygen-carbon chain-linked carboxyl side chain and an olefinic group. This ternary copolymer has a heparin-like structure and utilizes zwitterionic groups that mimic the hydrophilicity of cell membrane lecithin and negatively charged sulfonic acid groups with anticoagulant activity to inhibit both intrinsically and exogenously induced coagulation reactions. Anticoagulant coatings prepared from this copolymer can largely address the problem of zwitterionic polymers alone having poor anticoagulant properties and being unable to meet the anticoagulant requirements of biomedical devices. It also provides an effective solution to the multiple side effects and adverse reactions that systemic anticoagulants are prone to.

Description

Technical Field

[0001] The present invention belongs to the field of blood-compatible surface modification of medical devices, and relates to a blood-compatible polymer having the function of inhibiting both intrinsic and exogenous coagulation pathways and its anti-coagulation coating, which has the functions of resisting blood component adhesion and activating the anti-coagulation system. Background Art

[0002] Medical devices such as hemodialyzers, oxygenators, extracorporeal circulation circuits, and central venous cannulas often have poor blood compatibility due to the inherently poor blood compatibility of the polymer materials used in contact with blood. This leads to plasma protein adsorption, platelet adhesion, and activation on the material surface upon contact with blood. This activates a series of coagulation factors in the blood and induces the conversion of prothrombin to thrombin. Thrombin, in turn, converts previously soluble components such as albumin and fibrinogen into insoluble substances, which then adsorb on the material surface, ultimately causing blood coagulation and thrombosis, which poses serious risks to the patient. Current use of anticoagulants also carries numerous risks. For example, hemodialysis requires the infusion of anticoagulants such as heparin to achieve systemic anticoagulation, but achieving a balance between low and high heparinization can be challenging, which can easily lead to coagulation in the extracorporeal circulation circuit and internal bleeding. Therefore, biomedical materials that come into contact with blood urgently need improved blood compatibility and the ability to resist coagulation. The development of a multifunctional anticoagulant coating with excellent blood compatibility and the ability to inhibit both intrinsic and exogenous coagulation is particularly important.

[0003] Covalently binding heparin to the surface of biomaterials to construct an anticoagulant heparin coating is the most common method for achieving a good anticoagulant effect. However, heparin coatings achieve their anticoagulant effect by indirectly inhibiting the activity of thrombin, thereby preventing thrombin from converting soluble fibrinogen into insoluble fibrin. The degree and duration of anticoagulation are greatly affected by the way the heparin chains are fixed. Multi-point covalently bound heparin coatings are stable and have a long service life, but their anticoagulant properties are significantly reduced. Single-point or end-point bound heparin has stronger anticoagulant properties, but is easily degraded by blood components and has a short anticoagulant lifespan. In addition, the strong negative surface charge of the heparin coating can cause protein adsorption and platelet adhesion activation, which can also induce thrombosis. These limitations of heparin coatings make it impossible to meet the application requirements of longer-term antithrombotic effects.

[0004] Studies have found that zwitterionic polymers exhibit excellent blood compatibility due to the mutual neutralization of their surface charges. This near-zero charge design is inspired by the zwitterionic structure of the outer membrane of red blood cells. Polymers with zwitterionic structures similar to those on the cell membrane surface, such as carboxybetaines, sulfobetaines, and phosphorylcholines, when bound to the material surface, can undergo ionic solvation with water molecules, polarizing the surrounding water molecules and forming a dense hydration layer. This hydration layer gives the material excellent hydrophilic properties, effectively inhibiting the adhesion of blood components such as proteins, platelets, and red blood cells to the material surface, thereby improving the material's blood compatibility. By covalently bonding a bioinert phosphorylcholine zwitterionic polymer coating to a polydopamine pre-coated material, the intrinsic coagulation cascade caused by protein adsorption and platelet adhesion can be almost completely eliminated. However, this bioinert coating is not yet effective in preventing the coagulation cascade caused by exogenous pathways such as puncture surgery and blood cell damage.

[0005] Therefore, in order to overcome the limitations of existing anticoagulant coatings, the present invention provides a heparin-like ternary copolymer having both biological inertness and biologically active anticoagulant ability and a method for preparing its coating, hoping that this coating can not only avoid activation with blood components, but also effectively inactivate coagulation factors produced by exogenous pathways. Compared with the PMPCC polymer reported in J.Mater.Chem.B, 2020, 8, 4299-4309, this heparin-like ternary copolymer, on the basis of making full use of the excellent hydrophilicity and blood compatibility of zwitterionic polymers, its third negatively charged group with sulfonic acid group gives the ternary copolymer heparin-like anticoagulant function. We expect that this new polymer coating can simultaneously inhibit the occurrence of coagulation induced by endogenous and exogenous pathways, thereby achieving all-round resistance to coagulation, and providing technical methods for the design and application of blood-contact medical materials. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a heparin-like terpolymer with excellent blood compatibility and anticoagulant properties. This terpolymer utilizes hydrophilic zwitterionic groups that mimic the hydrophilic properties of cell membrane lecithin and negatively charged sulfonic acid groups with anticoagulant activity to inhibit both intrinsic and exogenous coagulation reactions.

[0007] The second technical problem to be solved by the present invention is to provide an efficient method for synthesizing a heparin-like terpolymer. The monomers of the terpolymer are dissolved in saline according to the designed ratio. An ATRP initiator, catalyst, and reducing agent are added in sequence. A closed reaction is then conducted, followed by impurity removal and freeze-drying to obtain the heparin-like terpolymer. Compared to conventional free radical copolymerization methods in solution, this ATRP method significantly improves the yield of zwitterionic heparin-like terpolymers.

[0008] The third technical problem to be solved by the present invention is to provide a method for preparing a heparin-like terpolymer coating, by covalently bonding multiple side chain carboxyl groups in the terpolymer to the surface of the material, thereby obtaining a more durable anti-coagulant property of the coating.

[0009] The fourth technical problem to be solved by the present invention is to provide a heparin-like terpolymer coating combining zwitterions with negative charges of sulfonic acid groups for application in a variety of common medical devices and other biomedical engineering fields.

[0010] The present invention is achieved through the following technical solutions:

[0011] To solve one of the above technical problems, the heparin-like ternary copolymer containing zwitterion, sulfonic acid group and carboxyl side chain of the present invention includes zwitterion as a hydrophilic group mimicking cell membrane blood compatibility and sulfonic acid group and carboxyl group as heparin-like anticoagulant groups.

[0012] Specifically, the heparin-like terpolymer has the following structure of general formula (I):

[0013]

[0014] In the formula, R1, R2 and R3 are CH3 or H, R4 is a negatively charged side chain containing a sulfonic acid group, R5 is a zwitterionic group connected by an oxygen-carbon chain, and R6 is a carboxyl side chain group connected by an oxygen-carbon chain.

[0015] The zwitterionic groups include sulfobetaine (SB), phosphorylcholine (PC), and carboxybetaine (CB).

[0016] Specifically, a variety of heparin-like terpolymers with different monomer ratios were designed to meet a variety of different application requirements, with the proportions of x, y, and z being 20% ​​to 60%, 20% to 60%, and 10% to 30%, respectively.

[0017] More specifically, excellent blood compatibility requires that y accounts for ≥40%; when x accounts for >40%, the anticoagulant ability will be stronger, but the adhesion of blood components to the coating surface will increase.

[0018] The second technical problem to be solved by the present invention is to provide an efficient method for synthesizing a heparin-like terpolymer. This method is characterized by dissolving the monomers of the terpolymer in saline according to a designed ratio, sequentially adding a water-soluble ATRP initiator, a catalyst, and a reducing agent, sealing the solution for reaction, and then purifying and freeze-drying the resulting heparin-like terpolymer. Compared with conventional free radical copolymerization methods in solution, this ATRP preparation method utilizes a larger feed volume, a higher yield, and a uniform molecular weight distribution of the copolymer. Compared with conventional ATRP copolymerization methods, this ATRP preparation method is simple, does not require a cumbersome deoxygenation step, and has a copper ion catalyst concentration of only ppm.

[0019] Furthermore, to solve the third of the above technical problems, a method for constructing a heparin-like ternary copolymer coating is provided, which includes a polydopamine-mediated layer that mimics the universal adhesion of mussels and a covalently bonded ternary copolymer anti-coagulation layer.

[0020] The surface of the mediating layer is covalently bonded with a copolymer containing zwitterions, sulfonic acid groups and carboxyl side chains in a multi-point anchoring manner to construct an anti-coagulation coating capable of inhibiting intrinsic and exogenous coagulation.

[0021] The method for preparing the heparin-like ternary copolymer coating containing zwitterions, sulfonic acid groups and carboxyl side chains of the present invention comprises the following steps:

[0022] (1) mixing the mussel-like adhesive material dopamine A and the low molecular weight crosslinking agent polyethyleneimine B in a mass ratio of A:B = 10:3 to prepare an A+B aqueous solution, immersing the material / device to be modified in the aqueous solution to obtain a material / device with a surface-deposited mediating layer;

[0023] (2) The synthesized heparin-like terpolymer is mixed with the activating agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a ratio of copolymer:EDC:NHS = 5:15:10 to prepare a terpolymer carboxyl-activated solution. The material coated with the mediating layer is immersed in the activation solution and reacted at 50-60°C for 5-10 hours to form a heparin-like terpolymer layer.

[0024] Specifically, in step (2), the heparinoid terpolymer layer is prepared in a PBS buffer solution (pH 5.6) in which the carboxyl groups of the heparinoid terpolymer are activated by EDC and NHS.

[0025] Specifically, in step (2), heparin-like terpolymers with different unit ratios can be selected to prepare the coating according to the emphasis on blood compatibility and anticoagulant performance requirements.

[0026] To solve the fourth technical problem mentioned above, the present invention provides a method for coating a ternary copolymer of mussel-like adhesion mediation combined with heparin, because the mediation layer has the characteristics of mussel-like universal adhesion, and can be easily applied to the surface of biomedical materials and devices that are in direct contact with blood, such as hemodialyzers, oxygenators, extracorporeal circulation circuits, central venous catheters, etc., to obtain anticoagulation and enhance the blood compatibility of materials. In aqueous solution, the coating of anticoagulant coatings on the surfaces of various biomedical materials and devices can be achieved. On the surface of the firm and stable mediation layer formed by the universal adhesion of mussels, the multiple side chain carboxyl groups of the ternary copolymer are covalently bonded through surface amidation coupling, so that the multiple side chain carboxyl groups are anchored at multiple points to form an anticoagulant coating with good stability, which can meet the complex blood environment and medical devices facing various environments.

[0027] The present invention is particularly suitable for coating and modification treatment of the surface of hemodialyzer hollow fiber membranes (inner surface), blood oxygenator hollow fiber membranes (outer surface), and blood toxin adsorbents, which have strict requirements on coating thickness. The modified coating is ultra-thin (5-10 nanometers), has little effect on pore size and structure, and can simultaneously inhibit coagulation by both intrinsic and exogenous pathways.

[0028] The heparin-like ternary copolymer anticoagulant coating of the present invention can largely solve the problem that the anticoagulant ability of a single zwitterionic polymer is poor and cannot be used in biomedical devices that meet the anticoagulant environment alone, and provides an effective solution to the various side effects and adverse reactions that are easily caused by systemic anticoagulants. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings are intended to further illustrate the details and features of the present invention. They are important components of this application and do not constitute an undue limitation of the present invention. In the drawings:

[0030] Figure 1 SEM images of the surface morphology of PU substrates modified with a polydopamine (PDA)-mediated layer and P2, P4, and P6 coatings, as well as unmodified PU. The unmodified PU surface is clean and free of microstructures. However, some nanoscale particles appear on the modified surfaces, and the increase in the size or number of these particles with increasing coating thickness indicates that PDA and the three P(SSNa-SBMA-MSA) copolymers have been successfully bonded to the PU surface.

[0031] Figure 2 A graph showing the thrombin inactivation effect of PU substrates modified with P2, P4, P6, and unfractionated heparin (UFH) coatings, as well as unmodified PU samples, over a 40-minute period. As the sulfonic acid unit content in the terpolymer increases, the coating's thrombin inactivation rate and percentage improves compared to conventional heparin coatings.

[0032] Figure 3 Comparison of protein adsorption on the surfaces of PU / P2, PU / P4, PU / P6, PU / UFH and endpoint-bound heparin coating (PU / EPA-Hep) with that of unmodified PU sample.

[0033] Figure 4 .SEM photos of platelet adhesion on the surface of P2, P4, and P6 modified PU sheets and unmodified PU samples, as well as a comparison of platelet numbers.

[0034] Figure 5 .Comparison of SEM photos of thrombus formation after PU / P2, PU / P4, PU / P6, PU / UFH, PU / EPA-Hep and unmodified PU surfaces were exposed to human whole blood for 24 hours. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the technical solution of the present invention in conjunction with the accompanying drawings. The described embodiments are only part of the present invention and are used to better understand the technical solution of the present invention. They do not constitute the entire scope of implementation of the present invention, nor do they limit the scope of protection of the present invention. Any adjustments or improvements made based on the inventive concept of the present invention fall within the scope of protection of the present invention.

[0036] The heparinoid terpolymer of the present invention contains zwitterions, negative sulfonic acid charges, and carboxyl side chains and is synthesized by random copolymerization using ATRP in a saline solution. The heparinoid terpolymer has the following general formula (I):

[0037]

[0038] In the formula, R1, R2, and R3 are CH3 or H, R4 is a negatively charged side chain containing a sulfonic acid group, R5 is a zwitterionic group linked by an oxygen-carbon chain, and R6 is a carboxyl side chain group linked by an oxygen-carbon chain. The molar percentages of the sulfonic acid, zwitterionic, and carboxyl side chain units in the terpolymer are represented by x, y, and z, respectively, and range from 20% to 60%, 20% to 60%, and 10% to 30%, respectively. Heparin-like terpolymers with varying unit ratios can be designed to meet diverse application requirements. When high bioactive anticoagulant properties are required while also taking into account anti-biofouling properties, the x ratio should be ≥40%. When high blood compatibility is required, with a primary focus on bioinert anticoagulant properties while also maintaining some bioactive anticoagulant properties, the y ratio should be >40%.

[0039] In general formula (I), the zwitterionic groups include sulfobetaine (SB), phosphorylcholine (PC), and carboxybetaine (CB); the sulfonic acid side chains include sodium sulfonate, sodium p-phenylsulfonate, and amido-2-methylpropanesulfonic acid. Taking a copolymer P(SSNa-SBMA-SMA) of sodium styrenesulfonate (SSNa), methacrylic acid sulfobetaine (SBMA), and 2-methacryloyloxyethylsuccinic acid (MSA) as an example, heparin-like terpolymers with copolymer unit molar ratios x:y:z of 2:6:2, 4:4:2, and 6:2:2 in general formula (I) are abbreviated as P2, P4, and P6, respectively; and heparin-like terpolymers with x:y:z ratios of 4:3:3 and 4:5:1 are abbreviated as P433 and P451, respectively.

[0040] The present invention provides a highly efficient method for synthesizing a heparin-like terpolymer. The heparin-like terpolymer is synthesized by dissolving the monomers in saline according to the designed unit ratio, sequentially adding a water-soluble ATRP initiator, a catalyst, and a reducing agent, sealing the solution, and reacting the resulting mixture. The terpolymer is then purified and freeze-dried to obtain the heparin-like terpolymer. The addition of salt, such as phosphate buffer, to the monomer solution enhances the reactivity of the zwitterionic monomers. Compared to a synthesis system without salt, the yield of the heparin-like terpolymer is increased by 20-30%.

[0041] Compared to conventional free radical-initiated copolymerization methods in solution, this ATRP preparation method has a high feed concentration, a uniform copolymer molecular weight distribution, and a yield of up to 94%. Compared to conventional ATRP copolymerization methods, this ATRP preparation method is simpler, does not require a cumbersome deoxygenation step, and uses a copper ion catalyst concentration of only ppm.

[0042] The present invention's method for preparing a heparinoid terpolymer coating involves modifying a material / device with a polydopamine-mediated layer that mimics the universal adhesion of mussels. Then, a layer of the heparinoid terpolymer is covalently bonded to the amino-modified surface at multiple points using an amidation coupling technique, forming an ultrathin anticoagulant coating of 5 to 10 nanometers. This polydopamine-mediated, multi-point bonded ultrathin coating exhibits excellent stability and is suitable for modifying the surfaces of various materials and devices with anticoagulant coatings. It is particularly well-suited for anticoagulant modification of blood oxygenators and hemodialyzers, which require high membrane pore structure and performance, resulting in an anticoagulant coating that simultaneously inhibits both intrinsic and exogenous coagulation.

[0043] The process for preparing the heparin-like ternary copolymer coating containing zwitterions, sulfonic acid groups and carboxyl side chains of the present invention comprises the following steps:

[0044] Step 1: Prepare a mediating layer with surface reactive amino groups. Dopamine A, a biomimetic universal adhesive material, and polyethyleneimine B, a low molecular weight crosslinker, are mixed uniformly in a mass ratio of A:B = 10:3 to form an aqueous solution with a pH of 8-8.5. The material to be modified is immersed in this aqueous solution to obtain a material with a surface-deposited mediating layer.

[0045] The application of biomimetic universal adhesive material A in the mediating layer is to show excellent biomimetic adhesion properties through its oxidative polymerization reaction, providing surface active amino groups for various inert surfaces. The biomimetic adhesion molecules can respond to dissolved oxygen or added oxidants in aqueous solution, triggering oxidative polymerization reactions, thereby spontaneously forming a layer of secondary modified coating on the surface of the material. It is worth noting that due to the special properties of these adhesion molecules in aqueous solution, they are difficult to penetrate into the hydrophobic micropores on the surface of the material, and therefore will not adversely affect the original hydrophobicity and chemical inertness of the micropores. The low molecular weight crosslinker B is polyethyleneimine (M = 600g / mol), which can promote dopamine to form a cross-linked network structure and enhance the stability of the polydopamine mediating layer.

[0046] This mediating layer exhibits excellent adhesion properties and can be applied to a wide range of materials and surfaces, including but not limited to metal, glass, polyurethane, polysulfone polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyvinylidene fluoride, polycarbonate, and polyethersulfone. These materials, despite their diverse compositions and shapes, can all be effectively adhered to and modified using this mediating layer.

[0047] Step 2: Providing an anticoagulant coating with excellent blood compatibility. Heparinoid terpolymers of varying composition ratios are prepared with carboxyl activators 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a ratio of 5:15:10 for copolymer:EDC:NHS. The material coated with the mediating layer is immersed in the activation solution at 50-65°C for 5-10 hours to form a heparinoid terpolymer layer.

[0048] The reaction of covalently bonding heparinoid terpolymer (I) to the surface of the material was carried out in a PBS buffer solution with a copolymer concentration of 5 mg / mL and catalyzed by carboxyl activators EDC (15 mg / mL) and NHS (10 mg / mL). One liter of PBS buffer solution contained 0.2 g of KH2PO4, 2.1 g of Na2HPO4, 8.0-24.0 g of NaCl, and 0.2 g of KCl. The PBS solvent in the polymer solution was mixed with deionized water at a ratio of 0% to 100% and the reaction was carried out at 50-65°C to optimize the reaction conditions. It was found that a 75% concentration of the PBS buffer solution at 60°C resulted in better polymer anchoring, and a more bioinert surface interface with a higher graft density of the heparinoid terpolymer and a tightly bound hydration layer was more easily obtained.

[0049] To address the application challenges of the present invention's heparin-like terpolymer anticoagulant coating on various biomaterial device surfaces, the present invention utilizes polydopamine, which mimics the universal adhesion of mussels, to initially form a mediating layer on the surfaces of biomedical devices that come into direct contact with blood, such as hemodialyzers, oxygenators, extracorporeal circulation circuits, and central venous catheters. The polydopamine mediating layer provides covalently bondable surface amino groups, which react with the multiple activated side-chain carboxyl groups in the heparin-like copolymer molecules through an amidation coupling reaction to form an ultrathin (5-10 nanometer) coating. Because the polydopamine mediating layer is constructed in aqueous solution, the hydrophilic polydopamine component cannot enter the interior of the highly hydrophobic polymer material. Consequently, the subsequently constructed multi-point covalently bonded heparin-like copolymer cannot enter and clog the pores of hydrophobic polymer membranes (such as polypropylene and polymethylpentene). This makes it particularly suitable for modifying anticoagulant coatings on blood oxygenator and hemodialyzer membranes. This multi-point covalently bound heparin copolymer anticoagulant coating constructed on the surface of various biomedical materials and devices in aqueous solution has good stability, can meet the anticoagulation requirements of medical device applications in complex blood environments, and can simultaneously inhibit the coagulation cascade reactions caused by both intrinsic and exogenous pathways.

[0050] Example 1

[0051] Heparin-like terpolymers P(SBMA-SSNa-MSA) were synthesized by thermally initiated free radical copolymerization in conventional aqueous solution. The monomer ratios of SSNa, SBMA, and MSA and the polymerization conditions are shown in Table 1. The terpolymers were designated P2 (2:6:2), P4 (4:4:2), P6 (6:2:2), P433 (4:3:3), and P451 (4:5:1) according to the different molar ratios of SSNa:SBMA:MSA. A fixed amount of SSNa, SBMA, and MSA was dissolved in 100 mL of deionized H2O and nitrogen was introduced at 70°C for 30 minutes with stirring to remove oxygen from the reaction system. K2S2O8 was then added as an initiator at a concentration of 4% by weight of the total monomer mass, and the reaction was continued at 70°C with continuous stirring for 24 hours. After exposing the solution to air to cool and stop the polymerization, 3 / 5 of the water was evaporated by rotary evaporation, and then it was dialyzed with deionized water using a dialysis bag with a molecular weight cutoff of 3500 (MWCO 3500Da). The dialysate was changed every 4 to 6 hours, and the dialysis was continued for 4 days. The dialyzed polymer liquid was freeze-dried to obtain a white powdery solid, which was a ternary polymer P(SBMA-SSNa-MSA) containing sulfobetaine zwitterions, carboxyl groups, and anticoagulant group side chains. The freeze-dried P(SBMA-SSNa-MSA) was dissolved in D2O and measured. 1 H NMR spectrum. Calculations confirmed that the ratios of benzenesulfonic acid, sulfonic acid, and carboxylic acid units in the polymer were essentially consistent with the feed ratio. The yields of heparinoid terpolymers with varying composition ratios synthesized by thermally initiated free radical copolymerization in conventional aqueous solution were relatively low (Table 1), ranging from 59% to 68%.

[0052] Table 1. Ratio, synthesis conditions and yield of copolymer P(SBMA-SSNa-MSA) with different monomer feed ratios

[0053]

[0054] Example 2

[0055] Heparin-like terpolymer P(SBMA-SSNa-MSA) was synthesized by ATRP copolymerization in saline solution. P4 (4:4:2) was synthesized according to the molar ratio and concentration of SSNa, SBMA, and MSA as shown in Table 2. SSNa, SBMA, and MSA were dissolved in 45 mL of deionized H2O or PBS (3NaCl) in a three-necked flask. The desired amount of water-soluble ATRP initiator, HEBiB, was then added. After stirring to dissolve the mixture at 30°C, 5.0 mL of a PBS catalyst solution containing 0.072 mg / mL CuBr2 and 0.304 mg / mL bpy was added. N2 was bubbled through the three-necked flask for 30 minutes. 200 mg of ascorbic acid was added to the mixture, stirred to dissolve, and then heated to 50°C. The mixture was sealed and reacted for 12 hours. After completion of the reaction, the reaction solution was dialyzed against deionized water in a 3500 Da dialysis bag for 3 days and freeze-dried to obtain terpolymer P4. 1 H NMR characterization results indicate that the unit ratio of the heparin-like terpolymer P4 synthesized by ATRP copolymerization in saline solution is consistent with the feed ratio. A PBS (3NaCl) buffer solution with a sodium chloride concentration threefold was prepared by adding sodium chloride to the aforementioned PBS buffer to triple the NaCl concentration. The results in Table 2 show that when the total polymerizable monomer feed concentration is 120 mg / mL and the aqueous solution contains a PBS (3NaCl) buffer solution with a sodium chloride concentration threefold, the ATRP copolymer yield reaches 92%, a 20-30% increase over the yield of the conventional free radical copolymerization method shown in Table 1.

[0056] Table 2. Feed ratio, polymerization conditions and yield of the synthesis of ternary polymer P4 (4:4:2) with different concentrations

[0057]

[0058] Example 3

[0059] Step 1: Cut the PU substrate into samples of 1 cm×0.5 cm, immerse them in ethanol and deionized water for ultrasonic cleaning for 10 minutes, and then take them out and place them in deionized water for later use.

[0060] Step 2: Dissolve 200 mg of DA and 50 mg of PEI-600 in 100 mL of 1× PBS buffer (pH 8.3). Stir thoroughly and quickly place the cleaned PU sample into the solution. Incubate the mixture in a 30°C water bath shaker for 6 h. After the reaction, rinse three times with deionized water to obtain PU / PDA.

[0061] Step 3: EDC and NHS were added to a 5 mg / mL PBS solution of the heparinoid terpolymers (P2, P4, and P6) to a concentration of 16 mg / mL and 12 mg / mL, respectively. The pH of the PBS buffer was adjusted to 5.6. The PU / PDA samples were immersed in each of the three solutions and reacted in a 60°C shaker water bath for 12 hours. The samples were then rinsed three times alternately with deionized water and ethanol to obtain the PU / P2, PU / P4, and PU / P6 samples, respectively.

[0062] Step 4: The surface morphology of each sample was observed using a SEM (Hitachi SU8010, Japan). After drying each sample at room temperature, it was cut into 0.4 cm × 0.4 cm pieces. The sample to be tested was fixed with conductive adhesive and then gold-sprayed on the surface to ensure that it has excellent conductive properties. The SEM images of the surface morphology of each sample at different magnifications were observed and analyzed. The results are shown in Figure 1 .

[0063] Example 4

[0064] This example compares the thrombin inactivation effect of a heparin-like terpolymer coating sample with that of an unfractionated heparin coating (PU / UFH), demonstrating that the terpolymer coating has comparable anticoagulant properties to the heparin coating.

[0065] Step 1: Antithrombin (AT, 10 IU = 1.5 mg per vial, 58,000 MW): Dissolve one vial of AT in 33 mL of Hepes to make a 774 nM solution. Thrombin (Thrombin, 50 IU = 16.7 μg per vial, 37,000 MW): Dissolve three vials of Thrombin in 20 mL of Hepes to make a 67.5 nM solution. Thrombin chromogenic substrate S-2238 (8 mg per vial, 625.6 MW): Dissolve one vial of S-2238 in 10 mL of deionized water to make a 1.25 mM solution.

[0066] Step 2: EDC and NHS were added to a 5 mg / mL unfractionated heparin (UFH) solution in PBS to a concentration of 16 mg / mL and 12 mg / mL, respectively. The pH of the PBS buffer was adjusted to 5.6. The PU / PDA prepared in Example 3 was immersed in the heparin solution and reacted in a shaking water bath at 60°C for 12 h. The PU / PDA sample was then rinsed three times with deionized water and ethanol, alternating between rinses.

[0067] Step 3: The PU, PU / P2, PU / P4, and PU / P6 samples obtained in Example 3 were immersed in 37°C PBS buffer (pH 7.4) for 2 hours along with PU / UFH. They were then placed in 24-well plates, 1.0 mL of Hepes buffer was added, followed by 55 μL of AT, and the reaction was incubated at 37°C for 5 minutes. Finally, 300 μL of Thrombin solution was added and the timer was started. At a set series of time points, 100 μL of the mixed solution was taken out of each 96-well plate, 50 μL of Tris-EDTA buffer (pH 7.4) was added, and then 28 μL of the color developer S-2238 solution (1.25 mM) was added. The color reaction was terminated at 4 minutes using 50% glacial acetic acid solution. Immediately after addition, the absorbance at 405 nm was measured using a microplate reader. The absorbance values ​​of each sample solution were used to calculate and analyze the inactivation effects of PU, PU / P2, PU / P4, PU / P6, and PU / UFH samples on thrombin. Figure 2 It was found that the terpolymer coating had an anticoagulant property comparable to that of the heparin coating.

[0068] Example 5

[0069] To illustrate the advantages of the heparin-like terpolymer coating of the present invention in its anti-protein adsorption performance, the most representative adsorption amounts of serum albumin (BSA) and fibrinogen (Fg) were measured in comparison with those of ordinary heparin coating and endpoint-bound heparin (EPA-Hep) coating samples.

[0070] Step 1: Dissolve 1 g of unfractionated heparin (UFH) in 300 mL of distilled water at 0°C, add 10 mg of NaNO2, adjust the pH of the reaction system to 2.7 with 1 mol / L HCl, and stir in an ice bath at 0°C for 2 hours. Adjust the pH to 7.0 with 1 mol / L NaOH, and dialyze the resulting reaction mixture using a 1000 Da molecular weight cutoff dialysis bag. Concentrate and freeze-dry to obtain aldehyde-modified heparin (OHC-Hep).

[0071] Step 2: The PU / PDA sample prepared in Example 3 was immersed in an aqueous solution containing OHC-Hep (0.2 mg / ml), NaBH3CN (0.01 mg / ml), and NaCl (0.15 mol / L) and reacted at 50°C and pH 3.5 for two hours. The sample was then rinsed alternately with deionized water and ethanol three times each to obtain an endpoint-bound heparin-coated sample (PU / EPA-Hep).

[0072] Step 3: PU, PU / P2, PU / P4, PU / P6, PU / UFH, and PU / EPA-Hep samples of 1.0 cm x 0.5 cm were immersed in PBS buffer (pH 7.4) at 37°C for 2 hours. The equilibrated samples were immersed in 1 mg / mL BSA and 1 mg / mL Fg solutions and incubated for 2 hours. After incubation, the sample surface was gently rinsed three times with PBS buffer (pH 7.4) to remove weakly adsorbed proteins. After rinsing, the sample was placed in a 24-well plate and immersed in 600 μL 1 wt% sodium dodecyl sulfate (SDS) solution and shaken for 45 minutes to obtain a protein desorption solution. The amount of protein desorbed in the SDS solution was determined using the Bradford method. 100 μL of the desorption solution of different samples was pipetted into a 96-well plate, and 60 μL of G250 colorimetric solution was added to each well. The color development time was 5 min at room temperature. The absorbance of the protein desorption solution of different samples was measured at 595 nm using a microplate reader (Tecan Infinite 200, Switzerland). Three parallel samples were taken for each sample to determine the structure of the protein. Figure 3 shown. Figure 3 The comparison results show that the heparin-like ternary copolymer coating has significantly better anti-protein adsorption performance than PU / UFH and PU / EPA-Hep heparin coatings, is less likely to activate coagulation reactions, and has better blood compatibility.

[0073] Example 6

[0074] In step 1, 3.8% sodium citrate (w / v) was used as an anticoagulant, and human whole blood and the anticoagulant were mixed at a ratio of 90:10 vol%, and then centrifuged at 1500 rpm for 15 minutes to obtain platelet-rich plasma (PRP).

[0075] Step 2: The PU, PU / P2, PU / P4, and PU / P6 samples obtained in Example 3 were immersed in PBS buffer (1×, pH 7.4) at 37°C for equilibration for 2 hours. The equilibrated samples were then immersed in PRP and incubated together at 37°C for 2 hours. After incubation, the samples were gently rinsed three times with PBS buffer (1×, pH 7.4) to remove the PRP remaining on the surface of the material and the platelets that were not stably adhered to the surface of the sample. At 4°C, the samples were immersed in a 2.5wt% glutaraldehyde solution for 24 hours to fix the platelets on the surface of the sample. Finally, the samples were immersed in a series of ethanol-PBS mixed solutions (30, 50, 70, 80, 90, 95, 100%) to dehydrate the samples. After the samples were dried, the platelet adhesion was observed using SEM. All samples were tested three times, and the statistically significant differences between the samples were evaluated using one-way ANOVA analysis of variance. Platelet adhesion results and platelet counts on the surfaces of PU, PU / P2, PU / P4, and PU / P6 samples are shown in Figure 2. Figure 4 shown.

[0076] Example 7

[0077] In step 1, 3.8% sodium citrate (w / v) was used as the anticoagulant. Human whole blood and the anticoagulant were mixed at a ratio of 90:10 vol%. During use, 54 μL of 3% CaCl2 (w / v) was added to each mL of the blood-anticoagulant mixture to balance the anticoagulant effect of the 75% equivalent sodium citrate.

[0078] In step 2, the PU, PU / P2, PU / P4, PU / P6, PU / UFH, and PU / EPA-Hep samples obtained in Examples 3, 4, and 5 were immersed in PBS buffer (pH 7.4) at 37°C for 2 hours. The samples were then immersed in human whole blood at 37°C for 24 hours. After immersion, the samples were gently rinsed three times with PBS buffer (pH 7.4) to remove any loosely adhered thrombi. At 4°C, the samples were immersed in a 2.5 wt% glutaraldehyde solution to crosslink and fix the thrombi on the sample surfaces. Finally, the samples were sequentially immersed in a series of ethanol-PBS mixtures with varying ethanol concentrations (30, 50, 70, 80, 90, 95, and 100%) to dehydrate the samples. After drying, the whole blood adhesion was observed using an upright microscope (Nikon Eclipse Ti-U, Japan) and a scanning electron microscope (SEM). All samples were tested three times, and the statistically significant differences between the samples were evaluated using the one-way ANOVA method. Figure 5 shown. Figure 5The comparison results show that the anti-whole blood thrombosis performance of the heparin-like terpolymer coating is significantly better than that of the PU / UFH and PU / EPA-Hep heparin coatings.

Claims

1. A heparin-like ternary copolymer containing zwitterions, sulfonic acid groups and carboxyl side chains, characterized in that: Has the following structure: In formula (I), R1, R2 and R3 are -CH3 or -H, R4 is a group containing a sulfonic acid group, R5 is a zwitterionic group connected by an oxygen-carbon chain, and R6 is a terminal carboxyl side chain group connected by an oxygen-carbon chain; x, y, and z are the molar percentages of the three units, x = 20% to 60%, y = 20% to 60%, and z = 10% to 30%; The zwitterionic groups include groups containing sulfobetaine, phosphorylcholine or carboxybetaine structures.

2. The copolymer according to claim 1, wherein The copolymer is synthesized from sodium styrene sulfonate, methacrylic acid sulfobetaine and 2-methacryloyloxyethyl succinic acid, and the molar ratio of the sodium styrene sulfonate, methacrylic acid sulfobetaine and 2-methacryloyloxyethyl succinic acid corresponds to the unit molar ratio x:y:z of the copolymer.

3. The method for synthesizing the copolymer according to claim 1 or 2, characterized in that: The following steps are involved: The first monomer, the second monomer, and the third monomer are dissolved in a saline solvent according to the ratio of x, y, and z, and mixed evenly, and a water-soluble ATRP initiator, a catalyst, and a reducing agent are added to carry out an atom transfer radical polymerization reaction to obtain the copolymer; The first monomer is a reactive monomer containing a sulfonic acid group and an alkenyl group in its structure; The second monomer is a reactive monomer containing zwitterionic groups and alkenyl groups connected by oxygen-carbon chains in its structure; The third monomer is a reactive monomer containing a terminal carboxyl side chain group and an olefinic group connected by an oxygen-carbon chain in its structure; The zwitterionic groups include groups containing sulfobetaine, phosphorylcholine or carboxybetaine structures.

4. The method according to claim 3, characterized in that The first monomer is sodium styrene sulfonate, the second monomer is methacrylic acid sulfobetaine, and the third monomer is 2-methacryloyloxyethyl succinic acid; the salt in the saline solvent is one or more of sodium chloride, potassium chloride, sulfate, and phosphate; the saline solvent is a buffer solution obtained by adding sodium chloride to a PBS buffer solution to triple the sodium chloride concentration; the initiator is 2-hydroxyethyl 2-bromo-2-methylpropionate; the catalyst is copper bromide and bipyridine; and the reducing agent is ascorbic acid.

5. A method for regulating the properties of a copolymer obtained by the method according to claim 3 or 4, characterized in that: The steps include: When the blood compatibility of the copolymer needs to be improved, the y value ratio of the second monomer is increased; When the anticoagulant ability of the copolymer needs to be improved, the x value ratio of the first monomer is increased; When it is necessary to reduce the adhesion of the copolymer to blood components, the x value ratio of the first monomer is reduced; When it is necessary to ensure the bioactive anticoagulant properties of the copolymer and take into account the anti-biofouling properties, the x value of the first monomer should account for ≥40%; When the blood compatibility of the copolymer needs to be ensured, and the copolymer is mainly bioinert anticoagulant and also has some bioactive anticoagulant properties, the y value of the second monomer should account for more than 40%.

6. A heparin-like terpolymer coating comprising a mediating layer and an anticoagulant layer, characterized in that: The main material of the mediating layer is a mussel-like adhesive material; The main material of the anti-coagulation layer is the copolymer material according to claim 1 or 2.

7. The method for preparing the copolymer coating according to claim 6, characterized in that: The following steps are involved: constructing a mussel-like adhesion material mediating layer on the substrate surface; Through surface amidation coupling covalent bonding, multiple side chain carboxyl groups of the copolymer according to claim 1 or 2 are anchored at multiple points on the surface of the mediating layer to form a stable anti-coagulation layer.

8. The method according to claim 7, wherein The following steps are involved: Dopamine and polyethyleneimine are mixed evenly to prepare an aqueous solution, and the substrate material to be modified is immersed in the aqueous solution to construct a mussel-mimicking adhesion material mediating layer; The copolymer material according to claim 1 or 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are uniformly mixed in a PBS buffer solution to obtain an activation solution; the material with a mussel-mimicking adhesion material mediating layer constructed on the surface is immersed in the activation solution, and the mixture is reacted at 50-60° C. for 5-10 hours to form a heparin-like terpolymer anti-coagulation layer.

9. The method according to claim 8, wherein The mass ratio of dopamine to polyethyleneimine is 10:

3. After preparing the aqueous solution, the pH value is adjusted to 8-8.

5. The PBS buffer solution contains 0.2g of KH2PO4, 2.1g of Na2HPO4, 8.0-24.0g of NaCl and 0.2g of KCl per 1L of the PBS buffer solution. The PBS buffer solution can be used alone or mixed with deionized water. In the activation solution, the concentration of the copolymer material according to claim 1 or 2 is 5 mg / mL; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 15 mg / mL; and the concentration of N-hydroxysuccinimide is 10 mg / mL. The substrate is made of metal, glass, polyurethane, polysulfone polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyvinylidene fluoride, polycarbonate and / or polyethersulfone.

10. Use of the coating according to claim 6 in preparing an anti-coagulation surface of a biomedical material or device and / or improving the blood compatibility of the biomedical material or device.

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