Anticoagulant biomaterials for blood purification and methods of making and using the same

Abstract

The application discloses an anticoagulant biomaterial for blood purification and a preparation method and application thereof, and comprises the following steps: providing an amphiphilic copolymer P(AA-MMA); blending polyethersulfone, P(AA-MMA) and a solvent, stirring at a set temperature to obtain a casting solution, and preparing a blending membrane from the casting solution; soaking the blending membrane in a 2-morpholine-4-yl ethanesulfonic acid buffer solution containing argatroban, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and hydroxy succinimide, and reacting at room temperature to obtain an argatroban grafted blending membrane, namely the anticoagulant biomaterial. The method improves the hydrophilicity of the membrane, reduces the albumin adsorption of the membrane, reduces the adhesion of platelets on the membrane, prolongs the coagulation time APTT, PT and TT of the membrane, and is better in biocompatibility and enhanced in anticoagulation performance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to an anticoagulant biomaterial for blood purification, its preparation method, and its application. Background Technology

[0002] Chronic kidney disease has a high incidence rate, and some patients will progress to end-stage renal disease. Hemodialysis is the main treatment for end-stage renal disease. Currently, the commonly used blood purification membrane materials are polysulfone and polyethersulfone. However, polymer materials are hydrophobic and have poor biocompatibility, often leading to coagulation and other phenomena in clinical use. To reduce the risk of coagulation, heparin or low molecular weight heparin is routinely used for anticoagulation, but this also carries the risk of bleeding, and some patients have contraindications to anticoagulation, making blood purification therapy difficult to implement.

[0003] To address these issues, researchers have grafted or coated anticoagulant drugs onto the surface of polyethersulfone membranes. For example, they have used a polydopamine coating method to introduce the anticoagulant argatroban onto the polyethersulfone membrane surface. However, the biocompatibility and anticoagulant performance of existing technologies still need improvement. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide an anticoagulant biomaterial for blood purification, its preparation method and application, so as to improve biocompatibility and anticoagulant performance.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] A method for preparing an anticoagulant biomaterial for blood purification includes the following steps:

[0007] (1) Provide an amphiphilic copolymer P(AA-MMA);

[0008] (2) Polyethersulfone, P(AA-MMA) and solvent are mixed and stirred at a set temperature to obtain casting solution, and the casting solution is used to make a blended membrane;

[0009] (3) The blend membrane obtained in step (2) is immersed in a 2-morpholin-4-ylethanesulfonic acid buffer solution containing argatroban, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide, and reacted at room temperature to obtain an argatroban-grafted blend membrane, namely the anticoagulant biomaterial.

[0010] As a further improvement, the solvent in step (2) is one of dimethylacetamide, DMSO or DMF.

[0011] As a further improvement, in step (2), polyethersulfone accounts for 14-15% of the casting solution mass, P(AA-MMA) accounts for 0.5-2% of the casting solution mass, and solvent accounts for 83-85.5% of the casting solution mass.

[0012] As a further improvement, the stirring in step (2) is carried out at a temperature of 60-70°C for 12-24 hours.

[0013] As a further improvement, step (2) uses the immersion precipitation phase inversion method to prepare the membrane.

[0014] As a further improvement, in step (3), the concentration of argatroban in the buffer solution is 0.8–1.2 mg / mL.

[0015] As a further improvement, the concentrations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide in step (3) are both 1-2 mg / mL.

[0016] As a further improvement, the reaction conditions for step (3) are 20-30°C for 20-24 hours.

[0017] The present invention also provides an anticoagulant biomaterial for blood purification prepared by the method described above.

[0018] The present invention also provides the application of the aforementioned anticoagulant biomaterial in the preparation of blood purification membranes.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention uses a blending method to blend the amphiphilic copolymer P(AA-MMA) with polyethersulfone to form a film, and then uses a surface grafting technique to graft the anticoagulant drug argatroban onto the surface of the blended film to exert an anticoagulant effect.

[0021] The introduction of the amphiphilic copolymer P(AA-MMA) allows the hydrophobic MMA segment to exhibit good compatibility with polyethersulfone (PES). Through hydrophobic interactions, MMA anchors to PES, making it less susceptible to elution. The acrylic AA segment, when blended with the PES membrane, enhances the membrane's hydrophilicity, forming a hydration layer on the blended membrane surface, reducing protein adsorption, and improving biocompatibility. Acrylic acid not only enhances the membrane's hydrophilicity but, more importantly, can also connect with the amino groups on argatroban, thus better utilizing its anticoagulant properties.

[0022] The preparation method using blending is simple and has fewer steps compared to coating. After blending and forming a film, the grafted drug is more uniform, which is more conducive to the drug exerting its anticoagulant effect.

[0023] The method of this invention improves the hydrophilicity of the membrane, reduces albumin adsorption, decreases platelet adhesion to the membrane, maintains better platelet morphology, and extends the membrane's clotting times (APTT, PT, TT). Therefore, it exhibits better biocompatibility and enhanced anticoagulant properties.

[0024] The raw materials used in this invention are inexpensive, safe and non-toxic, and the experimental methods are mild, the process is simple, and it is easy to industrialize. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The infrared spectrum of the argatroban-grafted blend membrane in Example 1;

[0027] Figure 2 This is a SEM cross-sectional view of the argatroban-grafted blend membrane from Example 1;

[0028] Figure 3 The following is a comparison diagram of the water contact angle of the membranes: (a) ordinary polyethersulfone membrane, (b) blend membrane of Comparative Example 2, and (c) blend membrane grafted with argatroban in Example 1.

[0029] Figure 4 A comparison chart of albumin adsorption amounts on the membrane;

[0030] Figure 5 The image shows the number and morphology of platelets adsorbed on a common polyethersulfone membrane.

[0031] Figure 6 Platelet adsorption quantity and morphology of argatroban-grafted blend membrane;

[0032] Figure 7 A comparison chart of APTT (application time to clotting time) of the membrane;

[0033] Figure 8 A comparison chart of PT (prothrombin time) for membrane clotting;

[0034] Figure 9 This is a comparison chart of membrane clotting time (TT). Detailed Implementation

[0035] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0037] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0038] The anticoagulant biomaterial for blood purification according to some specific embodiments of the present invention is prepared by the following steps:

[0039] 1. Provides amphiphilic copolymer P(AA-MMA)

[0040] In some specific embodiments, the amphiphilic copolymer P(AA-MMA) is copolymerized from acrylic acid and methyl methacrylate.

[0041] In some specific embodiments, acrylic acid and methyl methacrylate are added in a molar ratio of 1:(1-4), along with 2-(dodecyltrithiocarbonate)-2-methylpropionic acid and azobisisobutyronitrile (AIBN). The mixture is reacted at 75-85 degrees Celsius for 4-6 hours under nitrogen protection. The resulting product is precipitated with ethyl acetate, filtered, washed, and dried to obtain P(AA-MMA).

[0042] The copolymer is formed by copolymerizing acrylic acid and methyl methacrylate. The raw materials are widely used, easy to obtain and inexpensive. The synthesized amphiphilic copolymer is safe and non-toxic, and is suitable for membrane modification.

[0043] 2. Film preparation by blending amphiphilic copolymer P(AA-MMA) with polyethersulfone

[0044] Polyethersulfone, amphiphilic copolymer P(AA-MMA) and solvent are blended and stirred at a certain temperature for a period of time to obtain a casting solution. The casting solution is then used to prepare a film by immersion precipitation phase inversion method to obtain a blended film.

[0045] In some specific embodiments, the solvent is one of dimethylacetamide (DMAc), DMSO, or DMF. In the casting solution, polyethersulfone accounts for 14-15% of the casting solution mass, the amphiphilic copolymer P(AA-MMA) accounts for 0.5-2% of the casting solution mass, and the solvent DMAc accounts for 83-85.5% of the casting solution mass. If the proportion of P(AA-MMA) is too small, the amount of grafted drug will be too low, and the hydrophilicity of the membrane will be insufficient; since the miscibility between P(AA-MMA) and polyethersulfone is limited, if the proportion is too large, they will be difficult to miscible, resulting in poor film formation.

[0046] In some specific embodiments, the stirring is carried out at 60–70°C for 12–24 hours. Stirring at this temperature ensures a more uniform distribution of P(AA-MMA) in the blend membrane, more even grafting of the drug, and the best anticoagulant effect.

[0047] In some specific embodiments, the immersion precipitation phase inversion method involves drawing the casting solution onto a glass plate, using a tool to scrape a liquid membrane to create a liquid membrane, allowing the liquid membrane to remain in air, and then placing it in a water coagulation bath to obtain a blended membrane. In some specific embodiments, the liquid membrane is created by scraping a layer of 100-200 micrometers thick using a doctor blade or coating rod. In some specific embodiments, after allowing the liquid membrane to remain in air for 10-15 seconds, the membrane is completely immersed in pure water. This 10-15-second interval allows a dense separation layer to form on the upper layer of the membrane.

[0048] 3. Grafting argatroban onto the blended membrane

[0049] The blend membrane obtained above was immersed in a 2-morpholino-4-ylethanesulfonic acid (MES) buffer solution containing argatroban, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxysuccinimide (NHS), and reacted at room temperature to obtain an argatroban-grafted blend membrane.

[0050] EDC and NHS are catalysts for the amide reaction, while the MES buffer acts as a buffer.

[0051] In some specific embodiments, the concentration of argatroban in the buffer solution is 0.8–1.2 mg / mL, and the concentrations of EDC and NHS are both 1–2 mg / mL. Within these concentration ranges, argatroban can be grafted more effectively, resulting in optimal anticoagulation.

[0052] In some specific embodiments, the reaction conditions are 20-30°C for 20-24 hours, under which argatroban can be successfully grafted onto the surface of the blend membrane.

[0053] The hydrophilic acrylic acid segment in the amphiphilic copolymer P(AA-MMA) provides carboxyl groups that can be linked to argatroban, allowing argatroban to be bonded to the acrylic acid on the blend membrane via amide bonds. This strong bond ensures a secure connection that is difficult to wash off. Argatroban grafted onto the membrane surface enhances its anticoagulant properties. Linking argatroban to the carboxyl groups of acrylic acid does not affect the carboxyl groups on argatroban, thus preserving its anticoagulant activity. If other modification methods are used, linking the anticoagulant to the carboxyl groups of the anticoagulant via amino groups, the anticoagulant activity will be affected, resulting in inferior anticoagulant performance compared to this invention.

[0054] Example 1

[0055] The preparation method of the coagulation biomaterial in this embodiment includes the following steps:

[0056] (1) Preparation of amphiphilic copolymer P(AA-MMA): Acrylic acid and methyl methacrylate in a molar ratio of 1:1, 18g of acrylic acid, 25g of methyl methacrylate, 0.5g of 2-(dodecyltrithiocarbonate)-2-methylpropionic acid, and azobisisobutyronitrile (AIBN) were weighed and added to a three-necked flask. Nitrogen gas was purged for 30 minutes to remove gas. The reaction was carried out at 80 degrees Celsius for 5 hours under nitrogen protection. The product was precipitated with ethyl acetate, and then filtered, washed and dried to obtain the amphiphilic copolymer poly(acrylic acid-methyl methacrylate).

[0057] (2) Preparation of the blend membrane: Preparation of casting solution: Dissolve the above-obtained amphiphilic copolymer P(AA-MMA) and polyethersulfone in DMAc, wherein the weight ratio of polyethersulfone, P(AA-MMA) and DMAc is 14:2:84. Stir thoroughly at 65°C for 24 hours, degas under vacuum for 10 minutes, pour the casting solution onto a clean glass plate, and slowly and uniformly scrape out a liquid membrane using a 100μm scraper. After holding the membrane in the air for 10-15 seconds, immerse the membrane completely in a coagulation bath of pure water to obtain the blend membrane. After washing with pure water, dry and store.

[0058] (3) Grafting Argatroban onto Blended Membrane: Prepare a MES buffer containing 1.5 mg / mL EDC and NHS. Dissolve argatroban in DMSO first, and then dissolve it in the above MES buffer to make the final concentration of argatroban in the MES buffer 1 mg / mL. Immerse the blended membrane prepared in step (2) in the MES buffer containing 1 mg / mL argatroban (containing 1.5 mg / mL EDC and NHS), and shake and react for 24 hours at 25°C to obtain the argatroban-grafted blended membrane. Clean the membrane with pure water and then dry and store it.

[0059] The infrared spectrum of the argatroban-grafted blend film in this embodiment is as follows: Figure 1 This indicates that argatroban was successfully linked to the blend membrane via the carboxyl group of P(AA-MMA). The SEM cross-sectional image looks like... Figure 2 The membrane cross-section exhibits an asymmetrical three-layer finger structure, with a dense separation layer on top, a finger structure in the middle, and a loose support layer on the bottom.

[0060] Example 2

[0061] The preparation method of the coagulation biomaterial in this embodiment includes the following steps:

[0062] (1) Same as Example 1;

[0063] (2) Preparation of the blend membrane: Preparation of casting solution: Dissolve the above-obtained amphiphilic copolymer P(AA-MMA) and polyethersulfone in DMAc, wherein the weight ratio of polyethersulfone, P(AA-MMA) and DMAc is 15:1:84. Stir thoroughly at 65°C for 24 hours, degas under vacuum for 10 minutes, pour the casting solution onto a clean glass plate, and slowly and uniformly scrape out a layer of liquid membrane using a 100μm film scraper. After standing in the air for 10-15 seconds, completely immerse the membrane in a coagulation bath of pure water to obtain the blend membrane. After washing with pure water, dry and store.

[0064] (3) Grafting Argatroban onto Blended Membrane: Prepare a MES buffer containing 1.5 mg / mL EDC and NHS. Dissolve argatroban in DMSO first, and then dissolve it in the above MES buffer to make the final concentration of argatroban in the MES buffer 0.8 mg / mL. Immerse the blended membrane prepared in step (2) in the MES buffer containing 0.8 mg / mL argatroban (containing 1.5 mg / mL EDC and NHS), and shake and react at 25°C for 24 hours to obtain the argatroban-grafted blended membrane. Clean the membrane with pure water and then dry and store it.

[0065] Comparative Example 1

[0066] Ordinary polyethersulfone film.

[0067] Comparative Example 2

[0068] The blend membrane without argatroban grafting was prepared using steps (1) and (2) of Example 1.

[0069] Performance testing

[0070] 1. Measurement of water contact angle

[0071] The water contact angles of the membranes prepared in Example 1 and Comparative Examples 1-2 are as follows: Figure 3 Compared to ordinary polyethersulfone membranes, blended membranes have a lower water contact angle, and argatroban-grafted blended membranes have a further lower contact angle, indicating improved hydrophilicity.

[0072] 2. Bovine serum albumin adsorption experiment

[0073] Experimental procedure: Cut the membrane into 1*4cm pieces 2 The small pieces were first soaked in PBS for 60 minutes, and then the membrane was placed in 1 mg / mL BSA solution and shaken at 37°C for 24 hours. The BSA concentration in the solution before and after adsorption was determined by BCA method, and the amount of protein adsorbed per unit area of ​​membrane was calculated.

[0074] The albumin adsorption capacity of the membranes prepared in Example 1 and Comparative Examples 1-2 is as follows: Figure 4The amount of albumin adsorbed by the argatroban-grafted blend membrane decreased to 33.7 ug / cm. 2 This indicates that biocompatibility has been improved.

[0075] 3. Platelet adsorption experiment

[0076] Experimental procedure: Cut the membrane into 1*1cm pieces 2 Small pieces of platelets were first soaked in PBS for 60 minutes, then the membrane was placed in platelet-rich plasma and shaken for 2 hours for adsorption. The membrane was washed with PBS, and then soaked in 2.5% glutaraldehyde for 24 hours to fix the adsorbed platelets. The membrane was then dehydrated sequentially with 25%, 50%, 75% ethanol, and anhydrous ethanol for 10 minutes each. The morphology of the platelets adhering to the membrane was observed using scanning electron microscopy.

[0077] The platelet adsorption quantity and morphology of the membranes prepared in Example 1 and Comparative Example 1 are as follows: Figure 5 and Figure 6 On ordinary polyethersulfone membranes, a large number of platelets adhered, and the platelets aggregated to form microthrombi. The platelets extended long pseudopodia, and the platelets were severely deformed. On argatroban-grafted blend membranes, fewer platelets adhered, and their morphology was well maintained. No morphological changes or pseudopodia formation occurred, indicating improved biocompatibility.

[0078] 4. Measurement of clotting time

[0079] Measurement procedure: Cut the membrane into 0.5*0.5cm pieces. 2 The small pieces were first soaked in PBS for 60 minutes, and then the membrane was placed in 1200 μL of anemic platelet plasma and shaken for 30 minutes to adsorb. The membrane was then removed, and the plasma coagulation time (APTT, PT, TT) was measured using a fully automated coagulation analyzer.

[0080] The APTT, PT, and TT clotting times of the membranes prepared in Example 1 and Comparative Examples 1-2 were compared. Figures 7-9 This indicates that after grafting argatroban, the coagulation times APTT, PT, and TT of the blended membrane were all prolonged, and its anticoagulant performance was significantly improved.

[0081] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for preparing an anticoagulant biomaterial for blood purification, characterized in that, Includes the following steps: (1) Provide an amphiphilic copolymer P(AA-MMA) formed by copolymerization of acrylic acid and methyl methacrylate; (2) Polyethersulfone, P(AA-MMA) and solvent are mixed and stirred at a set temperature to obtain casting solution, and the casting solution is used to make a blended membrane; (3) The blend membrane obtained in step (2) is immersed in a 2-morpholin-4-ylethanesulfonic acid buffer solution containing argatroban, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide, and reacted at room temperature to obtain an argatroban-grafted blend membrane, namely the anticoagulant biomaterial.

2. The method for preparing anticoagulant biomaterials for blood purification according to claim 1, characterized in that, The solvent in step (2) is one of dimethylacetamide, DMSO or DMF.

3. The method for preparing anticoagulant biomaterials for blood purification according to claim 1, characterized in that, In step (2), polyethersulfone accounts for 14-15% of the casting solution mass, P(AA-MMA) accounts for 0.5-2% of the casting solution mass, and solvent accounts for 83-85.5% of the casting solution mass.

4. The method for preparing anticoagulant biomaterials for blood purification according to claim 1 or 3, characterized in that, The stirring in step (2) is carried out at a temperature of 60~70℃ for 12~24 hours.

5. The method for preparing anticoagulant biomaterials for blood purification according to claim 1, characterized in that, Step (2) The membrane is prepared by immersion precipitation phase inversion method.

6. The method for preparing anticoagulant biomaterials for blood purification according to claim 1, characterized in that, Step (3) The concentration of argatroban in the buffer solution is 0.8~1.2 mg / mL.

7. The method for preparing anticoagulant biomaterials for blood purification according to claim 1 or 6, characterized in that, In step (3), the concentrations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide are both 1~2 mg / mL.

8. The method for preparing anticoagulant biomaterials for blood purification according to claim 1 or 6, characterized in that, The reaction conditions for step (3) are 20~30℃ for 20~24 hours.

9. An anticoagulant biomaterial for blood purification prepared by the method according to any one of claims 1 to 8.

10. The use of the anticoagulant biomaterial of claim 9 in the preparation of a blood purification membrane.

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