Anticoagulation modification method of hemodialyzer

Abstract

The invention relates to the technical field of hemodialysis, in particular to an anticoagulation modification method of a hemodialyzer, which comprises the following steps: preparing heparinoid, putting the heparinoid into a buffer solution, adding absolute ethyl alcohol and dicyclohexylcarbodiimide, uniformly stirring, adding 3-aminopropyltriethoxysilane and N, N-dimethylacetamide, heating and stirring for reaction, adding a catalyst, stirring for reaction, adding a catalyst, and stirring for reaction to obtain the anticoagulation modified hemodialyzer. Cooling, adding lysine, and continuously stirring to react, so as to obtain an anticoagulant modified solution; and injecting the anticoagulation modification liquid into a dialysis membrane pipeline of the hemodialyzer, and after reaction modification is finished, cleaning and drying the dialysis membrane pipeline to obtain the hemodialyzer with the anticoagulation function. The heparinoid polymer with a sulfonic acid group and a carboxylic acid group is synthesized, and the heparinoid polymer and lysine are fixed in the dialysis membrane through amidation reaction of the heparinoid polymer with lysine and 3-aminopropyltriethoxysilane and self-polycondensation of a reaction product. Therefore, the dialysis membrane has good anticoagulation, thrombus solubility and use stability.

Description

Technical Field

[0001] The invention relates to the technical field of hemodialysis, in particular to an anticoagulation modification method for a hemodialyzer. Background Art

[0002] During hemodialysis, blood inevitably comes into contact with the dialysis membrane. The contact between the dialysis membrane and blood will activate a series of interrelated processes to promote coagulation reactions, including protein adsorption, platelet and red blood cell adhesion, and thrombin generation. Therefore, heparin needs to be added during hemodialysis to reduce the occurrence of coagulation reactions. With the in-depth study of the anticoagulation mechanism of heparin, people have found that the anticoagulation function of heparin is achieved thanks to the presence of two functional groups, carboxylic acid and sulfonic acid. Studies have confirmed that macromolecules containing carboxylic acid or sulfonic acid groups have anticoagulation functions, and such macromolecules are called heparinoids.

[0003] However, how to achieve stable loading of heparinoids on the inner surface of hemodialysis membrane, especially for the anticoagulant modification of encapsulated hemodialyzers; and although hemodialysis technology is constantly improving, it is still impossible to completely avoid the formation of thrombi on the inner wall of hemodialysis membranes. Based on this, a method for anticoagulant modification of hemodialyzers is proposed. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an anticoagulant modification method for a hemodialyzer, which fixes heparinoids and lysine in a dialysis membrane, thereby making the membrane have good anticoagulant properties, thrombus solubility and stability in use.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for anticoagulation modification of a hemodialyzer, comprising the following steps:

[0006] (1) Preparation of heparinoids using lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid;

[0007] (2) placing heparinoid in a buffer solution, adding anhydrous ethanol and dicyclohexylcarbodiimide, stirring evenly, adding 3-aminopropyltriethoxysilane and N,N-dimethylacetamide, first heating and stirring to react, then cooling, adding lysine and continuing to stir to react, to obtain an anticoagulant modified solution;

[0008] (3) Injecting the anticoagulant modified liquid into the dialysis membrane pipeline of the hemodialyzer, and after the reaction modification is completed, the dialysis membrane pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0009] Preferably, in step (1), the preparation method of the heparinoid is as follows: S1, dissolving lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid in N,N-dimethylformamide, and stirring for 50-60 minutes under a nitrogen atmosphere; S2, adding azobisisobutyronitrile, and continuing to stir at 75-80°C for 20-22 hours; after terminating the reaction, rotary evaporation and grinding are performed to obtain the heparinoid.

[0010] Preferably, in step S1, the mass ratio of lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid is (4-5):(3-4):(1-2); the material-liquid ratio of lauryl methacrylate to N,N-dimethylformamide is 1:(10-12) g / mL.

[0011] Preferably, in step S2, the amount of azobisisobutyronitrile added is 0.5-1% of the total mass of lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid.

[0012] Preferably, in step (2), the buffer solution is 0.06 mol / L 3-morpholinepropanesulfonic acid; and the volume ratio of anhydrous ethanol to the buffer solution is 1:10.

[0013] Preferably, in step (2), the mass ratio of dicyclohexylcarbodiimide, 3-aminopropyltriethoxysilane and heparin is (6-7): (4-5): (25-30).

[0014] Preferably, in step (2), the solid-liquid ratio of the heparinoid to the buffer is 1:(50-60) g / mL; and the volume ratio of N,N-dimethylacetamide to the buffer is 1:(100-110).

[0015] Preferably, in step (2), the temperature is first raised to 65-70° C. and stirred for reaction for 25-35 min, then the temperature is lowered to 35-40° C. and lysine is added and stirred for reaction for 20-30 min.

[0016] Preferably, in step (3), the anticoagulant modification liquid is injected into the dialysis membrane pipeline and retained for 4-6 hours for reaction modification treatment; or, the anticoagulant modification liquid is circulated through the dialysis membrane pipeline for 4-6 hours for reaction modification treatment.

[0017] Preferably, in step (3), the dialysis membrane is selected from polysulfone membrane, polyamide membrane and polylactic acid membrane.

[0018] The present invention provides an anticoagulation modification method for a hemodialyzer, which has the following beneficial effects compared with the prior art:

[0019] The invention uses lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid as basic raw materials to synthesize a heparinoid polymer with a sulfonic acid group and a carboxylic acid group, and fixes the heparinoid polymer and lysine on the inner wall of a dialysis membrane through an amidation reaction with lysine and 3-aminopropyltriethoxysilane, that is, the amino groups of lysine and 3-aminopropyltriethoxysilane react with the carboxyl groups of the heparinoid polymer, and the self-condensation of the reaction product, so that the dialysis membrane has good anticoagulant property, thrombus solubility and stability in use.

[0020] The present invention allows the anticoagulant modified liquid to circulate in the dialysis membrane pipeline, which is beneficial for the anticoagulant modified substance to be uniformly and fully and sufficiently loaded in the dialysis membrane, thereby improving the overall performance of the dialysis membrane.

[0021] In the amidation reaction process of the present invention, 3-aminopropyltriethoxysilane and the heparin-like polymer are first allowed to fully react at a relatively high temperature, and then the temperature is lowered to add lysine to continue the reaction. This is because a relatively low temperature can ensure that the properties of lysine are not affected, thereby ensuring the performance of the anticoagulant modified dialysis membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0023] Figure 1 Graph showing the relationship between the shaking treatment time of the dialysis membrane in physiological saline and the amount of thrombus adhesion in Example 6 of the present invention. DETAILED DESCRIPTION

[0024] The following examples are used to explain the implementation methods of the present application in detail, so that the implementation process of how the present application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0025] Example 1

[0026] The preparation method of heparinoids is as follows:

[0027] S1. Dissolve lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid in N,N-dimethylformamide and stir for 60 min under nitrogen atmosphere;

[0028] The mass ratio of the lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid is 4:3:1. The material-liquid ratio of lauryl methacrylate to N,N-dimethylformamide is 1:10 g / mL.

[0029] S2. Then, add azobisisobutyronitrile and continue stirring at 80° C. for 20 h. After terminating the reaction, perform rotary evaporation and grind to obtain heparin-like substance.

[0030] The amount of azobisisobutyronitrile added is 0.5% of the total mass of lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid.

[0031] Example 2

[0032] The preparation method of heparinoids is as follows:

[0033] S1. Dissolve lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid in N,N-dimethylformamide and stir for 50 min under nitrogen atmosphere;

[0034] The mass ratio of the above-mentioned lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid is 5:4:2. Among them, the material-liquid ratio of lauryl methacrylate to N,N-dimethylformamide is 1:12 g / mL.

[0035] S2. Then, add azobisisobutyronitrile and continue stirring at 75° C. for 22 h. After terminating the reaction, perform rotary evaporation and grind to obtain heparinoid.

[0036] The amount of azobisisobutyronitrile added is 1% of the total mass of lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid.

[0037] Example 3

[0038] A method for anticoagulation modification of a hemodialyzer comprises the following steps:

[0039] (1) placing heparinoid in a 0.06 mol / L 3-morpholinepropanesulfonic acid buffer, adding anhydrous ethanol and dicyclohexylcarbodiimide, stirring evenly, adding 3-aminopropyltriethoxysilane and N,N-dimethylacetamide, first heating to 65° C. and stirring for 35 min, then cooling to 35° C. and adding lysine and stirring for 30 min to obtain an anticoagulant modified solution;

[0040] The volume ratio of the above-mentioned anhydrous ethanol to 3-morpholinepropanesulfonic acid buffer is 1:10; the volume ratio of N,N-dimethylacetamide to 3-morpholinepropanesulfonic acid buffer is 1:110;

[0041] The mass ratio of the above-mentioned dicyclohexylcarbodiimide, 3-aminopropyltriethoxysilane and heparinoid is 7:5:30; the solid-liquid ratio of heparinoid to 3-morpholinepropanesulfonic acid buffer is 1:50 g / mL.

[0042] (2) The anticoagulant modified liquid is injected into the dialysis membrane (polylactic acid membrane) pipeline of the hemodialyzer and left for 4 hours for reaction modification treatment. After the reaction modification is completed, the dialysis membrane (polylactic acid membrane) pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0043] In this example, the heparinoid obtained in Example 1 was used.

[0044] Example 4

[0045] A method for anticoagulation modification of a hemodialyzer comprises the following steps:

[0046] (1) placing heparinoid in a 0.06 mol / L 3-morpholinepropanesulfonic acid buffer, adding anhydrous ethanol and dicyclohexylcarbodiimide, stirring evenly, adding 3-aminopropyltriethoxysilane and N,N-dimethylacetamide, first heating to 70° C. and stirring for 25 min, then cooling to 40° C. and adding lysine and stirring for 20 min to obtain an anticoagulant modified solution;

[0047] The volume ratio of the above-mentioned anhydrous ethanol to 3-morpholinepropanesulfonic acid buffer is 1:10; the volume ratio of N,N-dimethylacetamide to 3-morpholinepropanesulfonic acid buffer is 1:100;

[0048] The mass ratio of the above-mentioned dicyclohexylcarbodiimide, 3-aminopropyltriethoxysilane and heparinoid is 6:4:25; the solid-liquid ratio of heparinoid to 3-morpholinepropanesulfonic acid buffer is 1:60 g / mL.

[0049] (2) The anticoagulant modified liquid is injected into the dialysis membrane (polyamide membrane) pipeline of the hemodialyzer and left for 6 hours for reaction modification treatment. After the reaction modification is completed, the dialysis membrane (polyamide membrane) pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0050] In this example, the heparinoid obtained in Example 2 was used.

[0051] Example 5

[0052] A method for anticoagulation modification of a hemodialyzer comprises the following steps:

[0053] (1) placing heparinoid in a 0.06 mol / L 3-morpholinepropanesulfonic acid buffer, adding anhydrous ethanol and dicyclohexylcarbodiimide, stirring evenly, adding 3-aminopropyltriethoxysilane and N,N-dimethylacetamide, first heating to 70° C. and stirring for 25 min, then cooling to 35° C. and adding lysine and stirring for 20 min to obtain an anticoagulant modified solution;

[0054] The volume ratio of the above-mentioned anhydrous ethanol to 3-morpholinepropanesulfonic acid buffer is 1:10; the volume ratio of N,N-dimethylacetamide to 3-morpholinepropanesulfonic acid buffer is 1:100;

[0055] The mass ratio of the above-mentioned dicyclohexylcarbodiimide, 3-aminopropyltriethoxysilane and heparinoid is 6:5:27; the solid-liquid ratio of heparinoid to 3-morpholinepropanesulfonic acid buffer is 1:55 g / mL.

[0056] (2) The anticoagulant modified liquid is injected into the dialysis membrane (polysulfone membrane) pipeline of the hemodialyzer and left for 5 hours for reaction modification treatment. After the reaction modification is completed, the dialysis membrane (polysulfone membrane) pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0057] In this example, the heparinoid obtained in Example 2 was used.

[0058] Example 6

[0059] A method for anticoagulant modification of a hemodialyzer, wherein step (1) is the same as that of Example 5, except that in step (2), an anticoagulant modification liquid is circulated through a dialysis membrane (polysulfone membrane) pipeline for 5 hours for reaction modification treatment, and after the reaction modification is completed, the dialysis membrane (polysulfone membrane) pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0060] Comparative Example 1

[0061] A method for anticoagulation modification of a hemodialyzer comprises the following steps:

[0062] (1) placing heparinoid in a 0.06 mol / L 3-morpholinepropanesulfonic acid buffer, adding anhydrous ethanol, first heating to 70° C. and stirring for reaction for 25 min, then cooling to 35° C. and adding lysine and continuing stirring for reaction for 20 min to obtain an anticoagulant modified solution;

[0063] The volume ratio of the anhydrous ethanol to the 3-morpholinepropanesulfonic acid buffer is 1:10; the solid-liquid ratio of the heparinoid to the 3-morpholinepropanesulfonic acid buffer is 1:55 g / mL.

[0064] (2) The anticoagulant modified liquid is circulated through the dialysis membrane (polysulfone membrane) pipeline for 5 hours for reaction modification treatment. After the reaction modification is completed, the dialysis membrane (polysulfone membrane) pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0065] In this comparative example, the heparinoid obtained in Example 2 was used.

[0066] Comparative Example 2

[0067] A method for anticoagulation modification of a hemodialyzer comprises the following steps:

[0068] (1) placing heparinoid in a 0.06 mol / L 3-morpholinepropanesulfonic acid buffer, adding anhydrous ethanol and dicyclohexylcarbodiimide, stirring evenly, adding 3-aminopropyltriethoxysilane, N,N-dimethylacetamide and lysine, stirring and reacting at 70° C. for 45 min, to obtain an anticoagulant modified solution;

[0069] The volume ratio of the above-mentioned anhydrous ethanol to 3-morpholinepropanesulfonic acid buffer is 1:10; the volume ratio of N,N-dimethylacetamide to 3-morpholinepropanesulfonic acid buffer is 1:100;

[0070] The mass ratio of the above-mentioned dicyclohexylcarbodiimide, 3-aminopropyltriethoxysilane and heparinoid is 6:5:27; the solid-liquid ratio of heparinoid to 3-morpholinepropanesulfonic acid buffer is 1:55 g / mL.

[0071] (2) The anticoagulant modified liquid is circulated through the dialysis membrane (polysulfone membrane) pipeline for 5 hours for reaction modification treatment. After the reaction modification is completed, the dialysis membrane (polysulfone membrane) pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

[0072] In this comparative example, the heparinoid obtained in Example 2 was used.

[0073] Performance Testing

[0074] 1. Coagulation test: Take 10m 2 The dialysis membranes after anticoagulant modification in Examples 5-6 and Comparative Examples 1-2 were cut into pieces of about 0.5 cm × 0.5 cm and placed in a 24-well culture plate; 20 mL of whole blood from healthy volunteers was taken and centrifuged at 3500 r / min for 10 min to obtain platelet-poor plasma, 700 μL of platelet-poor plasma was added to each well, incubated in a 37°C constant temperature water bath for 2 h, and the plasma after incubation was extracted to measure the thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). The specific test results are shown in Table 1.

[0075] Table 1 Coagulation test results

[0076] type APTT(s) PT(s) Example 5 206 30 Example 6 220 32 Comparative Example 1 217 29 Comparative Example 2 210 25

[0077] 2. Thrombus adhesion test: Take 10m 2 The dialysis membranes after anticoagulant modification in Examples 5-6 and Comparative Examples 1-2 were vacuum dried and weighed to obtain a dry weight W1, respectively placed in 24-well culture plates, and 1.5 mL of whole blood from healthy volunteers was quickly added to each well, incubated in a 37°C constant temperature water bath for 1 h, taken out and rinsed with PBS, fixed with 3% glutaraldehyde, vacuum dried after treatment with gradient ethanol, and weighed again to obtain a dry weight W2. The difference between W2-W1 was calculated to characterize the amount of thrombus adhesion. The specific test results are shown in Table 2.

[0078] Table 2 Thrombus adhesion amount

[0079] type Thrombus adhesion amount (mg) Example 5 8.76 Example 6 7.82 Comparative Example 1 7.85 Comparative Example 2 9.04

[0080] From the above table, we can see that:

[0081] (1) The amount of thrombus adhesion in Example 6 is slightly lower than that in Example 5, indicating that the circulation of the anticoagulant modification liquid in the dialysis membrane pipeline is conducive to its uniform and comprehensive adhesion to the inner wall of the membrane, thereby improving the modification effect of the dialysis membrane;

[0082] (2) The amount of thrombus adhesion in Comparative Example 1 and Example 6 is almost the same, and the dialysis membrane performance of both is good in the early stage;

[0083] (3) Compared with Example 6, the amount of thrombus adhesion in Comparative Example 2 increased significantly, indicating that when the reaction was stirred at a higher temperature, lysine would be affected, which would eventually affect the quality of the dialysis membrane.

[0084] 3. Stability test: take 10m 2 The dialysis membranes after anticoagulant modification in Example 6 and Comparative Example 1 were placed in physiological saline and shaken for 8 hours, taken out and dried, and then subjected to coagulation test and thrombus adhesion test. The specific test results are shown in Table 3.

[0085] Table 3 Stability

[0086]

[0087]

[0088] Combining Table 1, Table 2 and Table 3, it can be seen that compared with before treatment, after the treatment with physiological saline shock soaking, the amount of thrombus adhesion in Example 6 did not increase significantly, but the amount of thrombus adhesion in Comparative Example 1 increased significantly, indicating that self-condensation can increase the binding force between the anticoagulant modified substance and the dialysis membrane, thereby improving the stability of the anticoagulant dialysis membrane. Similarly, the significant reduction in APTT and PT time in Comparative Example 1 can also explain the stability deviation of the anticoagulant dialysis membrane in Comparative Example 1.

[0089] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for anticoagulation modification of a hemodialyzer, characterized in that: The following steps are involved: (1) Preparation of heparinoids using lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid; (2) placing heparinoid in a buffer solution, adding anhydrous ethanol and dicyclohexylcarbodiimide, stirring evenly, adding 3-aminopropyltriethoxysilane and N,N-dimethylacetamide, first heating and stirring to react, then cooling, adding lysine and continuing to stir to react, to obtain an anticoagulant modified solution; (3) Injecting the anticoagulant modified liquid into the dialysis membrane pipeline of the hemodialyzer, and after the reaction modification is completed, the dialysis membrane pipeline is cleaned and dried to obtain a hemodialyzer with anticoagulant function.

2. The anticoagulation modification method of the hemodialyzer according to claim 1, characterized in that: In step (1), the preparation method of the heparinoid is as follows: S1. Dissolve lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid in N,N-dimethylformamide and stir for 50-60 min under nitrogen atmosphere; S2. Then, add azobisisobutyronitrile and continue stirring at 75-80° C. for 20-22 hours; after terminating the reaction, perform rotary evaporation and grind to obtain heparinoid.

3. The anticoagulation modification method of the hemodialyzer according to claim 2, characterized in that: In step S1, the mass ratio of lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid is (4-5):(3-4):(1-2); the material-liquid ratio of lauryl methacrylate to N,N-dimethylformamide is 1:(10-12) g / mL.

4. The anticoagulation modification method of the hemodialyzer according to claim 2, characterized in that: In step S2, the amount of azobisisobutyronitrile added is 0.5-1% of the total mass of lauryl methacrylate, sodium p-styrene sulfonate and acrylic acid.

5. The anticoagulation modification method of the hemodialyzer according to claim 1, characterized in that: In step (2), the buffer solution is 0.06 mol / L 3-morpholinepropanesulfonic acid; and the volume ratio of anhydrous ethanol to the buffer solution is 1:

10.

6. The anticoagulation modification method of the hemodialyzer according to claim 1, characterized in that: In step (2), the mass ratio of dicyclohexylcarbodiimide, 3-aminopropyltriethoxysilane and heparin is (6-7): (4-5): (25-30).

7. The anticoagulation modification method of the hemodialyzer according to claim 1, characterized in that: In step (2), the solid-liquid ratio of the heparinoid to the buffer solution is 1:(50-60) g / mL; the volume ratio of N,N-dimethylacetamide to the buffer solution is 1:(100-110).

8. The anticoagulation modification method of the hemodialyzer according to claim 1, characterized in that: In step (2), the temperature is first raised to 65-70° C. and stirred for reaction for 25-35 min, then the temperature is lowered to 35-40° C. and lysine is added and stirred for reaction for 20-30 min.

9. The anticoagulation modification method of the hemodialyzer according to claim 1, characterized in that: In step (3), the anticoagulant modified liquid is injected into the dialysis membrane pipeline and left for 4-6 hours for reaction modification treatment; or, The anticoagulant modified liquid is circulated through the dialysis membrane pipeline for 4-6 hours to perform reaction modification treatment.

10. The anticoagulation modification method of a hemodialyzer according to claim 1, characterized in that: In step (3), the dialysis membrane is selected from polysulfone membrane, polyamide membrane and polylactic acid membrane.

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