Adsorbent for removing protein binding toxins and medium and macromolecular toxins and preparation method thereof

Abstract

The invention discloses an adsorbent for removing protein binding toxins and medium and macromolecular toxins and a preparation method thereof, and belongs to the technical field of high polymer materials, and the preparation method comprises the following steps: preparation of polystyrene-based white balls, amination immobilization and enveloping. In the presence of glycidyl methacrylate and 4, 4 '-bis (methacrylamido)-azobenzene, a pore-foaming agent is added to prepare the adsorbent capable of adsorbing protein binding toxins and medium and macromolecular toxins, and the adsorbent is good in adsorption effect and relatively high in selectivity; the process method is simple, green and environment-friendly; glycidyl methacrylate and modified cyclodextrin are added, so that the hydrophilicity of the adsorbent is enhanced, and the blood compatibility of the adsorbent is improved; and through immobilization of amido, the adsorption effect of the adsorbent is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials technology, and more specifically relates to adsorbents for removing protein-bound toxins and medium-to-large molecular weight toxins, and their preparation methods. Background Technology

[0002] Chronic kidney disease (CKD) is a chronic disease caused by various factors, including diabetes, hypertension, and various primary kidney diseases, leading to impaired kidney function. Currently, CKD is a global public health problem that threatens human health. CKD stage 5 patients have a high incidence of cardiovascular disease and mortality. Cardiovascular disease (CVD) is a common complication and leading cause of death in CKD patients, and uremic toxins are a specific risk factor for CVD in CKD patients.

[0003] Studies have shown that uremia can lead to complications such as electrolyte and acid-base imbalances, cardiovascular diseases, nervous system diseases, and pruritus, seriously affecting patients' physical and mental health and safety. It is known that uremia patients have higher concentrations of more than 200 substances in their bodies than normal people. These are mainly classified into three categories: small molecule toxins, medium and large molecule toxins, and protein-bound toxins. Small molecule water-soluble solutes have molecular weights less than 500 Da, such as creatinine and urea; medium molecule solutes have molecular weights greater than or equal to 500 Da, with representative solutes being β2-microglobulin and parathyroid hormone; protein-bound toxins, represented by p-cresol sulfate (PCS) and indophenol sulfate (IS), account for 24% of uremic toxins. They can bind to serum albumin to form protein-bound uremic toxins. Numerous studies have confirmed that PCS and IS are positively correlated with the all-cause mortality rate of CKD patients.

[0004] Blood purification plays a vital role in removing uremic toxins as a primary treatment for patients with end-stage renal disease. However, the preparation process of adsorbents in existing blood purification systems is cumbersome, and the adsorption selectivity for medium and large molecular toxins and protein-bound toxins is relatively low and non-renewable.

[0005] Shape memory polymers are a new type of functional polymer materials that can adapt to their surrounding environment by changing their physical or chemical properties in response to external stimuli. External stimuli include light, temperature, pH, electromagnetic fields, etc. Azobenzene and its derivatives are common shape memory polymers. Under ultraviolet light, they can be converted from trans to cis, realizing the regeneration of adsorption resins.

[0006] Current shape memory polymers have regenerable properties and can be used as adsorbents to remove protein-bound toxins and medium-to-large molecular weight toxins. However, although existing shape memory polymers have good regenerability, they suffer from poor adsorption selectivity and poor blood compatibility. To solve these problems, this invention prepares an adsorbent for removing protein-bound toxins and medium-to-large molecular weight toxins and its preparation method. Summary of the Invention

[0007] To address the aforementioned problems and overcome the shortcomings of existing technologies, this invention provides an adsorbent for removing protein-bound toxins and medium-to-large molecular weight toxins, and its preparation method, thereby solving the problems of cumbersome preparation processes, poor adsorption selectivity, and poor blood compatibility of existing adsorbents.

[0008] The specific technical solution of this invention to solve the above-mentioned technical problems is as follows: a method for preparing an adsorbent for removing protein-bound toxins and medium-to-large molecular weight toxins, comprising the following steps:

[0009] (1) Preparation of polystyrene-based white spheres:

[0010] Styrene monomers, polyvinylbenzene compounds, glycidyl methacrylate, 4,4'-bis(methacrylamide)-azobenzene, porogen and initiator were mixed as the oil phase, and dispersant, inorganic salt and pure water were mixed as the aqueous phase. The two phases were mixed and subjected to suspension polymerization at a certain temperature and stirring rate to obtain polystyrene white spheres, which were then washed, extracted and dried.

[0011] (2) Amination immobilization;

[0012] The polystyrene-based white spheres obtained in step (1) are added to an amination agent for amination treatment, and after cleaning, amination polystyrene resin is obtained.

[0013] (3) Encapsulation:

[0014] Amine polystyrene resin was dissolved in N,N-dimethylformamide (DMF) solution, sodium carbonate was added, modified dextrin 6-OTs-β-CD was stirred and dissolved, N2 was introduced, the temperature was raised to react for a period of time, filtered, DMF-anhydrous ethanol was precipitated and washed with water to obtain the adsorbent;

[0015] Preferably, the styrene monomer is one or more selected from styrene, methylstyrene, ethylstyrene, or 4-vinylbiphenyl; the polyvinylbenzene series is one or more selected from divinylbenzene, divinylbenzenetoluene, divinylethylbenzene, and triallyl isocyanurate.

[0016] Preferably, the pore-forming agent is one or a mixture of two or three of toluene, xylene, chlorobenzene, 3-12 carbon atom alcohols, 5-12 carbon atom alkanes, 200# gasoline or liquid paraffin, and the amount of pore-forming agent added is 50-300%.

[0017] Preferably, the initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile, benzoyl peroxide, tert-butyl peroxide-2-ethylhexanoate, tert-amyl peroxide-2-ethylhexanoate, dodecanoyl peroxide, or alkyl hydrogen peroxide.

[0018] The amount of initiator used is 0.5%-5% of the sum of the mass of styrene monomers, polyvinylbenzene compounds, glycidyl methacrylate, and 4,4'-bis(methacrylamido)-azobenzene;

[0019] Preferably, the dispersant is a water-soluble polymer, and its content is 0.1-8% of the mass of the aqueous phase;

[0020] The water-soluble polymer is one or two of polyvinyl alcohol, polyvinylpyrrolidone, gelatin, polyethylene glycol, carboxymethyl cellulose or hydroxyethyl cellulose;

[0021] Preferably, in step (1), the mass ratio of oil phase to water phase is 1:1-1:5; the reaction time is 10-20h; and the reaction temperature is 55-90℃.

[0022] In particular: the preparation method of the modified dextrin 6-OTs-β-CD:

[0023] After adding cyclodextrin (β-CD) to water, sodium hydroxide dissolved in water was slowly added dropwise to the flask. Under ice-water bath conditions, p-toluenesulfonyl chloride dissolved in acetonitrile was slowly added dropwise to the reaction solution. After the reaction, the pH was adjusted, the mixture was filtered, recrystallized twice, and then dried under vacuum to obtain modified cyclodextrin 6-OTs-β-CD.

[0024] In particular: the preparation method of 4,4'-bis(methacrylamido)-azobenzene in step (1) is as follows:

[0025] A solution of 4,4-diaminoazobenzene was added to pyridine and stirred at room temperature. Methacryl chloride was gradually added. After complete addition, the reaction mixture was heated for a period of time. After cooling, the mixture was poured into ice, acidified with hydrochloric acid, filtered, washed with saturated sodium bicarbonate solution, and then washed with water. The crude product was recrystallized from ethanol.

[0026] In particular, the amination agent is at least one selected from ethylenediamine, hexamethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine, and polylysine;

[0027] An adsorbent for removing protein-bound toxins and medium- to large molecular weight toxins is prepared using the above-mentioned adsorbent preparation method.

[0028] The beneficial effects of this invention are:

[0029] This invention utilizes styrene monomers and polyvinylbenzene compounds, with the participation of glycidyl methacrylate and 4,4'-bis(methacrylamido)-azobenzene, to prepare an adsorbent capable of adsorbing protein-bound toxins and medium- to large-molecule toxins by adding a porogen. The adsorption effect is good and has high selectivity. The process of this invention is simple, green and environmentally friendly.

[0030] This invention creatively incorporates glycidyl methacrylate and modified cyclodextrin, which enhances the hydrophilicity of the adsorbent and improves its blood compatibility.

[0031] This invention creatively enhances the adsorption effect of the adsorbent by immobilizing amine groups. Attached Figure Description

[0032] Appendix Figure 1 These are hemolysis experiment diagrams related to the embodiments and comparative examples in this invention;

[0033] Appendix Figure 2 These are platelet count diagrams related to the embodiments and comparative examples in this invention;

[0034] Appendix Figure 3 These are coagulation experiment diagrams related to the embodiments and comparative examples in this invention;

[0035] Appendix Figure 4 This is a diagram illustrating the preparation of polystyrene white spheres in this invention;

[0036] Appendix Figure 5 This is a structural diagram of the resin microspheres after amino immobilization in this invention;

[0037] Appendix Figure 6 This is a structural diagram of the coated resin microspheres in this invention; Detailed Implementation

[0038] Specific details in the description of this invention are merely to provide a thorough understanding of the embodiments thereof; however, those skilled in the art should understand that the implementation of this invention is not limited to these details. Furthermore, well-known structures and functions have not been described or shown in detail to avoid obscuring the key points of the embodiments of this invention. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0039] Specific embodiments of the present invention:

[0040] To better understand the present invention, specific embodiments are described. It is worth emphasizing that the effects of these embodiments are not substantially different from those of various embodiments within the scope of protection of the present invention, including their respective reagents and reagent content ratios. All of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here.

[0041] Example 1

[0042] (1) Preparation of 4,4'-bis(methacrylamido)-azobenzene:

[0043] Add 4 g of 4,4-diaminoazobenzene solution to 40 ml of pyridine and stir at room temperature. Gradually add 7 ml of methacryloyl chloride. After complete addition, heat the reaction mixture to 60 °C for 1.5 h. After cooling, pour the mixture into ice and acidify it to pH 4 with hydrochloric acid. Filter, wash with saturated sodium bicarbonate solution, and then wash with water. The crude product is recrystallized from ethanol.

[0044] (2) Preparation of polystyrene-based white spheres

[0045] Add styrene (18g), divinylbenzene (6g), dodecyl peroxide (0.45g), glycidyl methacrylate (6g), 4,4'-bis(methacrylamido)-azobenzene (2g), n-heptane (12g), and toluene (20g) to a beaker to prepare an oil phase. Stir until the solids are completely dissolved. Add 110g of an aqueous phase (containing 1.5% PVA and 0.5% NaCl). Disperse the oil phase in the aqueous phase under stirring to prepare an O / W emulsion. Stir at 120 rpm. Raise the temperature to 55-60℃ to start polymerization. The polymerization reaction is carried out for 5 hours. Then raise the temperature to 85-90℃ and continue the reaction for another 5 hours. Wash several times with hot water and ethanol, and then extract with ethanol for 6 hours to remove oligomers in the microspheres. Vacuum dry for 5 hours before use.

[0046] (3) Immobilization of tetraethylenepentamine

[0047] Take 50 ml of polystyrene-based white spheres and add them to a 500 ml three-necked flask. Add 200 ml of aqueous solution and tetraethylenepentamine. Stir mechanically at 60 °C for 12 h to stop the reaction. Wash with water to obtain the grafted tetraethylenepentamine polystyrene resin.

[0048] (4) Encapsulation

[0049] Weigh 6g of β-CD into a 50ml round-bottom flask, add 25mL of water, and slowly add 0.68g of sodium hydroxide dissolved in 2mL of water to the flask. Under ice-water bath conditions, slowly add 1.5g of p-toluenesulfonyl chloride dissolved in 3mL of acetonitrile to the reaction solution. After reacting for 2.5h, adjust the pH to 6, filter, recrystallize twice in hot water at 85℃, and dry under vacuum at 60℃ to obtain 6-OTs-β-CD with p-toluenesulfonyl groups.

[0050] Take 10 ml of tetraethylenepentamine polystyrene resin obtained in step (3) and dissolve it in 30 ml of DMF solution. Add 0.5 g of sodium carbonate and 5 g of 6-OTs-β-CD and stir to dissolve. Pass N2 in, heat to 60-65℃ and react for 18 h. Filter, precipitate DMF-anhydrous ethanol and wash with water to obtain the target adsorbent.

[0051] Example 2

[0052] (1) Preparation of 4,4'-bis(methacrylamido)-azobenzene:

[0053] Add 10 g of 4,4-diaminoazobenzene solution to 100 ml of pyridine and stir at room temperature. Gradually add 17.5 ml of methacryloyl chloride. After complete addition, heat the reaction mixture to 60 °C for 1 h. After cooling, pour the mixture into ice and acidify it to pH 4 with hydrochloric acid. Filter, wash with saturated sodium bicarbonate solution, and then wash with water. The crude product is recrystallized from ethanol.

[0054] (2) Preparation of polystyrene-based white spheres

[0055] Add styrene (6g), divinylbenzene (18g), benzoyl peroxide (1g), glycidyl methacrylate (7g), 4,4'-bis(methacrylamido)-azobenzene (6g), n-butanol (30g), and toluene (40g) to a beaker to prepare an oil phase. Stir until the solids are completely dissolved. Add 315g of aqueous phase (containing 2% PVP and 3% NaCl). Disperse the oil phase in the aqueous phase under stirring to prepare an O / W emulsion. Stir at 60 rpm and raise the temperature to 70-75℃ to start polymerization. The polymerization reaction is carried out for 5 hours, and then the temperature is raised to 80℃ to continue the reaction for 12 hours. Wash several times with hot water and ethanol, and then extract with ethanol for 6 hours to remove oligomers in the microspheres. Vacuum dry for 5 hours before use.

[0056] (3) Immobilization of polyethyleneimine

[0057] Take 50 ml of polystyrene-based white spheres and add them to a 500 ml three-necked flask. Add 200 ml of aqueous solution and polyethyleneimine. Stir mechanically at 60 °C for 12 h to stop the reaction. Wash with water to obtain grafted polyethyleneimine polystyrene resin.

[0058] (4) Encapsulation

[0059] Weigh 12g of β-CD into a 50ml round-bottom flask, add 50mL of water, and slowly add 1.36g of sodium hydroxide dissolved in 4mL of water to the flask. Under ice-water bath conditions, slowly add 3g of p-toluenesulfonyl chloride dissolved in 6mL of acetonitrile to the reaction solution. After reacting for 2.5h, adjust the pH to 6, filter, recrystallize twice in hot water at 85℃, and dry under vacuum at 60℃ to obtain 6-OTs-β-CD with p-toluenesulfonyl groups.

[0060] Take 10 ml of the ethyleneimine polystyrene resin obtained in step (3) and dissolve it in 30 ml of DMF solution. Add 0.5 g of sodium carbonate and 5 g of 6-OTs-β-CD and stir to dissolve. Pass N2 in, heat to 60℃ and react for 24 h. Filter, precipitate DMF-anhydrous ethanol and wash with water to obtain the target adsorbent.

[0061] Example 3

[0062] (1) Preparation of 4,4'-bis(methacrylamido)-azobenzene:

[0063] Add 20 g of 4,4-diaminoazobenzene solution to 200 ml of pyridine and stir at room temperature. Gradually add 35 ml of methacryloyl chloride. After complete addition, heat the reaction mixture to 60 °C for 1 h. After cooling, pour the mixture into ice and acidify it to pH 4 with hydrochloric acid. Filter, wash with saturated sodium bicarbonate solution, and then wash with water. The crude product is recrystallized from ethanol.

[0064] (2) Preparation of polystyrene-based white spheres

[0065] Add styrene (12g), divinylbenzene (12g), azobisisobutyronitrile (2g), glycidyl methacrylate (6g), 4,4'-bis(methacrylamido)-azobenzene (10g), liquid paraffin (80g), and xylene (30g) to a beaker to prepare an oil phase. Stir until the solids are completely dissolved. Add 720g of aqueous phase (containing 3% gelatin and 5% NaCl). Disperse the oil phase in the aqueous phase under stirring to prepare an O / W emulsion. Stir at 100 rpm. Raise the temperature to 70℃ to start polymerization. The polymerization reaction is carried out for 6 hours. Then raise the temperature to 85-90℃ and continue the reaction for 5 hours. Wash several times with hot water and ethanol, and then extract with ethanol for 6 hours to remove oligomers in the microspheres. Vacuum dry for 5 hours before use.

[0066] (3) Immobilization of ethylenediamine

[0067] Take 50 ml of polystyrene-based white spheres and add them to a 500 ml three-necked flask. Add 200 ml of aqueous solution and ethylenediamine. Stir mechanically at 60 °C for 12 h to stop the reaction. Wash with water to obtain grafted ethylenediamine polystyrene resin.

[0068] (4) Encapsulation

[0069] Weigh 24g of β-CD into a 50ml round-bottom flask, add 100mL of water, and slowly add 2.72g of sodium hydroxide dissolved in 8mL of water to the flask. Under ice-water bath conditions, slowly add 6g of p-toluenesulfonyl chloride dissolved in 12mL of acetonitrile to the reaction solution. After reacting for 2.5h, adjust the pH to 6, filter, recrystallize twice in hot water at 85℃, and dry under vacuum at 60℃ to obtain 6-OTs-β-CD with p-toluenesulfonyl groups.

[0070] Take 10 ml of the ethylenediamine polystyrene resin obtained in step (3) and dissolve it in 30 ml of DMF solution. Add 0.5 g of sodium carbonate and 10 g of 6-OTs-β-CD and stir to dissolve. Pass N2 in, heat to 65℃ and react for 24 h. Filter, precipitate DMF-anhydrous ethanol and wash with water to obtain the target adsorbent.

[0071] To more intuitively demonstrate the technological advantages of this invention, a comparison is made between the preparation method of the adsorbent for removing protein-bound toxins and medium-to-large molecular weight toxins of this invention and a method using equivalent substitution in the same process.

[0072] Comparative Example 1

[0073] In the preparation of polystyrene-based white spheres, no modified cyclodextrin is added, and the remaining steps are the same as in Example 2;

[0074] Comparative Example 2

[0075] In the preparation of polystyrene-based white spheres, cyclodextrin was added, and the remaining steps were the same as in Example 2; the reduced adsorption performance may be due to blockage of the pore structure.

[0076] Comparative Example 3

[0077] In the preparation of polystyrene-based white spheres, modified cyclodextrin as described in the reference was added, and the remaining steps were the same as in Example 2; the modified cyclodextrin was used by reference, and the preparation method was based on the modified cyclodextrin preparation method disclosed in 202110645891.6 "Vinyl Monomer-Polyvinyl Crosslinking Agent Copolymer Non-porous Microspheres and Their Preparation Method and Application".

[0078] Comparative Example 4

[0079] In the preparation of polystyrene-based white spheres, glycidyl methacrylate was replaced with epichlorohydrin;

[0080] Comparative Example 5

[0081] The preparation method of polystyrene-based white spheres is the same as in Example 2, except that the second step of amino immobilization is not performed;

[0082] Comparative Example 6

[0083] In the preparation of polystyrene-based white spheres, the amination agent is replaced with octanediamine;

[0084] Comparative Example 7

[0085] The preparation method for polystyrene-based white spheres is the same as in Example 2, except that no pore-forming agent is added;

[0086] Comparative Example 8

[0087] The preparation method of polystyrene-based white spheres is the same as in Example 2, except that the pore-forming agent is ethyl acetate;

[0088] The specific operating procedures for evaluating adsorption performance are as follows:

[0089] Take multiple 10 mL portions containing 40 mg / L indophenol sulfate and p-cresol sulfate, add 1 mL of the adsorbent obtained in the examples and control examples respectively, and shake at 37 °C for 2 h. Then, use high performance liquid chromatography to test the adsorption effect of the adsorbent on indophenol sulfate and p-cresol sulfate. The test results are shown in Table 2.

[0090] The adsorption performance of the adsorbent for parathyroid hormone was evaluated using ELISA; the adsorption performance of the adsorbent for β2-microglobulin was evaluated using immunoturbidimetry. The test results are shown in Table 2.

[0091] Table 1: Comparison of differences in adsorption properties and biocompatibility between different embodiments and comparative examples:

[0092]

[0093] The results are shown in Table 2 below:

[0094] Table 2. Scavenging effect of adsorbents on protein-bound toxins and medium- to large molecular weight toxins

[0095]

[0096]

[0097] (1) Examples 2 and Comparative Examples 1-3 are analyses of the inventiveness of the "modified cyclodextrin" technical features on the adsorption effect of the present invention.

[0098] Comparative Example 1 did not add modified cyclodextrin; Comparative Example 2 added ordinary cyclodextrin; and Comparative Example 3 added the modified cyclodextrin from the reference. The experimental data comparison shows that…

[0099] Compared with Comparative Example 2 and Comparative Example 3, cyclodextrin did not participate in the reaction, but the adsorption effect of cresol sulfate, β2-microglobulin and parathyroid hormone in Comparative Example 2 was significantly reduced. This may be because cyclodextrin itself has poor water solubility and is difficult to wash out, thus clogging the pores. In contrast, the modified cyclodextrin in Example 3 has significantly enhanced hydrophilicity and is easily washed out. The test results showed almost no change in adsorption performance compared with Comparative Example 1 without the addition of cyclodextrin.

[0100] Compared with Comparative Examples 1-3, Example 2 shows that the adsorbent's effect on protein-bound toxins and medium-to-large molecular weight toxins is much lower than that of the present invention in terms of its adsorption effect on indophenol sulfate, acetophenol sulfate, β2-microglobulin, and parathyroid hormone. Therefore, it can be seen that the double-bond modified cyclodextrin with p-toluenesulfonyl group prepared in the present invention has a positive effect on the adsorption of indophenol sulfate, acetophenol sulfate, β2-microglobulin, and parathyroid hormone compared with no addition, addition of ordinary cyclodextrin, and addition of other double-bond modified cyclodextrin.

[0101] (2) Example 2 and Comparative Example 4 are analyses of the inventive impact of the "ring-opening reagent" technical feature on the adsorption effect of the present invention.

[0102] Among them, glycidyl methacrylate, as a ring-opening reagent, can enhance the hydrophilicity of the adsorbent and improve its blood compatibility. However, epichlorohydrin, which is also a ring-opening reagent, cannot participate in the reaction, and its decreased hydrophilicity leads to a lower adsorption effect, especially since the adsorption effect of indophenol sulfate and cresol sulfate is only 57-60%.

[0103] (3) Examples 2 and Comparative Examples 5-6 are analyses of the impact of the "amine group immobilization" technical feature on the inventive adsorption effect of the present invention.

[0104] The difference in Comparative Example 5 is that it does not perform the second step of amino group immobilization, while Comparative Example 6, although it performs amino group immobilization, replaces octanediamine with amination reagent. Through comparison of experimental data, it can be seen that Comparative Examples 5 and 6 have lower adsorption effects compared with the present invention. Therefore, it can be seen that amino group immobilization, especially the immobilization of polyethyleneimine amino group, has a positive promoting effect on adsorption effect in the present invention.

[0105] (4) Examples 2 and Comparative Examples 7-8 are examples of the inventive effects of the "porogen" technical features on the adsorption effect of the present invention.

[0106] Comparative Example 7 did not add a porogen, while Comparative Example 8 used ethyl acetate as a porogen. The experimental data comparison shows that the addition of porogens, especially toluene and n-butanol, has a positive promoting effect on the adsorption effect compared with ethyl acetate.

[0107] It is worth noting that, compared with Comparative Examples 7 and 8, although Comparative Example 8 added ethyl acetate as a porogen, the adsorption effect on indophenol sulfate, β2-microglobulin and parathyroid hormone was improved to a certain extent. However, the adsorption capacity for paracresol sulfate had the opposite effect compared with the absence of porogen, decreasing from 59% to 55%. This shows that the addition of porogen does not have a completely positive correlation with the adsorption capacity.

[0108] In summary, the factors of "modified cyclodextrin", "amino group immobilization", and "porogen" have a positive promoting effect on the adsorption effect of the present invention.

[0109] To more intuitively demonstrate the technological advantages of this invention, further research and investigation were conducted on biocompatibility, and the biocompatibility test results are as follows:

[0110] The hemolysis test method refers to YY / T1651.1-2019 Medical Device Hemolysis Test Part 1: Material-Mediated Hemolysis Test ( Figure 3 );

[0111] Hematological testing methods refer to YY / T1649.1-2019 Medical Devices and Platelet Interaction Tests Part 1: In Vitro Platelet Counting Method (…). Figure 4 );

[0112] Coagulation test method for medical devices (YY / T1911-2023) Figure 5 )

[0113] The results of the blood compatibility tests are shown in Table 3 above.

[0114] Table 3. Blood compatibility test data

[0115] Experimental group number sample hemolysis rate Platelet adhesion rate Clotting time ratio 1# Example 1 1.3％ 4.2％ 98.7％ 2# Example 2 0.9％ 7.1％ 95.4％ 3# Example 3 0.5％ 6.2％ 93.6％ 4# Comparative Example 1 2.1％ 15.3％ 81.2％ 5# Comparative Example 2 1.8％ 14.7％ 84.3％ 6# Comparative Example 3 2.2％ 15.9％ 80.7％ 7# Comparative Example 4 4.8％ 17.8％ 78.0％ 8# Comparative Example 5 2.2％ 13.6％ 75.9％ 9# Comparative Example 6 3.4％ 18.4％ 83.0％ 10# Comparative Example 7 3.3％ 15.2％ 81.2％ 11# Comparative Example 8 2.7％ 22.1％ 82.3％ index Less than 5% Greater than 80%

[0116] (1) Examples 2 and Comparative Examples 1-3 are analyses of the impact of the "modified cyclodextrin" technical features on the inventiveness of the biocompatibility of the present invention.

[0117] Comparative Example 1 was without modified cyclodextrin; Comparative Example 2 was with ordinary cyclodextrin; and Comparative Example 3 was with modified cyclodextrin from the reference. Through comparison of experimental data, it can be seen that the blood compatibility of the adsorbent was examined, focusing on the hemolysis rate, platelet adhesion rate, and clotting time ratio. Although the present invention outperformed Comparative Examples 1-3 in terms of hemolysis rate, platelet adhesion rate, and clotting time, and Comparative Examples 1-3 also generally met the requirements of a hemolysis rate of less than 5% and a clotting time ratio of greater than 80%, it is evident that the "modified cyclodextrin" technical feature has a certain impact on the biocompatibility of the present invention, but it is not the dominant factor.

[0118] (2) Example 2 and Comparative Example 4 are aimed at the impact of the "ring-opening reagent" technical feature on the inventiveness of the biocompatibility of the present invention. Glycidyl methacrylate, as a ring-opening reagent, can enhance the hydrophilicity of the adsorbent and is beneficial to improve the blood compatibility of the adsorbent. However, epichlorohydrin, which is also a ring-opening reagent, does not meet the requirements in terms of hemolysis rate, platelet adhesion rate and coagulation time ratio in terms of blood compatibility. The hemolysis rate is close to the critical value of 5%, and the coagulation time ratio is below 80%.

[0119] Therefore, it can be seen that glycidyl methacrylate, as a ring-opening agent, has a significant impact on the coagulation time ratio index of the biocompatibility of this invention.

[0120] (3) Examples 2 and Comparative Examples 5-6 address the impact of the "amino group immobilization" technical feature on the biocompatibility and inventiveness of this invention.

[0121] The difference in Comparative Example 5 is that it does not perform the second step of amino group immobilization, while Comparative Example 6, although it performs amino group immobilization, replaces octanediamine with amination reagent. Through comparison of experimental data, it can be seen that the hemolysis rate of the present invention is better than that of Comparative Examples 5-6, and the overall rate meets the requirement of less than 5%. However, the clotting time ratio of Comparative Examples 5-6 is already below 80%, which does not meet the requirements.

[0122] Therefore, it can be seen that the technical feature of "amine group immobilization" has a significant impact on the biocompatibility clotting time ratio index of this invention.

[0123] (4) Examples 2 and Comparative Examples 7-8 are analyses of the impact of the "porogen" technical features on the inventiveness of the biocompatibility of the present invention.

[0124] Comparative Example 7 did not use a porogen, while Comparative Example 8 used ethyl acetate as the porogen. Comparison of experimental data shows that, although the performance of this invention is superior to that of Comparative Examples 7-8, the overall hemolysis rate is less than 5%, and the clotting time ratio is generally greater than 80%. Therefore, the "porogen" technology has a certain influence on the biocompatibility of this invention, but it is not the dominant factor.

[0125] Therefore, the factors of "modified cyclodextrin", "amino group immobilization" and "porogen" have a positive promoting effect on the biocompatibility of the present invention. Among them, glycidyl methacrylate as a ring-opening agent and "amino group immobilization" have a significant promoting effect on biocompatibility.

[0126] In summary:

[0127] (1) The adsorbent and preparation method of the present invention can simultaneously adsorb protein-bound toxins and medium-to-large molecular toxins. Compared with traditional adsorbents, it has better adsorption effect and higher selectivity. The addition of glycidyl methacrylate and cyclodextrin during the reaction process enhances the hydrophilicity of the adsorbent and is beneficial to improving the blood compatibility of the adsorbent. In addition, the process of the present invention is simple, green and environmentally friendly.

[0128] (2) The present invention creatively adds glycidyl methacrylate as a ring-opening reagent to prepare an adsorbent, which can enhance the hydrophilicity of the adsorbent and improve its blood compatibility. In particular, the adsorbent prepared by adding double bond modified cyclodextrin has a significant positive effect on the adsorption of indophenol sulfate, cresol sulfate, β2-microglobulin and parathyroid hormone. At the same time, the "amino group immobilization" and "porogen" methods have a positive promoting effect on the adsorption effect of the present invention.

[0129] (3) The present invention creatively adds glycidyl methacrylate as a ring-opening reagent to prepare an adsorbent. The adsorbent has a significant impact on the biocompatibility of hemolysis and the clotting time ratio. The "amino-immobilization" technical feature has a significant impact on the biocompatibility of the adsorbent and the clotting time ratio, but has little impact on hemolysis.

Claims

1. A method for preparing an adsorbent for removing protein-bound toxins and medium- to macromolecular toxins, characterized in that, Includes the following steps: (1) Preparation of polystyrene-based white spheres: Styrene monomers, polyvinylbenzene compounds, glycidyl methacrylate, 4,4'-bis(methacrylamide)-azobenzene, porogen and initiator were mixed as the oil phase, and dispersant, inorganic salt and pure water were mixed as the aqueous phase. The two phases were mixed and subjected to suspension polymerization at a certain temperature and stirring rate to obtain polystyrene white spheres, which were then washed, extracted and dried. (2) Amination immobilization; The polystyrene-based white spheres obtained in step (1) are added to an amination agent for amination treatment, and after cleaning, amination polystyrene resin is obtained. (3) Encapsulation: Amine polystyrene resin was dissolved in DMF solution, sodium carbonate was added, and modified dextrin 6-OTs-β-CD was stirred and dissolved. N2 was introduced, the temperature was raised and the reaction was carried out. After filtration, DMF-anhydrous ethanol was precipitated and washed with water to obtain the adsorbent.

2. The method for preparing the adsorbent according to claim 1, characterized in that... The styrene monomers are one or more of styrene, methylstyrene, ethylstyrene, or 4-vinylbiphenyl; the polyvinylbenzene series are one or more of divinylbenzene, divinylbenzenetoluene, divinylethylbenzene, and triallyl isocyanurate.

3. The method for preparing the adsorbent according to claim 1, characterized in that... The pore-forming agent is one or a mixture of two or three of the following: toluene, xylene, chlorobenzene, 3-12 carbon atom alcohols, 5-12 carbon atom alkanes, 200# gasoline or liquid paraffin, and the amount of pore-forming agent added is 50%-300%.

4. The method for preparing the adsorbent according to claim 1, characterized in that... The initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile, benzoyl peroxide, tert-butyl peroxide-2-ethylhexanoate, tert-amyl peroxide-2-ethylhexanoate, dodecanoyl peroxide, or alkyl hydrogen peroxide. The amount of initiator used is 0.5%-5% of the sum of the masses of styrene monomers, polyvinylbenzene compounds, glycidyl methacrylate, and 4,4'-bis(methacrylamido)-azobenzene.

5. The method for preparing the adsorbent according to claim 1, characterized in that... The dispersant is a water-soluble polymer, with a content of 0.1%-8% of the water phase mass; The water-soluble polymer is one or two of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), gelatin, polyethylene glycol, carboxymethyl cellulose, or hydroxyethyl cellulose.

6. The method for preparing the adsorbent according to claim 1, characterized in that... In step (1), the mass ratio of oil phase to water phase is 1:1-1:5; the reaction time is 10-20h; and the reaction temperature is 55-90℃.

7. The method for preparing the adsorbent according to claim 1, characterized in that... The preparation method of the modified dextrin 6-OTs-β-CD: After adding cyclodextrin (β-CD) to water, sodium hydroxide dissolved in water was slowly added dropwise to the flask. Under ice-water bath conditions, p-toluenesulfonyl chloride dissolved in acetonitrile was slowly added dropwise to the reaction solution. After the reaction, the pH was adjusted, the mixture was filtered, recrystallized twice, and then dried under vacuum to obtain modified cyclodextrin 6-OTs-β-CD.

8. The method for preparing the adsorbent according to claim 1, characterized in that... In step (1), the preparation method of 4,4'-bis(methacrylamido)-azobenzene is as follows: A solution of 4,4-diaminoazobenzene was added to pyridine and stirred at room temperature. Methacryl chloride was gradually added. After complete addition, the reaction mixture was heated, cooled, poured into ice, acidified with hydrochloric acid, filtered, washed with saturated sodium bicarbonate solution, and then washed with water. The crude product was recrystallized from ethanol.

9. The method for preparing the adsorbent according to claim 1, characterized in that... The amination agent includes at least one of ethylenediamine, hexamethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine, and polylysine.

10. An adsorbent for removing protein-bound toxins and medium- to macromolecular toxins, prepared according to the method of any one of claims 1-9.

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