Blood perfusion adsorbent for removing protein-binding toxins and preparation method of blood perfusion adsorbent

Abstract

The invention relates to a blood perfusion adsorbent for removing protein-binding toxins and a preparation method of the blood perfusion adsorbent. The adsorbent comprises a carrier, a raw material of the carrier has an epoxy group, the carrier is fixedly loaded with modified hyperbranched polyamide-amine through the epoxy group, and the modified hyperbranched polyamide-amine is obtained by modifying a half-generation product of hyperbranched polyamide-amine with 1, 6-hexamethylenediamine. Fatty acid is immobilized on the modified hyperbranched polyamide-amine through condensation reaction of amine and carboxylic acid. Hyperbranched polyamide-amine contains a large number of positively charged amido groups to adsorb negatively charged protein binding toxins under the electrostatic interaction, and fatty acid is combined with albumin through the hydrophobic interaction to capture albumin in protein binding toxin molecules, so that the toxins are dissociated into free-state small molecules. Free toxins can be quickly adsorbed by the ligands with positive charges and can also be removed by the hemodialysis membrane, and the removal effect of the protein binding toxins is greatly improved through the synergistic effect of the ligands with positive charges and the hemodialysis membrane.

Description

Technical Field

[0001] This invention relates to the field of adsorbent technology, and in particular to a blood perfusion adsorbent for removing protein-bound toxins and its preparation method. Background Technology

[0002] In patients with chronic kidney disease (CKD), the glomerular filtration rate decreases, preventing the kidneys from effectively removing metabolic waste products from the blood. Additionally, impaired kidney function can lead to increased solute production due to intestinal dysbiosis and local tissue inflammation. This results in the retention of some solutes in the body, which can have toxic effects. These retained solutes, negatively impacting bodily functions, are called uremic toxins. In patients with end-stage renal disease (ESRD), multiple uremic toxins accumulate, affecting multiple systems throughout the body, leading to a decline in quality of life and an increased risk of death.

[0003] Uremic toxins are generally classified into three main categories: 1. Small molecule water-soluble solutes with a relative molecular mass of less than 500 Da, such as urea, creatinine, and uric acid, can be effectively removed by conventional hemodialysis due to their small molecular weight. 2. Medium and large molecular weight toxins, with a relative molecular mass greater than 500 Da, such as β2 microglobulin, leptin, parathyroid hormone, etc. These toxins can only be removed by dialysis membranes with larger pore sizes due to their large molecular weight. 3. Protein-bound toxins (PBUTs): These toxins are usually small-molecule organic anions. Among them, organic anions with aromatic benzene rings mainly bind tightly to the site II site of serum albumin in the blood circulation to form PBUTs. Examples include p-cresol sulfate (PCS), indophenol sulfate (IS), indole-3-acetic acid (IAA), and hippuric acid (HA).

[0004] Although protein-bound uremic toxins have a relative molecular mass of less than 500 Da, their high binding to serum albumin (such as IS, pCS, and IAA, which have a binding rate of over 90%) makes most blood purification methods ineffective in removing them.

[0005] Studies have shown that PBUTs can lead to oxidative stress and inflammatory responses in the body, promote renal interstitial fibrosis and the progression of CKD, and have certain cardiovascular toxicity, which is closely related to poor prognosis in ESRD patients. PBUTs with high protein binding rates are not easily removed by routine hemodialysis, posing a challenge in current clinical management. Currently, low-flux dialysis in blood purification can hardly remove PBUTs, while high-flux dialysis only slightly increases the clearance rate. Hemoperfusion (HP) adsorbs PBUTs through neutral macroporous resin, but its removal efficiency is still insufficient to meet clinical needs due to limitations in the adsorption capacity of the adsorbent.

[0006] Both p-cresol sulfate (PCS) and indophenol sulfate (IS) contain sulfonic acid groups (-SO3H), carrying a stable negative charge at physiological pH in vivo. Indole-3-acetic acid (IAA) and hippuric acid (HA) contain carboxyl groups (-COOH), exhibiting weak negative charge at physiological pH in vivo, and can bind to positively charged ligands through electrostatic interactions. Therefore, immobilizing positively charged ligands on an adsorbent support to adsorb PBUTs is currently a mainstream technical approach. However, this approach suffers from the drawback of excessively strong positive charges directly contacting blood cells, potentially causing cell damage or coagulation, while weak positive charges have poor clearance effects. Furthermore, since the binding of toxins such as PCS, IS, IAA, and HA to proteins is a reversible process primarily driven by van der Waals forces, according to Le Chatelier's principle, when some free toxins are removed, the equilibrium shifts towards enhanced dissociation of the complex, leading to the presence of more free IS in the solution. Some scholars have proposed competitive binding methods to remove PBUTs, such as adding competitive binding drugs to the dialysate to competitively bind albumin, thereby increasing the free level of PBUTs and facilitating their removal via hemodialysis. Competitive binding drugs include furosemide, ibuprofen, tryptophan, and salvianolic acid derivatives. However, these substances typically require high plasma concentrations to competitively inhibit the binding of PBUTs to albumin. Furthermore, these drugs enter the body through the dialysis membrane and accumulate in the dialysis patient, potentially causing new side effects. This approach presents a trade-off and is currently difficult to apply clinically, requiring further research.

[0007] Based on the principle of competitive binding, an existing patent (CN118384862A) reports a protein-bound toxin adsorbent. This adsorbent uses ultra-highly cross-linked styrene-divinylbenzene resin as a carrier, immobilizing positively charged polyethyleneimine. Furthermore, the polyethyleneimine adsorbs oleic acid and linoleic acid via electrostatic interactions, with the oleic and linoleic acids acting as adsorption ligands. The adsorption ligands detach from the protein-bound toxin adsorbent and enter the bloodstream. These ligands possess the same albumin-binding sites as p-cresol sulfate and indophenol sulfate, allowing them to compete with protein-bound toxins for adsorption on albumin. This causes the toxins to detach from the albumin, leaving them in a free state, which facilitates adsorption by the ultra-highly cross-linked styrene-divinylbenzene resin and the polyethyleneimine immobilized thereon, thus improving the clearance rate of protein-bound toxins. However, this patented technology still suffers from the drawback of ligand detachment and entry into the human body. Additionally, the surface of the adsorbent, carrying a large number of positive charges, directly contacts blood cells, which can also cause some damage. Therefore, this technology still has room for further optimization. Summary of the Invention

[0008] The purpose of this invention is to disclose a blood perfusion adsorbent for removing protein-bound toxins and its preparation method, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0009] The first aspect of the present invention is to provide an adsorbent.

[0010] A second aspect of the present invention is to provide a method for preparing the adsorbent described in the first aspect of the present invention.

[0011] A third aspect of the present invention is to provide the use of the adsorbent described in the second aspect of the present invention.

[0012] The adsorbent described in the first aspect of this invention includes a support, the raw material of which has epoxy groups. The support is immobilized with modified hyperbranched polyamide-amine via these epoxy groups. The modified hyperbranched polyamide-amine is obtained by modifying a half-generation product of hyperbranched polyamide-amine (PAMAM) with 1,6-hexanediamine (DH). The modified hyperbranched polyamide-amine is immobilized with fatty acids (FA) through a condensation reaction of amine and carboxylic acid. PAMAM contains a large number of positively charged amine groups that electrostatically adsorb negatively charged protein-bound glycoproteins (PBUTs), while FA binds to albumin through hydrophobic interactions, abstracting albumin from PBUT molecules and causing the toxins to dissociate into free small molecules. These free toxins can be rapidly adsorbed by positively charged ligands and also removed by hemodialysis membranes; the synergistic effect of both greatly enhances the removal efficiency of protein-bound toxins. The spatial arm of the 1,6-hexanediamine can reduce steric hindrance and help to better immobilize FA on PAMAM. FA and PAMAM are covalently connected, so they are not easy to fall off and enter the human body.

[0013] In a further application embodiment, the carrier is a porous microsphere with a particle size of 200-1000 μm and a pore size of 2-50 nm. Its raw materials include methyl methacrylate (MMA), hydroxyethyl methacrylate (HEMA), glycidyl methacrylate (GMA), and the crosslinking agent ethylene glycol dimethacrylate (EDMA). The adsorbent carrier with good blood compatibility is prepared by copolymerization of MMA, HEMA, GMA, and EDMA monomers. The copolymer product is in the form of porous microspheres, and the epoxy groups provided by the GMA monomer enable the adsorbent carrier to subsequently immobilize and adsorb PBUTs and to extract functional groups from albumin in PBUT molecules.

[0014] In a further application embodiment, the mass ratio of PAMAM to GMA monomer in the adsorbent carrier is (0.5~5):1. Sufficient hyperbranched polyamide-amine ensures a highly complete reaction with GMA.

[0015] In a further application embodiment, the methyl methacrylate monomer accounts for 1-40% of the total monomer mass, the hydroxyethyl methacrylate monomer accounts for 40-85% of the total monomer mass, the glycidyl methacrylate monomer accounts for 1-20% of the total monomer mass, and the crosslinking agent ethylene glycol dimethacrylate accounts for 1-5% of the total monomer mass.

[0016] In a further application embodiment, the PAMAM is a half-generation product, with a generation number of 0.5, 1.5, 2.5, or 3.5. Increasing the generation number will decrease the solubility in the reaction solvent and increase the viscosity, which is not conducive to the subsequent immobilization reaction. In addition, the increase in the number of positive charges is also not conducive to the blood compatibility of the adsorbent. Therefore, considering all factors, 1.5-generation PAMAM is considered more suitable.

[0017] In a further application embodiment, the mass ratio of the 1,6-hexanediamine to the PAMAM is (0.5~5):1.

[0018] In a further application embodiment, the FA is selected from one or more of saturated fatty acids (SFA), unsaturated fatty acids (UFA), straight-chain fatty acids (LFA), branched-chain fatty acids (BCFA), medium-chain fatty acids (MCFA), long-chain fatty acids (LCFA), or their corresponding fatty acid salts; preferably, the hydrocarbon chain of the FA is C6~C6. 22 .

[0019] In a further application embodiment, the mass ratio of fatty acid to 1,6-hexanediamine is (0.5~4):1. Sufficient FA is used to ensure the most complete reaction with 1,6-hexanediamine. The FA ligands are immobilized on the outermost surface of the adsorbent, spontaneously forming hydrophobic regions due to the hydrophobic interactions between their ligands. This prevents direct contact between the blood cell surface and the positively charged PAMAM, thereby reducing damage to blood cells. Furthermore, FA and PAMAM are covalently linked, making it difficult for FA to detach and enter the human body.

[0020] The preparation method described in the second aspect of the present invention includes the following steps: (1) Synthetic carrier: A certain amount of polyvinyl alcohol (PVA) and polyethylene glycol (PEG) were dissolved in purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form a stable aqueous phase system. MMA monomer, HEMA monomer, and GMA monomer were added to the aqueous phase system in a specific ratio, along with a certain amount of crosslinking agent EDMA. The total amount of monomers and crosslinking agent accounted for 10-50% of the reaction solvent. A certain amount of toluene and xylene (volume ratio 1:1) were added as porogens, accounting for 10-50% of the total monomer mass. Subsequently, a certain amount of benzoyl peroxide (BPO) was added as an initiator. The reaction was stirred at 65°C under nitrogen protection for 4-8 hours. After the reaction was completed, a carrier (PMMA-HEMA-GMA microspheres) was obtained. These microspheres were washed sequentially with anhydrous ethanol and purified water, and then sieved to obtain a carrier with a suitable particle size distribution for later use.

[0021] (2) Fixed load PAMAM: The carrier was placed in a three-necked flask, and a certain amount of DMSO and 1 mol / L NaOH solution were added sequentially. After stirring evenly, an appropriate amount of semi-generated PAMAM was added, and the mixture was stirred at 40°C for 8-16 hours. After the reaction was completed, the reactants were poured off and filtered to obtain a carrier immobilized with PAMAM. The product was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0022] (3) PAMAM modification treatment: The product from step (2) above was placed in a three-necked flask with an appropriate amount of methanol. The mixture was thoroughly stirred under ice-water bath and nitrogen conditions, and then cooled to 0°C. An appropriate amount of 1,6-hexanediamine (DH) dissolved in an appropriate amount of methanol was added dropwise to the reaction system, maintaining the reaction system temperature at 0-2°C throughout the process. After the addition was complete, the temperature was gradually increased to 25°C, and the reaction continued for 24 hours. After the reaction was complete, the remaining ethylenediamine and solvent were evaporated using a rotary evaporator to obtain an intermediate product (PMMA-HEMA-GMA-PAMAM-DH microspheres) immobilized with modified hyperbranched polyamide-amine.

[0023] (4) Fixed FA: The intermediate product was placed in a three-necked flask, and appropriate amounts of FA, acetone, and 0.1 mol / L MES buffer (pH 4.8) were added. After mixing thoroughly, appropriate amounts of 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) condensing agents were added to the reaction system. The reaction was carried out in a water bath at 37°C with shaking for 8 to 16 hours. After the reaction was completed, the product was washed with anhydrous ethanol and purified water in sequence to obtain the adsorbent.

[0024] The application described in the third aspect of this invention refers to the use of the above-mentioned adsorbent in the preparation of blood perfusion products.

[0025] The beneficial effects of this invention are as follows: This invention addresses the shortcomings of existing technologies by providing a novel blood purification adsorbent for removing protein-bound toxins (PBUTs). When a hemoperfusion device loaded with this adsorbent is used in conjunction with hemodialysis to remove protein-bound toxins from patients with end-stage renal disease, the adsorbent can directly bind to toxins such as indophenol sulfate, p-cresol sulfate, and hippuric acid through electrostatic adsorption. Simultaneously, it can competitively bind to albumin with its ligands, promoting the dissociation of toxins from albumin and increasing the amount of free toxins, thereby enhancing the toxin removal effect of hemodialysis. This dual action significantly improves the removal efficiency of PBUTs. Furthermore, it avoids damage to blood cells and improves blood compatibility. The adsorbent developed in this invention utilizes a blood-compatible carrier prepared by copolymerization of various hydrophilic olefin monomers. This carrier is then modified to immobilize positively charged ligands capable of adsorbing protein-bound toxins and hydrophobic ligands that compete with albumin for binding. The outermost hydrophobic ligands form protective hydrophobic groups that isolate blood cells from contact with the charged groups, thus reducing blood cell damage. Furthermore, the hydrophobic ligands are covalently bonded to the carrier, ensuring a strong connection and minimizing the risk of ligands entering the bloodstream. Therefore, this hemoperfusion adsorbent exhibits excellent blood compatibility and significant toxin removal efficacy, making it suitable for removing protein-bound toxins from patients with end-stage renal disease via hemoperfusion, prolonging their lives and improving their quality of life. Detailed Implementation

[0026] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0027] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0028] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of the present invention.

[0030] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0031] Example 1 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 8.00 g MMA monomer, 24.00 g HEMA monomer, and 4.00 g GMA were added to the aqueous phase system, bringing the total monomer mass to 36.00 g. 0.80 g of crosslinking agent EDMA was added, along with 3.00 mL toluene and 3.00 mL xylene as porogens. Subsequently, 0.50 g BPO was added as an initiator. The reaction was stirred at 65°C under nitrogen protection for 6 hours. After the reaction was complete, the support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0032] (2) Fixed load PAMAM: The carrier described in step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 3.0 g of 1.5 generation PAMAM was added, and the mixture was stirred and reacted at 40°C for 2 hours. After the reaction was completed, the PAMAM-supported carrier was obtained by filtration. The product was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0033] (3) Modification treatment of PAMAM: The product from step (2) and 100 mL of methanol were placed in a three-necked flask and thoroughly stirred under ice-water bath and nitrogen conditions, then cooled to 0°C. 3.0 g of 1,6-hexanediamine was dissolved in 50 mL of methanol and added dropwise to the reaction system, maintaining the reaction system temperature at 0–2°C throughout the process. After the addition was complete, the temperature was gradually increased to 25°C and the reaction continued for 24 hours. After the reaction was complete, the remaining ethylenediamine and solvent were evaporated using a rotary evaporator to obtain the intermediate product (PMMA-HEMA-GMA-PAMAM-DH microspheres).

[0034] (4) Fixed FA: The intermediate product obtained in step (3) was placed in a three-necked flask, and 3.0 g of sodium octanoate, 100 mL of acetone and 100 mL of MES buffer (pH 4.8) with a concentration of 0.1 mol / L were added. After mixing evenly, 5.0 g of HOBT and 5.0 g of EDC condensing agent were added to the reaction system. The reaction was carried out in a water bath at 37°C with shaking for 10 hours. After the reaction was completed, the product was washed with anhydrous ethanol and purified water in sequence. After cleaning, the final protein-bound toxin hemoperfusion adsorbent was obtained.

[0035] Example 2 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 8.00 g MMA monomer, 24.00 g HEMA monomer, and 6.00 g GMA were added to the aqueous phase system, bringing the total monomer mass to 38.00 g. 0.80 g of crosslinking agent EDMA was added, along with 3.00 mL toluene and 3.00 mL xylene as porogens. Subsequently, 0.50 g BPO was added as an initiator. The reaction was stirred at 65°C under nitrogen protection for 6 hours. After the reaction was complete, the support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0036] (2) Fixed load PAMAM: The carrier from step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 6.0 g of 1.5 generation PAMAM was added, and the mixture was stirred at 40 °C for 2 hours. After the reaction was completed, the PAMAM-supported carrier was obtained by filtration. The product was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0037] (3) Modification treatment of PAMAM: The product from step (2) and 150 mL of methanol were placed in a three-necked flask and thoroughly stirred under ice-water bath and nitrogen conditions, then cooled to 0°C. 6.0 g of 1,6-hexanediamine was dissolved in 50 mL of methanol and added dropwise to the reaction system, maintaining the reaction system temperature at 0–2°C throughout the process. After the addition was complete, the temperature was gradually increased to 25°C and the reaction continued for 24 hours. After the reaction was complete, the remaining ethylenediamine and solvent were evaporated using a rotary evaporator to obtain the intermediate product (PMMA-HEMA-GMA-PAMAM-DH microspheres).

[0038] (4) Fixed FA: The intermediate product obtained in step (3) was placed in a three-necked flask, and 6.0 g of decenoic acid, 100 mL of acetone and 100 mL of MES buffer (pH 4.8) with a concentration of 0.1 mol / L were added. After mixing evenly, 5.0 g of HOBT and 5.0 g of EDC condensing agent were added to the reaction system. The reaction was carried out in a water bath at 37°C with shaking for 10 hours. After the reaction was completed, the product was washed with anhydrous ethanol and purified water in sequence. After cleaning, the final protein-bound toxin hemoperfusion adsorbent was obtained.

[0039] Example 3 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 8.00 g MMA monomer, 20.00 g HEMA monomer, and 2.00 g GMA were added to the aqueous phase system, bringing the total monomer mass to 30.00 g. 0.50 g of crosslinking agent EDMA was added, along with 3.00 mL of toluene and 3.00 mL of xylene as porogens. Subsequently, 0.50 g of BPO was added as an initiator. The reaction was stirred at 65°C under nitrogen protection for 4 hours. After the reaction was complete, a support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0040] (2) Fixed load PAMAM: The carrier from step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 3.0 g of 1.5 generation PAMAM was added, and the mixture was stirred at 40 °C for 2 hours. After the reaction was completed, the PAMAM-supported carrier was obtained by filtration. The product was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0041] (3) Modification treatment of PAMAM: Take the product from step (2) and 150 mL of methanol and place them in a three-necked flask. Stir thoroughly under ice-water bath and nitrogen conditions, and then cool to 0°C. Dissolve 3.0 g of 1,6-hexanediamine in 50 mL of methanol and add it dropwise to the reaction system, maintaining the reaction system temperature at 0-2°C throughout the process. After the addition is complete, gradually raise the temperature to 25°C and continue the reaction for 24 hours. After the reaction is complete, evaporate the remaining ethylenediamine and solvent using a rotary evaporator to obtain the intermediate product.

[0042] (4) FA fixed load: The intermediate product obtained in step (3) was placed in a three-necked flask, and 8.0 g of linoleic acid (octadecadienoic acid), 100 mL of acetone and 100 mL of MES buffer (pH 4.8) with a concentration of 0.1 mol / L were added. After mixing evenly, 5.0 g of HOBT and 5.0 g of EDC condensing agent were added to the reaction system. The reaction was carried out in a water bath at 37°C with shaking for 12 hours. After the reaction was completed, the product was washed with anhydrous ethanol and purified water in sequence. After cleaning, the final protein-bound toxin hemoperfusion adsorbent was obtained.

[0043] Example 4 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 8.00 g MMA monomer, 18.00 g HEMA monomer, and 4.00 g GMA were added to the aqueous phase system, bringing the total monomer mass to 30.00 g. 0.50 g of crosslinking agent EDMA was added, along with 3.00 mL toluene and 3.00 mL xylene as porogens. Subsequently, 0.50 g of BPO was added as an initiator. The reaction was stirred at 65°C under nitrogen protection for 4 hours. After the reaction was complete, a support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0044] (2) Fixed load PAMAM: The carrier described in step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 3.0 g of 1.5 generation PAMAM was added, and the mixture was stirred and reacted at 40°C for 2 hours. After the reaction was completed, the PAMAM-supported carrier was obtained by filtration. The product was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0045] (3) Modification treatment of PAMAM: The product from step (2) and 150 mL of methanol were placed in a three-necked flask and thoroughly stirred under an ice-water bath and nitrogen atmosphere, then cooled to 0°C. 3.0 g of 1,6-hexanediamine was dissolved in 50 mL of methanol and added dropwise to the reaction system, maintaining the reaction system temperature at 0–2°C throughout the process. After the addition was complete, the temperature was gradually increased to 25°C and the reaction continued for 24 hours. After the reaction was complete, the remaining ethylenediamine and solvent were distilled off using a rotary evaporator to obtain the intermediate product.

[0046] (4) FA fixed load: 10.0 g of the intermediate product obtained in step (3) was placed in a three-necked flask, and 4.0 g of docosahexaenoic acid (DHA), 100 mL of acetone and 100 mL of MES buffer (pH 4.8) with a concentration of 0.1 mol / L were added. After mixing evenly, 5.0 g of HOBT and 5.0 g of EDC condensing agent were added to the reaction system. The reaction was carried out in a water bath at 37°C with shaking for 12 hours. After the reaction was completed, the product was washed with anhydrous ethanol and purified water in sequence. After cleaning, the final protein-bound toxin hemoperfusion adsorbent was obtained.

[0047] Example 5 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 10.00 g MMA monomer, 20.00 g HEMA monomer, and 6.00 g GMA were added to the aqueous phase system, bringing the total monomer mass to 36.00 g. 0.80 g of crosslinking agent EDMA was added, along with 3.00 mL toluene and 3.00 mL xylene as porogens. Subsequently, 0.50 g BPO was added as an initiator. The reaction was stirred at 65°C under nitrogen protection for 6 hours. After the reaction was complete, a support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0048] (2) Fixed load PAMAM: The carrier described in step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 4.0 g of 1.5 generation PAMAM was added, and the mixture was stirred and reacted at 40°C for 2 hours. After the reaction was completed, the PAMAM-supported carrier was obtained by filtration. The product was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0049] (3) Modification treatment of PAMAM: The product from step (2) and 150 mL of methanol were placed in a three-necked flask and thoroughly stirred under an ice-water bath and nitrogen atmosphere, then cooled to 0°C. 4.0 g of 1,6-hexanediamine was dissolved in 50 mL of methanol and added dropwise to the reaction system, maintaining the reaction system temperature at 0–2°C throughout the process. After the addition was complete, the temperature was gradually increased to 25°C and the reaction continued for 24 hours. After the reaction was complete, the remaining ethylenediamine and solvent were distilled through a rotary evaporator to obtain the intermediate product.

[0050] (4) Fixed FA: The intermediate product obtained in step (3) was placed in a three-necked flask, and 8.0 g of sodium myristate, 100 mL of acetone and 100 mL of MES buffer (pH 4.8) with a concentration of 0.1 mol / L were added. After mixing evenly, 5.0 g of HOBT and 5.0 g of EDC condensing agent were added to the reaction system. The reaction was carried out in a water bath at 37°C with shaking for 12 hours. After the reaction was completed, the product was washed with anhydrous ethanol and purified water in sequence. After cleaning, the final protein-bound toxin hemoperfusion adsorbent was obtained.

[0051] Example 6 The adsorbents prepared in Examples 1-5 above were subjected to static adsorption tests of protein-bound toxins.

[0052] IS, pCS, Ha, and IAA were used as representative protein-bound toxins (PBUTs) in the experiment. 1.0 mL of each of the wet adsorbents from Examples 1, 2, 3, 4, and 5 were placed in conical flasks, and 10.0 mL of plasma containing 50 mg / L IS, 50 mg / L pCS, 100 mg / L Ha, and 2 mg / L IAA were added to each flask. The flasks were shaken for 2 hours (37°C, 100±10 rpm) to adsorb the adsorbents. The concentrations of IS, pCS, Ha, and IAA in the plasma after adsorption were measured, and the adsorption rates of the adsorbents for IS, pCS, Ha, and IAA were calculated.

[0053] Table 1. Results of Static Adsorption Test

[0054] The adsorption test results are shown in Table 1. The amount of PAMAM fed and the adsorbents with different fatty acids FA immobilized on it have a slight effect on the adsorption effect of IS, pCS and Ha.

[0055] Example 7 The adsorbents prepared in Examples 1-5 above were used in a hemoperfusion combined with hemodialysis experiment.

[0056] A 1000 mL albumin phosphate solution containing 50 mg / L IS, 50 mg / L pCS, 100 mg / L Ha, and 2 mg / L IAA was prepared as the test solution. 50 mL of the adsorbents from Examples 1, 2, 3, 4, and 5 were used to prepare hemoperfusion devices. The hemoperfusion device was connected in series with a hemodialysis machine using a blood circuit tube, forming a loop with the test solution. Dialysis solution prepared with bicarbonate dialysis powder was used to simulate hemoperfusion in a series with hemodialysis to remove protein-bound toxins. The system was circulated at 37°C for 2 hours. The concentration of PBUTs in the test solution was measured, and the clearance rate was calculated. High-flux hemodialysis machines (without the hemoperfusion device) HD-1, HD-2, and HD-3 were tested using the same method, and the concentration of PBUTs in the test solution was measured. The results were compared between the two methods.

[0057] Table 2. Results of protein-bound toxin clearance assay

[0058] The experimental results are shown in Table 2. The effect of hemoperfusion combined with hemodialysis on the removal of protein-bound toxins is better than that of high-flux hemodialysis alone.

[0059] Example 8 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 8.00 g MMA monomer, 24.00 g HEMA monomer, and 4.00 g GMA were added to the aqueous phase system, along with 0.80 g of crosslinking agent EDMA, bringing the total monomer mass to 36.00 g. 3.00 mL toluene and 3.00 mL xylene were added as porogens, followed by 0.50 g BPO as an initiator. The reaction was carried out at 65°C under nitrogen protection with stirring for 6 hours. After the reaction was complete, the support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0060] (2) Fixed load PAMAM: The carrier described in step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 3.0 g of 1.5 generation PAMAM was added, and the mixture was stirred and reacted at 40℃ for 2 hours. After the reaction was completed, the adsorbent immobilized with PAMAM was obtained by filtration. The adsorbent was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0061] Example 9 (1) Synthetic carrier: 0.50 g PVA and 1.00 g PEG were dissolved in 100 mL of purified water and placed in a three-necked flask. Nitrogen gas was introduced, and the mixture was stirred at 65°C for 30 minutes to form an aqueous phase system. 8.00 g MMA monomer, 24.00 g HEMA monomer, and 4.00 g GMA were added to the aqueous phase system, along with 0.80 g of crosslinking agent EDMA, bringing the total monomer mass to 36.00 g. 3.00 mL of toluene and 3.00 mL of xylene were added as porogens, followed by 0.50 g of BPO as an initiator. The reaction was carried out at 65°C under nitrogen protection with stirring for 6 hours. After the reaction was complete, the support was obtained. The support was washed successively with anhydrous ethanol and purified water, and then sieved to obtain a support with a suitable particle size distribution for later use.

[0062] (2) Immobilized polyethyleneimine: The carrier described in step (1) was placed in a three-necked flask, and 50 mL of DMSO and 100 mL of 0.8 mol / L NaOH solution were added sequentially. After stirring evenly, 2.0 g of polyethyleneimine was added, and the mixture was stirred and reacted at 40°C for 2 hours. After the reaction was completed, the adsorbent with polyethyleneimine immobilized on its surface was obtained by filtration. The adsorbent was washed sequentially with purified water, anhydrous ethanol, and purified water, and then dried for later use.

[0063] Example 10 The hemolysis rate of the adsorbents in cases 1, 2, 3, 4, 5, 8, and 9 was examined according to the hemolysis test method specified in GB / T 14233.2. The test results are shown in Table 3.

[0064] Table 3. Adsorbent Hemolysis Test

[0065] The hemolysis rate of the adsorbents was less than 5%, which meets the requirements of standard GB / T 16886.4, indicating that the adsorbents have good blood compatibility. However, the hemolysis rate of adsorbents with a large number of positive charges on their surface (Examples 8 and 9) was relatively high when they came into direct contact with blood cells.

[0066] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. An adsorbent, characterized in that, The invention includes a carrier, the raw material of which has epoxy groups, and the carrier is supported by the epoxy groups on which modified hyperbranched polyamide-amine is immobilized. The modified hyperbranched polyamide-amine is obtained by modifying a half-generation product of hyperbranched polyamide-amine with 1,6-hexanediamine. The modified hyperbranched polyamide-amine is supported by fatty acids through a condensation reaction of amine and carboxylic acid.

2. The adsorbent according to claim 1, characterized in that, The raw materials for the carrier include methyl methacrylate, hydroxyethyl methacrylate and glycidyl methacrylate. The porous microspheres copolymerized from the raw materials have a particle size of 200~1000μm and a pore size of 2~50 nm.

3. The adsorbent according to claim 2, characterized in that, The mass ratio of the hyperbranched polyamide-amine to the glycidyl methacrylate monomer is (0.5~5):

1.

4. The adsorbent according to claim 2 or 3, characterized in that, The methyl methacrylate monomer accounts for 1-40% of the total monomer mass, the hydroxyethyl methacrylate monomer accounts for 40-85% of the total monomer mass, the glycidyl methacrylate monomer accounts for 1-20% of the total monomer mass, and the crosslinking agent ethylene glycol dimethacrylate accounts for 1-5% of the total monomer mass.

5. The adsorbent according to claim 1, characterized in that, The hyperbranched polyamide-amine has a generation number of 0.5, 1.5, 2.5 or 3.5, preferably 1.

5.

6. The adsorbent according to claim 1, characterized in that, The mass ratio of the 1,6-hexanediamine to the hyperbranched polyamide-amine is (0.5~5):

1.

7. The adsorbent according to claim 1, characterized in that, The fatty acid is selected from one or more of saturated fatty acids, unsaturated fatty acids, straight-chain fatty acids, branched-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, or their corresponding fatty acid salts; preferably, the carbon chain of the fatty acid is C6~C6. 22 .

8. The adsorbent according to claim 6 or 7, characterized in that, The mass ratio of the fatty acid to the 1,6-hexanediamine is (0.5~4):

1.

9. The method for preparing the adsorbent according to claim 1, characterized in that, Including the following steps: (1) Synthetic carrier; (2) Immobilized hyperbranched polyamide-amine; (3) Modification of hyperbranched polyamide-amine: 1,6-hexanediamine was added to the product of step (2) under ice-water bath and protective atmosphere, and then the temperature was gradually increased to 25°C until the reaction was completed. (4) Immobilized fatty acids: Add fatty acids and condensing agents to the product of step (3), and wash the product after the reaction is complete to obtain the adsorbent.

10. The use of the adsorbent according to any one of claims 1 to 8 in the preparation of hemoperfusion products.

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