A blood perfusion adsorbent for adsorbing endotoxins by dual-action mechanism, its preparation method and application

Abstract

This invention discloses a hemoperfusion adsorbent for adsorbing endotoxins through a dual-action mechanism, its preparation method, and its application. The adsorbent (PS-DVB@p(CD-His)) is produced by Raft polymerization of 4-cyano-4-(thiobenzoic acid)valeric acid grafted onto the surface of PS-DVB to generate a glycidyl methacrylate brush polymer. It is then combined with L-histidine-modified cyclodextrin functional molecules, utilizing the positive charge of the histidine groups and the hydrophobic cavities of the cyclodextrin to achieve synergistic and efficient adsorption of endotoxins through electrostatic and hydrophobic interactions. The hemoperfusion adsorbent material of this invention is readily available, inexpensive, and exhibits good blood compatibility and endotoxin adsorption performance. The development of this adsorbent contributes to the extracorporeal hemoperfusion treatment of sepsis patients, reducing clinical treatment costs.

Description

Technical Field

[0001] This invention belongs to the field of blood perfusion adsorbent technology, and specifically relates to a blood perfusion adsorbent for adsorbing endotoxins by dual-action mechanisms, its preparation method, and its application. Background Technology

[0002] Sepsis is a life-threatening acute organ dysfunction syndrome caused by a dysregulation of the response to bloodstream bacterial infection, and is a major global health problem. Endotoxin (also known as lipopolysaccharide LPS), a component of the cell wall of Gram-negative bacteria, is composed of lipid A, core oligosaccharides, and O-antigen. It is a key pathogenic factor in inducing sepsis. LPS infection further activates Kupffer cells in the liver, inducing the production of TNF-α, interferon-γ (INF-γ), IL-1β, IL-6, IL-8, and other cytokines, resulting in an uncontrollable cascade reaction. This leads to systemic inflammation, cell necrosis, and apoptosis, and exacerbates microcirculatory disturbances and oxidative stress, resulting in multiple organ failure. In severe cases, it can develop into septic shock and even death.

[0003] Hemoperfusion is a common treatment for sepsis. Its core principle lies in the specific adsorption of blood toxins by the adsorbent in the perfusion column. Depending on the toxin type, the adsorption forces include covalent bonds, electrostatic interactions, hydrophobic interactions, hydrogen bonds, metal coordination bonds, π-π and cation-π interactions, etc. However, traditional ordinary resin perfusion columns (polystyrene resin) rely solely on hydrophobic interactions, resulting in poor specific adsorption of endotoxins.

[0004] Polymyxin B, as a polypeptide antibiotic, possesses a large number of amino groups that can effectively electrostatically adsorb endotoxins. Although it can effectively and specifically bind to endotoxin phosphate anion lipid A through electrostatic interactions, polymyxin B, as an antibiotic, has extremely strong nephrotoxicity and neurotoxicity. Leakage can exacerbate kidney damage or neurological dysfunction in sepsis patients. In addition, the widespread use of polymyxin B may induce drug-resistant bacteria, which makes the application of polymyxin B perfusion columns fraught with many potential risks.

[0005] Although many adsorbent materials with excellent adsorption properties have been developed, such as chitosan, polylysine, peptides, polyethyleneimine, and polycaprolactone, which have hydrophobic or positively charged effects, they all have limitations, including poor blood compatibility, low clearance efficiency, and poor specificity.

[0006] To overcome these difficulties, there is an urgent clinical need for novel adsorbent molecules that can efficiently adsorb endotoxins and possess excellent biocompatibility, making sepsis hemoperfusion safer and more efficient. Most current research focuses on polymyxin B and the development of positively charged surface materials, lacking research on the molecular design of molecules that specifically bind to endotoxins. Summary of the Invention

[0007] In view of the current situation where hemoperfusion adsorbents for the clinical treatment of sepsis are mainly positively charged electrostatic adsorption (polymyxin B), the primary objective of this invention is to provide a hemoperfusion adsorbent for adsorbing endotoxins by dual forces and its preparation method.

[0008] Another objective of this invention is to provide an application of a hemoperfusion adsorbent that adsorbs endotoxins through dual-action mechanisms, offering an innovative and efficient application scheme for histidine-esterified cyclodextrin molecular adsorbents in sepsis hemoperfusion adsorption.

[0009] The objective of this invention is achieved through the following scheme.

[0010] A method for preparing a blood perfusion adsorbent includes the following steps:

[0011] (1) Polystyrene divinylbenzene microspheres, photoinitiator, β-mercaptoethylamine and solvent are mixed and subjected to ultraviolet light-initiated reaction to obtain aminated polystyrene divinylbenzene microspheres;

[0012] (2) The aminated polystyrene divinylbenzene microspheres, chain transfer reagent and solvent are mixed, and a crosslinking agent is added to catalyze the grafting reaction to obtain polystyrene divinylbenzene microspheres grafted with chain transfer reagent;

[0013] (3) The polystyrene divinylbenzene microspheres with the grafted chain transfer reagent are placed in a solvent containing glycidyl methacrylate, and a thermal initiator is added to initiate free radical polymerization to obtain polystyrene divinylbenzene microspheres with glycidyl methacrylate chain brushes on the surface.

[0014] (4) The polystyrene divinylbenzene microspheres with glycidyl methacrylate chain brushes on the surface, modified cyclodextrin and solvent are mixed, a catalyst is added, and the mixture is heated to obtain histidine esterified cyclodextrin modified polystyrene divinylbenzene microspheres, which are used as blood perfusion adsorbents.

[0015] The modified cyclodextrin is histidine-esterified cyclodextrin.

[0016] Furthermore, the cyclodextrin in the modified cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0017] Furthermore, in step (1),

[0018] The polystyrene-divinylbenzene microspheres are porous microspheres with nanopores, residual double bonds on the surface, and a particle size of 100~1000 μm, preferably 200~800 μm;

[0019] The photoinitiator is any one of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoin dimethyl ether, and benzophenone;

[0020] The solvent is any one of N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane, preferably N,N-dimethylformamide;

[0021] The mass ratio of polystyrene divinylbenzene microspheres, β-mercaptoethylamine, and photoinitiator in the reaction was (1~10):(1~10):1, which was optimized to be 8:(1~10):1.

[0022] The reaction temperature is 20~60℃ and the reaction time is 0.5~2 h, optimized to 30~50℃ and the reaction time is 0.5~1 h.

[0023] Furthermore, in step (2),

[0024] The chain transfer reagent is 4-cyano-4-(thiobenzoic acid)valerate;

[0025] The crosslinking agent is at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide;

[0026] The solvent is any one of N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane;

[0027] The molar ratio of amino groups to chain transfer reagent and crosslinking agent in the aminated polystyrene divinylbenzene microspheres of the reaction is 1:(1~4):(1~10), preferably 1:(1~2):(1~5).

[0028] Furthermore, the concentration of the chain transfer reagent is 10~40 mg / mL;

[0029] The reaction temperature is 20~50 ℃ and the reaction time is 1~48 h, optimized to 20~40 ℃ and 1~24 h.

[0030] Furthermore, in step (3),

[0031] The thermal initiator is any one of benzoyl peroxide, dibenzoyl peroxide, and azobisisobutyronitrile;

[0032] The solvent is any one of N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide, and is optimized to be N,N-dimethylformamide;

[0033] The mass ratio of the grafted chain transfer reagent, polystyrene divinylbenzene microspheres, glycidyl methacrylate, and thermal initiator is 1:(1~5):(0.01~0.05), preferably 1:(1~2):(0.01~0.02).

[0034] Furthermore, the molar concentration of glycidyl methacrylate was optimized to be 1~5 mol / L;

[0035] Furthermore, the molar concentration of the thermal initiator is 1~10 mmol / L;

[0036] The reaction temperature is optimized from 40~100℃ to 50~80℃, and the reaction time is 12~36 h, preferably 12~24 h.

[0037] Furthermore, in step (4),

[0038] The catalyst is any one of triethylamine, triethanolamine, and benzyldimethylamine;

[0039] The mass ratio of polystyrene divinylbenzene microspheres with glycidyl methacrylate chain brushes on the surface, modified cyclodextrin, and catalyst is 1:(0.5~2):(0.5~2), optimized to 1:(0.5~1):(0.5~1).

[0040] Furthermore, the mass concentration of the modified cyclodextrin is optimized from (1~100) g / L to (10~40) g / L.

[0041] Preferably, the volume concentration of the catalyst is 2 v / v.

[0042] The reaction temperature is 20~60℃, optimized to 20~40℃, and the reaction time is 1~24 h, preferably 12~24 h.

[0043] Furthermore, the preparation of the modified cyclodextrin includes the following steps:

[0044] (1) The three cyclodextrins α, β and γ were respectively mixed and dissolved with N,N'-di-tert-butoxycarbonyl-L-histidine containing a protecting group and a solvent. After adding a catalyst, the mixture was stirred and heated to react, and three tert-butoxycarbonylhistidine esterified cyclodextrins were obtained.

[0045] (2) Mix the α, β and γ tert-butoxycarbonyl histidine esterified cyclodextrins with solvent, add a deprotecting agent to remove the tert-butoxycarbonyl group, and obtain the three histidine esterified cyclodextrins.

[0046] Furthermore, in step (1),

[0047] The molar ratio of the three cyclodextrins (α, β, and γ) to N,N'-di-tert-butoxycarbonyl-L-histidine is (0.1~1):(1~3); it is further optimized to (0.1~0.2):(1~2).

[0048] The solvent is any one of N,N-dimethylformamide, dichloromethane, and dimethyl sulfoxide; it is further optimized to be N,N-dimethylformamide.

[0049] The catalyst is at least two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine, in a mass ratio of (1~10):(0.1~1); optimized to (2~4):(0.1~0.5).

[0050] The reaction temperature is 20~60 ℃, and the reaction time is 1~48 h, preferably 16~32 h.

[0051] In step (2),

[0052] The solvent is any one of N,N-dimethylformamide, dichloromethane, and dimethyl sulfoxide; it is optimized to be N,N-dimethylformamide.

[0053] The deprotecting agent is either a trifluoroacetic acid-dichloromethane mixed solvent or a dioxane-hydrochloric acid mixed solvent; preferably, it is a dioxane-hydrochloric acid mixed solvent, with a mass ratio of hydrochloric acid to dioxane of (0.5~1.5):6.

[0054] The volume ratio of the solvent to the deprotection reagent is (0.1~1):1; optimized to 1:1.

[0055] The reaction temperature is 0~20 ℃ and the reaction time is 0.5~4 h; the reaction temperature is optimized to 0~4 ℃ and the reaction time is optimized to 1~3 h.

[0056] A blood perfusion adsorbent prepared by any of the above preparation methods.

[0057] The above-described application of a blood perfusion adsorbent in the preparation of drugs for removing endotoxins.

[0058] This invention is based on the properties of endotoxin anionic lipid A, utilizing highly blood-compatible substances such as natural proteins or polysaccharides, combined with electrostatic and hydrophobic interactions to design adsorbent molecules. Therefore, based on the above analysis, this invention inspires the design of a molecule that synergistically adsorbs endotoxins through hydrophobic and electrostatic interactions. This molecule possesses excellent endotoxin neutralization capabilities and, when grafted onto the surface of an adsorbent material, also achieves highly efficient adsorption of endotoxins. This adsorbent molecule and its adsorbent material can effectively remove endotoxins from the blood, achieving effective treatment for sepsis patients, and eliminating the potential risks associated with polymyxin B by replacing it.

[0059] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0060] 1. This invention addresses the potential risks of neurotoxicity and nephrotoxicity associated with traditional polymyxin B fiber membrane adsorption columns by designing a hemoperfusion adsorbent that combines cyclodextrin polysaccharide molecules and basic amino acids as adsorbent molecules, exhibiting excellent cell compatibility and blood compatibility.

[0061] 2. This invention is the first to design and synthesize this blood perfusion adsorbent. Compared with traditional adsorbents that bind to endotoxins only through electrostatic interactions, this adsorbent utilizes an esterification reaction to consume the negative charge of the carboxyl group, forming a hydrophobic ester group. It combines the hydrophobic interaction force of the hydrophobic cavity of the cyclodextrin molecule with the electrostatic interaction force of the residual amino group of histidine and imidazole, achieving a dual-force binding to the phosphorylated lipid A of the toxic center of the endotoxin molecule to neutralize its toxicity. It has the potential to be used as an adsorbent for the treatment of sepsis.

[0062] 3. This invention utilizes polystyrene-divinylbenzene microspheres as a substrate and employs a novel method to prepare a blood perfusion adsorbent. First, aminated microspheres are prepared via an olefin-thiol click chemistry reaction. Then, Raft chain transfer reagent is grafted via an amidation reaction, followed by in-situ polymerization of glycidyl methacrylate molecular brushes using Raft free radical polymerization. Finally, a ring-opening reaction is used to achieve modified molecular grafting, yielding the microsphere adsorbent. This modification method maintains the blood compatibility of the microspheres and imparts excellent hydrophilicity, reducing protein adsorption and significantly enhancing their endotoxin adsorption capacity, demonstrating great potential in the treatment of sepsis. Attached Figure Description

[0063] The following is a brief introduction to the required drawings in the implementation examples to more clearly illustrate the technical solution of the present invention. The drawings are only a part of the embodiments of the present invention and are not a limitation of the scope. Therefore, other related drawings can be obtained by other people skilled in the art based on these drawings without creative design.

[0064] Figure 1This diagram illustrates the synthetic route of an adsorbent molecule that synergistically adsorbs endotoxins through electrostatic and hydrophobic interactions, and the preparation of its blood perfusion adsorbent. The degree of polymerization (x) of the polyglycidyl methacrylate (pGMA) generated by Raft polymerization on the surface of the microspheres is approximately 100-150.

[0065] Figure 2 This is the 1H NMR spectrum of the histidine esterified cyclodextrin adsorbed molecules in Example 1 of the present invention.

[0066] Figure 3 This is a diagram illustrating the endotoxin toxicity neutralization effect using β-CD-His as an example in Example 2 of the present invention.

[0067] Figure 4 The images show the Fourier transform infrared spectra of the modified microsphere adsorbents obtained in Examples 3-5 of this invention.

[0068] Figure 5 The X-ray photoelectron spectra of the modified microsphere adsorbents obtained in Examples 3-5 of this invention are shown.

[0069] Figure 6 The X-ray photoelectron spectroscopy (XPS) C1s peak fitting spectra of the modified microsphere adsorbents obtained in Examples 3-5 of this invention are shown.

[0070] Figure 7 The X-ray photoelectron spectroscopy (XPS) N1s peak fitting spectra of the modified microsphere adsorbents obtained in Examples 3-5 of this invention are shown.

[0071] Figure 8 The graph shows the zeta potential results of the modified microsphere adsorbents obtained in Examples 3-5 of this invention.

[0072] Figure 9 This is a graph showing the endotoxin adsorption results of the modified microsphere adsorbent in Example 6 of the present invention.

[0073] Figure 10 This is a graph showing the anti-protein adsorption results of the modified microsphere adsorbent in Example 7 of the present invention.

[0074] Figure 11 This is a graph showing the hemolysis rate of the modified microsphere adsorbent in Example 8 of the present invention.

[0075] Figure 12 This is a graph showing the clotting time results of the modified microsphere adsorbent in Example 9 of the present invention.

[0076] Figure 13 This is a diagram showing the cell compatibility results of the modified microsphere adsorbent in Example 10 of the present invention.

[0077] Figure 14 This is a graph showing the endotoxin and protein competitive adsorption results of the modified microspheres in Example 11 of this invention. Detailed Implementation

[0078] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below with reference to implementation examples and accompanying drawings. However, the described implementation examples are only a part and not all embodiments. Therefore, based on the implementation examples of the present invention, all other implementation examples obtained by other skilled in the art without innovative labor results should fall within the scope of protection of the present invention.

[0079] All the raw materials used in the following examples are commercially available.

[0080] This invention relates to the synthesis of a histidine esterified cyclodextrin molecule that synergistically adsorbs endotoxins through hydrophobic and electrostatic interactions, and its preparation and application as a blood perfusion adsorbent. The adsorbent uses polyethylene divinylbenzene microspheres (PS-DVB) as a substrate, a brush-like polymer as an adsorption layer, and histidine esterified cyclodextrin as the adsorbent molecule to form the final perfusion material.

[0081] In this embodiment of the invention, polystyrene divinylbenzene microspheres undergo reactions at different temperatures, including click chemistry of mercaptoolefins to achieve amination, amidation to achieve chain transfer reagent grafting, Raft polymerization to generate brush-like chains in situ, and ring-opening reaction to graft adsorbent molecules to obtain the final adsorption layer. The finished product is washed with anhydrous ethanol and endotoxin-free water, then vacuum dried and stored in a sealed drying oven.

[0082] Example 1: Synthesis of histidine-esterified cyclodextrin (α, β, γ-CD-His)

[0083] 1. Weigh 1.46 g α-CD, 1.74 g β-CD, and 1.95 g γ-CD into round-bottom flasks, respectively, and add 3.74 g N,N'-di-tert-butoxycarbonyl-L-histidine to each flask. Stir and dissolve the solutions in 50 mL of DMF.

[0084] 2. Weigh 2.61 g of dicyclohexylcarbodiimide and 0.242 g of 4-dimethylaminopyridine and add them to the reaction flasks respectively, and continue stirring until completely dissolved.

[0085] 3. The mixture was stirred at 40℃ for 24 h, and the white precipitate was removed by vacuum filtration. The supernatant was transferred to a dialysis bag and dialyzed for 3 days, with the deionized water (DIW) being changed 3 times a day. The product in the dialysis bag was collected, freeze-dried for 72 h, and then ground to obtain tert-butoxycarbonylhistidine esterified cyclodextrin (α,β,γ-CD-His(Boc)2).

[0086] 4. Weigh 2 g of α, β, and γ-CD-His(Boc)2 into beakers respectively, add 10 mL of DCM to each and stir to dissolve. Then, add 10 mL of 4 M dioxane hydrochloride under ice bath conditions, stir for 1 h, precipitate the product with diethyl ether, filter and collect the precipitate, and dry it under vacuum at 40 °C for 24 h to obtain three L-histidine esterified cyclodextrins (α-CD-His, β-CD-His, and γ-CD-His).

[0087] A small amount of the above product powder was dissolved in deuterated DMSO and analyzed by 1H NMR spectroscopy. Figure 2 The results of the 1H NMR spectrum (using β-CD as an example to prove the effectiveness of the synthesis process) show that after grafting N,N'-di-tert-butoxycarbonyl-L-histidine, β-CD-His(Boc)2 exhibits characteristic hydrogen peaks of histidine and the Boc protecting group. After deprotection, the hydrogen peak of the Boc group disappears significantly, while the characteristic peaks of histidine and cyclodextrin are retained, proving that β-CD-His was successfully synthesized. The same applies to the other two cyclodextrins.

[0088] Example 2: Endotoxin neutralization experiment using β-CD-His as an example

[0089] 1. Weigh 2 mg of β-CD-His into a 1.5 mL pyrogen-free centrifuge tube, add 1 mL of bacterial endotoxin test water to dissolve it, transfer it to a 15 mL centrifuge tube, and then add 9 mL of bacterial endotoxin test water to dilute it 10 times to obtain a 200 μg / mL neutralization solution. In addition, prepare a polymyxin B (PMB) solution of the same mass concentration as a control to evaluate the endotoxin toxicity neutralization ability of β-CD-His.

[0090] 2. Take 100 EU endotoxin (LPS) standard (Xiamen Limulus Amebocyte Lysate Reagent Biotechnology Co., Ltd.) and dissolve it according to the instructions to prepare a 5 EU / mL endotoxin solution for later use.

[0091] 3. Take a new pyrogen-free 1.5 mL centrifuge tube, add 300 μL of LPS solution, then add 300 μL of β-CD-His solution and mix well. The PMB group follows the same steps as above. Use 600 μL of pure LPS solution as a blank control. All groups are incubated in a constant temperature shaker at 37℃ for 1 h.

[0092] 4. Take 10 μL of each incubated solution and dilute it 10 times in a new pyrogen-free centrifuge tube. Then take 10 μL of the diluted solution and use the endotoxin detection kit (Beyotime) to determine its activity. The activity and toxicity of LPS are evaluated by measuring the absorbance after color development using the kit.

[0093] Figure 3The figure shows the results of the LPS toxicity neutralization experiment. It can be seen that there is almost no significant difference between the blank group and the PMB control group. Although the high positive charge of PMB can electrostatically bind the negative charge of LPS phosphate, enabling it to have an LPS adsorption effect on the material surface, it cannot mask the lipid A toxicity center of LPS. The absorbance of the β-CD-His group is significantly lower than that of the blank group and the PMB group, indicating that β-CD-His effectively binds the phosphorylated lipid A toxicity center of LPS, neutralizes the toxicity of LPS, and has the potential to act as an LPS adsorbent molecule.

[0094] Example 3: Preparation of histidine-esterified cyclodextrin-modified polystyrene-divinylbenzene microsphere adsorbent (PS@p(α-CD-His))

[0095] 1. Place 1 g PS-DVB microspheres, 0.8 g mercaptoethylamine, and 0.13 g I2959 photoinitiator into a 150 mL round-bottom flask, add 20 mL DMF, and stir to dissolve the mercaptoethylamine and photoinitiator to obtain the reaction solution.

[0096] 2. The reaction solution was sealed and protected from light. Then, a vacuum pump was used to remove oxygen from the reaction flask. After vacuuming, nitrogen gas was purged through the reaction solution. This process was repeated 3 times, followed by purging with nitrogen gas for 30 minutes. Finally, the reaction was stirred and reacted for 1 hour under 500 W UV light. The supernatant was removed, and the mixture was washed several times with DMF, anhydrous ethanol, and ultrapure water. After vacuum drying, microspheres 1 (PS@NH2) were obtained.

[0097] 3. Place 1 g of microsphere 1, 100 mg of 4-cyano-4-(thiobenzoic acid)valeric acid (CPPA), EDC and NHS at a molar ratio of 5 times that of CPPA in a 50 mL round-bottom flask, add 15 mL of DMF and stir to dissolve CPPA and crosslinking agent, stir and react at room temperature for 24 h, wash with DMF until the solution is clear, wash several times with ultrapure water, and collect microsphere 2 (PS@CPPA) by vacuum drying.

[0098] 4. Dissolve 10 mmol of glycidyl methacrylate (GMA) in 15 mL of DMF, then add 20.5 mg of azobisisobutyronitrile and stir to dissolve to obtain a polymerization reaction solution. Add 0.5 g of microspheres 2 and seal. Vacuum-nitrogen purging cycle 3 times, then purge with nitrogen for 30 min to remove oxygen. React in an oil bath at 70 ℃ under nitrogen atmosphere for 24 h with stirring. Wash 2-3 times with DMF, anhydrous ethanol and ultrapure water alternately. Collect microspheres 3 (PS@p(GMA)) after vacuum drying for 24 h.

[0099] 5. Place 0.2 g α-CD-His in a 50 mL round-bottom flask, dissolve it by sonication in 10 mL DMF, add 0.2 g microspheres 3 and 0.2 mL triethylamine catalyst, stir the reaction for 24 h, wash 2-3 times with DMF, anhydrous ethanol and ultrapure water, and vacuum dry for 24 h to collect the histidine esterified cyclodextrin modified polystyrene divinylbenzene microsphere adsorbent (PS@p(α-CD-His)).

[0100] Example 4: Preparation of histidine-esterified cyclodextrin-modified polystyrene divinylbenzene microsphere adsorbent (PS@p(β-CD-His))

[0101] 1. Place 1 g PS-DVB microspheres, 0.8 g mercaptoethylamine, and 0.13 g I2959 photoinitiator into a 150 mL round-bottom flask, add 20 mL DMF, and stir to dissolve the mercaptoethylamine and photoinitiator to obtain the reaction solution.

[0102] 2. The reaction solution was sealed and protected from light. Then, a vacuum pump was used to remove oxygen from the reaction flask. After vacuuming, nitrogen gas was purged through the reaction solution. This process was repeated 3 times, followed by purging with nitrogen gas for 30 minutes. Finally, the reaction was stirred and reacted for 1 hour under 500 W UV light. The supernatant was removed, and the mixture was washed several times with DMF, anhydrous ethanol, and ultrapure water. After vacuum drying, microspheres 1 (PS@NH2) were obtained.

[0103] 3. Place 1 g of microsphere 1, 100 mg of 4-cyano-4-(thiobenzoic acid)valeric acid (CPPA), EDC and NHS at a molar ratio of 5 times that of CPPA in a 50 mL round-bottom flask, add 15 mL of DMF and stir to dissolve CPPA and crosslinking agent, stir and react at room temperature for 24 h, wash with DMF until the solution is clear, wash several times with ultrapure water, and collect microsphere 2 (PS@CPPA) by vacuum drying.

[0104] 4. Dissolve 10 mmol glycidyl methacrylate (GMA) in 15 mL DMF, then add 20.5 mg azobisisobutyronitrile and stir to dissolve to obtain a polymerization reaction solution. Add 0.5 g microspheres 2 and seal. Vacuum-nitrogen purging cycle 3 times, then purge with nitrogen for 30 min to remove oxygen. React in an oil bath at 70 ℃ under nitrogen atmosphere for 24 h with stirring. Wash with DMF, anhydrous ethanol and ultrapure water 2-3 times alternately. Collect microspheres 3 (PS@p(GMA)) after vacuum drying for 24 h.

[0105] 5. Place 0.2 g β-CD-His in a 50 mL round-bottom flask, dissolve it by sonication in 10 mL DMF, add 0.2 g microspheres 3 and 0.2 mL triethylamine catalyst, stir the reaction for 24 h, wash with DMF, anhydrous ethanol and ultrapure water 2-3 times, and vacuum dry for 24 h to collect the histidine esterified cyclodextrin modified polystyrene divinylbenzene microsphere adsorbent (PS@p(β-CD-His)).

[0106] Example 5: Preparation of histidine-esterified cyclodextrin-modified polystyrene-divinylbenzene microsphere adsorbent (PS@p(γ-CD-His))

[0107] 1. Place 1 g PS-DVB microspheres, 0.8 g mercaptoethylamine, and 0.13 g I2959 photoinitiator into a 150 mL round-bottom flask, add 20 mL DMF, and stir to dissolve the mercaptoethylamine and photoinitiator to obtain the reaction solution.

[0108] 2. The reaction solution was sealed and protected from light. Then, a vacuum pump was used to remove oxygen from the reaction flask. After vacuuming, nitrogen gas was purged through the reaction solution. This process was repeated 3 times, followed by purging with nitrogen gas for 30 minutes. Finally, the reaction was stirred and reacted for 1 hour under 500 W UV light. The supernatant was removed, and the mixture was washed several times with DMF, anhydrous ethanol, and ultrapure water. After vacuum drying, microspheres 1 (PS@NH2) were obtained.

[0109] 3. Place 1 g of microsphere 1, 100 mg of 4-cyano-4-(thiobenzoic acid)valeric acid (CPPA), EDC and NHS at a molar ratio of 5 times that of CPPA in a 50 mL round-bottom flask, add 15 mL of DMF and stir to dissolve CPPA and crosslinking agent, stir and react at room temperature for 24 h, wash with DMF until the solution is clear, wash several times with ultrapure water, and collect microsphere 2 (PS@CPPA) by vacuum drying.

[0110] 4. Dissolve 10 mmol glycidyl methacrylate (GMA) in 15 mL DMF, then add 20.5 mg azobisisobutyronitrile and stir to dissolve to obtain a polymerization reaction solution. Add 0.5 g microspheres 2 and seal. Vacuum-nitrogen purging cycle 3 times, then purge with nitrogen for 30 min to remove oxygen. React in an oil bath at 70 ℃ under nitrogen atmosphere for 24 h with stirring. Wash with DMF, anhydrous ethanol and ultrapure water 2-3 times alternately. Collect microspheres 3 (PS@p(GMA)) after vacuum drying for 24 h.

[0111] 5. Place 0.2 g γ-CD-His in a 50 mL round-bottom flask, dissolve it by sonication in 10 mL DMF, add 0.2 g microspheres 3 and 0.2 mL triethylamine catalyst, stir the reaction for 24 h, wash with DMF, anhydrous ethanol and ultrapure water 2-3 times, and vacuum dry for 24 h to collect the histidine esterified cyclodextrin modified polystyrene divinylbenzene microsphere adsorbent (PS@p(γ-CD-His)).

[0112] In this embodiment of the invention, histidine-modified microspheres (PS@p(His)) and polymyxin B-modified microspheres (PS@p(PMB)) were set as controls, that is, a total of 6 groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), and PS@p(α, β, γ-CD-His), were set up to compare and evaluate the coagulation, hemolysis, anti-protein adsorption, endotoxin adsorption, and competitive adsorption capacity of the histidine esterified cyclodextrin molecule-PS-DVB modified microsphere hemoperfusion adsorbent of the present invention.

[0113] Preparation of PS@p(His): 0.2 g His (histidine) was placed in a 50 mL round-bottom flask and dissolved by sonication in 10 mL DMF. 0.2 g microspheres and 0.2 mL triethylamine catalyst were added and the mixture was stirred for 24 h. The mixture was washed 2-3 times with DMF, anhydrous ethanol, and ultrapure water and then vacuum dried for 24 h to collect the histidine-modified polystyrene divinylbenzene microspheres (PS@p(His)).

[0114] Preparation of PS@p(PMB): 0.2 g PMB was placed in a 50 mL round-bottom flask and dissolved by sonication in 10 mL DMF. 0.2 g microspheres and 0.2 mL triethylamine catalyst were added and the mixture was stirred for 24 h. The mixture was washed 2-3 times with DMF, anhydrous ethanol, and ultrapure water and then vacuum dried for 24 h to collect polymyxin B modified polystyrene divinylbenzene microspheres (PS@p(PMB)).

[0115] Appropriate amounts of PS-DVB, PS@NH2, PS@p(GMA), PS@p(His), PS@p(PMB), and PS@p(β-CD-His) microspheres were ground into powder, mixed with potassium bromide powder, and pressed into transparent sheets. The sheets were then scanned at room temperature using a Fourier transform infrared spectroscopy spectrometer in the range of 400–4000 cm⁻¹. -1 The effectiveness of the modification method of this invention was analyzed by observing the characteristic absorption peaks of chemical bonds in the spectrophotometer. The results are as follows: Figure 4 .

[0116] Figure 4 In the middle, amination of PS-DVB is at 3300~3500 cm⁻¹ -1The enhanced characteristic peak of the -NH2 stretching vibration at the position indicates successful amination. Then, Raft polymerization is performed in situ to generate glycidyl methacrylate chains. PS@p(GMA) at 920 cm⁻¹ -1 Characteristic peaks of epoxy groups appeared at 1730 cm⁻¹. -1 The presence of characteristic C=O peaks on both sides indicates successful polymerization. After the ring-opening reaction and grafting of histidine, PMB, and CD-His adsorbed molecules, the characteristic peaks of the epoxy groups disappeared, while the characteristic peak of C=O was enhanced, and it appeared at 1235 cm⁻¹. -1 The presence of enhanced characteristic peaks belonging to amide bonds and imidazole ring CN bonds on both sides indicates that the ring-opening reaction grafting adsorption of molecules was successful.

[0117] X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition, chemical bond composition, and content of PS-DVB, PS@p(GMA), PS@p(His), PS@p(PMB), and PS@p(α, β, γ-CD-His). The X-ray source was an Al K monochromator (hν = 1486.71 eV, 5 mA, 15 kV) with a vacuum degree less than 5 x 10⁻⁶. -8 mbar, results are shown Figure 5 , Figure 6 , Figure 7 .

[0118] Figure 5 Compared with unmodified PS-DVB microspheres, PS@p(GMA), PS@p(His), PS@p(PMB), and PS@p(α, β, γ-CD-His) have added nitrogen element, and the oxygen element is significantly enhanced with the modification. The nitrogen element is also significantly enhanced after grafting α, β, γ-CD-His adsorbent molecules with the ring-opening reaction compared with PS@p(GMA), indicating that the blood perfusion adsorbent was successfully prepared. Figure 6 In the study, the C1s peak of PS@p(GMA) showed an increase in binding energy peaks for C=O and CN bonds compared to the unmodified PS-DVB microspheres, indicating that p(GMA) was successfully polymerized in situ onto the PS-DVB surface. Subsequently, PS@p(His), PS@p(PMB), and PS@p(α, β, γ-CD-His) were obtained through ring-opening grafting. The C=O, CN, and CO peaks of the C1s peak were enhanced, and PS@p(His) and PS@p(α, β, γ-CD-His) showed binding energy peaks for C=N bonds belonging to the imidazole ring, indicating that CD-His was successfully grafted onto the microspheres.

[0119] Figure 7The N1s peak further proves that from PS@p(GMA) to PS@p(His), PS@p(PMB), and PS@p(α, β, γ-CD-His), the microspheres show obvious characteristic peaks belonging to protonated N and CN=C peaks belonging to the imidazole ring, which also indicates that the modification of CD-His was successful.

[0120] PS-DVB, PS@p(His), PS@p(PMB), and PS@p(α, β, γ-CD-His) microspheres were ground into powders smaller than 10 micrometers. The powders were mixed with DIW to prepare a 0.1 mg / mL dispersion solution. The potential of the microspheres before and after modification was measured using a nanoparticle size analyzer. The results are shown in [Figure number missing]. Figure 8 .

[0121] Figure 8 The results show that the unmodified PS-DVB microspheres have a potential close to 0, while the modified microspheres have a significantly increased potential of 25~35 mV due to the positive charge of the basic amino acids His, PMB and CD-His, which is beneficial for adsorbing endotoxin molecules.

[0122] Example 6: Endotoxin Adsorption Experiment

[0123] 1. Take the endotoxin standard, reconstitute it with water for endotoxin testing, vortex it on a vortex mixer for 15 min to ensure complete dispersion of LPS molecules, and then dilute it to a 20 EU / mL standard solution.

[0124] 2. Weigh 10 mg of each of the six groups of microspheres (PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His)) and place them in a 1.5 mL pyrogen-free centrifuge tube. The microsphere-free group was used as a positive control, and the water group used for LPS testing was used as a negative control. Four parallel samples were set up for each group.

[0125] 3. Add 1 mL of LPS solution to each centrifuge tube and incubate in a shaker at 37 °C for 2 h.

[0126] 4. Dilute the incubated solution 40 times, measure the absorbance using an endotoxin detection kit, and calculate the concentration and adsorption capacity based on the standard curve. The adsorption results are shown in [Figure number missing]. Figure 9 .

[0127] Figure 9In the study, unmodified PS-DVB showed low LPS adsorption capacity and weak adsorption ability. After grafting histidine, the adsorption capacity improved, and it could adsorb about 12 EU LPS in a 20 EU / mL LPS solution, with an adsorption rate of about 60%. The adsorption level of the α-CD-His modified group was comparable to that of histidine. It is possible that due to the smaller hydrophobic cavity, the adsorption performance in high-concentration LPS solution was not significantly different from that of the histidine group. However, the LPS adsorption level of the hemoperfusion microspheres grafted with β-CD-His and γ-CD-His could reach the same effect as PMB (the main adsorbent molecule in clinical perfusion therapy for sepsis). The adsorption capacity of 10 mg sample in 1 mL of 20 EU / mL high-concentration LPS solution reached 18-19 EU, and the LPS removal efficiency reached more than 90%, which showed excellent endotoxin adsorption performance.

[0128] Example 7: Anti-protein adsorption experiment

[0129] The anti-protein adsorption experiment selected the two components with the highest content in human serum for determination, namely human serum albumin (HSA) and human fibrinogen (FIB).

[0130] HSA adsorption resistance experiment:

[0131] 1. Take human serum albumin reagent and dissolve it in 1×PBS to prepare a 1 mg / mL HSA solution.

[0132] 2. Weigh 10 mg of each of the six groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), and place them in a 1.5 mL centrifuge tube. The group without microspheres is the control. Each group has 4 parallel samples.

[0133] 3. Add 1 mL of HSA solution to each group of microspheres and incubate in a shaker at 37 ℃ for 2 h.

[0134] 4. Take 20 μL of the adsorbed sample and use the BCA protein kit (Solepro) to detect its concentration, and calculate the protein adsorption amount and anti-protein adsorption rate.

[0135] Anti-FIB adsorption experiment:

[0136] 1. Take human fibrinogen reagent and dissolve it in 1×PBS to prepare a 1 mg / mL FIB solution.

[0137] 2. Weigh 10 mg of each of the six groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), and place them in a 1.5 mL centrifuge tube. The group without microspheres is the control. Each group has 4 parallel samples.

[0138] 3. Add 1 mL of FIB solution to each group of microspheres and incubate in a shaker at 37 ℃ for 2 h.

[0139] 4. Take 20 μL of the adsorbed sample and use the BCA protein kit (Solepro) to detect its concentration. Calculate the protein adsorption amount and anti-protein adsorption rate. The results are as follows: Figure 10 .

[0140] Figure 10 The results showed that the unmodified PS-DVB microspheres adsorbed 11.98 mg / g of HSA and 14.80 mg / g of FIB. After modification, the protein adsorption capacity decreased significantly, with the adsorption capacity of proteins grafted with CD-His molecules decreasing to around 5 mg / g. The HSA adsorption capacity was much lower than that of the PMB group, and the anti-HSA and anti-FIB protein adsorption rates reached over 95%, demonstrating excellent anti-protein adsorption performance.

[0141] Example 8: Hemolysis Rate Determination Experiment

[0142] 1. Take 2 mL of heparin sodium anticoagulated rabbit whole blood into a 15 mL centrifuge tube, centrifuge at 3000 rpm for 10 min, then remove the upper plasma layer to obtain rabbit red blood cells. Take an appropriate amount of rabbit red blood cells and mix with 1×PBS to prepare a 2% (v / v) rabbit red blood cell solution, which will be used as experimental blood.

[0143] 2. Weigh 10 mg of each of the six groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), and place them in a 1.5 mL centrifuge tube. Add 1 mL of experimental blood. Use 2% (v / v) rabbit red blood cell solution prepared by DIW as a positive control and the pure experimental blood as a negative control. Each group has 4 parallel samples.

[0144] 3. Incubate at 37 ℃ in a constant temperature shaker for 1 h.

[0145] 4. Centrifuge each group of microspheres and red blood cells at 3000 rpm for 10 min, take 100 μL and place it in a 96-well plate, and measure the OD value at 545 nm using an ELISA reader.

[0146] 5. Calculate the hemolysis rate based on the OD value. If the hemolysis rate is <5%, the material is considered safe and meets the requirements for hemolysis testing. If the hemolysis rate is >5%, the material can induce the rupture of red blood cells and is not suitable for clinical use. See the results below. Figure 11 .

[0147] Figure 11 The results showed that the hemolysis rate of each group of microspheres, including PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), was only 1%, with most of them less than 1%. This meets the standard of less than 5% for biomedical materials that come into contact with blood, as required by the national standard ISO 10993-5, and does not lead to hemolysis.

[0148] Example 9: Coagulation Test

[0149] 1. Weigh 10 mg of each of the six groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), and place them in a 1.5 mL centrifuge tube.

[0150] 2. Each group was given 1 mL of rabbit anemic platelet plasma (PPP) (Guangzhou Hongquan Biotechnology) anticoagulated with heparin sodium. Plasma without microsphere incubation was set up as a blank control group.

[0151] 3. Incubate at 37 ℃ in a constant temperature shaker for 0.5 h.

[0152] 4. Collect the supernatant serum and use a coagulation time analyzer to measure plasma activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). The control group consisted of fresh rabbit plasma that was not co-incubated with the microspheres. Results are shown below. Figure 12 .

[0153] Figure 12 In the study, the clotting times (APTT, PT, TT) of plasma after incubation with microspheres in the PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His) groups were not significantly different from those of unincubated normal plasma, indicating that the modified microsphere adsorbent does not induce coagulation upon contact with blood. Furthermore, the fibrinogen levels in the plasma before and after incubation did not decrease significantly, indicating that the adsorbent material does not affect normal coagulation after perfusion surgery. This demonstrates that the adsorbent material exhibits excellent blood compatibility.

[0154] Example 10: Cytotoxicity Experiment

[0155] 1. Weigh 100 mg of each of the six groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), and place them in 15 mL centrifuge tubes. Soak the tubes in 75% ethanol for 6 h, then vacuum dry them and sterilize them overnight under UV light.

[0156] 2. Add 1 mL of complete culture medium (DMEM:FBS:antibiotic = 90:10:1) to each tube and incubate in a 37 ℃ incubator for 24 h to prepare microsphere extract.

[0157] 3. Aspirate the extracted complete culture medium and filter it once using a 0.22 μm PES sterile filter membrane to remove bacteria and impurities, obtaining the corresponding microsphere extract.

[0158] 4. Seed L929 cells at a density of 5000 cells / well in 96-well plates and culture overnight to allow the cells to adhere completely. There were 4 parallel wells per group. Then, the old culture medium was aspirated and 100 μL of microsphere extract was added. The cells were cultured in a 37 ℃, 5% CO2 cell culture incubator for 24 h.

[0159] 5. Aspirate the microsphere extract and wash twice with PBS.

[0160] 6. Add 150 μL of cck-8 working solution (cck-8 stock solution: DMEM=1:9) to each well, wrap the well plate with aluminum foil to ensure complete protection from light, and incubate in a 37 ℃, 5% CO2 cell culture incubator for 2 h.

[0161] 7. After incubation, aspirate 100 μL of the developed solution from each well and place it in a new 96-well plate. Measure the OD value at 450 nm using a microplate reader. The results are then... Figure 13 show.

[0162] Figure 13 In the study, after L929 cells were cultured with extracts of PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His) for 24 h, the cell viability was close to 100%, and there was no significant difference between the groups, indicating that the adsorbent material has excellent cell compatibility.

[0163] Example 11: Endotoxin & Human Serum Albumin Competitive Adsorption Experiment

[0164] 1. Reconstitute the endotoxin (LPS) standard with pyrogen-free 1×PBS, vortex for 15 min to ensure complete dispersion, and then dilute to 10 EU / mL.

[0165] 2. Weigh an appropriate amount of human serum albumin, add 10 EU / mL LPS solution to dissolve the albumin, so that the HSA concentration is 1 mg / mL, and obtain a mixed LPS & HSA solution.

[0166] 3. Weigh 10 mg of each of the six groups of microspheres, namely PS-DVB, PS@p(His), PS@p(PMB), PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His), and place them in a 1.5 mL pyrogen-free centrifuge tube. Each group has 4 parallel samples.

[0167] 4. Add 1 mL of LPS & HSA mixed solution to each group and incubate in a shaker at 37 ℃ for 2 h.

[0168] 5. After diluting an appropriate amount of sample from each group 20 times, the endotoxin concentration was determined using an endotoxin detection kit. The endotoxin adsorption performance under protein interference was calculated and evaluated. The results are as follows: Figure 14 As shown.

[0169] Figure 14 In a mixed solution of 10 EU / mL LPS and 1 mg / mL HSA, with protein interference, the adsorption efficiency of unmodified PS-DVB and PS@p(His) for endotoxin decreased by approximately 20% compared to the protein-free solution, and the overall adsorption capacity was also low. However, the adsorption efficiency of the PS@p(α-CD-His), PS@p(β-CD-His), and PS@p(γ-CD-His) microspheres decreased by only 10%, maintaining a high adsorption efficiency of approximately 80%, showing no significant difference from the PMB group. This demonstrated excellent LPS adsorption performance even under competitive adsorption conditions in complex environments.

Claims

1. A method for producing a blood perfusion adsorbent, characterized by, Includes the following steps: (1) Polystyrene divinylbenzene microspheres, photoinitiator, β-mercaptoethylamine and solvent are mixed and subjected to ultraviolet light-initiated reaction to obtain aminated polystyrene divinylbenzene microspheres; (2) The aminated polystyrene divinylbenzene microspheres, chain transfer reagent and solvent are mixed, and a crosslinking agent is added to catalyze the grafting reaction to obtain polystyrene divinylbenzene microspheres grafted with chain transfer reagent; (3) The polystyrene divinylbenzene microspheres with the grafted chain transfer reagent are placed in a solvent containing glycidyl methacrylate, and a thermal initiator is added to initiate free radical polymerization to obtain polystyrene divinylbenzene microspheres with glycidyl methacrylate chain brushes on the surface. (4) The polystyrene divinylbenzene microspheres with glycidyl methacrylate chain brushes on the surface, modified cyclodextrin and solvent are mixed, a catalyst is added, and the mixture is heated to obtain histidine esterified cyclodextrin modified polystyrene divinylbenzene microspheres, which are used as blood perfusion adsorbents. The modified cyclodextrin is histidine-esterified cyclodextrin; The preparation of the modified cyclodextrin includes the following steps: (1) Three cyclodextrins, namely α, β, and γ, are respectively mixed and dissolved with N,N'-di-tert-butoxycarbonyl-L-histidine containing a protecting group and a solvent. After adding a catalyst, the mixture is stirred and heated to obtain three tert-butoxycarbonylhistidine esterified cyclodextrins. The molar ratio of the three cyclodextrins, namely α, β, and γ, to N,N'-di-tert-butoxycarbonyl-L-histidine is (0.1~1):(1~3). The solvent is any one of N,N-dimethylformamide, dichloromethane, and dimethyl sulfoxide. The catalyst is at least two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine, with a mass ratio of (1~10):(0.1~1). The reaction temperature is 20~60 °C, and the reaction time is 1~48 h. (2) Mix the α, β and γ tert-butoxycarbonyl histidine esterified cyclodextrins with a solvent, add a deprotecting agent to remove the tert-butoxycarbonyl group, and obtain the three histidine esterified cyclodextrins; the solvent is any one of N,N-dimethylformamide, dichloromethane and dimethyl sulfoxide; The deprotecting agent is either a trifluoroacetic acid-dichloromethane mixed solvent or a dioxane hydrochloride mixed solvent; the volume ratio of the solvent to the deprotecting agent is (0.1~1):1; the reaction temperature is 0~20 ℃, and the reaction time is 0.5~4 h.

2. The method of claim 1, wherein the blood perfusion adsorbent is prepared by the steps of: In step (1), The polystyrene-divinylbenzene microspheres have a particle size of 100~1000 μm; The photoinitiator is any one of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoin dimethyl ether, and benzophenone; The solvent is any one of N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane; The mass ratio of polystyrene divinylbenzene microspheres, β-mercaptoethylamine, and photoinitiator in the reaction is (1~10):(1~10):1; The reaction temperature is 20~60℃, and the reaction time is 0.5~2 h.

3. The method for preparing a blood perfusion adsorbent according to claim 1, characterized in that, In step (2), The chain transfer reagent is 4-cyano-4-(thiobenzoic acid)valerate; The crosslinking agent is at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The solvent is any one of N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane; The molar ratio of amino groups to chain transfer reagent and crosslinking agent in the aminated polystyrene divinylbenzene microspheres of the reaction is 1:(1~4):(1~10). The reaction temperature is 20~50 ℃, and the reaction time is 1~48 h.

4. The method for preparing a blood perfusion adsorbent according to claim 1, characterized in that, In step (3), The thermal initiator is any one of benzoyl peroxide, dibenzoyl peroxide, and azobisisobutyronitrile; The solvent is any one of N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide; The mass ratio of the grafted chain transfer reagent, polystyrene divinylbenzene microspheres, glycidyl methacrylate, and thermal initiator is 1:(1~5):(0.01~0.05). The reaction temperature is 40~100 ℃, and the reaction time is 12~36 h.

5. The method for preparing a blood perfusion adsorbent according to claim 1, characterized in that, In step (4), The catalyst is any one of triethylamine, triethanolamine, and benzyldimethylamine; The mass ratio of the polystyrene divinylbenzene microspheres with glycidyl methacrylate chain brushes on the surface, the modified cyclodextrin, and the catalyst is 1:(0.5~2):(0.5~2). The reaction temperature is 20~60℃, and the reaction time is 1~24 h.

6. A blood perfusion adsorbent prepared by the preparation method according to any one of claims 1-5.

7. The use of the hemoperfusion adsorbent according to claim 6 in the preparation of drugs for removing endotoxins.

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