Agarose microsphere adsorbent with double adsorption function, its preparation method and application

Abstract

The present application relates to the technical field of blood purification, in particular to a kind of agarose microsphere adsorbent with double adsorption and its preparation method and application, the adsorbent with crosslinked agarose microsphere as matrix, hydroxyl group on polysaccharide skeleton covalently grafts hydrophobic group, while skeleton also grafts the cationic macromolecule containing polyamine group, and hydrophobic group is not grafted on cationic macromolecule;Its preparation method is first prepared crosslinked agarose base ball, then carries out hydrophobic modification, finally carries out amination modification, and agarose microsphere adsorbent is obtained.The adsorbent of the present application can simultaneously adsorb endotoxin and proinflammatory cytokines in blood, and the total protein adsorption rate is low, and after heparin preflush, the anticoagulation performance is excellent, suitable for blood purification treatment of severe conditions such as sepsis.

Description

Technical Field

[0001] This invention relates to the field of blood purification technology, specifically to an agarose microsphere adsorbent with dual adsorption properties and its preparation method. This adsorbent can simultaneously adsorb endotoxins (LPS) and pro-inflammatory cytokines (such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)) in the blood, and can be applied to blood purification treatment of severe diseases such as sepsis. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by host immune dysregulation triggered by infection. It is also one of the most common critical illnesses in the intensive care unit (ICU), characterized by high morbidity and high mortality, and is regarded as the "number one killer" in the field of critical care.

[0003] Existing technologies mostly achieve the adsorption and removal of LPS or cytokines by modifying the substrate surface with affinity ligands. For example, CN113351185A discloses a hemoperfusion microsphere adsorbent using polystyrene-divinylbenzene microspheres as a carrier and linking polymyxin B via molecular arms. It has good adsorption performance for bilirubin and LPS, but cannot remove pro-inflammatory cytokines. CN1864755A discloses an LPS adsorbent using agarose gel as a matrix and activated with epichlorohydrin and hexamethylenediamine to bind polymyxin B ligands, which also targets only the single LPS target. CN104525151A discloses a... LPS adsorbents using styrene-divinylbenzene or polymethyl methacrylate porous resin as carriers are used to immobilize polylysine, polyethyleneimine (PEI) and other ligands after epoxidation modification of the residual double bonds of the carrier. By controlling the pore size and specific surface area, some cytokines can also be adsorbed. CN113509919A discloses a mesoporous adsorbent with nano-calcium carbonate-styrene-divinylbenzene as the backbone and grafted polyamine amino acids or peptide ligands, which can simultaneously remove LPS and inflammatory factors such as TNF-α and IL-6.

[0004] However, the aforementioned materials capable of dual adsorption all use highly hydrophobic polymers such as polystyrene as substrates. The non-specific hydrophobic interactions between the hydrophobic substrate and plasma proteins are strong, resulting in poor blood compatibility and a high risk of causing a significant decrease in total protein and platelets, leading to adverse reactions such as coagulation. On the other hand, adsorption materials modified with recombinant peptides, polycationic electrolytes, histidine, and other ligands using substrates such as chitosan, polyethersulfone, cellulose, and silica can generally only achieve single clearance of LPS and cannot simultaneously clear pro-inflammatory cytokines.

[0005] Agarose is a natural neutral polysaccharide extracted from red algae. It is highly hydrophilic, biocompatible, non-toxic, and does not easily activate the complement system. Its molecular chain is rich in hydroxyl groups, making it easy to introduce functional groups through chemical modification, which makes it an ideal substrate for constructing blood purification adsorbent materials. However, natural agarose gel has low mechanical strength and poor pressure resistance. When used directly as a blood perfusion adsorbent, it is prone to deformation and breakage under perfusion pressure. It must be reinforced by covalent cross-linking to meet the requirements. Furthermore, there is no mature solution in the current technology for how to simultaneously construct adsorption sites for LPS (electrostatic and hydrophobic synergistic effect) and pro-inflammatory cytokines (hydrophobic effect) on the hydrophilic polysaccharide backbone, while maintaining low total protein nonspecific adsorption and good blood compatibility in the modified material.

[0006] Therefore, there is an urgent need to develop a blood purification adsorbent that can simultaneously and efficiently remove LPS and pro-inflammatory cytokines from the blood, has low non-specific adsorption of total protein, and good blood compatibility. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings and provide an agarose microsphere adsorbent with dual adsorption effects of LPS and pro-inflammatory cytokines. This adsorbent can simultaneously and efficiently remove LPS and pro-inflammatory cytokines from the blood, and has low non-specific adsorption of total protein and good blood compatibility. This invention also provides a method for preparing this adsorbent and its application.

[0008] To achieve the above objectives, the specific solution of the present invention is as follows: The first aspect of this invention provides an agarose microsphere adsorbent with dual adsorption capabilities, comprising a covalently cross-linked agarose microsphere matrix, wherein hydrophobic groups are covalently grafted onto the hydroxyl groups of the polysaccharide backbone of the agarose microsphere matrix, and a cationic macromolecule containing polyamine groups is also covalently grafted onto the polysaccharide backbone of the agarose microsphere matrix; the hydrophobic groups are not grafted onto the cationic macromolecules, and the adsorbent can simultaneously adsorb LPS and pro-inflammatory cytokines in the blood.

[0009] Preferably, the hydrophobic group is linked to the hydroxyl group of the agarose polysaccharide backbone via an ether bond or an ester bond.

[0010] Preferably, the hydrophobic group connected by an ether bond is at least one of phenyl or C4-C12 alkyl; the hydrophobic group connected by an ester bond is at least one of C6-C12 acyl.

[0011] Preferably, the polyamine-containing cationic macromolecule is selected from at least one of PEI and polylysine.

[0012] Preferably, the amine density of the adsorbent is 150-250 μmol per milliliter of wet microspheres; the degree of grafting substitution of the hydrophobic group is 0.5-3.0 mmol per gram of cross-linked agarose dry weight, more preferably 1.0-2.0 mmol / g.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned agarose microsphere adsorbent, comprising the following steps in sequence: A1. Prepare an aqueous solution of agarose with a mass concentration of 4%-8%, heat to dissolve, add oil phase for emulsification and dispersion, cool and solidify into spheres, and then covalently crosslink with epichlorohydrin to obtain crosslinked agarose microspheres. A2. The cross-linked agarose microspheres obtained in step A1 are reacted with a hydrophobic reagent to covalently graft hydrophobic groups onto the hydroxyl groups of the agarose polysaccharide backbone, thereby obtaining hydrophobically modified microspheres. A3. First, the hydrophobic modified microspheres obtained in step A2 are epoxy activated, and then grafted with cationic macromolecules containing polyamine groups to obtain the agarose microsphere adsorbent.

[0014] Preferably, when the hydrophobicating agent in step A2 is butyl glycidyl ether, octyl glycidyl ether or phenyl glycidyl ether, the reaction is carried out using a mixed solvent of water and dimethyl sulfoxide (DMSO), with sodium hydroxide as the catalyst, at a reaction temperature of 30-60°C, and for a reaction time of 8-16 hours; after the reaction is completed, the mixture is washed alternately with water and ethanol until neutral.

[0015] Preferably, when the hydrophobicating agent in step A2 is an acyl chloride with a carbon chain length of 6-12, the reaction is carried out using pyridine, toluene or tetrahydrofuran as solvent, and triethylamine is added as an acid-binding agent; after the reaction is completed, the mixture is washed with pyridine, ethanol and water in sequence.

[0016] The third aspect of this invention provides the application of the above-mentioned agarose microsphere adsorbent in the preparation of blood purification adsorbent materials; the blood purification adsorbent materials are used to remove LPS and pro-inflammatory cytokines from the blood and can be used for blood purification treatment of sepsis patients.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention introduces hydrophobic groups and polyamine cationic macromolecules onto an agarose microsphere framework, enabling the simultaneous adsorption of LPS and pro-inflammatory cytokines such as IL-6 and TNF-α from the blood. Within the preferred range of amine density and hydrophobic substitution degree (amine density ≥170 μmol / mL, hydrophobic substitution degree ≥1.0 mmol / g), the adsorption capacity for pro-inflammatory cytokines is comparable to that of the Chinese Jianfan CA filter, while the LPS adsorption advantage is significant, far exceeding the 20%-30% adsorption level of the American Baxter oXiris filter. It should be noted that oXiris is a filtration-type blood purification product, and its working principle differs from that of the adsorption-type product of this invention. The LPS adsorption rate comparison here is only used to illustrate the performance advantage of this invention in LPS removal.

[0018] This invention uses natural hydrophilic agarose as a substrate, which significantly reduces the non-specific adsorption of plasma proteins compared to polystyrene-based strongly hydrophobic substrates. The total protein reduction rate is less than 10%, which is lower than the 13% of the Chinese Jianfan CA, and can effectively reduce the loss of normal blood components during treatment.

[0019] The grafted polyamine cationic macromolecules of this invention carry a large number of positive charges and can adsorb negatively charged heparin through electrostatic interaction. After pre-filling with heparin before clinical use, a stable heparin layer can be bound to the surface of the microspheres, which can effectively prolong the activated partial thromboplastin time (APTT), and has good anticoagulant properties, which can reduce the risk of coagulation during blood purification. Attached Figure Description

[0020] Figure 1 An optical microscope image of the agarose microsphere adsorbent prepared according to the present invention. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments, but this is not intended to limit the scope of the invention to this.

[0022] Example 1: Preparation of octyl glycidyl ether hydrophobicated + PEI-amined agarose microspheres (1) Preparation of agarose-based microspheres: Prepare a 4% (w / w) agarose aqueous solution and heat and stir until completely dissolved; slowly add the agarose solution to the oil phase (using a liquid paraffin system with Span 80 added, the amount of Span 80 added is 2% of the mass of liquid paraffin) preheated to 70℃, control the oil-water volume ratio to 3:1, and emulsify at a high speed of 500 rpm for 20 minutes to form a uniform droplet system. Control the particle size distribution of the target microspheres to 300-800 μm by adjusting the stirring speed, cool to room temperature to allow the droplets to solidify and form, and sieve to obtain agarose microspheres with uniform particle size; then disperse the microspheres in a 1 mol / L sodium hydroxide solution, and add 1.0 g of epichlorohydrin per gram of dry agarose. Epichlorohydrin was added at a ratio of mL, and the mixture was reacted at 50°C for 8 hours to carry out covalent cross-linking. After the reaction, the mixture was washed with deionized water until neutral to obtain cross-linked agarose microspheres. The volume swelling ratio of the cross-linked microspheres with pure water as the medium was measured to be 5.2, which meets the pressure resistance requirements for blood perfusion.

[0023] (2) Hydrophobic modification: Take the cross-linked agarose microspheres obtained in step (1), add a mixture of water and DMSO (water to DMSO volume ratio of 2:1) at a solid-liquid ratio of 1:8 (g dry basis: mL), then add sodium hydroxide with a final concentration of 1 mol / L, add 1.5 mL of octyl glycidyl ether per gram of agarose dry basis, mix well, and heat to 45℃ and keep warm for 12 hours; after the reaction is completed, wash the microspheres with deionized water and ethanol alternately until neutral to obtain hydrophobic modified microspheres.

[0024] (3) Amination modification: The hydrophobic modified microspheres obtained in step (2) were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. An alkaline catalytic system was used, with a molar ratio of epichlorohydrin to microsphere hydroxyl groups of 1.2:1. The reaction temperature was controlled at 40℃, and the reaction time was 4 hours. After the reaction was completed, the microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 2.0 g of PEI with a molecular weight of 70000 Da was added per gram of agarose dry basis. The temperature was raised to 60℃ and kept at that temperature for 12 hours. After the reaction was completed, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and deionized water until neutral to remove the uncovalently grafted PEI and obtain the target agarose microsphere adsorbent, such as Figure 1 As shown. The amine density of this adsorbent was measured to be approximately 175 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.6 mmol / g dry microspheres.

[0025] Example 2: Preparation of hydrophobicated n-octanoyl chloride + 3000 Da polylysine amino-modified agarose microspheres (1) Preparation of agarose-based microspheres: Prepare a 6% (w / w) agarose aqueous solution and heat and stir until completely dissolved; slowly add the agarose solution to the oil phase (using a liquid paraffin system with Span 80 added, the amount of Span 80 added is 2% of the mass of liquid paraffin) preheated to 70℃, control the oil-water volume ratio to 3:1, and emulsify at a high speed of 500 rpm for 20 minutes to form a uniform droplet system. Control the particle size distribution of the target microspheres to 300-800 μm by adjusting the stirring speed, cool to room temperature to allow the droplets to solidify and form, and sieve to obtain agarose microspheres with uniform particle size; then disperse the microspheres in a 1 mol / L sodium hydroxide solution, and add 1.2 g of epichlorohydrin per gram of dry agarose. Epichlorohydrin was added at a ratio of mL, and the mixture was reacted at 55°C for 10 hours to carry out covalent cross-linking. After the reaction, the mixture was washed with deionized water until neutral to obtain cross-linked agarose microspheres. The volume swelling ratio of the cross-linked microspheres with pure water as the medium was measured to be 4.8, which meets the pressure resistance requirements for blood perfusion.

[0026] (2) Hydrophobic modification: Take the cross-linked agarose microspheres obtained in step (1) and add them to pyridine solvent at a solid-liquid ratio of 1:8 (g dry basis: mL). Then add triethylamine as an acid-binding agent. The amount of triethylamine is 1.1 times the molar amount of n-octanoyl chloride. Slowly add n-octanoyl chloride at a dosage of 1.5 mmol / g agarose dry basis and react at room temperature for 8 hours. After the reaction is completed, wash the microspheres with pyridine, ethanol and deionized water in sequence until neutral to obtain hydrophobic modified microspheres.

[0027] (3) Amination modification: The hydrophobic modified microspheres obtained in step (2) were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. An alkaline catalytic system was used, with a molar ratio of epichlorohydrin to microsphere hydroxyl groups of 2:1. The reaction temperature was controlled at 35℃, and the reaction time was 4 hours. After the reaction was completed, the microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 2.0 g of polylysine with a molecular weight of 3000 Da was added per gram of agarose dry basis. The temperature was raised to 60℃ and kept at that temperature for 12 hours. After the reaction was completed, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and deionized water until neutral to remove uncovalently grafted polylysine, thus obtaining the target agarose microsphere adsorbent. The amino group density of the adsorbent was found to be approximately 180 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.4 mmol / g dry microspheres.

[0028] Polylysine molecules have a large number of primary and secondary amine groups, which can be protonated under physiological pH conditions to form positively charged sites, and have a similar electrostatic adsorption mechanism to PEI.

[0029] Microspheres were prepared using an 8% agarose solution according to the same process as in this embodiment. The volume swelling ratio was measured to be 4.2, and the column operation flow rate that it could tolerate was higher than that of the 6% concentration sample. Under the same test conditions, the adsorption rates of IL-6, TNF-α, and LPS, as well as the decrease rate of total protein, were basically equivalent to those of the 6% concentration sample.

[0030] Example 3: Low-substitution-degree octyl hydrophobication + low-amine-density PEI-modified agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 1, to obtain cross-linked agarose microspheres with a volume swelling ratio of 5.2.

[0031] (2) Hydrophobic modification: Take cross-linked agarose microspheres and add a mixture of water and DMSO (water to DMSO volume ratio of 2:1) at a solid-liquid ratio of 1:8 (g dry basis: mL). Then add sodium hydroxide with a final concentration of 1 mol / L and 0.45 mL of octyl glycidyl ether per gram of agarose dry basis. After mixing, heat to 45℃ and keep warm for 12 hours. After the reaction is completed, wash the microspheres with deionized water and ethanol alternately until neutral to obtain hydrophobic modified microspheres.

[0032] (3) Amination modification: The hydrophobically modified microspheres were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. The molar ratio of epichlorohydrin to the hydroxyl groups of the microspheres was 0.8:1. The reaction temperature was 25℃, and the reaction time was 2 hours. The microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 1.2 g of PEI with a molecular weight of 10000 Da was added per gram of agarose dry basis. The temperature was raised to 60℃ and kept at that temperature for 12 hours. After the reaction, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and then with deionized water until neutral to obtain the target adsorbent. The amino group density of the adsorbent was found to be approximately 152 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 0.52 mmol / g dry microspheres.

[0033] Example 4: Highly substituted octyl hydrophobic + high amino density PEI modified agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 1, to obtain cross-linked agarose microspheres with a volume swelling ratio of 5.2.

[0034] (2) Hydrophobic modification: Take cross-linked agarose microspheres and add a mixture of water and DMSO (water to DMSO volume ratio of 2:1) at a solid-liquid ratio of 1:8 (g dry basis: mL). Then add sodium hydroxide with a final concentration of 1 mol / L and 2.8 mL of octyl glycidyl ether per gram of agarose dry basis. After mixing, heat to 50℃ and keep warm for 16 hours. After the reaction is completed, wash the microspheres with deionized water and ethanol alternately until neutral to obtain hydrophobic modified microspheres.

[0035] (3) Amination modification: The hydrophobically modified microspheres were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. The molar ratio of epichlorohydrin to the hydroxyl groups of the microspheres was 1.5:1. The reaction temperature was 35℃, and the reaction time was 3 hours. The microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 3.0 g of PEI with a molecular weight of 60,000 Da was added per gram of agarose dry basis. The temperature was raised to 65℃ and kept at that temperature for 16 hours. After the reaction, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and then with deionized water until neutral to obtain the target adsorbent. The amino group density of the adsorbent was found to be approximately 247 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 2.94 mmol / g dry microspheres.

[0036] Example 5: Hydrophobicated phenyl glycidyl ether + PEI-amined agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 1, to obtain cross-linked agarose microspheres with a volume swelling ratio of 5.2.

[0037] (2) Hydrophobic modification: Take cross-linked agarose microspheres and add a mixture of water and DMSO (water to DMSO volume ratio of 1:1) at a solid-liquid ratio of 1:8 (g dry basis: mL). Then add sodium hydroxide with a final concentration of 1.2 mol / L and add 1.8 mmol (about 0.24 mL) of phenyl glycidyl ether per gram of agarose dry basis. After mixing, heat to 50℃ and keep warm for 12 hours. After the reaction is completed, wash the microspheres with deionized water and ethanol alternately until neutral to obtain phenyl hydrophobic modified microspheres.

[0038] (3) Amination modification: Same as step (3) in Example 1, grafting PEI with a molecular weight of 30,000 Da. The amino group density of this adsorbent was found to be approximately 170 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.5 mmol / g dry microspheres. Due to the electron-withdrawing effect of the benzene ring, phenyl glycidyl ether has higher epoxy ring reactivity than alkyl glycidyl ether. Furthermore, this example used a higher alkali concentration of 1.2 mol / L and a reaction temperature of 50℃, thus achieving a higher degree of grafting substitution with a lower feed amount.

[0039] Example 6: Butyl glycidyl ether hydrophobication + PEI-amined agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 1, to obtain cross-linked agarose microspheres with a volume swelling ratio of 5.2.

[0040] (2) Hydrophobic modification: Take cross-linked agarose microspheres and add a mixture of water and DMSO (water to DMSO volume ratio of 2:1) at a solid-liquid ratio of 1:8 (g dry basis: mL). Then add sodium hydroxide with a final concentration of 1 mol / L and 1.2 mL of butyl glycidyl ether per gram of agarose dry basis. After mixing, heat to 45℃ and keep warm for 10 hours. After the reaction is completed, wash the microspheres with deionized water and ethanol alternately until neutral to obtain butyl hydrophobic modified microspheres.

[0041] (3) Amination modification: Same as step (3) in Example 1, grafting PEI with a molecular weight of 20000 Da. The test results showed that the amino group density of the adsorbent was about 178 μmol / mL wet microspheres, and the degree of substitution of hydrophobic groups was about 1.4 mmol / g dry microspheres.

[0042] Example 7: Hydrophobicated dodecyl chloride + 2000 Da polylysine amino-modified agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 2, to obtain cross-linked agarose microspheres with a volume swelling ratio of 4.8.

[0043] (2) Hydrophobic modification: Take cross-linked agarose microspheres and add them to pyridine solvent at a solid-liquid ratio of 1:8 (g dry basis: mL). Then add triethylamine as an acid-binding agent. The amount of triethylamine is 1.1 times the molar amount of dodecyl chloride. Slowly add dodecyl chloride at a dosage of 1.6 mmol / g agarose dry basis and react at room temperature for 10 hours. After the reaction is completed, wash the microspheres with pyridine, ethanol and deionized water in sequence until neutral to obtain dodecyl hydrophobic modified microspheres.

[0044] (3) Amination modification: Same as step (3) in Example 2, grafting polylysine with a molecular weight of 2000 Da. The amino group density of the adsorbent was found to be approximately 175 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.3 mmol / g dry microspheres.

[0045] Example 8: Hydrophobicated n-octanoyl chloride + 1000 Da polylysine amino-modified agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 2, prepare a 6% agarose aqueous solution, and after emulsification and curing and epichlorohydrin crosslinking, obtain crosslinked agarose microspheres with a volume swelling ratio of 4.8.

[0046] (2) Hydrophobic modification: Same as step (2) in Example 2, using pyridine as solvent, triethylamine as acid binding agent, and n-octanoyl chloride at a dosage of 1.5 mmol / g agarose dry base, reacted at room temperature for 8 hours to obtain octanoyl hydrophobic modified microspheres.

[0047] (3) Amination modification: The hydrophobic modified microspheres obtained in step (2) were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. An alkaline catalytic system was used, with a molar ratio of epichlorohydrin to microsphere hydroxyl groups of 2:1. The reaction temperature was controlled at 30℃, and the reaction time was 3 hours. After the reaction was completed, the microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 2.0 g of polylysine with a molecular weight of 1000 Da was added per gram of agarose dry basis. The temperature was raised to 60℃ and kept at that temperature for 12 hours. After the reaction was completed, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and deionized water until neutral to remove uncovalently grafted polylysine, thus obtaining the target agarose microsphere adsorbent. The amino group density of the adsorbent was found to be approximately 185 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.4 mmol / g dry microspheres.

[0048] Example 9: Hydrophobicated n-octanoyl chloride + 2000 Da polylysine amino-modified agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 2, prepare a 6% agarose aqueous solution, and after emulsification and curing and epichlorohydrin crosslinking, obtain crosslinked agarose microspheres with a volume swelling ratio of 4.8.

[0049] (2) Hydrophobic modification: Same as step (2) in Example 2, using pyridine as solvent, triethylamine as acid binding agent, and n-octanoyl chloride at a dosage of 1.5 mmol / g agarose dry base, reacted at room temperature for 8 hours to obtain octanoyl hydrophobic modified microspheres.

[0050] (3) Amination modification: The hydrophobic modified microspheres obtained in step (2) were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. An alkaline catalytic system was used, with a molar ratio of epichlorohydrin to microsphere hydroxyl groups of 2:1. The reaction temperature was controlled at 35℃, and the reaction time was 4 hours. After the reaction was completed, the microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 2.0 g of polylysine with a molecular weight of 2000 Da was added per gram of agarose dry basis. The temperature was raised to 60℃ and kept at that temperature for 12 hours. After the reaction was completed, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and deionized water until neutral to remove uncovalently grafted polylysine, thus obtaining the target agarose microsphere adsorbent. The amino group density of the adsorbent was found to be approximately 182 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.4 mmol / g dry microspheres.

[0051] Example 10: Hydrophobicated n-octanoyl chloride + 1500 Da polylysine amino-modified agarose microspheres (1) Preparation of agarose-based spheres: Same as step (1) in Example 2, prepare a 6% agarose aqueous solution, and after emulsification and curing and epichlorohydrin crosslinking, obtain crosslinked agarose microspheres with a volume swelling ratio of 4.8.

[0052] (2) Hydrophobic modification: Same as step (2) in Example 2, using pyridine as solvent, triethylamine as acid binding agent, and n-octanoyl chloride at a dosage of 1.5 mmol / g agarose dry base, reacted at room temperature for 8 hours to obtain octanoyl hydrophobic modified microspheres.

[0053] (3) Amination modification: The hydrophobic modified microspheres obtained in step (2) were placed in a 1 mol / L sodium hydroxide solution with a solid-liquid ratio of 1:8 (g dry basis: mL). Epichlorohydrin was added for epoxy activation. An alkaline catalytic system was used, with a molar ratio of epichlorohydrin to microsphere hydroxyl groups of 2:1. The reaction temperature was controlled at 40℃, and the reaction time was 4 hours. After the reaction was completed, the microspheres were washed until neutral. The activated microspheres were then added to deionized water, and the pH of the system was adjusted to 10 with sodium hydroxide. 2.0 g of polylysine with a molecular weight of 1500 Da was added per gram of agarose dry basis. The temperature was raised to 60℃ and kept at that temperature for 12 hours. After the reaction was completed, the microspheres were washed thoroughly with 2 mol / L sodium chloride solution and deionized water until neutral to remove uncovalently grafted polylysine, thus obtaining the target agarose microsphere adsorbent. The amino group density of the adsorbent was found to be approximately 188 μmol / mL wet microspheres, and the degree of hydrophobic group grafting substitution was approximately 1.4 mmol / g dry microspheres.

[0054] For example, the amino group density is determined by acid-base back titration. The specific steps are as follows: (1) Preparation of wet microspheres: Take the microspheres to be tested and place them in a sand core funnel. Wash them with pure water three times and filter them under negative pressure until no liquid drops fall. Accurately measure 1.0 mL of wet microspheres and place them in an iodine flask; (2) Protonation reaction: Accurately add 10.0 mL of 0.1 mol / L hydrochloric acid standard solution and shake the reaction at 200 rpm for 2 hours at room temperature to fully protonate the amino groups on the microspheres; (3) Back titration: Transfer the reaction solution to a conical flask and wash the iodine flask three times with a small amount of pure water. Combine the washing solution into the conical flask and add 3 drops of methyl red-bromocresol green mixed indicator (color change point pH 5.1). Titrate with 0.1 mol / L sodium hydroxide standard solution until the solution changes from wine red to green, which is the endpoint. Record the volume V of sodium hydroxide standard solution consumed. 样品 (4) Blank control: Replace the wet microspheres with 1.0 mL of pure water, and perform the same operation. Record the volume V of sodium hydroxide standard solution consumed. 空白 (5) Calculation: Amine density (μmol / mL wet microspheres) = (V 空白 -V 样品 )×C _NaOH ×1000 / V 微球 In the formula: Vblank is the volume (mL) of NaOH standard solution consumed by the blank control; Vblank 样品 The volume (mL) of NaOH standard solution consumed by the sample; C _NaOH V represents the concentration (mol / L) of the NaOH standard solution. 微球 The volume (mL) of the wet microspheres to be tested.

[0055] The degree of grafting substitution of hydrophobic groups was determined by elemental analysis. The specific steps are as follows: (1) Take unmodified cross-linked agarose microspheres and final product microspheres respectively, wash them with pure water 3 times, dry them in vacuum at 60℃ to constant weight, and grind them into fine powder; at the same time, accurately measure a certain volume of final product wet microspheres (consistent with the state of wet microspheres used for amino density determination), dry them to constant weight under the same conditions, and determine the dry matter concentration of wet microspheres (unit: g dry basis / mL wet microspheres); (2) Elemental analysis: use an elemental analyzer to determine the carbon mass fraction C0 (%) of unmodified cross-linked agarose and the carbon mass fraction C1 (%) of the final product respectively; (3) Calculation of carbon contribution of cationic macromolecules: divide the amino density (μmol / mL wet microspheres) measured by acid-base back titration by dry matter concentration to convert it into dry basis amino density (μmol / g dry basis); determine the carbon mass corresponding to each mole of titrable amino group according to the type of grafted cationic macromolecules: polyethyleneimine (PEI) is 24 g / mol, polylysine is 72 g / mol. g / mol (if two types are grafted simultaneously, the weighted average is calculated using molar ratios); then the carbon mass fraction contributed by the cationic macromolecule: C poly=Dry-based amine group density × Carbon mass per mole of amine group × 10 -4 (4) Calculation of carbon contribution of hydrophobic groups: carbon mass fraction C contributed by hydrophobic groups hydro =(C1-C0)-C poly (5) Degree of substitution calculation: Determine the carbon content M per mole of hydrophobic group based on the type of hydrophobic group grafted. C (g / mol): Butyl is 48, Octyl is 96, Phenyl is 72, Octanoyl is 96, Dodecanoyl is 144 (if multiple grafts are used, the weighted average is calculated by molar ratio), degree of substitution of hydrophobic groups (mmol / g dry group) = C hydro ×10 / M C In the formula: C hydro Mass fraction of carbon contributed to hydrophobic groups (%); M C The carbon content per mole of hydrophobic groups (g / mol).

[0056] Adsorption performance test The adsorbent samples prepared in Examples 1, 2, 3, 4, 5, and 7 were used for performance testing, while commercially available Chinese Jianfan CA adsorption columns and Baxter oXiris filters were used as control samples.

[0057] 1. Cytokine and total protein adsorption test IL-6 and TNF-α were added to calf plasma to achieve initial concentrations of 1000 pg / mL and 500 pg / mL, respectively, to prepare an adsorption solution. 10 mL of the adsorption solution was added to 1 mL of microsphere sample and incubated in a 37℃ shaker for 2 hours to complete adsorption. The concentrations of IL-6 and TNF-α before and after adsorption were detected using a chemiluminescence immunoassay analyzer, and the concentration of total protein in the plasma before and after adsorption was detected using a biochemical analyzer. The adsorption rate of each indicator and the decrease rate of total protein were calculated.

[0058] 2. LPS Adsorption Test Bovine serum albumin (BSA) and LPS were added to physiological saline to prepare an adsorption solution containing 1 mg / mL BSA and 10 μg / mL LPS. 5 mL of the adsorption solution was added to 1 mL of microsphere sample and incubated in a shaker at 37°C for 2 hours to complete adsorption. The concentration of LPS in the solution before and after adsorption was determined using dynamic turbidimetry, and the LPS adsorption rate was calculated.

[0059] 3. Test Results The test results are shown in the table below: ; ;

[0060] It should be noted that: Baxter oXiris is a filtration-type blood purification product that mainly removes substances through a sieving mechanism. Its removal efficiency for medium-molecular cytokines such as IL-6 and TNF-α is generally lower than that of adsorption-type products, and it also suffers from the problem of inherent protein loss. Therefore, parallel adsorption tests were not conducted for comparison here.

[0061] The results showed that the preferred embodiment of the present invention (amine density ≥170 μmol / mL, hydrophobic substitution degree ≥1.0 mmol / g) had a pro-inflammatory cytokine adsorption capacity comparable to that of Jianfan CA, while also possessing excellent LPS adsorption capacity and a significantly better total protein reduction rate; compared with the filtration product Baxter oXiris, it had obvious advantages in LPS adsorption and better overall blood purification performance.

[0062] Coagulation performance test The adsorbent samples prepared in Examples 1, 2, 3, 4, 5, and 7 were tested for APTT in the untreated state and after heparin pre-purge treatment (the heparin pre-purge treatment method was as follows: the microsphere sample was soaked in an equal volume of heparin saline solution (heparin concentration of 100 U / mL) at 37°C for 30 minutes, and after pre-purge, it was washed 3 times with saline solution, each time with a volume 5 times the volume of the microspheres, to remove unbound free heparin). Blank plasma and plasma treated with the CA adsorption column of China Jianfan were used as controls.

[0063] The test results are shown in the table below:

[0064] The results show that the adsorbent of the present invention, after being pre-treated with heparin, significantly prolongs the APTT time and has good anticoagulant properties, which can effectively reduce the coagulation risk during blood purification treatment.

[0065] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included within the protection scope of this patent application.

Claims

1. An agarose microsphere adsorbent with dual adsorption properties, characterized in that, The adsorbent comprises a covalently cross-linked agarose microsphere matrix, wherein hydrophobic groups are covalently grafted onto the hydroxyl groups of the polysaccharide backbone of the agarose microsphere matrix, and cationic macromolecules containing polyamine groups are also covalently grafted onto the polysaccharide backbone of the agarose microsphere matrix; the hydrophobic groups are not grafted onto the cationic macromolecules, and the adsorbent can simultaneously adsorb endotoxins and pro-inflammatory cytokines in the blood.

2. The agarose microsphere adsorbent according to claim 1, characterized in that, The hydrophobic groups are linked to the hydroxyl groups of the agarose polysaccharide backbone via ether or ester bonds.

3. The agarose microsphere adsorbent according to claim 2, characterized in that, The hydrophobic group connected by an ether bond is at least one of phenyl or C4-C12 alkyl; the hydrophobic group connected by an ester bond is at least one of C6-C12 acyl.

4. The agarose microsphere adsorbent according to claim 1, characterized in that, The polyamine-containing cationic macromolecule is selected from at least one of polyethyleneimine and polylysine.

5. The agarose microsphere adsorbent according to claim 1, characterized in that, The adsorbent has an amine density of 150-250 μmol per milliliter of wet microspheres; the degree of grafting substitution of the hydrophobic groups is 0.5-3.0 mmol per gram of cross-linked agarose dry weight.

6. A method for preparing the agarose microsphere adsorbent as described in any one of claims 1 to 5, characterized in that, The steps, in sequence, are as follows: A1. Prepare an aqueous solution of agarose with a mass concentration of 4%-8%, heat to dissolve, add oil phase for emulsification and dispersion, cool and solidify into spheres, and then covalently crosslink with epichlorohydrin to obtain crosslinked agarose microspheres. A2. The cross-linked agarose microspheres obtained in step A1 are reacted with a hydrophobic reagent to covalently graft hydrophobic groups onto the hydroxyl groups of the agarose polysaccharide backbone, thereby obtaining hydrophobically modified microspheres. A3. First, the hydrophobic modified microspheres obtained in step A2 are epoxy activated, and then grafted with cationic macromolecules containing polyamine groups to obtain the agarose microsphere adsorbent.

7. The preparation method according to claim 6, characterized in that, When the hydrophobicating agent mentioned in step A2 is butyl glycidyl ether, octyl glycidyl ether or phenyl glycidyl ether, the reaction is carried out using a mixed solvent of water and dimethyl sulfoxide, with sodium hydroxide as the catalyst, at a reaction temperature of 30-60℃ and a reaction time of 8-16 hours; after the reaction is completed, the mixture is washed alternately with water and ethanol until neutral.

8. The preparation method according to claim 6, characterized in that, When the hydrophobicating agent mentioned in step A2 is an acyl chloride with a carbon chain length of 6-12, the reaction is carried out using pyridine, toluene or tetrahydrofuran as solvent, and triethylamine is added as an acid-binding agent; after the reaction is completed, the mixture is washed with pyridine, ethanol and water in sequence.

9. The preparation method according to claim 6, characterized in that, The conditions for covalent crosslinking of epichlorohydrin in step A1 are as follows: the cured microspheres are dispersed in a 1-2 mol / L sodium hydroxide solution, and 0.5-1.5 mL of epichlorohydrin is added per gram of agarose dry base. The reaction is carried out at 40-70℃ for 4-16 hours. The volume swelling ratio of the resulting crosslinked microspheres in pure water is 3-8.

10. The application of the agarose microsphere adsorbent according to any one of claims 1 to 5 in the preparation of blood purification adsorbent materials; the blood purification adsorbent materials are used to remove endotoxins and pro-inflammatory cytokines from the blood and can be used for blood purification treatment of sepsis patients.

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