Biligand-modified cellulose-based adsorbents, methods of making and using the same

Abstract

The present application relates to a double-ligand modified cellulose-based adsorbent and its preparation method and application, and belongs to the technical field of biological medical adsorption materials.The technical problem solved by the present application is to provide a double-ligand modified cellulose-based adsorbent capable of efficiently removing a variety of toxins in a broad spectrum.The adsorbent comprises a cellulose microsphere matrix, the surface of the cellulose microsphere matrix is simultaneously grafted with long-chain alkyl amine ligands and nitrogen-containing heterocyclic ligands, the nitrogen-containing heterocyclic ligands simultaneously contain at least one amino group or quaternary ammonium group, and one nitrogen-containing five-membered heterocyclic ring or nitrogen-containing six-membered heterocyclic ring.The present application realizes simultaneous and efficient removal of beta2-microglobulin, interleukin-6, indoxyl sulfate and p-cresol sulfate through double-ligand collaborative design, and the adsorption rate of albumin is less than 10%, which has good selectivity, can be used for preparing a hemoperfusion device, and is used for blood purification treatment of patients with end-stage renal disease, and has a broad market prospect and clinical application value.

Description

Technical Field

[0001] This invention relates to dual-ligand modified cellulose-based adsorbents, their preparation methods, and applications, belonging to the field of biomedical adsorption materials technology. Background Technology

[0002] End-stage renal disease (ESRD) is the final stage of chronic kidney disease, where patients rely on maintenance hemodialysis to survive. However, long-term hemodialysis patients accumulate various uremic toxins, primarily including β2-microglobulin (β2-MG, molecular weight approximately 11.8 kDa), interleukin-6 (IL-6, molecular weight approximately 26 kDa), and protein-bound uremic toxins (PBUTs) such as indoxyl sulfate (IS) and p-cresyl sulfate (PCS). The accumulation of these toxins is closely associated with serious complications such as dialysis-associated amyloidosis, cardiovascular disease, renal osteodystrophy, and systemic inflammation.

[0003] Hemoperfusion is an extracorporeal circulation therapy that utilizes adsorbent materials to bind with pathogenic substances in blood or plasma, thereby removing these pathogens from the blood. Compared to conventional hemodialysis, hemoperfusion has significant advantages in removing medium-molecular-weight toxins and protein-bound toxins. Cellulose-based adsorbents are widely used in hemoperfusion due to their good biocompatibility, mechanical strength, and chemical stability. Several existing patents disclose cellulose-based adsorbents for hemoperfusion and their preparation methods.

[0004] Chinese patent CN114405483A discloses a porous cellulose microsphere adsorbent with a core-shell structure, its preparation method, and its application. This porous cellulose microsphere adsorbent has a core-shell structure, forming a channel structure with macropores in the core and micropores in the outer shell through the combination of a mixed solvent and a mixed porogen. This adsorbent exhibits a high adsorption rate for β2-MG. Chinese patent CN116284964A discloses a cellulose microsphere adsorbent for adsorbing β2-microglobulin, which achieves selective adsorption of β-MG by grafting hexadecylamine onto the surface of the cellulose microspheres.

[0005] However, the aforementioned existing technologies only focus on the clearance of β2-MG and fail to effectively address the clearance of protein-bound toxoids (IS and PCS) and the inflammatory factor IL-6. Protein-bound toxoids possess unique physicochemical properties: they all contain aromatic ring structures and negatively charged sulfate groups, primarily binding to adsorbents through electrostatic interactions and π-π stacking interactions. IL-6 is a glycoprotein, and its surface also carries a negative charge. Existing single hydrophobic ligands (such as hexadecaneamine) mainly rely on hydrophobic interactions, exhibiting limited adsorption capacity for IS, PCS, and IL-6.

[0006] Therefore, developing a cellulose-based adsorbent that can simultaneously and efficiently remove β2-MG, IL-6, IS, and PCS has significant clinical application value and market prospects. Summary of the Invention

[0007] To address the above deficiencies, the first technical problem solved by this invention is to provide a dual-ligand modified cellulose-based adsorbent that can broadly and efficiently remove a variety of toxins, overcoming the shortcomings of existing cellulose-based adsorbents that can only selectively adsorb a single type of toxin (such as β2-MG) and cannot simultaneously and effectively remove protein-bound toxins (IS, PCS) and inflammatory factors (IL-6).

[0008] The present invention relates to a dual-ligand modified cellulose-based adsorbent, comprising a cellulose microsphere matrix, wherein the surface of the cellulose microsphere matrix is ​​simultaneously grafted with the following two types of ligands:

[0009] (a) Long-chain alkylamine ligands with 12 to 18 carbon atoms;

[0010] (b) A nitrogen-containing heterocyclic ligand, wherein the nitrogen-containing heterocyclic ligand contains: (i) at least one amino or quaternary ammonium group, and (ii) at least one nitrogen-containing five-membered heterocycle or nitrogen-containing six-membered heterocycle.

[0011] In one embodiment of the present invention, the nitrogen-containing heterocyclic ligand is selected from aminoimidazolium compounds, aminopyridine compounds, aminotriazole compounds, or aminotetrazole compounds. Preferably, the nitrogen-containing heterocyclic ligand is selected from at least one of 1-(3-aminopropyl)imidazole, histamine, 4-aminomethylimidazole, 2-aminoimidazole, and 1-(2-aminoethyl)imidazole. More preferably, the nitrogen-containing heterocyclic ligand is selected from 1-(3-aminopropyl)imidazole.

[0012] The long-chain alkylamine ligand is selected from at least one of dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, N,N-dimethyldodecylamine, and oleylamine. Preferably, the long-chain alkylamine ligand is selected from hexadecylamine.

[0013] In one embodiment of the present invention, the grafting molar ratio of long-chain alkylamine ligands to nitrogen-containing heterocyclic ligands is 1:0.3-6.

[0014] In one embodiment of the present invention, the particle size of the cellulose microspheres is 300–800 μm.

[0015] The second technical problem solved by this invention is to provide a method for preparing a dual-ligand modified cellulose-based adsorbent.

[0016] The preparation method of the dual-ligand modified cellulose-based adsorbent of the present invention includes the following steps:

[0017] (1) Preparation of cellulose acetate microspheres by emulsification and evaporation method: cellulose acetate is dissolved in an organic solvent, and a pore-forming agent and an emulsifier are added to obtain a cellulose mixed solution; the cellulose mixed solution is dispersed in an aqueous solution containing hydrophilic polymers, stirred and emulsified to form droplets, and the solvent is evaporated to obtain cellulose acetate microspheres;

[0018] (2) Alkaline hydrolysis regeneration: The cellulose acetate microspheres obtained in step (1) are hydrolyzed in an alkaline solution to obtain regenerated cellulose microspheres;

[0019] (3) Crosslinking: The regenerated cellulose microspheres obtained in step (2) are reacted with epichlorohydrin under alkaline conditions to crosslink the cellulose chains with ether bonds, thereby obtaining crosslinked cellulose microspheres;

[0020] (4) First activation: The cross-linked cellulose microspheres obtained in step (3) are reacted with epichlorohydrin under alkaline conditions to introduce epoxy groups on the surface of the cellulose microspheres, thus obtaining cellulose microspheres with first epoxy activation;

[0021] (5) Grafting hexadecylamine: The cellulose microspheres obtained in step (4) with the first epoxy activation are reacted with long-chain alkylamine ligands, so that the long-chain alkylamine ligands are covalently grafted onto the surface of the cellulose microspheres through epoxy ring opening, and cellulose microspheres grafted with long-chain alkylamine ligands are obtained.

[0022] (6) Third epoxy treatment - second activation: The cellulose microspheres grafted with long-chain alkylamine ligands obtained in step (5) are reacted with epichlorohydrin under alkaline conditions to introduce new epoxy groups into the remaining hydroxyl sites on the surface of the cellulose microspheres, thus obtaining cellulose microspheres with second epoxy activation.

[0023] (7) First grafting of nitrogen-containing heterocyclic ligands: The second epoxy-activated cellulose microspheres obtained in step (6) are reacted with nitrogen-containing heterocyclic ligands, so that the nitrogen-containing heterocyclic ligands are covalently grafted onto the surface of the cellulose microspheres through epoxy ring-opening, and the first nitrogen-containing heterocyclic ligand grafted cellulose microspheres are obtained.

[0024] (8) Fourth epoxy treatment - third activation: The cellulose microspheres grafted with nitrogen-containing heterocyclic ligands obtained in step (7) are reacted with epichlorohydrin under alkaline conditions, and new epoxy groups are introduced again at the remaining hydroxyl sites on the surface of the cellulose microspheres to obtain the cellulose microspheres with third epoxy activation.

[0025] (9) Second grafting of nitrogen-containing heterocyclic ligands: The cellulose microspheres obtained in step (8) after the third epoxy activation are reacted with nitrogen-containing heterocyclic ligands again, so that the nitrogen-containing heterocyclic ligands are further grafted onto the surface of the cellulose microspheres to obtain a dual-ligand modified cellulose-based adsorbent.

[0026] In one embodiment of the present invention, in step (1), cellulose acetate is cellulose triacetate or cellulose diacetate; the pore-forming agent is ethylene glycol diacetate and isooctanol, and the volume ratio of ethylene glycol diacetate to isooctanol is 1:0.5-2; the emulsifier is Tween 80; the organic solvent is dichloromethane; the aqueous solution containing hydrophilic polymers is a polyvinyl alcohol aqueous solution; the volume ratio of Tween 80 to the pore-forming agent is 0.01-0.1:1; and the mass fraction of cellulose acetate in the cellulose acetate solution is 5%-15%.

[0027] In one embodiment of the present invention, in step (2), the alkaline solution is a 1-3 mol / L NaOH solution, the hydrolysis temperature is 40-60°C, and the hydrolysis time is 2-4 hours;

[0028] In one embodiment of the present invention, in steps (3) to (9), the reaction temperature is 35 to 60°C and the reaction time is 8 to 24 hours; the volume ratio of epichlorohydrin to cellulose microspheres is 0.2 to 1:1; the concentration of long-chain alkylamine ligands is 5 to 20 mg / mL and the concentration of nitrogen-containing heterocyclic ligands is 100 to 500 mg / mL.

[0029] In a preferred embodiment, the reaction temperature in step (5) is 50–70°C, and the reaction temperatures in steps (3), (4), (6), (7), (8), and (9) are 35–50°C. More preferably, the reaction temperature in step (5) is 60°C, the reaction temperatures in steps (3), (4), (6), and (8) are 50°C, and the reaction temperatures in steps (7) and (9) are 40°C.

[0030] The present invention also provides the application of the ligand-modified cellulose-based adsorbent described in the present invention or the dual-ligand-modified cellulose-based adsorbent prepared by the method of the present invention in the simultaneous removal of β2-microglobulin, interleukin-6, indophenol sulfate and p-cresol sulfate.

[0031] The present invention provides a dual-ligand modified cellulose-based adsorbent that can efficiently and simultaneously remove β2-microglobulin, interleukin-6, indophenol sulfate, and p-cresol sulfate from plasma.

[0032] The present invention also provides a blood perfusion device.

[0033] The hemoperfusion device of the present invention is filled with the dual-ligand modified cellulose-based adsorbent as described in the present invention, or the dual-ligand modified cellulose-based adsorbent prepared by the method described in the present invention.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] Broad-spectrum and highly efficient: This invention is the first to achieve the simultaneous and efficient removal of three types of toxins with different physicochemical properties—β2-MG, IL-6, and PBUTs (IS, PCS, etc.)—by cellulose microspheres. Experiments show that the adsorbent of this invention can achieve an adsorption rate of up to 87.1% for β2-MG, up to 39.3% for IL-6, up to 86.5% for IS, and up to 90.7% for PCS.

[0036] Synergistic mechanism: This invention utilizes a dual-ligand synergistic design, where long-chain alkylamines provide hydrophobic interactions to adsorb β2-MG and IL-6, while nitrogen-containing heterocyclic ligands simultaneously provide electrostatic interactions and π-π stacking interactions to adsorb IS and PCS. These three adsorption mechanisms complement and synergistically achieve broad-spectrum scavenging of multiple toxins.

[0037] Inventiveness of Ligand Screening: The inventors systematically screened five small molecule ligands with different structures (ammonia, 1,6-hexanediamine, diethylenetriamine, quaternary ammonium glycidyl ether, and 1-(3-aminopropyl)imidazole) and found that only 1-(3-aminopropyl)imidazole, which contains both an amino group and an imidazole ring, could achieve simultaneous and efficient removal of all four toxins, while the other four ligands could not achieve simultaneous and efficient adsorption. These results indicate that the synergistic effect of electrostatic adsorption provided by the amino group and π-π stacking provided by the imidazole ring is key to achieving the technical effect of this invention, and this synergistic effect cannot be replaced by ligands containing only amino groups or only quaternary ammonium groups. Therefore, the ligand selection in this invention is inventive.

[0038] Good selectivity: The adsorbent of this invention has an adsorption rate of ≤10% for albumin, indicating that it has good selectivity for the target toxin and will not excessively consume beneficial proteins in the blood.

[0039] Whole blood compatibility: The adsorbent of this invention has a particle size range of 300-800μm and sufficient packing gaps to ensure smooth passage of red blood cells; the adsorbent has good mechanical strength and does not break or fall off under dynamic perfusion conditions, has good safety, is suitable for whole blood perfusion, and has good blood compatibility and clinical application prospects.

[0040] Process innovation: This invention adopts a combination of low-temperature long-time crosslinking (40℃, 16h), temperature-controlled grafting (hexadecylamine 60℃, aminoimidazole 40℃), asymmetric grafting (hexadecylamine once, aminoimidazole twice) and four ECH treatments, forming a unique preparation technology route. Attached Figure Description

[0041] Figure 1 This is a process flow diagram for preparing the dual-ligand modified cellulose-based adsorbent of the present invention.

[0042] Figure 2 Photograph of the dual-ligand modified cellulose-based adsorbent prepared in Example 1 of this invention.

[0043] Figure 3 These are scanning electron microscope (SEM) images of the regenerated cellulose microspheres prepared in step two of Example 1 of the present invention, wherein a and b are SEM images of the surface and cross-section, respectively, and c and d are magnified SEM images of the surface and cross-section, respectively.

[0044] Figure 4 The bar chart shows the adsorption performance comparison between the embodiments and comparative examples of the present invention and existing products. Detailed Implementation

[0045] The present invention relates to a dual-ligand modified cellulose-based adsorbent, comprising a cellulose microsphere matrix, wherein the surface of the cellulose microsphere matrix is ​​simultaneously grafted with the following two types of ligands:

[0046] (a) Long-chain alkylamine ligands with 12 to 18 carbon atoms;

[0047] (b) A nitrogen-containing heterocyclic ligand, wherein the nitrogen-containing heterocyclic ligand contains: (i) at least one amino or quaternary ammonium group, and (ii) at least one nitrogen-containing five-membered heterocycle or nitrogen-containing six-membered heterocycle.

[0048] This invention relates to a dual-ligand modified cellulose-based adsorbent. The synergistic effect of long-chain alkylamine ligands and nitrogen-containing heterocyclic ligands enables the adsorbent to simultaneously and efficiently statically adsorb β2-microglobulin, interleukin-6, indophenol sulfate, and p-cresol sulfate from plasma, while exhibiting low adsorption rate for albumin. This adsorbent can be used to prepare hemoperfusion devices for blood purification treatment of end-stage renal disease patients, effectively removing β2-microglobulin, interleukin-6, indophenol sulfate, and p-cresol sulfate simultaneously and efficiently, demonstrating broad market prospects and clinical application value.

[0049] In one embodiment of the present invention, the nitrogen-containing heterocyclic ligand is selected from aminoimidazolium compounds, aminopyridine compounds, aminotriazole compounds, or aminotetrazole compounds. Preferably, the nitrogen-containing heterocyclic ligand is selected from at least one of 1-(3-aminopropyl)imidazole, histamine, 4-aminomethylimidazole, 2-aminoimidazole, and 1-(2-aminoethyl)imidazole. More preferably, the nitrogen-containing heterocyclic ligand is selected from 1-(3-aminopropyl)imidazole.

[0050] The long-chain alkylamine ligand is selected from at least one of dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, N,N-dimethyldodecylamine, and oleylamine. Preferably, the long-chain alkylamine ligand is selected from hexadecylamine.

[0051] In one embodiment of the present invention, the grafting molar ratio of long-chain alkylamine ligands to nitrogen-containing heterocyclic ligands is 1:0.3-6.

[0052] In one embodiment of the present invention, the particle size of the cellulose microspheres is 300–800 μm.

[0053] The preparation method of the dual-ligand modified cellulose-based adsorbent of the present invention includes the following steps:

[0054] (1) Preparation of cellulose acetate microspheres by emulsification and evaporation method: cellulose acetate is dissolved in an organic solvent, and a pore-forming agent and an emulsifier are added to obtain a cellulose mixed solution; the cellulose mixed solution is dispersed in an aqueous solution containing hydrophilic polymers, stirred and emulsified to form droplets, and the solvent is evaporated to obtain cellulose acetate microspheres;

[0055] (2) Alkaline hydrolysis regeneration: The cellulose acetate microspheres obtained in step (1) are hydrolyzed in an alkaline solution to obtain regenerated cellulose microspheres;

[0056] (3) Crosslinking: The regenerated cellulose microspheres obtained in step (2) are reacted with epichlorohydrin under alkaline conditions to crosslink the cellulose chains with ether bonds, thereby obtaining crosslinked cellulose microspheres;

[0057] (4) First activation: The cross-linked cellulose microspheres obtained in step (3) are reacted with epichlorohydrin under alkaline conditions to introduce epoxy groups on the surface of the cellulose microspheres, thus obtaining cellulose microspheres with first epoxy activation;

[0058] (5) Grafting hexadecylamine: The cellulose microspheres obtained in step (4) with the first epoxy activation are reacted with long-chain alkylamine ligands, so that the long-chain alkylamine ligands are covalently grafted onto the surface of the cellulose microspheres through epoxy ring opening, and cellulose microspheres grafted with long-chain alkylamine ligands are obtained.

[0059] (6) Third epoxy treatment - second activation: The cellulose microspheres grafted with long-chain alkylamine ligands obtained in step (5) are reacted with epichlorohydrin under alkaline conditions to introduce new epoxy groups into the remaining hydroxyl sites on the surface of the cellulose microspheres, thus obtaining cellulose microspheres with second epoxy activation.

[0060] (7) First grafting of nitrogen-containing heterocyclic ligands: The second epoxy-activated cellulose microspheres obtained in step (6) are reacted with nitrogen-containing heterocyclic ligands, so that the nitrogen-containing heterocyclic ligands are covalently grafted onto the surface of the cellulose microspheres through epoxy ring-opening, and the first nitrogen-containing heterocyclic ligand grafted cellulose microspheres are obtained.

[0061] (8) Fourth epoxy treatment - third activation: The cellulose microspheres grafted with nitrogen-containing heterocyclic ligands obtained in step (7) are reacted with epichlorohydrin under alkaline conditions, and new epoxy groups are introduced again at the remaining hydroxyl sites on the surface of the cellulose microspheres to obtain the cellulose microspheres with third epoxy activation.

[0062] (9) Second grafting of nitrogen-containing heterocyclic ligands: The cellulose microspheres obtained in step (8) after the third epoxy activation are reacted with nitrogen-containing heterocyclic ligands again, so that the nitrogen-containing heterocyclic ligands are further grafted onto the surface of the cellulose microspheres to obtain a dual-ligand modified cellulose-based adsorbent.

[0063] In one embodiment of the present invention, in step (1), cellulose acetate is cellulose triacetate or cellulose diacetate; the pore-forming agent is ethylene glycol diacetate and isooctanol, and the volume ratio of ethylene glycol diacetate to isooctanol is 1:0.5-2; the emulsifier is Tween 80; the organic solvent is dichloromethane; the aqueous solution containing hydrophilic polymers is a polyvinyl alcohol aqueous solution; the volume ratio of Tween 80 to the pore-forming agent is 0.01-0.1:1; and the mass fraction of cellulose acetate in the cellulose acetate solution is 5%-15%.

[0064] In one embodiment of the present invention, in step (2), the alkaline solution is a 1-3 mol / L NaOH solution, the hydrolysis temperature is 40-60°C, and the hydrolysis time is 2-4 hours;

[0065] In one embodiment of the present invention, in steps (3) to (9), the reaction temperature is 35 to 60°C and the reaction time is 8 to 24 hours; the volume ratio of epichlorohydrin to cellulose microspheres is 0.2 to 1:1; the concentration of long-chain alkylamine ligands is 5 to 20 mg / mL and the concentration of nitrogen-containing heterocyclic ligands is 100 to 500 mg / mL.

[0066] In a preferred embodiment, the reaction temperature in step (5) is 50–70°C, and the reaction temperatures in steps (3), (4), (6), (7), (8), and (9) are 35–50°C. More preferably, the reaction temperature in step (5) is 60°C, the reaction temperatures in steps (3), (4), (6), and (8) are 50°C, and the reaction temperatures in steps (7) and (9) are 40°C.

[0067] The present invention also provides the application of the ligand-modified cellulose-based adsorbent described in the present invention or the dual-ligand-modified cellulose-based adsorbent prepared by the method of the present invention in the simultaneous removal of β2-microglobulin, interleukin-6, indophenol sulfate and p-cresol sulfate.

[0068] The present invention provides a dual-ligand modified cellulose-based adsorbent that can efficiently and simultaneously remove β2-microglobulin, interleukin-6, indophenol sulfate, and p-cresol sulfate from plasma.

[0069] The hemoperfusion device of the present invention is filled with the dual-ligand modified cellulose-based adsorbent as described in the present invention, or the dual-ligand modified cellulose-based adsorbent prepared by the method described in the present invention.

[0070] Preferably, the outlet of the irrigation device is provided with a filter screen with a pore size ≤200μm.

[0071] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0072] Example 1

[0073] like Figure 1 As shown, a dual-ligand modified cellulose-based adsorbent was prepared according to the following method:

[0074] Step 1: Preparation of cellulose acetate microspheres by emulsification and evaporation method

[0075] Weigh 30 g of cellulose acetate powder and add it to 200 mL of dichloromethane. Stir until completely dissolved. Measure 130 mL of ethylene glycol diacetate (EGDA) and 70 mL of isooctanol and add them to the above mixture. After shaking and mixing evenly, add 10 mL of Tween 80 to obtain a clear and transparent cellulose mixture. Add the obtained mixture to 2.5 L of a 2% (w / w) polyvinyl alcohol (PVA) aqueous solution and emulsify and disperse at 350 rpm for 20 minutes at 25°C to form cellulose droplets. After emulsification, heat in a 35°C water bath for 24 hours to evaporate the organic solvent. Filter, wash with water, and sieve through 800 μm and 300 μm sieves respectively to collect cellulose acetate microspheres with a particle size of 300–800 μm.

[0076] Step 2: Alkali hydrolysis regeneration

[0077] The cellulose acetate microspheres obtained in step one were added to a 1.5 mol / L NaOH solution and hydrolyzed at 40°C for 3 hours. The mixture was then filtered and thoroughly washed with water until neutral to obtain regenerated cellulose microspheres. A scanning electron microscope image of the regenerated cellulose microspheres is shown below. Figure 3 .

[0078] Step 3: First epoxy treatment – ​​crosslinking

[0079] Take 100 mL of the regenerated cellulose microspheres obtained in step two, add 100 mL of 2 mol / L NaOH solution and 20 mL of epichlorohydrin (ECH), and react at 50 °C for 16 hours to induce ether crosslinking between the cellulose chains. After the reaction is complete, filter and wash thoroughly with water to obtain crosslinked cellulose microspheres.

[0080] Step 4: Second Epoxy Treatment – ​​First Activation

[0081] Take 100 mL of the cross-linked cellulose microspheres obtained in step three, add 50 mL of dimethyl sulfoxide and 50 mL of epichlorohydrin, stir at room temperature for 30 minutes, then add 50 mL of 2 mol / L NaOH solution, and shake and react at 50 °C for 8 hours to introduce epoxy groups onto the surface of the cellulose microspheres. After the reaction is complete, wash thoroughly with water to obtain the first epoxy-activated cellulose microspheres.

[0082] Step 5: Grafting hexadecylamine

[0083] The cellulose microspheres obtained in step four, which underwent initial epoxy activation, were mixed with 400 mL of a 20 mg / mL hexadecylamine ethanol solution and reacted at 60 °C for 16 hours, allowing hexadecylamine to be covalently grafted onto the surface of the cellulose microspheres via epoxy ring-opening. After the reaction was complete, the microspheres were thoroughly washed with ethanol to obtain hexadecylamine-grafted cellulose microspheres.

[0084] Step Six: Third Epoxy Treatment – ​​Second Activation

[0085] Take 100 mL of the hexadecylamine-grafted cellulose microspheres obtained in step five, add 50 mL of dimethyl sulfoxide and 50 mL of epichlorohydrin, stir at room temperature for 30 minutes, then add 50 mL of 2 mol / L NaOH solution, and shake and react at 50 °C for 8 hours to introduce epoxy groups onto the surface of the cellulose microspheres. After the reaction is complete, wash thoroughly with water to obtain the second epoxy-activated cellulose microspheres.

[0086] Step 7: First grafting of 1-(3-aminopropyl)imidazolium

[0087] Take 100 mL of the second epoxy-activated cellulose microspheres obtained in step six, add 50 mL of 10 mol / L NaOH solution and 125 mL of 100 mg / mL 1-(3-aminopropyl)imidazole, and mix at 40 °C with shaking for 16 hours to allow 1-(3-aminopropyl)imidazole to be covalently grafted onto the cellulose microspheres via epoxy ring-opening. After the reaction is complete, wash thoroughly with water to obtain the first aminoimidazole-grafted cellulose microspheres.

[0088] Step 8: Fourth Epoxy Treatment – ​​Third Activation

[0089] Take 100 mL of the first aminoimidazole-grafted cellulose microspheres obtained in step seven, add 50 mL of dimethyl sulfoxide and 50 mL of epichlorohydrin, stir at room temperature for 30 minutes, then add 50 mL of 2 mol / L NaOH solution, and shake and react at 50 °C for 8 hours to introduce epoxy groups again onto the surface of the cellulose microspheres. After the reaction is complete, wash thoroughly with water to obtain the third epoxy-activated cellulose microspheres.

[0090] Step Nine: Second grafting of 1-(3-aminopropyl)imidazolium

[0091] The cellulose microspheres obtained in step eight, after the third epoxy activation, were mixed with 100 mL of 50 mL of 10 mol / L NaOH solution and 125 mL of 100 mg / mL 1-(3-aminopropyl)imidazole. The mixture was shaken and reacted at 40 °C for 16 hours to further graft 1-(3-aminopropyl)imidazole onto the surface of the cellulose microspheres. After the reaction, the mixture was thoroughly washed with water to obtain the dual-ligand modified cellulose-based adsorbent. A photograph of this dual-ligand modified cellulose-based adsorbent is shown below. Figure 2 .

[0092] Performance testing

[0093] Prepare a bovine plasma solution containing β2-MG (approximately 10 mg / L), IL-6 (approximately 1800 pg / mL), IS (approximately 30 mg / L), and PCS (approximately 30 mg / L). Take 1 mL of the above adsorbent and add it to 10 mL of the prepared plasma solution. Shake and adsorb at 37℃ and 130 rpm for 2 hours. Measure the concentrations of each toxin before and after adsorption and calculate the adsorption rate.

[0094] Test results: β2-MG adsorption rate 87.1%, IL-6 adsorption rate 39.3%, IS adsorption rate 86.5%, PCS adsorption rate 90.7%, albumin (TP) adsorption rate 5.6%.

[0095] Example 2 (using histamine)

[0096] It is basically the same as Example 1, except that 1-(3-aminopropyl)imidazole is replaced with histamine.

[0097] Test results: β2-MG adsorption rate 72.2%, IL-6 adsorption rate 27.1%, IS adsorption rate 85.4%, PCS adsorption rate 86.6%, TP adsorption rate 6.5%, see Table 1 for details.

[0098] Example 3 (using 4-aminomethylimidazole)

[0099] The example is basically the same as in Example 1, except that 1-(3-aminopropyl)imidazole is replaced with 4-aminomethylimidazole.

[0100] Test results: β2-MG adsorption rate 78.4%, IL-6 adsorption rate 37.2%, IS adsorption rate 81.4%, PCS adsorption rate 85.9%, TP adsorption rate 6.9%, see Table 1 for details.

[0101] Comparative Example 1 (hexadecylamine only, no biligands)

[0102] The process is basically the same as in Example 1, except that only hexadecylamine is grafted onto it, and nitrogen-containing heterocyclic ligands are not grafted onto it, that is, only steps one to five are performed.

[0103] Test results: β2-MG adsorption rate 90.2%, IL-6 adsorption rate 76.2%, IS adsorption rate 20.3%, PCS adsorption rate 28.7%, TP adsorption rate 8.2%, see Table 1 for details.

[0104] The results showed that when only hexadecylamine was grafted, the adsorption rate for IS and PCS was less than 30%, proving that the adsorption capacity of a single long-chain alkylamine ligand for protein-bound toxoids was limited.

[0105] Comparative Example 2 (containing only nitrogen heterocyclic ligands, without biligands)

[0106] It is basically the same as Example 1, except that only 1-(3-aminopropyl)imidazole is grafted and hexadecylamine is not grafted, that is, only steps one to four and steps seven to nine are performed.

[0107] Test results: β2-MG adsorption rate 24.5%, IL-6 adsorption rate 14.3%, IS adsorption rate 54.6%, PCS adsorption rate 47.3%, TP adsorption rate 5.8%, see Table 1 for details.

[0108] The results showed that when only nitrogen-containing heterocyclic ligands were grafted, the adsorption rate of β2-MG and IL-6 was less than 30%, proving that the adsorption capacity of a single nitrogen-containing heterocyclic ligand for protein toxins was insufficient.

[0109] Comparative Example 3 (ammonia as the second ligand)

[0110] It is basically the same as Example 1, except that 1-(3-aminopropyl)imidazole is replaced with ammonia.

[0111] Test results: β2-MG adsorption rate 80.2%, IL-6 adsorption rate 34.6%, IS adsorption rate 41.8%, PCS adsorption rate 23.5%, TP adsorption rate 8.2%, see Table 1 for details.

[0112] Comparative Example 4 (1,6-hexanediamine as the second ligand)

[0113] It is basically the same as Example 1, except that 1-(3-aminopropyl)imidazole is replaced with 1,6-hexanediamine.

[0114] Test results: β2-MG adsorption rate 79.5%, IL-6 adsorption rate 35.0%, IS adsorption rate 57.8%, PCS adsorption rate 57.5%, TP adsorption rate 7.5%, see Table 1 for details.

[0115] Comparative Example 5 (Diethylenetriamine as the second ligand)

[0116] It is basically the same as Example 1, except that 1-(3-aminopropyl)imidazole is replaced with diethylenetriamine.

[0117] Test results: β2-MG adsorption rate 75.8%, IL-6 adsorption rate 32.4%, IS adsorption rate 48.3%, PCS adsorption rate 38.2%, TP adsorption rate 6.9%, see Table 1 for details.

[0118] Comparative Example 6 (2,3-epoxypropyltrimethylammonium chloride as the second ligand)

[0119] The example is basically the same as Example 1, except that 1-(3-aminopropyl)imidazole is replaced with 2,3-epoxypropyltrimethylammonium chloride.

[0120] Test results: β2-MG adsorption rate 72.3%, IL-6 adsorption rate 32.5%, IS adsorption rate 50.8%, PCS adsorption rate 37.7%, TP adsorption rate 7.5%, see Table 1 for details.

[0121] Table 1

[0122] Example 1 Hexadecylamine 1-(3-aminopropyl)imidazolium 87.1% 39.3% 86.5% 90.7% 5.6% Example 2 Hexadecylamine histamine 72.2% 27.1% 85.4% 86.6% 6.5% Example 3 Hexadecylamine 4-Aminomethylimidazolium 78.4% 37.2% 81.4% 85.9% 6.9% Comparative Example 1 Hexadecylamine none 90.2% 76.2% 20.3% 28.7% 8.2% Comparative Example 2 none 1-(3-aminopropyl)imidazolium 24.5% 14.3% 54.6% 47.3% 5.8% Comparative Example 3 Hexadecylamine ammonia 80.2% 34.6% 41.8% 23.5% 8.2% Comparative Example 4 Hexadecylamine 1,6-Hexamethylenediamine 79.5% 35.0% 57.8% 57.5% 7.5% Comparative Example 5 Hexadecylamine Diethylenetriamine 75.8% 32.4% 48.3% 38.2% 6.9% Comparative Example 6 Hexadecylamine 2,3-Epoxypropyltrimethylammonium chloride 72.3% 32.5% 50.8% 37.7% 7.5% PHA / / 73.3% 28.1% 83.8% 94.7% 10.5% HA / / 76.0% 37.6% 20.7% 21.3% 9.7% Lixelle / / 85.4% 45.6% 8.2% 11.9% 2.9%

[0123] Note: The concentration of IL-6 was 1800 pg / mL, the concentration of β2-MG was 10 mg / L, and the concentrations of IS and PCS were 30 mg / L; the latter three were commercially available adsorbents. Among them, PHA is PHA-130, a product of Chienfan Biotech Group Co., Ltd., HA is HA-130, a product of Chienfan Biotech Group Co., Ltd., and Lixelle is a product of Kaneka Corporation.

[0124] The results analysis shows that:

[0125] (1) The combined adsorption effect of the dual-ligand system (Examples 1-3) is significantly better than that of any single-ligand system (Comparative Examples 1-2), which proves the synergistic effect between long-chain alkylamines and nitrogen-containing heterocyclic ligands.

[0126] (2) Among the five second ligands—1-(3-aminopropyl)imidazole, ammonia, 1,6-hexanediamine, diethylenetriamine, and 2,3-epoxypropyltrimethylammonium chloride—only 1-(3-aminopropyl)imidazole (Example 1) could effectively adsorb the four target toxins. The other four ligands (Comparative Examples 3-6) had adsorption rates of less than 60% for IS and PCS, and less than 75% for IL-6. This indicates that the synergistic effect of the amino group providing electrostatic adsorption and the imidazole ring providing π-π stacking is the key to achieving the technical effect of this invention, and this synergistic effect cannot be replaced by ligands containing only amino groups or only quaternary ammonium groups.

Claims

1. A cellulose-based adsorbent modified with two ligands, characterized in that: The matrix includes a cellulose microsphere matrix, the surface of which is simultaneously grafted with the following two types of ligands: (a) Long-chain alkylamine ligands with 12 to 18 carbon atoms; (b) A nitrogen-containing heterocyclic ligand, wherein the nitrogen-containing heterocyclic ligand contains: (i) at least one amino or quaternary ammonium group, and (ii) at least one nitrogen-containing five-membered heterocycle or nitrogen-containing six-membered heterocycle.

2. The dual-ligand modified cellulose-based adsorbent according to claim 1, characterized in that: The nitrogen-containing heterocyclic ligand is selected from aminoimidazolium compounds, aminopyridine compounds, aminotriazole compounds, or aminotetrazole compounds; preferably, the nitrogen-containing heterocyclic ligand is selected from at least one of 1-(3-aminopropyl)imidazolium, histamine, 4-aminomethylimidazolium, 2-aminoimidazolium, and 1-(2-aminoethyl)imidazolium; more preferably, the nitrogen-containing heterocyclic ligand is selected from 1-(3-aminopropyl)imidazolium; The long-chain alkylamine ligand is selected from at least one of dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, N,N-dimethyldodecylamine, and oleylamine, with hexadecylamine being the preferred long-chain alkylamine ligand.

3. The dual-ligand modified cellulose-based adsorbent according to claim 1, characterized in that: The grafting molar ratio of long-chain alkylamine ligands to nitrogen-containing heterocyclic ligands is 1:0.3–6.

4. The dual-ligand modified cellulose-based adsorbent according to claim 1, characterized in that: The particle size of the cellulose microspheres is 300–800 μm.

5. The method for preparing the dual-ligand modified cellulose-based adsorbent according to any one of claims 1 to 4, characterized in that: Includes the following steps: (1) Preparation of cellulose acetate microspheres by emulsification and evaporation method: cellulose acetate is dissolved in an organic solvent to obtain a cellulose acetate solution, a pore-forming agent and an emulsifier are added to obtain a cellulose mixed solution; the cellulose mixed solution is dispersed in an aqueous solution containing hydrophilic polymers, stirred and emulsified to form droplets, and the solvent is evaporated to obtain cellulose acetate microspheres; (2) Alkaline hydrolysis regeneration: The cellulose acetate microspheres obtained in step (1) are hydrolyzed in an alkaline solution to obtain regenerated cellulose microspheres; (3) Crosslinking: The regenerated cellulose microspheres obtained in step (2) are reacted with epichlorohydrin under alkaline conditions to crosslink the cellulose chains with ether bonds, thereby obtaining crosslinked cellulose microspheres; (4) First activation: The cross-linked cellulose microspheres obtained in step (3) are reacted with epichlorohydrin under alkaline conditions to introduce epoxy groups on the surface of the cellulose microspheres, thus obtaining cellulose microspheres with first epoxy activation; (5) Grafting hexadecylamine: The cellulose microspheres obtained in step (4) with the first epoxy activation are reacted with long-chain alkylamine ligands, so that the long-chain alkylamine ligands are covalently grafted onto the surface of the cellulose microspheres through epoxy ring opening, and cellulose microspheres grafted with long-chain alkylamine ligands are obtained. (6) Third epoxy treatment - second activation: The cellulose microspheres grafted with long-chain alkylamine ligands obtained in step (5) are reacted with epichlorohydrin under alkaline conditions to introduce new epoxy groups into the remaining hydroxyl sites on the surface of the cellulose microspheres, thus obtaining cellulose microspheres with second epoxy activation. (7) First grafting of nitrogen-containing heterocyclic ligands: The second epoxy-activated cellulose microspheres obtained in step (6) are reacted with nitrogen-containing heterocyclic ligands, so that the nitrogen-containing heterocyclic ligands are covalently grafted onto the surface of the cellulose microspheres through epoxy ring-opening, and the first nitrogen-containing heterocyclic ligand grafted cellulose microspheres are obtained. (8) Fourth epoxy treatment - third activation: The cellulose microspheres grafted with nitrogen-containing heterocyclic ligands obtained in step (7) are reacted with epichlorohydrin under alkaline conditions, and new epoxy groups are introduced again at the remaining hydroxyl sites on the surface of the cellulose microspheres to obtain the cellulose microspheres with third epoxy activation. (9) Second grafting of nitrogen-containing heterocyclic ligands: The cellulose microspheres obtained in step (8) after the third epoxy activation are reacted with nitrogen-containing heterocyclic ligands again, so that the nitrogen-containing heterocyclic ligands are further grafted onto the surface of the cellulose microspheres to obtain a dual-ligand modified cellulose-based adsorbent.

6. The method for preparing the dual-ligand modified cellulose-based adsorbent according to claim 5, characterized in that: In step (1), cellulose acetate is cellulose triacetate or cellulose diacetate; the pore-forming agent is ethylene glycol diacetate and isooctanol, and the volume ratio of ethylene glycol diacetate to isooctanol is 1:0.5-2; the emulsifier is Tween 80; the organic solvent is dichloromethane; the aqueous solution containing hydrophilic polymers is a polyvinyl alcohol aqueous solution; the volume ratio of Tween 80 to the pore-forming agent is 0.01-0.1:1; and the mass fraction of cellulose acetate in the cellulose acetate solution is 5%-15%.

7. The method for preparing the dual-ligand modified cellulose-based adsorbent according to claim 5, characterized in that: In step (2), the alkaline solution is a 1-3 mol / L NaOH solution, the hydrolysis temperature is 40-60℃, and the hydrolysis time is 2-4 hours.

8. The method for preparing the dual-ligand modified cellulose-based adsorbent according to claim 5, characterized in that: In steps (3) to (9), the reaction temperature is 35–60℃ and the reaction time is 8–24 hours; the volume ratio of epichlorohydrin to cellulose microspheres is 0.2–1:1; the concentration of long-chain alkylamine ligands is 5–20 mg / mL and the concentration of nitrogen-containing heterocyclic ligands is 100–500 mg / mL. The preferred reaction temperature in step (5) is 50-70°C, and the reaction temperature in steps (3), (4), (6), (7), (8), and (9) is 35-50°C; more preferably, the reaction temperature in step (5) is 60°C, the reaction temperature in steps (3), (4), (6), and (8) is 50°C, and the reaction temperature in steps (7) and (9) is 40°C.

9. The use of the dual-ligand modified cellulose-based adsorbent according to any one of claims 1 to 4, or the dual-ligand modified cellulose-based adsorbent prepared by the method according to any one of claims 5 to 8, in the simultaneous removal of β2-microglobulin, interleukin-6, indophenol sulfate, and p-cresol sulfate.

10. A blood perfusion device, characterized in that, The hemoperfusion device is filled with the dual-ligand modified cellulose-based adsorbent as described in any one of claims 1 to 4, or the dual-ligand modified cellulose-based adsorbent prepared by the method described in any one of claims 5 to 8.

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