Cellulose microsphere adsorbent for blood purification and method for preparing the same

Abstract

The present application relates to the technical field of blood purification, and particularly relates to a cellulose microsphere adsorbent for blood purification and a preparation method thereof; the adsorbent takes cellulose microspheres as a base material, and simultaneously grafts hydrophobic ligands and positive charge ligands; the hydrophobic ligands are alkyl amines containing 6-14 carbon atoms; the positive charge ligands are selected from cationic small molecule compounds containing amine groups, including one or more of small molecule polyamines, quaternary ammonium salt type amine compounds and amine compounds containing aromatic heterocyclic rings; the cellulose acetate microspheres are prepared by an emulsification solvent evaporation method, and after hydrolysis and crosslinking of epichlorohydrin, the hydrophobic ligands and the positive charge ligands are grafted in two steps, so that the synergistic effect of the two ligands is utilized, and uremia-related middle molecule toxins (such as beta2-microglobulin and interleukin-6) and protein-bound toxins (such as indoxyl sulfate and p-cresol sulfate) are simultaneously removed, the total protein adsorption rate is less than 7%, the biocompatibility is good, and the adsorbent has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of blood purification technology, and in particular to a cellulose microsphere adsorbent for blood purification and its preparation method. The adsorbent can be used for hemoperfusion and simultaneously removes uremia-related toxins such as β2-microglobulin, some inflammatory factors, and protein-bound toxins. Background Technology

[0002] Uremia is a clinical syndrome characterized by systemic poisoning symptoms caused by the near-complete loss of kidney function in the end-stage of chronic renal failure, leading to the accumulation of metabolic waste and water, severe electrolyte and acid-base imbalances. Hemodialysis can help uremia patients regulate acid-base balance, correct water and electrolyte imbalances, and remove small molecule toxins. However, because hemodialysis primarily relies on diffusion to remove small molecules, it cannot effectively remove toxins with slightly larger molecular weights, such as uremia-related small to medium molecule toxins (e.g., β2-microglobulin (β2-MG)) and medium to medium molecule toxins (e.g., interleukin-6 (IL-6)). Furthermore, protein-bound toxins (PBUTs), which are relatively small in molecular weight but readily bind to serum proteins (e.g., indophenol sulfate (IS) and p-cresol sulfate (PCS), are difficult to remove directly using conventional dialysis methods. The inability to separate toxins from proteins can lead to long-term accumulation of toxins in the patient's body, or severe albumin loss after removal, resulting in various complications.

[0003] Hemoperfusion is a relatively novel blood purification technique that has been implemented in the treatment of various diseases with good clinical results. For uremia, hemoperfusion can remove small to medium molecular weight and larger toxins that cannot be removed by hemodialysis through adsorption.

[0004] Current research and application of adsorbent technology for uremic perfusion are extensive, but it still faces some limitations in two aspects: First, regarding the removal of target substances, most adsorbent research focuses on small to medium molecular weight proteins such as β2-microglobulin, while research on the removal of protein-bound toxoids is relatively limited. However, recent studies have gradually confirmed that protein-bound toxoids are a class of highly hazardous and difficult-to-remove uremic toxins, closely related to complications such as cardiovascular disease and renal osteodystrophy. Second, regarding adsorbent substrates, current mainstream research and application focus on porous polystyrene microspheres. While the preparation process of these microspheres is mature and they have a high basic loading capacity, some problems exist. For example, the synthesis process uses toxic raw materials and hazardous processes, and the strong hydrophobicity of the microspheres leads to poor biocompatibility, easily causing adverse reactions such as thrombocytopenia and complement activation.

[0005] Cellulose is a naturally abundant macromolecule with advantages such as low cost, environmental friendliness, good biocompatibility, and ease of functionalization. Some related studies have used cellulose as a substrate and, after specific hydrophobic treatment, achieved effective adsorption of certain uremic-related toxins (such as β2-MG).

[0006] However, existing cellulose-based adsorbents are mainly targeted at small and medium molecular weight toxins, such as β2-MG, and their removal effect on PBUTs is very limited, which cannot meet clinical needs.

[0007] Therefore, developing a blood perfusion adsorbent with good biocompatibility, based on cellulose, that can simultaneously and efficiently remove small and medium molecular weight toxins (such as β2-MG), some inflammatory factors and PBUTs, and with low total protein adsorption rate is of great clinical significance. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a cellulose microsphere adsorbent and its preparation method that can simultaneously remove small and medium molecular weight toxins (such as β2-MG), some inflammatory factors and PBUTs, and has a low total protein adsorption rate, for use in blood purification therapy.

[0009] To achieve the above objectives, the specific solution of the present invention is as follows: A cellulose microsphere adsorbent for blood purification includes a cellulose microsphere substrate, wherein a hydrophobic ligand and a positively charged ligand are grafted onto the cellulose microsphere substrate; the hydrophobic ligand is an alkylamine containing 6 to 14 carbon atoms, and the positively charged ligand is selected from cationic small molecule compounds containing amine groups, including one or more of small molecule polyamines, quaternary ammonium salt amine compounds, and amine compounds containing aromatic heterocycles; based on the wet microsphere volume, the grafting amount of the hydrophobic ligand is 20 to 100 μmol / mL, and the grafting amount of the positively charged ligand is 100 to 400 μmol / mL.

[0010] Furthermore, the cellulose microsphere substrate is prepared by hydrolysis and epichlorohydrin crosslinking of cellulose acetate microspheres.

[0011] Furthermore, the epichlorohydrin crosslinking is a single crosslinking reaction or a double crosslinking reaction.

[0012] Furthermore, the hydrophobic ligand is selected from n-hexylamine, n-octylamine, or tetradecylamine.

[0013] Further, the positively charged ligand is one or two of 1,3-propanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, p-phenylenediamine, 3-(3-aminopropyl)pyridine, and 3-aminopropyltrimethylammonium chloride.

[0014] Furthermore, when incubated with bovine plasma at 37°C and with a wet microsphere volume to adsorption liquid volume ratio of 1:10, the adsorbent exhibits an adsorption rate of not less than 80% for β2-MG, not less than 70% for IS, not less than 75% for PCS, not less than 30% for IL-6, and a total protein adsorption rate not higher than 7%.

[0015] The present invention also provides a method for preparing the above-mentioned cellulose microsphere adsorbent, comprising the following steps: (1) Cellulose acetate was dissolved in an organic solvent, a pore-forming agent was added, and cellulose acetate microspheres were prepared by emulsification solvent evaporation method; (2) The cellulose acetate microspheres are hydrolyzed and then crosslinked with epichlorohydrin to obtain cellulose microsphere substrate; (3) Activate the hydroxyl groups on the cellulose microsphere substrate with an activator and graft hydrophobic ligands to obtain hydrophobically modified microspheres; (4) The remaining hydroxyl groups on the hydrophobic modified microspheres are activated again with an activator and positively charged ligands are grafted to obtain the cellulose microsphere adsorbent.

[0016] Further, in step (1), the organic solvent is one or two of dichloromethane, ethanol, acetone or ethyl acetate, the pore-forming agent is one or two of n-octanol, n-heptol, butyl acetate or ethylene glycol diacetate, and the aqueous phase used for emulsification contains polyvinyl alcohol.

[0017] Furthermore, in step (2), the hydrolysis treatment uses an aqueous solution of sodium hydroxide or a mixed solution of sodium hydroxide in ethanol and water.

[0018] Further, in step (1), the cellulose acetate is cellulose diacetate or cellulose triacetate; in steps (3) and (4), the activator is epichlorohydrin.

[0019] The core principle of this invention lies in the synergistic effect of hydrophobic ligands and positively charged ligands: (1) Hydrophobic ligands (alkylamines) can effectively adsorb medium-molecular-weight protein toxins with hydrophobic domains, such as β2-MG and IL-6, through hydrophobic interactions.

[0020] (2) Positively charged ligands (amine compounds) can bind to negatively charged PBUTs (such as IS and PCS) through electrostatic interactions. More importantly, positively charged ligands can competitively displace toxin molecules bound to albumin, causing them to change from a bound state to a free state.

[0021] (3) When hydrophobic ligands and positively charged ligands are present at the same time, the free PBUTs displaced by the positively charged ligands can be further captured by the hydrophobic ligands through hydrophobic interactions, thereby forming a "displacement-capture" synergistic mechanism, which effectively improves the adsorption rate of PBUTs.

[0022] Experimental data show that when only hydrophobic ligands are grafted, the adsorption rates for β2-MG and IL-6 are high (93.20% and 82.41%, respectively), but the adsorption rates for PBUTs are low, with 20.30% for IS and 28.70% for PCS. When only positively charged ligands are grafted, the adsorption rates for β2-MG and IL-6 are relatively low, at 34.62% and 23.12%, respectively, while the adsorption rates for IS and PCS are 53.76% and 56.12%, respectively. However, when both hydrophobic and positively charged ligands are grafted simultaneously, the adsorbent prepared by the method of this invention can increase the adsorption rate for PBUTs to over 70%, with adsorption rates for IS reaching 70.50%–78.50% and for PCS reaching 75.30%–85.40%, while the adsorption rate for β2-MG remains stable above 80%, fully demonstrating the synergistic effect of the two ligands on PBUTs.

[0023] Compared with the prior art, the present invention has the following beneficial effects: Broad-spectrum clearance of multiple toxins: One adsorbent can simultaneously clear small- to medium-molecular-weight protein toxins (such as β2-MG), protein-bound uremic toxins (such as IS and PCS), and also clear inflammatory factors (such as IL-6), covering the main categories of uremic toxins. All embodiments of this invention exhibit adsorption rates exceeding 80% for β2-MG and clearance rates of 31.30%–42.70% for IL-6, generally comparable to or better than commercially available HA130 (35.18%). Clinically, excessive clearance of IL-6 is not sought to avoid immunosuppression; this clearance level meets clinical application requirements. By selecting different types of positively charged ligands, the focus of toxin clearance can be flexibly adjusted while maintaining broad-spectrum efficacy.

[0024] Significant synergistic effect: Through the "displacement-capture" synergistic effect of hydrophobic and positively charged ligands, the scavenging efficiency of this invention for PBUTs is far higher than that of hydrophobic modification alone, and significantly better than that of positively charged modification alone. Specifically, the adsorption rate for IS is more than 3 times higher than that of hydrophobic modification alone, and about 30%–40% higher than that of positively charged modification alone; the adsorption rate for PCS is more than 1.7 times higher than that of hydrophobic modification alone, and about 37%–47% higher than that of positively charged modification alone. This synergistic effect can be stably achieved on various types of positively charged ligands, including aliphatic polyamines, quaternary ammonium salts, and nitrogen-containing heterocyclic amines.

[0025] Good biocompatibility: Made from natural cellulose, the raw materials are widely available and environmentally friendly. Its biocompatibility is superior to that of synthetic polymer materials (such as polystyrene), which can reduce the risk of adverse reactions during blood perfusion.

[0026] Low total protein adsorption rate: By controlling the pore size through cross-linking and using a ligand grafting strategy, the total protein adsorption rate is controlled below 7% while ensuring toxin adsorption efficiency. This is significantly lower than that of commercially available HA130 (9.90%) and PHA130 (10.20%) products, which is beneficial for maintaining patients' plasma protein levels. Further control of the microsphere pore size through a secondary cross-linking process can reduce the total protein adsorption rate to 5.80%, further reducing the loss of beneficial proteins while ensuring toxin clearance capacity.

[0027] High process controllability: By adjusting parameters such as the type and ratio of porogen, the amount of dispersant, the number of cross-linking steps, and the type and amount of ligands, the pore size distribution and adsorption performance of microspheres can be flexibly controlled. For example, choosing quaternary ammonium salts or polyamine ligands can focus on improving PBUTs clearance efficiency, using a secondary cross-linking process can focus on reducing protein adsorption, and using mixed ligands can take into account multiple dimensions of performance, flexibly adapting to different clinical application needs. Attached Figure Description

[0028] Figure 1 An optical microscope image of the cellulose microsphere adsorbent prepared in this invention; Figure 2 This is a SEM image of the cellulose microsphere adsorbent prepared in this invention; Figure 3 This is a low-magnification SEM image of the fracture cross-section of the cellulose microsphere adsorbent prepared in this invention; Figure 4 This is a high-magnification SEM image of the interior cross-section of the cellulose microsphere adsorbent prepared in this invention. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this is not to limit the scope of the invention to this.

[0030] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available analytical grade reagents.

[0031] The cellulose microsphere adsorbent prepared in this invention is as follows: Figures 1 to 4 As shown, by Figure 1 It is evident that the microspheres exhibit good sphericity and excellent particle dispersibility. Figure 2 A single, intact microsphere is shown, with a relatively flat outer surface; the cross-section obtained by breaking the microsphere is shown below. Figure 3 The microspheres have an outer shell and an inner porous matrix; further magnification can be seen in [the image / description]. Figure 4 The interior of the microspheres is a three-dimensional porous mesh framework that is interconnected.

[0032] General rules for testing methods: 1. Standard for measuring the volume of wet microspheres: Place the microspheres to be measured in a G3 sand core funnel and filter continuously under a vacuum of -0.08 MPa for 5 min until there is no free water or obvious flowing liquid film on the surface of the microspheres. Then, read the volume of the microspheres in the graduated cylinder as the measurement standard for grafting amount and adsorption test.

[0033] 2. Ligand Grafting Amount Determination: Microspheres were loaded into a chromatography column and connected to a protein purification chromatography device. A 0.1M hydrochloric acid solution was prepared and flowed through the chromatography column. The conductivity displayed on the device was used to calculate the amount of hydrochloric acid adsorbed by the microspheres, and then the amino group content of the microspheres was calculated. For microsphere samples that had undergone two grafting processes, the grafting amount of the second grafting was calculated by subtracting the amino group content after the first grafting from the total amino group content after the second grafting.

[0034] 3. Determination of degree of hydrolysis: The residual amount of acetyl groups in the microspheres before and after hydrolysis was determined by acid-base titration, and the degree of hydrolysis was calculated.

[0035] 4. Pore size determination: The pore size distribution of the dried microspheres was determined by nitrogen adsorption-desorption method, and the average pore size was calculated by BJH method.

[0036] Example 1: Cellulose microsphere adsorbent grafted with n-octylamine and 1,6-hexanediamine The method for preparing a cellulose microsphere adsorbent for blood purification described in this embodiment specifically includes the following steps: Step (1) Preparation of cellulose acetate-based spheres: Cellulose diacetate (acetyl content 39.8%, viscosity 250 mPa·s) was dissolved in dichloromethane to prepare a 10% cellulose acetate solution. Octanol was added to the solution as a pore-forming agent, with a mass ratio of octanol to cellulose acetate of 1.5:1. The mixture was stirred until homogeneous to obtain the oil phase.

[0037] A 2% (w / w) aqueous solution of polyvinyl alcohol (PVA, degree of polymerization 1700±50) was prepared as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase, with a volume ratio of oil phase to water phase of 1:5. The mixture was stirred and emulsified at 300 rpm for 30 minutes to form a stable O / W (oil-in-water) emulsion.

[0038] The emulsion was heated to 35°C and stirred at 200 rpm for 12 hours to allow the dichloromethane to completely evaporate and the microspheres to solidify. The microspheres were collected, washed with deionized water until no PVA residue was found, and cellulose acetate microspheres with a particle size of 300–600 μm were sieved for later use.

[0039] Step (2) Hydrolysis and cross-linking: The cellulose acetate microspheres were added to a 1 mol / L sodium hydroxide aqueous solution at a volume ratio of 1:10. The mixture was stirred at 50°C for 4 hours to completely hydrolyze the cellulose acetate into cellulose microspheres. Titration showed that the residual acetyl group content of the hydrolyzed microspheres was less than 1%, indicating complete hydrolysis. After the reaction, the microspheres were washed with deionized water until neutral.

[0040] Hydrolyzed cellulose microspheres were added to an aqueous solution containing 2 mol / L sodium hydroxide and 0.5 g / L sodium borohydride, followed by epichlorohydrin at a volume ratio of 1:1 to the microspheres. The mixture was stirred at 45°C for 6 hours to induce crosslinking. The higher amount of epichlorohydrin and longer reaction time during the crosslinking stage promoted the opening of the bi-terminal rings of epichlorohydrin, leading to intermolecular crosslinking of cellulose molecules. After the reaction, the microspheres were washed with deionized water until neutral to obtain the cellulose microsphere substrate. The average pore size of the substrate was determined to be approximately 18 nm by nitrogen adsorption.

[0041] Step (3) Hydrophobic modification (grafting n-octylamine): The aforementioned cellulose microsphere substrate was added to an aqueous solution containing 1 mol / L sodium hydroxide, followed by epichlorohydrin at a volume ratio of 1:2. The mixture was stirred at 40°C for 4 hours to activate the hydroxyl groups on the microspheres. During the activation phase, the reaction temperature and feed ratio were controlled to ensure that epichlorohydrin was grafted onto the microsphere surface primarily in a single-end ring-opening manner, introducing epoxy active groups. After the reaction was complete, the microspheres were washed with deionized water until neutral.

[0042] The activated microspheres were added to an ethanol-water solution (volume ratio 1:1) containing 5 mol / L n-octylamine, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50°C with stirring for 12 hours to graft hydrophobic ligands. After the reaction was completed, the microspheres were washed successively with ethanol and deionized water to obtain hydrophobically modified microspheres. The grafting amount of hydrophobic ligands was determined to be approximately 50 μmol / mL (based on the volume of wet microspheres).

[0043] Step (4) Positive charge conversion (grafting 1,6-hexanediamine): The hydrophobic modified microspheres were activated again with epichlorohydrin according to step (3).

[0044] The activated microspheres were added to an aqueous solution containing 1 mol / L 1,6-hexanediamine, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50°C with stirring for 12 hours to graft positively charged ligands. After the reaction was completed, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 250 μmol / mL (based on the wet volume of the microspheres).

[0045] Parallel verification showed that replacing the sodium hydroxide aqueous solution with a mixed solution of sodium hydroxide in ethanol and water (volume ratio 1:1) in the hydrolysis and crosslinking steps, while keeping other process parameters unchanged, resulted in an adsorbent with a grafting amount deviation of less than 5%, and the adsorption performance was basically consistent with that in this embodiment.

[0046] Example 2: Cellulose microsphere adsorbent grafted with n-hexylamine and diethylenetriamine In this embodiment, cellulose diacetate in step (1) of Example 1 is replaced with cellulose triacetate, the organic solvent is a dichloromethane-ethanol mixture (volume ratio 4:1), n-heptanol is used as a pore-forming agent, and n-octylamine in step (3) is replaced with n-hexylamine. The remaining process parameters and steps are the same as those in Example 1. The amount of hydrophobic ligand grafted was determined to be approximately 55 μmol / mL (based on the volume of wet microspheres).

[0047] Step (4) Positive charge conversion (grafting diethylenetriamine): The hydrophobically modified microspheres were reactivated with epichlorohydrin and then added to an aqueous solution containing 1 mol / L diethylenetriamine. The pH was adjusted to 10 with sodium carbonate, and the reaction was carried out at 50°C with stirring for 12 hours to graft positively charged ligands. After the reaction was completed, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 280 μmol / mL (based on the wet volume of the microspheres).

[0048] Example 3: Secondarily cross-linked cellulose microsphere adsorbent In the preparation method of this embodiment, step (1) is the same as the preparation method of Example 1.

[0049] Step (2) Hydrolysis and secondary crosslinking: The hydrolysis step is the same as that in Example 1. The acetyl residue after hydrolysis is less than 1%.

[0050] The hydrolyzed cellulose microspheres underwent a first crosslinking reaction: they were added to an aqueous solution containing 2 mol / L sodium hydroxide and 0.5 g / L sodium borohydride, along with epichlorohydrin (volume ratio 1:1), and reacted at 45°C for 4 hours. After washing with water until neutral, a second crosslinking reaction was carried out: epichlorohydrin was added again under the same conditions, and the reaction was carried out for another 4 hours. After the reaction was completed, the microspheres were washed with water until neutral, yielding the cellulose microsphere substrate after the second crosslinking reaction. Nitrogen adsorption analysis showed that the average pore size of the substrate after the second crosslinking was approximately 12 nm, a significant reduction compared to the first crosslinking reaction.

[0051] Steps (3) to (4) of this embodiment are the same as those of the preparation method in Example 1. It was determined that the grafting amount of the hydrophobic ligand was approximately 48 μmol / mL, and the grafting amount of the positively charged ligand was approximately 220 μmol / mL (based on the volume of wet microspheres).

[0052] The secondary cross-linking reduces the pore size of the microspheres, further decreasing the total protein adsorption rate.

[0053] Example 4: Cellulose microsphere adsorbent grafted with tetradecylamine and triethylenetetramine The difference between this preparation method and Example 1 is that dichloromethane in step (1) of Example 1 is replaced with acetone, butyl acetate is used as a porogen, and n-octylamine in step (3) is replaced with tetradecylamine. The remaining process parameters and steps are the same as those in Example 1. The amount of hydrophobic ligand grafted was determined to be approximately 45 μmol / mL (based on the volume of wet microspheres).

[0054] Step (4) Positive charge conversion (grafting triethylenetetramine): The hydrophobic modified microspheres were activated again with epichlorohydrin according to step (3).

[0055] The activated microspheres were added to an aqueous solution containing 1 mol / L triethylenetetramine, and the pH was adjusted to 10 with sodium carbonate. The mixture was stirred at 50°C for 12 hours to graft positively charged ligands. After the reaction was complete, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 260 μmol / mL (based on the wet volume of the microspheres).

[0056] Example 5: Cellulose microsphere adsorbent grafted with n-octylamine and 1,3-propanediamine The difference between this preparation method and Example 1 is that the organic solvent in step (1) of Example 1 is a mixture of ethyl acetate and acetone, and ethylene glycol diacetate is used as the porogen. The remaining process parameters and steps are the same as those in Example 1. The amount of hydrophobic ligand grafted was determined to be approximately 52 μmol / mL (based on the volume of wet microspheres).

[0057] Step (4) Positive charge conversion (grafting 1,3-propanediamine): The hydrophobic modified microspheres were activated again with epichlorohydrin according to step (3).

[0058] The activated microspheres were added to an aqueous solution containing 1 mol / L 1,3-propanediamine, and the pH was adjusted to 10 with sodium carbonate. The mixture was stirred at 50°C for 12 hours to graft positively charged ligands. After the reaction was complete, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 220 μmol / mL (based on the wet volume of the microspheres).

[0059] Example 6: Cellulose microsphere adsorbent grafted with n-octylamine and 3-(3-aminopropyl)pyridine In this embodiment, steps (1) to (3) are the same as those in Example 1. The amount of hydrophobic ligand grafted was determined to be approximately 50 μmol / mL (based on the volume of wet microspheres).

[0060] Step (4) Positive charge conversion (grafting 3-(3-aminopropyl)pyridine): The hydrophobic modified microspheres were activated again with epichlorohydrin according to step (3).

[0061] The activated microspheres were added to an aqueous solution containing 1 mol / L 3-(3-aminopropyl)pyridine, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50 °C with stirring for 12 hours to graft positively charged ligands. After the reaction was completed, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 210 μmol / mL (based on the wet volume of the microspheres).

[0062] Example 7: Cellulose microsphere adsorbent grafted with n-octylamine and 3-aminopropyltrimethylammonium chloride In this embodiment, steps (1) to (3) are the same as those in Example 1. The amount of hydrophobic ligand grafted was determined to be approximately 53 μmol / mL (based on the volume of wet microspheres).

[0063] Step (4) Positive charge conversion (grafting 3-aminopropyltrimethylammonium chloride): The hydrophobic modified microspheres were activated again with epichlorohydrin according to step (3).

[0064] The activated microspheres were added to an aqueous solution containing 1 mol / L 3-aminopropyltrimethylammonium chloride, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50°C with stirring for 12 hours to graft positively charged ligands. After the reaction was completed, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 200 μmol / mL (based on the wet volume of the microspheres).

[0065] Example 8: Cellulose microsphere adsorbent grafted with n-octylamine, p-phenylenediamine and diethylenetriamine In this embodiment, steps (1) to (3) are the same as those in Example 1. The amount of hydrophobic ligand grafted was determined to be approximately 50 μmol / mL (based on the volume of wet microspheres).

[0066] Step (4) Positive charge conversion (co-grafting of p-phenylenediamine and diethylenetriamine): The hydrophobic modified microspheres were activated again with epichlorohydrin according to step (3).

[0067] The activated microspheres were added to a mixed aqueous solution containing 0.5 mol / L p-phenylenediamine and 0.5 mol / L diethylenetriamine. The pH was adjusted to 10 with sodium carbonate, and the reaction was carried out at 50°C with stirring for 12 hours, simultaneously grafting two positively charged ligands. After the reaction, the microspheres were washed with deionized water until neutral to obtain a multi-ligand composite functionalized cellulose microsphere adsorbent. The total grafting amount of positively charged ligands was determined to be approximately 270 μmol / mL (based on the volume of wet microspheres).

[0068] Example 9: Cellulose microsphere adsorbent grafted with n-octylamine and 1,6-hexanediamine The difference between this embodiment and Embodiment 1 is that the process parameters for hydrophobic modification in step (3) are adjusted as follows, while the remaining steps are the same as in Embodiment 1: Step (3) Hydrophobic modification (grafting n-octylamine): The cellulose microsphere substrate was added to an aqueous solution containing 1 mol / L sodium hydroxide, and then epichlorohydrin was added. The volume ratio of epichlorohydrin to microspheres was 1:5. The mixture was stirred at 40°C for 2 hours to activate the hydroxyl groups on the microspheres. After the reaction was completed, the microspheres were washed with deionized water until neutral.

[0069] The activated microspheres were added to an ethanol-water solution (volume ratio 1:1) containing 1 mol / L n-octylamine, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50°C with stirring for 6 hours to graft hydrophobic ligands. After the reaction was completed, the microspheres were washed successively with ethanol and deionized water to obtain hydrophobically modified microspheres. The grafting amount of hydrophobic ligands was determined to be approximately 20 μmol / mL (based on the volume of wet microspheres).

[0070] Step (4) The positive charge conversion process is completely consistent with that in Example 1, with a positive charge ligand grafting amount of approximately 245 μmol / mL (based on the volume of wet microspheres).

[0071] Example 10: Cellulose microsphere adsorbent grafted with n-octylamine and 1,6-hexanediamine The difference between this embodiment and Embodiment 1 is that the process parameters for hydrophobic modification in step (3) are adjusted as follows, while the remaining steps are the same as in Embodiment 1: Step (3) Hydrophobic modification (grafting n-octylamine): The cellulose microsphere substrate was added to an aqueous solution containing 1 mol / L sodium hydroxide, and then epichlorohydrin was added. The volume ratio of epichlorohydrin to microspheres was 1:1. The mixture was stirred at 40°C for 8 hours to activate the hydroxyl groups on the microspheres. After the reaction was completed, the microspheres were washed with deionized water until neutral.

[0072] The activated microspheres were added to an ethanol-water solution (volume ratio 1:1) containing 8 mol / L n-octylamine, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50°C with stirring for 24 hours to graft hydrophobic ligands. After the reaction was completed, the microspheres were washed successively with ethanol and deionized water to obtain hydrophobically modified microspheres. The grafting amount of hydrophobic ligands was determined to be approximately 100 μmol / mL (based on the volume of wet microspheres).

[0073] Step (4) The positive charge conversion process is completely consistent with that in Example 1, with a positive charge ligand grafting amount of approximately 240 μmol / mL (based on the volume of wet microspheres).

[0074] Example 11: Cellulose microsphere adsorbent grafted with n-octylamine and 1,6-hexanediamine The difference between this embodiment and Embodiment 1 is that the process parameters for positive charge conversion in step (4) are adjusted as follows, while the remaining steps are the same as in Embodiment 1: Step (3) The hydrophobic modification process is completely consistent with that in Example 1, and the amount of hydrophobic ligand grafting is about 50 μmol / mL (based on the volume of wet microspheres).

[0075] Step (4) Positive Charge Conversion (Grafting with 1,6-Hexanediamine): The hydrophobically modified microspheres were reactivated with epichlorohydrin, with the activation time shortened to 2 hours and the volume ratio of epichlorohydrin to microspheres being 1:5. The activated microspheres were added to an aqueous solution containing 0.3 mol / L 1,6-hexanediamine, and the pH was adjusted to 10 with sodium carbonate. The reaction was carried out at 50°C with stirring for 6 hours to graft positively charged ligands. After the reaction was completed, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 100 μmol / mL (based on the wet volume of the microspheres).

[0076] Example 12: Cellulose microsphere adsorbent grafted with n-octylamine and 1,6-hexanediamine The difference between this embodiment and Embodiment 1 is that the process parameters for positive charge conversion in step (4) are adjusted as follows, while the remaining steps are the same as in Embodiment 1: Step (3) The hydrophobic modification process is completely consistent with that in Example 1, and the amount of hydrophobic ligand grafting is about 50 μmol / mL (based on the volume of wet microspheres).

[0077] Step (4) Positive charge conversion (grafting 1,6-hexanediamine): The hydrophobically modified microspheres were reactivated with epichlorohydrin for 8 hours, with a volume ratio of epichlorohydrin to microspheres of 1:1. The activated microspheres were added to an aqueous solution containing 2.5 mol / L 1,6-hexanediamine, and the pH was adjusted to 10 with sodium carbonate. The mixture was stirred at 50°C for 24 hours to graft positively charged ligands. After the reaction, the microspheres were washed with deionized water until neutral to obtain bifunctionalized cellulose microsphere adsorbents. The grafting amount of positively charged ligands was determined to be approximately 400 μmol / mL (based on the wet volume of the microspheres).

[0078] Comparative Example 1: Cellulose microspheres modified only with hydrophobic properties The preparation method of this comparative example is the same as the preparation method of Example 1, steps (1) to (3) are the same, but the positive charge modification in step (4) is not performed, and hydrophobically modified cellulose microspheres with only octylamine grafted are obtained.

[0079] Comparative Example 2: Cellulose microspheres modified with only positive charge The preparation method of this comparative example is the same as steps (1) to (2) of Example 1. The hydrophobic modification in step (3) is skipped, and the positive charge modification in step (4) is carried out directly to obtain positive charge modified cellulose microspheres grafted only with 1,6-hexanediamine.

[0080] Comparative Example 3: Commercially available HA130 perfusion device adsorbent Microspheres were removed from a commercially available disposable blood perfusion device (HA130 type, the main component of which is neutral macroporous resin) as a control.

[0081] Comparative Example 4: Commercially available PHA130 perfusion device adsorbent Microspheres were removed from a commercially available disposable blood perfusion device (PHA130 model, whose main component is polystyrene-based adsorption resin) as a control.

[0082] Adsorption performance test Test method: Test baseline description: The background total protein concentration of the fresh bovine plasma used is 60-65 g / L, and it is sterilized by a 0.22μm filter membrane before use; all adsorption tests are performed using wet microspheres that have undergone standardized vacuum filtration, and the measurement method is the same as the general rule mentioned above.

[0083] Test 1: Determination of adsorption rate of PBUTs (IS, PCS) IS and PCS were added to fresh bovine plasma to prepare an adsorption solution with a final concentration of 50 μg / mL for IS and 20 μg / mL for PCS.

[0084] Take 1 mL of wet microspheres (with surface moisture removed) and add them to 10 mL of adsorption solution. Incubate at 37℃ and 150 rpm for 2 hours with shaking. After incubation, take the supernatant, precipitate the protein with acetonitrile, extract IS and PCS with the organic phase, and determine the concentration of IS and PCS in the supernatant by high performance liquid chromatography (HPLC).

[0085] Adsorption rate calculation formula: Adsorption rate (%) = (Initial concentration - Concentration after incubation) / Initial concentration × 100% Test 2, Determination of β2-MG and IL-6 Adsorption Rates β2-MG and IL-6 were added to fresh bovine plasma to prepare an adsorption solution with a final concentration of 20 μg / mL for β2-MG and 500 pg / mL for IL-6.

[0086] Take 1 mL of wet microspheres and add them to 10 mL of adsorption solution. Incubate at 37℃ and 150 rpm for 2 hours with shaking. After incubation, take the supernatant and detect the concentration of IL-6 using a chemiluminescence immunoassay analyzer and the concentration of β2-MG using a fully automated biochemical analyzer.

[0087] Test 3: Determination of total protein adsorption rate Take the supernatant after incubation and use a fully automated biochemical analyzer to detect the total protein (TP) concentration using the biuret method, and calculate the total protein adsorption rate.

[0088] Test Results The adsorption performance test results of each embodiment and comparative example are shown in the table below:

[0089]

[0090] Results Analysis The data in the table above shows that: (1) Dual ligands achieve complementarity and broaden the toxin scavenging spectrum: Hydrophobic ligands mainly scavenge toxins such as β2-MG and IL-6 through hydrophobic interactions (comparative example 1 showed adsorption rates of 93.20% and 82.41% for β2-MG and IL-6, respectively), but have limited scavenging ability for anionic PBUTs such as IS and PCS (20.30% for IS and 28.70% for PCS); Positively charged ligands have better scavenging effects on IS and PCS through electrostatic interactions (comparative example 2 showed 53.76% for IS and 56.12% for PCS), but have weaker scavenging effects on β2-MG and IL-6 (34.62% for β2-MG and 23.12% for IL-6). It can be seen that any single modification scheme can only cover a part of the toxin spectrum. Examples 1-12 simultaneously grafted hydrophobic ligands and different types of positively charged ligands, achieving adsorption rates of over 80%, over 70%, and over 75% for β2-MG, IS, and PCS, respectively. This enabled broad-spectrum scavenging of small- to medium-molecular-weight protein toxins and PBUTs on the same adsorbent, which cannot be achieved simultaneously by any single modification scheme.

[0091] (2) Synergistic effect on PBUTs (IS, PCS): The adsorption rates of IS and PCS in Examples 1-12 were not only significantly higher than those of Comparative Example 1, which was modified only by hydrophobic modification, but also significantly higher than those of Comparative Example 2, which was modified only by positive charge modification, i.e., better than any single modification scheme. This indicates that the hydrophobic ligand and the positively charged ligand do not simply add up when scavenging PBUTs, but rather produce a synergistic effect: the positively charged ligand enhances the enrichment of anionic toxins through electrostatic interaction, while the hydrophobic ligand further promotes the binding of toxin molecules to adsorption sites. The combined effect of the two significantly improves the scavenging efficiency of PBUTs.

[0092] Among them, aliphatic polyamines (such as diethylenetriamine and triethylenetetramine) and quaternary ammonium salt ligands (such as 3-aminopropyltrimethylammonium chloride) have a more prominent synergistic effect on PBUTs due to their higher positive charge density and stronger electrostatic interaction; ligands containing aromatic heterocycles (such as 3-(3-aminopropyl)pyridine) also show a clear synergistic effect.

[0093] (3) Highly efficient removal of β2-MG: The adsorption rates of β2-MG in Examples 1-12 remained in the range of 81.20% to 89.60%. Although the adsorption rates were slightly lower than those of Comparative Example 1 (which only underwent hydrophobic modification) due to the occupation of some adsorption sites by positively charged ligands, they were still at a high level and significantly better than those of commercially available HA130 (73.10%) and PHA130 (68.23%). In Example 3, which used a secondary crosslinking process, the adsorption rate of β2-MG only decreased slightly due to the moderate reduction in the pore size of the microspheres, and still maintained a highly efficient removal level of over 84%. The above results indicate that the adsorbent of the present invention achieves broad-spectrum removal while maintaining good removal ability for small and medium molecular weight protein toxins.

[0094] (4) Description of IL-6 clearance level: The adsorption rates of IL-6 in Examples 1-12 ranged from 31.30% to 42.70%, all not lower than 30%, which is comparable to or better than that of commercially available HA130 (35.18%). IL-6 is mainly adsorbed through hydrophobic interactions, so hydrophobic modification alone resulted in a higher clearance rate (82.41%). After the introduction of positively charged ligands, some hydrophobic adsorption sites were replaced, and the IL-6 clearance rate decreased accordingly. Among them, Example 6, which contains a pyridine heterocycle, has a slightly higher IL-6 adsorption level than the aliphatic amine series because the ligand itself has a hydrophobic aromatic ring structure. Considering that the adsorbent of this invention is designed to broadly clear a variety of PBUTs while reducing the loss of beneficial proteins, and that excessive clearance of IL-6 is not pursued clinically to avoid immunosuppression, the entire series of examples maintains a moderate clearance level of IL-6, which can meet the needs of practical applications.

[0095] (5) Low loss rate of beneficial proteins and high safety: The total protein adsorption rate of Examples 1-12 is no higher than 7%, which is significantly lower than that of commercially available HA130 (9.90%) and PHA130 (10.20%). This indicates that the adsorbent of the present invention effectively removes various toxins while having less non-specific adsorption of beneficial proteins in plasma, which can reduce the loss of beneficial proteins during treatment and has higher safety. Among them, Example 3, which adopts a secondary cross-linking process, further reduces the entry and non-specific adsorption of large molecular proteins by controlling the pore size, and the total protein adsorption rate is as low as 5.80%, which verifies that optimizing the number of cross-linking can further improve the safety of the product; the total protein adsorption of the heterocyclic ligand series is also at a lower level due to the relatively low positive charge density.

[0096] (6) Overall Performance: Considering the overall toxin removal spectrum, removal efficiency, beneficial protein loss rate, and substrate biocompatibility: Compared with single modifications that are only hydrophobic or only positively charged, Examples 1-12 all achieved broad-spectrum and efficient removal of small- to medium-molecular-weight protein toxins and PBUTs; compared with commercially available HA130 and PHA130, the entire series of examples maintained comparable or better toxin removal effects while significantly lowering the beneficial protein loss rate; and the present invention uses cellulose as a substrate, which has better biocompatibility than PHA130 using polystyrene as a substrate. Meanwhile, different ligands and process schemes can create differentiated focuses: for high PBUT removal, triethylenetetramine or 3-aminopropyltrimethylammonium chloride ligands can be selected; for lower protein loss, a secondary cross-linking process or heterocyclic ligands can be selected; mixed ligand schemes can take into account multiple dimensions of performance. Therefore, the bifunctionalized cellulose microsphere adsorbent prepared in this invention has comprehensive advantages in broad-spectrum removal, removal safety, and biocompatibility, and has good prospects for clinical application.

[0097] 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. A cellulose microsphere adsorbent for blood purification, characterized in that, The substrate includes cellulose microspheres, on which hydrophobic ligands and positively charged ligands are grafted. The hydrophobic ligand is an alkylamine containing 6 to 14 carbon atoms, and the positively charged ligand is selected from cationic small molecule compounds containing amine groups, including one or more of small molecule polyamines, quaternary ammonium salt amine compounds, and amine compounds containing aromatic heterocycles; Based on the volume of the wet microspheres, the grafting amount of the hydrophobic ligand is 20–100 μmol / mL, and the grafting amount of the positively charged ligand is 100–400 μmol / mL.

2. The cellulose microsphere adsorbent according to claim 1, characterized in that, The cellulose microsphere substrate is prepared by hydrolysis and epichlorohydrin crosslinking of cellulose diacetate or cellulose triacetate microspheres.

3. The cellulose microsphere adsorbent according to claim 2, characterized in that, The epichlorohydrin crosslinking is either a single crosslinking reaction or a double crosslinking reaction.

4. The cellulose microsphere adsorbent according to claim 1, characterized in that, The hydrophobic ligand is selected from n-hexylamine, n-octylamine, or tetradecanamine.

5. The cellulose microsphere adsorbent according to claim 1, characterized in that, The positively charged ligand is one or two of 1,3-propanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, p-phenylenediamine, 3-(3-aminopropyl)pyridine, and 3-aminopropyltrimethylammonium chloride.

6. The cellulose microsphere adsorbent according to claim 1, characterized in that, When incubated with bovine plasma at 37°C and a wet microsphere volume to adsorption liquid volume ratio of 1:10 for 2 hours, the adsorbent showed an adsorption rate of not less than 80% for β2-microglobulin, not less than 70% for indophenol sulfate, not less than 75% for p-cresol sulfate, not less than 30% for interleukin-6, and a total protein adsorption rate not higher than 7%.

7. A method for preparing the cellulose microsphere adsorbent according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Cellulose acetate was dissolved in an organic solvent, a pore-forming agent was added, and cellulose acetate microspheres were prepared by emulsification solvent evaporation method; (2) The cellulose acetate microspheres are hydrolyzed and then crosslinked with epichlorohydrin to obtain cellulose microsphere substrate; (3) Activate the hydroxyl groups on the cellulose microsphere substrate with an activator and graft hydrophobic ligands to obtain hydrophobically modified microspheres; (4) The remaining hydroxyl groups on the hydrophobic modified microspheres are activated again with an activator and positively charged ligands are grafted to obtain the cellulose microsphere adsorbent.

8. The method according to claim 7, characterized in that, In step (1), the organic solvent is one or two of dichloromethane, ethanol, acetone or ethyl acetate, the pore-forming agent is one or two of n-octanol, n-heptol, butyl acetate or ethylene glycol diacetate, and the aqueous phase used for emulsification contains polyvinyl alcohol.

9. The method according to claim 7, characterized in that, In step (2), the hydrolysis treatment uses an aqueous solution of sodium hydroxide or a mixed solution of sodium hydroxide in ethanol and water.

10. The method according to claim 7, characterized in that, In step (1), the cellulose acetate is cellulose diacetate or cellulose triacetate; in steps (3) and (4), the activator is epichlorohydrin.

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