Layered functional modified core-shell cellulose microspheres

Abstract

The present application relates to a kind of layered function modified core-shell cellulose microspheres, the layered function modified core-shell cellulose microspheres are core-shell structure, including inner core and the shell layer covering the inner core;The inner core has radial through hole with average pore size 5-8 μm, the shell layer has sponge-like mesoporous channel with average pore size 0.2-0.4 μm;The outermost outer surface of the cellulose microspheres is covalently grafted blood compatibility ligand;The inner wall of the radial through hole, the inner wall of the sponge-like mesoporous channel are all covalently grafted target adsorption ligand.The layered function modified core-shell cellulose microspheres provided by the present application, comprehensive adsorption performance, biosafety, clinical adaptability are significantly better than single global modification traditional cellulose perfusion microspheres, are high-performance, high-safety new type blood perfusion adsorption material.

Description

Technical Field

[0001] This invention belongs to the fields of medical blood purification adsorption materials and biopolymer functional modification technology. Specifically, it relates to a layered functional modified core-shell cellulose microsphere, which can be applied to the efficient and selective removal of macromolecular toxins in β2 microglobulin in hemoperfusion systems. It is suitable for clinical scenarios such as uremic toxin removal in maintenance dialysis patients and prevention of dialysis-related amyloidosis. Background Technology

[0002] β2-microglobulin (β2-MG) is a major medium-to-large molecular weight uremic toxin in the human body, with a molecular weight of approximately 11.8 kDa. It cannot be effectively removed from patients with renal failure through conventional hemodialysis. Long-term accumulation can lead to deposition in bones, joints, skin, and cardiovascular tissues, causing serious dialysis complications such as dialysis-related amyloidosis, intractable pruritus, bone and joint pain, and cardiovascular calcification. It is a key risk factor affecting the quality of life and survival of long-term dialysis patients.

[0003] Hemoperfusion technology relies on porous adsorption materials to specifically remove medium and large molecular weight toxins, and is currently the core method for clinical removal of β2-microglobulin. Existing mainstream hemoperfusion materials mainly include three categories: activated carbon, synthetic polymer resins, and natural cellulose microspheres. Among them, activated carbon materials have broad adsorption spectrum but extremely poor selectivity, a high risk of microparticle detachment, and poor blood compatibility, easily inducing coagulation and inflammatory reactions; synthetic resin adsorption materials have strong hydrophobicity and high adsorption capacity, but severe non-specific adsorption, which can significantly deplete beneficial proteins such as albumin in the blood. Furthermore, the hydrophobic surface easily causes platelet adhesion and complement activation, posing safety risks in clinical use.

[0004] Natural cellulose microspheres have become the safest substrate for whole blood perfusion due to their excellent hydrophilicity, bioinertness, extremely low hemolysis rate, low complement activation, and lack of cytotoxicity. However, existing cellulose microspheres rely solely on physical pores for non-specific adsorption, resulting in low adsorption capacity, slow adsorption rate, and easy adsorption saturation for β2-microglobulin, which cannot meet the clinical demand for efficient toxin removal.

[0005] Currently, the industry generally faces a technical bottleneck where adsorption performance and blood compatibility cannot be simultaneously achieved: high-adsorption materials have poor safety, and high-safety materials have weak adsorption capacity. There is a lack of modified cellulose adsorption microspheres that have strong targeting specificity, high adsorption capacity, are fully compatible with whole blood perfusion, and have an extremely high level of biosafety. Summary of the Invention

[0006] This invention provides a layered functional modified core-shell cellulose microsphere to solve the above-mentioned technical problems.

[0007] To address the aforementioned technical problems, this invention provides a layered functionally modified core-shell cellulose microsphere. The layered functionally modified core-shell cellulose microsphere has a core-shell structure, comprising a core and a shell covering the core. The core has radially interconnected channels with an average pore size of 5–8 μm, and the shell has sponge-like mesoporous channels with an average pore size of 0.2–0.4 μm. The outermost surface of the layered functionally modified core-shell cellulose microsphere is covalently grafted with a blood-compatible ligand. The inner walls of the radially interconnected channels and the inner walls of the sponge-like mesoporous channels are all covalently grafted with targeted adsorption ligands.

[0008] In another embodiment, the blood compatibility ligand is selected from any one of the following: bi-terminated amino polyethylene glycol, glycine, ethanolamine, phosphorylcholine zwitterion ligand, and low molecular weight heparin.

[0009] Among them, the long PEG chain of the double-amino-terminated polyethylene glycol (NH2-PEG 2000-NH2) forms a hydrophilic hydration layer, which isolates blood cells from direct contact with hydrophobic adsorption ligands, greatly reducing platelet activation, non-specific albumin adsorption, and coagulation risk; the primary amino group at the molecular end can stably open the ring and covalently bind with the epoxy group, making it non-toxic and non-dissolving for medical use.

[0010] Glycine and ethanolamine are low-cost, small-molecule hydrophilic ligands; phosphorylcholine zwitterionic ligands have ultra-low protein adsorption; and low-molecular-weight heparin has anticoagulant activity.

[0011] In another embodiment, the targeted adsorption ligand is selected from any one of 4-aminoanisole, hexadecylamine, aniline, phenolic aromatic small molecule ligands, and anti-β2-MG nanobody.

[0012] Among them, the aromatic ether structure of 4-aminoanisole can specifically bind to β2 microglobulin through hydrophobic / hydrogen bonds, which is suitable for the removal of molecular toxins in dialysis; it is only distributed on the inner wall of the radial through-pore channel and the inner wall of the spongy mesoporous channel, providing adsorption sites with ultra-large specific surface area; the layered functional modification of the core-shell cellulose microsphere surface does not expose hydrophobic ligands, so as to achieve both high adsorption capacity and blood safety.

[0013] In another embodiment, the covalent grafting density of the blood-compatible ligand on the layered functionally modified core-shell cellulose microspheres is 15-60 μmol / g.

[0014] In another embodiment, the covalent grafting density of the targeted adsorption ligand on the layered functional modified core-shell cellulose microspheres is 20-40 μmol / g.

[0015] In another embodiment, the blood-compatible ligand forms a CN covalent bond with the layered functional modified core-shell cellulose microspheres.

[0016] In another embodiment, the targeted adsorption ligand forms a CN covalent bond with the layered functional modified core-shell cellulose microspheres.

[0017] In another embodiment, the shell thickness is 30-120 μm.

[0018] In another embodiment, the diameter of the core is 40-300 μm.

[0019] To address the aforementioned technical problems, the present invention also provides a blood perfusion device comprising the aforementioned layered functionally modified core-shell cellulose microspheres.

[0020] The layered functionally modified core-shell cellulose microspheres provided by this invention achieve targeted toxin adsorption and blood compatibility protection functions in separate zones through a hierarchical core-shell pore structure combined with differentiated layered functional modifications. While retaining excellent β2 microglobulin adsorption capacity and meeting the clinical dialysis toxin removal requirements, it significantly reduces safety hazards such as platelet adhesion, hemolysis, and non-specific albumin adsorption. At the same time, the material has high mechanical strength, no particle shedding, no ligand dissolution, and a stable preparation process that is easy to mass-produce. Its comprehensive adsorption performance, biosafety, and clinical suitability are significantly better than traditional cellulose perfusion microspheres with single global modification, making it a high-performance and highly safe novel blood perfusion adsorption material. Detailed Implementation

[0021] To illustrate the technical solutions of the invention in more detail, specific embodiments are listed below to demonstrate the technical effects; it should be emphasized that these embodiments are used to illustrate the invention and not to limit the scope of the invention.

[0022] Example 1

[0023] A layered functionally modified core-shell cellulose microsphere is disclosed. The microsphere has a core-shell structure, comprising a core and a shell covering the core. The core has radially interconnected channels with an average pore size of 8 μm, and the shell has sponge-like mesoporous channels with an average pore size of 0.4 μm. A blood-compatible ligand is covalently grafted onto the outermost surface of the microsphere. Targeted adsorption ligands are covalently grafted onto the inner walls of both the radially interconnected channels and the sponge-like mesoporous channels. The preparation method of the layered functionally modified core-shell cellulose microsphere is as follows:

[0024] Step S1: Preparation of kernel precursor solution

[0025] Raw material mass ratio: 9 parts cellulose (nanocellulose: hydroxypropyl methylcellulose = 2:1 compound), 2 parts halloysite nanotubes, 5 parts solubilizer N-methylmorpholine-N-oxide, 0.4 parts dispersant polyethylene glycol, and 16 parts water / ethanol mixed solvent; stir at 70℃ for 2 h until the material is completely dissolved and dispersed, degas under vacuum for 30 min, and cool to room temperature to obtain a uniform, bubble-free core precursor liquid.

[0026] Step S2: Low-temperature cryogenic molding + solvent replacement shaping of the core with radially penetrating channels

[0027] A micro-injection pump drips the core precursor solution into liquid nitrogen at a uniform rate of 1 drop / s for 4 min to rapidly pre-freeze it and form spherical gel precursor particles.

[0028] The spherical gel precursor particles were transferred into a tert-butanol solvent replacement system and soaked at -20°C four times for six hours each time to completely replace the water in the pores and prevent the freeze-drying pores from collapsing.

[0029] The solvent was completely removed by transferring the product into a freeze dryer at a temperature of -40 to -60°C for 96 hours to obtain a loose and porous native core.

[0030] Low temperature and mild chemical crosslinking

[0031] The lyophilized native kernel was immersed in a crosslinking curing solution: 5 wt% NaOH aqueous solution + 3 vol% ethylene glycol diglycidyl ether (EGDE, a mild crosslinking agent), and crosslinked at low speed at 40°C for 4 hours. Crosslinking mechanism: The hydroxyl groups of cellulose and the epoxy groups at both ends of EGDE undergo etherification, forming a stable three-dimensional network of ether bonds between molecules, without destroying the hydroxyl groups of glucose units, while locking in radial interconnected channels with an average pore size of 8 μm.

[0032] The filtrate was repeatedly washed with deionized water until it was neutral, and then dried under vacuum at 40°C to obtain a high-strength, hydroxyl-rich core with radially interconnected channels having an average pore size of 8 μm.

[0033] Step S3: Preparation of shell coating precursor solution and core-shell coating molding

[0034] The shell precursor solution was prepared by mass ratio as follows: 10 parts cellulose (a blend of nanocellulose and hydroxypropyl methylcellulose, with a higher concentration than the core precursor solution), 5 parts N-methylmorpholine-N-oxide, 0.4 parts polyethylene glycol, and 16 parts water / ethanol mixed solvent. The solution was stirred and dissolved at 70°C, degassed, and cooled for later use. The S2 cross-linked core was completely immersed in the shell precursor solution and allowed to stand at room temperature for 10 min, resulting in a uniform coating of cellulose liquid film on the core surface. The microspheres were then removed, pre-frozen in liquid nitrogen for 1 min, replaced with tert-butanol, and freeze-dried to obtain core-shell composite gel microspheres.

[0035] Step S4: Low-temperature gradient crosslinking of the shell layer to prepare blank core-shell cellulose microspheres.

[0036] The composite gel microspheres were placed in a low-temperature crosslinking autoclave and subjected to two stages of gentle crosslinking to shape the mesoporous shell layer:

[0037] Stage 1: At 30℃, a system of 2 wt% NaOH + 1 vol% EGDE was stirred at low speed for 1.5 h to initially cross-link and shape the sponge pores.

[0038] Stage 2: Add 2 vol% EGDE at 40℃, keep warm and stir for 3 h to complete the three-dimensional cross-linking of the entire shell layer;

[0039] After crosslinking, residual crosslinking agent and alkali were removed by alternating washing with pure water and ethanol; vacuum drying was performed at 40℃ to obtain blank core-shell cellulose microspheres: the average pore diameter of the radially interconnected channels in the core was 8 μm, and the average pore diameter of the sponge mesopores in the shell was 0.4 μm. The skeleton of the blank core-shell cellulose microspheres was rich in a large number of free hydroxyl groups, and the mechanical strength met the requirements of blood perfusion circulation.

[0040] S5: Global epoxy activation (epoxy groups are generated on the outer surface of the microspheres and the inner walls of all pores).

[0041] Take 10 g of blank S4 core-shell microspheres, sonicate with 200 mL of 0.8 mol / L NaOH for 30 min; add 15 mL of epichlorohydrin (ECH), stir at 45℃ for 4 h; wash with water and dry, the microspheres are fully epoxy activated, with an epoxy value of 0.15~0.50 mmol / g dry gel.

[0042] S6: First step of modification: Only the outermost surface of the microspheres is grafted with aminoPEG (blood-compatible ligand, the pore epoxy is protected by PEG and does not react).

[0043] Prepare a 40 wt% PEG 6000 aqueous solution and immerse the entire epoxy microsphere in it. Impregnate at 50℃ under negative pressure for 30 min. The pores are completely filled with macromolecular PEG, and the epoxy on the inner wall of the pores is physically shielded; only the outermost epoxy layer of the sphere is exposed.

[0044] Prepare the surface grafting reaction solution: 10 g of aminoPEG 2000 + 200 mL of anhydrous ethanol; add microspheres to the grafting solution and stir at 50℃ for 12 h in a sealed environment; only the outer epoxy layer undergoes ring-opening with the aminoPEG primary amine, covalently grafting a blood-compatible ligand; the epoxy within the pores is isolated by PEG and does not react. Rapidly filter the solution, then briefly rinse the surface with cold ethanol to remove free aminoPEG, without soaking to prevent PEG dissolution.

[0045] S7: Second step modification: Elution of pore protectant, epoxy-grafted 4-aminoanisole adsorbent on the inner wall of the pore.

[0046] Microspheres were soaked in a large amount of room temperature pure water for 12 h, with the water changed every 2 h to thoroughly wash away PEG 6000 from the pores, fully exposing the epoxy on the inner walls of the pores. An adsorption ligand reaction solution was prepared: 8 g of 4-aminoanisole + 200 mL of anhydrous ethanol + 2 mL of triethylamine. The microspheres were then added to the grafting solution and stirred at 60℃ for 24 h. The epoxy on the inner walls of the radially interconnected pores and the sponge-like mesoporous pores covalently bonded to 4-aminoanisole, thus loading the pores with targeted adsorption ligands. The outer layer, already grafted with aminoPEG, had no residual epoxy and no longer bound to adsorption ligands. The microspheres were washed three times with hot ethanol and then soaked in PBS to remove free 4-aminoanisole.

[0047] S8: Residual site capping, purification, and sterilization

[0048] Glycine 0.5 mol / L aqueous solution was stirred at 50℃ for 6 h: only a trace amount of unreacted epoxy was sealed; the outer layer was already PEG-coated, and there was no excess epoxy, so no end-capping was required; pure water was continuously soaked for 12 h, and no ligand dissolution was detected by UV; freeze-drying and ethylene oxide sterilization were performed to obtain the target layered functional microspheres.

[0049] Example 2: 4-Aminoanisole grafted onto the entire domain

[0050] Steps S1-S5 of Example 2 are the same as those of Example 1. The difference between Example 2 and Example 1 is that:

[0051] S6: Global one-step grafting of targeted adsorption ligands

[0052] Preparation of 4-aminoanisole grafting solution: 8 g 4-aminoanisole + 200 mL ethanol + 2 mL triethylamine; all epoxy microspheres were added and stirred at 60℃ for 24 h; 4-aminoanisole was covalently grafted onto the outer surface of the microspheres and the inner walls of all pores.

[0053] S7: End-capping purification and sterilization (same as step S8 in Example 1)

[0054] Product features of Example 2: The entire microsphere (outer surface + all pores) contains only the adsorbent ligand 4-aminoanisole, and has no hydrophilic compatibility ligands.

[0055] Example 3: Fully grafted amino PEG 2000 (without targeted adsorption ligands)

[0056] Steps S1-S5 of Example 3 are the same as those of Example 1. The difference between Example 3 and Example 1 is that:

[0057] S6: One-step grafting of aminoPEG across the entire domain

[0058] 10 g of aminoPEG 2000 was dissolved in 200 mL of anhydrous ethanol; the entire epoxy microspheres were added and stirred at 50 °C for 12 h; hydrophilic PEG ligands were grafted onto the inside and outside of the microspheres, and no 4-aminoanisole was used.

[0059] S7: Glycine capping, purification, and sterilization

[0060] Product features: The entire microsphere contains only blood-compatible amino PEG, and there are no β2 microsphere protein-specific adsorption sites.

[0061] Comparative Example 1: Cellulose Microspheres

[0062] The cellulose microspheres of Comparative Example 1 have no covalent ligands on their surface, in their radially interconnected channels, or inside their sponge-like channels. The difference between the preparation steps of the cellulose microspheres of Comparative Example 1 and those of Example 1 is that Comparative Example 1 only includes steps S1 to S4 of Example 1, and other steps are omitted.

[0063] Performance testing of cellulose microspheres in Examples 1-3 and Comparative Example 1

[0064] The microsphere samples of Examples 1-3 and Comparative Example 1 were freeze-dried under vacuum, and 50 mg of dried microspheres were accurately weighed. The test medium was simulated plasma at a constant temperature of 37°C. Each group was tested in parallel 3 times, and the average value was taken.

[0065] I. Balanced adsorption capacity of β2 microglobulin

[0066] Test methods

[0067] Prepare a simulated plasma solution containing β2-microglobulin: Take plasma from healthy individuals, add purified β2-microglobulin standard, and adjust the volume to an initial concentration C0 = 15 mg / L. Keep the solution at a constant temperature of 37°C in a water bath for later use.

[0068] Adsorption procedure: 50 mg of dried microspheres were placed in a 100 mL Erlenmeyer flask, and 50 mL of the above-mentioned simulated plasma was added. The Erlenmeyer flask was sealed and shaken at a constant temperature of 37℃ at a low speed of 120 r / min for 24 h to reach adsorption equilibrium. After equilibrium, the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane to remove microsphere particles. The filtrate was collected for analysis.

[0069] Detection and Calculation: The equilibrium concentration C in the filtrate was determined using an enzyme-linked immunosorbent assay (ELISA) kit. e (mg / L).

[0070] Adsorption capacity calculation formula:

[0071] In the formula:

[0072] Q e —Equilibrium adsorption capacity, mg / g dry microspheres;

[0073] C0—Initial β2-microglobulin concentration, mg / L;

[0074] C e —Concentration of the supernatant after equilibration, mg / L;

[0075] V – Simulated blood plasma volume, L;

[0076] m — mass of dried microspheres, g.

[0077] II. Platelet adhesion rate

[0078] Test methods

[0079] 1. Reagent pretreatment:

[0080] Fresh anticoagulated human whole blood (3.8% sodium citrate anticoagulant, blood:anticoagulant = 9:1);

[0081] Preparation of platelet-rich plasma (PRP) by low-speed centrifugation: Centrifuge whole blood at 1000 r / min for 10 min, and the supernatant liquid is PRP;

[0082] The initial platelet concentration N0 (cells / mL) of PRP was determined using a hemocytometer.

[0083] 2. Adhesion reaction: Accurately weigh 100 mg of dried microspheres, add 10 mL of PRP, and shake in a shaker at 37℃ for 2 h at a speed of 80 r / min; after the reaction is complete, let stand for 3 min, aspirate the upper PRP layer, filter through a 0.22 μm filter membrane, and count the remaining platelet concentration N. e .

[0084] Formula for calculating platelet adhesion rate:

[0085] III. Hemolysis Rate Test

[0086] Test method: GB / T 16886.4 Standard for hemolysis test of medical devices

[0087] 1. Reagent preparation: Anticoagulated fresh rabbit blood (or human blood) was diluted with physiological saline to prepare a 2% red blood cell suspension; Positive control: Deionized water (complete hemolysis); Negative control: 0.9% physiological saline (no hemolysis).

[0088] 2. Grouping operations

[0089] Experimental group: 0.5 g dried microspheres + 10 mL physiological saline, pretreated by soaking in a water bath at 37℃ for 30 min; 10 mL 2% red blood cell suspension was added and incubated at 37℃ for 2 h;

[0090] Positive control group: 10 mL deionized water + 10 mL red blood cell suspension;

[0091] Negative control group: 10 mL physiological saline + 10 mL red blood cell suspension.

[0092] 3. Detection and calculation:

[0093] Centrifuge the entire system at 3000 r / min for 5 min, take the supernatant, and measure the absorbance at 545 nm using a UV spectrophotometer: Sample A, A positive, and A negative.

[0094] Hemolysis rate formula:

[0095] National standard for qualified medical materials: hemolysis rate <5%.

[0096] IV. Non-specific adsorption capacity of plasma albumin (HSA)

[0097] 1. Test solution preparation: Prepare human serum albumin solution using PBS buffer (pH 7.4), initial concentration... H 0 =4000mg / L, simulating plasma albumin level.

[0098] 2. Adsorption reaction: 50 mg of dried microspheres were added to 50 mL of albumin solution and shaken at 120 r / min for 24 h at 37℃ until adsorption equilibrium was reached; the supernatant was collected, filtered, and the equilibrium albumin concentration was determined using a BCA protein quantification kit. H e .

[0099] 3. Adsorption capacity calculation formula

[0100] Q 白蛋白 Albumin adsorption capacity, mg / g dry microspheres;

[0101] V: Solution volume, L;

[0102] m: dry weight of microspheres, g.

[0103] The lower the value, the less the patient loses plasma nutrients.

[0104] V. Characterization Tests

[0105] 1. Epoxy value titration (sodium thiosulfate titration method, to verify ligand grafting).

[0106] Accurately weigh 0.5 g of activated microspheres and add 25 mL of 0.1 mol / L sodium thiosulfate aqueous solution; shake at room temperature in the dark for 30 min to allow the epoxy ring to open and generate an equivalent amount of OH⁻; titrate with 0.1 mol / L standard HCl, using blank microspheres as a control, and calculate the epoxy value (mmol / g dry glue).

[0107] 2. XPS + EDS elemental scanning of frozen sections (proving the hierarchical distribution of ligands, providing core evidence for the microsphere structure in Example 1)

[0108] (1) XPS full spectrum analysis of the intact outer surface of microspheres: detection of nitrogen element (characteristic element of aminoPEG and 4-aminoanisole);

[0109] (2) Liquid nitrogen freezing of brittle microspheres, SEM-EDS scanning cross section:

[0110] The outermost region of the sphere has a high N signal (aminoPEG); the inner wall regions of the core and shell pores have only aromatic amine N signals and no long-chain PEG characteristic elements; this clearly shows that the outermost surface is covalently grafted with blood-compatible ligands; the inner walls of the radially penetrating pores and the inner walls of the sponge-like mesoporous pores are all covalently grafted with targeted adsorption ligands, creating a layered modification structure.

[0111] Pore ​​size and specific surface area (BET nitrogen adsorption method): The sample was degassed under vacuum for 12 h and subjected to nitrogen adsorption test at 77 K to determine the specific surface area and average pore size, verifying the core 8 μm radial macropore and shell 0.4 μm sponge mesoporous structure.

[0112] Table 1. Performance test data of Examples 1-3 and Comparative Example 1

[0113] β2 microglobulin equilibrium adsorption capacity mg / g dry microspheres 53 65 4 8 Platelet adhesion rate % 3.2 22.4 3.6 10.2 hemolysis rate % 0.5 2.6 0.4 1.3 Nonspecific adsorption of albumin mg / g dry microspheres 3.1 16.3 1.8 7.1

[0114] As can be seen from Table 1:

[0115] β2 microglobulin adsorption capacity (toxin removal capacity)

[0116] Example 2: The entire surface area is covered with hydrophobic adsorption sites of 4-aminoanisole, making full use of the specific surface area and achieving the highest adsorption capacity of up to 65 mg / g.

[0117] In Example 1, the targeted adsorption ligand was loaded only on the inner wall of the pores, and there were no adsorption sites on the surface. The adsorption capacity of β2 microglobulin decreased slightly to 53 mg / g, which was only about 15% lower than that in Example 2. It still had the clinically efficient clearance capacity and fully met the needs of dialysis perfusion therapy.

[0118] Example 3: The entire domain is grafted with hydrophilic amino PEG. There are no specific affinity sites, only weak physical adsorption, and the capacity is only 4 mg / g, which has no clinical therapeutic value.

[0119] Comparative Example 1 relied solely on physical retention through cellulose pores, resulting in weak adsorption capacity (only 8 mg / g), which was insufficient to effectively reduce the accumulation of β2 microglobulin in patients.

[0120] Platelet adhesion rate (a core indicator of coagulation risk; the lower the value, the safer the coagulation).

[0121] Example 2: The outer surface of the microspheres is exposed with a large amount of hydrophobic 4-aminoanisole, which can easily induce platelet adhesion and aggregation, with an adhesion rate as high as 22.4%. Clinical use poses risks of thrombosis and tubing blockage.

[0122] Example 1: The outermost layer of the microspheres was grafted with hydrophilic amino PEG to form a hydration isolation layer on the material surface, which isolated the blood from the internal hydrophobic ligands. The platelet adhesion rate was only 3.2%, and the blood compatibility was close to that of Example 3 (3.6%) with pure PEG modification.

[0123] Comparative Example 1 blank microspheres had no hydrophobic ligands but lacked hydrophilic stealth modification, resulting in moderate platelet adhesion (10.2%) and weaker safety than Example 1.

[0124] Hemolysis rate (risk of cell damage, national standard acceptable threshold <5%)

[0125] All four groups of samples met medical standards for hemolysis rates, but there were significant differences in the rates of hemolysis.

[0126] Example 3 (full-domain PEG) showed the lowest hemolysis (0.4%); Example 1 showed the second lowest (0.5%).

[0127] Comparative Example 1: The blank cellulose skeleton showed moderate hemolysis (1.3%).

[0128] Example 2: The hydrophobic ligand caused mild erythrocyte damage with a hemolysis rate of 2.6%, which was significantly higher than that in Example 1. Long-term dialysis would worsen the patient's anemia.

[0129] Non-specific albumin adsorption (an indicator of nutrient loss; the lower the better).

[0130] Example 2: The global hydrophobic aromatic ligand can indiscriminately adsorb plasma albumin, with an adsorption capacity as high as 16.3 mg / g. Long-term use can cause hypoalbuminemia and malnutrition in patients.

[0131] Example 1: The surface hydrophilic PEG significantly inhibited non-specific protein binding, with only a small amount of weak adsorption in the pores, and the albumin adsorption amount was only 3.1 mg / g;

[0132] Example 3: The structure is hydrophilic throughout, with the lowest albumin adsorption (1.8 mg / g).

[0133] Comparative Example 1 blank microspheres showed moderate nonspecific adsorption (7.1 mg / g).

[0134] Comprehensive clinical suitability

[0135] Example 1 possesses both high toxin adsorption capacity and excellent blood compatibility, balancing therapeutic efficacy and safety. Through layered controllable modification, the surface hydrophilic PEG isolates blood cells from hydrophobic ligands, while the pores retain a large number of adsorption sites, thus resolving the contradiction between adsorption efficiency and blood compatibility. Its overall performance is significantly better than that of Example 2, Example 3 and Comparative Example 1.

[0136] Example 2 only grafts targeted adsorption ligands across the entire domain. Although the toxin adsorption capacity is large, the outer hydrophobic aromatic ligands come into direct contact with the blood, which can easily cause platelet aggregation, coagulation, and a large loss of albumin. It is not suitable for dialysis patients with low platelet counts or who are prone to bleeding.

[0137] Example 3 only grafts PEG-compatible ligands, which has good blood safety, but no targeted adsorption site and cannot effectively remove β2 microglobulin, so it does not have dialysis therapeutic value;

[0138] Comparative Example 1 has no functional ligands and relies solely on the weak adsorption of toxins through physical pores, resulting in extremely poor therapeutic effects.

[0139] The above description is merely a preferred embodiment of the invention and is not intended to limit the scope of this application in any way. Although the preferred embodiment has been disclosed above, it is not intended to limit the application. Any person skilled in the art can make some modifications or alterations to the disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of this application. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the scope of the technical solution of this application.

Claims

1. A layered functionally modified core-shell cellulose microsphere, characterized in that, The layered functional modified core-shell cellulose microspheres have a core-shell structure, comprising a core and a shell covering the core; the core has radially interconnected channels with an average pore size of 5–8 μm, and the shell has sponge-like mesoporous channels with an average pore size of 0.2–0.4 μm; the outermost surface of the layered functional modified core-shell cellulose microspheres is covalently grafted with a blood-compatible ligand; the inner walls of the radially interconnected channels and the inner walls of the sponge-like mesoporous channels are all covalently grafted with targeted adsorption ligands.

2. The layered functional modified core-shell cellulose microspheres according to claim 1, characterized in that, The blood compatibility ligand is selected from any one of the following: bi-terminated amino polyethylene glycol, glycine, ethanolamine, phosphorylcholine zwitterion, and low molecular weight heparin.

3. The layered functional modified core-shell cellulose microspheres according to claim 1, characterized in that, The targeted adsorption ligand is selected from any one of 4-aminoanisole, hexadecylamine, aniline, phenolic aromatic small molecule ligands, and anti-β2-MG nanobody.

4. The layered functional modified core-shell cellulose microspheres according to claim 1, characterized in that, The covalent grafting density of the blood-compatible ligand on the layered functionally modified core-shell cellulose microspheres is 15-60 μmol / g.

5. The layered functional modified core-shell cellulose microspheres according to claim 1, characterized in that, The covalent grafting density of the targeted adsorption ligand on the layered functional modified core-shell cellulose microspheres is 20-40 μmol / g.

6. The layered functional modified core-shell cellulose microspheres according to claim 1, characterized in that, The blood-compatible ligand forms a CN covalent bond with the layered functionally modified core-shell cellulose microspheres.

7. The layered functional modified core-shell cellulose microspheres according to claim 1, characterized in that, The targeted adsorption ligand forms a CN covalent bond with the layered functional modified core-shell cellulose microspheres.

8. The layered functional modified core-shell cellulose microspheres as described in claim 1, characterized in that, The shell thickness is 30-120 μm.

9. The layered functional modified core-shell cellulose microspheres as described in claim 1, characterized in that, The diameter of the core is 40-300 μm.

10. A blood perfusion device, characterized in that, The product comprises the layered functional modified core-shell cellulose microspheres as described in any one of claims 1 to 9.