An adsorbent for removing excess hepcidin and preparation method thereof

Abstract

The present invention discloses an adsorbent for removing excess hepcidin and a preparation method thereof. The adsorbent for removing excess hepcidin is made by using EDTA as a cross-linking agent to form cross-linked chitosan microspheres embedded with zirconium phosphate and / or zirconium oxide, and L-glutathione is bonded to the surface of the cross-linked chitosan microspheres. The adsorbent for removing excess hepcidin of the present invention uses chitosan microspheres as a carrier, and while the microspheres are cross-linked, further bonding ligands are introduced, eliminating the need for additional cross-linking agents and ligands. The reaction conditions are mild, the preparation method is simple, and the cross-linked microspheres have suspended carboxyl groups, which can be further coupled with peptides and amino acids to achieve functional modification. The microspheres can withstand high-temperature steam sterilization and effectively remove excess hepcidin and urea nitrogen from the blood.

Description

Technical Field

[0001] The present invention relates to the field of blood purification, in particular to an adsorbent for removing excess hepcidin in blood and a preparation method thereof. Background Art

[0002] Hepcidin (Hepc) is a cysteine-rich antimicrobial polypeptide synthesized and secreted by the liver. It can be expressed in large quantities during the immune process and participate in the immune response. It plays a negative regulatory role in the regulation of iron balance in the body and has certain therapeutic effects in related clinical applications of iron metabolism diseases.

[0003] Human hepcidin also has a wide range of antibacterial and antiprotozoal effects. Injury, infection, and inflammatory stimuli can strongly increase hepcidin gene expression, and hepcidin itself may be an important downstream effector molecule affecting iron transport between different iron pools.

[0004] The hepcidin molecule consists of eight cysteine ​​residues forming a single hairpin structure containing four disulfide bonds. Its amino acid sequence is highly conserved in different mammals. Three types of hepcidin have been discovered, namely Hepc22, Hepc25 and Hepc20. The latter two are the main forms of Hepc, and Hepc25 exists in human blood and urine.

[0005] Most of the human body's iron comes from the recycling of heme iron after aging red blood cells are phagocytosed by macrophages. The other part comes from the absorption of food iron. The small intestine is the only part that absorbs iron. Cytochrome b in the stomach and duodenum first reduces Fe3+ in the food to Fe2+, which is then transported to the small intestinal epithelial cells by divalent metal ion transporters. At the same time, heme can also be absorbed and degraded by intestinal epithelial cells to release the Fe2+ therein. The Fe2+ absorbed by the above pathways is stored in the liver, small intestine and macrophages in the form of ferritin for use by the body when needed. On the other hand, under the joint action of ferroportin 1 and ferroportin, it passes through the basement membrane of intestinal epithelial cells, is released into the blood, and is oxidized into Fe3+ and combined with transferrin (Tf), and transported in the form of Tf-Fe2, reaching the liver through the portal system. Hepatocytes take up these bound irons under the mediation of transferrin receptor 2. Some of the iron reaches the bone marrow through the blood for hemoglobin synthesis and red blood cell production, or combines with transferrin receptor 1 on immature red blood cells to enter reticulocytes for hemoglobin synthesis during red blood cell differentiation. Finally, the iron is used in various tissues, organs and cells.

[0006] Regardless of the organism, under physiological conditions, all lack a mechanism for iron excretion. Therefore, the body's iron homeostasis critically depends on the balance between intestinal iron absorption and the body's iron needs. The iron metabolism process described above demonstrates that ferroportin 1 is the sole pathway for transporting iron from cells to the blood. Studies have shown that hepcidin can bind to ferroportin 1, promoting its internalization and degradation, thereby indirectly regulating iron homeostasis. When the body is iron overloaded, hepcidin gene expression is enhanced, increasing hepcidin synthesis and secretion in the liver. This accelerates the degradation of ferroportin 1, closing the iron export port and reducing iron transport from intestinal epithelial cells and macrophages to the blood. When the body is iron deficient, these processes reverse, maintaining iron homeostasis.

[0007] However, when chronic kidney disease occurs, metabolic disorders occur in the body, and the ubiquitous micro-inflammatory state stimulates long-term overexpression of hepcidin. The level of hepcidin increases significantly, and it excessively binds to ferroportin 1, causing excessive degradation of ferroportin 1, hindering the transport of iron from cells to blood, thereby causing iron metabolism disorders and inflammatory anemia.

[0008] Hepcidin levels in healthy individuals are (14.4±16.2) ng / mL, while those on maintenance hemodialysis (MHD) exceed the normal range, reaching as high as (44.8±48.2) ng / mL. Traditional treatments and medications are ineffective for treating iron metabolism disorders. Hepcidin is a medium-to-large molecule, and high-throughput hemodialysis has limited therapeutic efficacy. Therefore, it is imperative to develop a therapeutic approach that can effectively inhibit hepcidin overexpression or effectively eliminate hepcidin from the blood. Patent publication number CN102549439A discloses an adsorbent for hepcidin adsorption. This adsorbent chemically bonds an antibody to an agarose or polyacrylate or copolymer carrier to achieve binding to the hepcidin antigen. However, prior to antibody bonding, the carrier must be activated with ethylene oxide, which results in low activation efficiency. Furthermore, the antibody bonded to the chemical carrier cannot withstand high-temperature sterilization, making it unsuitable for practical application. Summary of the Invention

[0009] The purpose of the present invention is to provide an adsorbent for removing excess hepcidin from the blood; the specific technical solution is:

[0010] An adsorbent for removing excess hepcidin is prepared by using EDTA as a cross-linking agent to form cross-linked chitosan microspheres embedded with zirconium phosphate and / or zirconium oxide, and L-glutathione is bonded to the surface of the cross-linked chitosan microspheres.

[0011] Furthermore, the amount of L-glutathione bonded to the surface of the cross-linked chitosan microspheres is 0.4-1.0 mg / g.

[0012] Furthermore, the mass ratio of the zirconium phosphate and / or zirconium oxide to the microspheres before cross-linking is 1:10 to 1:20.

[0013] The present invention also discloses a method for preparing the above-mentioned adsorbent for removing excess hepcidin, comprising the following steps:

[0014] Add EDTA solution of zirconium phosphate to the acetic acid solution of chitosan and stir to form a suspension, pour the suspension into the pre-prepared oil phase of n-hexane and liquid paraffin, heat to 40°C, stir for 1 hour, then add 3 mol / L sodium hydroxide solution to pH ≥ 9, heat to 70°C and continue stirring for 3 hours, filter, extract with petroleum ether for 8 hours, extract with ethanol for 8 hours, and wash with water until neutral to obtain chitosan microspheres;

[0015] Add the product from step 1 to an EDTA solution, then add an excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, heat to 37°C, and stir for 2 hours. Then, add the L-glutathione solution and continue stirring for 2 hours to obtain cross-linked chitosan microspheres.

[0016] The product of step 2 was washed with EDTA solution and then with water. The zirconium ion concentration of the eluate was detected to be less than 0.001 mol / L and the pH of the eluate was neutral. The product was dried under ventilation at room temperature and then placed in a 0.9% sodium chloride solution.

[0017] The present invention also discloses three new applications of the above-mentioned adsorbent for removing excess hepcidin, namely, for removing excess hepcidin; for assisting in removing urea nitrogen; and for whole blood perfusion, which can realize partial dialyzer functions and regulate acid-base balance.

[0018] The adsorbent for removing excess hepcidin of the present invention uses chitosan microspheres as carriers, and introduces the ligands required for further bonding while cross-linking the microspheres. There is no need to add more cross-linking agents and ligands, and no need to add organic cross-linking agents. The reaction conditions are mild and the preparation method is simple. In addition, the cross-linked microspheres have suspended carboxyl groups, which can be further coupled with peptides and amino acids to achieve functional modification. The microspheres can withstand high-temperature steam sterilization and can effectively remove excess hepcidin in the blood. Implementation Method

[0019] The present invention is described in more detail below using the following examples. The present invention can be embodied in many different forms and should not be construed as being limited to the exemplary embodiments described herein. Example 1

[0020] Step 1: Place 20g of chitosan with a 98% degree of deacetylation in a beaker, add 5mL of 1% acetic acid solution, and stir until completely dissolved. Add 1.5g of zirconium phosphate to 4mL of EDTA solution and vortex for 1 minute. Then add it to the chitosan solution and stir to form a suspension. The suspension is then added to a three-necked flask containing a pre-prepared mixture of n-hexane and liquid paraffin (2mL of n-hexane, 6mL of liquid paraffin). Heat to 40°C and stir at 300 rpm for 1 hour. Then, add 3mol / L sodium hydroxide solution to a pH ≥ 9, heat to 70°C, and continue stirring for 3 hours. Filter, extract with petroleum ether for 8 hours, then extract with ethanol for 8 hours, and wash with water until neutral to obtain chitosan microspheres.

[0021] Step 2: Take 10g of the product from Step 1, add 3mL of EDTA solution, 4mL of 1mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and heat to 37°C and stir for 2h. Then, add 5mL of 0.04mol / L L-glutathione solution and continue stirring for 2h to obtain cross-linked chitosan microspheres.

[0022] Step 3: Wash the product of step 2 with EDTA solution, then wash with water until the zirconium ion detection amount is less than 0.001 mol / L, and the pH of the eluent is neutral. Place the product at room temperature for ventilation and drying, and then store it in 0.9% sodium chloride solution. Example 2

[0023] Step 1: Place 20g of chitosan with a 98% deacetylation degree in a beaker, add 4mL of a 2% acetic acid solution, and stir until completely dissolved. Add 1.1g of zirconium phosphate to 3mL of EDTA solution and vortex for 1 minute. Then add it to the chitosan solution and stir to form a suspension. The suspension is then added to a three-necked flask containing a pre-prepared mixture of n-hexane and liquid paraffin (3mL of n-hexane, 6mL of liquid paraffin). Heat to 40°C and stir at 300 rpm for 1 hour. Then, add 3mol / L sodium hydroxide solution to a pH ≥ 9, heat to 70°C, and continue stirring for 3 hours. Filter, extract with petroleum ether for 8 hours, then extract with ethanol for 8 hours, and wash with water until neutral to obtain chitosan microspheres.

[0024] Step 2: Take 10g of the product from Step 1, add 3mL of EDTA solution, 4mL of 1mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and heat to 37°C and stir for 2h. Then, add 4mL of 0.04mol / L L-glutathione solution and continue stirring for 2h to obtain cross-linked chitosan microspheres.

[0025] Step 3: Wash the product of step 2 with EDTA solution, then wash with water until the zirconium ion detection amount is less than 0.001 mol / L, and the pH of the eluent is neutral. Place the product at room temperature for ventilation and drying, and then store it in 0.9% sodium chloride solution. Example 3

[0026] Step 1: Place 20g of chitosan with a 98% deacetylation degree in a beaker, add 5mL of 1% acetic acid solution, and stir until completely dissolved. Add 1.0g of zirconium phosphate and 0.5g of zirconium oxide to 5mL of EDTA solution, vortex on a shaker for 1 minute, then add to the chitosan solution and stir to form a suspension. The suspension is then added to a three-necked flask containing a pre-prepared mixture of n-hexane and liquid paraffin (4mL of n-hexane, 6mL of liquid paraffin). Heat to 40°C and stir at 300 rpm for 1 hour. Then, add 3mol / L sodium hydroxide solution to a pH ≥ 9, heat to 70°C, and continue stirring for 3 hours. Filter, extract with petroleum ether for 8 hours, then extract with ethanol for 8 hours, and wash with water until neutral to obtain chitosan microspheres.

[0027] Step 2: Take 8 g of the product from Step 1, add 3 mL of EDTA solution, 3 mL of 1 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and heat to 37°C and stir for 2 hours. Then, add 4 mL of 0.04 mol / L L-glutathione solution and continue stirring for 2 hours to obtain cross-linked chitosan microspheres.

[0028] Step 3: Wash the product of step 2 with EDTA solution, then wash with water until the zirconium ion detection amount is less than 0.001 mol / L, and the pH of the eluent is neutral. Place the product at room temperature for ventilation and drying, and then store it in 0.9% sodium chloride solution. Example 4

[0029] Step 1: Place 20g of chitosan with a 97% deacetylation degree in a beaker, add 5mL of 1% acetic acid solution, and stir until completely dissolved. Add 1.0g of zirconium phosphate to 3mL of EDTA solution and vortex for 1 minute. Then add it to the chitosan solution and stir to form a suspension. The suspension is then added to a three-necked flask containing a pre-prepared mixture of n-hexane and liquid paraffin (2mL of n-hexane, 6mL of liquid paraffin). Heat to 40°C and stir at 300 rpm for 1 hour. Then, add 3mol / L sodium hydroxide solution to a pH ≥ 9, heat to 70°C, and continue stirring for 3 hours. Filter, extract with petroleum ether for 8 hours, then extract with ethanol for 8 hours, and wash with water until neutral to obtain chitosan microspheres.

[0030] Step 2: Take 8 g of the product from Step 1, add 3 mL of EDTA solution, 3 mL of 1 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and heat to 37°C and stir for 2 hours. Then, add 4 mL of 0.04 mol / L L-glutathione solution and continue stirring for 2 hours to obtain cross-linked chitosan microspheres.

[0031] Step 3: Wash the product of step 2 with EDTA solution, then wash with water until the zirconium ion detection amount is less than 0.001 mol / L, and the pH of the eluent is neutral. Place the product at room temperature for ventilation and drying, and then store it in 0.9% sodium chloride solution.

[0032] Comparative Example 1

[0033] Place 20g of chitosan with a 98% deacetylation degree in a beaker, add 5mL of 1% acetic acid solution, and stir until completely dissolved. Add 1.2g of zirconium phosphate to 5mL of EDTA solution and vortex for 1 minute. Then add the chitosan solution and stir to form a suspension. The suspension is then added to a three-necked flask containing a pre-prepared mixture of n-hexane and liquid paraffin (4mL of n-hexane, 6mL of liquid paraffin). Heat the mixture to 40°C and stir at 300 rpm for 1 hour. Then, add 3mol / L sodium hydroxide solution until the pH is ≥9. Heat the mixture to 70°C and continue stirring for 3 hours. Filter the mixture, extract with petroleum ether for 8 hours, then with ethanol for 8 hours, and then wash with water until neutral. Add 3mL of EDTA solution and 3mL of 1mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution. Heat the mixture to 37°C and stir for 2 hours. Extract with ethanol for 8 hours, and then wash with water until neutral.

[0034] Clearance effect test:

[0035] After treatment, 2 ml of peripheral blood was collected from the subjects and centrifuged at 3000 × g for 10 min to quickly and carefully separate the serum and red blood cells. The serum was collected, 20 g of adsorbent was added, and the blood was shaken in a constant temperature shaker at 37°C for 2 h. The supernatant was collected and the concentrations of hepcidin and urea nitrogen before and after adsorption were detected by enzyme-linked immunosorbent assay, and the clearance rate was calculated.

[0036] Table 1 Comparison of cleaning effects

[0037] adsorbent Hepcidin concentration after adsorption (ng / mL) Hepcidin clearance (%) Urea nitrogen concentration after adsorption (mmol / L) Urea nitrogen clearance rate (%) Example 1 70.6 28.0 7.9 75.3 Example 2 71.3 27.2 14.3 55.3 Example 3 71.6 26.9 8.2 74.4 Example 4 70.9 27.7 17.1 46.6 Comparative Example 1 95.9 2.1 8.5 73.4 Comparative samples 89.7 8.5 30.5 4.7

[0038] Note: The hepcidin concentration before adsorption was 98 ng / mL, and the urea nitrogen concentration before adsorption was 32 mmol / L. The comparison sample was a purchased polystyrene neutral macroporous resin (specific surface area 1050 m2 / g, average pore size 29 nm).

[0039] The results show that the adsorbents bonded with L-glutathione have a high hepcidin removal rate, exceeding 26%. Examples 1 and 3 exhibit superior adsorption of urea nitrogen compared to Examples 2 and 4, primarily due to the varying amounts of zirconium salt embedded within the adsorbents. Comparative Example 1 exhibits a low adsorption rate for hepcidin, but its adsorption of urea nitrogen remains unaffected. Polystyrene neutral macroporous resin exhibits a small amount of adsorption for both hepcidin and urea nitrogen, likely due to the presence of internal macropores that provide a certain degree of retention, but the adsorption is relatively small.

[0040] Blood compatibility test:

[0041] The effect of adsorbents on platelet count in human blood was evaluated using a platelet count method to assess the hemocompatibility of the adsorbents. 1.000 g of adsorbent was accurately weighed and added to a test tube containing 10 mL of blood with sodium citrate anticoagulant, calcium chloride, and sodium heparin. The tubes were incubated at (37 ± 1)°C for 1 hour to serve as the experimental group. 1 g of high-density polyethylene was added to a test tube containing 10 mL of blood with sodium citrate anticoagulant, calcium chloride, and sodium heparin and incubated at (37 ± 1)°C for 1 hour to serve as the negative control group. A blank (blank blood) was prepared in the same manner. Three parallel tubes of each adsorbent and blank were prepared. After incubation, EDTA was added to each tube to terminate the reaction at a final concentration of 5 mM. The platelet count in each tube was determined using an automated hematology analyzer, and the percentage of each group relative to the blank control was calculated.

[0042] Table 2 Platelet adhesion test results

[0043] adsorbent <![CDATA[Average platelet count (10 9 / L)]]> <![CDATA[Mean blank platelet count (10 9 / L)]]> <![CDATA[Negative control average value (10 9 / L)]]> Average platelet adhesion rate (%) Example 1 199.1 201.7 194.0 1.3 Example 2 198.7 201.7 194.0 1.5 Example 3 198.9 201.7 194.0 1.4 Example 4 198.5 201.7 194.0 1.6 Comparative Example 1 191.0 201.7 194.0 5.3 Comparative samples 171.2 201.7 194.0 15.1

[0044] Note: The platelet count of the negative control was 96.2% of the blank control, which is within the acceptable range of 80% to 120%.

[0045] The results show that the platelet adhesion rate of the chitosan-based adsorbent is much lower than that of the polystyrene-based adsorbent, demonstrating excellent blood compatibility. The platelet adhesion rate of the chitosan adsorbent bonded with L-glutathione is lower than that of the chitosan microspheres without L-glutathione, likely due to the increased hydrophilicity of the microsphere surface due to the introduction of amide bonds.

[0046] Hemolysis test:

[0047] A hemolysis test was performed on the adsorbent to evaluate the potential risk of adsorption-induced in vitro hemolysis. 1 g of adsorbent was weighed and added to 10 mL of 0.9% sodium chloride as the test group. For the negative control, 10 mL of 0.9% sodium chloride was added to each tube, and for the positive control, 10 mL of distilled water was added to each tube. Three replicate tubes were prepared for each group. All tubes were placed in a thermostatic shaker at 60 rpm in water at (37 ± 1)°C for 72 hours. Diluted rabbit blood was added to each tube at a ratio of 0.2 mL of diluted rabbit blood per 10 mL of test solution. The tubes were gently mixed and incubated in water at (37 ± 1)°C for another hour. The contents of the tubes were then decanted and centrifuged at 800 g for 5 minutes. The supernatant was aspirated and the absorbance measured at 545 nm. The hemolysis rate was calculated using the following formula.

[0048]

[0049] R——adsorbent hemolysis rate, %;

[0050] A——absorbance of adsorbent;

[0051] B – absorbance of negative control;

[0052] C – absorbance of positive control.

[0053] The results are shown in Table 3.

[0054] Table 3 Hemolysis rate of adsorbent

[0055] adsorbent Average absorbance (A) Average absorbance of negative control (B) Average absorbance of positive control (C) Hemolysis rate (%) Example 1 0.0197 0.0181 0.8269 0.2 Example 2 0.0189 0.0181 0.8269 0.1 Example 3 0.0189 0.0181 0.8269 0.1 Example 4 0.0205 0.0181 0.8269 0.3 Comparative Example 1 0.0221 0.0181 0.8269 0.5 Comparative samples 0.0585 0.0181 0.8269 5.0

[0056] From the results, it can be seen that the hemolysis rate of the adsorbents prepared with natural chitosan skeleton is 0.5%, which is much lower than that of the adsorbent with polystyrene skeleton.

[0057] The beneficial effects of the present invention are:

[0058] The adsorbent filled in the perfusion device with sodium chloride solution as the preservation solution contains a large amount of zirconium phosphate, which carries Na + and H + ions, when zirconium phosphate encounters a more active monovalent or divalent cation such as K + , Ca 2+ or Mg 2+ When the solution is filled with water, it will preferentially release Na + and H + In exchange, it absorbs other cations. These ions are then removed from the blood, regulating the charge and pH balance in the blood of patients with chronic kidney disease. Zirconium phosphate's honeycomb structure has both excellent ion exchange capacity and the ability to adsorb small molecules, enabling it to remove the small molecule toxin urea nitrogen from uremia.

[0059] Zirconium phosphate can improve the three-dimensional structure of chitosan microspheres and increase the adsorption surface area.

[0060] In addition, the L-glutathione bonded to the cross-linked chitosan microspheres has a strong reducing property, which can change the four disulfide bonds inside hepcidin. Through competition, it forms disulfide bonds with some of the sulfhydryl groups in it, breaking the disulfide bonds of hepcidin itself, causing the tertiary structure of the hepcidin protein to change from a tertiary globular structure to a linear chain, thereby increasing the contact opportunity between the chitosan microspheres and hepcidin, thereby enhancing the clearance effect on hepcidin.

[0061] The skeleton structure of chitosan itself has good biocompatibility and blood compatibility, and does not require coating or hydrophilic chemical modification. The adsorbent prepared with this skeleton has no toxic side effects and can be used for whole blood perfusion. It can also realize the functions of some dialyzers, reduce the use of dialysate, and improve dialysis efficiency.

[0062] The above examples are only used to illustrate the present invention. In addition, there are many different implementation methods. These implementation methods are all conceivable by those skilled in the art after understanding the concept of the present invention. Therefore, they are not listed here one by one.

Claims

1. A blood purification adsorbent, characterized in that: Cross-linked chitosan microspheres embedded with zirconium phosphate and / or zirconium oxide are prepared using EDTA as a cross-linking agent, and L-glutathione is bonded to the surface of the cross-linked chitosan microspheres; the mass ratio of the zirconium phosphate and / or zirconium oxide to the microspheres before cross-linking is 1:10 to 1:20; The preparation method comprises the following steps: 1) Adding an EDTA solution of zirconium phosphate to an acetic acid solution of chitosan and stirring to form a suspension, pouring the suspension into a pre-prepared oil phase of n-hexane and liquid paraffin, heating to 40°C, stirring for 1 hour, then adding a 3 mol / L sodium hydroxide solution to a pH ≥ 9, heating to 70°C and continuing to stir for 3 hours, filtering, extracting with petroleum ether for 8 hours, extracting with ethanol for 8 hours, and washing with water until neutral to obtain chitosan microspheres; 2) Add the product obtained in step 1 to an EDTA solution, then add an excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, heat to 37°C, and stir for 2 hours; then add L-glutathione solution and continue stirring for 2 hours to obtain cross-linked chitosan microspheres; 3) The product of step 2 was washed with EDTA solution and then washed with water. The zirconium ion concentration of the eluate was detected to be less than 0.001 mol / L and the pH of the eluate was neutral. The product was placed in a ventilated and dried at room temperature and then placed in a 0.9% sodium chloride solution.

2. The blood purification adsorbent according to claim 1, wherein The amount of L-glutathione bonded to the surface of the cross-linked chitosan microspheres is 0.4-1.0 mg / g.

3. The method for preparing a blood purification adsorbent according to claim 1 or 2, wherein: The following steps are involved: 1) Adding an EDTA solution of zirconium phosphate to an acetic acid solution of chitosan and stirring to form a suspension, pouring the suspension into a pre-prepared oil phase of n-hexane and liquid paraffin, heating to 40°C, stirring for 1 hour, then adding a 3 mol / L sodium hydroxide solution to a pH ≥ 9, heating to 70°C and continuing to stir for 3 hours, filtering, extracting with petroleum ether for 8 hours, extracting with ethanol for 8 hours, and washing with water until neutral to obtain chitosan microspheres; 2) Add the product obtained in step 1 to an EDTA solution, then add an excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, heat to 37°C, and stir for 2 hours; then add L-glutathione solution and continue stirring for 2 hours to obtain cross-linked chitosan microspheres; 3) The product of step 2 was washed with EDTA solution and then washed with water. The zirconium ion concentration of the eluate was detected to be less than 0.001 mol / L and the pH of the eluate was neutral. The product was placed in a ventilated and dried at room temperature and then placed in a 0.9% sodium chloride solution.

4. Use of the blood purification adsorbent according to claim 1 in the preparation of a hepcidin scavenger, wherein the hepcidin scavenger is used to remove excess hepcidin.

5. Use of the blood purification adsorbent according to claim 1 in the preparation of a urea nitrogen auxiliary scavenger, wherein the urea nitrogen auxiliary scavenger is used to assist in the removal of urea nitrogen.

6. Use of the blood purification adsorbent according to claim 1 in the preparation of a perfusion device, wherein the adsorbent in the perfusion device is used for whole blood perfusion, realizing partial dialyzer functions and regulating acid-base balance.