Preparation Of Hemoperfusion Adsorbent Cellulosebased MONOLITH And Its Specific Clearance Of β2-Microglobulin

China has the largest number of patients with chronic kidney disease.Chronic kidney failure,as an advanced manifestation of chronic kidney disease,has become one of the main threats to public health in recent years and has attracted increasing attention worldwide.Hemodialysis is currently the most commonly used clinical treatment.However,due to the relatively small pore size of dialysis membranes,some harmful macromolecules with large molecular weight,such asβ2-microglobulin（β2M）,cannot be effectively removed,and the accumulation ofβ2M in the body will induce dialysis-related amyloidosis（DRA）,leading to symptoms such as frozen shoulder,carpal tunnel syndrome and bone cyst.Hemoperfusion is one of the most effective methods for the prevention and treatment of DRA in clinical practice,as it can adsorb the target substance specifically and is not limited by its molecular size during blood purification.Currently commonly used hemoperfusion adsorption materials such as activated carbon aperture is small and have poor mechanical strength,so the material easy to fall off will cause hemolysis and other side effects,synthetic polymer material biocompatibility is low,so natural polysaccharide materials because of their excellent blood compatibility and bioinert,surface easy modification characteristics have been more and more widely used;In addition,these adsorption materials are generally granular form,usually have the disadvantages of large invalid volume and low adsorption efficiency.On the other hand,most functional ligands of adsorbent materials remove harmful substances by physical adsorption using hydrophobic interactions,van der Waals forces and hydrogen bonds,which leads to the disadvantage of poor adsorption specificity.Recently,antibody molecules and nanobodies have also been used as functional ligands for hemoperfusion,but their poor stability and high cost limit their use.Therefore,in this study,natural polysaccharide cellulose was used as the matrix material to prepare a MONOLITH monolithic column porous material as a new type of hemoperfusion material.This material has the characteristics of large specific surface area,high porosity and easy surface modification.It also has great application potential in the prevention and treatment of DRA by realizing the specific adsorption ofβ2M by fixing the functional ligand of the upper cyclic peptide molecule.This topic is mainly carried out in the following parts:1.Preparation of cellulose-based MONOLITH material with multi-level pore structure and its surface chemical modificationCellulose acetate-based MONOLITH（CA MONOLITH）with macropores and mesoporous structures was prepared by thermally induced phase separation using cellulose acetate with good solubility as the starting material.Cellulose-based（CE MONOLITH）was obtained by hydrolysis of acetate bonds under alkaline conditions.To realize the specific adsorption of proteins by CE MONOLITH,anhydride compounds with high reactivity need to be selected for chemical modification.In this study,ethylenediaminetetraacetic dianhydride（EDTAD）,succinic anhydride（SA）and pimelic acid anhydride（PAA）are respectively used to undergo esterification reaction with hydroxyl groups on CE MONOLITH for modification.The modified material was characterized by scanning electron microscope（SEM）,infrared spectroscopy（FT-IR）and nitrogen adsorption and desorption.The results showed that the EDTA-modified material（EDTA-CE MONOLITH）still maintained the original multi-level pore structure and had the largest specific surface area of 31.27 m²/g.2.Separation and purification of recombinantβ2M cultured from E.coli using Ni²⁺-CE MONOLITHThe dried EDTA-CE MONOLITH was fixed in a heat shrink tube,and the nickel ion solution was chelated to the material using a microinjection pump.Since nickel ions could specifically bind to the target proteinβ2M with His-tag,β2M could be purified from the crude protein solution obtained from the induced culture of E.coli.The result of SDS-PAGE showed that the recombinantβ2M had high purity,and the purity ofβ2M purified from EDTA-CE MONOLITH was 90.70%by using Image-J.Using the Bradford method,the purification capacity ofβ2M was 32.14 mg/g.Theβ2M was regenerated by gradient dialysis,and the regeneratedβ2M was measured by circular dichroism spectrogram and compared with a standard spectrogram.The second order results showed that the two had the same obviousβ-folding characteristic peak.Finally,the reusability of the material was investigated,and the results showed that the purification efficiency of the material remained at 92.06%after 5 cycles of use,showing excellent repurification ability.Renaturatedβ2M obtained by gradient dialysis was determined by circular dichroic spectrogram and compared with the standard spectrum to prove that the two had the same secondary structure of the obviousβ-folded characteristic peak.Finally,the reusability of the materials was investigated,and the results showed that after five cycles,the purification efficiency of the materials remained at92.06%,which had excellent reproducibility and purification ability.3.Preparation of hemoperfusion immunosorbent material CP-CE MONOLITH and its specific removal ofβ2MEDC/NHS reaction was used to activate the carboxyl group on the surface of MONOLITH material and conjugate the primary amino group on the lysine residues at the end of the peptide molecules to achieve the chemical bond fixation of the peptide on the surface of MONOLITH material.The EDX test results showed that the peptide molecules were successfully fixed on the surface of MONOLITH material.The Bradford method calculated that the ability of EDTA-CE MONOLITH to fix cyclic peptide was 44.65 mg/g,indicating that the material had high cyclic peptide fixation ability and successfully prepared the hemoperfusion immunoadsorption material CP-CE MONOLITH.The adsorption conditions of CP-CE MONOLITH were optimized by static adsorption methods,including adsorption time,adsorption temperature,initial concentration ofβ2M,and the amount of adsorbed material to achieve the best removal effect.The elution experiment ofβ2M adsorbed on the material was carried out under different p H values.According to the results of the Bradford method,p H 1.5 is the best elution condition.On this basis,the reutilization of the removal ofβ2M by CP-CE MONOLITH was investigated.The result shows that the material still has a clearance rate of 63.54%after 5 times reuse,indicating that the material has a good repeated adsorption capacity.To simulate the actual hemoperfusion process,the dynamic adsorption ofβ2M by CP-CE MONOLITH was investigated.The breakthrough curve showed a typical"S"shape with high steepness,indicating that CP-CE MONOLITH has a high adsorption efficiency forβ2M.To verify the specificity of CP-CE MONOLITH forβ2M adsorption,a mixed solution ofβ2M,HSA,Ig G and human serum with a certain amount ofβ2M added were adsorbed by the material.Characterization methods such as SDS-PAGE and immunoturbidimetry have demonstrated that CP-CE MONOLITH has high specificity forβ2M clearance.