Blood purification adsorbent and preparation method thereof

Abstract

The invention provides a blood purification adsorbent and a preparation method thereof, the blood purification adsorbent takes macroporous resin containing an epoxy group as a carrier, the epoxy group on the carrier reacts with polyethyleneimine, the polyethyleneimine is immobilized on the carrier, the polyethyleneimine is taken as a spacer arm, and the epoxy group on the carrier reacts with the polyethyleneimine to prepare the blood purification adsorbent. Superoxide dismutase and lipoic acid are connected to the spacer arm through a dehydration condensation reaction, so that the superoxide dismutase and the lipoic acid are immobilized on the carrier. The blood purification adsorbent can remove superoxide, free radicals and inflammatory factors at the same time, and can effectively improve oxidative stress and microinflammation states.

Description

Technical Field

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

[0002] Oxidative stress refers to a state in which the body's oxidation and anti-oxidation are out of balance, tending toward oxidation, leading to inflammatory infiltration of neutrophils, increased secretion of proteases, and the production of a large number of oxidative intermediates. This state is mainly caused by the negative effects of free radicals in the body and is considered to be an important factor leading to aging and disease. Free radicals, such as superoxide anions (O 2- ), hydroxyl radicals (OH) and hydrogen peroxide (H2O2), etc., when produced in excess in organisms, can damage the lipids of cell membranes, leading to cell dysfunction, while also affecting the structure of DNA and increasing the risk of mutation.

[0003] Micro-inflammatory state refers to a state in which the patient has no systemic or local acute clinical infection signs, but has a persistent low-level inflammatory state, which is manifested by a slight increase in inflammatory factors. It is characterized by persistence and relative concealment, and its essence is immune inflammation. This state is particularly common in dialysis patients. Although they may not have obvious systemic or local clinical infection signs, the levels of inflammatory cytokines and inflammatory factors such as interleukin-1, interleukin-6, and tumor necrosis factor a in the body continue to increase.

[0004] Oxidative stress and micro-inflammatory states are two physiological states that are harmful to human health. Oxidative stress and micro-inflammatory reactions are common in hemodialysis patients. These two are one of the important reasons for the occurrence of various complications in patients undergoing blood purification treatment, which may lead to health problems such as metabolic syndrome, fatty liver, atherosclerosis, cognitive dysfunction and sleep disorders. Moreover, oxidative stress and micro-inflammatory states not only cause harm to the body independently, but also aggravate the development of human cell damage and inflammatory reactions through interaction. However, existing blood purification adsorbents cannot remove oxygen free radicals and inflammatory factors at the same time. Summary of the invention

[0005] The present invention aims to solve the above-mentioned defects of the prior art and provide a blood purification adsorbent and a preparation method thereof, wherein the blood purification adsorbent can simultaneously remove superoxides, free radicals and inflammatory factors, and can effectively improve oxidative stress and micro-inflammatory states.

[0006] To solve the above problems, the first aspect of the present invention provides a blood purification adsorbent, which uses a macroporous resin containing epoxy groups as a carrier, the epoxy groups on the carrier react with polyethyleneimine, and the polyethyleneimine is immobilized on the carrier. The polyethyleneimine is used as a spacer arm, and superoxide dismutase and lipoic acid are connected to the spacer arm through a dehydration condensation reaction, so that superoxide dismutase and lipoic acid are immobilized on the carrier.

[0007] The second aspect of the present invention provides a method for preparing a blood purification adsorbent, which is used to prepare the blood purification adsorbent as described in the first aspect, and the preparation method comprises the following steps:

[0008] preparing a macroporous resin containing epoxy groups;

[0009] The macroporous resin containing epoxy groups is reacted with polyethyleneimine to immobilize the polyethyleneimine on the macroporous resin containing epoxy groups to obtain a macroporous resin containing a spacer arm, wherein the spacer arm is polyethyleneimine;

[0010] The macroporous resin containing the spacer arm is reacted with superoxide dismutase and lipoic acid, so that the superoxide dismutase and the lipoic acid are connected to the spacer arm through a dehydration condensation reaction, thereby preparing the blood purification adsorbent.

[0011] The blood purification adsorbent of the present invention uses a macroporous resin containing epoxy groups as a carrier, and the epoxy groups on the carrier react with polyethyleneimine to immobilize the polyethyleneimine on the carrier, thereby forming a large number of amino active groups and spacer arms with different molecular chain lengths on the carrier, and then connecting superoxide dismutase and lipoic acid to the spacer arm through a dehydration condensation reaction between the amino group and the carboxyl group on the spacer arm, so that the superoxide dismutase and lipoic acid are simultaneously immobilized on the carrier through the spacer arm, so that the blood purification adsorbent has both superoxide dismutase that can quickly decompose superoxide and lipoic acid that has anti-free radical oxidation performance. At the same time, the macroporous resin containing epoxy groups itself has rich medium and macroporous structures, and can effectively adsorb inflammatory factors such as interleukin-1, interleukin-6, and tumor necrosis factor α, thereby achieving the function of the blood purification adsorbent to simultaneously remove superoxide, free radicals and inflammatory factors, and can effectively improve oxidative stress and micro-inflammatory states. In addition, the epoxy groups contained on the surface of the carrier in the present invention can improve the hydrophilicity of the carrier, making the surface of the carrier hydrophilic, and the immobilized polyethyleneimine makes the carrier rich in long-chain active amine groups. The synergistic effect of the two is not only conducive to the grafting of large molecular weight superoxide dismutase, but also can prevent the superoxide dismutase from being adsorbed by the macroporous resin and causing it to lose its activity, which is conducive to maintaining the superoxide dismutase's scavenging activity on superoxide, and the rich long-chain active amine groups on the carrier have a large number of amine groups, which is conducive to increasing the immobilization amount of lipoic acid on the carrier and improving the scavenging ability of free radicals.

[0012] The preparation method of the blood purification adsorbent of the present invention has a simple preparation process, mild reaction conditions, high safety during the reaction process, low production cost, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The present invention provides a process flow chart for preparing a blood purification adsorbent according to an embodiment of the present invention. DETAILED DESCRIPTION

[0014] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0015] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0016] In addition, the terms "comprising", "including", "containing", and "having" are not restrictive, that is, other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are commercially available.

[0017] In addition, although the present invention describes the steps in the preparation in the form of S110, S120, S130, etc., this description is only for ease of understanding, and the form of S110, S120, S130, etc. does not limit the sequence of the steps.

[0018] The first aspect of the embodiments of the present application provides a blood purification adsorbent, which uses a macroporous resin containing epoxy groups as a carrier, and the epoxy groups on the carrier react with polyethyleneimine to immobilize the polyethyleneimine on the carrier. The polyethyleneimine is used as a spacer arm, and superoxide dismutase and lipoic acid are connected to the spacer arm through a dehydration condensation reaction, so that the superoxide dismutase and lipoic acid are immobilized on the carrier.

[0019] The blood purification adsorbent provided in the embodiment of the present application uses a macroporous resin containing epoxy groups as a carrier, and the epoxy groups on the carrier react with polyethyleneimine to immobilize the polyethyleneimine on the carrier, thereby forming a large number of amino active groups and spacer arms with different molecular chain lengths on the carrier, and then through the dehydration condensation reaction between the amino groups and carboxyl groups on the spacer arms, superoxide dismutase and lipoic acid are connected to the spacer arms, so that superoxide dismutase and lipoic acid are simultaneously immobilized on the carrier through the spacer arms, so that the blood purification adsorbent has both superoxide dismutase that can quickly decompose superoxide and lipoic acid that has anti-free radical oxidation performance. At the same time, the macroporous resin containing epoxy groups itself has rich medium and macroporous structures, which can effectively adsorb inflammatory factors such as interleukin-1, interleukin-6, and tumor necrosis factor α, thereby achieving the effect of the blood purification adsorbent in simultaneously removing superoxide, free radicals and inflammatory factors, and can effectively improve oxidative stress and micro-inflammatory states. In addition, the epoxy groups contained on the surface of the carrier in this embodiment can improve the hydrophilicity of the carrier, making the surface of the carrier hydrophilic, and the immobilized polyethyleneimine makes the carrier rich in long-chain active amine groups. The synergistic effect of the two is not only conducive to the grafting of large molecular weight superoxide dismutase, but also can prevent the superoxide dismutase from being adsorbed by the macroporous resin and causing it to lose its activity, which is conducive to maintaining the superoxide dismutase scavenging activity against superoxide, and the rich long-chain active amine groups on the carrier have a large number of amine groups, which is conducive to increasing the immobilization amount of lipoic acid on the carrier, and is conducive to improving the scavenging ability of free radicals.

[0020] On the basis of the above embodiment, as an optional implementation, the immobilized amount of superoxide dismutase on the blood purification adsorbent is 0.1mg / mL to 1mg / mL, and the immobilized amount of lipoic acid is 0.5mg / mL to 1.5mg / mL, that is, the immobilized amount of superoxide dismutase per 1mL of blood purification adsorbent is 0.1mg to 1mg, and the immobilized amount of lipoic acid is 0.5mg to 1.5mg. Thus, the immobilized amount of superoxide dismutase and lipoic acid on the blood purification adsorbent is within the above range, which can have a good scavenging effect on superoxide and a good scavenging ability on free radicals.

[0021] Superoxide dismutase has antioxidant and anti-aging effects, and it can catalyze superoxide free radicals to undergo dismutation reactions to remove superoxide anion free radicals that are harmful to the body. Based on the above embodiments, as an optional embodiment, superoxide dismutase is one or more of copper / zinc-superoxide dismutase (containing copper and zinc, also known as copper- and zinc-containing superoxide dismutase), manganese-superoxide dismutase (containing manganese, also known as manganese-containing superoxide dismutase) and iron-superoxide dismutase (containing iron, also known as iron-containing superoxide dismutase).

[0022] Figure 1 The process flow chart of preparing the blood purification adsorbent provided in the examples of this application is shown in FIG. Figure 1 As shown, the second aspect of the embodiment of the present application provides a method for preparing a blood purification adsorbent, which is used to prepare the blood purification adsorbent as described in the first aspect, and the preparation method comprises the following steps:

[0023] Step S110, preparing a macroporous resin containing epoxy groups.

[0024] Specifically, a polystyrene-based macroporous resin is prepared, and the polystyrene-based macroporous resin is epoxy-modified by an epoxy compound to obtain a macroporous resin containing epoxy groups.

[0025] More specifically, styrene monomers and polyvinyl crosslinking agents are subjected to suspension polymerization in a dispersion medium under the action of a porogen and an initiator, the reaction temperature of the suspension polymerization is 50°C to 100°C, and the reaction time is 12h to 20h, so as to obtain a polystyrene-based macroporous resin; an epoxy compound is added to the polystyrene-based macroporous resin, and the polystyrene-based macroporous resin is subjected to epoxy modification reaction to obtain a macroporous resin containing epoxy groups.

[0026] On the basis of the above embodiments, as a preferred implementation mode, an epoxy compound is added to the polystyrene-based macroporous resin after the oil phase is fixed and before the skeleton is solidified to carry out an epoxy modification reaction, specifically: a styrene monomer and a polyvinyl crosslinking agent are subjected to suspension polymerization in a dispersion medium under the action of a porogen and an initiator, wherein the reaction temperature of the suspension polymerization is 70°C to 80°C, and the reaction time is 1h to 3h, to obtain a polystyrene-based macroporous resin after the oil phase is fixed and before the skeleton is solidified. The polystyrene-based macroporous resin obtained at this stage shows that the oil phase droplets have been partially converted into polymers and the oil droplets can be fixed without breaking; an epoxy compound is added to the polystyrene-based macroporous resin after the oil phase is fixed and before the skeleton is solidified, and the reaction is continued at 70°C to 80°C for 3h to 5h to obtain an intermediate product; the reaction temperature is increased to 80°C to 85°C, and the intermediate product is allowed to continue to react for 2h to 6h to obtain a solidified macroporous resin containing epoxy groups. Therefore, the use of this method to prepare a macroporous resin containing epoxy groups can ensure that there are enough double bonds to participate in the epoxy modification reaction, so that the outer skeleton of the polystyrene-based macroporous resin contains active epoxy groups, and prevent the epoxy groups from entering the interior of the polystyrene-based macroporous resin skeleton, thereby avoiding affecting the hydrophobicity of the polystyrene-based macroporous resin skeleton and the stability of the pore structure.

[0027] On the basis of the above-mentioned embodiment, as a preferred embodiment, the epoxy compound is a compound containing an epoxy group and an olefin group at the same time, wherein the epoxy group is an epoxy reactive group and the olefin group is an olefin reactive group. Thus, the olefin group in the epoxy compound can undergo a cross-linking reaction with a polystyrene-based macroporous resin, and the epoxy group can subsequently connect to polyethyleneimine, so that polyethyleneimine is immobilized on the macroporous resin, thereby enabling polyethyleneimine to be more firmly connected to the macroporous resin containing the epoxy group, which is conducive to improving the structural stability of the macroporous resin containing the spacer arm. As an optional embodiment, the epoxy compound is methacrylic acid glycidyl ether or allyl glycidyl ether. The amount of the epoxy compound is 0.1 to 0.5 times the total mass of the monomer, wherein the monomer is composed of a styrene monomer and a polyvinyl cross-linking agent.

[0028] The styrene monomer is one or a combination of two of styrene, methyl styrene and ethyl styrene. When the styrene monomer is a mixture of two, the two are mixed in any proportion. In some preferred embodiments, the styrene monomer is styrene or ethyl styrene. The styrene monomer accounts for 20% to 45% of the total weight of the monomer, wherein the monomer is composed of a styrene monomer and a polyvinyl crosslinking agent.

[0029] The polyvinyl crosslinking agent is one or a combination of divinylbenzene (DVB), divinyltoluene, divinylxylene and divinylethylbenzene. When the polyvinyl crosslinking agent is a mixture of several kinds, they are mixed with each other in any proportion. In some preferred embodiments, the polyvinyl crosslinking agent is divinylbenzene. The polyvinyl crosslinking agent accounts for 55% to 80% of the total weight of the monomer, wherein the monomer is composed of a styrene monomer and a polyvinyl crosslinking agent.

[0030] The porogen is a combination of one or more of aromatic hydrocarbons, alkanes, higher alcohols, higher ketones and esters, wherein the aromatic hydrocarbons are toluene and / or xylene; the alkanes are a combination of one or more of n-heptane, 200# gasoline, liquid paraffin and solid paraffin; the higher alcohols are a combination of one or more of butanol, hexanol, cyclohexanol, isooctyl alcohol, n-octanol and methyl isobutyl carbinol; the higher ketones are a combination of one or more of methyl isobutyl ketone, 2-hexanone, diisobutyl ketone and methyl tert-butyl ketone; in some preferred embodiments, the porogen is obtained by combining aromatic hydrocarbons, alkanes, higher alcohols and higher ketones, and the mass ratio of aromatic hydrocarbons, alkanes, higher alcohols and higher ketones is 1:0.1:0.1:0.1 to 1:2:2:2. The ratio of the total mass of the porogen to the monomer is 40% to 200%, wherein the monomer is composed of a styrene monomer and a polyvinyl crosslinking agent.

[0031] The initiator is an organic peroxide. As an optional embodiment, the organic peroxide is one or a combination of benzoyl peroxide, tert-butyl peroxy-2-hexylhexanoate and tert-amyl peroxy-2-ethylhexanoate. When the initiator is a mixture of several kinds, they are mixed with each other in any proportion. In some preferred embodiments, the initiator is tert-butyl peroxy-2-hexylhexanoate. The amount of the initiator is 0.5% to 2% of the total mass of the monomer, wherein the monomer is composed of a styrene monomer and a polyvinyl crosslinking agent.

[0032] The volume ratio of the dispersion medium to the mixed organic phase is 1:1 to 3:1, wherein the mixed organic phase refers to a mixture of styrene monomers, polyvinyl crosslinking agents, porogens and initiators. The dispersion medium includes a dispersant and water, and the dispersant is dissolved in water to form the dispersion medium, wherein the dispersant includes any one of gelatin, polyvinyl alcohol or methyl cellulose, and in some preferred embodiments, the dispersant is gelatin. The amount of the dispersant is 0.5% to 2% of the mass of the dispersion medium.

[0033] After the epoxidation modification reaction is completed, the reaction mixture is cooled to room temperature, the mother liquor is filtered out, the mixture is extracted with acetone, and then the mixture is washed with water until there is no acetone smell, filtered, dried, and sieved to obtain a macroporous resin containing epoxy groups. The epoxy group content in the macroporous resin containing epoxy groups is measured to be 0.5 μmol / mL to 5 μmol / mL. In some preferred embodiments, the epoxy group content in the macroporous resin containing epoxy groups is 0.8 μmol / mL to 3 μmol / mL.

[0034] The specific surface area of ​​the macroporous resin containing epoxy groups (measured by BET method) ranges from 300 m 2 / g to 1000m 2 / g, pore volume range is 0.5cm 3 / g to 2.3cm 3 / g, the average pore size ranges from 5nm to 100nm, and the particle size ranges from 0.3mm to 1.5mm; in some preferred embodiments, the specific surface area (measured by BET method) of the macroporous resin containing epoxy groups ranges from 400m 2 / g to 800m 2 / g, pore volume range is 0.7cm 3 / g to 1.7cm 3 / g, the average pore size ranges from 6nm to 15nm, and the particle size ranges from 0.4mm to 1.0mm.

[0035] Step S120, reacting a macroporous resin containing epoxy groups with polyethyleneimine to immobilize the polyethyleneimine on the macroporous resin containing epoxy groups, thereby obtaining a macroporous resin containing a spacer arm, wherein the spacer arm is polyethyleneimine.

[0036] Specifically, a polyethyleneimine solution is added to a macroporous resin containing epoxy groups, and the mixture is reacted at 70° C. to 80° C. for 2 to 8 hours to allow the polyethyleneimine to react with the epoxy groups, thereby fixing the polyethyleneimine on the macroporous resin containing epoxy groups and forming a plurality of spacer arms on the macroporous resin to obtain a macroporous resin containing spacer arms.

[0037] In this embodiment, polyethyleneimine reacts with active epoxy groups so that polyethyleneimine and active epoxy groups are connected together through chemical bonds, thereby improving the stability of the connection between the two, which is beneficial to improving the structural stability of the macroporous resin containing the spacer arm. Polyethyleneimine has a long-chain branched polyamine matrix, and when it is immobilized on the macroporous resin containing the epoxy group, a large number of amino active groups and spacer arms with different molecular chain lengths can be formed on the macroporous resin containing the epoxy group, which is subsequently beneficial to increasing the immobilization amount of superoxide dismutase and lipoic acid.

[0038] Based on the above embodiment, as an optional implementation manner, the mass fraction of the polyethyleneimine solution is 3% to 15%, and the polyethyleneimine can be polyethyleneimine with different molecular weights, and the molecular weight of the polyethyleneimine is 275 to 10,000.

[0039] After the reaction between polyethyleneimine and epoxy group is completed and before the macroporous resin containing the spacer arm is prepared, the process further includes: washing the polyethyleneimine not participating in the grafting reaction with a large amount of pure water to avoid the polyethyleneimine not participating in the grafting reaction remaining in the pores of the macroporous resin.

[0040] In the examples of the present application, the immobilized amount of polyethyleneimine on the macroporous resin containing the spacer arm can be adjusted by adjusting the concentration of the polyethyleneimine solution, the reaction temperature and the reaction time, but in some preferred embodiments, the immobilized amount of polyethyleneimine on the macroporous resin containing the spacer arm is 0.3mmol / mL to 0.8mmol / mL based on the amine group. It should be noted that the specific amount of polyethyleneimine solution used in the examples of the present application is not further limited, and those skilled in the art can adjust it according to the actual situation, as long as the immobilized amount of polyethyleneimine on the macroporous resin containing the spacer arm can be guaranteed to be 0.3mmol / mL to 0.8mmol / mL.

[0041] Step S130, reacting a macroporous resin containing a spacer arm with superoxide dismutase and lipoic acid, so that the superoxide dismutase and the lipoic acid are connected to the spacer arm through a dehydration condensation reaction, thereby preparing a blood purification adsorbent.

[0042] Wherein, superoxide dismutase and lipoic acid are immobilized on a macroporous resin containing a spacer arm by a stepwise reaction. Specifically, step S130 comprises:

[0043] Step S131, reacting a macroporous resin containing a spacer arm with superoxide dismutase, connecting the carboxyl group on the superoxide dismutase with the amine group on the spacer arm through a dehydration condensation reaction, to obtain a macroporous resin immobilized with superoxide dismutase;

[0044] Step S132, reacting the macroporous resin immobilizing superoxide dismutase with lipoic acid, connecting the carboxyl group on the lipoic acid with the remaining amine group on the spacer arm through a dehydration condensation reaction, so that the lipoic acid is grafted onto the macroporous resin immobilizing superoxide dismutase.

[0045] In this embodiment, by first immobilizing superoxide dismutase on a macroporous resin containing a spacer arm, superoxide dismutase can preferentially react with an amine group having a longer spacer arm and being farther from the macroporous resin skeleton, thereby avoiding the situation that a superoxide dismutase having a larger molecular weight is difficult to react with an amine group closer to the macroporous resin due to a steric hindrance effect, resulting in a situation where grafting cannot occur, which is beneficial to increasing the immobilization amount of superoxide dismutase on the macroporous resin, and superoxide dismutase is immobilized on an amine group farther from the macroporous resin skeleton, so that superoxide dismutase Far away from the macroporous resin skeleton, the performance of the macroporous resin itself can be avoided to affect the activity of superoxide dismutase; then lipoic acid is immobilized on the macroporous resin containing the spacer arm, lipoic acid is a small molecule, and it has no special requirements for the length and position of the spacer arm, and it is easier to participate in the reaction. After the superoxide dismutase is immobilized on the macroporous resin containing the spacer arm, lipoic acid reacts with the remaining amine group on the spacer arm, and lipoic acid can be immobilized on the amine group closer to the macroporous resin skeleton, which is conducive to improving the immobilized amount of lipoic acid. In the present embodiment, the above-mentioned step-by-step reaction method is adopted to immobilize superoxide dismutase and lipoic acid on the macroporous resin containing the spacer arm, which can simultaneously improve the immobilized amount of superoxide dismutase and lipoic acid on the macroporous resin, and can maintain the activity of superoxide dismutase.

[0046] Based on the above embodiment, as an optional implementation, step S131 specifically includes: under the action of an activator, reacting a macroporous resin containing a spacer arm with superoxide dismutase in an acidic environment of 30° C. to 40° C. for 12 h to 24 h, so that the carboxyl group on the superoxide dismutase and the amine group on the spacer arm are connected by a dehydration condensation reaction, thereby obtaining a macroporous resin immobilizing superoxide dismutase.

[0047] Based on the above embodiment, as an optional implementation, the acidic environment in step S131 refers to a buffer solution with a pH value of 4.5 to 6. Therefore, the macroporous resin containing the spacer arm reacts with the superoxide dismutase in an acidic environment with a pH value of 4.5 to 6, which is beneficial to maintaining the activity of the activator and improving the immobilization efficiency of the superoxide dismutase on the one hand, and can avoid being too acidic and reducing the activity of the superoxide dismutase on the other hand.

[0048] Based on the above embodiment, as an optional implementation, the activator in step S131 is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Therefore, the above activator has good activity performance.

[0049] On the basis of the above embodiment, as an optional implementation, step S132 specifically includes: under the action of an activator, reacting the macroporous resin immobilizing superoxide dismutase with lipoic acid in an acidic environment of 30°C to 40°C for 12h to 24h, so that the carboxyl group on the lipoic acid is connected with the remaining amine group on the spacer arm through a dehydration condensation reaction, and superoxide dismutase and lipoic acid are simultaneously immobilized on the macroporous resin to obtain a blood purification adsorbent.

[0050] On the basis of the above embodiment, as an optional implementation, the acidic environment in step S132 refers to a buffer solution with a pH value of 4.5 to 6. Thus, the macroporous resin immobilizing superoxide dismutase reacts with lipoic acid in an acidic environment with a pH value of 4.5 to 6, which is beneficial to maintaining the activity of the activator and improving the immobilization efficiency of lipoic acid on the one hand, and can avoid being too acidic, reducing the activity of superoxide dismutase on the other hand. On the basis of the above embodiment, as a preferred implementation, the pH value of the acidic environment in step S131 and step S132 remains consistent.

[0051] Based on the above embodiment, as an optional implementation, the activator in step S132 is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Therefore, the above activator has good activity performance.

[0052] In this embodiment, after the blood purification adsorbent is prepared, the blood purification adsorbent is further rinsed with a large amount of purified water to remove the acidic solution and ungrafted reaction reagents in the blood purification adsorbent, which is beneficial to improving the safety of the blood purification adsorbent.

[0053] The immobilized amount of superoxide dismutase on the blood purification adsorbent prepared in this embodiment is 0.1 mg / mL to 1 mg / mL, and the immobilized amount of lipoic acid is 0.5 mg / mL to 1.5 mg / mL, that is, the immobilized amount of superoxide dismutase per 1 mL of blood purification adsorbent is 0.1 mg to 1 mg, and the immobilized amount of lipoic acid is 0.5 mg to 1.5 mg.

[0054] The preparation method of the blood purification adsorbent provided in the embodiment of the present application first introduces active epoxy groups into the outer skeleton of the polystyrene-based macroporous resin through epoxidation modification, which can improve the hydrophilicity of the surface of the macroporous resin without affecting the adsorption performance of the macroporous resin itself, so that it can effectively adsorb inflammatory factors such as interleukin-1, interleukin-6, and tumor necrosis factor α. Subsequently, the macroporous resin containing epoxy groups is used as a carrier, and the epoxy groups on the surface of the carrier react with polyethyleneimine to immobilize the polyethyleneimine on the carrier, forming a large number of amino active groups and spacer arms with different molecular chain lengths on the carrier, and then through the dehydration condensation reaction between the amino group and the carboxyl group on the spacer arm, the superoxide dismutase is adsorbed on the carrier. The superoxide dismutase and lipoic acid are connected to the spacer arm, so that superoxide dismutase and lipoic acid are stably connected to the spacer arm through chemical bonds, and the spacer arm is also stably connected to the carrier through chemical bonds, thereby improving the structural stability of the blood purification adsorbent. At the same time, the blood purification adsorbent has both superoxide dismutase that can quickly decompose superoxide and lipoic acid with anti-free radical oxidation performance, and the macroporous resin containing epoxy groups itself has rich medium and macroporous structures, which can effectively adsorb inflammatory factors such as interleukin-1, interleukin-6, and tumor necrosis factor a, thereby achieving the effect of the blood purification adsorbent in simultaneously removing superoxide, free radicals and inflammatory factors, and can effectively improve oxidative stress and micro-inflammatory states. In addition, the epoxy groups contained on the surface of the carrier in the present embodiment can improve the hydrophilicity of the carrier, making the surface of the carrier hydrophilic, and the immobilized polyethyleneimine makes the carrier rich in long-chain active amine groups, and the two act synergistically, which is not only conducive to grafting superoxide dismutase with large molecular weight, but also can avoid superoxide dismutase from being adsorbed by macroporous resin and causing it to lose activity, which is conducive to maintaining the scavenging activity of superoxide dismutase to superoxide, and the long-chain active amine groups rich in the carrier have a large number of amine groups, which is conducive to improving the immobilized amount of lipoic acid on the carrier, and is conducive to improving the scavenging ability of free radicals. The preparation method provided by the present invention has simple preparation process, mild reaction conditions, high safety in the reaction process, low production cost, and is suitable for industrial production.

[0055] In order to further explain the present invention in detail, the present invention will be further explained in conjunction with specific examples. The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified; the materials, reagents, etc. used in the examples of the present invention are purchased from the market unless otherwise specified.

[0056] Example 1

[0057] This embodiment provides a method for preparing a blood purification adsorbent, comprising the following steps:

[0058] (1) Preparation of macroporous resin containing epoxy groups: 600 mL of an aqueous solution containing 1.5 wt% gelatin was added to a 1000 mL three-necked flask, and a mixed organic phase consisting of 40 g of divinylbenzene, 10 g of ethylstyrene, 25 g of porogen toluene, 10 g of n-heptane, 25 g of n-octanol, 20 g of diisobutyl ketone, and 0.5 g of initiator tert-butyl peroxy-2-hexylhexanoate was added. Under mechanical stirring, the size of the oil droplets was controlled, and the temperature was raised to 70°C for reaction for 2 h. After sampling and observation, the oil droplets were fixed. 10 g of allyl glycidyl ether was added dropwise to a three-necked flask, and the reaction was continued at 70°C for 5 hours. The reaction temperature was then raised to 80°C and the reaction was continued for 4 hours to achieve sufficient crosslinking and curing. After the reaction was completed, the mixture was cooled, the mother liquor was filtered out, and acetone was extracted for 12 hours. The mixture was washed with water until there was no acetone smell, filtered, dried, and sieved. The resin with a particle size of 0.6 mm to 1.0 mm was selected to obtain the desired macroporous resin containing epoxy groups in the outer layer skeleton. The specific surface area (BET) of the macroporous resin containing epoxy groups was 806 m 2 / g, pore volume 2.1cm 3 / g, the average pore diameter is 9.9nm, and the content of grafted epoxy groups in the outer layer skeleton of the macroporous resin containing epoxy groups is measured to be 1.3μmol / mL.

[0059] (2) Preparation of macroporous resin containing spacer arms: 100 mL of the macroporous resin containing epoxy groups in the outer skeleton prepared in step (1) in a water-wet state was added to a 500 mL three-necked flask, 200 mL of water was added, and then 20 g of polyethyleneimine with a molecular weight of 275 was added. After stirring and dissolving, the temperature was raised to 75° C. and the reaction was carried out for 4 hours. The immobilized amount of polyethyleneimine was sampled and tested. After the reaction was completed, the polyethyleneimine that did not participate in the grafting reaction was washed with a large amount of water to obtain a macroporous resin immobilized with polyethyleneimine, i.e., a macroporous resin containing spacer arms. The grafted amount of polyethyleneimine on the macroporous resin containing spacer arms was 0.45 mmol / mL (based on amine groups).

[0060] (3) Preparation of blood purification adsorbent: 100 mL of pH 5.4 buffer solution was added to a 500 mL three-necked flask, 1 g of copper / zinc-superoxide dismutase was added, and then 3 g of N-hydroxysuccinimide and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation. Finally, 100 mL of the macroporous resin containing a spacer arm prepared in step (2) was added, and the mixture was reacted at 37° C. for 24 h. After the reaction was completed, the solution was drained to obtain a macroporous resin immobilized with superoxide dismutase, and samples were taken to detect the immobilized amount of copper / zinc-superoxide dismutase. Subsequently, 100 mL of a buffer solution with a pH value of 5.4 was added to another 500 mL three-necked flask, 2 g of lipoic acid was added, and then 3 g of N-hydroxysuccinimide and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation for 30 minutes. After activation, it was poured into the aforementioned macroporous resin containing immobilized superoxide dismutase, and the reaction was continued at 37° C. for 24 hours. After the reaction was completed, the reaction liquid was removed and the reagents not involved in the reaction were rinsed with a large amount of water to obtain a macroporous adsorption resin simultaneously immobilizing superoxide dismutase and lipoic acid, i.e., a blood purification adsorbent.

[0061] It was determined that the immobilized amount of superoxide dismutase on the blood purification adsorbent was 0.54 mg / mL, and the immobilized amount of lipoic acid was 0.75 mg / mL.

[0062] Example 2

[0063] This embodiment provides a method for preparing a blood purification adsorbent, comprising the following steps:

[0064] (1) Preparation of macroporous resin containing epoxy groups: 600 mL of an aqueous solution containing 1.5 wt% gelatin was added to a 1000 mL three-necked flask, and a mixed organic phase consisting of 31.5 g of divinylbenzene, 18.5 g of ethylstyrene, 35 g of porogen toluene, 15 g of n-heptane, 15 g of methyl isobutyl carbinol, 15 g of methyl isobutyl ketone, and 0.5 g of initiator tert-butyl peroxy-2-hexylhexanoate was added. Under mechanical stirring, the size of the oil droplets was controlled, and the temperature was raised to 70°C for reaction for 2 h, and then the oil was sampled and observed. After the beads are shaped, 15 g of allyl glycidyl ether is added dropwise to a three-necked flask, and the reaction is continued at 70°C for 5 hours. The reaction temperature is then raised to 80°C and the reaction is continued for 4 hours to achieve sufficient crosslinking and curing. After the reaction is completed, the mixture is cooled, the mother liquor is filtered out, and acetone is extracted for 12 hours. The mixture is washed with water until there is no acetone smell, filtered, dried, and sieved. The resin with a particle size of 0.6 mm to 1.0 mm is selected to obtain the desired macroporous resin containing epoxy groups in the outer skeleton. The specific surface area (BET) of the macroporous resin containing epoxy groups is 653 m 2 / g, pore volume 1.7cm 3 / g, the average pore diameter is 10.4nm, and the content of grafted epoxy groups in the outer layer skeleton of the macroporous resin containing epoxy groups is measured to be 1.8μmol / mL.

[0065] (2) Preparation of macroporous resin containing spacer arms: 100 mL of the macroporous resin containing epoxy groups in the outer skeleton prepared in step (1) in a water-wet state was added to a 500 mL three-necked flask, 200 mL of water was added, and then 20 g of polyethyleneimine with a molecular weight of 4500 was added. After stirring and dissolving, the temperature was raised to 75° C. and the reaction was carried out for 4 hours. The polyethyleneimine immobilized amount was sampled and tested. After the reaction was completed, the polyethyleneimine that did not participate in the grafting reaction was washed with a large amount of water to obtain a macroporous resin immobilized with polyethyleneimine, i.e., a macroporous resin containing spacer arms. The grafting amount of polyethyleneimine on the macroporous resin containing spacer arms was 0.55 mmol / mL (based on amine groups).

[0066] (3) Preparation of blood purification adsorbent: 100 mL of a buffer solution with a pH value of 5.4 was added to a 500 mL three-necked flask, 1 g of manganese-superoxide dismutase was added, and then 3 g of N-hydroxysuccinimide and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation. Finally, 100 mL of the macroporous resin containing a spacer arm prepared in step (2) was added, and the mixture was reacted at 37° C. for 24 h. After the reaction was completed, the solution was drained to obtain a macroporous resin immobilized with superoxide dismutase, and a sample was taken to detect the immobilized amount of manganese-superoxide dismutase. Subsequently, 100 mL of a buffer solution with a pH value of 5.4 was added to another 500 mL three-necked flask, 3 g of lipoic acid was added, and then 3 g of N-hydroxysuccinimide and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation for 30 minutes. After activation, it was poured into the aforementioned macroporous resin containing immobilized superoxide dismutase, and the reaction was continued at 37° C. for 24 hours. After the reaction was completed, the reaction liquid was removed and the reagents not involved in the reaction were washed with a large amount of water to obtain a macroporous adsorption resin simultaneously immobilized with superoxide dismutase and lipoic acid, i.e., a blood purification adsorbent.

[0067] It was determined that the immobilized amount of superoxide dismutase on the blood purification adsorbent was 0.59 mg / mL, and the immobilized amount of lipoic acid was 0.88 mg / mL.

[0068] Example 3

[0069] This embodiment provides a method for preparing a blood purification adsorbent, comprising the following steps:

[0070] (1) Preparation of macroporous resin containing epoxy groups: 600 mL of an aqueous solution containing 1.5 wt% gelatin was added to a 1000 mL three-necked flask, and a mixed organic phase consisting of 27.5 g of divinylbenzene, 21.5 g of ethylstyrene, 50 g of porogen toluene, 10 g of n-heptane, 10 g of isooctyl alcohol, 10 g of methyl tert-butyl ketone, and 0.5 g of initiator peroxy-2-hexylhexanoate was added. Under mechanical stirring, the size of the oil droplets was controlled, the temperature was raised to 70°C, and the reaction was continued for 2 h, and then a sample was taken to observe the formation of the oil droplets. After that, 25 g of allyl glycidyl ether was added dropwise to the three-necked flask, and the reaction was continued at 70°C for 5 hours, and then the reaction temperature was raised to 80°C and the reaction was continued for 4 hours to achieve sufficient crosslinking and curing. After the reaction was completed, the mixture was cooled, the mother liquor was filtered out, and acetone was extracted for 12 hours. The mixture was washed with water until there was no acetone smell, filtered, dried, and sieved. The resin with a particle size of 0.6 mm to 1.0 mm was selected to obtain the desired macroporous resin containing epoxy groups in the outer skeleton. The specific surface area (BET) of the macroporous resin containing epoxy groups was 540 m 2 / g, pore volume 1.5cm 3 / g, the average pore diameter is 10.9nm, and the content of grafted epoxy groups in the outer layer skeleton of the macroporous resin containing epoxy groups is measured to be 2.8μmol / mL.

[0071] (2) Preparation of macroporous resin containing spacer arms: 100 mL of the macroporous resin containing epoxy groups in the outer skeleton prepared in step (1) in a water-wet state was added to a 500 mL three-necked flask, 200 mL of water was added, and then 20 g of polyethyleneimine with a molecular weight of 10,000 was added. After stirring and dissolving, the temperature was raised to 75° C. and the reaction was carried out for 4 hours. The immobilized amount of polyethyleneimine was sampled and tested. After the reaction was completed, the polyethyleneimine that did not participate in the grafting reaction was washed with a large amount of water to obtain a macroporous resin immobilized with polyethyleneimine, i.e., a macroporous resin containing spacer arms. The grafted amount of polyethyleneimine on the macroporous resin containing spacer arms was 0.67 mmol / mL (based on amine groups).

[0072] (3) Preparation of blood purification adsorbent: 100 mL of pH 5.4 buffer solution was added to a 500 mL three-necked flask, 1 g of iron-superoxide dismutase was added, and then 3 g of N-hydroxysuccinimide and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation. Finally, 100 mL of the macroporous resin containing the spacer arm prepared in step (2) was added, and the mixture was reacted at 37° C. for 24 h. After the reaction was completed, the solution was drained to obtain a macroporous resin immobilized with superoxide dismutase, and samples were taken to detect the immobilized amount of iron-superoxide dismutase. Subsequently, 100 mL of a buffer solution with a pH value of 5.4 was added to another 500 mL three-necked flask, 4 g of lipoic acid was added, and then 3 g of N-hydroxysuccinimide and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation for 30 minutes. After activation, it was poured into the aforementioned macroporous resin containing immobilized superoxide dismutase, and the reaction was continued at 37° C. for 24 hours. After the reaction was completed, the reaction liquid was removed, and the reagents not involved in the reaction were rinsed with a large amount of water to obtain a macroporous adsorption resin simultaneously immobilized with superoxide dismutase and lipoic acid, i.e., a blood purification adsorbent.

[0073] It was determined that the immobilized amount of superoxide dismutase on the blood purification adsorbent was 0.64 mg / mL, and the immobilized amount of lipoic acid was 1.03 mg / mL.

[0074] Test Example 1: In vitro adsorption performance test of interleukin-1, interleukin-6 and tumor necrosis factor-a

[0075] 1 mL of the blood purification adsorbent prepared in Examples 1 to 3 was accurately measured respectively, and each group of blood purification adsorbent was added to 10 mL of plasma containing interleukin-1, interleukin-6, and tumor necrosis factor-a. After shaking at 37° C. for 2 hours, the adsorption was completed, and the plasma was taken for detection. The concentrations of interleukin-1, interleukin-6, and tumor necrosis factor-a were tested by chemiluminescence, and the adsorption rate was calculated. The adsorption performance results of the blood purification adsorbent in each embodiment for interleukin-1, interleukin-6, and tumor necrosis factor-a are shown in Table 1.

[0076] Table 1 Adsorption performance results of various inflammatory factors

[0077]

[0078] It can be seen from Table 1 that the blood purification adsorbents prepared in Examples 1 to 3, after introducing spacer arms on the macroporous resin and then immobilizing superoxide dismutase and lipoic acid through the spacer arms, still have excellent adsorption capacity for various inflammatory factors such as interleukin-1, interleukin-6 and tumor necrosis factor α, indicating that the blood purification adsorbent provided in this embodiment can effectively adsorb various inflammatory factors.

[0079] Experimental Example 2: Perfusion experiment of the big-eared rabbit model with micro-inflammation and micro-oxidative stress

[0080] The blood purification adsorbents prepared in Examples 1 to 3 were aseptically treated and assembled into 20 mL / branch adsorption columns. The adsorption column was connected to the arteries and veins of the modeling rabbits in micro-inflammation and micro-oxidation stress states by a peristaltic pump and a pipeline. After the anticoagulant was given, the peristaltic pump was started, and blood perfusion was performed at a blood flow rate of 20 mL / min for 1 h, wherein the modeling rabbits in micro-inflammation and micro-oxidation stress states can be constructed by feeding high-sugar and high-fat feed and injecting low-dose endotoxins (specifically, the dosage range can be set by those skilled in the art according to actual conditions). Rabbit blood was collected before and after perfusion, and superoxide dismutase activity (SOD) and malondialdehyde (MDA), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor a (TNF-a) were detected. The adsorption performance results of the blood purification adsorbents in each embodiment are shown in Tables 2 and 3.

[0081] Table 2 Test results of anti-oxidative stress performance in experimental animals

[0082]

[0083] It can be seen from Table 2 that after the blood purification adsorbents prepared in Examples 1 to 3 were assembled into a micro-adsorption column and blood perfusion was performed on the model rabbits induced to form micro-inflammation and micro-oxidative stress, the superoxide dismutase level in the body was significantly increased, and the malondialdehyde level in the body was significantly decreased, indicating that the oxidative stress level in the model rabbit model was significantly improved. Therefore, it can be explained that the blood purification adsorbent provided in this embodiment can significantly improve the oxidative stress level in the body, and its use in the body can effectively improve the oxidative stress state.

[0084] Table 3 Results of adsorption performance of various inflammatory factors in animals

[0085]

[0086] It can be seen from Table 3 that after the blood purification adsorbents prepared in Examples 1 to 3 were assembled into a micro-adsorption column and blood perfusion was performed on the model rabbits induced to form micro-inflammation and micro-oxidative stress, the levels of various inflammatory factors such as interleukin-1, interleukin-6 and tumor necrosis factor α in the rabbit model were rapidly reduced after blood perfusion, indicating that the blood purification adsorbent provided in this embodiment can effectively adsorb various inflammatory factors and can quickly and effectively improve the micro-inflammatory state.

[0087] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A blood purification adsorbent, characterized in that: The blood purification adsorbent uses a macroporous resin containing epoxy groups as a carrier, the epoxy groups on the carrier react with polyethyleneimine, the polyethyleneimine is immobilized on the carrier, the polyethyleneimine is used as a spacer arm, superoxide dismutase and lipoic acid are connected to the spacer arm through a dehydration condensation reaction, so that the superoxide dismutase and lipoic acid are immobilized on the carrier.

2. The blood purification adsorbent according to claim 1, characterized in that: The immobilized amount of the superoxide dismutase on the blood purification adsorbent is 0.1 mg / mL to 1 mg / mL, and the immobilized amount of the lipoic acid is 0.5 mg / mL to 1.5 mg / mL.

3. The blood purification adsorbent according to claim 1, characterized in that: The superoxide dismutase is one or a combination of copper / zinc-superoxide dismutase, manganese-superoxide dismutase and iron-superoxide dismutase.

4. A method for preparing a blood purification adsorbent, characterized in that: Used for preparing the blood purification adsorbent according to any one of claims 1 to 3, the preparation method comprises the following steps: preparing a macroporous resin containing epoxy groups; The macroporous resin containing epoxy groups is reacted with polyethyleneimine to immobilize the polyethyleneimine on the macroporous resin containing epoxy groups to obtain a macroporous resin containing a spacer arm, wherein the spacer arm is polyethyleneimine; The macroporous resin containing the spacer arm is reacted with superoxide dismutase and lipoic acid, so that the superoxide dismutase and the lipoic acid are connected to the spacer arm through a dehydration condensation reaction, thereby preparing the blood purification adsorbent.

5. The method for preparing a blood purification adsorbent according to claim 4, characterized in that: The method of reacting the macroporous resin containing the spacer arm with superoxide dismutase and lipoic acid so that the superoxide dismutase and lipoic acid are connected to the spacer arm through a dehydration condensation reaction comprises: The macroporous resin containing the spacer arm is reacted with the superoxide dismutase, and the carboxyl group on the superoxide dismutase is connected with the amine group on the spacer arm through a dehydration condensation reaction to obtain a macroporous resin immobilized with superoxide dismutase; The superoxide dismutase-immobilized macroporous resin is reacted with the lipoic acid, and the carboxyl group on the lipoic acid is connected with the remaining amine group on the spacer arm through a dehydration condensation reaction, so that the lipoic acid is grafted onto the superoxide dismutase-immobilized macroporous resin.

6. The method for preparing a blood purification adsorbent according to claim 5, characterized in that: The macroporous resin containing the spacer arm is reacted with the superoxide dismutase, and the carboxyl group on the superoxide dismutase is connected with the amine group on the spacer arm through a dehydration condensation reaction to obtain the macroporous resin immobilizing superoxide dismutase, comprising: Under the action of an activator, the macroporous resin containing a spacer arm and the superoxide dismutase are reacted in an acidic environment of 30° C. to 40° C. for 12 h to 24 h, so that the carboxyl group on the superoxide dismutase and the amine group on the spacer arm are connected through a dehydration condensation reaction to obtain a macroporous resin immobilizing superoxide dismutase.

7. The method for preparing a blood purification adsorbent according to claim 5, characterized in that: The step of reacting the superoxide dismutase-immobilized macroporous resin with the lipoic acid, and connecting the carboxyl group on the lipoic acid with the remaining amine group on the spacer arm through a dehydration condensation reaction, comprises: Under the action of an activator, the macroporous resin immobilizing superoxide dismutase and the lipoic acid are reacted in an acidic environment of 30° C. to 40° C. for 12 h to 24 h, so that the carboxyl group on the lipoic acid is connected with the remaining amine group on the spacer arm through a dehydration condensation reaction, and the superoxide dismutase and the lipoic acid are simultaneously immobilized on the macroporous resin to obtain the blood purification adsorbent.

8. The method for preparing a blood purification adsorbent according to claim 4, characterized in that: The step of reacting the macroporous resin containing epoxy groups with polyethyleneimine to immobilize the polyethyleneimine on the macroporous resin containing epoxy groups comprises: A polyethyleneimine solution is added to the macroporous resin containing epoxy groups, and the mixture is reacted at 70° C. to 80° C. for 2 to 8 hours to allow the polyethyleneimine to react with the epoxy groups, thereby fixing the polyethyleneimine on the macroporous resin containing epoxy groups and forming a plurality of spacer arms on the macroporous resin.

9. The method for preparing a blood purification adsorbent according to claim 8, characterized in that: The mass fraction of the polyethyleneimine solution is 3% to 15%, and the molecular weight of the polyethyleneimine is 275 to 10,000.

10. The method for preparing a blood purification adsorbent according to claim 4, characterized in that: The method for preparing a macroporous resin containing epoxy groups comprises: Preparation of polystyrene-based macroporous resin; Performing epoxy modification on the polystyrene-based macroporous resin by using an epoxy compound to obtain the macroporous resin containing epoxy groups; The epoxy compound is a compound containing both an epoxy group and an olefin group.

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