Low-density lipoprotein adsorbent and preparation method thereof
By introducing a disulfide bond crosslinking agent into the polystyrene-based resin and performing reduction and grafting reactions, an efficient low-density lipoprotein adsorbent was prepared, which solved the problem of low removal efficiency of existing adsorbents and achieved higher adsorption rate and adsorption rate.
Patent Information
- Application Number
- CN202411528056.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-02-28
AI Technical Summary
Existing LDL adsorbents cannot effectively and quickly remove LDL from patients.
By adding a crosslinking agent with disulfide bonds to a disulfide bond in the synthesis process of the polystyrene-based resin, a first adsorption resin containing disulfide bonds is prepared, and a second adsorption resin containing sulfhydride bonds is reduced by a reducing agent to a sulfhydride group to a sulfhydride group is prepared. Then, the organic acid monomer is grafted onto the second adsorption resin through a thiol-ene click reaction to prepare a low-density lipoprotein adsorbent.
The adsorption rate and adsorption rate of low-density lipoprotein adsorbents are improved, and effective and rapid removal of low-density lipoprotein in patients' bodies is achieved.
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Figure CN119524812A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of blood purification, and in particular to a preparation method of a low-density lipoprotein adsorbent and the low-density lipoprotein adsorbent. Background Art
[0002] Hemoperfusion refers to the process of introducing blood into a perfusion device containing fixed adsorbents to absorb harmful substances in the blood through physical or chemical adsorption, thereby achieving the purpose of blood purification. Low-density lipoprotein is a lipoprotein particle that carries cholesterol into peripheral tissue cells and can be oxidized into oxidized low-density lipoprotein. When low-density lipoprotein or oxidized low-density lipoprotein is excessive, the cholesterol it carries accumulates on the arterial wall, which can easily cause arteriosclerosis.
[0003] However, the existing low-density lipoprotein adsorbents have low adsorption rates and adsorption speeds and are unable to effectively and quickly remove low-density lipoprotein from the patient's body. Summary of the invention
[0004] The present invention provides a low-density lipoprotein adsorbent and a preparation method thereof, aiming to solve the problem that the existing low-density lipoprotein adsorbent cannot effectively and quickly remove low-density lipoprotein in a patient's body.
[0005] In a first aspect, the present invention provides a method for preparing a low-density lipoprotein adsorbent, the preparation method comprising:
[0006] During the synthesis of the polystyrene resin, a cross-linking agent having a disulfide bond is added to react to obtain a first adsorption resin containing a disulfide bond;
[0007] Using a reducing agent to reduce the disulfide bonds in the first adsorption resin to thiol groups, thereby obtaining a second adsorption resin containing thiol groups;
[0008] The organic acid monomer containing carbon-carbon double bonds is grafted onto the second adsorption resin through a thiol-ene click reaction to prepare a low-density lipoprotein adsorbent.
[0009] In a second aspect, the present invention provides a low-density lipoprotein adsorbent, wherein the low-density lipoprotein adsorbent is prepared by using the above-mentioned method for preparing the low-density lipoprotein adsorbent.
[0010] The embodiment of the present application provides a low-density lipoprotein adsorbent and a preparation method thereof, wherein a cross-linking agent having a disulfide bond is added to a polystyrene resin during the synthesis process to react, thereby obtaining a first adsorption resin containing a disulfide bond; a reducing agent is used to reduce the disulfide bond in the first adsorption resin to a thiol group, thereby obtaining a second adsorption resin containing a thiol group; an organic acid monomer containing a carbon-carbon double bond is grafted onto the second adsorption resin through a thiol-ene click reaction, thereby obtaining a low-density lipoprotein adsorbent. In the process of preparing a low-density lipoprotein adsorbent, the present invention introduces a cross-linking agent having a disulfide bond, so that after the disulfide bond is reduced to a thiol group, a specific ligand can be grafted to perform modification, thereby improving the adsorption rate and adsorption rate of the low-density lipoprotein adsorbent, thereby effectively and quickly removing low-density lipoprotein in the patient's body. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.
[0012] Figure 1 The present invention provides a schematic flow chart of the steps of a method for preparing a low-density lipoprotein adsorbent;
[0013] Figure 2 The embodiment of the present invention provides a schematic flow chart of the steps of another method for preparing a low-density lipoprotein adsorbent. DETAILED DESCRIPTION
[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0015] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.
[0016] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.
[0017] It should be further understood that the term "and / or" used in the present description and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
[0018] In conjunction with the accompanying drawings, some embodiments of the present application are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.
[0019] The present application provides a low-density lipoprotein adsorbent and a preparation method thereof. In the process of preparing the low-density lipoprotein adsorbent, a cross-linking agent having a disulfide bond can be introduced so that the disulfide bond can be reduced to a thiol group and then grafted with a specific ligand for modification, thereby improving the adsorption rate and adsorption speed of the low-density lipoprotein adsorbent, thereby effectively and quickly removing the low-density lipoprotein in the patient's body.
[0020] See also Figure 1 , Figure 1 The present invention provides a schematic flow chart of a method for preparing a low-density lipoprotein adsorbent.
[0021] like Figure 1 As shown, the method for preparing the low-density lipoprotein adsorbent includes: step S101 to step S103.
[0022] S101. During the synthesis process of a polystyrene resin, a cross-linking agent having a disulfide bond is added to react to obtain a first adsorption resin containing a disulfide bond.
[0023] The first adsorption resin is a once-crosslinked polystyrene resin, and the first adsorption resin contains a disulfide bond provided by a crosslinking agent.
[0024] Since a cross-linking agent having a disulfide bond is present during the synthesis of the polystyrene resin and a polymerization reaction is performed, the first adsorption resin obtained after the polymerization reaction also contains a disulfide bond.
[0025] For example, the cross-linking agent may be a material such as N,N'-bis(acryl)cystamine that can provide disulfide bonds and carbon-carbon double bonds for cross-linking, and is not specifically limited here.
[0026] Preferably, the cross-linking agent can be N,N'-bis(acryl)cystamine. N,N'-bis(acryl)cystamine not only has a disulfide bond, but also has two carbon-carbon double bonds at the end for cross-linking. Therefore, N,N'-bis(acryl)cystamine used as a cross-linking agent can ensure that the disulfide bond will not fall off when it is subsequently reduced to a thiol group, thereby effectively improving the preparation stability of the low-density lipoprotein adsorbent.
[0027] S102, using a reducing agent to reduce the disulfide bonds in the first adsorption resin to thiol groups, to obtain a second adsorption resin containing thiol groups.
[0028] The second adsorption resin is a primary cross-linked polystyrene resin, and the second adsorption resin contains thiol groups generated by disulfide bond reduction.
[0029] Exemplarily, the reducing agent can be selected from one or more of dithiothreitol, 2-mercaptoethanol, and tris(2-formylethyl)phosphine hydrochloride, which are not specifically limited herein. The reducing agent can effectively reduce the disulfide bonds in the first adsorption resin to sulfhydryl groups, so the second adsorption resin obtained after the reduction reaction will contain sulfhydryl groups.
[0030] In some embodiments, the second adsorption resin has a thiol content of 0.05-0.8 mmol / mL.
[0031] The content of thiol groups in the second adsorption resin can be determined according to the content of the cross-linking agent.
[0032] Generally, the content of thiol groups can be controlled by controlling the amount of cross-linking agent with disulfide bonds added. Generally, the more the amount of cross-linking agent with disulfide bonds added, the more thiol groups content of the second adsorption resin obtained after the reduction reaction, the more the amount of subsequent ligand grafting, the faster the adsorption rate of the low-density lipoprotein adsorbent, and the more the adsorption amount.
[0033] If the thiol content of the second adsorption resin is too low, it will affect the grafting efficiency; if the thiol content of the second adsorption resin is too high, it means that the amount of crosslinking agent added is too much, which is not conducive to the sphering of the resin. Therefore, it is necessary to adjust the proportion of each material in the synthesis process of the polystyrene resin to re-prepare a crosslinked resin. Therefore, the grafting effect is best when the thiol content of the second adsorption resin is controlled to 0.05-0.8 mmol / mL.
[0034] S103, grafting an organic acid monomer containing a carbon-carbon double bond onto a second adsorption resin through a thiol-ene click reaction to prepare a low-density lipoprotein adsorbent.
[0035] Among them, low-density lipoprotein adsorbent can effectively adsorb low-density lipoprotein.
[0036] Since there are many positively charged amino acids in the ApoB domain on the surface of low-density lipoprotein, anionic ligands can be designed on the surface of low-density lipoprotein adsorbent to achieve effective adsorption of low-density lipoprotein. Organic acid monomers have anionic groups, so the surface of the second adsorbent resin can be modified to graft the anionic groups of the organic acid monomers onto the thiol groups in the second adsorbent resin through a thiol-ene click reaction to prepare a low-density lipoprotein adsorbent.
[0037] Specifically, the organic acid monomer may be a carboxylic acid monomer containing a carbon-carbon double bond and / or a sulfonic acid monomer containing a carbon-carbon double bond.
[0038] For example, the carboxylic acid monomer containing a carbon-carbon double bond can be selected from one or more of acrylic acid and methacrylic acid, and the sulfonic acid monomer containing a carbon-carbon double bond can be selected from one or more of 2-acrylamide-2-methylpropane sulfonic acid and propylene sulfonic acid.
[0039] The low-density lipoprotein adsorbent prepared by the above preparation method. Performance tests show that the specific surface area of the low-density lipoprotein adsorbent (BET) is 400-600m 2 / g; pore volume is 1-1.8cm 3 / g; the average pore size is between 12-25nm. Compared with the low-density lipoprotein adsorbent provided by the related technology, the adsorption rate and adsorption speed of low-density lipoprotein are significantly improved.
[0040] The embodiment of the present application provides a method for preparing a low-density lipoprotein adsorbent. During the preparation of the low-density lipoprotein adsorbent, a cross-linking agent having a disulfide bond is introduced so that the disulfide bond can be reduced to a thiol group and then modified to graft a specific ligand, thereby improving the adsorption rate and adsorption rate of the low-density lipoprotein adsorbent, thereby effectively and quickly removing the low-density lipoprotein in the patient's body.
[0041] See also Figure 2 , Figure 2 It is a schematic flow chart of another method for preparing a low-density lipoprotein adsorbent provided in an embodiment of the present invention.
[0042] like Figure 2 As shown, the method for preparing the low-density lipoprotein adsorbent includes the following steps: step S201 to step S203.
[0043] S201, polymerizing styrene monomers, polyvinyl monomers, N,N'-bis(acryl)cystamine, porogens and initiators to obtain a first adsorption resin containing disulfide bonds.
[0044] Among them, styrene monomers are the main raw materials for the production of polystyrene, and polyvinyl monomers are materials containing two or more vinyl groups in the molecule. Polyvinyl monomers can form cross-linked structures during the polymerization process, thereby enhancing the performance of the polymer. N,N'-bis(acryl)cystamine can provide disulfide bonds, which can be subsequently reduced to thiol groups for subsequent grafting of thiol groups and organic acid monomers.
[0045] Exemplarily, if the total mass of styrene monomers, polyvinyl monomers and N,N'-bis(acryl)cystamine is Z, the mass of styrene monomers is 15%-40% of Z, for example: 15%, 22%, 28%, 40%, etc.; the mass of polyvinylbenzene monomers is 20%-80% of Z, for example: 20%, 40%, 55%, 80%, etc.; the mass of N,N'-bis(acryl)cystamine is 5%-50% of Z, for example: 5%, 15%, 30%, 50%, etc.
[0046] For example, the styrene monomer may be one or more of styrene, methylstyrene, ethylstyrene, dimethylstyrene and trimethylstyrene, which are not specifically limited herein.
[0047] For example, the polyvinyl monomer may be one or more of divinylbenzene, trivinylbenzene, and divinyltoluene, without any specific limitation herein.
[0048] If methyl styrene is used as the styrene monomer and trivinyl benzene is used as the polyvinyl monomer, the mass of methyl styrene can be 20% of Z, the mass of trivinyl benzene can be 55% of Z, and the mass of N,N'-bis(acryl)cystamine can be 25% of Z.
[0049] If trimethylstyrene is used as the styrene monomer and divinyltoluene is used as the polyvinyl monomer, the mass of trimethylstyrene can be 15% of Z, the mass of divinyltoluene can be 45% of Z, and the mass of N,N'-bis(acryl)cystamine can be 40% of Z.
[0050] Exemplarily, the porogen is mainly composed of long-chain alkanes, aromatic hydrocarbons, and higher fatty alcohols, wherein long-chain alkanes refer to alkanes with more carbon atoms, usually 20 or more carbon atoms, and a long and complex molecular structure. The mass of the porogen can be 50%-150% of Z.
[0051] Specifically, if the total mass of the porogen is M, the mass of the long-chain alkane is 0-50% of M, for example: 0%, 15%, 25%, 50%, etc.; the mass of the aromatic hydrocarbon is 30-60% of M, for example: 30%, 35%, 45%, 60%, etc.; the mass of the higher fatty alcohol is 20-50% of M, for example: 20%, 25%, 40%, 50%, etc.
[0052] For example, the long-chain alkane may be one or more of white oil and gasoline, which are not specifically limited herein.
[0053] For example, the aromatic hydrocarbon may be one or more of toluene, ethylbenzene, and xylene, which are not specifically limited herein.
[0054] For example, the higher fatty alcohol may be one or more of isooctyl alcohol, methyl isobutyl carbinol, and n-heptanol, which are not specifically limited herein.
[0055] If xylene and isooctyl alcohol are selected as the porogen, the mass of xylene can be 60% of M, and the mass of isooctyl alcohol can be 40% of M.
[0056] If the porogens are white oil, ethylbenzene and methyl isobutyl carbinol, the mass of the white oil can be 20% of M, the mass of the ethylbenzene can be 50% of M, and the mass of the methyl isobutyl carbinol can be 30% of M.
[0057] For example, the initiator can be selected from one or more of azobisisobutyronitrile, benzoyl peroxide and tert-butyl perbenzoate, which are not specifically limited here. The mass of the initiator can be 0.5%-5% of Z, for example: 0.5%, 1%, 3%, 5%, etc.
[0058] Specifically, a suspension polymerization method is used to uniformly mix styrene monomers, polyvinyl monomers, N,N'-bis(acryl)cystamine, a porogen and an initiator to obtain an oil phase solution; the oil phase solution is added to an aqueous phase solution to allow the styrene monomers, polyvinyl monomers, N,N'-bis(acryl)cystamine, a porogen and an initiator to undergo a suspension polymerization reaction to obtain a first adsorption resin containing a disulfide bond; the reaction conditions of the polymerization reaction include: a reaction stirring speed of 120-180rpm, a reaction temperature of 70-90°C, and a reaction time of 6-18 hours.
[0059] The oil phase solution is a mixture of styrene monomers, polyvinyl monomers, N,N'-bis(acryl)cystamine, porogen and initiator, and the water phase solution is a water-soluble organic polymer solution, which serves as a dispersion medium.
[0060] For example, the water-soluble organic polymer can be selected from one or more of hydroxymethyl cellulose, hydroxyethyl cellulose, chitosan, polyacrylic acid, sodium polyacrylate and gelatin as a dispersant, which is not specifically limited here. The concentration of the water-soluble organic polymer in the aqueous phase solution can be 0.05%-0.5%wt, for example: 0.05%wt, 0.2%wt, 0.3%wt%, 0.5%wt, etc.
[0061] Exemplarily, it is assumed that the reaction conditions of the polymerization reaction include: a reaction stirring speed of 150 rpm, a reaction temperature of 70°C, and a reaction time of 10 hours. Specifically, the oil phase solution and the aqueous phase solution are mixed, and the reaction is continuously stirred at a speed of 150 rpm for 10 hours at a reaction temperature of 75°C. After the reaction is completed, the mixture generated after the polymerization reaction is washed with clean water to remove excess impurities, and the mixture is continuously subjected to resin extraction treatment with acetone using a Soxhlet extractor for 24 hours, and then dried, and a resin with a particle size between 0.4-0.6 mm is selected as a primary cross-linked resin containing a disulfide bond, i.e., the first adsorption resin.
[0062] S202, using a reducing agent to reduce the disulfide bonds in the first adsorption resin to thiol groups, to obtain a second adsorption resin containing thiol groups.
[0063] Specifically, the reducing solution is mixed with the first adsorption resin to perform a reduction reaction, the reducing solution is prepared from a reducing agent and a buffer solution, and the reaction conditions of the reduction reaction include: a reaction temperature of 25-60° C. and a reaction time of 8-48 hours.
[0064] The volume ratio of the reducing solution to the first adsorption resin is 1:1 to 3:1.
[0065] In the synthesis process of polystyrene resin, a cross-linking agent with a disulfide bond is added in the form of a reactive monomer, and then the disulfide bond is reduced to a thiol group, so that a thiol group can be introduced on the skeleton surface of the polystyrene resin, providing a thiol group and other reactive groups for subsequent grafting. The thiol group introduced in this way has good stability, and by controlling the amount of the cross-linking agent with a disulfide bond added, the thiol content of the resin can be effectively adjusted. Compared with the method of grafting thiol groups in the form of ligands, the present application can effectively avoid the problems of unstable grafting and uncontrollable grafting amount.
[0066] Exemplarily, the buffer solution and the reducing agent are mixed evenly to obtain a reducing solution, the pH value of the buffer solution is 7-8, and the concentration of the reducing solution is 1-10%wt.
[0067] The buffer solution may be a phosphate buffered saline solution (PBS buffer solution), and the concentration of the reducing solution may be 1-10% wt, for example, 1% wt, 2.5% wt, 10% wt, and the like.
[0068] Exemplarily, the buffer solution can be prepared from materials such as potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride in different proportions, and the pH value of the buffer solution is controlled to be between 7 and 8.
[0069] Exemplarily, the reducing agent may be one or more of dithiothreitol, 2-mercaptoethanol and tris(2-formylethyl)phosphine hydrochloride, which is not specifically limited herein.
[0070] For example, it is assumed that the reaction conditions of the reduction reaction include: a reaction temperature of 40°C and a reaction time of 30 hours. Under a reaction temperature of 40°C, the volume ratio of the reducing solution to the first adsorbent resin is controlled to be 2:1, and the first adsorbent resin is dissolved in the reducing solution and reacted for 30 hours. After the reaction is completed, the excess buffer and reducing agent are washed with clean water to avoid the residual buffer and reducing agent from affecting the subsequent grafting, thereby obtaining a thiol-containing primary cross-linked resin, i.e., the second adsorbent resin.
[0071] It should be noted that it is generally necessary to control the use of excessive reducing solution to reduce all disulfide bonds on the first adsorption resin to thiol groups.
[0072] S203, after swelling the second adsorption resin, in the presence of an initiator, utilizing the carbon-carbon double bonds of the organic acid monomers to react with the thiol groups in the second adsorption resin to produce a thiol-ene click reaction, thereby preparing a low-density lipoprotein adsorbent.
[0073] Among them, low-density lipoprotein adsorbent can effectively adsorb low-density lipoprotein.
[0074] Specifically, the second adsorption resin is swelled with an alcohol solvent, and the swollen second adsorption resin is mixed with an organic acid monomer and a thermal initiator, so that the carbon-carbon double bond of the organic acid monomer undergoes a thiol-ene click reaction with the thiol group in the second adsorption resin to prepare a low-density lipoprotein adsorbent.
[0075] The volume ratio of the alcohol solvent to the second adsorption resin is 1:1 to 2:1, and the reaction conditions of the thiol-ene click reaction include: a reaction temperature of 40-80° C. and a reaction time of 6-36 hours.
[0076] Taking into account the swelling effect of the second adsorption resin, since the polarity of the thiol group is similar to that of the alcohol solvent, the thiol group can be better expanded in the alcohol solvent, which is beneficial to the subsequent reaction. In order to make the second adsorption resin swell better, it is preferred to swell the second adsorption resin with an alcohol solvent.
[0077] For example, the alcohol solvent may be one or more of methanol, ethanol, isopropanol and n-butanol, which are not specifically limited herein. The volume ratio of the alcohol solvent to the second adsorption resin may be controlled between 1:1 and 2:1, which is conducive to full swelling.
[0078] For example, the thermal initiator can be selected from one or more of di-tert-butyl peroxide, azobisisoheptonitrile and diisopropyl peroxydicarbonate, which are not specifically limited herein. The mass of the thermal initiator accounts for 0.5-5% of the total mass of the second adsorption resin, for example, 0.5%, 3%, 5%, etc.
[0079] Specifically, the organic acid-based monomers may include carboxylic acid-based monomers containing a carbon-carbon double bond and sulfonic acid-based monomers containing a carbon-carbon double bond.
[0080] For example, the carboxylic acid monomer containing a carbon-carbon double bond can be selected from one or more of acrylic acid and methacrylic acid, and the sulfonic acid monomer containing a carbon-carbon double bond can be selected from one or more of 2-acrylamide-2-methylpropane sulfonic acid and propylene sulfonic acid.
[0081] Exemplarily, it is assumed that the reaction conditions of the thiol-ene click reaction include: a reaction temperature of 70°C and a reaction time of 30 hours. The volume ratio of the alcohol solvent to the second adsorbent resin is controlled to be 2:1, and the second adsorbent resin is added to the alcohol solvent to swell the second adsorbent resin. After the second adsorbent resin is swollen, the reaction temperature is controlled to be 70°C, and the swollen second adsorbent resin is mixed with a thermal initiator and a carboxylic acid monomer or a sulfonic acid monomer containing a carbon-carbon double bond, and the thiol-ene click reaction is carried out for 30 hours. After the reaction is completed, the anionic group of the organic acid monomer can be grafted onto the thiol group in the second adsorbent resin. Finally, the reaction product is washed with water several times and dried to obtain a low-density lipoprotein adsorbent. Since there are grafted anionic groups in the prepared low-density lipoprotein adsorbent, these anionic groups can effectively adsorb the positively charged amino acids on the ApoB domain on the surface of low-density lipoprotein, thereby achieving effective adsorption of low-density lipoprotein.
[0082] The low-density lipoprotein adsorbent was prepared by the above preparation method. Performance tests showed that the specific surface area of the low-density lipoprotein adsorbent was 400-600m 2 / g; pore volume is 1-1.8cm 3 / g; the average pore size is between 12-25nm. Compared with the low-density lipoprotein adsorbent provided by the related technology, the adsorption rate and adsorption speed of low-density lipoprotein are significantly improved.
[0083] The preparation method and performance of the low-density lipoprotein adsorbent of the present application are described below through specific examples.
[0084] Embodiment 1:
[0085] (1) Add 400 mL of 0.2% wt gelatin aqueous solution to a 1000 mL three-necked flask as an aqueous solution. Add reaction monomers, porogens and initiators to a beaker, stir evenly, and prepare an oil phase solution, wherein the reaction monomers include 15 g of styrene, 6 g of ethyl styrene, 24 g of divinyl benzene and 40 g of N, N'-bis (acryl) cystamine; the porogen includes 60 g of toluene and 60 g of methyl isobutyl carbinol; and the initiator includes 1 g of benzoyl peroxide. Add the oil phase solution to the aqueous solution, and stir the reaction at a reaction temperature of 75°C and a speed of 140 rpm for 8 hours. After the reaction is completed, wash the resin with water to remove excess impurities, and then use a Soxhlet extractor to extract the resin mixture with acetone for 24 hours, and then dry and sieve to select a resin with a particle size between 0.4 and 0.6 mm, thereby obtaining a primary cross-linked resin containing disulfide bonds, i.e., the first adsorption resin.
[0086] (2) Take 100 mL of the first adsorption resin obtained in step (1) in a three-necked flask; prepare 200 mL of PBS buffer solution with a pH of 7.4, add 5 g of dithiothreitol, add it to the three-necked flask after it is completely dissolved, and react at 40° C. for 24 hours. After the reaction is completed, remove the remaining dithiothreitol and PBS buffer solution with a large amount of water to obtain a thiol-containing primary cross-linked resin, i.e., the second adsorption resin, wherein the thiol content in the second adsorption resin is 0.5 mmol / mL.
[0087] (3) Take 30 g of the second adsorption resin obtained in step (2) in another three-necked flask, add 200 mL of ethanol, stir and fully swell, then add 10 g of methacrylic acid and 0.3 g of di-tert-butyl peroxide, and react at 80° C. for 18 hours. After the reaction is completed, the reaction product is washed with water several times and dried to obtain a low-density lipoprotein adsorbent.
[0088] Embodiment 2:
[0089] (1) Add 400 mL of 0.08% wt hydroxyethyl cellulose aqueous solution to a 1000 mL three-necked flask as an aqueous solution. Add reaction monomers, porogens and initiators to a beaker, stir evenly, and prepare an oil phase solution, wherein the reaction monomers include 18 g of styrene, 24 g of divinylbenzene, 16 g of ethyl styrene and 32 g of N, N'-bis(acryl)cystamine; the porogen includes 30 g of white oil, 45 g of toluene and 35 g of isooctyl alcohol; the initiator includes 1.5 g of azobisisobutyronitrile. Add the oil phase solution to the aqueous solution, and stir the reaction at a reaction temperature of 80°C and a rotation speed of 160 rpm for 12 hours. After the reaction is completed, the resin is washed with water to remove excess impurities, and then the resin mixture is extracted with acetone using a Soxhlet extractor for 24 hours, and then dried and sieved, and the resin with a particle size between 0.4-0.6 mm is selected to obtain a primary cross-linked resin containing disulfide bonds, namely the first adsorption resin.
[0090] (2) Take 150 mL of the first adsorption resin obtained in step (1) in a three-necked flask; prepare 250 mL of PBS buffer with a pH of 7.4, add 6.3 g of dithiothreitol, add it to the three-necked flask after it is completely dissolved, and react at 35° C. for 36 hours. After the reaction is completed, remove the remaining dithiothreitol and PBS buffer with a large amount of water to obtain a thiol-containing primary cross-linked resin, i.e., the second adsorption resin, wherein the thiol content in the second adsorption resin is 0.37 mmol / mL.
[0091] (3) Take 50 g of the second adsorption resin obtained in step (2) in another three-necked flask, add 280 mL of methanol, stir and fully swell, then add 15 g of 2-acrylamide-2-methylpropane sulfonic acid and 0.4 g of di-tert-butyl peroxide, and react at 75° C. for 12 hours. After the reaction is completed, the reaction product is washed with water several times and dried to obtain a low-density lipoprotein adsorbent.
[0092] Embodiment 3:
[0093] (1) Add 400 mL of polyacrylic acid aqueous solution with a concentration of 0.2% wt to a 1000 mL three-necked flask as an aqueous solution. Add reaction monomers, porogens and initiators to a beaker, stir evenly, and prepare an oil phase solution, wherein the reaction monomers include 30 g of methyl styrene, 16 g of divinylbenzene, 4 g of ethyl styrene and 48 g of N, N'-bis (acryl) cystamine; the porogen includes 30 g of gasoline, 45 g of xylene and 35 g of n-heptanol; the initiator includes 1.5 g of azobisisobutyronitrile. Add the oil phase solution to the aqueous solution, and stir the reaction at a reaction temperature of 85°C and a rotation speed of 120 rpm for 8 hours. After the reaction is completed, the resin is washed with water to remove excess impurities, and then the resin mixture is extracted with acetone using a Soxhlet extractor for 24 hours, and then dried and sieved, and the resin with a particle size between 0.4-0.6 mm is selected to obtain a primary cross-linked resin containing disulfide bonds, namely the first adsorption resin.
[0094] (2) 180 mL of the first adsorption resin obtained in step (1) was placed in a three-necked flask; 300 mL of PBS buffer solution with a pH of 7.4 was prepared, and 5.5 g of dithiothreitol was added, and after it was completely dissolved, it was added to the three-necked flask, and reacted at 50° C. for 48 hours. After the reaction was completed, the remaining dithiothreitol and PBS buffer solution were removed with a large amount of water to obtain a thiol-containing primary cross-linked resin, i.e., the second adsorption resin, wherein the thiol content in the second adsorption resin was 0.43 mmol / mL.
[0095] (3) Take 35 g of the second adsorption resin obtained in step (2) in another three-necked flask, add 200 mL of isopropanol, stir and fully swell, then add 16 g of propylene sulfonic acid and 0.24 g of azobisisoheptanenitrile, and react at 60° C. for 8 hours. After the reaction is completed, dry and obtain a low-density lipoprotein adsorbent.
[0096] It should be noted that the resin of Comparative Example 1 is the second cross-linked resin obtained in step (2) of Example 1, the resin of Comparative Example 2 is the second cross-linked resin obtained in step (2) of Example 2, and the resin of Comparative Example 3 is the second cross-linked resin obtained in step (2) of Example 3.
[0097] The performance test was performed using the low-density lipoprotein adsorbent provided in the above embodiment and the second cross-linked resin provided in the comparative example. Specifically, 1 mL of the low-density lipoprotein adsorbent provided in the above embodiment and 1 mL of the second cross-linked resin provided in the comparative example were added to 10 mL of high-fat plasma with a concentration of 28 mmol / mL, and the mixture was shaken at 37°C. The plasma adsorbed by the low-density lipoprotein adsorbent was taken after 0.5 hour, 1 hour, 1.5 hours and 2 hours, respectively, to detect the concentration of low-density lipoprotein in the plasma. The adsorption results of the low-density lipoprotein adsorbent provided in the above embodiment and the second cross-linked resin provided in the comparative example on blood lipids are shown in Table 1 below.
[0098] Table 1
[0099]
[0100] The test results in Table 1 show that by comparing the adsorption rates of low-density lipoprotein in each embodiment and each comparative example, it can be seen that the low-density lipoprotein adsorbent obtained after grafting of the present invention has better adsorption performance for low-density lipoprotein, which indicates that grafting a specific ligand to the second cross-linked resin has the beneficial effect of improving the adsorption rate of low-density lipoprotein. At the same time, due to the excellent performance parameters (large specific surface area, pore volume, average pore size) of the low-density lipoprotein adsorbent, the low-density lipoprotein adsorbent prepared by the present invention has improved adsorption rate and adsorption rate compared with the existing low-density lipoprotein adsorbent, thereby being able to effectively and quickly remove low-density lipoprotein in the patient's body.
[0101] The present application also provides a low-density lipoprotein adsorbent, which is prepared by using the preparation method provided in any of the above embodiments.
[0102] The specific method of preparing the low-density lipoprotein adsorbent can refer to the corresponding embodiment recorded in the specification of this application, and this embodiment will not be repeated here. The low-density lipoprotein adsorbent provided in the embodiment of this application can improve the adsorption rate and adsorption speed of the low-density lipoprotein adsorbent, thereby effectively and quickly removing low-density lipoprotein in the patient's body.
[0103] Specifically, the specific surface area of the low-density lipoprotein adsorbent provided in the embodiment of the present application is 400-600m 2 / g; pore volume is 1-1.8cm 3 / g; the average pore size is between 12-25nm. Compared with the low-density lipoprotein adsorbent provided by the related technology, the adsorption rate and adsorption speed of the low-density lipoprotein adsorbent are improved, thereby being able to effectively and quickly remove the low-density lipoprotein in the patient's body.
[0104] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.
Claims
1. A method for preparing a low-density lipoprotein adsorbent, characterized in that: include: During the synthesis of the polystyrene resin, a cross-linking agent having a disulfide bond is added to react to obtain a first adsorption resin containing a disulfide bond; Using a reducing agent to reduce the disulfide bonds in the first adsorption resin to thiol groups, thereby obtaining a second adsorption resin containing thiol groups; The organic acid monomer containing carbon-carbon double bonds is grafted onto the second adsorption resin through a thiol-ene click reaction to prepare a low-density lipoprotein adsorbent.
2. The preparation method according to claim 1, characterized in that: The organic acid monomers are carboxylic acid monomers and / or sulfonic acid monomers containing carbon-carbon double bonds.
3. The preparation method according to claim 1, characterized in that: The cross-linking agent is N,N'-bis(acryl)cystamine.
4. The preparation method according to claim 1, characterized in that: The reducing agent is selected from one or more of dithiothreitol, 2-mercaptoethanol, and tris(2-formylethyl)phosphine hydrochloride.
5. The preparation method according to claim 1, characterized in that: The thiol content of the second adsorption resin is 0.05-0.8 mmol / mL.
6. The preparation method according to any one of claims 1 to 5, characterized in that: The steps include: The first adsorption resin containing disulfide bonds is prepared by polymerizing styrene monomers, polyvinyl monomers, N,N'-bis(acryl)cystamine, porogens and initiators; Using a reducing agent to reduce the disulfide bonds in the first adsorption resin to thiol groups, thereby obtaining a second adsorption resin containing thiol groups; After the second adsorption resin is swollen, in the presence of an initiator, a thiol-ene click reaction is carried out between the carbon-carbon double bond of the organic acid monomer and the thiol group in the second adsorption resin to prepare a low-density lipoprotein adsorbent.
7. The preparation method according to claim 6, characterized in that: The polymerization reaction is a suspension polymerization reaction, and the reaction conditions of the polymerization reaction include: a reaction stirring speed of 120-180 rpm, a reaction temperature of 70-90° C., and a reaction time of 6-18 hours.
8. The preparation method according to claim 6, characterized in that: The method of reducing the disulfide bonds in the first adsorption resin to thiol groups by using a reducing agent to obtain a second adsorption resin containing thiol groups comprises: The reducing solution is mixed with the first adsorption resin to perform a reducing reaction, wherein the reducing solution is prepared from a reducing agent and a buffer solution, and the reaction conditions of the reducing reaction include: a reaction temperature of 25-60° C. and a reaction time of 8-48 hours.
9. The preparation method according to claim 6, characterized in that: The step of swelling the second adsorption resin comprises: using an alcohol solvent to swell the second adsorption resin; The reaction conditions of the thiol-ene click reaction include: a reaction temperature of 40-80° C. and a reaction time of 6-36 hours. 10 . A low-density lipoprotein adsorbent, wherein the low-density lipoprotein adsorbent is prepared by the preparation method according to claim 1 .