Endotoxin adsorption material with sphingomyelin-like structure, preparation method of endotoxin adsorption material and application of endotoxin adsorption material in sepsis blood purification

Abstract

The invention discloses an endotoxin adsorption material with a sphingomyelin-like structure, a preparation method of the endotoxin adsorption material and application of the endotoxin adsorption material in sepsis blood purification. The endotoxin adsorption material with the sphingomyelin-like structure has chemical structures of polyethylene polyamine and phosphocholine; the adsorbent has good affinity to endotoxin, also has excellent blood compatibility, and can resist adhesion of blood cells and plasma proteins, so that the endotoxin can be effectively removed in a whole blood environment; the sphingomyelin-like structure ligand can be grafted on the surfaces of various substrates through covalent bonds, and has stability and universality; the endotoxin adsorbent provided by the invention has both endotoxin adsorbability and blood compatibility, is simple to prepare and low in cost, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of blood purification, and in particular to a sphingomyelin-like endotoxin adsorption material, a preparation method thereof and an application thereof in sepsis blood purification. Background Art

[0002] Sepsis is a complex disease caused by the body's dysregulated immune response to microbial (such as bacteria, viruses, etc.) infection, which leads to organ dysfunction and even death. It is a common complication in critically ill patients. According to statistics, as of 2019, approximately 13.9 million people died from sepsis-related infectious syndromes worldwide.

[0003] Endotoxins released by the proliferation and death of Gram-negative bacteria are the main cause of sepsis. After being recognized by the host's Toll-like receptors, endotoxins will induce the release of downstream cytokines (such as interleukin-1β, interleukin-18, etc.), activating the immune response. Unlike the normal immune process, the release of a large amount of endotoxins will make it difficult for the host to restore normal immune balance. The excessive immune response caused by endotoxins can lead to adverse reactions such as activation of the complement system and coagulation system and cytokine storm, which can cause patients to faint, shock, multiple organ failure, and even death. Therefore, rapid removal of endotoxins from the blood is the key to treating sepsis.

[0004] Adsorbing endotoxins from the blood through blood purification is an important means of treating sepsis. Endotoxins contain hydrophilic negatively charged phosphate groups and hydrophobic alkyl long chains. Based on their structural characteristics, the endotoxin adsorbent materials currently developed are mostly designed based on cationic or hydrophobic groups. However, this structural design often brings about blood safety issues: cationic groups carry a large amount of positive charge, which may cause blood cell aggregation, platelet activation, and induce coagulation cascade reactions; hydrophobic groups are easy to adsorb proteins, resulting in the formation of microthrombi on the surface of the adsorbent, and may cause protein loss. Therefore, in the development of endotoxin adsorbent materials for blood purification, how to ensure that the adsorbent has both excellent blood compatibility and good adsorption performance has always been a difficult problem that needs to be solved urgently.

[0005] Currently, many types of endotoxin adsorption materials have been reported.

[0006] For example, the Japanese Toray Company produces The adsorption column uses polymyxin B (PMB) as an endotoxin affinity ligand. However, PMB has a limited adsorption capacity for endotoxin and a low adsorption rate. It is easily contaminated by blood cells in a whole blood environment, which reduces the adsorption effect. This seriously affects its effect in sepsis blood purification treatment. In addition, PMB is also nephrotoxic and may cause serious adverse effects on patients once it falls off.

[0007] Another commercial endotoxin adsorbent material is produced by Baxter International, USA The filter has a three-layer structure of modified AN69 membrane, polyethyleneimine coating and heparin coating. The polyethyleneimine layer adsorbs endotoxins through electrostatic action, and the heparin coating improves the blood compatibility of the filter and prevents the risk of coagulation during treatment. However, The multi-layer design of the filter increases the production process. In addition, the polyethyleneimine layer and the heparin layer are combined in the form of a coating, which also poses a potential risk of falling off and entering the circulatory system. Summary of the invention

[0008] One of the purposes of the present invention is to provide a sphingomyelin-like endotoxin adsorption material with good blood compatibility to solve the above-mentioned problems.

[0009] In order to solve the above problems, the technical solution adopted is a sphingomyelin-like endotoxin adsorbent material, wherein the adsorbent has a chemical structure of polyethylene polyamine and phosphorylcholine, and its chemical structure is:

[0010]

[0011] Wherein, 0 is a substrate, the substrate is a polymer having a cyano chemical structure, or is polyvinyl alcohol, polymethyl methacrylate, polyurethane, polyvinylidene chloride, cellulose and its derivatives, and n ranges from 1 to 5.

[0012] In the process of endotoxin binding to the cell membrane, due to the molecular charge characteristics of endotoxin, it will preferentially bind to the sphingomyelin site rich in phosphorylcholine in the phospholipid layer of the cell membrane; however, sphingomyelin cannot be materialized and has never been used to manufacture endotoxin adsorbents. The present application proposes an endotoxin adsorption ligand with a sphingomyelin-like structure. The present invention uses ethyleneamine polymers as connecting arms, which can enhance the hydrophilicity of the adsorption ligand and improve blood compatibility on the one hand; on the other hand, it can better capture endotoxins through hydrogen bonding. The phosphorylcholine functional group modified on the outside of the ethyleneamine polymer chain has a good affinity for endotoxins on the one hand, and on the other hand, it avoids adhesion to blood cells and plasma proteins through the hydration layer, thereby effectively reducing the damage to blood cells, while avoiding the interference of nonspecific protein adsorption on endotoxin removal, and improving the adsorbent's endotoxin removal performance in the complex environment of whole blood.

[0013] Regarding the value of n, if the value of n is too large, the grafting reaction will be unfavorable due to the large steric hindrance of the ethyleneamine polymer, but if the value of n is too small, cross-linking will easily occur during the grafting process. Therefore, as a preferred technical solution, the value of n is in the range of 1-3, and more preferably 2.

[0014] As a further preferred technical solution, the substrate is selected from one of polyacrylonitrile, acrylonitrile-styrene copolymer, acrylonitrile-butadiene-styrene copolymer, cyano-modified styrene-divinylbenzene copolymer; or a blended composite of the above polymers with polyethersulfone, polysulfone, polyamide, or polyester.

[0015] As a further preferred technical solution, the substrate is a polyacrylonitrile / polyethersulfone blended composite.

[0016] As a preferred technical solution, the substrate morphology is selected from one of random particles, microspheres, flat membranes, hollow fiber membranes, nanofiber membranes or nanoparticles.

[0017] A second object of the present invention is to provide a method for preparing the above-mentioned sphingomyelin-like endotoxin adsorbing material, which comprises:

[0018] (1) Soaking the substrate in an appropriate amount of deionized water, adding ethyleneamine polymer, preferably in an amount of 1 to 1.5 times the molar amount of cyano groups on the substrate surface; then reacting at 50 to 70° C. for 10 to 14 hours; preferably, washing the substrate with anhydrous ethanol and deionized water alternately 5 times after the reaction; and fully drying the obtained substrate-polyethylene polyamine and storing it in a dry environment;

[0019] (2) soaking the substrate obtained in step (1) - polyethylene polyamine in an appropriate amount of anhydrous organic solvent, cooling to -25 to -15°C; adding 2-chloro-1,3,2-dioxaphospholane 2-oxide thereto, preferably wherein the amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide added is 1.5 to 2 times the molar amount of amino groups on the surface of the substrate - polyethylene polyamine; reacting at -25 to -15°C for 3 to 5 hours; and then continuing the reaction at room temperature for 1 to 2 hours;

[0020] (3) Soak the product obtained in step (2) in an anhydrous organic solvent, add trimethylamine, preferably in an amount of 1.5 to 2 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide, react at 50 to 70° C. for 22 to 28 hours; after the reaction, wash the substrate 5 times with anhydrous ethanol and deionized water alternately to obtain the sphingomyelin-like endotoxin adsorbent material, and store it in sterile saline.

[0021] The third object of the present invention is to provide an application of the above-mentioned sphingomyelin-like endotoxin adsorption material in the treatment of sepsis.

[0022] In the process of applying endotoxin adsorbent materials to treat sepsis, adsorption performance and blood compatibility are equally important. The published patent "An adsorption resin, its preparation method and application" also discloses an endotoxin adsorbent material for the treatment of sepsis, but only verifies the adsorption performance of the material in a plasma environment, and no whole blood adsorption experiment is conducted. Many adsorbents with positive charges or hydrophobic components are difficult to apply to a whole blood environment due to poor blood compatibility and easy contamination by plasma proteins. They can only adopt a strategy of purifying plasma after plasma separation, which will generate additional operating procedures and reduce blood purification efficiency. The endotoxin adsorbent of the present application has been proven to have excellent blood compatibility and anti-blood cell and anti-plasma protein adhesion capabilities, and can exert endotoxin adsorption capabilities in a whole blood environment without the need for plasma separation.

[0023] Another disclosed patent, "An endotoxin adsorbent, its preparation method and application", discloses an endotoxin adsorbent based on betaine zwitterion modification. However, the ligand synthesis step of the adsorbent is relatively complicated, involving multiple separation and purification steps, and the preparation cost is relatively high. The endotoxin adsorbent described in the present invention adopts a strategy of gradual surface modification, does not require additional separation and purification steps, is relatively simple to prepare, and has a low production cost. In terms of adsorption performance, the examples of the disclosed patent "An endotoxin adsorbent, its preparation method and application" have a clearance rate of more than 78% for endotoxins in plasma, and have not been applied to whole blood endotoxin removal. The adsorbent proposed in the present invention can achieve an endotoxin clearance rate of more than 90% in plasma, and can still maintain a clearance rate of more than 74% in a whole blood environment, and has better endotoxin adsorption performance.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] (1) The endotoxin adsorbent of the present invention has a high affinity for endotoxin and excellent blood compatibility, can resist the adhesion of blood cells and plasma proteins, and avoid damage to blood cells, so it can be directly used for whole blood purification of sepsis patients.

[0026] (2) The endotoxin adsorbent described in the present invention is easy to prepare and has a lower cost than adsorbents coupled with affinity ligands such as polymyxin B and polypeptides, which can greatly reduce the economic burden of sepsis patients.

[0027] (3) The endotoxin affinity ligand described in the present invention is connected to the surface of various substrates through covalent bonds, thereby reducing the risk of shedding. Compared with the adsorbent prepared by the coating method, it has better universality and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The data are data on the endotoxin clearance rate in plasma of a commercial immobilized polymyxin B endotoxin adsorbent, the adsorbents obtained in Examples 1 to 3 of the present invention, and Comparative Examples 1 to 3.

[0029] Figure 2 The data are data on the clearance rate of endotoxin in whole blood of a commercial endotoxin adsorbent immobilized with polymyxin B, the adsorbents obtained in Examples 1 to 3 of the present invention, and Comparative Examples 1 to 3.

[0030] Figure 3 The data are the adsorption amount of plasma protein by the commercial endotoxin adsorbent of immobilized polymyxin B, the adsorbent obtained by Examples 1 to 3 of the present invention and Comparative Examples 1 to 3.

[0031] Figure 4 The data are the hemolysis rate data of the commercial immobilized polymyxin B endotoxin adsorbent, the adsorbents obtained in Examples 1 to 3 of the present invention, and Comparative Examples 1 to 3.

[0032] Figure 5 The figures are statistical data of the number of apoptotic cells (HK-2) induced by the extracts of polymyxin B and the adsorbents obtained in Examples 1 to 3 of the present invention. DETAILED DESCRIPTION

[0033] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is described in detail below in conjunction with the accompanying drawings, but it should not be understood as limiting the scope of implementation of the present invention.

[0034] Unless otherwise specified, the reagents, materials and equipment used in the present invention are commercially available.

[0035] The preparation process of a sphingomyelin-like endotoxin adsorption material provided by the present invention is as follows:

[0036] S1: The cyano groups on the surface of the substrate are reacted with the vinylamine polymer under heating conditions, so that the vinylamine polymer is connected to the substrate by reacting with the cyano groups to obtain the substrate-polyethylene polyamine.

[0037] S2: Immerse the substrate-polyethylene polyamine in anhydrous organic solvent A, add 2-chloro-1,3,2-dioxaphospholane 2-oxide to react at -25 to -15°C, and then heat to room temperature to continue the reaction.

[0038] S3: Immerse the above product in anhydrous organic solvent B, add trimethylamine and react at 50-70° C. After the obtained product is fully washed, the sphingomyelin-like endotoxin adsorption material is obtained.

[0039] In some specific embodiments, the substrate is generally any material of any shape containing cyano groups on the surface. In the present invention, the carrier is preferably polyacrylonitrile, acrylonitrile-styrene copolymer, acrylonitrile-butadiene-styrene copolymer, cyano-modified styrene-divinylbenzene copolymer, and includes a blended composite of the above polymers with polyethersulfone, polysulfone, polyamide, and polyester. More preferably, it is a polyacrylonitrile / polyethersulfone blended composite. In addition, other common materials used in blood perfusion can also be selected as the substrate of the adsorption ligand in the present invention, such as: polyvinyl alcohol, polymethyl methacrylate, polyurethane, polyvinylidene chloride, cellulose and its derivatives, etc., as long as it can be used in a blood contact environment and the adsorption ligand of the present invention can be introduced in a suitable manner.

[0040] In some specific embodiments, the ethyleneamine polymer can be one or more of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, preferably one or more of ethylenediamine, diethylenetriamine and triethylenetetramine, more preferably diethylenetriamine.

[0041] In some specific embodiments, the organic solvent A is preferably tetrahydrofuran, dioxane and acetone, more preferably tetrahydrofuran. The organic solvent B is preferably tetrahydrofuran, acetonitrile and ethyl acetate, more preferably acetonitrile.

[0042] In some specific embodiments, the addition of an acid binding agent to neutralize the generated hydrogen chloride during the reaction with 2-chloro-1,3,2-dioxaphospholane 2-oxide in step S2 is conducive to the forward reaction and can increase the grafting rate of phosphorylcholine functional groups. The type and amount of the acid binding agent can be determined by a person skilled in the art based on relevant chemical knowledge, and pyridine or triethylamine is generally selected, and the added amount is 1 to 1.2 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide.

[0043] It should be noted that the connection between the substrate, the ethyleneamine polymer and the phosphorylcholine in the present invention may not be carried out in the manner described above of "the substrate first reacts with the ethyleneamine polymer to form the substrate-polyethylene polyamine, and then reacts with 2-chloro-1,3,2-dioxaphospholane 2-oxide and trimethylamine in sequence to form the phosphorylcholine group", but may also be connected in the following manner:

[0044] (1) Ethyleneamine polymer first reacts with 2-chloro-1,3,2-dioxaphospholane 2-oxide, and then reacts with trimethylamine to generate phosphorylcholine structure. Finally, the synthesized ligand is grafted onto the surface of the substrate.

[0045] (2) The ethyleneamine polymer first reacts with 2-chloro-1,3,2-dioxaphospholane 2-oxide, is then grafted onto the substrate surface, and then reacts with trimethylamine to form a phosphorylcholine structure.

[0046] The above methods are all equivalent to the methods claimed in the present invention.

[0047] It should be noted that "substrate" can be understood as the raw material used to prepare endotoxin adsorbent; "substrate-polyethylene polyamine" can be understood as the product of the reaction between the substrate and the ethyleneamine polymer; "adsorption ligand" can be understood as the part after the ethyleneamine polymer is connected to the phosphorylcholine structure.

[0048] Example 1

[0049] This embodiment provides an endotoxin adsorbent, which is prepared according to the following method:

[0050] S1: polyacrylonitrile, polyethersulfone and N,N-dimethylformamide are mixed in a mass ratio of 8:8:84, and mechanically stirred to a transparent homogeneous solution; a polyacrylonitrile / polyethersulfone composite precursor solution is obtained, wherein the solid content accounts for 16%, and the mass ratio of polyacrylonitrile to polyethersulfone is 1:1;

[0051] S2: The precursor solution is dropped into a 20% (v / v) alcohol solution by a syringe to solidify into microspheres by phase inversion, and the prepared microspheres are alternately rinsed with anhydrous ethanol and deionized water for 5 times, and vacuum dried; the prepared microspheres have a diameter of 800-1000 μm;

[0052] S3: The prepared microspheres were used as substrates, immersed in deionized water, and diethylenetriamine (the amount was 1.3 times the molar amount of cyano groups of the substrate) was added, and the reaction was carried out at 60°C for 14 hours; after the reaction, the substrates were washed alternately with anhydrous ethanol and deionized water for 5 times, and vacuum dried for 12 hours;

[0053] S4: Determine the number of amino groups on the surface of the substrate-polyethylene polyamine by titration; soak the above product in anhydrous tetrahydrofuran, cool to -25°C, add 2-chloro-1,3,2-dioxaphospholane 2-oxide (the amount is 1.2 times the molar amount of surface amino groups) and triethylamine (the amount is 1.1 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide), react at -25°C for 5 hours and then at room temperature for 1 hour;

[0054] S5: The above product is washed with anhydrous acetonitrile, then immersed in an anhydrous acetonitrile solution, trimethylamine (the amount is 1.2 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide), and reacted at 70°C for 24 hours; after the reaction, it is washed alternately with anhydrous ethanol and deionized water for 5 times, and stored in sterile physiological saline to obtain the endotoxin adsorbent provided in Example 1.

[0055] Example 2

[0056] This embodiment provides an endotoxin adsorbent, which is prepared according to the following method:

[0057] S1: polyacrylonitrile, polyethersulfone and N,N-dimethylformamide are mixed in a mass ratio of 5:10:85, and mechanically stirred to a transparent homogeneous solution; a polyacrylonitrile / polyethersulfone composite precursor solution is obtained, wherein the solid content accounts for 15%, and the mass ratio of polyacrylonitrile to polyethersulfone is 1:2;

[0058] S2: The precursor solution is dropped into a 20% (v / v) ethanol solution by a syringe to solidify into microspheres by phase inversion, and the prepared microspheres are alternately rinsed with anhydrous ethanol and deionized water for 5 times, and vacuum dried; the prepared microspheres have a diameter of 800-1000 μm;

[0059] S3: The prepared microspheres were used as substrates, immersed in deionized water, and diethylenetriamine (the amount was 1.3 times the molar amount of cyano groups of the substrate) was added, and the reaction was carried out at 60°C for 14 hours; after the reaction, the substrates were washed alternately with anhydrous ethanol and deionized water for 5 times, and vacuum dried for 12 hours;

[0060] S4: Determine the number of amino groups on the surface of the substrate-polyethylene polyamine by titration. Soak the above product in anhydrous tetrahydrofuran, cool to -25°C, add 2-chloro-1,3,2-dioxaphospholane 2-oxide (the amount is 1.1 times the molar amount of surface amino groups) and triethylamine (the amount is 1.05 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide), react at -25°C for 5 hours and then at room temperature for 1 hour;

[0061] S5: The above product was washed with anhydrous acetonitrile, then immersed in an anhydrous acetonitrile solution, trimethylamine (the amount was 1.1 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide) was added, and the reaction was carried out at 70° C. for 24 hours. After the reaction, it was washed alternately with anhydrous ethanol and deionized water for 5 times, and stored in sterile physiological saline to obtain the endotoxin adsorbent provided in Example 2.

[0062] Example 3

[0063] This embodiment provides an endotoxin adsorbent, which is prepared according to the following method:

[0064] S1: Mix polyacrylonitrile, polyethersulfone and N,N-dimethylformamide in a mass ratio of 6:9:85, and mechanically stir until a transparent homogeneous solution is obtained. A polyacrylonitrile / polyethersulfone composite precursor solution is obtained, in which the solid content accounts for 15%, and the mass ratio of polyacrylonitrile to polyethersulfone is 2:3;

[0065] S2: The precursor solution is dropped into a 20% (v / v) ethanol solution by a syringe to solidify into microspheres by phase inversion, and the prepared microspheres are alternately rinsed with anhydrous ethanol and deionized water for 5 times, and vacuum dried; the prepared microspheres have a diameter of 800-1000 μm;

[0066] S3: The prepared microspheres were used as substrates, soaked in deionized water, and diethylenetriamine (the amount was 1.2 times the molar amount of cyano groups in the substrate) was added, and the reaction was carried out at 60°C for 12 hours. After the reaction, the substrates were washed alternately with anhydrous ethanol and deionized water for 5 times, and vacuum dried for 14 hours;

[0067] S4: Determine the number of amino groups on the surface of the substrate-polyethylene polyamine by titration. Soak the above product in anhydrous tetrahydrofuran, cool to -25°C, add 2-chloro-1,3,2-dioxaphospholane 2-oxide (the amount is 1.15 times the molar amount of surface amino groups) and triethylamine (the amount is 1.15 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide), react at -20°C for 5 hours and then at room temperature for 2 hours;

[0068] S5: The above product was washed with anhydrous acetonitrile, then immersed in an anhydrous acetonitrile solution, trimethylamine (the amount was 1.2 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide) was added, and the reaction was carried out at 80° C. for 22 hours. After the reaction, it was washed alternately with anhydrous ethanol and deionized water for 5 times, and stored in sterile physiological saline to obtain the endotoxin adsorbent provided in Example 3.

[0069] Comparative Example 1

[0070] This comparative example provides an adsorbent. Compared with Example 1, the adsorbent only has polyethylene polyamine chains grafted onto the surface of the substrate, and no phosphorylcholine groups are grafted. The adsorbent is prepared according to the following method:

[0071] S1: Mix polyacrylonitrile, polyethersulfone and N,N-dimethylformamide in the mass proportion of 8:8:84, and mechanically stir until a transparent homogeneous solution is obtained. A polyacrylonitrile / polyethersulfone composite precursor solution is obtained, in which the solid content accounts for 16%, and the mass ratio of polyacrylonitrile to polyethersulfone is 1:1;

[0072] S2: The precursor solution is dropped into a 20% (v / v) ethanol solution by a syringe to solidify into microspheres by phase inversion, and the prepared microspheres are alternately rinsed with anhydrous ethanol and deionized water for 5 times, and vacuum dried; the prepared microspheres have a diameter of 800-1000 μm;

[0073] S3: The prepared microspheres were used as substrates, soaked in deionized water, and diethylenetriamine (the amount was 1.3 times the molar amount of cyano groups of the substrate) was added, and reacted at 60°C for 14 hours. After the reaction, the substrates were washed alternately with anhydrous ethanol and deionized water for 5 times. The substrates were stored in sterile saline to obtain the endotoxin adsorbent provided in Comparative Example 1.

[0074] Comparative Example 2

[0075] This comparative example provides an adsorbent. Compared with Example 2, the adsorbent only has polyethylene polyamine chains grafted onto the surface of the substrate, and no phosphorylcholine groups are grafted. The adsorbent is prepared according to the following method:

[0076] S1: polyacrylonitrile, polyethersulfone and N,N-dimethylformamide are mixed in a mass ratio of 5:10:85, and mechanically stirred to a transparent homogeneous solution; a polyacrylonitrile / polyethersulfone composite precursor solution is obtained, wherein the solid content accounts for 15%, and the mass ratio of polyacrylonitrile to polyethersulfone is 1:2;

[0077] S2: The above precursor solution was dripped into 20% (v / v) ethanol solution by syringe, and solidified into microspheres by phase inversion. The prepared microspheres were alternately rinsed with anhydrous ethanol and deionized water for 5 times, and vacuum dried. The diameter of the prepared microspheres was 800-1000μm;

[0078] S3: The prepared microspheres were used as substrates, soaked in deionized water, and diethylenetriamine (the amount was 1.3 times the molar amount of cyano groups of the substrate) was added, and reacted at 60°C for 14 hours; after the reaction, the substrates were washed alternately with anhydrous ethanol and deionized water for 5 times. The substrates were stored in sterile saline to obtain the endotoxin adsorbent provided in Comparative Example 2.

[0079] Comparative Example 3

[0080] This comparative example provides an adsorbent. Compared with Example 3, the adsorbent only has polyethylene polyamine chains grafted onto the surface of the substrate, and no phosphorylcholine groups are grafted. The adsorbent is prepared according to the following method:

[0081] S1: polyacrylonitrile, polyethersulfone and N,N-dimethylformamide are mixed in a mass ratio of 6:9:85, and mechanically stirred to a transparent homogeneous solution; a polyacrylonitrile / polyethersulfone composite precursor solution is obtained, wherein the solid content accounts for 15%, and the mass ratio of polyacrylonitrile to polyethersulfone is 2:3;

[0082] S2: The above precursor solution was dripped into 20% (v / v) ethanol solution by syringe, and solidified into microspheres by phase inversion. The prepared microspheres were alternately rinsed with anhydrous ethanol and deionized water for 5 times, and vacuum dried. The diameter of the prepared microspheres was 800-1000μm;

[0083] S3: Use the prepared microspheres as the substrate, soak them in deionized water, add diethylenetriamine (the amount is 1.2 times the molar amount of cyanide of the substrate), and react at 60°C for 12 hours; after the reaction, wash the substrate alternately with anhydrous ethanol and deionized water for 5 times; store in sterile saline to obtain the endotoxin adsorbent provided in Comparative Example 3.

[0084] Test Case

[0085] (1) Elimination of endotoxins in human plasma system

[0086] The endotoxin adsorbents obtained in Examples 1-3 and Comparative Examples 1-3 were used to test the endotoxin removal performance in a human whole blood system, and a commercial endotoxin agent (Detoxi-Gel TM ) As a comparison,

[0087] It should be noted that: the above-mentioned background technology It has not been approved for use in China, and it is not legal to purchase this filter in China. Moreover, the filter is made of fiber braided membrane material, which is different from the material form of the present invention. Therefore, the inventor used another adsorbent Detoxi-Gel for protein purification and adsorption of endotoxin. TM For comparison, although this adsorbent is not used for blood purification, it also has immobilized polymyxin B, so this material is used as a comparison;

[0088] The specific test method is as follows:

[0089] Take 100mg Detoxi-Gel TM The adsorbent, the endotoxin adsorbent obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were added to 1 mL of human plasma with an initial concentration of 3 to 10 EU / mL. The plasma used heparin as an anticoagulant and was obtained by centrifugation of fresh anticoagulated whole blood at 1800 × g. Each adsorbent was incubated with plasma at 37°C for 3 h with shaking. The concentration of endotoxin in the plasma before and after incubation was determined using the Limulus amebocyte lysate method, and the clearance rate of endotoxin in the plasma was calculated as follows:

[0090] R p =(C p-0 -C p-t ) / C p-0 ×100%

[0091] Among them, R p (%) represents the clearance rate of endotoxin in plasma by adsorbent, C p-0 (EU / mL) represents the initial endotoxin concentration in plasma, C p-t (EU / mL) represents the endotoxin concentration in plasma after adsorption.

[0092] The experimental results are as follows Figure 1As shown, Detoxi-Gel TM The clearance rate of the adsorbent was 80.7%. The clearance rates of Examples 1 to 3 were 92.6%, 90.3% and 90.7%, respectively; the clearance rates of Comparative Examples 1 to 3 were 15.0%, 14.5% and 16.7%, respectively, proving that the adsorption effect of the sphingomyelin-like endotoxin adsorbent in plasma is better than that of Detoxi-Gel immobilized with polymyxin B. TM The adsorbent can effectively remove endotoxins in plasma. The removal rate of each embodiment is above 90%, while the removal rate of the adsorbent only connected with polyethylene polyamine is lower, and the removal rate of each comparative example is less than 20%, which illustrates the effectiveness of sphingomyelin-like structure as endotoxin adsorption ligand.

[0093] (2) Elimination of endotoxins in human whole blood system

[0094] Take Detoxi-Gel TM The adsorbent, the endotoxin adsorbent obtained in Examples 1-3 and Comparative Examples 1-3 were tested for endotoxin removal performance in a human whole blood system. The specific test method is as follows:

[0095] Take 100mg Detoxi-Gel TM The adsorbent, the endotoxin adsorbent obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were added to 1 mL of human whole blood with an initial concentration of 3 to 10 EU / mL, and incubated at 37° C. for 3 h. The whole blood before and after incubation was first centrifuged to separate the plasma, and then the endotoxin concentration therein was determined using the Limulus amebocyte lysate method. The clearance rate of endotoxin in whole blood was calculated as follows:

[0096] R b =(C b-0 -C b-t ) / C b-0 ×100%

[0097] Among them, R b (%) represents the clearance rate of endotoxin in whole blood by adsorbent, C b-0 (EU / mL) represents the initial endotoxin concentration in whole blood, C b-t (EU / mL) represents the endotoxin concentration in whole blood after adsorption.

[0098] The experimental results are as follows Figure 2 As shown, Detoxi-Gel TM The clearance rate of the adsorbent in whole blood decreased significantly to only 62.4%. The clearance rates of Examples 1 to 3 were 85.1%, 77.3% and 72.3%, respectively, which were better than Detoxi-Gel immobilized with polymyxin B. TMAdsorbent; the clearance rates of comparative examples 1 to 3 are 12.7%, 12.9% and 15.2% respectively, proving that the sphingomyelin-like endotoxin adsorbent proposed in the present invention can effectively remove endotoxins in whole blood, and the clearance rates of each embodiment are above 74%, while the clearance rate of the adsorbent only connected with polyethylene polyamine is lower, and the clearance rate of each comparative example is less than 20%. The above analysis shows that the sphingomyelin-like endotoxin adsorbent proposed in the present invention has good adsorption performance for endotoxins in blood and can be used for endotoxin removal in human whole blood environment.

[0099] (3) Adsorption degree of plasma proteins

[0100] Take 500mg Detoxi-Gel TM The adsorbent, the endotoxin adsorbent obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were added to 1 mL of human plasma and incubated at 37° C. for 3 h. The incubated adsorbent was separated and washed three times with phosphate buffer (pH=7.2-7.4) and deionized water alternately and lightly buffered to remove the soft-bound plasma proteins on the surface. The adsorbent was then soaked in a 2% sodium dodecyl sulfate solution and vigorously shaken for 2 h to obtain hard-bound plasma proteins on the surface of the adsorbent, which were defined as plasma proteins adsorbed by the adsorbent. The amount of plasma proteins eluted from the adsorbent was measured by BCA colorimetric method.

[0101] The experimental results are as follows Figure 3 As shown, Detoxi-Gel TM The protein adsorption capacity of the adsorbent was 23.2 mg / g; the plasma protein adsorption capacities of Examples 1-3 were 7.5, 8.7 and 7.6 mg / g, respectively; the protein adsorption capacities of Comparative Examples 1-3 were 32.7, 36.3 and 32.2 mg / g, respectively. The plasma protein adsorption capacities of Examples 1-3 were significantly lower than those of Detoxi-Gel immobilized with polymyxin B. TM Adsorbent and Control Examples 1-3. The above analysis shows that the sphingomyelin-like endotoxin adsorbent proposed in the present invention has good anti-plasma protein adsorption performance and can greatly reduce the adverse effects such as protein contamination and protein loss that occur during the blood purification process.

[0102] (4) Hemolysis rate test

[0103] Take 500mg Detoxi-Gel TM The adsorbent, the endotoxin adsorbent obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were added to 1 mL of a human red blood cell suspension diluted to a concentration of 1%, and incubated at 37° C. for 3 h. After centrifugation, the absorbance of the supernatant at a wavelength of 545 nm was tested, and the hemolysis rate was calculated according to the following formula:

[0104] Hemolysis rate (%) = (OD 实验组 -OD 阴性组) / (OD 阳性组 -OD 阴性组 )×100%

[0105] OD 实验组 The absorbance of the supernatant incubated with 1% red blood cell suspension of Examples 1 to 3 and Comparative Examples 1 to 3, OD 阴性组 The OD value represents the absorbance of the supernatant of 1% red blood cell suspension without any treatment. 阳性组 It indicates the absorbance after 1% red blood cells are completely hemolyzed by adding deionized water.

[0106] The experimental results are as follows Figure 4 As shown, the hemolysis rate is used to evaluate the degree of damage to blood cells by the material. TM The hemolysis rate of the adsorbent was 2.0%, the hemolysis rates of Examples 1 to 3 were 1.2%, 1.7% and 1.5% respectively; the hemolysis rates of Control Examples 1 to 3 were 2.2%, 2.0% and 2.4% respectively. TM The hemolysis rates of the adsorbent, each embodiment and the control example were all lower than 5%, and the hemolysis rates of Examples 1 to 3 were all lower than those of Detoxi-Gel immobilized with polymyxin B. TM Adsorbent and Comparative Examples 1 to 3. The above analysis shows that the sphingomyelin-like endotoxin adsorbent proposed by the present invention has good blood compatibility, does not damage blood cells during the blood purification process, and can be applied to whole blood purification.

[0107] (5) Cell apoptosis test

[0108] Human renal proximal tubular cells (HK-2) were used as a model to investigate the potential renal toxicity of adsorbent shedding. 3 g of the endotoxin adsorbent obtained in Examples 1 to 3 was added to 10 mL of HK-2 complete medium and extracted at 37°C for 24 h. HK-2 cells were then cultured with the extraction medium for 24 h, and HK-2 cells to which 200 μM polymyxin B was added to the complete medium were set as a control. Cell apoptosis was detected by Annexin V / PI double-labeling flow cytometry, and the proportion of cells that were double-positive for Annexin V and PI was counted.

[0109] The experimental results are as follows Figure 5As shown, Annexin V can specifically bind to phosphatidylserine flipped out of the apoptotic cell membrane, so Annexin V positivity represents apoptotic cells. PI is a nucleic acid dye, but it cannot pass through the intact cell membrane and can only pass through the middle and late stages of apoptosis and necrotic cell membranes. Therefore, Annexin V and PI double positivity represent cells in the late stages of apoptosis. A trace amount (200 μM) of polymyxin B will cause apoptosis in 42.6% of cells, indicating that it has potential nephrotoxicity and will damage kidney cells. However, the cell apoptosis rates of the extracts of Examples 1 to 3 were 4.2%, 3.2% and 3.6%, which were significantly lower than polymyxin B. The above analysis shows that the adsorbent proposed in the present invention will not release toxic substances and has higher safety than the polymyxin B ligand with potential nephrotoxicity.

Claims

1. A sphingomyelin-like endotoxin adsorption material, characterized in that: The adsorbent has a chemical structure of polyethylene polyamine and phosphorylcholine, and its chemical structure is: Wherein, 0 is a substrate, the substrate is a polymer having a cyano chemical structure, or is polyvinyl alcohol, polymethyl methacrylate, polyurethane, polyvinylidene chloride, cellulose and its derivatives, and n ranges from 1 to 5.

2. The sphingomyelin-like endotoxin adsorption material according to claim 1, characterized in that: The value range of n is 1-3.

3. The sphingomyelin-like endotoxin adsorption material according to claim 1, characterized in that: The substrate is selected from one of polyacrylonitrile, acrylonitrile-styrene copolymer, acrylonitrile-butadiene-styrene copolymer, cyano-modified styrene-divinylbenzene copolymer; or a blended composite of the above polymers with polyethersulfone, polysulfone, polyamide, and polyester.

4. The sphingomyelin-like endotoxin adsorption material according to claim 3, characterized in that: The substrate is a polyacrylonitrile / polyethersulfone blended composite.

5. The sphingomyelin-like endotoxin adsorption material according to claim 1, characterized in that: The substrate morphology is selected from one of random particles, microspheres, flat membranes, hollow fiber membranes, nanofiber membranes or nanoparticles.

6. The method for preparing the sphingomyelin-like endotoxin adsorbing material according to any one of claims 1 to 5, characterized in that: The steps include: (1) soaking the substrate in deionized water, adding ethyleneamine polymer; then reacting at 50-70° C. for 10-14 hours, and then drying to obtain the substrate-polyethylene polyamine; (2) soaking the substrate obtained in step (1) - polyethylene polyamine in an anhydrous solvent, reacting with 2-chloro-1,3,2-dioxaphospholane 2-oxide at -25 to -15°C for 3 to 5 hours, and then continuing the reaction at room temperature for 1 to 2 hours; (3) soaking the product obtained in step (2) in an anhydrous solvent, and reacting with trimethylamine or trimethylamine hydrochloride at 50-70° C. for 22-28 hours to obtain the product.

7. The method according to claim 6, characterized in that In step (1), the amount of the ethyleneamine polymer added is 1 to 1.5 times the molar amount of cyano groups on the substrate surface, and after the reaction is completed, the substrate is washed alternately with anhydrous ethanol and deionized water; the ethyleneamine polymer is selected from at least one of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and pentaethylenehexamine.

8. The method according to claim 6, characterized in that In step (2), the amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide added is 1.5 to 2 times the molar amount of amino groups on the surface of the substrate-polyethylene polyamine.

9. The method according to claim 6, characterized in that In step (3), the amount of trimethylamine or trimethylamine hydrochloride added is 1.5 to 2 times the molar amount of 2-chloro-1,3,2-dioxaphospholane 2-oxide, and after the reaction is completed, the mixture is washed alternately with anhydrous ethanol and deionized water.

10. Use of the sphingomyelin-like endotoxin adsorption material according to any one of claims 1 to 5 in the preparation of a blood purification material for the treatment of sepsis.

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