Toxin adsorption protein complex, toxin removal method and toxin adsorbent made therefrom

Abstract

This invention discloses a toxin adsorption protein complex, a toxin removal method, and a fabricated toxin adsorbent, belonging to the field of toxin adsorption technology. It includes a toxin adsorption protein and a positively charged microporous filter membrane medium. The toxin adsorption protein is immobilized on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex. The positively charged microporous filter membrane medium is composed of a neutral microporous filter membrane and a polymer treatment liquid. In this invention, the toxin adsorption protein is mixed with the positively charged polymer treatment liquid to obtain a treatment liquid. Then, using a neutral microporous filter membrane as a substrate, the treatment liquid is sprayed onto its surface. After drying at room temperature, the toxin adsorption protein is immobilized on the positively charged microporous filter membrane medium, forming the toxin adsorption protein complex. The toxin adsorption protein complex is then used to fill the interior of the adsorbent body to prepare a toxin adsorbent, which can effectively adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids.

Description

Technical Field

[0001] This invention relates to the field of toxin adsorption technology, specifically to toxin adsorption protein complexes, toxin removal methods, and toxin adsorbers manufactured therefrom. Background Technology

[0002] Severe bacterial sepsis is often accompanied by organ dysfunction, shock, and high mortality. The main causative factor of sepsis is bacterial endotoxin, with the median plasma level in patients (515 pg / mL) being about 100 times higher than the normal average (5.1 pg / mL) (Opal SM et al, J. Infect. Dis. 1999; 180(5):1584-9). Endotoxin induces the body to produce excessive nitric oxide, leading to severe hypotension and potentially triggering systemic inflammatory response syndrome. Plasma exchange to remove toxins is a common but costly emergency treatment for sepsis, and a safe, efficient, and low-cost alternative is urgently needed in clinical practice.

[0003] Animal-derived hemoglobin is inexpensive, and studies have been conducted on its binding to bacterial endotoxins or nitric oxide. For example, in endotoxin adsorption studies, Bahl N. et al. used surface plasmon resonance (SPR) technology to find that both the alpha and beta subunits of hemoglobin have endotoxin binding sites with high affinity (KD in the nanomolar range), which may be related to evolutionarily conserved positively charged surface regions (J. Biol. Chem. 2011; 286(43):37793-803). Similarly, in nitric oxide adsorption studies, Angelo M. et al. reviewed the interaction between nitric oxide and hemoglobin, noting that binding sites include the thiol groups of heme and cysteine ​​(Methods Enzymol. 2008; 436:131-68). In rats injected intraperitoneally with bacterial endotoxin, the formation of S-nitrosohemoglobin after hemoglobin binds to nitric oxide was 25 times higher than in the control group [Jourd'heuil D et al, Biochem. Biophys. Res. Commun. 2000; 273(1):22-6]. Active free cysteine ​​in hemoglobin has important antioxidant functions under physiological or pathological conditions. Mice with a cysteine ​​gene knockout at position 93 of the beta subunit showed greater hypotension and lung damage after bacterial endotoxin injection (Vitturi DA et al, Free Radic. Biol. Med. 2013; 55:119-29).

[0004] Based on this, the present invention designs a toxin adsorption protein complex, a toxin removal method, and a toxin adsorbent to solve the above problems. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a toxin adsorption protein complex, a toxin removal method, and a toxin adsorbent made therefrom.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A toxin-adsorbing protein complex, comprising a toxin-adsorbing protein and a positively charged microporous filter membrane medium, wherein the toxin-adsorbing protein is immobilized on the positively charged microporous filter membrane medium to form a toxin-adsorbing protein complex; the positively charged microporous filter membrane medium is composed of a neutral microporous filter membrane and a polymer treatment solution.

[0008] Furthermore, the preparation method of the neutral microporous filter membrane is as follows: polyvinyl chloride, polysulfone, hydroxyethyl cellulose and a pore-forming agent are dissolved in an organic solvent to obtain a casting solution; nano-hydrophilic silica and konjac glucomannan microspheres are added to the casting solution, the particle size of the konjac glucomannan microspheres being 200-300 μm; after stirring under a pressure of 4-5.5 MPa for 12-25 min, a modified casting solution is obtained; the modified casting solution is coated on a non-woven fabric to obtain a neutral microporous filter membrane.

[0009] Furthermore, the mass ratio of polyvinyl chloride, polysulfone, hydroxyethyl cellulose and porogen is 25-40:15-16:6-11:6-8.

[0010] Furthermore, the mass ratio of nano-hydrophilic silica to konjac glucomannan microspheres is 3–5:4–7.

[0011] Furthermore, the pore-forming agent is polyethylene glycol with a molecular weight of 2000-5000.

[0012] Furthermore, the positively charged polymer treatment liquid comprises the following raw materials in parts by weight: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch, 10-25% of sodium hydroxide with a pH of 8.8-9.6, and the balance being chitosan.

[0013] To better achieve the objectives of this invention, this invention also provides a toxin removal method, employing the aforementioned toxin adsorption protein complex, comprising the following steps:

[0014] I. Preparation of toxin-adsorbing proteins;

[0015] II. Preparation of neutral microporous filter membrane: Dissolve 25-40 parts of polyvinyl chloride, 15-16 parts of polysulfone, 6-11 parts of hydroxyethyl cellulose and 6-8 parts of pore-forming agent in 1.3-1.5 times their volume of organic solvent to obtain a casting solution; add 3-5 parts of nano-hydrophilic silica and 4-7 parts of konjac glucomannan microspheres (with a particle size of 200-300 μm) to the casting solution, and stir under a pressure of 4-5.5 MPa for 12-25 min to obtain a modified casting solution. Coat the modified casting solution onto a nonwoven fabric to obtain a neutral microporous filter membrane.

[0016] III. Preparation of positively charged polymer treatment solution: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 10-25% of sodium hydroxide with a pH of 8.8-9.6, and the balance chitosan are mixed and stirred at 45-50℃ for 1-2 hours to obtain the solution.

[0017] IV. The toxin-adsorbing protein and the positively charged polymer treatment liquid are mixed at a weight ratio of 1:70 to 110 to obtain the treatment liquid. Then, the treatment liquid is sprayed onto the surface of a neutral microporous filter membrane as a substrate.

[0018] 5. Drying at room temperature fixes the toxin-adsorbing proteins onto the positively charged microporous filter membrane medium, forming a toxin-adsorbing protein complex.

[0019] VI. It adsorbs and removes bacterial endotoxins and nitric oxide from blood or body fluids through a toxin adsorption protein complex.

[0020] Furthermore, in step four, the spraying amount is controlled at 105–130 g / m². 2 .

[0021] Furthermore, step one specifically involves:

[0022] 1.1. Made from whole animal blood;

[0023] 1.2. Remove plasma components from whole blood by methods such as centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution;

[0024] 1.3 Add water or hypotonic solution to the red blood cell components and allow it to react for 0.5 to 8 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing them to absorb water, swell, and burst, releasing hemoglobin.

[0025] The hypotonic solution can be selected from 5% glucose solution, 5% glucose saline or 10% mannitol injection;

[0026] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0027] The hypertonic solution can be selected from 50% glucose, 10% glucose, or 20% mannitol;

[0028] 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0029] 1.6. Impurities are removed by methods such as ultrafiltration or chromatography to obtain highly purified toxin-adsorbing proteins;

[0030] 1.7 Then, heat the system to 80-90℃, treat for 15-30 minutes to inactivate, and cool to room temperature;

[0031] 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0032] To better achieve the objectives of this invention, this invention also provides a toxin adsorbent, which is prepared using the aforementioned toxin adsorption protein complex, comprising an adsorbent body and a toxin adsorption protein complex filled inside the adsorbent body.

[0033] Compared with the prior art, the beneficial effects of this invention are as follows: This invention prepares a toxin-adsorbing protein, a neutral microporous filter membrane, and a positively charged polymer treatment liquid. The toxin-adsorbing protein and the positively charged polymer treatment liquid are mixed to obtain a treatment liquid. Then, the neutral microporous filter membrane is used as a substrate, and the treatment liquid is sprayed onto its surface, with the spraying amount controlled at 120 g / m³. 2 The toxin-adsorbing proteins are fixed on a positively charged microporous filter membrane medium by room temperature drying, forming a toxin-adsorbing protein complex. This complex is then used to fill the interior of the adsorber body, thus preparing a toxin adsorber that can effectively adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids. It can serve as a safe, efficient, and low-cost alternative to plasma exchange. Attached Figure Description

[0034] Figure 1 The image shows the characteristic absorption spectrum of hemoglobin prepared in step one of Example 1. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] Example 1: In some embodiments, the toxin adsorbs the protein complex, and the preparation steps are as follows:

[0037] I. Preparation of toxin-adsorbing proteins

[0038] 1.1. Made from whole animal blood;

[0039] 1.2. Remove plasma components from whole blood by methods such as centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution;

[0040] 1.3 Add water or hypotonic solution to the red blood cell components and let it act for 5 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water, swell and burst, and release hemoglobin.

[0041] The hypotonic solution may be a 5% glucose solution;

[0042] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0043] The hypertonic solution can be selected from 50% glucose;

[0044] 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0045] 1.6. Impurities are removed by methods such as ultrafiltration or chromatography to obtain highly purified toxin-adsorbing protein (hemoglobin);

[0046] 1.7 Then, heat the system to 80°C, treat for 30 minutes to inactivate, and cool to room temperature;

[0047] 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0048] II. Preparation of Neutral Microporous Filter Membranes

[0049] 25 parts of polyvinyl chloride, 16 parts of polysulfone, 6 parts of hydroxyethyl cellulose, and 8 parts of pore-forming agent (polyethylene glycol with a molecular weight of 4000-5000) were dissolved in 1.3 times their volume of organic solvent to obtain a casting solution. 5 parts of nano-hydrophilic silica and 4 parts of konjac glucomannan microspheres with a particle size of 200 μm were added to the casting solution. After stirring at a pressure of 5.5 MPa for 12 min, a modified casting solution was obtained. The modified casting solution was then coated onto a nonwoven fabric to obtain a neutral microporous filter membrane.

[0050] III. Preparation of Positively Charged Polymer Treatment Liquid

[0051] A mixture of 65% polydiallyldimethylammonium chloride and cationic etherified starch in a 1:1 mass ratio, 10% sodium hydroxide with a pH of 9.6, and the remainder chitosan was mixed and stirred at 50°C for 1 hour to obtain the final product.

[0052] IV. A treatment solution is prepared by mixing toxin-adsorbing proteins and positively charged polymeric treatment liquid at a weight ratio of 1:70. Then, a neutral microporous filter membrane is used as the substrate, and the treatment solution is sprayed onto the surface at a coating density of 130 g / m³. 2 ;

[0053] 5. Drying at room temperature fixes the toxin-adsorbing proteins onto the positively charged microporous filter membrane medium, forming a toxin-adsorbing protein complex.

[0054] It can adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids through a toxin adsorption protein complex.

[0055] A toxin adsorbent is prepared by filling the body of the adsorbent with the aforementioned toxin adsorption protein complex.

[0056] Hemoglobin is the most abundant protein molecule in blood and can bind to certain harmful substances, such as bacterial endotoxins and excess nitric oxide. This invention immobilizes animal-derived hemoglobin onto a positively charged microporous filter membrane medium to form a toxin-adsorbing protein complex, which can effectively adsorb and remove harmful components such as bacterial endotoxins and nitric oxide from blood / body fluids. The toxin-adsorbing protein complex can be filled inside the adsorber body to form a toxin adsorber.

[0057] Example 2: In some examples, the toxin adsorbs the protein complex, and the preparation steps are as follows:

[0058] I. Preparation of toxin-adsorbing proteins

[0059] 1.1. Made from whole animal blood;

[0060] 1.2. Remove plasma components from whole blood by methods such as centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution;

[0061] 1.3 Add water or hypotonic solution to the red blood cell components and let it act for 8 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water, swell and burst, and release hemoglobin.

[0062] The hypotonic solution may be selected as 5% glucose saline;

[0063] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0064] The hypertonic solution can be selected as 10% glucose;

[0065] 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0066] 1.6. Impurities are removed by methods such as ultrafiltration or chromatography to obtain highly purified toxin-adsorbing protein (hemoglobin);

[0067] 1.7 Then, heat the system to 90°C, treat for 15 minutes to inactivate, and cool to room temperature;

[0068] 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0069] II. Preparation of Neutral Microporous Filter Membranes

[0070] 40 parts of polyvinyl chloride, 15 parts of polysulfone, 11 parts of hydroxyethyl cellulose, and 6 parts of pore-forming agent (polyethylene glycol with a molecular weight of 2000-3000) were dissolved in 1.5 times their volume of organic solvent to obtain a casting solution. 3 parts of nano-hydrophilic silica and 7 parts of konjac glucomannan microspheres with a particle size of 300 μm were added to the casting solution. After stirring under a pressure of 4 MPa for 25 min, a modified casting solution was obtained. The modified casting solution was then coated onto a nonwoven fabric to obtain a neutral microporous filter membrane.

[0071] III. Preparation of Positively Charged Polymer Treatment Liquid

[0072] A mixture of 55% polydiallyldimethylammonium chloride and cationic etherified starch in a 1:1 mass ratio, 25% sodium hydroxide with a pH of 8.8, and the remainder chitosan was mixed and stirred at 45°C for 2 hours to obtain the final product.

[0073] IV. A treatment solution is prepared by mixing toxin-adsorbing proteins with a positively charged polymeric treatment liquid at a weight ratio of 1:110. Then, a neutral microporous filter membrane is used as the substrate, and the treatment solution is sprayed onto the surface, with the spraying amount controlled at 105 g / m³. 2 ;

[0074] 5. Drying at room temperature fixes the toxin-adsorbing proteins onto the positively charged microporous filter membrane medium, forming a toxin-adsorbing protein complex.

[0075] It can adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids through a toxin adsorption protein complex.

[0076] A toxin adsorbent is prepared by filling the body of the adsorbent with the aforementioned toxin adsorption protein complex.

[0077] Example 3: In some examples, the toxin adsorbs the protein complex, and the preparation steps are as follows:

[0078] I. Preparation of toxin-adsorbing proteins

[0079] 1.1. Made from whole animal blood;

[0080] 1.2. Remove plasma components from whole blood by methods such as centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution;

[0081] 1.3 Add water or hypotonic solution to the red blood cell components and let it act for 7 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water, swell and burst, and release hemoglobin.

[0082] The hypotonic solution may be selected as 10% mannitol injection;

[0083] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0084] The hypertonic solution may be selected from 20% mannitol;

[0085] 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0086] 1.6. Impurities are removed by methods such as ultrafiltration or chromatography to obtain highly purified toxin-adsorbing protein (hemoglobin);

[0087] 1.7 Then, the system is heated to 85°C, treated for 20 minutes to inactivate, and then cooled to room temperature;

[0088] 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0089] II. Preparation of Neutral Microporous Filter Membranes

[0090] 30 parts of polyvinyl chloride, 15.5 parts of polysulfone, 8 parts of hydroxyethyl cellulose, and 7 parts of pore-forming agent (polyethylene glycol with a molecular weight of 3000-5000) were dissolved in 1.4 times their volume of organic solvent to obtain a casting solution. 4 parts of nano-hydrophilic silica and 5 parts of konjac glucomannan microspheres with a particle size of 250 μm were added to the casting solution. After stirring at a pressure of 4.5 MPa for 18 min, a modified casting solution was obtained. The modified casting solution was then coated onto a nonwoven fabric to obtain a neutral microporous filter membrane.

[0091] III. Preparation of Positively Charged Polymer Treatment Liquid

[0092] A mixture of 60% polydiallyldimethylammonium chloride and cationic etherified starch in a 1:1 mass ratio, 20% sodium hydroxide with a pH of 9, and the remainder chitosan was mixed and stirred at 48°C for 1.7 h to obtain the final product.

[0093] IV. A treatment solution is prepared by mixing toxin-adsorbing proteins and positively charged polymeric treatment liquid at a weight ratio of 1:90. Then, a neutral microporous filter membrane is used as the substrate, and the treatment solution is sprayed onto the surface, with the spraying amount controlled at 120 g / m³. 2 ;

[0094] 5. Drying at room temperature fixes the toxin-adsorbing proteins onto the positively charged microporous filter membrane medium, forming a toxin-adsorbing protein complex.

[0095] It can adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids through a toxin adsorption protein complex.

[0096] A toxin adsorbent is prepared by filling the body of the adsorbent with the aforementioned toxin adsorption protein complex.

[0097] Comparative Example 1

[0098] The toxin-adsorbing protein complex is prepared using the following steps:

[0099] I. Preparation of toxin-adsorbing proteins

[0100] 1.1. Made from whole animal blood;

[0101] 1.2. Remove plasma components from whole blood by methods such as centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution;

[0102] 1.3 Add water or hypotonic solution to the red blood cell components and let it act for 7 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water, swell and burst, and release hemoglobin.

[0103] The hypotonic solution may be selected as 10% mannitol injection;

[0104] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0105] The hypertonic solution may be selected from 20% mannitol;

[0106] 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0107] 1.6. Impurities are removed by methods such as ultrafiltration or chromatography to obtain highly purified toxin-adsorbing protein (hemoglobin);

[0108] 1.7 Then, the system is heated to 85°C, treated for 20 minutes to inactivate, and then cooled to room temperature;

[0109] 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0110] II. Preparation of Neutral Microporous Filter Membranes

[0111] 30 parts of polyvinyl chloride, 15.5 parts of polysulfone, 8 parts of hydroxyethyl cellulose, and 7 parts of pore-forming agent (polyethylene glycol with a molecular weight of 3000-5000) were dissolved in 1.4 times their volume of organic solvent to obtain a casting solution. 4 parts of nano-hydrophilic silica and 5 parts of konjac glucomannan microspheres with a particle size of 250 μm were added to the casting solution. After stirring at a pressure of 4.5 MPa for 18 min, a modified casting solution was obtained. The modified casting solution was then coated onto a nonwoven fabric to obtain a neutral microporous filter membrane.

[0112] 3. A treatment solution is prepared by mixing toxin-adsorbing protein and water at a weight ratio of 1:90. Then, a neutral microporous filter membrane is used as the substrate, and the treatment solution is sprayed onto the surface, with the spraying amount controlled at 120 g / m³. 2 ;

[0113] Fourth, drying at room temperature fixes the toxin-adsorbing proteins onto the neutral microporous filter membrane medium, forming a toxin-adsorbing protein complex.

[0114] A toxin adsorbent is prepared by filling the body of the adsorbent with the aforementioned toxin adsorption protein complex.

[0115] Comparative Example 2

[0116] The toxin-adsorbing protein complex is prepared using the following steps:

[0117] I. Preparation of toxin-adsorbing proteins

[0118] 1.1. Made from whole animal blood;

[0119] 1.2. Remove plasma components from whole blood by methods such as centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution;

[0120] 1.3 Add water or hypotonic solution to the red blood cell components and let it act for 7 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water, swell and burst, and release hemoglobin.

[0121] The hypotonic solution may be selected as 10% mannitol injection;

[0122] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0123] The hypertonic solution may be selected from 20% mannitol;

[0124] 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0125] 1.6. Impurities are removed by methods such as ultrafiltration or chromatography to obtain highly purified toxin-adsorbing protein (hemoglobin);

[0126] 1.7 Then, the system is heated to 85°C, treated for 20 minutes to inactivate, and then cooled to room temperature;

[0127] 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0128] II. Preparation of Positively Charged Polymer Treatment Liquid

[0129] A mixture of 60% polydiallyldimethylammonium chloride and cationic etherified starch in a 1:1 mass ratio, 20% sodium hydroxide with a pH of 9, and the remainder chitosan was mixed and stirred at 48°C for 1.7 h to obtain the final product.

[0130] 3. A treatment solution is prepared by mixing toxin-adsorbing proteins with a positively charged polymeric treatment liquid at a weight ratio of 1:90. Then, a commercially available nylon microporous filter membrane is used as the substrate, and the treatment solution is sprayed onto its surface at a coating density of 120 g / m². 2 ;

[0131] Fourth, drying at room temperature fixes the toxin adsorption proteins onto the positively charged microporous filter membrane medium, forming a toxin adsorption protein complex.

[0132] It can adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids through a toxin adsorption protein complex.

[0133] A toxin adsorbent is prepared by filling the body of the adsorbent with the aforementioned toxin adsorption protein complex.

[0134] In experimental examples, the adsorption effects of bacterial endotoxins and nitric oxide were tested using the toxin adsorbers prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0135] Experimental methods: (1) Preparation of a solution containing bacterial endotoxin (2 EU / mL) and excess nitric oxide (70 μg / L nitrate ions NO2) - / NO3 -(1) Plasma; (2) Plasma was adsorbed using a toxin adsorber, and the endotoxin, colony count and nitric oxide content at the outlet of the toxin adsorber were detected. The results are shown in Table 1.

[0136] Table 1. Results of endotoxin, bacterial count, and nitric oxide content detection.

[0137]

[0138] This invention involves preparing a toxin-adsorbing protein, a neutral microporous filter membrane, and a positively charged polymer treatment solution. The toxin-adsorbing protein is mixed with the positively charged polymer treatment solution to obtain the treatment solution. Then, using the neutral microporous filter membrane as a substrate, the treatment solution is sprayed onto its surface at a spraying rate controlled at 120 g / m³. 2 The toxin-adsorbing proteins are fixed on a positively charged microporous filter membrane medium by room temperature drying, forming a toxin-adsorbing protein complex. This complex is then used to fill the interior of the adsorber body, thus preparing a toxin adsorber that can effectively adsorb and remove bacterial endotoxins and nitric oxide from blood or body fluids. It can serve as a safe, efficient, and low-cost alternative to plasma exchange.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A toxin-adsorbing protein complex, characterized in that, It includes a toxin-adsorbing protein and a positively charged microporous filter membrane medium. The toxin-adsorbing protein is immobilized on the positively charged microporous filter membrane medium to form a toxin-adsorbing protein complex. The positively charged microporous filter membrane medium is composed of a neutral microporous filter membrane and a polymer treatment solution. The toxin-adsorbing protein is hemoglobin; Preparation of neutral microporous filter membrane: 25-40 parts by weight of polyvinyl chloride, 15-16 parts by weight of polysulfone, 6-11 parts by weight of hydroxyethyl cellulose, and 6-8 parts by weight of a pore-forming agent are dissolved in 1.3-1.5 times their volume of organic solvent to obtain a casting solution. 3-5 parts by weight of nano-hydrophilic silica and 4-7 parts by weight of konjac glucomannan microspheres (with a particle size of 200-300 μm) are added to the casting solution. The mixture is stirred at 4-5.5 MPa for 12-25 min to obtain a modified casting solution. The modified casting solution is then coated onto a nonwoven fabric to obtain a neutral microporous filter membrane. The polymer treatment solution includes the following raw materials in parts by weight: polydiallyldimethylammonium chloride, cationic etherified starch... The mixture consists of 55-65% polydiallyldimethylammonium chloride and cationic etherified starch in a 1:1 mass ratio, 10-25% sodium hydroxide with a pH of 8.8-9.6, and the remainder chitosan. A positively charged polymeric treatment solution is prepared by mixing 55-65% polydiallyldimethylammonium chloride and cationic etherified starch in a 1:1 mass ratio, 10-25% sodium hydroxide with a pH of 8.8-9.6, and the remainder chitosan. The mixture is stirred at 45-50°C for 1-2 hours to obtain the final product. The toxin-adsorbing protein complex is prepared by mixing the toxin-adsorbing protein with the positively charged polymeric treatment solution in a 1:70-110 weight ratio. The treatment solution is then sprayed onto a neutral microporous filter membrane as a substrate and dried at room temperature.

2. The toxin-adsorbing protein complex according to claim 1, characterized in that, The pore-forming agent is polyethylene glycol with a molecular weight of 2000-5000.

3. A toxin removal method, employing the toxin adsorption protein complex according to any one of claims 1-2, characterized in that, Includes the following steps: I. Preparation of toxin-adsorbing proteins; II. Preparation of neutral microporous filter membrane: Dissolve 25-40 parts by weight of polyvinyl chloride, 15-16 parts by weight of polysulfone, 6-11 parts by weight of hydroxyethyl cellulose and 6-8 parts by weight of pore-forming agent in 1.3-1.5 times the volume of organic solvent to obtain casting solution; add 3-5 parts by weight of nano-hydrophilic silica and 4-7 parts by weight of konjac glucomannan microspheres to casting solution. The particle size of konjac glucomannan microspheres is 200-300 μm. Stir under a pressure of 4-5.5 MPa for 12-25 min to obtain modified casting solution. Coat the modified casting solution onto non-woven fabric to obtain neutral microporous filter membrane. III. The polymer treatment solution comprises the following raw materials in parts by weight: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch, 10-25% of sodium hydroxide with a pH of 8.8-9.6, and the balance being chitosan; Preparation of a positively charged polymer treatment solution: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 10-25% of sodium hydroxide with a pH of 8.8-9.6, and the balance chitosan are mixed and stirred at 45-50°C for 1-2 hours to obtain the solution; 4. The toxin adsorbing protein and the positively charged polymer treatment liquid are mixed at a weight ratio of 1:70~110 to obtain the treatment liquid. Then, the treatment liquid is sprayed onto the surface of the neutral microporous filter membrane as the substrate.

5. Drying at room temperature fixes the toxin-adsorbing proteins onto the positively charged microporous filter membrane medium, forming a toxin-adsorbing protein complex. VI. It adsorbs and removes bacterial endotoxins and nitric oxide from blood or body fluids through a toxin adsorption protein complex.

4. The toxin removal method according to claim 3, characterized in that, In step four, the spraying amount is controlled at 105~130g / m². 2 .

5. The toxin removal method according to claim 3, characterized in that, Step one is as follows: 1.

1. Made from whole animal blood; 1.

2. Remove plasma components from whole blood by centrifugation or microfiltration, and then wash red blood cell components with physiological sodium chloride solution; 1.3 Add water or hypotonic solution to the red blood cell components and allow it to react for 0.5 to 8 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing them to absorb water, swell, and burst, releasing hemoglobin. The hypotonic solution is selected from 5% glucose solution, 5% glucose saline or 10% mannitol injection; 1.

4. Add hypertonic solution to restore the overall isotonic environment; The hypertonic solution can be selected from 50% glucose, 10% glucose or 20% mannitol; 1.5 Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin; 1.

6. Remove impurities by ultrafiltration or chromatography to obtain highly purified toxin-adsorbing proteins; 1.7 Then, heat the system to 80-90℃, treat for 15-30 minutes to inactivate, and cool to room temperature; 1.8 Then, using 0.9% sodium chloride as the dialysate, the solution was replaced with a 30kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

6. A toxin adsorbent, prepared using the toxin adsorption protein complex according to any one of claims 1-2, characterized in that, It includes the adsorber body and the toxin adsorption protein complex filling the interior of the adsorber body.

Images