A blood purification membrane for removing proinflammatory cytokines and a method for preparing the same

Abstract

The application discloses a blood purification membrane for removing proinflammatory cytokines and a preparation method thereof. The method comprises the following steps: dissolving a film-forming material and an additive in a solvent to obtain a homogeneous casting solution; preparing a hollow fiber plasma separation membrane by using a non-solvent phase inversion method; and constructing a proinflammatory cytokine adsorption functional layer on the outer surface of the hollow fiber plasma separation membrane to obtain a homogeneous blood purification membrane. The blood purification membrane prepared by the method has the plasma separation function, can separate inflammatory cytokines from the inner side of the membrane to the outer side of the membrane, and has excellent blood compatibility on the inner surface of the membrane and the adsorption and removal function of pathogenic toxins and proinflammatory cytokines on the outer surface of the membrane. In the construction process, the two functions do not interfere with each other, the blood compatibility of the material is improved, the removal rate of the pathogenic toxins is not affected, and the problem of the reduction of the toxin adsorption and removal effect caused by the existing technology for improving the blood compatibility of the plasma perfusion adsorption resin is solved.

Description

Technical Field

[0001] This invention belongs to the field of blood purification, specifically a blood purification membrane for removing pro-inflammatory cytokines and its preparation method. Background Technology

[0002] Severe pneumonia and other diseases seriously impact human life and health, with high mortality rates in critical cases. One reason for this is multiple organ failure caused by cytokine storms, for which there is currently no specific treatment. Blocking cytokine storms is a crucial step in treating severe pneumonia and other diseases. Plasma perfusion is an important technique for removing pro-inflammatory cytokines in vitro. By clearing pro-inflammatory cytokines from the plasma, it further blocks cytokine storms and plays a vital role in the treatment of severe pneumonia and other diseases.

[0003] Plasma perfusion adsorption resins for clearing pro-inflammatory cytokines primarily utilize activated carbon or macroporous resins as carriers, modified through physical or chemical methods, such as incorporating nano-calcium carbonate into polystyrene resin or modifying polytetrafluoroethylene with graphene nanosheets. However, current research mainly focuses on developing novel adsorption materials with high pro-inflammatory cytokine clearance efficiency, with limited research on the blood compatibility of these materials. Since adsorption materials used in plasma perfusion need to come into contact with human blood, their blood compatibility is crucial.

[0004] In the existing technological field, there are many methods to enhance the blood compatibility of plasma perfusion adsorbent materials, mainly including blending modification, coating modification, and surface grafting modification. Most existing studies share a common problem: improving the blood compatibility of the adsorbent material often leads to a decrease in its clearance rate of pathogenic toxins. This is because methods to improve blood compatibility can alter the pore size of the resin or reduce the number of toxin adsorption functional groups on the resin surface. Therefore, there is a "trade-off" effect between traditional methods of improving the blood compatibility of hemoperfusion resins and improving toxin clearance rates.

[0005] Clinically, to compensate for the insufficient blood compatibility of plasma perfusion adsorption materials, a plasma separator is needed. First, the plasma containing toxins is separated, and then the separated plasma is brought into contact with the plasma perfusion adsorption material to adsorb and remove the toxins, avoiding direct contact between the adsorption material and blood cells. However, existing clinical treatment solutions also introduce new problems, such as increased risk of infection and increased treatment costs for patients due to the introduction of plasma separators.

[0006] Plasma separation membranes are a rapidly developing blood purification technology in recent years. Current plasma separation membranes are mainly made of polysulfone, ethylene-vinyl alcohol polymers, and polyethersulfone. During use, plasma separation membranes can directly contact blood, possessing plasma screening properties and blood compatibility, and are now widely used in the clinical treatment of various diseases, such as liver failure and hyperbilirubinemia. However, current plasma separation membranes only have the function of plasma separation and cannot simultaneously remove pathogenic toxins. When these plasma separation membranes are used in clinical plasma exchange therapy, the plasma containing pathogenic toxins separated by the membrane is directly discarded, while fresh, toxin-free plasma is replenished to complete the treatment. However, the source of fresh plasma is limited, and this treatment method carries the risk of infection with viruses in the donor plasma, such as hepatitis B virus, leading to new diseases.

[0007] Another approach to plasma exchange membrane therapy involves combining the membrane with a plasma perfusion adsorption resin. The plasma containing the pathogenic toxin separated by the membrane is brought into contact with the resin, where it is adsorbed and removed. The treated plasma is then reinfused into the body to complete the treatment. This approach addresses the limitations of plasma exchange therapy due to the availability of plasma and the risks of viral infection. However, the introduction of the plasma perfusion adsorption resin complicates the process, increasing the difficulty of the procedure and the amount of blood circulating outside the body. Therefore, the risks and costs of the treatment increase.

[0008] Therefore, at present, the treatment of diseases such as severe pneumonia can only be carried out using existing plasma exchange therapy and hemoperfusion therapy. It is not possible to directly remove pro-inflammatory cytokines in the blood of patients with severe pneumonia and other diseases in one step and block the cytokine storm. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is to provide a blood purification membrane for clearing pro-inflammatory cytokines and its preparation method.

[0010] The technical solution of this invention to solve the aforementioned problem is to provide a method for preparing a blood purification membrane for clearing pro-inflammatory cytokines, characterized in that the method includes the following steps:

[0011] Step 1: Prepare the casting solution: Dissolve the film-forming material and additives in a solvent to obtain a homogeneous casting solution;

[0012] The film-forming material is polyethersulfone, polyvinylidene fluoride, or polysulfone;

[0013] Step 2: Prepare hollow fiber plasma separation membrane using a solvent-free phase inversion method;

[0014] Step 3: Construct a pro-inflammatory cytokine adsorption functional layer on the outer surface of the hollow fiber plasma separation membrane to obtain a homogeneous blood purification membrane;

[0015] I. Construction of the adsorption functional layer of polyethersulfone hollow fiber plasma separation membrane:

[0016] A3.1. Dissolve polyethersulfone in concentrated sulfuric acid to form a homogeneous solution; then add chlorosulfonic acid dropwise to carry out the sulfonation reaction of polyethersulfone to obtain a reaction mixture; then solidify the reaction mixture, wash to remove impurities, and dry to obtain sulfonated polyethersulfone;

[0017] A3.2. Sulfonated polyethersulfone is dissolved in dichloromethane solution to obtain sulfonated polyethersulfone solution; then MES, EDC, NHS and polyethyleneimine are added to carry out amidation reaction to obtain amination polyethersulfone.

[0018] A3.3. Amination polyethersulfone, polyvinylpyrrolidone and polyethylene glycol are dissolved in dimethylacetamide to prepare amination polyethersulfone casting solution; a polyethersulfone hollow fiber plasma separation membrane with both ends sealed is placed in the amination polyethersulfone casting solution so that the casting solution is evenly coated on the base membrane; after the membrane is formed and cured, it is washed to remove impurities and then dried to obtain a polyethersulfone blood purification membrane;

[0019] II. Construction of the adsorption functional layer of polyvinylidene fluoride hollow fiber plasma separation membrane:

[0020] B3.1 After sealing both ends of the polyvinylidene fluoride hollow fiber plasma separation membrane, immerse it in a diethylenetriamine solution to carry out a Michael addition reaction; after the reaction is completed, remove the membrane and wash it to remove impurities.

[0021] B3.2. Place the membrane in a MES buffer solution containing tryptophan, EDC, and NHS for amidation reaction; after the reaction is complete, remove the membrane, wash to remove impurities, and then dry to obtain a polyvinylidene fluoride blood purification membrane.

[0022] III. Construction of the adsorption functional layer of polysulfone hollow fiber plasma separation membrane:

[0023] C3.1 After sealing both ends of the polysulfone hollow fiber plasma separation membrane, immerse it in a dopamine solution for impregnation coating; after coating, remove the membrane, cure it, and wash it to remove impurities;

[0024] C3.2. The membrane is placed in an acetic acid solution containing ciprofloxacin (MES, EDC, and NHS) for esterification. After the reaction is complete, the membrane is removed, washed to remove impurities, and then dried to obtain a polysulfone blood purification membrane.

[0025] The technical solution of this invention to solve the aforementioned problem of blood purification membrane technology is to provide a blood purification membrane prepared by a method for preparing a blood purification membrane for clearing pro-inflammatory cytokines, characterized in that the blood purification membrane has an inner diameter of 100~400μm, a wall thickness of 10~50μm, and a packing density of 5~20cm³. 2 / cm 3 The pore size of the blood purification membrane is 0.2~2μm.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The blood purification membrane prepared by the present invention not only has the function of separating plasma and separating pro-inflammatory cytokines from the inner side of the membrane to the outer side of the membrane, but also has excellent blood compatibility on the inner surface of the membrane and adsorption and removal function of virulent pro-inflammatory cytokines on the outer surface of the membrane.

[0028] (2) The present invention constructs excellent blood compatibility and high toxin specific adsorption function on the inner and outer sides of the membrane respectively, and they do not interfere with each other during the construction process. While improving the blood compatibility of the material, it does not affect the clearance rate of virulent toxins, thus solving the problem of reduced toxin adsorption and clearance effect caused by existing technologies for improving the blood compatibility of plasma perfusion adsorption resin.

[0029] (3) Unlike the two-step method of removing toxins in traditional plasma perfusion therapy, which requires the simultaneous use of a plasma separator and a plasma perfusion device, this invention uses a single membrane to complete the filtration-adsorption process in one step to remove virulent toxins. Therefore, this invention integrates the functions of two Class III medical devices into one Class III medical device, combining the separation function of the plasma separator and the toxin removal function of the plasma perfusion device in traditional plasma perfusion therapy, significantly simplifying the treatment process and reducing the risk of infection during treatment; at the same time, it reduces the amount of extracorporeal blood during treatment, thereby avoiding the risk of hypotension caused by a decrease in the patient's blood volume; and it also reduces the number of Class III medical devices and extracorporeal circulation tubing used, greatly reducing the patient's treatment costs.

[0030] (4) The blood purification membrane prepared by the present invention has the advantages of small inner diameter (1 / 2 to 1 / 5 of that of traditional water treatment membrane), thin wall thickness (1 / 2 to 1 / 10 of that of traditional water treatment membrane) and large pore size, which improves the packing density in the effective area and enhances the blood purification effect. Attached Figure Description

[0031] Figure 1 This is an electron microscope image of the blood purification membrane obtained in Example 1 of the present invention;

[0032] Figure 2 Here is an electron microscope image of the blood purification membrane obtained in Example 2 of the present invention;

[0033] Figure 3 This is an electron microscope image of the blood purification membrane obtained in Example 3 of the present invention. Detailed Implementation

[0034] Specific embodiments of the present invention are given below. These specific embodiments are only used to further illustrate the present invention in detail and do not limit the scope of protection of the claims of the present invention.

[0035] This invention provides a method for preparing a blood purification membrane for clearing pro-inflammatory cytokines (hereinafter referred to as the method), characterized in that the method includes the following steps:

[0036] Step 1: Prepare the casting solution: Dissolve the film-forming material and additives in a solvent to obtain a homogeneous casting solution;

[0037] The film-forming material is made of a material with good blood compatibility and has little impact on blood cells, specifically polyethersulfone, polyvinylidene fluoride or polysulfone;

[0038] Preferably, in step 1, the film-forming material accounts for 10-30 wt% of the total mass of the casting solution, and the additives account for 1-10 wt% of the total mass of the casting solution.

[0039] Preferably, in step 1, the additive is polyvinyl alcohol, polyethylene glycol, or polyvinylpyrrolidone, used to create pores and enhance blood compatibility; the solvent is dimethylformamide, dimethylacetamide, or dimethyl sulfoxide.

[0040] Preferably, in step 1, the dissolution process is: stirring at 60~120℃ for 6~24h.

[0041] Step 2: Prepare hollow fiber plasma separation membrane using a solvent-free phase inversion method;

[0042] Preferably, step 2 specifically involves: placing the casting solution in a reaction vessel and allowing it to stand to remove bubbles; placing the core solution in a core solution vessel; then starting spinning, under pressure, the casting solution and the core solution flow together into the spinneret and are extruded into the air through the spinneret. After passing through an air bath, the solution is immersed in a coagulation bath to solidify, forming a hollow fiber membrane; then, deionized water is used to remove additives and solvents to obtain a hollow fiber plasma separation membrane.

[0043] Preferably, in step 2, the temperature of the reactor in the hollow fiber spinning machine is 60~120℃, and the pressure is 0.1~1 MPa. The settling and degassing time is 1~24 h. The core solution is an aqueous solution of dimethylformamide, dimethylacetamide, or dimethyl sulfoxide with a mass fraction of 40~90 wt%. The temperature of the core solution reactor is 60~120℃. The core solution flow rate is 25~250 mL / min. The length of the air bath is 1~10 cm. The temperature of the coagulation bath is 20~60℃, and the coagulation bath is an aqueous solution of dimethylformamide, dimethylacetamide, or dimethyl sulfoxide with a mass fraction of 0~30 wt%.

[0044] Preferably, in step 2, the pore size of the hollow fiber plasma separation membrane is 0.25~2.5μm (preferably 0.3~1μm).

[0045] Step 3: Construct a pro-inflammatory cytokine adsorption functional layer on the outer surface of the hollow fiber plasma separation membrane to obtain a homogeneous blood purification membrane;

[0046] I. Construction of the adsorption functional layer of polyethersulfone hollow fiber plasma separation membrane:

[0047] A3.1 Dissolve polyethersulfone in concentrated sulfuric acid to form a homogeneous solution; then add chlorosulfonic acid dropwise to avoid uneven reaction caused by excessively rapid local reaction, and carry out the sulfonation reaction of polyethersulfone to obtain a reaction mixture; then solidify the reaction mixture, wash to remove impurities, and dry to obtain sulfonated polyethersulfone;

[0048] Preferably, in step A3.1, the mass ratio of polyethersulfone to the volume of concentrated sulfuric acid is 1g:1~5ml; the dissolution temperature is 5~40℃; and the dissolution time is 1~48h.

[0049] Preferably, in step A3.1, the mass ratio of polyethersulfone to chlorosulfonic acid is 1:2~6; the sulfonation reaction temperature is 5~40℃; and the sulfonation reaction time is 1~48h.

[0050] Preferably, in step A3.1, curing involves pouring the reaction mixture onto a glass plate and curing it into a film. Washing involves using deionized water to remove concentrated sulfuric acid and unreacted chlorosulfonic acid until neutral. The drying process involves drying at 20~40℃ for 24~48 hours.

[0051] A3.2. Sulfonated polyethersulfone is dissolved in dichloromethane solution to obtain sulfonated polyethersulfone solution; then MES (2-(N-morpholino)ethanesulfonic acid), EDC (ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and polyethyleneimine are added to carry out amidation reaction to obtain amination polyethersulfone;

[0052] Preferably, in step A3.2, the mass ratio of sulfonated polyethersulfone to the volume of dichloromethane solution is 1g:5~10ml.

[0053] Preferably, in step A3.2, the volume ratio of the dichloromethane solution to the mass of MES is 100 ml: 1~5 g; the concentration of EDC is 0.05~1 mol / L, and the concentration ratio of EDC to NHS is 1:1~3; the polyethyleneimine is a 20~50 wt% polyethyleneimine aqueous solution, the volume of which is 1~40% of the volume of the sulfonated polyethersulfone solution.

[0054] Preferably, in step A3.2, the amidation reaction temperature is 30~60℃ and the reaction time is 2~12h.

[0055] A3.3. Amination polyethersulfone, polyvinylpyrrolidone and polyethylene glycol are dissolved in dimethylacetamide to prepare amination polyethersulfone casting solution; a polyethersulfone hollow fiber plasma separation membrane with both ends sealed is placed in the amination polyethersulfone casting solution so that the casting solution is evenly coated on the base membrane; after coating is completed, the membrane is removed, cured, washed to remove impurities, and then dried to obtain a polyethersulfone blood purification membrane.

[0056] Preferably, in step A3.3, the aminated polyethersulfone casting solution contains 5-20 wt% aminated polyethersulfone, 1-10 wt% polyvinylpyrrolidone, and 1-10 wt% polyethylene glycol.

[0057] Preferably, in step A3.3, the time spent in the aminated polyethersulfone casting solution is 5 to 30 minutes.

[0058] Preferably, in step A3.3, the washing process involves soaking in deionized water at room temperature for 2-48 hours. The drying process involves placing the product in a constant temperature drying oven at 25-60℃ (preferably 45℃) for 12-24 hours until completely dried, which facilitates long-term storage and sterilization after manufacturing the blood purifier, and inhibits bacterial growth.

[0059] II. Construction of the adsorption functional layer of polyvinylidene fluoride hollow fiber plasma separation membrane:

[0060] B3.1 After sealing both ends of the polyvinylidene fluoride hollow fiber plasma separation membrane, immerse it in a diethylenetriamine solution to carry out a Michael addition reaction; after the reaction is completed, remove the membrane and wash it to remove impurities.

[0061] Preferably, in step B3.1, the area of ​​the polyvinylidene fluoride hollow fiber plasma separation membrane to the mass ratio of the diethylenetriamine solution is 10 cm². 2 :0.1~10g; the concentration of diethylenetriamine solution is 0.1~10mol / L.

[0062] Preferably, in step B3.1, the Michael addition reaction takes place in an alkaline environment (preferably pH=11~13, more preferably pH=12), at a temperature of 40~80℃, for a time of 1~24h.

[0063] Preferably, in step B3.1, the washing process involves using deionized water to remove the unreacted diethylenetriamine solution.

[0064] B3.2. Place the membrane in a MES buffer solution containing tryptophan, EDC, and NHS for amidation reaction; after the reaction is complete, remove the membrane, wash to remove impurities, and then dry to obtain a polyvinylidene fluoride blood purification membrane.

[0065] Preferably, in step B3.2, the ratio of membrane area to tryptophan mass is 100 cm². 2 1~5g; the ratio of membrane area to MES buffer volume is 1cm². 2 5~20mL; In the MES buffer, the concentration of MES is 0.04~0.05mol / L, the concentration of EDC is 0.05~1mol / L, and the concentration ratio of EDC to NHS is 1:1~3.

[0066] Preferably, in step B3.2, the amidation reaction temperature is 30~60℃ and the time is 6~72h.

[0067] Preferably, in step B3.2, washing involves using deionized water to remove unreacted buffer solution. The drying process involves placing the product in a constant temperature drying oven at 25~60℃ (preferably 45℃) for 12~24 hours until completely dried, facilitating long-term storage and sterilization after manufacturing a blood purifier, and inhibiting bacterial growth.

[0068] III. Construction of the adsorption functional layer of polysulfone hollow fiber plasma separation membrane:

[0069] C3.1 After sealing both ends of the polysulfone hollow fiber plasma separation membrane, immerse it in a dopamine solution for impregnation coating; after coating, remove the membrane, cure it, and wash it to remove impurities;

[0070] Preferably, in step C3.1, the ratio of the area of ​​the polysulfone hollow fiber plasma separation membrane to the mass of the solute (i.e., dopamine) in the dopamine solution is 100 cm². 2 :1.53~3.06g; the concentration of dopamine solution is 0.01~2mol / L.

[0071] Preferably, in step C3.1, the coating temperature is 20~40℃ and the time is 1~24h.

[0072] Preferably, in step C3.1, the washing process involves using deionized water to remove unreacted dopamine solution.

[0073] C3.2. The membrane is placed in an acetic acid solution containing ciprofloxacin (MES, EDC, and NHS) for esterification. After the reaction is complete, the membrane is removed, washed to remove impurities, and then dried to obtain a polysulfone blood purification membrane.

[0074] Preferably, in step C3.2, the ratio of the membrane area to the mass of the solute (i.e., ciprofloxacin) in the acetic acid solution of ciprofloxacin is 100 cm². 20.1~5g; In the acetic acid solution of ciprofloxacin, the concentration of MES is 0.04~0.05mol / L, the concentration of EDC is 0.05~1mol / L, and the concentration of NHS is 0.05~1mol / L.

[0075] Preferably, in step C3.2, the esterification reaction temperature is 30~60℃ and the time is 6~72h.

[0076] Preferably, in step C3.2, the washing process involves using deionized water to remove unreacted substances. The drying process involves placing the product in a constant temperature drying oven at 25~60℃ (preferably 45℃) for 12~24 hours until completely dried, which facilitates long-term storage and sterilization after manufacturing a blood purifier, and inhibits bacterial growth.

[0077] This invention also provides a method for preparing a blood purification membrane for clearing pro-inflammatory cytokines, characterized in that the blood purification membrane has an inner diameter of 100-400 μm (preferably 200-300 μm), a wall thickness of 10-50 μm (preferably 20-30 μm), and a packing density of 5-20 cm³. 2 / cm 3 The pore size of the blood purification membrane is 0.2~2μm (preferably 0.3~1μm).

[0078] Example 1

[0079] Step 1: Dissolve 180g of polyethersulfone, 50g of polyvinylpyrrolidone, and 20g of polyethylene glycol in 750mL of dimethylacetamide to prepare a uniformly dissolved casting solution.

[0080] Step 2: Prepare a polyethersulfone hollow fiber plasma separation membrane with excellent blood compatibility using a solvent-free phase inversion method;

[0081] Step 3: Construct a pro-inflammatory cytokine adsorption functional layer on the outer surface of the polyethersulfone hollow fiber plasma separation membrane to obtain a homogeneous polyethersulfone blood purification membrane;

[0082] A3.1 Dissolve 100g of polyethersulfone in 100mL of concentrated sulfuric acid solution at 20℃ for 10h to form a homogeneous solution; then slowly add 200g of chlorosulfonic acid and sulfonate at 20℃ for 24h to obtain a reaction mixture; then pour the reaction mixture onto a glass plate to form a film, wash with deionized water until neutral, and dry at 40℃ for 48h to obtain sulfonated polyethersulfone.

[0083] A3.2 Dissolve 80g of sulfonated polyethersulfone in 400mL of dichloromethane solution, then add 4g of MES, 19g of EDC, 12g of NHS and 100mL of 50wt% polyethyleneimine aqueous solution, and carry out amidation reaction at 40℃ for 8h to obtain amination polyethersulfone.

[0084] A3.3. Dissolve 20g of amination polyethersulfone, 5g of polyvinylpyrrolidone, and 5g of polyethylene glycol in 70mL of dimethylacetamide to prepare an amination polyethersulfone casting solution; 100cm of end-sealed film... 2 After the polyethersulfone hollow fiber plasma separation membrane was placed in the aminated polyethersulfone casting solution for 30 minutes, it was transferred to deionized water at room temperature and soaked for 24 hours, with the water being changed continuously during the process. After thorough washing to remove impurities, it was placed in a constant temperature drying oven at 45°C and dried for 24 hours until completely dried, thus obtaining a homogeneous polyethersulfone blood purification membrane.

[0085] Depend on Figure 1 As can be seen, the obtained polyethersulfone blood purification membrane has a finger-like pore structure on the inner side, which facilitates the flow of toxic plasma from the inner side to the outer side of the membrane. The outer surface of the membrane has a thin skin structure, which facilitates the efficient separation of toxic plasma.

[0086] Example 2

[0087] Step 1: Dissolve 200g of polyvinylidene fluoride, 70g of polyvinylpyrrolidone, and 30g of polyethylene glycol in 700mL of dimethylacetamide to prepare a uniformly dissolved casting solution.

[0088] Step 2: Prepare a polyvinylidene fluoride hollow fiber plasma separation membrane with excellent blood compatibility using a solvent-free phase inversion method;

[0089] Step 3: Construct a pro-inflammatory cytokine adsorption functional layer on the outer surface of the polyvinylidene fluoride hollow fiber plasma separation membrane to obtain a homogeneous polyvinylidene fluoride blood purification membrane.

[0090] B3.1, 100cm 2 After sealing both ends of the polyvinylidene fluoride hollow fiber plasma separation membrane, it is immersed in 100 mL of 2 mol / L diethylenetriamine solution and reacted at pH=12 and 60℃ for 3 h. Then the membrane is taken out and thoroughly washed with deionized water to remove impurities.

[0091] B3.2. Place the membrane in 1L of MES buffer containing 1g tryptophan, 9.6g EDC and 5.8g NHS (where the concentration of MES is 0.04mol / L), and after amidation reaction at 60℃ for 12h, remove the membrane, wash it thoroughly with deionized water to remove impurities, and dry it at 40℃ for 24h to obtain a homogeneous polyvinylidene fluoride blood purification membrane.

[0092] Depend on Figure 2 As can be seen, the obtained polyvinylidene fluoride blood purification membrane has a finger-like pore structure on the inner side, which facilitates the flow of toxic plasma from the inner side to the outer side of the membrane. The outer surface of the membrane has a thin skin structure, which facilitates the efficient separation of toxic plasma.

[0093] Example 3

[0094] Step 1: Dissolve 220g polysulfone, 80g polyvinylpyrrolidone, and 20g polyethylene glycol in 780mL dimethyl sulfoxide to prepare a uniformly dissolved casting solution.

[0095] Step 2: Prepare a polysulfone hollow fiber plasma separation membrane with excellent blood compatibility using a solvent-free phase inversion method;

[0096] Step 3: Construct a pro-inflammatory cytokine adsorption functional layer on the outer surface of the polysulfone hollow fiber plasma separation membrane to obtain a homogeneous polysulfone blood purification membrane;

[0097] C3.1, 100cm 2 After sealing both ends of the polysulfone hollow fiber plasma separation membrane, it is immersed in 1L of 0.01mol / L dopamine solution and coated at 40℃ for 3h. Then, the membrane is taken out and thoroughly washed with deionized water to remove impurities.

[0098] C3.2. The membrane was placed in 1L of acetic acid solution containing 8.5g MES, 9.6g EDC and 5.8g NHS in 0.1g / L ciprofloxacin. After reacting at 60℃ for 6h, the membrane was removed, thoroughly washed with deionized water to remove impurities, and dried at 40℃ for 24h to obtain a homogeneous polysulfone blood purification membrane.

[0099] Depend on Figure 3 As can be seen, the obtained polysulfone blood purification membrane has a finger-like pore structure on the inner side, which facilitates the flow of toxic plasma from the inner side to the outer side of the membrane. The outer surface of the membrane has a thin skin structure, which facilitates the efficient separation of toxic plasma.

[0100] Example 4

[0101] After the blood purification membranes obtained in Examples 1-3 are used to make a blood purifier, blood flows on the inner side of the membrane, making it less likely to cause adverse reactions such as thrombosis and hemolysis. Simultaneously, plasma containing pro-inflammatory cytokines can pass through the membrane and contact the toxin-specific adsorption functional layer constructed on the outer side of the membrane. The pro-inflammatory cytokines are adsorbed and cleared, reducing the content of pro-inflammatory cytokines (interleukin-6) in the plasma by 30%–99%, while the content of anti-inflammatory cytokines (interleukin-1Ra, etc.) decreases by 1–15%. Then, the purified plasma merges with the blood on the inner side of the membrane and is reinfused into the body. Due to the effective reduction of pro-inflammatory cytokines, the generation and development of cytokine storms can be further effectively blocked, thereby achieving effective treatment for patients with severe pneumonia.

[0102] The blood purification membranes prepared in Examples 1-3 above were characterized using the following methods:

[0103] 1. Adsorption assay method for pro-inflammatory cytokines:

[0104] The adsorption capacity of pro-inflammatory cytokines was studied according to Example 4. A blood purification membrane was fabricated into a blood purifier and connected to a vessel containing a pro-inflammatory cytokine solution (interleukin-6) to form a circulation pathway. The membrane was circulated at 200 ml / min for 2 hours at 37°C. The solution from the filtration side was then collected. The concentration of the pro-inflammatory cytokine solution in the collected solution from the filtration side before and after 2 hours of circulation was determined by enzyme-linked immunosorbent assay (ELISA). The adsorption rate of the pro-inflammatory cytokine (interleukin-6) on the outer surface of the blood purification membrane was calculated as R = (C0 - C...). t ) / C0×100%, where C0 is the initial concentration of pro-inflammatory cytokines, C t The concentration of pro-inflammatory cytokines after the blood purification membrane of this invention adsorbs for a certain period of time.

[0105] Table 1 shows the removal effects of different membranes on interleukin-6 using the one-step "filtration-adsorption" method in Examples 1-3.

[0106] Table 1

[0107]

[0108] As shown in Table 1, in Examples 1-3, for different types of plasma separation membranes, the adsorption rate of the pro-inflammatory cytokine interleukin-6 via a one-step "filtration-adsorption" method was significantly enhanced after constructing a functional layer on the outer surface of the membrane. In Example 1, the adsorption rate increased from 15.5% to 89.3%; in Example 2, it increased from 6.1% to 92.8%; and in Example 3, it increased from 11.5% to 85.3%. This indicates that the construction of a functional layer on the outer surface of the membrane in Examples 1-3 effectively achieved the one-step "filtration-adsorption" method for clearing pro-inflammatory cytokines.

[0109] 2. Coagulation properties:

[0110] The blood purification membrane of this invention was immersed in fresh human blood and incubated at 37°C for 2 hours. After thorough mixing, the blood was transferred to centrifuge tubes and centrifuged at 3000 rpm for 15 minutes at 4°C. Fresh plasma was then collected, and the activated partial thromboplastin time (APT) was tested using a coagulation analyzer. To evaluate the coagulation properties of the outer membrane surface, both ends of the membrane were sealed. To evaluate the coagulation properties of the inner membrane surface, fresh blood was injected into the membrane, and both sides of the membrane were sealed.

[0111] The effects of different membranes on coagulation in Examples 1-3 are shown in Table 2.

[0112] Table 2

[0113]

[0114] As shown in Table 2, compared with the blank control, the inner and outer surfaces of the membranes in Examples 1-3 had no significant effect on the activation partial thromboplastin time. Furthermore, the activation partial thromboplastin time of the blood purification membrane did not change much compared with that of the plasma separation membrane. This indicates that neither the plasma separation membrane nor the blood purification membrane in Examples 1-3 had any effect on coagulation and would not induce a coagulation reaction.

[0115] 3. Hemolytic:

[0116] The damage to red blood cells caused by the blood purification membrane was determined by a hemolysis rate assay, which was detected using an ELISA reader. When evaluating hemolysis on the outer surface of the membrane, both ends of the membrane were sealed. When evaluating hemolysis on the inner surface of the membrane, diluted fresh blood was injected into the membrane, and both ends were then sealed.

[0117] The formula for calculating hemolysis rate is as follows:

[0118]

[0119] The effects of different membranes on hemolysis rate in Examples 1-3 are shown in Table 3.

[0120] Table 3

[0121]

[0122] As shown in Table 3, in Examples 1-3, the hemolysis rate on the inner surface of the blood purification membrane was not significantly different from that on the inner surface of the plasma separation membrane, and the hemolysis rate was less than 5% in all cases. However, the hemolysis rate on the outer surface of the blood purification membrane was higher than that on the outer surface of the plasma separation membrane, and the hemolysis rate was greater than 5% in all cases. The national standard GB / T 16886 clearly stipulates that when the hemolysis rate is greater than 5%, the biomaterial causes significant damage to red blood cells and has poor blood compatibility. When the blood purification membrane is in use, the blood comes into contact with the inner surface of the membrane, and the inner surface does not induce a hemolytic reaction. The blood passes through the membrane, filtering the toxic plasma to the outer surface. Although the outer surface of the membrane exhibits a slight hemolytic reaction, it does not come into contact with red blood cells. Therefore, the blood purification membrane of this invention, using a one-step "filtration-adsorption" method to remove toxins, can simultaneously utilize the advantages of excellent blood compatibility on the inner surface of the membrane and high toxin removal capacity on the outer surface of the membrane.

[0123] The working principle and workflow of this invention are as follows: Patient blood is drawn out of the body through one lumen of a dual-lumen cannula and connected to a blood purifier made from the blood purification membrane of this invention via tubing. Simultaneously, an anticoagulant injection line is inserted before the blood purifier. During clinical treatment, the anticoagulant is continuously injected into the blood purifier by the anticoagulant injection pump. Blood, driven by the pump, flows from the body into the inner side of the blood purification membrane. Through the pump's action, a pressure difference exists between the inner and outer sides of the membrane. Under this pressure difference, utilizing the sieving mechanism of the membrane pores, smaller plasma molecules containing toxins permeate through the pores and are separated to the outer side of the blood purification membrane. There, they come into contact with the adsorption functional layer on the outer surface of the membrane. The toxins are adsorbed onto the outer surface of the membrane through various mechanisms such as hydrogen bonding and van der Waals forces, achieving clearance. Larger blood cells are retained on the inner side of the membrane. Then, the purified plasma on the outer side of the blood purification membrane merges with the blood cells on the inner side and is reinfused into the body through the other lumen of the dual-lumen cannula, thus completing the treatment process.

[0124] Any aspects not covered in this invention are applicable to existing technologies.

Claims

1. A method for preparing a blood purification membrane for clearing pro-inflammatory cytokines, characterized in that, The method includes the following steps: Step 1: Prepare the casting solution: Dissolve the film-forming material and additives in a solvent to obtain a homogeneous casting solution; The film-forming material is polyethersulfone, polyvinylidene fluoride, or polysulfone; the additive is polyvinyl alcohol, polyethylene glycol, or polyvinylpyrrolidone. Step 2: Place the casting solution in the reaction vessel and allow it to stand to remove bubbles; place the core solution in the core solution vessel; then start spinning. Under pressure, the casting solution and core solution flow together into the spinneret and are extruded into the air. After passing through an air bath, they are immersed in a coagulation bath to solidify and form a hollow fiber membrane; then use deionized water to remove additives and solvents to obtain polyethersulfone hollow fiber plasma separation membrane, polyvinylidene fluoride hollow fiber plasma separation membrane, or polysulfone hollow fiber plasma separation membrane, respectively. Step 3: Construct a pro-inflammatory cytokine adsorption functional layer on the outer surface of each hollow fiber plasma separation membrane obtained in Step 2 to obtain a homogeneous blood purification membrane; wherein, The construction steps of the pro-inflammatory cytokine adsorption functional layer of the polyethersulfone hollow fiber plasma separation membrane include: A3.

1. Dissolve polyethersulfone in concentrated sulfuric acid to form a homogeneous solution; then add chlorosulfonic acid dropwise to carry out the sulfonation reaction of polyethersulfone to obtain a reaction mixture; then solidify the reaction mixture, wash to remove impurities, and dry to obtain sulfonated polyethersulfone; A3.

2. Sulfonated polyethersulfone is dissolved in dichloromethane solution to obtain sulfonated polyethersulfone solution; then MES, EDC, NHS and polyethyleneimine are added to carry out amidation reaction to obtain amination polyethersulfone. A3.

3. Amination polyethersulfone, polyvinylpyrrolidone, and polyethylene glycol are dissolved in dimethylacetamide to prepare amination polyethersulfone casting solution. A polyethersulfone hollow fiber plasma separator membrane with both ends sealed is placed in the amination polyethersulfone casting solution so that the casting solution is evenly coated on the polyethersulfone hollow fiber plasma separator membrane. After coating, the membrane is removed, cured, washed to remove impurities, and then dried to obtain a polyethersulfone blood purification membrane. II. The construction steps of the pro-inflammatory cytokine adsorption functional layer of polyvinylidene fluoride hollow fiber plasma separation membrane include: B3.1 After sealing both ends of the polyvinylidene fluoride hollow fiber plasma separation membrane, immerse it in a diethylenetriamine solution to carry out a Michael addition reaction; after the reaction is completed, remove the membrane and wash it to remove impurities. B3.

2. Place the membrane in a MES buffer solution containing tryptophan, EDC, and NHS for amidation reaction; after the reaction is complete, remove the membrane, wash to remove impurities, and then dry to obtain a polyvinylidene fluoride blood purification membrane. III. The construction steps of the pro-inflammatory cytokine adsorption functional layer of polysulfone hollow fiber plasma separation membrane include: C3.1 After sealing both ends of the polysulfone hollow fiber plasma separation membrane, immerse it in a dopamine solution for impregnation coating; after coating, remove the membrane, cure it, and wash it to remove impurities; C3.

2. The membrane is placed in an acetic acid solution containing ciprofloxacin (MES, EDC, and NHS) for esterification. After the reaction is complete, the membrane is removed, washed to remove impurities, and then dried to obtain a polysulfone blood purification membrane.

2. The method for preparing the blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step 1, the film-forming material accounts for 10-30 wt% of the total mass of the casting solution, and the additives account for 1-10 wt% of the total mass of the casting solution.

3. The method for preparing the blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step 1, the solvent is dimethylformamide, dimethylacetamide, or dimethyl sulfoxide.

4. The method for preparing the blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step 2, the temperature of the reactor is 60~120℃ and the pressure is 0.1~1MPa; the settling and degassing time is 1~24h; the core liquid is an aqueous solution of dimethylformamide, dimethylacetamide or dimethyl sulfoxide with a mass fraction of 40~90wt%; the temperature of the core liquid reactor is 60~120℃; the core liquid flow rate is 25~250mL / min; the length of the air bath is 1~10cm; the temperature of the coagulation bath is 20~60℃, and the coagulation bath is an aqueous solution of dimethylformamide, dimethylacetamide or dimethyl sulfoxide with a mass fraction of 0~30wt%.

5. The method for preparing a blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step 2, the pore size of the hollow fiber plasma separation membrane is 0.25~2.5μm.

6. The method for preparing the blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step A3.1, the mass ratio of polyethersulfone to the volume of concentrated sulfuric acid is 1g:1~5ml; the dissolution temperature is 5~40℃, and the dissolution time is 1~48h; In step A3.1, the mass ratio of polyethersulfone to chlorosulfonic acid is 1:2~6; the sulfonation reaction temperature is 5~40℃, and the sulfonation reaction time is 1~48h; In step A3.1, the washing process involves using deionized water to remove concentrated sulfuric acid and unreacted chlorosulfonic acid until neutral; the drying process is as follows: drying at 20~40℃ for 24~48h. In step A3.2, the mass ratio of sulfonated polyethersulfone to the volume of dichloromethane solution is 1g:5~10ml; In step A3.2, the volume ratio of dichloromethane solution to MES is 100 ml: 1~5 g; the concentration of EDC is 0.05~1 mol / L, and the concentration ratio of EDC to NHS is 1:1~3; the polyethyleneimine used is a 20~50 wt% polyethyleneimine aqueous solution, the volume of which is 1~40% of the volume of the sulfonated polyethersulfone solution. In step A3.2, the amidation reaction temperature is 30~60℃, and the reaction time is 2~12h; In step A3.3, the aminated polyethersulfone casting solution contains 5-20 wt% aminated polyethersulfone, 1-10 wt% polyvinylpyrrolidone, and 1-10 wt% polyethylene glycol. In step A3.3, the time spent in the aminated polyethersulfone casting solution is 5~30 min; In step A3.3, the washing process involves soaking in deionized water at room temperature for 2 to 48 hours; the drying process involves placing the product in a constant temperature drying oven at 25 to 60°C for 12 to 24 hours until it is completely dried.

7. The method for preparing a blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step B3.1, the area of ​​the polyvinylidene fluoride hollow fiber plasma separation membrane to the mass ratio of the diethylenetriamine solution is 1 cm². 2 0.1~10g; the concentration of the diethylenetriamine solution is 0.1~10mol / L; In step B3.1, the Michael addition reaction is carried out in an alkaline environment at a temperature of 40-80°C for 1-24 hours. In step B3.1, washing involves using deionized water to remove unreacted diethylenetriamine solution; In step B3.2, the ratio of membrane area to tryptophan mass is 100 cm². 2 1~5g; the ratio of membrane area to MES buffer volume is 1cm². 2 5~20mL; In the MES buffer, the concentration of MES is 0.04~0.05mol / L, the concentration of EDC is 0.05~1mol / L, and the concentration ratio of EDC to NHS is 1:1~3; In step B3.2, the amidation reaction temperature is 40~60℃ and the time is 6~72h; In step B3.2, washing involves using deionized water to remove unreacted buffer solution; the drying process involves placing the container in a constant temperature drying oven at 25~60℃ for 12~24 hours until completely dried.

8. The method for preparing a blood purification membrane for clearing pro-inflammatory cytokines according to claim 1, characterized in that, In step C3.1, the ratio of the area of ​​the polysulfone hollow fiber plasma separation membrane to the mass of dopamine is 100 cm². 2 : 1.53~3.06g; the concentration of dopamine solution is 0.01~2mol / L; In step C3.1, the coating temperature is 20~40℃ and the time is 1~24h; In step C3.1, washing involves using deionized water to remove unreacted dopamine solution; In step C3.2, the ratio of the membrane area to the mass of ciprofloxacin is 100 cm². 2 0.1~5g; the ratio of membrane area to MES mass is 100cm². 2 5~20g; In the acetic acid solution of ciprofloxacin, the concentration of MES is 0.04~0.05mol / L, the concentration of EDC is 0.05~1mol / L, and the concentration of NHS is 0.05~1mol / L; In step C3.2, the esterification reaction temperature is 40~60℃ and the time is 6~72h; In step C3.2, washing is done with deionized water to remove unreacted substances; the drying process is to place the product in a constant temperature drying oven at 25~60℃ for 12~24 hours until it is completely dried.

9. A blood purification membrane prepared by the method for preparing the blood purification membrane for clearing pro-inflammatory cytokines according to any one of claims 1-8, characterized in that, The blood purification membrane has an inner diameter of 100-400 μm, a wall thickness of 10-50 μm, and a packing density of 5-20 cm³. 2 / cm 3 The pore size of the blood purification membrane is 0.2~2μm.

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