Preparation method of hydrophobin-based bifunctional modified membrane and application of hydrophobin-based bifunctional modified membrane in field of artificial lungs
By constructing a hydrophobic protein coating on the PMP gas-blood exchange membrane, the problems of low CO2/O2 selectivity and thrombosis in ECMO and ECCO2R systems were solved, achieving efficient gas exchange and improved blood compatibility.
Patent Information
- Application Number
- CN202511456782.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-13
- Publication Date
- 2026-03-06
AI Technical Summary
Existing gas exchange membranes in ECMO and ECCO2R systems suffer from low CO2/O2 selectivity and a tendency to form thrombi, which affect patient health and equipment lifespan.
A biomimetic membrane coating based on hydrophobic proteins was constructed on the PMP gas exchange membrane. The surface was modified under mild conditions by utilizing the self-assembly properties of hydrophobic proteins to form a stable and uniform hydrophilic interface, thereby improving the blood compatibility and CO2 permeability of the membrane.
It significantly improves the membrane's CO2/O2 selectivity and blood compatibility, reduces the risk of thrombosis, and enhances gas exchange capacity, making it suitable for ECMO and ECCO2R systems.
Smart Images
Figure CN121607033A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of gas-liquid separation membrane technology, specifically to a method for preparing a bifunctional modified membrane based on hydrophobic proteins and its application in the field of artificial lungs. Background Technology
[0002] With the continuous advancement of medical technology, the demands for respiratory support technology are increasing. Extracorporeal life support (ECLS) is a life support technology that uses mechanical devices to provide cardiopulmonary support (partial or complete) for several days to several months in cases of severe cardiopulmonary failure, and is an effective way to reduce the mortality rate of patients with respiratory failure. Extracorporeal membrane oxygenation (ECMO) and extracorporeal carbon dioxide removal technology (ECCO2R) are both part of ECLS. Currently, the main indications for ECMO can be divided into two categories: one is cardiogenic indications, such as fulminant myocarditis, cardiogenic shock caused by myocardial infarction, difficulty in weaning from extracorporeal circulation after cardiac surgery, and transitional support before heart transplantation; the other is pulmonary indications, mainly severe acute respiratory failure caused by various reasons, such as severe ARDS (acute respiratory distress syndrome), especially when conventional mechanical ventilation cannot maintain adequate oxygenation. In addition, it can also be used for bridging before lung transplantation, certain fatal pulmonary embolism cases, and persistent pulmonary hypertension in the neonatal field. In summary, the core application of ECMO is to provide temporary life support for patients with reversible cardiopulmonary failure who do not respond to conventional treatments but have the potential for reversibility or cure, serving as a "bridge" to save lives. ECCO2R is mainly used for severe exacerbations of chronic obstructive pulmonary disease (COPD) (aiming to reduce intubation rates and duration of mechanical ventilation) and moderate ARDS (reducing ventilatory load to allow for "superprotective" ventilation settings).
[0003] The main principle of ECMO is to extract low-oxygen, high-carbon dioxide venous blood from the patient, exchange oxygen and carbon dioxide between the blood and air through a membrane oxygenator, and then return the arterial blood to the body, replacing the function of the lungs and providing the lungs with a rest and recovery time. Doctors then choose a treatment method based on the patient's cardiopulmonary failure. ECCO2R is an innovative extension of membrane oxygenation technology, aiming to maintain blood acid-base balance by reducing the concentration of carbon dioxide in the blood. During normal breathing, the lungs inhale oxygen into the blood and expel carbon dioxide. However, when patients suffer from severe respiratory failure or lung disease, lung function is impaired, and carbon dioxide cannot be effectively expelled. In this case, ECCO2R can serve as an auxiliary means of breathing, helping to expel the accumulated carbon dioxide normally. Therefore, one of the main functions of ECCO2R is to help reduce the concentration of carbon dioxide in the patient's blood, reducing the respiratory burden.
[0004] The key to the effectiveness of ECMO and ECCO2R lies in the performance of the gas exchange membrane. Poly(4-methyl-1-pentene) (PMP), a thermoplastic polyolefin, is used in the field of artificial lungs due to its good mechanical stability, thermal stability, air permeability, and resistance to plasma leakage. However, there are still two main problems in its clinical application: (1) The membrane lung (ML) has low permeability to carbon dioxide gas, that is, the CO2 / O2 selectivity of PMP is low, which leads to a slower rate of decrease in the concentration of carbon dioxide in the blood. This will prolong the service life of the device and increase the risk of complications such as hemolysis and thrombosis. (2) The gas exchange membrane is prone to thrombosis after contact with blood, which will have an adverse effect on the patient's health and the lifespan of the membrane lung. Therefore, in order to improve the effectiveness of ECMO and ECCO2R, it is necessary to improve the CO2 permeability and blood compatibility of PMP hollow fiber membrane through modification.
[0005] In recent years, researchers have optimized membrane performance by constructing biomimetic membrane interfaces in blood-gas exchange membranes. However, few studies have reported that biomimetic membrane interfaces simultaneously exhibit excellent blood compatibility, high CO2 permeability, and high CO2 / O2 selectivity. Summary of the Invention
[0006] The purpose of this invention is to address the problems existing in the prior art and provide a method for preparing a bifunctional modified membrane based on hydrophobic proteins. This method improves the CO2 resolution performance and blood compatibility of the original membrane by constructing a biomimetic membrane coating based on hydrophobic proteins on the membrane used in ECMO / ECCO2R systems. The core advantage of this method lies in the unique amphiphilic self-assembly properties of hydrophobic proteins. Their hydrophobic surface can be firmly anchored to the hydrophobic PMP membrane surface, while the hydrophilic surface naturally forms a stable and uniform hydrophilic interface. Compared with traditional protein modification techniques, this method does not rely on chemical crosslinking agents or other loading methods, avoiding problems such as loss of protein activity, increased material toxicity, and interface inhomogeneity that may be caused by crosslinking reactions. Hydrophobic proteins, with their inherent spontaneous adsorption and directional alignment capabilities, can achieve one-step, green, and efficient surface reconstruction under mild conditions, providing a simpler, safer, and more biocompatible innovative path for the functional modification of biomedical membrane materials. This biomimetic coating not only effectively improves the biocompatibility of the PMP membrane surface and significantly reduces the non-specific adsorption of proteins in the blood and platelet activation, thereby reducing the risk of thrombosis; more importantly, it can mimic the function of natural cell membranes and maintain efficient gas (oxygen and carbon dioxide) exchange capacity.
[0007] A method for preparing a bifunctional modified membrane based on hydrophobic proteins includes the following steps: Step 1: Prepare a buffer solution containing EAS protein; Step 2: Contact the polymer membrane with a buffer solution containing EAS protein, and dry it to obtain a bifunctional modified membrane.
[0008] The buffer solution is a PBS solution, and the concentration of EAS protein in the buffer solution containing EAS protein ranges from 10 to 200 μg / ml.
[0009] The EAS protein was extracted using the TSE method.
[0010] The extracted EAS protein was purified by nickel affinity chromatography. The steps were as follows: first, the dialysis column was equilibrated with binding buffer, then the sample was loaded and eluted with elution buffer. The target protein fraction was collected and filtered through a microporous membrane.
[0011] The binding buffer consists of 20-60 mM Tris-HCl, pH 6.0-8.0; the elution buffer contains 0.1-0.5 M imidazole, 10-50 mM Tris-HCl, 50-200 mM NaCl, pH 6.0-8.0.
[0012] In step 2, the contact time is 12-96 hours and the temperature range is 20-60℃.
[0013] In step 2, the contact time is 60-80 hours and the temperature range is 35-45℃.
[0014] An extracorporeal life support system wherein the gas-blood separation membrane is the aforementioned bifunctional modified membrane.
[0015] The extracorporeal life support system mentioned is an ECMO or ECCO2R system.
[0016] Bifunctional modified membranes are used for CO2 / O2 separation.
[0017] The beneficial effects of this invention are: 1. The modified membrane EAS / PMP disclosed in this application is made by self-assembling a hydrophobic protein coating on a PMP hollow fiber membrane used in ECMO / ECCO2R systems. The hydrophobic protein is a type I hydrophobic protein - EAS protein. By limiting the temperature of the modification solution and the soaking time of the original membrane, the performance of the original membrane is improved, and the blood compatibility of the membrane is significantly improved compared with the original PMP membrane. 2. The modified membrane disclosed in this application not only has excellent blood compatibility, but also high CO2 / O2 selectivity and excellent carbon dioxide removal performance. These properties make it highly suitable for ECMO and ECCO2R systems, helping to improve the membrane lung's ability to extract CO2 from human blood. 3. EAS proteins are amphiphilic and capable of self-assembly at interfaces. When secreted and dissolved in water, EAS proteins exist as monomers, mostly in a randomly coiled, relatively disordered but soluble state. This prevents premature and unnecessary aggregation within cells or in aqueous environments. When soluble EAS monomers encounter a hydrophobic-hydrophilic interface, a conformational change occurs, spontaneously arranging and assembling into highly ordered, insoluble amyloid hyphae, forming a thin film at the interface. This film exhibits significant amphiphilicity; the side of the film facing air or hydrophobic materials is highly hydrophobic, while the side facing water or hydrophilic materials is hydrophilic. This ensures the protein membrane can be stably loaded onto the membrane surface, allowing EAS proteins to adsorb onto hydrophobic surfaces, making their outer layer hydrophilic. In preparing the modified membrane, this application utilizes the self-assembly capability of hydrophobic proteins to spontaneously form a uniform coating on the membrane surface, ultimately constructing a stable and uniform hydrophilic interface that can significantly inhibit protein adsorption and platelet adhesion, thereby improving the antithrombotic performance of PMP hollow fiber membranes. 4. The preparation process of the modified membrane disclosed in this application is simple and controllable, providing a new approach for the preparation and application of bifunctional coatings, with broad application prospects. Attached Figure Description
[0018] Figure 1 These are electron microscope images of the original PMP membrane and the modified membrane prepared in Example 8 (immersion time of 72 h and modification temperature of 40 °C). Figure 2 This is a graph showing the gas permeability data of the original PMP membrane and the modified EAS / PMP composite membranes prepared in Examples 1-3 at different immersion times; Figure 3 This is a graph showing the gas permeability data of the original PMP membrane and the modified EAS / PMP composite membranes prepared in Examples 4-6 at different immersion times; Figure 4 This is a graph showing the gas permeability data of the original PMP membrane and the modified EAS / PMP composite membranes prepared in Examples 7-9 at different immersion times; Figure 5 This is a graph showing the protein adsorption data of the original PMP membrane and the modified EAS / PMP composite membranes prepared in Examples 1-3 at different soaking times; Figure 6 This is a graph showing the protein adsorption data of the original PMP membrane and the modified EAS / PMP composite membranes prepared under different soaking times in Examples 4-6. Figure 7 This is a graph showing the protein adsorption data of the original PMP membrane and the modified EAS / PMP composite membranes prepared in Examples 7-9 at different soaking times; Figure 8This is a graph showing the hemolysis rate data of the original PMP membrane and the modified membrane prepared in Example 8 (soaking time of 72 h and modification temperature of 40 °C). Figure 9 This is a graph showing the coagulation time data of the original PMP membrane and the modified membrane prepared in Example 8 (soaking time of 72 h and modification temperature of 40 °C). Figure 10 This is a graph showing the extracorporeal membrane oxygenation and carbon dioxide removal data of the modified membrane prepared in Example 8 (soaking time of 72 h and modification temperature of 40 ° C). Detailed Implementation
[0019] In some specific embodiments, the technical solutions in this patent include: A method for preparing a bifunctional modified membrane based on hydrophobic proteins, comprising the following steps: S1. Extract and purify the hydrophobic protein, denoted as EAS protein; S2. Preparation of modified solution: The EAS protein synthesized in step S1 is uniformly diluted in PBS solution and mixed in a shaker. S3. Immerse the PMP hollow fiber membrane in the modified solution prepared in step S2, remove it and dry it to obtain the composite membrane, denoted as EAS / PMP.
[0020] Further, in step S1, the method for extracting proteins is as follows: periplasmic proteins are extracted using the TSE method: the bacterial cells are treated with a Tris-sucrose solution containing EDTA to obtain the periplasmic extract.
[0021] Further, in step S1, the protein purification method is as follows: the component containing the target protein is purified by nickel affinity chromatography, and then analyzed and quantified; Further, in step S1, the protein purification step specifically involves: first equilibrating the dialysis column with binding buffer (40 mM Tris-HCl, 140 mM NaCl, pH 7.0), then eluting with elution buffer containing 0.25 M imidazole (20 mM Tris-HCl, 140 mM NaCl, pH 7.0), collecting the target protein fraction, and filtering it through a microporous membrane; Further, in step S1, the method for protein analysis and quantification is as follows: the Bradford method is used to determine the protein concentration, and the protein expression and purity are verified.
[0022] Furthermore, in step S2, the amount of PBS used depends on the concentration of the final protein modification solution (50 μg / ml), and the preparation conditions are: 25℃, 1h, 80rpm.
[0023] Furthermore, in step S3, the PMP hollow fiber membrane is immersed in the modification solution for 12-96 hours, and the modification temperature is 20-60℃.
[0024] Preferably, the protein concentration in the modification solution is 50 μg / ml, the PMP hollow fiber membrane is immersed in the modification solution for 72 h, and the modification temperature is 40 °C.
[0025] The modified membrane was prepared using the above method by self-assembling a hydrophobic protein coating on the gas-blood exchange membrane of the ECMO / ECCO2R system. The membrane of the ECMO / ECCO2R system is a PMP hollow fiber membrane, and the hydrophobic protein is a type I hydrophobic protein - EAS protein. This membrane can be used for CO2 / O2 separation, can improve CO2 / O2 selectivity, and significantly improve the blood compatibility of the membrane of the ECMO / ECCO2R system. Example 1
[0026] (1) Extraction and purification of EAS protein: The method can refer to existing techniques (e.g., Quan, S., Hiniker, A., Collet, JF., Bardwell, JCA (2013). Isolation of Bacteria Envelope Proteins. In: Delcour, A. (eds) Bacterial Cell Surfaces. Methods in Molecular Biology, vol 966. Humana Press, Totowa, NJ.、Cui L, Cheng C, Qiu Y, et al. Excretory overexpression of hydrophobins as multifunctional biosurfactants in E. coli[J]. International Journal of Biological Macromolecules, 2020, 165:1296-1302.); First, the Neurospora crassa cells were cultured and induced to express the protein. Periplasmic protein was extracted using the TSE method. Tris-sucrose solution containing EDTA (200 mM Tris-HCl, 500 mM sucrose, 1 mM EDTA was used to process bacterial cells according to a predetermined procedure to obtain periplasmic extracts. The fraction containing the target protein was purified by nickel affinity chromatography. The dialysis column was first equilibrated with binding buffer (40 mM Tris-HCl, 140 mM NaCl, pH 7.0), and after loading the sample, it was eluted with elution buffer containing 0.25 M imidazole (20 mM Tris-HCl, 140 mM NaCl, pH 7.0). The target protein fraction was collected and filtered through a microporous membrane. The protein concentration was determined using the Bradford method, and the protein expression and purity were verified.
[0027] (2) Preparation of protein modification solution: Take the protein stock solution prepared in step (1) and place it in sterile phosphate buffer solution. Shake it in a shaker for 1 hour to mix it. The final concentration of the protein modification solution is 50 μg / ml. (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 24 h, wherein the modification temperature is 20℃, and then dry it in the oven for 24 h to obtain the modified membrane. Example 2
[0028] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 24 h, wherein the modification temperature is 40℃, and then dry it in the oven for 24 h to obtain the modified membrane. Example 3
[0029] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut a 15 cm * 15 cm PMP hollow fiber membrane, immerse it in the modification solution for 24 h, wherein the modification temperature is 60 °C, and then dry it in an oven for 24 h to obtain the modified membrane. Example 4
[0030] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 48 h, the modification temperature is 20 °C, and then dry it in the oven for 24 h to obtain the modified membrane. Example 5
[0031] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 48 h, wherein the modification temperature is 40℃, and then dry it in the oven for 24 h to obtain the modified membrane. Example 6
[0032] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 48 h, wherein the modification temperature is 60 °C, and then dry it in the oven for 24 h to obtain the modified membrane. Example 7
[0033] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 72 h, wherein the modification temperature is 20℃, and then dry it in the oven for 24 h to obtain the modified membrane. Example 8
[0034] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 72 h, wherein the modification temperature is 40℃, and then dry it in the oven for 24 h to obtain the modified membrane. Example 9
[0035] (1) Preparation of EAS protein: Same as in Example 1; (2) Preparation of protein modification solution: Same as in Example 1; (3) Preparation of EAS / PMP modified membrane: Cut 15 cm * 15 cm PMP hollow fiber membrane, soak it in the modification solution for 72 h, wherein the modification temperature is 60℃, and then dry it in the oven for 24 h to obtain the modified membrane.
[0036] 1. The gas selectivity test results of the modified EAS / PMP membranes prepared in Examples 1-9 are shown in the figure. Figure 2-4 As can be seen from the corresponding figures, the CO2 / O2 selectivity of the modified membrane increases with time at different temperatures.
[0037] The protein adsorption capacity test results of the modified EAS / PMP membranes prepared in Examples 1-9 are shown below. Figure 5-7 As can be seen from the corresponding figures, when the temperature is 20~40℃, the anti-protein adsorption performance of the modified membrane increases with time; when the growth temperature is 60℃, the anti-protein adsorption performance of the modified membrane first increases and then decreases with the increase of growth time.
[0038] At a temperature of 40°C and a time of 72 hours (prepared in Example 8), the modified membrane exhibited the highest CO2 / O2 selectivity, reaching 4.23, which is 4.32 times that of the original PMP membrane; it also showed the lowest protein adsorption (3.3), a reduction of 93.2% compared to the original membrane (48.5). 2. Subsequent performance tests were conducted on the EAS / PMP modified membrane, which was prepared under the conditions of soaking time of 72 h and modification temperature of 60 °C as disclosed in Example 8, exhibiting the highest CO2 / O2 selectivity and the lowest protein adsorption (the modified membrane is referred to in the corresponding image).
[0039] Figure 1The images show electron micrographs of the original PMP membrane and the modified membrane. It can be seen from the images that a uniformly thick ultrathin coating is formed on the surface of the modified membrane without causing damage to the internal structure of the membrane.
[0040] Figure 8 The results of the hemolysis rate test of the original PMP membrane and the modified membrane are shown. The hemolysis rate of the modified membrane decreased by 64% compared with that of the original membrane.
[0041] Figure 9 The results of the coagulation time test of the original PMP membrane and the modified membrane are shown. The coagulation time of the modified membrane increased by 50% compared with that of the original membrane.
[0042] Figure 10 The results of oxygenation and carbon dioxide removal tests on the modified membrane clearly show that the pO2 and sO2 values of the blood treated with the modified membrane increase significantly over time, indicating that the membrane has good oxygenation capacity. Simultaneously, the pCO2 value of the blood treated with the modified membrane decreases significantly, demonstrating that the modified membrane possesses good carbon dioxide removal capacity. Therefore, the experiments prove that the hydrophobic protein-modified PMP hollow fiber membrane disclosed in this application has high selective adsorption performance for CO2 / O2, excellent carbon dioxide removal and oxygenation performance, and good blood compatibility, making it suitable for ECMO and ECCO2R systems. It is beneficial for improving the membrane lung's ability to remove CO2 from human blood and its antithrombotic capacity.
[0043] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.
Claims
1. A method for preparing a hydrophobin-based, bifunctional modified membrane, characterized by, The method comprises the following steps: Step 1, preparing a buffer solution containing EAS protein; Step 2, contacting the polymer membrane with the buffer solution containing EAS protein, and drying to obtain a bifunctional modified membrane.
2. The method for producing a hydrophobin-based bifunctional modified membrane according to claim 1, characterized by, The buffer solution is PBS solution, and the concentration of EAS protein in the buffer solution containing EAS protein ranges from 10 to 200 μg / ml.
3. The method for producing a hydrophobin-based bifunctional modified membrane according to claim 1, characterized by, The EAS protein is extracted by TSE method.
4. The method for producing a hydrophobin-based bifunctional modified membrane according to claim 1, characterized by, The extracted EAS protein is purified by nickel affinity chromatography, and the steps are as follows: first, equilibrating the column by dialysis with binding buffer, then, eluting with elution buffer after loading, collecting the target protein fraction, and filtering through a microporous filter.
5. The method for preparing a hydrophobin-based bifunctional modified membrane according to claim 1, characterized by, The composition of the binding buffer comprises 20-60 mM Tris-HCl, pH 6.0-8.0; and the elution buffer comprises 0.1-0.5 M imidazole, 10-50 mM Tris-HCl, 50-200 mM NaCl, pH 6.0-8.
0.
6. The method for preparing a hydrophobin-based bifunctional modified membrane according to claim 1, characterized by, In step 2, the contacting time is 12-96 h, and the temperature ranges from 20 to 60℃.
7. The method for producing a hydrophobin-based bifunctional modified membrane according to claim 1, characterized by, In step 2, the contacting time is 60-80 h, and the temperature ranges from 35 to 45℃.
8. An extracorporeal life support system, comprising: The gas-blood separation membrane is the bifunctional modified membrane.
9. The extracorporeal life support system of claim 8, wherein, The ECMO or ECCO2R system.
10. The extracorporeal life support system of claim 8, wherein, The bifunctional modified membrane is used for CO2 / O2 separation.