A biomimetic gradient composite membrane for hemodialysis and a preparation method thereof

Abstract

The present application relates to a kind of biomimetic gradient composite membrane for hemodialysis and its preparation method.The biomimetic gradient composite membrane includes dense layer, transition layer and loose layer in turn from blood contact side to dialysate side, and the preparation material is the combination of PES / PSBMA / CS.The present application prepares biomimetic gradient composite membrane by the following steps: PES and PSBMA mixed solution and CS solution are prepared dense layer, transition layer and loose layer respectively by overgradient casting method, and are crosslinked by glutaraldehyde vapor gradient.The scheme solves the problem of middle molecule toxin removal by triple synergistic innovation of material-structure-process, and provides core technical support for the development of new generation of hemodialysis device.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a biomimetic gradient composite membrane for hemodialysis and its preparation method. Background Technology

[0002] Hemodialysis has been widely used in the treatment of various diseases, including acute and chronic kidney disease, multiple organ failure, severe trauma, infection, systemic inflammatory response syndrome (SIRS), burns, acute pancreatitis, and chemical poisoning, becoming an important supportive measure in the treatment of various critical illnesses. Hemodialysis membranes utilize the principle of semipermeable membranes, relying on the combined effects of solute gradients, osmotic gradients, and pressure gradients across the membrane to remove toxins and excess water from the patient's body, while simultaneously replenishing necessary substances from the dialysate, thereby maintaining electrolyte and acid-base balance. To ensure optimal treatment, the development trend of hemodialysis membranes aims to further resemble the function of the human glomerular membrane. Therefore, the hemodialysis membrane is the core component of the hemodialysis process.

[0003] Traditional dialysis membranes, limited by their uniform pore size distribution and surface inertness, cannot simultaneously achieve high-efficiency clearance and biocompatibility. Dialysis membranes exhibit insufficient clearance (<40%) of medium-molecular-weight toxins (such as β2-microglobulin), easily leading to irreversible damage associated with dialysis-related amyloidosis and micro-inflammation triggered by the accumulation of molecular toxins. For example, existing polyethersulfone hemodialysis membrane materials suffer from steep retention characteristics, resulting in insufficient clearance of molecular toxins in the blood, thus limiting their application in hemodialysis and other related fields.

[0004] The glomerular basement membrane has a multi-layered structure with varying pore sizes, enabling selective filtration that allows medium-sized molecules to pass through while retaining large protein molecules. Biomimetic design needs to mimic this gradient pore size distribution, potentially transitioning from large pores on the blood-contact side to smaller pores on the other. Biomimetic gradient membranes could fundamentally reduce the incidence of dialysis-related amyloidosis (DRA), prolong patient survival, and lower healthcare costs (e.g., reducing DRA-related surgical costs by more than 30%), representing the next generation of technology for blood purification materials.

[0005] Simultaneously meeting the requirements of high retention rate (albumin retention) and high flux (removal of medium-molecular-weight toxins) in hemodialysis membranes, which necessitate a dense structure, presents a conflicting need in existing technologies. Therefore, a solution that balances the demands of the hemodialysis membrane field is urgently needed. This invention proposes, for the first time, a biomimetic gradient pore size design, which satisfies both requirements through structural innovation. Summary of the Invention

[0006] In view of the current state of technology, this invention provides a biomimetic gradient composite membrane for hemodialysis and its preparation method, which overcomes the problem that existing dialysis membranes are insufficient in removing molecular toxins from the blood, leading to dialysis-related amyloidosis (DRA), thus prolonging patient survival and reducing medical costs.

[0007] In a first aspect, the present invention provides a biomimetic gradient composite membrane, wherein the composite membrane comprises a dense layer, a transition layer and a porous layer sequentially from the blood contact side to the dialysate side, and the composite membrane is prepared from two or three of polyethersulfone (PES), polymethyl methacrylate sulfobetaine (PSBMA) or chitosan (CS).

[0008] The composite membrane is prepared using a combination of materials such as PES / CS, PES / PSBMA / CS, or PSBMA / CS, preferably a combination of PES / PSBMA / CS.

[0009] The mass concentration of the PES and PSBMA mixed solution is 12%–16%, and the mass concentration of CS is 4%–8%.

[0010] Furthermore, the PES and PSBMA mixed solution has a mass concentration of 12% in the loose layer, 14% in the transition layer, and 16% in the dense layer; the CS solution has a mass concentration of 8% in the loose layer, 6% in the transition layer, and 4% in the dense layer.

[0011] The composite membrane is also prepared using materials containing 0.1% sodium heparin, 0.5% nano-SiO2, and 1% Pluronic 127.

[0012] Secondly, the present invention provides a method for preparing a biomimetic gradient composite membrane, wherein a dense layer, a transition layer and a porous layer are prepared by gradient casting of a PES and PSBMA mixed solution and a CS solution, respectively, and then crosslinked by glutaraldehyde vapor gradient.

[0013] The preparation of the biomimetic gradient composite membrane according to this invention specifically includes the following steps:

[0014] (1) To prepare the loose layer, Pluronic F127 was pre-dissolved in deionized water at 60°C, and CS solution and PES / PSBMA solution were added. PEG6000 was added in three portions, with a 15-minute interval between each addition. The mixture was coated onto the nonwoven support layer and immersed in a 15°C deionized water coagulation bath. After a pre-gelling time of 10 seconds, the mixture was transferred to the main coagulation bath. The dip-coating method was used at a lifting speed of 2 mm / s to form a liquid film with a thickness of 80 ± 5 μm.

[0015] (2) Preparation of the transition layer: Nano-SiO2 was added to anhydrous ethanol and ultrasonically dispersed for 1 h at 40 kHz and 400 W to obtain a dispersion. The above dispersion was mixed with PES / PSBMA solution and CS solution, and PEG6000 was added. The mixture was stirred at 50 °C for 12 h to form a uniform milky white solution. The solution was then immersed in a 25 °C coagulation bath containing 5% acetic acid for 8 min. The solution was continuously coated on the surface of the pregel substrate using a doctor blade coating method, maintaining a gap height of 150 μm.

[0016] (3) To prepare a dense layer, CS solution and PES / PSBMA solution were mixed with 0.1% heparin sodium and magnetically stirred for 24 hours (500 rpm). Vacuum degassing was performed at -0.1 MPa for 30 minutes. The mixture was then immersed in a coagulation bath containing 10% ethanol at 35°C for 5 minutes. A liquid film with a thickness of 20 ± 2 μm was sprayed onto the surface of the transition layer using a pneumatic spraying process (nozzle diameter 0.3 mm, air pressure 0.2 MPa).

[0017] (4) Gradient crosslinking: glutaraldehyde vapor crosslinking (concentration gradient: 0.5% for the surface layer → 2% for the bottom layer), crosslinking time 2h, to stabilize pore size distribution.

[0018] Furthermore, the PES and PSBMA mixed solution has a mass concentration of 12% in the loose layer, 14% in the transition layer, and 16% in the dense layer; the CS solution has a mass concentration of 8% in the loose layer, 6% in the transition layer, and 4% in the dense layer; the PEG6000 mass concentration in the loose layer is 10%, and the PEG6000 mass concentration in the dense layer is 5%.

[0019] Thirdly, the application of the biomimetic gradient composite membrane of the present invention in the field of hemodialysis.

[0020] In summary, the present invention has the following beneficial effects:

[0021] 1. Structural innovation: A three-dimensional continuous gradient pore size (10-50nm) matching the glomerular basement membrane is established, and the pore size is precisely controlled (±3nm) through the synergistic effect of PES / PSBMA / CS concentration gradient and template agent;

[0022] 2. Improved Mechanical Properties: Existing technologies report that increased CS content inevitably leads to a decrease in mechanical strength. This invention breaks this perception through concentration gradient design, allowing the bottom high-CS (8%) region to provide flexible support, while the surface low-CS (4%) region maintains rigidity, thus improving the overall strength of the biomimetic gradient composite film.

[0023] 3. This solution systematically addresses the challenge of clearing medium-molecular-weight toxins through synergistic innovation in materials, structure, and process, providing core technological support for the development of next-generation hemodialysis machines. Attached Figure Description

[0024] Figure 1 Comparison of the β2-MG removal, albumin retention and lysozyme removal performance of the biomimetic gradient dialysis membrane of this invention. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. The embodiments provided by the present invention are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0026] This invention provides a biomimetic gradient composite membrane for hemodialysis that mimics the selective filtration characteristics of the glomerular basement membrane. It features a continuously changing pore size gradient and synergistic functional stratification. From the blood contact side to the dialysate side, it sequentially includes a dense layer for albumin retention, a transition layer, and a loose layer for removing medium-molecular-weight toxins.

[0027] Chitosan (CS) powder was purchased from Shaanxi Fubang Biotechnology Co., Ltd.; polyethersulfone (PES) particles were purchased from Shanghai Teflon New Materials Technology Co., Ltd.; methylpyrrolidone (NMP), dimethylacetamide (DMAC), polyethylene glycol 6000 (PEG6000), and polymethyl methacrylate sulfobetaine (PSBMA) powder were all purchased from Merck Life Sciences Materials Science; other reagents, solvents, and other experimental materials were all commercially available.

[0028] Example 1: Solution Preparation

[0029] CS powder was pre-dissolved in a 1% acetic acid aqueous solution and stirred at 40°C for 2 hours to prepare transparent CS solutions with mass concentrations of 2%, 5%, and 8%, respectively, for later use.

[0030] PES particles were added to a mixed solvent of NMP and DMAC and dissolved by ultrasonic vibration at 60°C (300W) for 3 hours to prepare PES solutions with mass concentrations of 12%, 15% and 18%, respectively, for later use.

[0031] Prepare PEG6000 concentrations of 5% and 10% by mass for later use;

[0032] PSBMA powder was added to a mixed solvent of methylpyrrolidone (NMP) and dimethylacetamide (DMAC) and dissolved by ultrasonic vibration at 60°C (300W) for 3 hours to prepare PES solutions with mass concentrations of 12%, 15% and 18%, respectively, for later use.

[0033] Example 2: Construction of a biomimetic gradient composite membrane

[0034] Table 1. Construction of PES / CS biomimetic gradient composite membrane

[0035]

[0036] 2.1 Preparation of a porous layer

[0037] Pluronic F127 (also known as poloxamer 407) was pre-dissolved in 10% deionized water at 60°C. An 8% CS solution and a 12% PES solution (prepared in Example 1) were slowly added. PEG6000 (10% concentration) was added in three portions, 15 minutes apart (to avoid excessively high local concentrations). The mixture was coated onto a nonwoven fabric support layer and immersed in a 15°C deionized water coagulation bath. After a pre-gel time of 10 seconds, it was transferred to the main coagulation bath. A liquid film with a thickness of 80±5 μm was formed using an dip-coating method at a speed of 2 mm / s. PEG6000 and Pluronic F127 synergistically created pores, and low-temperature delayed phase separation resulted in a 40-50 nm through-pore macroporous structure.

[0038] 2.2 Preparation of transition layer

[0039] Nano-SiO2 with a mass concentration of 0.5% and a particle size of 20 nm was added to anhydrous ethanol and ultrasonically dispersed for 1 h at 40 kHz and 400 W to obtain a dispersion. This dispersion was mixed with a 15% PES solution and a 5% CS solution, and then 5% PEG6000 was added. The mixture was stirred at 50 °C for 12 h to form a homogeneous milky white solution. The solution was then immersed in a 25 °C coagulation bath containing 5% acetic acid for 8 min. Using a doctor blade coating method, maintaining a gap height of 150 μm, the solution was continuously coated onto the surface of the pre-gelled substrate. The nano-SiO2 acted as nucleation sites during phase separation, and the pore size exhibited a Gaussian distribution (peak at 30 nm) by controlling the solvent diffusion rate.

[0040] 2.3 Preparation of dense layer

[0041] The 2% CS solution and 18% PES solution prepared in Example 1 were mixed at a volume ratio of 1:9, and 0.1% heparin sodium was added. The mixture was magnetically stirred for 24 hours (500 rpm). Vacuum degassing was then performed at -0.1 MPa for 30 minutes. The mixture was then immersed in a coagulation bath containing 10% ethanol at 35°C for 5 minutes. A liquid film with a thickness of 20 ± 2 μm was sprayed onto the transition layer surface using a pneumatic spraying process (nozzle diameter 0.3 mm, air pressure 0.2 MPa). Rapid non-solvent phase separation (NIPS) was initiated by a high ethanol content (50%) to form a dense, defect-free surface layer of 10-20 nm.

[0042] Stabilize the pore size distribution by using glutaraldehyde vapor crosslinking (concentration gradient: 0.5% for the surface layer → 2% for the bottom layer) for 2 hours.

[0043] Example 3: Construction of a biomimetic gradient composite membrane

[0044] Table 2. Construction of PES / PSBMA / CS biomimetic gradient composite membrane

[0045]

[0046] 3.1 Preparation of a loose layer

[0047] Pluronic F127 was pre-dissolved in 10% deionized water at 60°C. An 8% CS solution and a 12% PES / PSBMA solution prepared in Example 1 were slowly added. PEG6000 (10% concentration) was added in three portions, 15 minutes apart (to avoid excessively high local concentrations). The mixture was coated onto a nonwoven support layer and immersed in a 15°C deionized water coagulation bath. After a pre-gel time of 10 seconds, it was transferred to the main coagulation bath. A liquid film with a thickness of 80±5 μm was formed using an dip-coating method at a speed of 2 mm / s. PEG6000 and Pluronic F127 synergistically created pores, and low-temperature delayed phase separation resulted in a 40-50 nm through-pore macroporous structure.

[0048] 3.2 Preparation of transition layer

[0049] Nano-SiO2 with a mass concentration of 0.5% and a particle size of 20 nm was added to anhydrous ethanol and ultrasonically dispersed for 1 h at 40 kHz and 400 W to obtain a dispersion. This dispersion was mixed with a 14% PES / PSBMA solution and a 6% CS solution, and then 5% PEG6000 was added. The mixture was stirred at 50 °C for 12 h to form a homogeneous milky white solution. This solution was then immersed in a 25 °C coagulation bath containing 5% acetic acid for 8 min. Using a doctor blade coating method, maintaining a gap height of 150 μm, the nano-SiO2 was continuously coated onto the surface of the pre-gelled substrate. During phase separation, the nano-SiO2 acted as nucleation sites, and the pore size exhibited a Gaussian distribution (peak at 30 nm) by controlling the solvent diffusion rate.

[0050] 3.3 Preparation of dense layer

[0051] The 4% CS solution and 16% PES / PSBMA solution prepared in Example 1 were mixed with 0.1% heparin sodium and magnetically stirred for 24 hours (500 rpm). Vacuum degassing was then performed at -0.1 MPa for 30 minutes. The mixture was then immersed in a coagulation bath containing 10% ethanol at 35°C for 5 minutes. A liquid film with a thickness of 20 ± 2 μm was sprayed onto the transition layer surface using a pneumatic spraying process (nozzle diameter 0.3 mm, air pressure 0.2 MPa). Rapid non-solvent phase separation (NIPS) was initiated by a high ethanol content (50%) to form a dense, defect-free surface layer of 10-20 nm.

[0052] Stabilize the pore size distribution by using glutaraldehyde vapor crosslinking (concentration gradient: 0.5% for the surface layer → 2% for the bottom layer) for 2 hours.

[0053] Example 4: Construction of a biomimetic gradient composite membrane

[0054] Table 3. Construction of PSBMA / CS biomimetic gradient composite membrane

[0055]

[0056]

[0057] 4.1 Preparation of a loose layer

[0058] Pluronic F127 was pre-dissolved in 10% deionized water at 60°C. An 8% CS solution and a 12% PSBMA solution (prepared in Example 1) were slowly added. PEG6000 (10% concentration) was added in three portions, 15 minutes apart (to avoid excessively high local concentrations). The mixture was coated onto a nonwoven fabric support layer and immersed in a 15°C deionized water coagulation bath. After a pre-gel time of 10 seconds, it was transferred to the main coagulation bath. A liquid film with a thickness of 80±5 μm was formed using an dip-coating method at a speed of 2 mm / s. PEG6000 and Pluronic F127 synergistically created pores, and low-temperature delayed phase separation resulted in a 40-50 nm through-pore macroporous structure.

[0059] 2.2 Preparation of transition layer

[0060] Nano-SiO2 with a mass concentration of 0.5% and a particle size of 20 nm was added to anhydrous ethanol and ultrasonically dispersed for 1 h at 40 kHz and 400 W to obtain a dispersion. This dispersion was mixed with a 15% PSBMA solution and a 5% CS solution, and then 5% PEG6000 was added. The mixture was stirred at 50 °C for 12 h to form a homogeneous milky white solution. This solution was then immersed in a 25 °C coagulation bath containing 5% acetic acid for 8 min. Using a doctor blade coating method, maintaining a gap height of 150 μm, the nano-SiO2 was continuously coated onto the surface of the pre-gelled substrate. During phase separation, the nano-SiO2 acted as nucleation sites, and the pore size exhibited a Gaussian distribution (peak at 30 nm) by controlling the solvent diffusion rate.

[0061] 2.3 Preparation of dense layer

[0062] The 2% CS solution and 18% PSBMA solution prepared in Example 1 were mixed with 0.1% heparin sodium and magnetically stirred for 24 hours (500 rpm). Vacuum degassing was then performed at -0.1 MPa for 30 minutes. The mixture was then immersed in a coagulation bath containing 10% ethanol at 35°C for 5 minutes. A liquid film with a thickness of 20 ± 2 μm was sprayed onto the transition layer surface using a pneumatic spraying process (nozzle diameter 0.3 mm, air pressure 0.2 MPa). Rapid non-solvent phase separation (NIPS) induced by a high ethanol content (50%) was used to form a dense, defect-free surface layer of 10-20 nm.

[0063] Stabilize the pore size distribution by using glutaraldehyde vapor crosslinking (concentration gradient: 0.5% for the surface layer → 2% for the bottom layer) for 2 hours.

[0064] Example 5: Verification of Gradient Aperture

[0065] The pore size and distribution of the biomimetic gradient composite membrane of the present invention were determined using the bubble point method.

[0066] The biomimetic gradient composite membranes prepared in Examples 2-4 were vacuum impregnated (-0.1 MPa) in a Porefil wetting solution containing 0.1% Triton X-100 with a surface tension of 16 mN / m for 30 min to ensure that each layer of the gradient was completely wetted. The pore size data of each layer of the composite membrane were measured using a Porolux 1000 bubble point meter.

[0067] In Stage I, a pressure of 0-0.5 bar was applied to detect the macropores (40-50 nm) of the porous layer. Wetting fluid was injected into the porous layer chamber, followed by vacuum degassing (-0.1 MPa, 10 min). The pressure was gradually increased in 0.05 bar increments, and the flow rate was recorded after each pressure stabilization stage. When the flow rate suddenly increased to >10 μL / min, it was determined to be the bubble point (corresponding to the maximum pore size). The dominant pore size distribution was identified by the inflection point of the dQ / dP curve.

[0068] In stage II, a pressure of 0.5-2 bar was applied to detect the pores (20-40 nm) in the transition layer. The pressure was gradually increased in increments of 0.1 bar, and the flow rate after each pressure level stabilized was recorded.

[0069] The aperture distribution was calculated using differential analysis.

[0070] f(d i )=ΔQj / (∑ΔQi)×P i 2 / 4γcosθ (1)

[0071] The location of the main peak is determined by the extreme points of the second derivative.

[0072] In Stage III, a pressure of 2-5 bar is applied to detect the dense layer pores (10-20 nm), and a high-sensitivity mode (detection limit 0.1 μL / min) is used to capture minute flow rates.

[0073] Correcting gradient effects using the Washburn equation:

[0074] d=4γcosθ{1 / (1+0.05z1)}(z=0-1) (2)

[0075] Record the rupture pressure (the failure point of the membrane structure).

[0076] Record the pressure-flow curve and determine the dominant orifice diameter of each layer by the inflection point.

[0077]

[0078]

[0079] The bubble point method results show that the pore size of the dense layer of the biomimetic gradient composite membrane prepared by the present invention is 10-20 nm, the pore size of the transition layer is 20-40 nm, and the pore size of the loose layer is 40-50 nm.

[0080] Example 6 Mechanical Performance Verification

[0081] The burst strength was tested according to ISO 7198:2016 (burst pressure test of artificial blood vessels for cardiovascular implants). The burst strength of the biomimetic gradient composite membrane prepared by this invention is higher than 400 kPa. Moreover, the biomimetic gradient membrane prepared in Example 3 has a water flux increased by 50.0% compared with the traditional homogeneous membrane.

[0082] According to the pressure-driven permeation test of GB / T 32361-2015 "Test Methods for Ultrafiltration Membranes", the water flux of the biomimetic gradient composite membrane prepared in this invention is higher than 300 L / m³. 2 The biomimetic gradient membrane prepared in Example 3 has a water flux that is 38.9% higher than that of a conventional homogeneous membrane.

[0083] By simulating the mechanical fatigue damage to the membrane caused by the blood pump pulsation (0-0.5MPa, 1Hz) in clinical dialysis, the number of cycles of pressure resistance test was higher than 200 for the biomimetic gradient composite membrane prepared in this invention. Moreover, the biomimetic gradient membrane prepared in Example 3 has a water flux increased by 48.1% compared with the traditional homogeneous membrane.

[0084] Table 4 Comparison of mechanical properties of the biomimetic gradient composite membrane of the present invention with those of traditional homogeneous membranes, PES membranes, and CS membranes.

[0085]

[0086] According to existing technology reports (Reference 1: J.Membr.Sci.2020,600,117876), an increase in CS content inevitably leads to a decrease in mechanical strength. This invention breaks this perception through concentration gradient design. Compared with traditional homogeneous membranes, PES membranes, and CS membranes, the mechanical properties of Examples 2-4 of this invention are all improved to a certain extent. In particular, the biomimetic gradient composite membrane prepared in Example 3 has a bottom high-CS (8%) region providing flexible support, while the surface low-CS (4%) region maintains rigidity, resulting in an overall improved strength.

[0087] Example 7 Mass transfer performance verification

[0088] A 4-hour hemodialysis experiment was conducted on the biomimetic gradient composite membranes of Examples 2-4 and the traditional homogeneous membranes.

[0089] 7.1 Determination of β2-MG scavenging rate by HPLC

[0090] β2-MG clearance was determined by HPLC. The simulated dialysate was phosphate-buffered saline (PBS, pH 7.4) containing β2-MG (100 μg / mL) and supplemented with 0.1% BSA-simulated plasma environmental standard: recombinant human β2-MG (purity >95%).

[0091] First, plot the standard curve by diluting the β2-MG standard with PBS to concentrations of 0, 5, 10, 20, 50, and 100 μg / mL. Inject the standard solution sequentially and record the peak area (or peak height). Plot the standard curve (R²) with concentration on the x-axis and peak area on the y-axis. 2 ≥0.99).

[0092] The undiluted solution (C0) and residual blood solution (Cb) were injected separately. The β2-MG concentration in each sample was calculated based on the standard curve, using the formula shown below:

[0093] Sweep rate = (1-C0 / C) b )×100%(1)

[0094] 7.2 Determination of albumin retention rate using the BCA method

[0095] The albumin rejection rate of the biomimetic gradient composite membrane of this invention was determined using the BCA method. The simulated blood solution was phosphate-buffered saline (PBS, pH 7.4) containing bovine serum albumin (BSA, 40 g / L). First, a standard curve was plotted. 20 μL of each concentration of BSA standard solution (0, 0.2, 0.5, 1, 2, 4 mg / mL) was added to 2 mL of BCA working solution. After mixing, the mixture was incubated at 37°C for 30 minutes and then cooled to room temperature. A562 was measured using a spectrophotometer, and the data were recorded. A linear equation (R²) was fitted with BSA concentration as the x-axis and A562 as the y-axis. 2≥0.99).

[0096] The dialysis membrane was pretreated with PBS solution, and then a simulated dialysis process was performed on a simulated blood solution. The permeate was collected every 10 minutes for a total of 3 times (10, 20, and 30 minutes). Each time, 1 μL of the sample was labeled and the concentrations of the stock solution and the filtrate were determined. The albumin rejection rate was calculated.

[0097] The calculation formula is: Albumin retention rate (R%) = (1-C) / (R%) f / C0)×100%(2)

[0098] C0: BSA concentration in the stock solution (40 g / L, i.e., 40 mg / mL); Cf: BSA concentration in the filtrate (calculated using a standard curve).

[0099] 7.3 Determination of Lysozyme Scavenging Rate by Enzyme Activity Assay

[0100] The lysozyme clearance rate of the biomimetic gradient composite membrane of the present invention was determined by enzyme activity method. The lyophilized powder of Micrococcus lysodeikticus and the simulated blood solution were phosphate buffered saline (PBS, pH 7.4) containing lysozyme (0.5 mg / mL). The absorbance at 450 nm was measured by spectrophotometer.

[0101] The original concentration of the simulated blood solution was marked, the membrane was loaded into the ultrafiltration device, a transmembrane pressure of 100 mmHg was applied, the permeate was collected, and the residual liquid on the blood side after dialysis (C) was collected. b To determine the lysozyme concentration, dissolve the lyophilized micrococcus powder in buffer solution to a cell concentration of 0.25 mg / mL, and adjust the OD value accordingly. 600 Adjust to 0.6-0.8. Take 2.9 mL of substrate suspension and add 0.1 mL of the test sample (C0, C2). b Mix immediately and start timing; incubate at 37°C for 15 minutes. Stop the reaction by adding 1 mL of ice-cold buffer. Immediately measure the absorbance (OD) at 450 nm. 450 Record the decrease value (ΔOD) 450 ).

[0102] Plot a standard curve, prepare lysozyme standard solutions (0, 0.1, 0.2, 0.5, 1.0 μg / mL), and determine ΔOD following the steps described above. 450 Plotting lysozyme concentration on the x-axis, ΔOD 450 Plot the standard curve (R) with the ordinate as the vertical axis. 2 ≥0.99). The formula for calculating the lysozyme clearance rate is shown below:

[0103] Clearance rate (%) = (1-C) 0 / C b )×100%(3)

[0104] Wherein, C0: concentration of the original solution before dialysis (μg / mL); C b : Concentration of residual blood fluid after dialysis (μg / mL).

[0105] Table 5 Comparison of mass transfer performance of the biomimetic gradient composite membrane of the present invention with traditional homogeneous membranes, PES membranes, and CS membranes.

[0106]

[0107]

[0108] The biomimetic gradient composite membranes prepared by this invention all have a β2-MG scavenging rate higher than 60.0%, and the biomimetic gradient membrane prepared in Example 3 has a β2-MG scavenging rate of >80.0%, which is 84.7% higher than the β2-MG scavenging rate of traditional homogeneous membranes, 60.3% higher than PES membranes, and 67.7% higher than CS membranes.

[0109] The biomimetic gradient composite membranes prepared by this invention all have a β2-MG albumin retention rate higher than 90.0%, and the biomimetic gradient membrane prepared in Example 3 has an albumin retention rate of >95.0%, which is 10.6% higher than the albumin retention rate of traditional homogenized membranes, 8.7% higher than PES membranes, and 9.9% higher than CS membranes.

[0110] The biomimetic gradient composite membranes prepared by this invention all have a lysozyme removal rate of over 50.0%, and the biomimetic gradient membrane prepared in Example 3 has a lysozyme removal rate of >55.0%, which is 163.7% higher than that of traditional homogenized membranes, 42.9% higher than that of PES membranes, and 57.7% higher than that of CS membranes.

[0111] The biomimetic gradient composite membrane prepared by this invention exhibits improved β2-microglobulin clearance, albumin retention, and lysozyme clearance rates compared to traditional homogenized membranes. Furthermore, the biomimetic gradient composite membrane prepared in Example 3 demonstrates a β2-microglobulin clearance rate >80.0%, an albumin retention rate >95.0%, and a significantly improved lysozyme clearance rate compared to traditional homogenized membranes. Therefore, through synergistic innovation in materials, structure, and process, this invention systematically solves the problem of clearing medium-molecular-weight toxins.

[0112] The above embodiments are merely examples to clearly illustrate the present invention and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements derived from this invention should be included within the scope of protection of the claims of this invention.

Claims

1. A method for preparing a biomimetic gradient composite membrane, comprising the following steps: (1) To prepare a loose layer, Pluronic F127 was pre-dissolved in deionized water at 60°C, and chitosan CS solution and PES and / or PSBMA solution were added; PEG6000 with a mass concentration of 10% was added in three batches, with an interval of 15 min each time; the above mixed solution was coated on the non-woven fabric support layer and immersed in a 15°C deionized water coagulation bath. After a pre-gel time of 10 s, it was transferred to the main coagulation bath; the dip-lifting method was used with a lifting speed of 2 mm / s to form a liquid film with a thickness of 80 ± 5 μm. (2) Preparation of transition layer: Add nano-SiO2 to anhydrous ethanol and ultrasonically disperse for 1 h at 40 kHz and 400 W to obtain dispersion; mix the above dispersion with PES and / or PSBMA solution and CS solution, add 5% PEG6000 by mass, stir at 50℃ for 12 h to form a uniform milky white solution; use a doctor blade to coat the surface of the pregel bottom layer continuously while maintaining a gap height of 150 μm; immerse in a 25℃ coagulation bath containing 5% acetic acid for 8 min. (3) To prepare a dense layer, mix the CS solution with the PES and / or PSBMA solution, add 0.1% heparin sodium, and stir magnetically for 24 h; vacuum degassing treatment at -0.1 MPa pressure for 30 min; spray a liquid film with a thickness of 20±2 μm on the surface of the transition layer using a pneumatic spraying process; immerse in a coagulation bath containing 10% ethanol at 35℃ for 5 min. (4) Gradient crosslinking: Gradient crosslinking is performed using glutaraldehyde vapor. The concentration gradient of glutaraldehyde is 0.5% to 2% from the surface layer to the bottom layer. The crosslinking time is 2 hours to stabilize the pore size distribution and obtain the biomimetic gradient composite membrane.

2. The method for preparing the composite membrane according to claim 1, characterized in that, When using a mixed solution of PES and PSBMA and a CS solution to prepare a composite membrane, the mass concentration of the mixed solution of PES and PSBMA is 12% in the loose layer, 14% in the transition layer, and 16% in the dense layer; the mass concentration of the CS solution is 8% in the loose layer, 6% in the transition layer, and 4% in the dense layer.

3. The method for preparing the composite membrane according to claim 1, characterized in that: The mass concentration of the nano-SiO2 solution is 0.5%.

4. The method for preparing the composite membrane according to claim 1, characterized in that: The concentration of the Pluronic F127 solution is 1%.

5. The method for preparing a composite membrane according to claim 1, characterized in that: In step (3), the magnetic stirring speed is 500 rpm; and / or, the nozzle diameter is 0.3 mm and the air pressure is 0.2 MPa during pneumatic spraying.

6. A biomimetic gradient composite membrane, characterized in that, Prepared using the method described in any one of claims 1-5.

7. An application of the biomimetic gradient composite membrane according to claim 6 in the field of hemodialysis.

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