Functionalized nanofiber membrane and application thereof

Abstract

The invention provides a functionalized nanofiber membrane and application thereof, the surface of the functionalized nanofiber membrane contains functionalized protein, and the functionalized nanofiber membrane can at least remove one protein in a liquid substance; according to the functionalized nanofiber membrane disclosed by the invention, through collaborative design of nanofiber membrane structure advantages and functionalized protein specific effects, breakthrough is realized in the aspects of protein removal specificity, efficiency, stability and safety, the technical blank is effectively filled, the problems of an existing adsorption removal method are solved, and the application prospect is wide. The method has extremely high application value and market prospect in the fields of biomedicine, food industry, water purification, plasma filtration and the like.

Description

Technical Field

[0001] This invention relates to the field of membrane materials technology, and in particular to a functionalized nanofiber membrane and its applications. Background Technology

[0002] In autoimmune diseases, antibody-mediated transplant rejection, and some neurological diseases, abnormally elevated levels of pathogenic autoantibodies or allogeneic antibodies are key drivers of tissue damage and disease progression. While traditional plasma exchange can reduce circulating antibody levels, it indiscriminately loses important plasma components such as albumin and coagulation factors and requires large amounts of replacement fluid. Therefore, clinical practice tends to favor the development of in vitro immunoadsorption technology with high selectivity and the ability to continuously process large amounts of plasma to specifically remove antibodies from plasma.

[0003] To achieve efficient, safe, and specific antibody removal, research has shifted its focus to the preparation of "immunoadsorption membranes / membrane adsorbers" with proteins or biomimetic ligands as functional layers. This involves chemically grafting, coupling, or surface modification to covalently immobilize Protein A, autoantigens, or synthetic receptors on the surface or within the pores of a porous polymer membrane, constructing a protein-grafted separation membrane that combines rapid convective mass transfer with affinity recognition capabilities. This allows the membrane to achieve highly selective adsorption and controllable regeneration of IgG and specific pathogenic antibodies in plasma while maintaining high throughput and low pressure drop.

[0004] Currently, protein graft separation membranes have begun to be used in the fields of glomerular basement membrane disease, autoimmune encephalitis, Guillain-Barré syndrome, and organ transplant sensitization to specifically remove pathogenic antibodies from the patient's plasma. Related clinical and in vitro experiments have shown that these membrane adsorption devices can significantly reduce IgG and autoantibody titers, improve or stabilize clinical symptoms within a limited number of treatments, and have little impact on non-target proteins such as albumin and coagulation factors. This provides an important foundation for the future development of high-throughput, reusable, and even disease-personalized membrane immunoadsorption blood purification devices.

[0005] Currently, the biggest challenge for protein graft separation membranes to be truly used for "removing harmful antibody proteins from plasma" lies in: efficiently, stably, and repeatedly "capturing" pathogenic antibodies while avoiding activation of coagulation / complement and preventing secondary damage, and at the same time meeting the requirements of cost-effective, scalable production and strict clinical supervision.

[0006] CN120795166A discloses a bovine serum albumin-biotin-based immobilization carrier, its preparation method, and its application. The biological sample immobilization carrier includes a substrate and a composite layer of bovine serum albumin-conjugated biotin and neutral avidin immobilized on the substrate surface. The composite layer is attached to the substrate surface after co-incubation of the bovine serum albumin-conjugated biotin with the substrate, and then further incubated with neutral avidin to bind the neutral avidin to the biotin in the bovine serum albumin-conjugated biotin, thus obtaining the composite layer of bovine serum albumin-conjugated biotin and neutral avidin immobilized on the substrate surface. This carrier exhibits high biomolecule immobilization efficiency, good uniformity, strong adaptability, high signal-to-noise ratio, and simple operation.

[0007] CN105442319A discloses a method for preparing a nanofiber membrane material with a three-dimensional flower-like protein structure immobilized on its surface, comprising the following steps: (1) treating a PVA-co-PE nanofiber membrane with sodium hydroxide solution and then immersing it in a 1,4-dioxane solution of cyanuric chloride; (2) immersing the above-mentioned active PVA-co-PE nanofiber membrane in an aminopolyacid solution; (3) immersing the above-mentioned PVA-co-PE nanofiber membrane with aminopolyacid immobilized on its surface in a heavy metal solution; (4) immersing the above-mentioned PVA-co-PE nanofiber membrane containing metal ions on its surface in a protein solution to prepare a nanofiber membrane material with a three-dimensional flower-like protein structure immobilized on its surface. The protein immobilized on the surface of this nanofiber membrane not only has a special three-dimensional flower-like structure, but also retains the original biological activity of the protein, which is an effective protein immobilization method. The prepared material has important application value in the fields of biocatalysis and sensors. This membrane is mainly used in biocatalysis.

[0008] CN106693059B discloses a composite tissue repair patch, its preparation method, and its application. The composite tissue repair patch comprises: a nanofiber membrane, a woven mesh, and an adhesive material, wherein the adhesive material is located between the nanofiber membrane and the woven mesh; the adhesive material includes a hydrophilic substance, the nanofiber membrane and the woven mesh are bonded together by the adhesive material, and the adhesive material is embedded in the pores of the nanofiber membrane and the woven mesh; the hydrophilic substance includes: one or more protein compounds and their derivatives modified with carbodiimide, carbodiimide / N-hydroxysuccinimide, genipin, or aldehyde compounds; cellulose compounds and their derivatives modified with aldehyde compounds; and chitosan compounds and their derivatives modified with glycerol and water; the peel strength between the nanofiber membrane and the woven mesh is 20~75 cN / mm; the tensile strength of the composite tissue repair patch is 8~12.5 MPa; and the distance between the nanofiber membrane and the woven mesh is 0.1~3 mm. However, this invention mainly improves the mechanical properties and biocompatibility of composite tissue repair patches, but does not have the function of removing related proteins. In addition, the method uses proteins, cellulose or chitosan as matrix modifiers, that is, loading additional functional groups, including EDC / NHS, aldehyde groups, hydroxyl groups, etc., onto these intermediate substances. These functional groups provide the interaction with the target, rather than using the protein itself as an adsorption site.

[0009] Currently, there are few reports on protein graft separation membrane technology. Among the existing publicly available technologies, the membrane performance cannot meet the higher regulatory requirements in application. Therefore, the development of a new type of membrane material is of great significance in clinical practice. Summary of the Invention

[0010] The technical problem to be solved by this invention is that there is no existing technology that combines nanofiber membranes with protein applications to remove related proteins.

[0011] To address the aforementioned technical problems, the present invention provides a functionalized nanofiber membrane, wherein the surface of the nanofiber membrane contains functionalized proteins, and the functionalized nanofiber membrane is capable of removing at least one protein from a liquid substance.

[0012] This invention utilizes multi-nozzle electrospinning technology to prepare specific electrospun nanofiber membranes. Then, through surface chemical modification, selected proteins are loaded onto the surface of the nanofiber membrane. Based on the precise coordination of the specific proteins (or peptides) loaded on the membrane with target harmful antibodies, it is possible to selectively remove harmful antibodies from plasma. This coordination should be understood as hydrogen bonding interaction, primarily conformational coordination, similar to the binding of antibodies to antigens, and not merely an interaction at the chemical bond scale; otherwise, selectivity would not be possible. Therefore, high adsorption capacity and good adsorption selectivity are achieved, enabling the removal of proteins in different liquid states, especially harmful antibodies.

[0013] This invention achieves precise removal of liquid substances by controlling the connectivity rate of functionalized proteins on nanofiber membranes, with a removal rate exceeding 90%. The proteins in these liquid substances are generally harmful antibodies.

[0014] In this field, common proteins used in nanofibers include zein solution protein, gelatin, soy protein isolate, and whey protein isolate. Proteins are amphiphilic, and these proteins primarily serve as a matrix in the spinning process, influencing the properties of the prepared fiber materials. The application of these proteins mainly involves regulating the spinning process and the properties of the formed fibers, such as the conductivity and surface tension of the spinning solution, and the toughness and mechanical properties of the fiber material; they do not affect the filtration and adsorption properties of the nanofibers.

[0015] Preferably, the raw materials for preparing the nanofiber membrane include polyacrylonitrile and / or polyvinyl alcohol.

[0016] The nanofiber membrane of the present invention is prepared by electrospinning. The preparation of electrospun nanofibers utilizes, but is not limited to, single-nozzle electrospinning, dual-nozzle electrospinning, and multi-nozzle electrospinning apparatuses. Preferably, the preparation of electrospun nanofibers employs a multi-nozzle electrospinning apparatus.

[0017] Preferably, the concentration of the electrospinning solution is 8% to 13%; more preferably, the concentration of the electrospinning solution is 10%; preferably, the voltage of the electrospinning is 10 to 20 kV; more preferably, the voltage of the electrospinning is 16 kV; preferably, the flow rate of the electrospinning is 10 to 30 μL / min; more preferably, the flow rate of the electrospinning is 20 μL / min; preferably, the distance of the electrospinning is 10 to 20 cm; more preferably, the distance of the electrospinning is 15 cm.

[0018] Preferably, the functionalized protein includes bovine serum albumin (BSA).

[0019] Bovine serum albumin (BSA) molecules possess unique biocompatibility, hydrophilicity, ligand binding ability, and bioactivity, overcoming the functional deficiencies of traditional fiber membranes in applications such as biomedicine, food processing, and water purification. This endows fiber membranes with diverse properties including biorecognition, protein adsorption, antibacterial and antifouling capabilities, and biosensing. BSA, a widely available, inexpensive, and structurally stable globular protein (molecular weight approximately 66.5 kDa, isoelectric point approximately 4.7), contains numerous active functional groups such as amino, carboxyl, and hydroxyl groups, providing ample reaction sites for grafting reactions with fiber membrane substrates. By precisely controlling grafting process parameters, uniform loading, strong binding, and activity retention of BSA on the fiber membrane surface can be achieved, ultimately preparing composite fiber membrane materials that combine the mechanical properties of the substrate with the biological functions of BSA.

[0020] Preferably, the functionalized protein is attached to the nanofiber membrane surface by any one or a combination of at least two of the following methods: hydrolysis, cross-linking, or grafting.

[0021] Preferably, the hydrolysis includes alkaline hydrolysis and / or acidic hydrolysis. Alkaline hydrolysis is preferred.

[0022] The hydrolysis process can be described as follows: BSA hydrolysis is a modification process in which intact BSA molecules are degraded into small peptide chains with active fragments through controlled hydrolysis, and then the peptide chains are attached to the surface of the fiber membrane. The entire process can improve the binding efficiency of BSA to the membrane substrate, increase the surface active sites, and retain the biological functions of the peptide chains. It is suitable for scenarios with higher requirements for binding strength and active site density (such as high-precision immunoadsorption and long-term biosensing). The core processes are hydrolysis reaction, membrane surface pretreatment, peptide chain attachment, and post-treatment.

[0023] In this invention, the hydrolysis temperature is 30~100℃; more preferably, the hydrolysis temperature is 60℃; the hydrolysis time is 1-24 hours; more preferably, the hydrolysis time is 45 minutes.

[0024] Preferably, the crosslinking method includes any one or a combination of at least two of polyamine crosslinking, polyacid crosslinking, polyaldehyde crosslinking, or polyhydroxy crosslinking. Polyamine crosslinking is preferred.

[0025] In this invention, BSA crosslinking is a modification process that constructs a dual covalent network between BSA molecules and between BSA and the fibrous membrane substrate using a crosslinking agent. Utilizing the dual / multifunctional characteristics of the crosslinking agent, it simultaneously binds to the active sites on the BSA molecules and the substrate surface, forming a dense and stable crosslinked layer. Compared to direct grafting and hydrolytic bonding, this process significantly improves the mechanical strength, elution resistance, and structural stability of the BSA layer on the membrane surface, especially increasing the overall grafting rate of the nanofiber membrane. For example, glutaraldehyde can be used for polyaldehyde crosslinking, hexamethylenediamine can be used for polyamine crosslinking, and glutaric acid can be used for polyacid crosslinking. Other crosslinking agents such as genipor can also be used.

[0026] Preferably, the grafting method includes any one or a combination of at least two of the following: free radical grafting, cation grafting, anion grafting, chemical reaction grafting, or redox reaction grafting. Chemical reaction grafting is preferred.

[0027] In this invention, chemical grafting can be an amino-carboxyl condensation reaction: when the surface of the fiber membrane substrate (such as polyester fiber membranes made of polylactic acid, polycaprolactone, etc.) contains carboxyl groups, an activator can be used to activate the carboxyl groups to generate an active ester intermediate, which then undergoes a condensation reaction with the amino group in the BSA molecule to form an amide bond, thus achieving BSA grafting. This pathway has mild reaction conditions, can retain the bioactivity of BSA to the greatest extent, and the grafting efficiency is controllable. Cationic grafting, anionic grafting, etc., can be achieved through epoxy ring-opening reactions: epoxy groups are introduced onto the surface of the fiber membrane through plasma treatment, chemical oxidation, etc. The epoxy groups undergo a ring-opening reaction under acidic or alkaline conditions to generate hydroxyl or amino groups, which then react with the carboxyl or amino groups in the BSA molecule to form covalent bonds. This can significantly improve the surface activity of the substrate and the grafting strength of BSA. Thiol-double bond addition reaction: If monomers containing double bonds (such as acrylic acid and methyl methacrylate) are grafted onto the surface of the fiber membrane, the thiol groups contained in the cysteine ​​residues of the BSA molecule can undergo Michael addition or free radical addition reactions with the double bonds to achieve BSA grafting fixation. This pathway has high reaction specificity and can effectively control the grafting density of BSA on the membrane surface.

[0028] Preferably, the liquid substance includes any one of water, a chemical solution, or blood plasma. Blood plasma is preferred.

[0029] Preferably, the thickness of the functionalized nanofiber membrane is 50~500μm, for example, it can be 50μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm or 500μm, etc., preferably 100μm; the fiber diameter is 100~500nm, for example, it can be 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm or 500nm, etc., preferably 200nm.

[0030] On the other hand, the present invention provides an application of the functionalized nanofiber membrane described above in the removal of proteins from liquid substances.

[0031] Implementing this invention has the following beneficial effects:

[0032] This invention innovatively introduces functionalized proteins onto the surface of nanofiber membranes, achieving highly efficient removal of target proteins from liquid substances. Specifically, the effects are as follows:

[0033] This invention utilizes the specific interaction between functionalized proteins and target proteins to precisely identify and bind to target proteins in liquid substances, significantly reducing non-specific adsorption of other beneficial proteins or components in the system. Compared to traditional adsorption membrane materials that rely on non-specific adsorption for protein removal, this invention effectively avoids co-adsorption of target substances and impurities, greatly improving the purity of the removed liquid substance. It is particularly suitable for scenarios with high purity requirements, such as plasma, biological agents, and food liquid phases, and fills a gap in existing technologies.

[0034] In this invention, the nanofiber membrane itself possesses an ultra-large specific surface area and high porosity. Combined with surface-fixed functionalized proteins, a high-density active site can be constructed on the membrane surface. This shortens the contact path between the target protein and the active site, increasing the removal rate per unit time. Furthermore, it significantly improves the membrane's adsorption capacity for the target protein, reducing the amount of membrane material used and the frequency of replacement. Compared to conventional planar membranes or particulate adsorption materials, the functionalized nanofiber membrane of this invention significantly improves treatment efficiency, achieving a protein removal rate of over 90% and an adsorption capacity 1.5-3 times that of existing materials. It can adapt to the needs of continuous, large-scale liquid substance processing, reducing equipment investment and operating costs in industrial applications.

[0035] The functionalized nanofiber membrane of this invention achieves breakthroughs in the specificity, efficiency, stability, and safety of protein removal through the synergistic design of the advantages of nanofiber membrane structure and the specific effects of functionalized proteins. It effectively fills the technological gap and solves the problems of existing adsorption removal methods. It has extremely high application value and market prospects in the fields of biomedicine, food industry, water purification, and plasma filtration. Attached Figure Description

[0036] Figure 1 This is a graph showing the nitrogen content of the functionalized nanofiber membrane prepared in Example 1 using XPS. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] This embodiment provides a functionalized nanofiber membrane.

[0040] 10 g of polyacrylonitrile was dissolved in 90 g of DMF (dimethylformamide) by heating at 60 °C for 24 hours to prepare a 10% spinning solution. Spinning was performed using 30 mL of the polyacrylonitrile solution on a multi-nozzle electrospinning machine. The spinning voltage was 16 kV, the flow rate was 20 μL / min, the spinning distance was 15 cm, and the collecting roller speed was 60 rpm. The spun electrospun nanofiber membrane was hydrolyzed in a 1 M sodium hydroxide aqueous solution for 45 minutes, washed with water, and vacuum dried. The dried membrane was then subjected to a crosslinking reaction in 500 mL of a 0.7% polyethyleneamine aqueous solution at room temperature for 24 hours. The crosslinked polyacrylonitrile electrospun membrane was then immersed in a 1% chlorocholine chloride aqueous solution for chemical grafting at room temperature for 24 hours. After washing with water, it was vacuum dried at 60 °C for 24 hours.

[0041] A 10 cm × 10 cm electrospun membrane was placed in 100 mL of 2000 ppm BSA aqueous solution and stirred at room temperature for 24 hours. The membrane was then washed five times with deionized water and vacuum dried for 24 hours to obtain the functionalized nanofiber membrane.

[0042] Example 2

[0043] This embodiment provides a functionalized nanofiber membrane.

[0044] 10 g of polyvinyl alcohol was dissolved in 90 g of water and heated at 120 °C for 24 hours to prepare a 10% spinning solution. Triton 100 (0.2%) was added before spinning. Spinning was performed using 30 mL of the polyvinyl alcohol solution on a multi-nozzle electrospinning machine. The spinning voltage was 20 kV, the flow rate was 20 μL / min, the spinning distance was 15 cm, and the collecting roller speed was 60 rpm. The spun electrospun nanofiber membrane was placed in acetone, a few drops of concentrated hydrochloric acid were added, and then an equal weight of 50% glutaraldehyde aqueous solution was added for a crosslinking reaction for 6 hours. The membrane was then washed with water and vacuum dried.

[0045] A 10 cm × 10 cm electrospun membrane was placed in 100 mL of 2000 ppm BSA aqueous solution and stirred at room temperature for 24 hours. It was then washed thoroughly with deionized water five times and vacuum dried for 24 hours to obtain the functionalized nanofiber membrane.

[0046] Performance testing: The protein-grafted nanofiber membrane prepared in Example 1 was characterized using XPS, and the nitrogen content on its surface was measured. Figure 1 As shown. Based on the elemental content of the membrane surface, the grafting rate of the protein in Example 1 can be estimated to be 55 mg / g.

[0047] Example 3

[0048] This embodiment provides a functionalized nanofiber membrane.

[0049] Eight grams of polyacrylonitrile (PA) were dissolved in 82 grams of dimethylformamide (DMF) by heating at 60°C for 24 hours to prepare a spinning solution. Spinning was performed using 25 mL of the PA solution on a multi-nozzle electrospinning machine. The spinning voltage was 14 kV, the flow rate was 25 μL / min, the spinning distance was 15 cm, and the collecting roller speed was 60 rpm. The spun electrospun nanofiber membrane was hydrolyzed in 0.8 M sodium hydroxide aqueous solution for 45 minutes, washed with water, and vacuum dried. The dried membrane was then subjected to a crosslinking reaction in 450 mL of 0.6% polyethyleneamine aqueous solution at room temperature for 24 hours. The crosslinked PA electrospun membrane was then immersed in a 1% chlorocholine chloride aqueous solution for chemical grafting at room temperature for 22 hours. After washing with water, it was vacuum dried at 60°C for 24 hours.

[0050] A 10 cm × 10 cm electrospun membrane was placed in 100 mL of 2000 ppm BSA aqueous solution and stirred at room temperature for 24 hours. The membrane was then washed five times with deionized water and vacuum dried for 24 hours to obtain the functionalized nanofiber membrane.

[0051] Example 4

[0052] This embodiment provides a functionalized nanofiber membrane.

[0053] 12 g of polyacrylonitrile was dissolved in 88 g of DMF (dimethylformamide) by heating at 58 °C for 24 hours to prepare a spinning solution. Spinning was performed using 28 mL of the polyacrylonitrile solution on a multi-nozzle electrospinning machine. The spinning voltage was 18 kV, the flow rate was 22 μL / min, the spinning distance was 15 cm, and the collecting roller speed was 60 rpm. The spun electrospun nanofiber membrane was hydrolyzed in 0.8 M sodium hydroxide aqueous solution for 45 minutes, washed with water, and vacuum dried. The dried membrane was placed in 480 mL of 0.6% polyethyleneamine aqueous solution for crosslinking at room temperature for 24 hours. The crosslinked polyacrylonitrile electrospun membrane was then immersed in 1.2% chlorocholine chloride aqueous solution for chemical grafting at room temperature for 22 hours. After washing with water, it was vacuum dried at 60 °C for 24 hours.

[0054] A 10 cm × 10 cm electrospun membrane was placed in 100 mL of 2000 ppm BSA aqueous solution and stirred at room temperature for 24 hours. The membrane was then washed five times with deionized water and vacuum dried for 24 hours to obtain the functionalized nanofiber membrane.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A functionalized nanofiber membrane, characterized in that, The functionalized nanofiber membrane has functionalized proteins on its surface, and the functionalized nanofiber membrane is capable of removing at least one protein from a liquid substance; the functionalized protein is attached to the surface of the nanofiber membrane by any one or a combination of at least two of the following methods: hydrolysis, cross-linking, or grafting.

2. The functionalized nanofiber membrane according to claim 1, characterized in that, The raw materials for preparing the nanofiber membrane include polyacrylonitrile and / or polyvinyl alcohol.

3. The functionalized nanofiber membrane according to claim 1, characterized in that, The functionalized protein includes bovine serum albumin.

4. The functionalized nanofiber membrane according to claim 1, characterized in that, The hydrolysis includes alkaline hydrolysis and / or acidic hydrolysis.

5. The functionalized nanofiber membrane according to claim 1, characterized in that, The crosslinking method includes any one or a combination of at least two of polyamine crosslinking, polyacid crosslinking, polyaldehyde crosslinking, or polyhydroxy crosslinking.

6. The functionalized nanofiber membrane according to claim 1, characterized in that, The grafting method includes any one or a combination of at least two of the following: free radical grafting, cation grafting, anion grafting, chemical reaction grafting, or redox reaction grafting.

7. The functionalized nanofiber membrane according to claim 1, characterized in that, The liquid substance includes any one of water, chemical solution, or blood plasma.

8. The functionalized nanofiber membrane according to claim 1, characterized in that, The functionalized nanofiber membrane has a thickness of 50~500μm and a fiber diameter of 100~500nm.

9. The application of the functionalized nanofiber membrane according to claim 1 in the removal of proteins from liquid substances.

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