Nanofiber porous material for exosome separation and extraction and preparation method thereof

Abstract

The invention provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof.The method comprises the steps that a plurality of nanofiber suspensions are sequentially coated according to the average diameter in a stepped mode, nanofiber gradient membranes are prepared, then the faces, with the minimum average diameter, of the two nanofiber gradient membranes are attached, and the nanofiber porous material is obtained. The nanofiber composite membrane material is prepared; mixing the pre-crosslinking modified nanofiber foam liquid with a crosslinking agent, a chitosan aqueous solution and a polyethyleneimine aqueous solution to prepare a secondary pre-crosslinking modified nanofiber foam liquid; the nanofiber composite membrane material is placed on the surface of the nanofiber foam liquid subjected to secondary pre-crosslinking modification for directional freeze drying, and a nanofiber porous material is prepared; a gradient screening pore channel and aerogel multistage pore channel structure is constructed, and tough compounding of the two pore channels is realized through a nanofiber transition layer, so that the problem that a nanofiber membrane or an aerogel material with a single pore diameter is difficult to synergistically improve stage-by-stage screening and adsorption performance is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a nanofiber porous material for separating and extracting exosomes and a preparation method thereof. Background Art

[0002] Exosomes play an important role between cells. Exosomes are a specific subclass of extracellular vesicles secreted by most cell types, which play an important role in intercellular communication by transporting a variety of contents including proteins, mRNA, miRNA and DNA. This substance is closely related to the pathogenesis of most human malignancies. Therefore, exosomes not only help to better understand cell physiology and pathology, but also show great potential in translation into clinical applications, involving various aspects from diagnosis and prognosis to nucleic acid delivery and cancer treatment. Therefore, how to separate exosomes in a simple, efficient and economical way has become an urgent need for scientific research and clinical diagnosis. At present, a variety of exosome separation and purification technologies have been developed based on the biophysical properties of exosomes such as density, size and shape, and biochemical properties such as surface antibodies and biofilms, but standardized and large-scale clinical-grade exosome separation and purification methods are still lacking, which limits their clinical application.

[0003] The most commonly used ultracentrifugation method currently uses multiple steps of centrifugation to remove cells of different sizes and densities, cell fragments and large-sized microvesicles in sequence, and the exosomes obtained are of high purity; however, the processing time is long and the yield is low, which makes the exosomes easily destroyed. Polymers such as PEG are used to coat and precipitate exosomes, and the yield is more than 8 times that of ultracentrifugation, but the impurity content is high. Size exclusion chromatography separates exosomes by size interception, and there are problems such as ultrafiltration membranes or gels that separation media are prone to protein adhesion and pore blockage. Since the surface of exosomes carries universal transmembrane proteins such as CD63, CD9, and CD81, the specific binding between antibodies or aptamers and membrane proteins of exosomes can distinguish exosomes from microvesicles, significantly improving the separation purity of exosomes. The disadvantage is that the specific binding requires a sufficiently long action time. It can be seen that methods based on a single separation mechanism are difficult to meet the multiple requirements of exosome separation for high purity, high yield, no loss and rapidity. Integrating multiple separation mechanisms on the same platform has become a bold and effective attempt to solve the above problems.

[0004] In existing exosome separation methods, the porous membranes used for interception and specific adsorption have the problems of single structure and low specific surface area.

[0005] In view of this, it is necessary to design a nanofiber porous material and a preparation method thereof for exosome separation and extraction to solve the above problems. Summary of the invention

[0006] In view of the defects of the above-mentioned prior art, the purpose of the present invention is to provide a nanofiber porous material for exosome separation and extraction and a preparation method thereof, by constructing a gradient screening channel and an aerogel multi-level channel structure, and by constructing a nanofiber transition layer to achieve a strong and tough composite of the above two channels, thereby solving the problem that a single nanofiber membrane or aerogel material is difficult to synergistically improve the step-by-step screening and adsorption performance.

[0007] To achieve the above object, the present invention provides a method for preparing a nanofiber porous material for separation and extraction of exosomes, comprising the following steps:

[0008] S1, subjecting the sea phase polymer to a melt extrusion process to prepare a plurality of copolymer nanofibers having different average diameters;

[0009] S2, placing several copolymer nanofibers having different average diameters, polyethylene glycol, and a first cross-linking agent in an isopropanol aqueous solution, stirring and dispersing them, to obtain several nanofiber suspensions;

[0010] S3, coating the prepared nanofiber suspensions layer by layer on the surface of the support in the order of increasing or decreasing average diameters, and obtaining a nanofiber gradient membrane 1, i.e., a gradient pore filtration layer, after drying;

[0011] S4, repeating step S3 to obtain a second nanofiber gradient membrane, i.e., a nanofiber transition layer; and then laminating the side with the smallest average diameter of the first nanofiber gradient membrane with the side with the smallest average diameter of the second nanofiber gradient membrane, to obtain a nanofiber composite membrane material;

[0012] S5, mixing one of the copolymer nanofibers with a second crosslinking agent and dispersing the mixture, and obtaining a pre-crosslinked modified nanofiber foam liquid after the reaction is completed;

[0013] S6, mixing and stirring the pre-crosslinked modified nanofiber foam liquid, the third crosslinking agent, the chitosan aqueous solution, and the polyethyleneimine aqueous solution to obtain a secondary pre-crosslinked modified nanofiber foam liquid;

[0014] S7, placing the secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 in a directional freezing device, and then placing the nanofiber composite membrane material obtained in step S4 on the surface of the secondary pre-crosslinked modified nanofiber foam liquid, and performing directional freeze drying to obtain a nanofiber porous material that is not affinity modified; wherein the secondary pre-crosslinked modified nanofiber foam liquid is made into a nanofiber aerogel; and the nanofiber transition layer is located between the gradient pore filter layer and the nanofiber aerogel;

[0015] S8. Using an affinity modifier to perform several grafting modifications on the non-affinity-modified nanofiber porous material obtained in step S7, thereby obtaining the nanofiber porous material for exosome separation and extraction.

[0016] Furthermore, the sea phase polymer in step S1 includes a mixture of copolymer masterbatch and cellulose acetate butyrate; the copolymer masterbatch includes polyethylene-vinyl alcohol copolymer particles; the mass ratio of the polyethylene-vinyl alcohol copolymer particles to the cellulose acetate butyrate is 2:8 to 3:7; the drawing rate of the melt extrusion process is 18 to 30 m / min; the average diameter of the copolymer nanofibers ranges from 100 to 1000 nm.

[0017] Furthermore, in step S2, the first cross-linking agent includes a glutaraldehyde solution; the mass ratio of the copolymer nanofibers, the polyethylene glycol with a concentration of 1 to 15wt.%, and the glutaraldehyde solution with a concentration of 1 to 5wt.% is 1:5:5 to 1:15:15; the mass of isopropanol in the isopropanol aqueous solution is 45 to 50%; and the stirring speed is 1000 to 2000 rpm.

[0018] Furthermore, in step S3 and step S4, in the nanofiber gradient membrane 1 and the nanofiber gradient membrane 2, the average diameter of the copolymer nanofibers increases gradually from 100 nm to 1000 nm along the thickness direction, and the gradient value of the pore size in the thickness direction is 1 to 190 nm / μm.

[0019] Furthermore, in step S3, the coating method includes one of spray coating and spin coating.

[0020] Furthermore, in step S5 and step S6, the second cross-linking agent and the third cross-linking agent both include glutaraldehyde solution; in step S5, the mass ratio of the copolymer nanofibers to the glutaraldehyde solution with a concentration of 1 to 5 wt.% is 1:20 to 1:30; and the dispersion process is performed by using an emulsifier for 10 to 20 minutes.

[0021] Furthermore, in step S6, the volume ratio of the pre-crosslinked modified nanofiber foam liquid, the glutaraldehyde solution with a concentration of 1 to 5wt.%, the chitosan aqueous solution with a concentration of 2.5 to 5wt.%, and the polyethyleneimine aqueous solution with a concentration of 0.1 to 10wt.% is 1:0.3:2:0.5 to 1:0.1:1:0.1, and the stirring process is uniform stirring for 18 to 25 minutes.

[0022] Furthermore, the directional freeze-drying process in step S7 is: placing the secondary pre-cross-linked modified nanofiber foam liquid with the nanofiber composite membrane material on the surface on a metal block, and immersing the metal block in a solution with a temperature of -209°C to -40°C; the solution includes one of ethanol, isopropanol, and liquid nitrogen; and the time for the directional freeze-drying is 46 to 50 hours.

[0023] Further, in step S8, the affinity modifier includes: a spacer arm aqueous solution, an aptamer aqueous solution;

[0024] The several grafting modification processes include: the first grafting modification process is: placing the non-affinity-modified nanofiber porous material obtained in step S7 in a closed container with an inlet and an outlet, placing the spacer arm aqueous solution in an open container, entering the closed container through the inlet of the closed container by a peristaltic pump, then passing through the non-affinity-modified nanofiber porous material, and then flowing out through the outlet of the closed container, to graft-modify the non-affinity-modified nanofiber porous material; the second grafting modification process is: using the aptamer aqueous solution to perform the second grafting modification on the non-affinity-modified nanofiber porous material that has undergone the first grafting modification according to the steps of the first grafting modification;

[0025] The concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1 to 10 wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, adipaldehyde, pentamethylenediamine, and hexamethylenediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, CD81 with an amino or aldehyde terminal group, protein A, and protein G.

[0026] The present invention also provides a nanofiber porous material for exosome separation and extraction prepared by the preparation method, wherein the nanofiber porous material for exosome separation and extraction comprises the gradient pore filtration layer, the nanofiber transition layer whose side with the smallest average diameter is bonded to the side with the smallest average diameter of the gradient pore filtration layer, and the nanofiber aerogel connected to the side with the largest average diameter of the nanofiber transition layer.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention provides a nanofiber porous material for separation and extraction of exosomes and a preparation method thereof, wherein a plurality of nanofiber suspensions prepared from copolymer nanofibers, a crosslinking agent, and polyethylene glycol are coated layer by layer in the order of increasing or decreasing average diameters to prepare a nanofiber gradient membrane, and then two nanofiber gradient membranes are laminated with the sides with the smallest average diameters to prepare a nanofiber composite membrane material; at the same time, a pre-crosslinked modified nanofiber foam liquid prepared from copolymer nanofibers and a crosslinking agent is mixed with a crosslinking agent chitosan aqueous solution and a polyethyleneimine aqueous solution to prepare a secondary pre-crosslinked modified nanofiber foam liquid; and The nanofiber composite membrane material is then placed on the surface of the secondary pre-crosslinked modified nanofiber foam liquid for directional freeze-drying, thereby obtaining a nanofiber porous material for exosome separation and extraction. In this way, by constructing a gradient screening channel (i.e., a gradient channel filtration layer) and an aerogel multi-level channel (i.e., a structure formed by directional freeze-drying of the secondary pre-crosslinked modified nanofiber foam liquid), and by constructing a nanofiber transition layer to achieve a strong and tough composite of the above two channels, the problem that it is difficult for nanofiber membranes or aerogel materials with a single pore size to synergistically improve the step-by-step screening and adsorption performance is solved, and at the same time, efficient and low-consumption separation of exosomes is also achieved.

[0029] 2. The present invention provides a nanofiber porous material for separation and extraction of exosomes and a preparation method thereof. The nanofiber composite membrane material is composed of two nanofiber gradient membranes, so that the nanofiber gradient membrane on one side of the material can not only separate and intercept the particles in the treated liquid step by step, but also allow the specific molecules that are not intercepted to pass through, thereby realizing the screening of particles and molecules; on the other hand, the nanofiber gradient membrane on the other side of the material can be used for the subsequent composite of nanofiber aerogel, so as to realize the stable and effective composite and penetration of the pores in the membrane material that play an intercepting role and the macropores in the aerogel, thereby integrating the interception function and the specific adsorption function in the same material.

[0030] 3. The present invention provides a nanofiber porous material for separation and extraction of exosomes and a preparation method thereof. The nanofiber gradient membrane and nanofiber aerogel material involved have different surface chemical structures. Through cross-linking, the gradient membrane pore surface contains a cross-linked network of polyethylene glycol, which has an anti-adhesion function, can achieve low adhesion pollution during the gradient membrane interception process, and improve the interception and separation efficiency; through cross-linking and subsequent grafting modification, a specific adsorption structure is formed on the pore surface of the nanofiber aerogel, which can achieve specific adsorption and separation of exosomes by aerogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the structure of a nanofiber porous material for separation and extraction of exosomes provided by the present invention;

[0032] Figure 2A schematic diagram of the specific process of extracting exosomes using a nanofiber porous material for exosome separation and extraction provided in Example 1. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] It should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the scheme of the present invention are shown in the drawings, while other details that are not closely related to the present invention are omitted.

[0035] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0036] A method for preparing a nanofiber porous material for separation and extraction of exosomes comprises the following steps:

[0037] S1, subjecting the sea phase polymer to a melt extrusion process to prepare a plurality of copolymer nanofibers having different average diameters;

[0038] The sea phase polymer comprises a mixture of copolymer masterbatch and cellulose acetate butyrate; the copolymer masterbatch comprises polyethylene-vinyl alcohol copolymer particles; the mass ratio of the polyethylene-vinyl alcohol copolymer particles to the cellulose acetate butyrate is 2:8-3:7; the drawing rate of the melt extrusion process is 18-30 m / min; the average diameter of the copolymer nanofibers ranges from 100 to 1000 nm;

[0039] S2, placing several copolymer nanofibers having different average diameters, polyethylene glycol, and a first cross-linking agent in an isopropanol aqueous solution and stirring them to uniformly disperse them to obtain several nanofiber suspensions;

[0040] The first cross-linking agent includes a glutaraldehyde solution; the mass ratio of the copolymer nanofibers, the polyethylene glycol with a concentration of 1 to 15 wt.%, and the glutaraldehyde solution with a concentration of 1 to 5 wt.% is 1:5:5 to 1:15:15; the mass of isopropanol in the isopropanol aqueous solution is 45 to 50%; the stirring speed is 1000 to 2000 rpm;

[0041] S3, coating the prepared nanofiber suspensions layer by layer on the surface of the support in the order of increasing or decreasing average diameters, and drying at room temperature to obtain a nanofiber gradient membrane 1, i.e., a gradient pore filtration layer;

[0042] The coating method includes one of spray coating and spin coating; the support body includes a PP substrate with a length and width of 20×20 cm;

[0043] S4, repeating step S3 to obtain a second nanofiber gradient membrane, i.e., a nanofiber transition layer; and then laminating the side with the smallest average diameter of the first nanofiber gradient membrane with the side with the smallest average diameter of the second nanofiber gradient membrane, to obtain a nanofiber composite membrane material;

[0044] In step S3 and step S4, in the nanofiber gradient membrane 1 and the nanofiber gradient membrane 2, the average diameter of the copolymer nanofibers increases gradually from 100 nm to 1000 nm along the thickness direction, and the gradient value of the pore size in the thickness direction is 1 to 190 nm / μm.

[0045] The nanofiber composite membrane material has a structure with large average diameters at the top and bottom and small average diameter in the middle;

[0046] S5, mixing one of the copolymer nanofibers with a second crosslinking agent, and dispersing the mixture for 10 to 20 minutes using an emulsifier, and obtaining a pre-crosslinked modified nanofiber foam liquid after the reaction is completed;

[0047] The second cross-linking agent includes a glutaraldehyde solution; the mass ratio of the copolymer nanofibers to the glutaraldehyde solution with a concentration of 1 to 5 wt.% is 1:20 to 1:30;

[0048] S6, mixing the pre-crosslinked modified nanofiber foam liquid, the third crosslinking agent, the chitosan aqueous solution, and the polyethyleneimine aqueous solution, and stirring at a uniform speed for 18 to 25 minutes to obtain a secondary pre-crosslinked modified nanofiber foam liquid;

[0049] The third cross-linking agent includes glutaraldehyde solution;

[0050] The volume ratio of the pre-crosslinked modified nanofiber foam liquid, the glutaraldehyde solution with a concentration of 1 to 5 wt.%, the chitosan aqueous solution with a concentration of 2.5 to 5 wt.%, and the polyethyleneimine aqueous solution with a concentration of 0.1 to 10 wt.% is 1:0.3:2:0.5 to 1:0.1:1:0.1;

[0051] S7, placing the secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 in a directional freezing device, and then placing the nanofiber composite membrane material obtained in step S4 on the surface of the secondary pre-crosslinked modified nanofiber foam liquid, and performing directional freeze drying for 46 to 50 hours to obtain a nanofiber porous material that is not affinity-modified; at this time, the secondary pre-crosslinked modified nanofiber foam liquid is treated to become a nanofiber aerogel; the nanofiber transition layer is located between the gradient pore filter layer and the nanofiber aerogel;

[0052] The directional freeze-drying process is as follows: placing the secondary pre-crosslinked modified nanofiber foam liquid with the nanofiber composite membrane material on the surface on a metal block, and immersing the metal block in a solution with a temperature of -209°C to -40°C; the solution includes one of ethanol, isopropanol, and liquid nitrogen;

[0053] S8. Using an affinity modifier to perform several grafting modifications on the non-affinity-modified nanofiber porous material obtained in step S7, thereby obtaining the nanofiber porous material for exosome separation and extraction.

[0054] Such a setting can solve the problem that nanofiber membranes or aerogel materials with a single pore size are difficult to synergistically improve the step-by-step screening and adsorption performance, and also achieve efficient and low-cost separation of exosomes. In addition, the nanofiber gradient membrane and nanofiber aerogel materials involved have different surface chemical structures. Through cross-linking, the surface of the gradient membrane pores contains a cross-linked network of polyethylene glycol, which has an anti-adhesion function and can achieve low adhesion pollution during the gradient membrane interception process and improve the interception and separation efficiency; through cross-linking and subsequent grafting modification, the pore surface of the nanofiber aerogel forms a specific adsorption structure, which can achieve specific adsorption and separation of aerogels on exosomes.

[0055] Specifically, in some embodiments of the present invention, in step S8, the affinity modifier includes: a spacer arm aqueous solution, an aptamer aqueous solution;

[0056] The several grafting modification processes include: the first grafting modification process is: placing the non-affinity-modified nanofiber porous material obtained in step S7 in a closed container with an inlet and outlet, placing the spacer arm aqueous solution in an open container, entering the closed container through the inlet of the closed container by a peristaltic pump, then passing through the non-affinity-modified nanofiber porous material, and then flowing out through the outlet of the closed container, and grafting the non-affinity-modified nanofiber porous material at room temperature for 0.5 to 2 hours; the second grafting modification process is: using the aptamer aqueous solution to perform the second grafting modification on the non-affinity-modified nanofiber porous material that has undergone the first grafting modification according to the steps of the first grafting modification for 0.5 to 2 hours;

[0057] The concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1 to 10 wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, adipaldehyde, pentamethylenediamine, and hexamethylenediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, CD81 with an amino or aldehyde terminal group, protein A, and protein G.

[0058] In this way, the non-affinity-modified nanofiber porous material is first grafted and modified using a spacer arm aqueous solution to facilitate further grafting and affinity adsorption in the later stage. Subsequently, a ligand molecule is grafted on the other end of the spacer arm molecule for a second grafting modification, thereby satisfying the specific adsorption and separation of specific exosomes.

[0059] The present invention also provides a nanofiber porous material for exosome separation and extraction prepared by the preparation method, wherein the nanofiber porous material for exosome separation and extraction comprises the gradient pore filtration layer, the nanofiber transition layer whose side with the smallest average diameter is bonded to the side with the smallest average diameter of the gradient pore filtration layer, and the pre-cross-linked modified nanofiber foam liquid connected to the side with the largest average diameter of the nanofiber transition layer.

[0060] Such an arrangement enables the nanofiber gradient membrane on one side of the material to not only separate and intercept the particles in the treated liquid step by step, but also allow the specific molecules that are not intercepted to pass through, thereby realizing the screening of particles and molecules; on the other hand, the nanofiber gradient membrane on the other side of the material can be used for the subsequent composite of nanofiber aerogel, thereby realizing the stable and effective composite and penetration of the pores in the membrane material that play an intercepting role and the macropores in the aerogel, thereby integrating the interception function and the specific adsorption function in the same material.

[0061] The following is a detailed description of the nanofiber porous material for exosome separation and extraction and the preparation method thereof provided by the present invention in conjunction with the embodiments:

[0062] Example 1

[0063] This embodiment provides a nanofiber porous material for separation and extraction of exosomes and a preparation method thereof, which specifically includes the following steps:

[0064] A nanofiber porous material for separation and extraction of exosomes and a preparation method thereof, comprising the following steps:

[0065] S1, subjecting the sea phase polymer to a melt extrusion process to prepare a plurality of copolymer nanofibers having different average diameters;

[0066] The sea phase polymer comprises a mixture of copolymer masterbatch and cellulose acetate butyrate; the copolymer masterbatch comprises polyethylene-vinyl alcohol copolymer particles; the mass ratio of the polyethylene-vinyl alcohol copolymer particles to the cellulose acetate butyrate is 2:8; the temperature distribution of each zone of the melt extrusion process is: 180°C, 215°C, 225°C, 220°C, 210°C, 180°C; the drawing rate of the melt extrusion process is 18-30m / min; the average diameter of the prepared copolymer nanofiber is: 100, 200nm, ... 900nm, 1000nm;

[0067] S2, placing several kinds of the copolymer nanofibers, polyethylene glycol with a concentration of 10wt.%, and glutaraldehyde solution with a concentration of 3wt.% in an isopropanol aqueous solution at a mass ratio of 1:10:10, stirring to make them uniformly dispersed, and obtaining several kinds of nanofiber suspensions;

[0068] The mass of isopropanol in the isopropanol aqueous solution is 48%; the stirring speed is 1500 rpm;

[0069] S3, spraying the prepared nanofiber suspensions layer by layer on one side of a PP substrate having a length and width of 20×20 cm in the order of increasing average diameter, and drying at room temperature to obtain a nanofiber gradient membrane 1, i.e., a gradient pore filtration layer;

[0070] S4, spraying the plurality of nanofiber suspensions on the other side of the PP substrate in a layer-by-layer coating manner as described in step S3, and drying at room temperature to obtain a second nanofiber gradient membrane, i.e., a nanofiber transition layer; and then laminating the side with the smallest average diameter of the first nanofiber gradient membrane with the side with the smallest average diameter of the second nanofiber gradient membrane to obtain a nanofiber composite membrane material;

[0071] In step S3 and step S4, in the nanofiber gradient membrane 1 and the nanofiber gradient membrane 2, the average diameter of the copolymer nanofibers increases gradually from 100 nm to 1000 nm along the thickness direction, and the gradient value of the pore size in the thickness direction is 100 nm / μm;

[0072] S5, mixing the copolymer nanofibers with an average diameter of 500 nm and a glutaraldehyde solution with a concentration of 3 wt.% at a mass ratio of 1:25, and dispersing them with an emulsifier for 15 minutes. After the reaction is completed, a pre-crosslinked modified nanofiber foam liquid is obtained;

[0073] S6, mixing the pre-crosslinked modified nanofiber foam liquid, a 3wt.% glutaraldehyde solution, a 3wt.% chitosan aqueous solution, and a 5wt.% polyethyleneimine aqueous solution in a volume ratio of 1:0.3:2:0.5, and stirring at a constant speed for 20 minutes to obtain a secondary pre-crosslinked modified nanofiber foam liquid;

[0074] S7, placing the secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 in a directional freezing device, and then placing the nanofiber composite membrane material obtained in step S4 on the surface of the secondary pre-crosslinked modified nanofiber foam liquid, and performing directional freeze drying for 48 hours to obtain a nanofiber porous material that is not affinity-modified; wherein the secondary pre-crosslinked modified nanofiber foam liquid is made into a nanofiber aerogel, and the nanofiber transition layer is located between the gradient pore filter layer and the nanofiber aerogel;

[0075] The directional freeze-drying process is as follows: placing the secondary pre-crosslinked modified nanofiber foam liquid with the nanofiber composite membrane material on the surface on a metal block, and immersing the metal block in liquid nitrogen at a temperature of -100°C;

[0076] S8, placing the non-affinity-modified nanofiber porous material obtained in step S7 in a closed container with inlets and outlets at both ends that completely match its shape, placing a 5wt.% glutaraldehyde aqueous solution in an open container, entering the closed container through the inlet of the closed container by a peristaltic pump, then passing through the non-affinity-modified nanofiber porous material, and then flowing out through the outlet of the closed container, to graft-modify the non-affinity-modified nanofiber porous material; then using a 5wt.% CD63 aptamer with an amine end group to perform the second grafting modification on the non-affinity-modified nanofiber porous material that has undergone the first grafting modification according to the aforementioned grafting modification steps; then rinsing it with pure water and drying it to obtain the nanofiber porous material for exosome separation and extraction.

[0077] In this embodiment, the nanofiber porous material for exosome separation and extraction is prepared by the above-mentioned preparation method, such as Figure 2 As shown, the specific process of the porous material for extracting exosomes is as follows: (1) the porous material is used to screen and adsorb the biological culture fluid containing exosomes to obtain the removal of impurity proteins and small-sized microvesicles; (2) cells, cell fragments and large-sized microvesicles are obtained by backwashing; (3) high-purity exosome components are obtained by elution. In this embodiment, exosomes with a particle size distribution in the range of 30-150nm can be obtained, the impurity content is 0.2wt.%, and the separation speed of the whole process is about 15min / L (biological culture fluid) / g (porous material).

[0078] Example 2

[0079] Example 2 provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof, which is different from Example 1 in that the diameter range of the copolymer nanofiber prepared in step S1 is different, and the range of the copolymer nanofiber prepared in Example 2 is 100, 200, 300, 400, 500 nm. The remaining steps are consistent with Example 1 and are not repeated here.

[0080] Example 3

[0081] Example 3 provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof, which is different from Example 1 in that the diameter of the copolymer nanofiber used in step S5 is different. The diameter of the copolymer nanofiber used in Example 3 is 100 nm. The remaining steps are consistent with Example 1 and are not repeated here.

[0082] Example 4

[0083] Example 4 provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof, which is different from Example 1 in that the diameter of the copolymer nanofiber used in step S1 is different. The diameter range of the copolymer nanofiber used in Example 4 is 200, 300, ..., 900, 1000 nm. The remaining steps are consistent with Example 1 and are not repeated here.

[0084] Comparative Example 1

[0085] This comparative example provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof, which is different from Example 1 in that the diameter range of the copolymer nanofiber prepared in step S1 is different, and the range of the copolymer nanofiber prepared in Comparative Example 1 is 100, 200, ... 1400, 1500 nm. The remaining steps are consistent with Example 1 and are not repeated here.

[0086] Comparative Example 2

[0087] Comparative Example 2 provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof, which is different from Example 1 in that the order of coating the average diameters of the several nanofiber suspensions described in step S3 is different. The several nanofiber suspensions described in Comparative Example 2 are coated in an irregular manner according to the average diameter. The remaining steps are consistent with Example 1 and are not repeated here.

[0088] Comparative Example 3

[0089] Comparative Example 3 provides a nanofiber porous material for exosome separation and extraction and a preparation method thereof, which is different from Example 1 in that the diameter range of the copolymer nanofiber used in step S1 is different. The diameter ranges of the copolymer nanofibers used in Comparative Example 3 are 50nm, 100nm, 200nm, ..., 900nm, and 1000nm. The remaining steps are consistent with Example 1 and are not repeated here.

[0090] The changes in parameters in each step of the embodiment and the comparative example and the effect on the extraction of exosomes are shown in Table 1. Comparing the experimental results of Examples 1 to 2 and Comparative Example 1, it is found that when the size range of the copolymer nanofibers used in step S1 is within the range of 100 to 1000 nm, and the pore size is reduced from top to bottom in the gradient membrane 1, and the smallest pore is about 100 nm, the purity of the obtained exosomes is high, and the particle size range is relatively complete (30-150 nm); the difference is that when the fiber diameter of the top layer is too small (<1000 nm, Example 2), the gradient membrane 1 has a poor degree of stepwise interception of particles of different sizes in the biological culture medium, and the particles are easily accumulated in one layer, which is easy to block the pores, resulting in a slow separation speed; and when the fiber diameter of this layer is too large (>1000 nm, Comparative Example 1), the amount of porous material increases, resulting in an increase in the mass of the porous material, which makes the separation speed of the unit mass of the porous material decrease.

[0091] By comparing the experimental results of Example 1, Example 4 and Comparative Example 3, it is found that when the minimum size of the copolymer nanofiber used in step S1 is less than 100 nm (Comparative Example 3), large-sized exosomes will be intercepted, and the diameter range of the separated exosomes will become smaller, which will increase the loss of exosomes, and the minimum pore size will become smaller, the flux will decrease, and the separation speed will be significantly reduced; when the minimum fiber diameter is greater than 100 nm (Example 4), more exosomes or cellular impurities of other sizes are not intercepted, which increases the impurity content in the exosomes. Although the separation speed is faster at this time, the purity of the exosomes is reduced, which is not worth the loss.

[0092] By comparing the experimental results of Example 1 and Example 3, it was found that when the diameter of the copolymer nanofiber used in step S5 was smaller, although the specific surface area of ​​the downstream aerogel increased and the functional groups that could specifically adsorb exosomes increased, the purity of the exosomes was not significantly improved, and the spacing between the fibers on the aerogel pore wall decreased (i.e., the size of the pores on the pore wall was smaller), which reduced the effect of liquid convection and thus reduced the separation speed.

[0093] By comparing the experimental results of Example 1 and Comparative Example 2, it is found that when the method of "average diameter coating in gradient order" is adopted in step S3, the gradient membrane of the nanofiber porous material obtained for separation and extraction of exosomes has a pore size from large to small from top to bottom, which can intercept large-sized particles first and then intercept small-sized particles, and it is not easy to block the pores of each layer, which not only realizes the step-by-step interception of particles of different sizes, but also makes it easier to keep the pores unobstructed, and the flux is not easy to decrease, that is, the separation is faster. However, when the size of the copolymer nanofiber is lower than this range when the method of "average diameter irregular coating" is adopted, it is difficult for the nanofiber porous material obtained for separation and extraction of exosomes to form a gradient pore size that gradually decreases from top to bottom, and it will not be able to better achieve the step-by-step interception of particles of different sizes, the pores are easily blocked, and the separation speed is greatly reduced.

[0094] This shows that the effects of the prepared fiber-based porous affinity separation material with a periodic necking channel structure are all the result of the effects of various parameters.

[0095] Table 1 Parameter comparison of the examples and comparative examples and the effect of exosome extraction

[0096]

[0097]

[0098] In summary, the present invention provides a nanofiber porous material for separation and extraction of exosomes and a preparation method thereof. The nanofiber gradient membrane is prepared by coating a plurality of nanofiber suspensions prepared by copolymer nanofibers, a crosslinking agent, and polyethylene glycol in the order of increasing or decreasing average diameters, and then the two nanofiber gradient membranes are laminated with the sides with the smallest average diameters to prepare a nanofiber composite membrane material; at the same time, a pre-crosslinked modified nanofiber foam liquid prepared by copolymer nanofibers and a crosslinking agent is mixed with a crosslinking agent chitosan aqueous solution and a polyethyleneimine aqueous solution to prepare a secondary pre-crosslinked nanofiber foam liquid. cross-linked modified nanofiber foam liquid; then placing the nanofiber composite membrane material on the surface of the secondary pre-cross-linked modified nanofiber foam liquid for directional freeze-drying, the nanofiber porous material for exosome separation and extraction can be obtained; in this way, by constructing a gradient screening channel (i.e., a gradient channel filtration layer) and an aerogel multi-level channel structure, and by constructing a nanofiber transition layer to achieve a strong and tough composite of the above two channels, the problem that nanofiber membranes or aerogel materials with a single pore diameter are difficult to synergistically improve the step-by-step screening and adsorption performance is solved, and at the same time, the efficient and low-consumption separation of exosomes is also achieved.

[0099] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a nanofiber porous material for separation and extraction of exosomes, characterized in that: The following steps are involved: S1, subjecting the sea phase polymer to a melt extrusion process to prepare a plurality of copolymer nanofibers having different average diameters; S2, placing several copolymer nanofibers having different average diameters, polyethylene glycol, and a first cross-linking agent in an isopropanol aqueous solution, stirring and dispersing them, to obtain several nanofiber suspensions; S3, coating the prepared nanofiber suspensions layer by layer on the surface of the support in the order of increasing or decreasing average diameters, and obtaining a nanofiber gradient membrane 1, i.e., a gradient pore filtration layer, after drying; S4, repeating step S3 to obtain a second nanofiber gradient membrane, i.e., a nanofiber transition layer; and then laminating the side with the smallest average diameter of the first nanofiber gradient membrane with the side with the smallest average diameter of the second nanofiber gradient membrane, to obtain a nanofiber composite membrane material; S5, mixing one of the copolymer nanofibers with a second crosslinking agent and dispersing the mixture, and obtaining a pre-crosslinked modified nanofiber foam liquid after the reaction is completed; S6, mixing and stirring the pre-crosslinked modified nanofiber foam liquid, the third crosslinking agent, the chitosan aqueous solution, and the polyethyleneimine aqueous solution to obtain a secondary pre-crosslinked modified nanofiber foam liquid; S7, placing the secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 in a directional freezing device, and then placing the nanofiber composite membrane material obtained in step S4 on the surface of the secondary pre-crosslinked modified nanofiber foam liquid, and performing directional freeze drying to obtain a nanofiber porous material that is not affinity modified; wherein the secondary pre-crosslinked modified nanofiber foam liquid is made into a nanofiber aerogel, and the nanofiber transition layer is located between the gradient pore filter layer and the nanofiber aerogel; S8. Using an affinity modifier to perform several grafting modifications on the non-affinity-modified nanofiber porous material obtained in step S7, thereby obtaining the nanofiber porous material for exosome separation and extraction.

2. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: The sea phase polymer in step S1 includes a mixture of copolymer masterbatch and cellulose acetate butyrate; the copolymer masterbatch includes polyethylene-vinyl alcohol copolymer particles; the mass ratio of the polyethylene-vinyl alcohol copolymer particles to the cellulose acetate butyrate is 2:8 to 3:7; the drawing rate of the melt extrusion process is 18 to 30 m / min; the average diameter of the copolymer nanofibers ranges from 100 to 1000 nm.

3. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: In step S2, the first cross-linking agent includes a glutaraldehyde solution; the mass ratio of the copolymer nanofibers, the polyethylene glycol with a concentration of 1 to 15wt.%, and the glutaraldehyde solution with a concentration of 1 to 5wt.% is 1:5:5 to 1:15:15; the mass of isopropanol in the isopropanol aqueous solution is 45 to 50%; and the stirring speed is 1000 to 2000 rpm.

4. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: In step S3 and step S4, in the nanofiber gradient membrane 1 and the nanofiber gradient membrane 2, the average diameter of the copolymer nanofibers increases gradually from 100 nm to 1000 nm along the thickness direction, and the gradient value of the pore size in the thickness direction is 1 to 190 nm / μm.

5. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: In step S3, the coating method includes one of spray coating and spin coating.

6. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: In step S5 and step S6, the second crosslinking agent and the third crosslinking agent both include glutaraldehyde solution; in step S5, the mass ratio of the copolymer nanofibers to the glutaraldehyde solution with a concentration of 1 to 5 wt.% is 1:20 to 1:30; and the dispersion process is performed by using an emulsifier for 10 to 20 minutes.

7. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 6, characterized in that: In step S6, the volume ratio of the pre-crosslinked modified nanofiber foam liquid, the glutaraldehyde solution with a concentration of 1 to 5wt.%, the chitosan aqueous solution with a concentration of 2.5 to 5wt.%, and the polyethyleneimine aqueous solution with a concentration of 0.1 to 10wt.% is 1:0.3:2:0.5 to 1:0.1:1:0.1, and the stirring process is uniform stirring for 18 to 25 minutes.

8. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: The directional freeze-drying process in step S7 is as follows: placing the secondary pre-crosslinked modified nanofiber foam liquid with the nanofiber composite membrane material on the surface on a metal block, and immersing the metal block in a solution with a temperature of -209°C to -40°C; the solution includes one of ethanol, isopropanol, and liquid nitrogen; and the directional freeze-drying time is 46 to 50 hours.

9. The method for preparing a nanofiber porous material for exosome separation and extraction according to claim 1, characterized in that: In step S8, the affinity modifier includes: a spacer arm aqueous solution and an aptamer aqueous solution; The several grafting modification processes include: the first grafting modification process is: placing the non-affinity-modified nanofiber porous material obtained in step S7 in a closed container with an inlet and an outlet, placing the spacer arm aqueous solution in an open container, entering the closed container through the inlet of the closed container by a peristaltic pump, then passing through the non-affinity-modified nanofiber porous material, and then flowing out through the outlet of the closed container, to graft-modify the non-affinity-modified nanofiber porous material; the second grafting modification process is: using the aptamer aqueous solution to perform the second grafting modification on the non-affinity-modified nanofiber porous material that has undergone the first grafting modification according to the steps of the first grafting modification; The concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1 to 10 wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, adipaldehyde, pentamethylenediamine, and hexamethylenediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, CD81 with an amino or aldehyde terminal group, protein A, and protein G.

10. The nanofiber porous material for separation and extraction of exosomes prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The nanofiber porous material for exosome separation and extraction includes the gradient pore filtration layer, the nanofiber transition layer whose side with the smallest average diameter is bonded to the side with the smallest average diameter of the gradient pore filtration layer, and the nanofiber aerogel connected to the side with the largest average diameter of the nanofiber transition layer.

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