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

Abstract

This invention provides a nanofiber porous material for exosome separation and extraction and its preparation method. The method involves coating several nanofiber suspensions in a stepped order according to their average diameter to obtain a nanofiber gradient membrane. Then, the sides of two nanofiber gradient membranes with the smallest average diameter are bonded together to obtain a nanofiber composite membrane material. A secondary pre-crosslinked modified nanofiber foam liquid is prepared by mixing a pre-crosslinked modified nanofiber foam liquid with a crosslinking agent, chitosan aqueous solution, and polyethyleneimine aqueous solution. The nanofiber composite membrane material is then placed on the surface of the secondary pre-crosslinked modified nanofiber foam liquid and subjected to directional freeze-drying to obtain a nanofiber porous material. By constructing a gradient sieving pore structure and an aerogel multi-level pore structure, and achieving a strong and robust composite of the two types of pores through a nanofiber transition layer, the problem that single-pore-size nanofiber membranes or aerogel materials are difficult to synergistically improve the stepwise sieving and adsorption performance is solved.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a nanofiber porous material for exosome separation and extraction and its preparation method. Background Technology

[0002] Exosomes play a vital role in intercellular communication. They are a specific subclass of extracellular vesicles secreted by most cell types, playing a crucial role in intercellular communication by transporting various contents, including proteins, mRNA, miRNA, and DNA. These substances are closely related to the pathogenesis of most human malignancies. Therefore, exosomes not only contribute to a better understanding of cell physiology and pathology but also show great potential for clinical applications, encompassing aspects from diagnosis and prognosis to nucleic acid delivery and cancer treatment. Consequently, the isolation of exosomes using a simple, efficient, and economical method has become an urgent need for scientific research and clinical diagnosis. Currently, various exosome isolation and purification techniques have been developed based on the biophysical properties of exosomes, such as density, size, and morphology, as well as their biochemical properties, such as surface antibodies and biomembranes. However, standardized and large-scale clinical-grade methods for the isolation and purification of exosomes are still lacking, thus limiting their clinical application.

[0003] Currently, the most commonly used ultracentrifugation method removes cells, cell debris, and large microvesicles of different sizes and densities sequentially through multi-step centrifugation, resulting in high exosome purity. However, it is time-consuming, has low yield, and is prone to exosome destruction. Using polymers such as PEG to coat and precipitate exosomes yields more than eight times that of ultracentrifugation, but with higher impurity content. Size exclusion chromatography separates exosomes by size interception, but suffers from problems such as protein adhesion and pore blockage of separation media like ultrafiltration membranes or gels. Since exosomes carry universal transmembrane proteins such as CD63, CD9, and CD81 on their surface, the specific binding of antibodies or aptamers to exosome membrane proteins can distinguish exosomes from microvesicles, significantly improving separation purity. However, this specific binding requires a sufficiently long reaction time. Therefore, methods based on a single separation mechanism are insufficient to meet the multiple requirements of high purity, high yield, zero loss, and speed in exosome separation. Integrating multiple separation mechanisms onto a single platform represents a bold and effective attempt to address these issues.

[0004] In existing exosome separation methods, porous membranes used for interception and specific adsorption suffer from problems such as simple structure and low specific surface area.

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

[0006] To address the shortcomings of the existing technology, the present invention aims to provide a nanofiber porous material for exosome separation and extraction and its preparation method. By constructing a gradient sieving pore structure and an aerogel multi-level pore structure, and by constructing a nanofiber transition layer, the present invention achieves a strong composite of the two types of pores, thereby solving the problem that neither single nanofiber membranes nor aerogel materials can synergistically improve the graded sieving and adsorption performance.

[0007] To achieve the above objectives, the present invention provides a method for preparing nanofiber porous materials for exosome separation and extraction, comprising the following steps:

[0008] S1. Several copolymer nanofibers with different average diameters were prepared by melt extrusion of marine polymers.

[0009] S2. Several types of copolymer nanofibers with different average diameters are respectively placed in an isopropanol aqueous solution with polyethylene glycol and a first crosslinking agent and stirred and dispersed to obtain several nanofiber suspensions.

[0010] S3. The prepared nanofiber suspensions are coated layer by layer on the surface of the support in order of increasing or decreasing average diameter. After drying, a nanofiber gradient membrane is obtained, namely a gradient pore filter layer.

[0011] S4. Repeat step S3 to obtain nanofiber gradient membrane II, i.e. nanofiber transition layer; then attach the side with the smallest average diameter of nanofiber gradient membrane I to the side with the smallest average diameter of nanofiber gradient membrane II to obtain nanofiber composite membrane material.

[0012] S5. After mixing one and the second crosslinking agent in the copolymer nanofibers, the mixture is dispersed, and after the reaction is completed, a pre-crosslinked modified nanofiber foam liquid is obtained.

[0013] S6. Mix and stir 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. The secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 is placed in a directional freezing device, and the nanofiber composite membrane material obtained in step S4 is placed on the surface of the secondary pre-crosslinked modified nanofiber foam liquid and directionally freeze-dried to obtain an unaffinity-modified nanofiber porous material; wherein the secondary pre-crosslinked modified nanofiber foam liquid is made into a nanofiber aerogel; the nanofiber transition layer is located between the gradient pore filter layer and the nanofiber aerogel;

[0015] S8. The unaffinity-modified nanofiber porous material obtained in step S7 is grafted and modified several times using an affinity modifier to obtain the nanofiber porous material for exosome separation and extraction.

[0016] Further, the marine polymer in step S1 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 to 3:7; the draw rate of the melt extrusion process is 18 to 30 m / min; and the average diameter of the copolymer nanofibers ranges from 100 to 1000 nm.

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

[0018] Furthermore, in steps S3 and S4, in the first and second nanofiber gradient films, the average diameter of the copolymer nanofibers along the thickness direction gradually increases from 100 nm to 1000 nm, 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 spraying or spin coating.

[0020] Further, in steps S5 and S6, both the second crosslinking agent and the third crosslinking agent include glutaraldehyde solution; in step S5, the mass ratio of the copolymer nanofibers to the glutaraldehyde solution with a concentration of 1-5 wt.% is 1:20-1:30; the dispersion process is carried out by dispersing with an emulsifier for 10-20 min.

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

[0022] Further, the directional freeze-drying process in step S7 is as follows: the secondary pre-crosslinked modified nanofiber foam liquid with the nanofiber composite membrane material on its surface is placed on a metal block, and the metal block is immersed in a solution with a temperature of -209℃ to -40℃; the solution includes one of ethanol, isopropanol, and liquid nitrogen; the directional freeze-drying time is 46 to 50 hours.

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

[0024] The grafting modification process includes: the first grafting modification process is as follows: the unaffected nanofiber porous material obtained in step S7 is placed in a sealed container with an inlet and outlet, the spacer arm aqueous solution is placed in an open container, and the solution is introduced into the sealed container through the inlet of the sealed container by a peristaltic pump, then passes through the unaffected nanofiber porous material, and then flows out through the outlet of the sealed container to graft modify the unaffected nanofiber porous material; the second grafting modification process is as follows: the aptamer aqueous solution is used to perform the second grafting modification on the unaffected nanofiber porous material after the first grafting modification, following 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 molecules in the spacer arm aqueous solution include one of glutaraldehyde, hexamethylenedialdehyde, pentanediamine, and hexanediamine. The ligand molecules in the aptamer aqueous solution include one of the aptamers of CD63, CD9, and 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 described above, wherein the nanofiber porous material for exosome separation and extraction comprises the gradient pore filter layer, the nanofiber transition layer having its smallest average diameter side attached to the smallest average diameter side of the gradient pore filter layer, and the nanofiber aerogel connected to the largest average diameter side of the nanofiber transition layer.

[0027] The beneficial effects of this invention are:

[0028] 1. This invention provides a nanofiber porous material for exosome separation and extraction and its preparation method. The method involves coating several nanofiber suspensions prepared from copolymer nanofibers, crosslinking agents, and polyethylene glycol layer by layer in order of increasing or decreasing average diameter to obtain a nanofiber gradient membrane. Then, the sides of two nanofiber gradient membranes with the smallest average diameter are bonded together to obtain a nanofiber composite membrane material. Simultaneously, a pre-crosslinked modified nanofiber foam liquid prepared from copolymer nanofibers and crosslinking agents is mixed with a crosslinking agent chitosan aqueous solution and a polyethyleneimine aqueous solution to prepare a secondary pre-crosslinked modified nanofiber foam liquid. After placing the nanofiber composite membrane material on the surface of the secondary pre-crosslinked modified nanofiber foam liquid and performing directional freeze-drying, a nanofiber porous material for exosome separation and extraction can be obtained. In this way, by constructing a gradient sieving channel (i.e., gradient channel filter layer) and an aerogel multi-level channel (i.e., formed after directional freeze-drying of the secondary pre-crosslinked modified nanofiber foam liquid), and by constructing a nanofiber transition layer, the strong composite of the two types of channels is achieved. This solves the problem that single-pore nanofiber membranes or aerogel materials are difficult to synergistically improve the graded sieving and adsorption performance, and also achieves efficient and low-consumption separation of exosomes.

[0029] 2. This invention provides a nanofiber porous material for exosome separation and extraction and its preparation method. The nanofiber composite membrane material, composed of two nanofiber gradient membranes, allows the nanofiber gradient membrane on one side of the material to not only separate and intercept particles in the treatment solution but also allow specific unintercepted molecules to pass through, achieving particle-molecule sieving. On the other hand, the nanofiber gradient membrane on the other side of the material can be used for subsequent composite with nanofiber aerogels, achieving stable and effective composite and interconnection between the pores in the membrane material that perform the interception function and the macropores in the aerogel, integrating the interception function and specific adsorption function into the same material.

[0030] 3. This invention provides a nanofiber porous material for exosome separation and extraction and its preparation method. The nanofiber gradient membrane and nanofiber aerogel material involved have different surface chemical structures. Through cross-linking, the pore surface of the gradient membrane contains a cross-linked network of polyethylene glycol, which has anti-adhesion function, can achieve low adhesion pollution in 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 realize the specific adsorption and separation of exosomes by the aerogel. Attached Figure Description

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

[0032] Figure 2This is a schematic diagram illustrating the specific process of exosome extraction using a nanofiber porous material for exosome separation and extraction, as provided in Example 1. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be 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 with unnecessary details, only the structures and / or processing steps closely related to the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0035] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0036] A method for preparing nanofiber porous materials for exosome separation and extraction includes the following steps:

[0037] S1. Several copolymer nanofibers with different average diameters were prepared by melt extrusion of marine polymers.

[0038] The marine 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 to 3:7; the draw 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.

[0039] S2. Several types of copolymer nanofibers with different average diameters are respectively placed in an isopropanol aqueous solution with polyethylene glycol and a first crosslinking agent and stirred to disperse them evenly, so as to obtain several types of nanofiber suspensions.

[0040] The first crosslinking agent comprises a glutaraldehyde solution; the mass ratio of the copolymer nanofibers, polyethylene glycol (1-15 wt.%), and glutaraldehyde solution (1-5 wt.%) is 1:5:5 to 1:15:15; the isopropanol aqueous solution contains 45-50% isopropanol; and the stirring speed is 1000-2000 rpm.

[0041] S3. The prepared nanofiber suspensions are coated layer by layer on the surface of the support in order of increasing or decreasing average diameter. After drying at room temperature, a nanofiber gradient membrane is obtained, namely a gradient pore filter layer.

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

[0043] S4. Repeat step S3 to obtain nanofiber gradient membrane II, i.e. nanofiber transition layer; then attach the side with the smallest average diameter of nanofiber gradient membrane I to the side with the smallest average diameter of nanofiber gradient membrane II to obtain nanofiber composite membrane material.

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

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

[0046] S5. After mixing one of the copolymer nanofibers and the second crosslinking agent, disperse the mixture in an emulsifier for 10-20 minutes. After the reaction is completed, a pre-crosslinked modified nanofiber foam liquid is obtained.

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

[0048] S6. Mix the pre-crosslinked modified nanofiber foam liquid, the third crosslinking agent, the chitosan aqueous solution, and the polyethyleneimine aqueous solution, and stir at a constant speed for 18-25 minutes to obtain the secondary pre-crosslinked modified nanofiber foam liquid.

[0049] The third crosslinking agent all includes glutaraldehyde solution;

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

[0051] S7. Place the secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 in a directional freezing device, and then place the nanofiber composite membrane material obtained in step S4 on the surface of the secondary pre-crosslinked modified nanofiber foam liquid. Perform directional freeze-drying for 46-50 hours to obtain unaffected nanofiber porous material. At this time, the secondary pre-crosslinked modified nanofiber foam liquid becomes nanofiber aerogel after treatment. 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: the secondary pre-crosslinked modified nanofiber foam liquid with the nanofiber composite membrane material on its surface is placed on a metal block, and the metal block is immersed in a solution with a temperature of -209℃ to -40℃; the solution includes one of ethanol, isopropanol, and liquid nitrogen;

[0053] S8. The unaffinity-modified nanofiber porous material obtained in step S7 is grafted and modified several times using an affinity modifier to obtain the nanofiber porous material for exosome separation and extraction.

[0054] This design addresses the challenge of synergistically improving the stepwise sieving and adsorption performance of nanofiber membranes or aerogel materials with a single pore diameter, while also achieving efficient and low-consumption separation of exosomes. Furthermore, the nanofiber gradient membrane and nanofiber aerogel materials involved possess different surface chemical structures. Through cross-linking, the gradient membrane pore surface contains a polyethylene glycol cross-linked network, which has anti-adhesion properties, enabling low adhesion contamination during the gradient membrane interception process and improving interception and separation efficiency. Through cross-linking and subsequent grafting modification, the pore surface of the nanofiber aerogel forms a specific adsorption structure, enabling the aerogel to specifically adsorb and separate exosomes.

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

[0056] The grafting modification process includes: the first grafting modification process is as follows: the unaffected nanofiber porous material obtained in step S7 is placed in a sealed container with an inlet and outlet, the spacer arm aqueous solution is placed in an open container, and the solution is introduced into the sealed container through the inlet of the sealed container by a peristaltic pump, then passes through the unaffected nanofiber porous material, and then flows out through the outlet of the sealed container. The unaffected nanofiber porous material is grafted and modified at room temperature for 0.5 to 2 hours; the second grafting modification process is as follows: the aptamer aqueous solution is used to perform the second grafting modification on the unaffected nanofiber porous material after the first grafting modification, following 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 molecules in the spacer arm aqueous solution include one of glutaraldehyde, hexamethylenedialdehyde, pentanediamine, and hexanediamine. The ligand molecules in the aptamer aqueous solution include one of the aptamers of CD63, CD9, and CD81 with an amino or aldehyde terminal group, protein A, and protein G.

[0058] This setup allows for the first grafting modification of the unaffinity-modified nanofiber porous material using a spacer arm aqueous solution, facilitating further grafting and affinity adsorption. Subsequently, a ligand molecule is grafted onto the other end of the spacer arm molecule for a second grafting modification, thereby enabling 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 described above, the nanofiber porous material for exosome separation and extraction comprising the gradient pore filter layer, the nanofiber transition layer having its smallest average diameter side attached to the smallest average diameter side of the gradient pore filter layer, and the pre-crosslinked modified nanofiber foam liquid connected to the largest average diameter side of the nanofiber transition layer.

[0060] This configuration allows the nanofiber gradient membrane on one side of the material to not only separate and intercept particles in the treatment solution step by step, but also to allow specific molecules that are not intercepted to pass through, thus achieving particle-molecule sieving. On the other hand, the nanofiber gradient membrane on the other side of the material can be used for subsequent composite with nanofiber aerogel, achieving stable and effective composite and interconnection between the pores in the membrane material that play an interception role and the macropores in the aerogel, integrating the interception function and the specific adsorption function into the same material.

[0061] The following examples illustrate the nanofiber porous material for exosome separation and extraction provided by the present invention and its preparation method:

[0062] Example 1

[0063] This embodiment provides a nanofiber porous material for exosome separation and extraction and its preparation method, specifically including the following steps:

[0064] A nanofiber porous material for exosome separation and extraction and its preparation method, comprising the following steps:

[0065] S1. Several copolymer nanofibers with different average diameters were prepared by melt extrusion of marine polymers.

[0066] The marine 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 in the melt extrusion process is: 180℃, 215℃, 225℃, 220℃, 210℃, 180℃; the draw rate of the melt extrusion process is 18-30 m / min; the average diameter of the prepared copolymer nanofibers is: 100, 200 nm, ... 900 nm, 1000 nm;

[0067] S2. Several types of copolymer nanofibers are respectively mixed with polyethylene glycol (10 wt.%) and glutaraldehyde (3 wt.%) in an isopropanol aqueous solution at a mass ratio of 1:10:10 and stirred to uniformly disperse them, thereby obtaining several types of nanofiber suspensions.

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

[0069] S3. The prepared nanofiber suspensions are sprayed layer by layer onto one side surface of a PP substrate with a length and width of 20×20cm in order of increasing average diameter. After drying at room temperature, a nanofiber gradient membrane is obtained, namely a gradient pore filter layer.

[0070] S4. Spray the several kinds of nanofiber suspensions onto the other side surface of the PP substrate in the manner described in step S3, and dry them at room temperature to obtain nanofiber gradient film two, i.e. nanofiber transition layer; then attach the side with the smallest average diameter of nanofiber gradient film one to the side with the smallest average diameter of nanofiber gradient film two to obtain nanofiber composite film material.

[0071] In steps S3 and S4, in nanofiber gradient membrane one and nanofiber gradient membrane two, the average diameter of the copolymer nanofibers along the thickness direction increases from 100 nm to 1000 nm, and the gradient value of the pore size in the thickness direction is 100 nm / μm.

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

[0073] S6. The pre-crosslinked modified nanofiber foam liquid, glutaraldehyde solution with a concentration of 3 wt.%, chitosan aqueous solution with a concentration of 3 wt.%, and polyethyleneimine aqueous solution with a concentration of 5 wt.% are mixed in a volume ratio of 1:0.3:2:0.5 and stirred at a constant speed for 20 min to obtain a secondary pre-crosslinked modified nanofiber foam liquid.

[0074] S7. The secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 is placed in a directional freezing device, and the nanofiber composite membrane material obtained in step S4 is placed on the surface of the secondary pre-crosslinked modified nanofiber foam liquid. Directional freeze-drying is performed for 48 hours to obtain an unaffected nanofiber porous material. 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: the secondary pre-crosslinked modified nanofiber foam liquid with the nanofiber composite membrane material on its surface is placed on a metal block, and the metal block is immersed in liquid nitrogen at a temperature of -100℃;

[0076] S8. The unaffinity-modified nanofiber porous material obtained in step S7 is placed in a sealed container with inlets and outlets at both ends that perfectly match its shape. A 5 wt.% glutaraldehyde aqueous solution is placed in an open container and introduced into the sealed container through the inlet using a peristaltic pump. The solution then passes through the unaffinity-modified nanofiber porous material and flows out through the outlet of the sealed container, thus performing graft modification on the unaffinity-modified nanofiber porous material. Subsequently, a 5 wt.% CD63 aptamer with an amino end group is used to perform a second graft modification on the unaffinity-modified nanofiber porous material after the first graft modification, following the aforementioned graft modification steps. The material is then rinsed with pure water and dried to obtain the nanofiber porous material for exosome separation and extraction.

[0077] This embodiment uses the above-described preparation method to prepare a nanofiber porous material for exosome separation and extraction, such as... Figure 2 As shown, the specific process for extracting exosomes from the porous material is as follows: (1) the porous material is sieved and adsorbed onto the biological culture medium containing exosomes to remove impurities such as proteins and small-sized microvesicles; (2) cells, cell debris, 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 30-150 nm can be obtained with an impurity rate of 0.2 wt.%, and the separation rate of the entire process is about 15 min / L (biological culture medium) / g (porous material).

[0078] Example 2

[0079] Example 2 provides a nanofiber porous material for exosome separation and extraction and its preparation method. The difference from Example 1 is that the diameter range of the copolymer nanofibers prepared in step S1 is different. The copolymer nanofibers prepared in Example 2 have a diameter range of 100, 200, 300, 400, and 500 nm. The remaining steps are the same as in Example 1 and will not be repeated here.

[0080] Example 3

[0081] Example 3 provides a nanofiber porous material for exosome separation and extraction and its preparation method. The difference between Example 3 and Example 1 lies in the diameter of the copolymer nanofibers used in step S5. The copolymer nanofibers used in Example 3 have a diameter of 100 nm. The remaining steps are the same as in Example 1 and will not be repeated here.

[0082] Example 4

[0083] Example 4 provides a nanofiber porous material for exosome separation and extraction and its preparation method. The difference between Example 4 and Example 1 lies in the diameter of the copolymer nanofibers used in step S1. The diameter range of the copolymer nanofibers used in Example 4 is 200, 300, ..., 900, 1000 nm. The remaining steps are the same as in Example 1 and will not be repeated here.

[0084] Comparative Example 1

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

[0086] Comparative Example 2

[0087] Comparative Example 2 provides a nanofiber porous material for exosome separation and extraction, and its preparation method. The difference between Comparative Example 2 and Example 1 lies in the order in which the average diameters of the various nanofiber suspensions are coated in step S3. In Comparative Example 2, the various nanofiber suspensions are coated in a random manner based on their average diameters. The remaining steps are the same as in Example 1 and will not be repeated here.

[0088] Comparative Example 3

[0089] Comparative Example 3 provides a nanofiber porous material for exosome separation and extraction and its preparation method. The difference between Comparative Example 3 and Example 1 lies in the diameter range of the copolymer nanofibers used in step S1. The diameter ranges of the copolymer nanofibers used in Comparative Example 3 are 50 nm, 100 nm, 200 nm, ..., 900 nm, and 1000 nm. The remaining steps are the same as in Example 1 and will not be repeated here.

[0090] The changes in parameters and the exosome extraction effects in each step of the examples and comparative examples are shown in Table 1. Comparing the experimental results of Examples 1-2 and Comparative Example 1, it was found that when the size range of the copolymer nanofibers used in step S1 is in the range of 100-1000 nm, and the pore size in the gradient membrane is reduced from top to bottom, with the smallest pore size being about 100 nm, the purity of the obtained exosomes is higher and the particle size range is more complete (30-150 nm). The difference is that when the diameter of the uppermost fiber is too small (<1000 nm, Example 2), the gradient membrane does not effectively intercept particles of different sizes in the biological culture medium, and the particles tend to accumulate in one layer, which easily blocks the pores and slows down the separation speed. When the diameter of the fiber in this layer is too large (>1000 nm, Comparative Example 1), the increased amount of porous material leads to an increased mass of porous material, which reduces the separation speed per unit mass of porous material.

[0091] Comparing the experimental results of Examples 1, 4, and 3, it was found that when the minimum size of the copolymer nanofibers used in step S1 is less than 100 nm (Comparative Example 3), large-sized exosomes are intercepted, resulting in a smaller diameter range of the separated exosomes, increased exosome loss, smaller minimum pore size, and reduced flux, thus significantly reducing the separation speed. When the minimum fiber diameter is greater than 100 nm (Example 4), more extravesicles or cellular impurities of other sizes are not intercepted, increasing 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 effort.

[0092] Comparing the experimental results of Example 1 and Example 3, it was found that when the diameter of the copolymer nanofibers used in step S5 is smaller, although the specific surface area of ​​the downstream aerogel increases and the number of functional groups that can specifically adsorb exosomes increases, the purity of exosomes is not significantly improved. Moreover, the spacing between fibers on the pore walls of the aerogel decreases (i.e., the pore size on the pore walls is smaller), which reduces the effect of liquid convection and thus reduces the separation speed.

[0093] Comparing the experimental results of Example 1 and Comparative Example 2, it was found that when the "average diameter coated in a gradient order" method was used in step S3, the resulting nanofiber porous material for exosome separation and extraction had a gradient membrane with pore sizes decreasing from top to bottom. This allowed for the interception of large particles first, followed by smaller particles, without easily causing pore blockage in each layer. This achieved step-by-step interception of particles of different sizes and made it easier to maintain pore unobstructed flow, resulting in faster separation. However, when the "average diameter randomly coated" method was used, and the size of the copolymer nanofibers was below this range, the resulting nanofiber porous material for exosome separation and extraction struggled to form a gradient pore size that gradually decreased from top to bottom. This prevented effective step-by-step interception of particles of different sizes, leading to easy pore blockage and a significant decrease in separation speed.

[0094] This demonstrates that the effects of the prepared fiber-based porous affinity separation material with a periodic necking pore structure are all dependent on the effects of various parameters.

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

[0096]

[0097]

[0098] In summary, the present invention provides a nanofiber porous material for exosome separation and extraction and its preparation method. This involves coating several nanofiber suspensions prepared from copolymer nanofibers, crosslinking agents, and polyethylene glycol layer by layer in order of increasing or decreasing average diameter to obtain a nanofiber gradient membrane. Then, the sides of two nanofiber gradient membranes with the smallest average diameter are bonded together to obtain a nanofiber composite membrane material. Simultaneously, a pre-crosslinked modified nanofiber foam prepared from copolymer nanofibers and crosslinking agents is mixed with a crosslinking agent chitosan aqueous solution and a polyethyleneimine aqueous solution to prepare a secondary pre-... Cross-linked modified nanofiber foam liquid; then, the nanofiber composite membrane material is placed on the surface of the secondary pre-cross-linked modified nanofiber foam liquid and subjected to directional freeze-drying to obtain a nanofiber porous material for exosome separation and extraction; thus, by constructing gradient sieving channels (i.e., gradient channel filter layer) and aerogel multi-level channel structure, and by constructing a nanofiber transition layer, the above two types of channels are combined to achieve a strong and tough composite, which solves the problem that nanofiber membranes or aerogel materials with a single pore diameter are difficult to synergistically improve the stepwise sieving and adsorption performance, and at the same time achieves efficient and low-consumption separation of exosomes.

[0099] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing nanofiber porous materials for exosome separation and extraction, characterized in that, Includes the following steps: S1. Several copolymer nanofibers with different average diameters were prepared by melt extrusion of marine polymers. S2. Several types of copolymer nanofibers with different average diameters are respectively placed in an isopropanol aqueous solution with polyethylene glycol and a first crosslinking agent and stirred and dispersed to obtain several nanofiber suspensions. S3. The prepared nanofiber suspensions are coated layer by layer on the surface of the support in order of increasing or decreasing average diameter. After drying, a nanofiber gradient membrane is obtained, namely a gradient pore filter layer. S4. Repeat step S3 to obtain nanofiber gradient membrane II, i.e. nanofiber transition layer; then attach the side with the smallest average diameter of nanofiber gradient membrane I to the side with the smallest average diameter of nanofiber gradient membrane II to obtain nanofiber composite membrane material. S5. After mixing one and the second crosslinking agent in the copolymer nanofibers, the mixture is dispersed, and after the reaction is completed, a pre-crosslinked modified nanofiber foam liquid is obtained. S6. Mix and stir 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. The secondary pre-crosslinked modified nanofiber foam liquid obtained in step S6 is placed in a directional freezing device, and the nanofiber composite membrane material obtained in step S4 is placed on the surface of the secondary pre-crosslinked modified nanofiber foam liquid and directionally freeze-dried to obtain an unaffinity-modified nanofiber porous material; 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. The unaffinity-modified nanofiber porous material obtained in step S7 is grafted and modified several times using an affinity modifier to obtain the nanofiber porous material for exosome separation and extraction.

2. The method for preparing nanofiber porous materials for exosome separation and extraction according to claim 1, characterized in that: The marine polymer in step S1 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 to 3:7; the draw rate of the melt extrusion process is 18 to 30 m / min; and the average diameter of the copolymer nanofibers ranges from 100 to 1000 nm.

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

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

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

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

7. The method for preparing nanofiber porous materials 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 1~5 wt.%, the chitosan aqueous solution with a concentration of 2.5~5 wt.%, and the polyethyleneimine aqueous solution with a concentration of 0.1~10 wt.% is 1:0.3:2:0.5~1:0.1:1:0.1, and the stirring process is uniform stirring for 18~25 min.

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

9. The method for preparing nanofiber porous materials 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 grafting modification process includes: the first grafting modification process is as follows: the unaffected nanofiber porous material obtained in step S7 is placed in a sealed container with an inlet and outlet, the spacer arm aqueous solution is placed in an open container, and the solution is introduced into the sealed container through the inlet of the sealed container by a peristaltic pump, then passes through the unaffected nanofiber porous material, and then flows out through the outlet of the sealed container to graft modify the unaffected nanofiber porous material; the second grafting modification process is as follows: the aptamer aqueous solution is used to perform the second grafting modification on the unaffected nanofiber porous material after the first grafting modification, following the steps of the first grafting modification. The concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1~10 wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, hexamethylenedialdehyde, pentanediamine, and hexanediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, and CD81 with an amino or aldehyde terminal group, protein A, and protein G.

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

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