A method for preparing a gradient fiber membrane and a gradient fiber membrane

Abstract

The application provides a preparation method of a gradient fiber membrane and the gradient fiber membrane. The preparation method comprises the following steps: electrospinning a high-molecular polymer solution, and then soaking the obtained electrospinning nanofiber membrane in a cation solution to clean the nanofiber membrane, and then soaking the cleaned nanofiber membrane in an anion solution to obtain a primary self-assembled fiber membrane; the liquid level of the cation solution and the anion solution is kept unchanged during the soaking, and the liquid level is continuously increased; the above operation is repeated, and after n times, an n-layer self-assembled gradient fiber membrane is obtained, and n is greater than or equal to 3. The gradient fiber membrane prepared by the application can keep good morphology, porosity and good orientation; the self-assembled layer number is increased, and more bioactive substances can be adsorbed on the nanofiber; the fiber membrane has extremely strong hydrophilicity, extremely high cell activity, good biocompatibility, and the cell behavior can be well controlled by designing a gradient structure of bioactive components on the electrospinning nanofiber, so that the cell migration is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, further relates to a preparation method of gradient fiber membrane and the gradient fiber membrane. BACKGROUND

[0002] Gradient-based biomaterials are a hot research field in the field of materials science and tissue engineering, and the design purpose is to imitate the naturally occurring gradients in composition, signaling and other components found in vivo. With the increasing application of functionally graded materials in tissue engineering, several methods have been developed to incorporate the concept of gradient into biomaterials, including light-based methods, 3D printing, microfluidics, electrospinning, freeze-drying and solvent casting / particulate leaching. But most of the research is based on the internal regulation of the bulk structure or the gradual superposition of multiple materials, and few involves the construction of surface gradient active substances.

[0003] The nanofiber membrane prepared by electrospinning has the advantages of high porosity and large specific surface area, similar nanostructure to natural extracellular matrix (ECM), wide range of raw material sources, strong combination of multiple technologies, strong feasibility of macro preparation, good adjustable fiber structure and diversified composite structure. So far, electrospinning is still the most suitable method for preparing porous fiber membranes. It can prepare fiber membranes with ultra-fine diameter, high specific surface area and high porosity, and its versatility and simplicity have great application in the preparation of porous materials and tissue engineering. But facing the increasing complexity of tissue structure and different requirements of its microenvironment, pure electrospinning fiber membranes cannot meet all performance requirements. To solve this problem, researchers mostly add bioactive substances (proteins, growth factors, etc.) to the spinning process, but this simple mixing and co-spinning can only get a uniform system, which cannot better simulate the gradient structure in tissue engineering and provide similar gradient biological signals. And this method is mostly based on mixing one bioactive substance, but since a single bioactive substance provides low or insufficient biological activity, multiple bioactive particles need to be loaded to assist.

[0004] At present, post-processing of the electrospun fiber membrane prepared is also an increasingly important research field. Due to its simplicity and controllability in nanoscale operation, layer-by-layer self-assembly (LBL) technology has received widespread attention in adjusting surface properties. It is a simple and convenient surface modification method with multiple functions, and the principle is mainly through electrostatic interaction, and polyanions and polycations are alternately deposited layer by layer to form self-assembled multilayer coatings or independent films. Its main advantages are that the thickness can be controlled at the nanometer-micrometer level; it can be applied to any complex substrate structure and the components of the self-assembled coating can be any combination; the whole experimental process is easy to operate and the conditions are mild.

[0005] But most of the prior art is based on the internal regulation of the body structure or the gradual superposition of multiple materials, and rarely involves the construction of surface gradient active substances, among which the mixed co-spinning based on electrospinning can obtain a uniform system, which cannot better simulate the gradient structure in tissue engineering and provide similar gradient biological signals. Like 3D printing and electrospinning, they use the adjusted syringe / extrusion components to design the gradient structure of the material body; the conventional addition of bioactive substances generally adopts blending / physical adsorption and the like, which cannot accurately design the construction of surface gradient bioactive substances of the material, and cannot better provide additional biochemical clues for the cells attached thereto to regulate the morphology and behavior of the cells. Moreover, this method is mostly based on the addition of one bioactive substance, and it is often difficult to realize the synchronous regeneration of different tissues, and finally it is difficult to achieve the ideal repair effect. SUMMARY

[0006] In order to solve the technical problems existing in the prior art, the application provides a preparation method of a gradient fiber membrane and a gradient fiber membrane.

[0007] The gradient fiber membrane prepared by the application is a fiber membrane loaded with double gradient active substances, which is a static spinning fiber membrane with double gradient of bioactive substances constructed on the nanofiber membrane by introducing layer-by-layer self-assembly (LBL) technology into the post-processing of the electrospinning membrane.

[0008] The application combines the advantages of electrospinning and the biochemical clues provided by the concentration gradient of bioactive components to design a gradient tissue engineering scaffold, that is, to construct a double gradient of bioactive substances on the electrospinning membrane by layer-by-layer self-assembly method, so as to regulate the cell behavior and to a certain extent simulate the gradient structure of the natural tissue interface. In addition, this simple strategy of generating double gradient of bioactive components can be extended to the preparation of different types of gradient scaffolds, providing a simple and economical universal method for the preparation of gradient scaffolds, and providing a new idea for constructing functional hierarchical scaffolds to better match the structure and morphology of natural tissues.

[0009] Electrospinning is a simple and universal technology that relies on electrostatic repulsion between surface charges to continuously draw nanofibers from a viscoelastic fluid under the action of an electric field, and its high porosity, large specific surface area and designable scaffold structure provide great potential for tissue engineering scaffolds. More importantly, the topological structure of electrospinning fibers can be used to regulate the directional migration and differentiation of stem cells. By combining the biochemical clues provided by the concentration gradient of bioactive components with the advantages of electrospinning, the cell behavior can be better guided, and the gradient structure of the natural tissue / interface can be simulated to a certain extent.

[0010] Layer-by-layer (LBL) self-assembly is mainly carried out through electrostatic interaction, and a gradient coating of self-assembled multilayer bioactive substances is formed through the layer-by-layer deposition of polyanions and polycations. The LBL self-assembly has the following advantages: 1) the thickness can be adjusted by changing the pH value or ionic strength of the polyelectrolyte solution, and the thickness can be accurately adjusted in the nanometer-micrometer range; 2) the LBL self-assembly can be applied to any complex substrate structure, and the raw materials of the LBL self-assembly can be arbitrarily combined; and 3) the experimental process is mild and the operation is convenient.

[0011] One of the purposes of the present application is to provide a preparation method of a gradient fiber membrane, comprising the following steps:

[0012] (1) adding a high molecular polymer into a solvent to obtain a solution after sufficient dissolution;

[0013] (2) electrospinning the solution obtained in step (1) to obtain an electrospun nanofiber membrane;

[0014] (3) respectively preparing a cation solution and an anion solution; the cation solution is a solution of a positively charged bioactive substance; and the anion solution is a solution of a negatively charged bioactive substance;

[0015] (4) vertically placing the electrospun nanofiber membrane obtained in step (2) in the height direction, first immersing the electrospun nanofiber membrane in the cation solution, immersing the electrospun nanofiber membrane in deionized water to wash away the positively charged bioactive substance that is not adsorbed, then immersing the electrospun nanofiber membrane in the anion solution, and immersing the electrospun nanofiber membrane in deionized water to wash away the negatively charged bioactive substance that is not adsorbed, to obtain a 1-time self-assembled fiber membrane; the cation solution and the anion solution have the same liquid level height, and the liquid level height is lower than the height of the electrospun nanofiber membrane;

[0016] (5) raising the liquid level height of the cation solution and the anion solution, still maintaining the same liquid level height, repeating the self-assembly process of step (4) by using the 1-time self-assembled fiber membrane obtained in step (4), to obtain a 2-time self-assembled fiber membrane;

[0017] (6) repeating step (5) for n-2 times, until step (4) is repeated by using an n-1-time fiber membrane, the liquid level height is higher than the electrospun nanofiber membrane, and the gradient fiber membrane is obtained after n-time self-assembly; n is greater than or equal to 3.

[0018] The self-assembly is performed at least 3 times, and a high-low gradient can be presented.

[0019] In a preferred embodiment of the present application,

[0020] step (1),

[0021] The high molecular polymer is at least one of a synthetic high molecular polymer and a natural high molecular polymer; the synthetic high molecular polymer is preferably at least one of polycaprolactone, polylactic acid, polyurethane, polyvinyl alcohol, polylactic acid-glycolic acid copolymer, and polylactic acid-glycolic acid-caprolactone copolymer; and the natural high molecular polymer is preferably at least one of gelatin, silk fibroin, collagen, chitosan, starch, cellulose, and elastin; wherein the cellulose can be cellulose acetate.

[0022] The solvent is at least one of trifluoroethanol, N,N'-dimethylformamide, dichloromethane, acetic acid, hexafluoroisopropanol, acetone, dimethyl sulfoxide, tetrahydrofuran, chloroform, methanol, and deionized water.

[0023] In a preferred embodiment of the present application,

[0024] Step (1),

[0025] The mass fraction of the high molecular polymer in the solution is 5-20%.

[0026] The solution is stirred at room temperature for 12-24 hours, and the stirring is stopped when the solution is uniform; the stirring rate is usually maintained at 200-600 rpm, and a low rate for a long time or a high rate for a short time can also be used.

[0027] In a preferred embodiment of the present application,

[0028] Step (2),

[0029] When the spinning starts, the voltage needs to be slowly adjusted to observe the spinning needle to determine the stable voltage.

[0030] The electrospinning solution feeding rate is 0.2-2.5 mL / h, preferably 0.5-2.0 mL / h; the spinning voltage is 10-25 kV; the receiving distance is 10-25 cm; the spinning temperature is 20-30°C; and the relative humidity of the spinning is 20-45%.

[0031] After the spinning is completed, the obtained electrospun nanofiber membrane is placed in a clean fume hood for 12-48 h to remove the unvolatilized solvent; preferably, after being dried for 12-48 h, the nanofiber membrane is soaked in a sodium hydroxide solution or subjected to plasma treatment; the concentration of the sodium hydroxide solution is 0.02-1.0 M, preferably 0.05-0.1 M; and the soaking time is 2-48 h, preferably 12-48 h.

[0032] The plasma treatment is to use a plasma treatment instrument (commonly known as Plasma on the surface of the material) to etch the surface of the material and bring negative charges, so as to bring corresponding negative charges to the fiber membrane and better perform layer-by-layer self-assembly.

[0033] The electrospun nanofiber membrane of the present application can also use the nanofiber membrane of the prior art with a core-shell structure. The core layer is generally selected from at least one of natural polymer polymers, and the shell layer is at least one of synthetic polymer polymers and natural polymer polymers. The range of natural polymer polymers and synthetic polymer polymers is as described above. The solvent is as described above. The mass fraction of the synthetic polymer polymers or natural polymer polymers in the solution is 5-20%. The electrospinning liquid feeding rate of the core layer is 0.2-2.5 mL / h, preferably 0.5-2.0 mL / h. The electrospinning liquid feeding rate of the shell layer is 0.2-3 mL / h, preferably 0.5-2.0 mL / h. The spinning voltage is 10-25 kV. The receiving distance is 10-25 cm.

[0034] In a preferred embodiment of the present application,

[0035] Step (3),

[0036] The positively charged bioactive substance is added to deionized water or a deionized water solution with a pH adjusted and stirred uniformly to obtain the cationic solution. The concentration of the cationic solution is 0.1-5 mg / mL, preferably 1-2 mg / mL. The deionized water solution is a deionized water solution of acetic acid and sodium chloride. The concentration of acetic acid is preferably 0.1-1 M. The concentration of the sodium chloride solution is preferably 0.1-1 M. The pH is preferably adjusted to 4-10 using a sodium hydroxide solution, more preferably to 4-6. The concentration of the sodium hydroxide solution is preferably 0.1-2 M. And / or,

[0037] The negatively charged bioactive substance is added to deionized water or a deionized water solution with a pH adjusted and stirred uniformly to obtain the anionic solution. The concentration of the anionic solution is 0.1-5 mg / mL, preferably 1-2 mg / mL. The deionized water solution is a deionized water solution of sodium chloride. The concentration of the sodium chloride solution is preferably 0.1-1 M. The pH is preferably adjusted to 4-10 using an acetic acid solution, more preferably to 4-6. The concentration of the acetic acid solution is preferably 0.1-1 M.

[0038] In a preferred embodiment of the present application,

[0039] Step (4),

[0040] The electrospun nanofiber membrane is placed vertically in a container, and the cationic solution is added. The liquid level height is 1 / n of the vertical height of the electrospun nanofiber membrane. The soaking time is 10-60 min. After soaking, the electrospun nanofiber membrane is taken out and washed in deionized water, ultrapure water, a sodium chloride solution, or a phosphate buffer PBS for 5-30 min. The washing is performed in three times, and each washing time is 2-10 min. The concentration of the sodium chloride solution is preferably 0.1-1 M.

[0041] Then the electrospun nanofiber membrane is placed vertically in a container, an anion solution of the same volume as the cation solution is added, the liquid level is also the same as the height of the cation solution, after soaking for the same time, it is taken out, washed in deionized water, ultrapure water, a sodium chloride solution or a phosphate buffer PBS for 5-30 min, and dried, such as washing 3 times, each time for 2-10 min; the concentration of the sodium chloride solution is preferably 0.1-1M.

[0042] The present application controls the self-assembly of bioactive substances on the surface of the fiber membrane by regulating the liquid level, and controls the liquid level of the anion solution and the cation solution to be the same, so as to better realize ordered self-assembly at the same part of the fiber membrane.

[0043] The value of n is the number of self-assembly, and the height of 1 / n refers to the liquid level during each self-assembly, for example: if a 5-layer concentration gradient is constructed on the fiber membrane, the vertical height of the electrospun nanofiber membrane is first divided into 5 equal parts, and the liquid level is at 1 / 5 of the height of the fiber membrane during the first self-assembly (bioactive substance soaking of cation / anion), the liquid level is increased to 2 / 5 during the second self-assembly, and so on, until the fifth self-assembly, which increases to 5 / 5 of the height of the fiber membrane, that is, the fiber membrane is completely immersed, at this time, the obtained fiber membrane is a gradient fiber membrane which has adsorbed 1 layer, 2 layers, 3 layers, 4 layers and 5 layers of double bioactive substances from top to bottom.

[0044] In a preferred embodiment of the present application,

[0045] The positively charged bioactive substance and the negatively charged bioactive substance are each independently selected from at least one of collagen, quaternary ammonium chitin, chondroitin sulfate, chitosan, phosphorescent protein, lysozyme, ovalbumin, polyallylamine hydrochloride, silk fibroin, hydroxyapatite, beta-tricalcium phosphate (β-TCP), bioglass, laminin, fibronectin, gelatin and growth factor;

[0046] The growth factor is at least one of vascular endothelial growth factor, nerve growth factor, brain-derived nerve growth factor and human acidic fibroblast growth factor.

[0047] The bioactive substance has positive or negative charge, which is related to the isoelectric point of the bioactive substance or the final surface charge, but the positive or negative nature and intensity (how much charge) of the charge of the bioactive substance can be adjusted by preparing a specific solution or adjusting the pH of the solution; for example, collagen sometimes has positive charge and sometimes has negative charge, and the skilled person can select the required bioactive substance according to the specific requirements and applications.

[0048] In a preferred embodiment of the present application,

[0049] Step (5),

[0050] The height of the cation solution and the anion solution is 2 / n of the vertical height of the electrospun nanofiber membrane, and the rest of the process is the same as step (4), to obtain 2 times of self-assembly electrospun fiber membrane; the liquid level height is increased to control the exposure time of different parts of the fiber membrane to the solution, so as to form a concentration gradient on the surface of the fiber membrane.

[0051] In a preferred embodiment of the present application,

[0052] Step (6),

[0053] In 3 times of self-assembly, the height of the cation solution and the anion solution is 3 / n of the vertical height of the electrospun nanofiber membrane; in n times of self-assembly, the cation solution and the anion solution completely submerge the vertical height of the electrospun nanofiber membrane; the process of each self-assembly is the same as step (4);

[0054] 3≤n≤15, preferably 3≤n≤10.

[0055] The second object of the present application is to provide a gradient fiber membrane prepared by the above method.

[0056] The present application can specifically adopt the following technical solutions:

[0057] Take the dried and pretreated electrospun nanofiber membrane (the current pretreatment method is mainly through NaOH solution immersion / plasma treatment), first immerse it in a certain initial liquid surface positively charged bioactive substance, then immerse it in deionized water to wash off the unabsorbed positively charged bioactive substance, and then immerse the nanofiber membrane with adsorbed positively charged bioactive substance in a negatively charged bioactive substance solution with the same liquid surface height. After the same immersion time, take it out and immerse it in deionized water to wash off the unabsorbed negatively charged bioactive substance. Then, the liquid surface of the positively / negatively charged bioactive substance solution is raised, and the above electrospun fiber membrane after one complete self-assembly is repeatedly immersed in turn. One complete self-assembly includes immersion in positively and negatively charged bioactive substance solutions, and the liquid surface of the two bioactive substance solutions is raised after each layer-by-layer self-assembly (the positively and negatively charged bioactive substances need to maintain the same liquid surface height), and the cycle is repeated in turn, so that the electrospun fiber membrane with a double bioactive substance gradient from dense to sparse in the direction from the initial immersion height to the termination immersion height is obtained. The liquid surface height raised during each layer-by-layer self-assembly, the number of times of layer-by-layer self-assembly, and the types of positively and negatively charged bioactive substances can be freely adjusted. And this method has strong universality and is suitable for the construction of multiple bioactive substance gradients on the surface of almost all membrane materials.

[0058] The preparation method of the present application specifically comprises the following steps:

[0059] Step (1), adding selected polymer / natural polymer into corresponding solvent, dissolving thoroughly to obtain solution;

[0060] Step (2), electrospinning the solution obtained in step (1) to obtain electrospun nanofiber membrane;

[0061] Step (3), respectively preparing cationic solution and anionic solution;

[0062] Step (4), immersing the electrospun nanofiber membrane obtained in step (2) into bioactive substance with positive charge at a certain initial liquid level, then immersing in deionized water to wash away the unabsorbed bioactive substance with positive charge, and then immersing the nanofiber membrane with absorbed bioactive substance with positive charge into bioactive substance with negative charge at the same liquid level. After immersing for the same time, taking out, and immersing in deionized water to wash away the unabsorbed bioactive substance with negative charge. Then, raising the liquid level of the bioactive substance solution with positive charge, one complete self-assembly includes immersing the bioactive substance solution with positive charge and the bioactive substance solution with negative charge, after each layer-by-layer self-assembly, raising the liquid level of the two bioactive substance solutions (the bioactive substance with positive charge and the bioactive substance with negative charge need to keep the same liquid level), and sequentially circulating, so that the electrospun fiber membrane with gradient structure of double bioactive substances from dense to sparse in the direction from the initial immersing height to the terminal immersing height can be obtained.

[0063] In order to generate double gradient of two bioactive substances on the surface of nanofiber membrane, the cationic solution and the anionic solution prepared in step (3) are respectively added into two containers, and then the membrane is sequentially placed upright in the containers. In the first cycle of self-assembly, a certain volume of cationic solution is added until the electrospun fiber membrane is immersed to 1 / n of the height, and the immersing time is 10-60 min. Then, after washing in deionized water / ultra-pure water for 2-10 min and drying, the electrospun fiber membrane is immersed in the same volume of anionic solution, and the liquid level is still kept at the above-mentioned height of 1 / n of the fiber membrane. After immersing for the same time, washing and drying, the first layer-by-layer self-assembled electrospun fiber membrane is obtained. In the second cycle, the volume of the solution is increased, so that 2 / n of the fiber membrane is immersed in the solution. In this way, the cycle is repeated n times, until the whole membrane is immersed in the cationic solution and the anionic solution respectively, so that the electrospun fiber membrane with double active substance gradient can be obtained, and different positions on the gradient membrane correspond to different self-assembly times of bioactive substances. One complete self-assembly process includes immersing the cationic solution and the anionic solution once respectively, and the number of layers of self-assembly for 1 time is recorded as 1 layer, and the first adsorption of bioactive substance with positive charge on the nanofiber membrane is abbreviated as LBL 0.5The self-assembled fiber membrane is marked as LBL1, LBL2, LBL3, …, LBLn according to the number n of self-assembled bioactive substances, wherein n is 1, 2, 3, …, n. n The fiber membrane with a gradient scaffold is formed by adsorbing 1 layer, 2 layers, 3 layers, …, n layers of double bioactive substances from top to bottom.

[0064] Compared with the prior art, the present application has the following beneficial effects:

[0065] In the construction of the gradient tissue engineering scaffold, the electrospun fiber is concerned due to the similar nano-scale structure, high porosity, large specific surface area, and designable scaffold structure to the natural extracellular matrix. However, the pure electrospun membrane cannot meet all the performance requirements in the face of more and more complex tissue structures and different requirements of the microenvironment. The blending of bioactive substances in the spinning process, the mixed electrospinning or the doping of the bioactive substances in the form of core-shell structure in the spinning process can only obtain a uniform system, and the provided bioactivity is low or cannot achieve the use effect, and the gradient structure in the tissue engineering and the similar gradient biological signal cannot be better simulated. Therefore, the present application constructs a hierarchical gradient structure by alternately depositing polyanions and polycations layer by layer through the surface modification method of layer-by-layer self-assembly, and to a certain extent, the gradient structure of the natural tissue / interface is simulated. In addition, the simple strategy of generating double gradient of bioactive components can be extended to the preparation of different types of gradient scaffolds, and a simple and economical universal method is provided for the preparation of the gradient scaffold.

[0066] The present application combines the advantages of electrospinning and the biochemical clues provided by the concentration gradient of bioactive components to design a gradient tissue engineering scaffold, that is, a double bioactive substance gradient is constructed on the electrospun membrane by the layer-by-layer self-assembly method, so as to regulate the cell behavior, and to a certain extent, the gradient structure of the natural tissue / interface is simulated. In addition, the simple strategy of generating double gradient of bioactive components can be extended to the preparation of different types of gradient scaffolds, and a simple and economical universal method is provided for the preparation of the gradient scaffold.

[0067] The present application combines the advantages of electrospinning and the biochemical clues provided by the concentration gradient of bioactive components to design a gradient tissue engineering scaffold, that is, a double bioactive substance gradient is constructed on the electrospun membrane by the layer-by-layer self-assembly method, so as to regulate the cell behavior, and to a certain extent, the gradient structure of the natural tissue / interface is simulated. In addition, the simple strategy of generating double gradient of bioactive components can be extended to the preparation of different types of gradient scaffolds, and a simple and economical universal method is provided for the preparation of the gradient scaffold.

[0068] The present application forms a self-assembled multilayer bioactive substance gradient coating by alternately depositing polyanions and polycations layer by layer, and has the following advantages: 1) the thickness can be adjusted by changing the pH value or ionic strength of the polyelectrolyte solution, and the nanometer-micrometer level can be accurately adjusted; 2) it can be applied to any complex substrate structure, and the raw materials for LBL self-assembly can be arbitrarily combined; 3) the experimental process is mild and the operation is convenient.

[0069] The gradient fiber membrane prepared by the present application has the following advantages:

[0070] 1. The fiber membrane can maintain good form, porosity and good orientation;

[0071] 2. The self-assembly layer number increases to adsorb more bioactive substances on the nanofiber;

[0072] 3. The number of self-assembly layers can be determined according to actual needs, and the gradient design and construction of the fiber membrane are well controlled by adjusting the deposition number of layer-by-layer self-assembly;

[0073] 4. The fiber membrane has strong hydrophilicity, which is conducive to cell adhesion and growth;

[0074] 5. The nanofiber membrane has high cell viability and good biocompatibility;

[0075] 6. By designing a gradient structure of bioactive components on the electrospun nanofiber, cell behavior can be well regulated to promote cell migration; BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 It is a comparison chart of Zeta potential test of the cationic solution and the anionic solution of Example 1;

[0077] Figure 2 It is a chart showing the change of adsorption thickness of different self-assembly layers in the self-assembly process of Example 1;

[0078] Figure 3 It is a chart showing the change of adsorption mass of different self-assembly layers in the self-assembly process of Example 1;

[0079] Figure 4 It is a scanning electron microscope image of the electrospun polycaprolactone (PCL) fiber membrane of Example 1;

[0080] As shown in the figure, the average diameter of the fiber is 460±150nm;

[0081] Figure 5 It is a scanning electron microscope image of the LBL1 fiber membrane of Example 1;

[0082] As shown in the figure, the average diameter of the fiber is 620±230nm;

[0083] Figure 6 It is a scanning electron microscope image of the LBL5 fiber membrane of Example 1;

[0084] As shown in the figure, the average diameter of the fiber is 940±370nm;

[0085] Figure 7Electrospun poly-caprolactone (PCL) fiber membrane and LBL for Example 1 n FTIR spectra of fiber membranes (n = 1-5);

[0086] Figure 8 Electrospun poly-caprolactone (PCL) fiber membrane and LBL for Example 1 self-assembly process n Variation of water contact angle of fiber membranes (n = 1-5);

[0087] Figure 9 Electrospun poly-caprolactone (PCL) fiber membrane, LBL for Example 1 n Proliferation results of BMSCs after 7 days of culture on fiber membranes (n = 1-5) and blank samples;

[0088] Figure 10 Fiber membranes with gradient scaffolds, electrospun poly-caprolactone (PCL) fiber membranes, LBL prepared for Example 1 n Migration distance of cells after 3 days of cell migration of fiber membranes (n = 1-5);

[0089] Figure 11 LBL for Example 2 10 Scanning electron microscope image of fiber membranes;

[0090] As shown in the figure, the average diameter of the fibers is 1130 ± 320 nm;

[0091] Figure 12 Scanning electron microscope image of LBL3 fiber membranes for Example 6;

[0092] As shown in the figure, the average diameter of the fibers is 680 ± 240 nm. DETAILED DESCRIPTION

[0093] The following specific description of the application is made in conjunction with the specific drawings and examples. It is necessary to point out that the following examples are only used to further illustrate the application and cannot be understood as limiting the scope of protection of the application. Some non-essential improvements and adjustments of the application made by those skilled in the art based on the content of the application are still within the scope of protection of the application.

[0094] The raw materials used in the examples are all conventional commercially available raw materials.

[0095] Test method:

[0096] Determination of Zeta potential: instrument model Malvern, Nano-ZS90;

[0097] Test of adsorption thickness and adsorption mass of different self-assembly layers: quartz crystal microbalance instrument model QCM, Qsense, Biolin Scientific AB;

[0098] Elemental analysis: The X-ray spectrometer is a Hitachi S4800.

[0099] Infrared spectroscopy analysis: The infrared spectrometer is a Bruker Nicolet 8700.

[0100] Water contact angle: The water contact angle measuring instrument is a Dataphysics OCA 15EC;

[0101] CCK-8 assay: After 7 days of co-culture of materials and cells, the CCK-8 assay kit was used, and the absorbance was read at a wavelength of 450 nm using a microplate reader; the microplate reader model was Model-680, Bio-Rad.

[0102] Cell migration assay: All fiber membranes were cut into rectangular strips and covered with polydimethylsiloxane (PDMS), leaving a 0.5 cm gap between each group of fiber membranes. After seeding BMSCs, PDMS was removed after overnight culture to allow cells to migrate to the distal end. After co-culturing the materials and cells for 3 days, the migration distance of cells on different membranes was measured and statistically analyzed.

[0103] Example 1

[0104] (1) According to the mass fraction of polycaprolactone (PCL) of 10%, a certain amount of PCL was dissolved in trifluoroethanol (TFE) and stirred magnetically at room temperature for 12 hours to obtain an electrospinning solution.

[0105] (2) The obtained electrospinning solution was subjected to electrospinning, and the voltage was slowly adjusted to ensure stable fiber output. The injection rate of the spinning solution was 1 mL / h, the spinning voltage was 15 kV, the receiving distance was 15 cm, the spinning temperature was 30 °C, and the relative humidity of the spinning was 30%. The electrospinned PCL fiber membrane was obtained and placed in a clean fume hood to dry at room temperature for 48 h to remove any unevaporated solvent. The radially arranged fiber membrane was collected using appropriate glass slides. The obtained fiber membrane was then treated with plasma for later use.

[0106] (3) Collagen was added to a deionized aqueous solution (containing 0.1 mol / L HAc and 0.2 mol / L NaCl), and the pH of the solution was adjusted to pH=4.5 with 1 mol / L NaOH to obtain a collagen solution with a mass concentration of 1 mg / mL, which was used as a cationic solution with a positively charged bioactive component; Chondroitin sulfate was added to a deionized aqueous solution (containing 0.2 mol / L NaCl), and the pH was adjusted to pH=4.5 with 0.1 mol / L HAc to obtain a chondroitin sulfate solution with a mass concentration of 1 mg / mL, which was used as an anionic solution with a negatively charged bioactive component.

[0107] (4) In the first cycle of self-assembly, the nanofiber membrane is sequentially added into the positively charged cation solution and the negatively charged anion solution until 1 / 5 of the nanofiber membrane is immersed, and the soaking time is 30 min. After each layer deposition, the nanofiber membrane is washed with deionized water for 3 times, each time for 5 min, to remove the residual solution, and after drying, a 1-time self-assembled fiber membrane is obtained.

[0108] (5) Then, during the second cycle, the volume of the solution is increased so that 2 / 5 of the height of the nanofiber membrane is immersed in the solution, and the rest of the self-assembly process is the same as step (4), to obtain a 2-time self-assembled electrospun fiber membrane.

[0109] (6) Repeat cycle step (5) for 3 times until the liquid level is higher than the electrospun nanofiber membrane, and the whole membrane is immersed in the two solutions respectively, and the rest of the self-assembly process is the same as step (4), to obtain a gradient fiber membrane after 5 times of self-assembly.

[0110] Example 2

[0111] (1) Dissolve the polyurethane in tetrahydrofuran and N,N'-dimethylformamide, with a volume ratio of tetrahydrofuran to N,N'-dimethylformamide being 1:1. Stir magnetically at room temperature for 12 h to obtain an electrospinning solution with a mass concentration of 20%.

[0112] (2) Perform electrospinning using the obtained solution, with a spinning solution propelling rate being 0.8 mL / h, a spinning voltage being 20 kV, a receiving distance being 20 cm, a spinning temperature being 30°C, and a spinning relative humidity being 30%, to obtain an electrospun polyurethane fiber membrane; place the obtained fiber membrane in a clean fume hood and dry at room temperature for 24 h, and then perform plasma treatment for standby.

[0113] (3) Add collagen into an acetic acid solution (containing 0.1 mol / L HAc and 0.5 mol / L NaCl), and use 1 mol / L NaOH to adjust the solution pH value to pH=5, to obtain a collagen solution with a mass concentration of 1 mg / mL, which is used as the positively charged cation solution of the biologically active component; add chondroitin sulfate into a deionized water solution (containing 0.5 mol / L NaCl), and use 0.1 mol / L HAc to adjust the pH value to 5, to obtain a chondroitin sulfate solution with a mass concentration of 1 mg / mL, which is used as the negatively charged anion solution of the biologically active component.

[0114] (4) In the first cycle of self-assembly, the nanofiber membrane is sequentially added into the positively charged cation solution and the negatively charged anion solution until 1 / 10 of the nanofiber membrane is immersed, and the soaking time is 30 min. After each layer deposition, the nanofiber membrane is washed with deionized water for 3 times, each time for 5 min, to remove the residual solution, and after drying, a 1-time self-assembled fiber membrane is obtained.

[0115] (5) After that, during the second cycle, the volume of the solution is increased so that 2 / 10 of the nanofiber membrane is immersed in the solution, and the rest of the self-assembly process is the same as step (4), obtaining 2 times of self-assembled electrospun fiber membranes.

[0116] (6) Repeat cycle step (5) 8 times until the liquid level is higher than the electrospun nanofiber membrane, and the entire membrane is immersed in the two solutions respectively, and the rest of the self-assembly process is the same as step (4), obtaining 10 times of self-assembled gradient fiber membranes.

[0117] Example 3

[0118] (1) Take polylactic acid (PLA) and dissolve it in trifluoroethanol solution, and magnetically stir at room temperature for 24 h to obtain an electrospinning solution with a mass concentration of 5%.

[0119] (2) Electrospinning is performed using the above solution, wherein the spinning solution propelling rate is 2 mL / h, the spinning voltage is 10 kV, the receiving distance is 10 cm, the spinning temperature is 30°C, and the relative humidity of the spinning is 30%, obtaining an electrospun polylactic acid fiber membrane; the obtained fiber membrane is dried in a clean fume hood at room temperature for 24 h, and then treated by plasma for standby.

[0120] (3) Select a deionized water solution (2 mg / mL) of quaternized chitin (QC) as the cationic solution of the positively charged bioactive component, and select a deionized water solution (2 mg / mL) of silk fibroin (SF) as the anionic solution of the negatively charged bioactive component.

[0121] (4) In the first cycle of self-assembly, the nanofiber membrane is sequentially added to the positively charged cationic solution and the negatively charged anionic solution until 1 / 8 of the nanofiber membrane is immersed, and the soaking time is 60 min; after each layer is deposited, the nanofiber membrane is washed with phosphate buffer PBS for 3 times, each time for 10 min, to remove the residual solution, and dried to obtain 1 time of self-assembled fiber membrane.

[0122] (5) After that, during the second cycle, the volume of the solution is increased so that 2 / 8 of the nanofiber membrane is immersed in the solution, and the rest of the self-assembly process is the same as step (4), obtaining 2 times of self-assembled electrospun fiber membranes.

[0123] (6) Repeat cycle step (5) 6 times until the liquid level is higher than the electrospun nanofiber membrane, and the entire membrane is immersed in the two solutions respectively, and the rest of the self-assembly process is the same as step (4), obtaining 8 times of self-assembled gradient fiber membranes.

[0124] Example 4

[0125] (1) Dissolve silk fibroin (SF) and PCL in hexafluoroisopropanol (HFIP) solution (mass concentration of 12wt%) at a mass ratio of 1:2 to obtain electrospinning solution A, and dissolve polyvinyl alcohol in deionized water (mass concentration of 8wt%) to obtain polyvinyl alcohol solution as electrospinning solution B.

[0126] (2) Electrospinning is performed with the obtained solutions, and the voltage is slowly adjusted to ensure stable fiber output. The flow rates of electrospinning solutions A and B are 0.5mL / h and 2mL / h, respectively, the spinning voltage is 20kV, the receiving distance is 20cm, the spinning temperature is 30℃, and the relative humidity of spinning is 30%, to obtain a nanofiber membrane with a core-shell structure; place it in a clean fume hood for room temperature drying for 48h to remove the unevaporated solvent, and collect the obtained radially arranged fiber membrane with the corresponding glass sheet; the obtained fiber membrane is reserved after plasma treatment.

[0127] (3) Select a deionized water solution of chitosan (1mg / mL) as a cationic solution of a positively charged bioactive component, and select a deionized water solution of connective tissue growth factor (CTGF, 100μg / mL) as an anionic solution of a negatively charged bioactive component.

[0128] (4) In the first cycle of self-assembly, the nanofiber membrane is sequentially immersed in the positively charged cationic solution and the negatively charged anionic solution until 1 / 6 of the nanofiber membrane is immersed, and the soaking time is 60min; after each layer deposition, the nanofiber membrane is washed with deionized water for 3 times, each time for 10min, to remove the residual solution, and a 1-time self-assembled fiber membrane is obtained after drying.

[0129] (5) Then, during the second cycle, the volume of the solution is increased so that 2 / 6 of the nanofiber membrane is immersed in the solution, and the remaining self-assembly process is the same as step (4), to obtain a 2-time self-assembled electrospun fiber membrane.

[0130] (6) Repeat cycle step (5) 4 times until the liquid level is higher than the electrospun nanofiber membrane, and the entire membrane is immersed in the two solutions, respectively, and the remaining self-assembly process is the same as step (4), to obtain a gradient fiber membrane after 6 times of self-assembly.

[0131] Example 5

[0132] (1) Dissolve cellulose acetate (CA) in a solvent with a volume ratio of acetone and N,N-dimethylacetamide of 1:1, and magnetically stir at room temperature for 12h to obtain an electrospinning solution with a CA mass concentration of 15%;

[0133] (2) Electrospinning was performed using the obtained electrospinning solution. The voltage was slowly adjusted to ensure stable fiber production; the electrospinning solution was fed at a rate of 1 mL / h, the electrospinning voltage was 15 kV, the receiving distance was 25 cm, and the ambient temperature and relative humidity were maintained at 30 °C and 40%, respectively. The obtained electrospun fiber membrane was dried at room temperature for 12 h and then immersed in a 0.1 M NaOH solution for 24 h before use.

[0134] (3) A deionized water solution of lysozyme (LY, 1 mg / mL) was selected as the cationic solution of the positively charged bioactive component, and a deionized water solution of ovalbumin (AE, 1 mg / mL) was selected as the anionic solution of the negatively charged bioactive component.

[0135] (4) In the first cycle of self-assembly, the nanofiber membrane was sequentially immersed in the positively charged cationic solution and the negatively charged anionic solution until 1 / 10 of the nanofiber membrane was immersed, and the immersion time was 20 min; after each layer was deposited, the nanofiber membrane was washed with a 0.1 M NaCl solution for 3 times, each time for 2 min, to remove the residual solution, and a 1-time self-assembled fiber membrane was obtained after drying.

[0136] (5) Subsequently, in the second cycle, the volume of the solution was increased so that 2 / 10 of the nanofiber membrane was immersed in the solution, and the remaining self-assembly process was the same as step (4), and a 2-time self-assembled electrospun fiber membrane was obtained.

[0137] (6) The cycle of step (5) was repeated 8 times until the liquid level was higher than the electrospun nanofiber membrane, and the entire membrane was immersed in the two solutions, respectively, and the remaining self-assembly process was the same as step (4), and a gradient fiber membrane after 10 times of self-assembly was obtained.

[0138] Example 6

[0139] (1) PCL was dissolved in dichloromethane and N,N-dimethylacetamide at a volume ratio of 3:1 and magnetically stirred at room temperature for 12 h to obtain an electrospinning solution with a PCL mass concentration of 10%.

[0140] (2) Electrospinning was performed using the obtained electrospinning solution. The voltage was slowly adjusted to ensure stable fiber production; the electrospinning solution was fed at a rate of 1.5 mL / h, the electrospinning voltage was 16 kV, the receiving distance was 18 cm, and the receiver was a roller with a rotation speed of 1000 rpm, and the ambient temperature and relative humidity were maintained at 25 °C and 30%, respectively, to obtain an oriented PCL nanofiber membrane; which was dried at room temperature for 24 h and then immersed in a 0.05 M NaOH solution for 48 h before use.

[0141] (3) Deionized water solution of hydroxyapatite (HA, 2 mg / mL) was chosen as the cationic solution of the positively charged bioactive component, and deionized water solution of chitosan (CS, 2 mg / mL) was chosen as the anionic solution of the negatively charged bioactive component.

[0142] (4) In the first cycle of self-assembly, the nanofiber membrane was sequentially immersed in the positively charged cationic solution and the negatively charged anionic solution until 1 / 3 of the nanofiber membrane was immersed, and the soaking time was 30 min; after each layer deposition, the nanofiber membrane was washed with deionized water for 3 times, each time for 5 min, to remove the residual solution, and after drying, a 1-time self-assembled fiber membrane was obtained.

[0143] (5) Then, in the second cycle, the volume of the solution was increased so that 2 / 3 of the nanofiber membrane was immersed in the solution, and the rest of the self-assembly process was the same as step (4), to obtain a 2-time self-assembled electrospun fiber membrane.

[0144] (6) Repeat cycle step (5) once until the liquid level is higher than the electrospun nanofiber membrane, and the entire membrane is immersed in the two solutions respectively, and the rest of the self-assembly process is the same as step (4), to obtain a gradient fiber membrane after 3 times of self-assembly.

[0145] Figure 1 is the Zeta potential determination of the cationic solution and the anionic solution prepared in Example 1, and the Zeta potentials of collagen and chondroitin sulfate are +4.5±0.7 mV and -10.8±0.9 mV respectively, which confirms the opposite charges required for the layer-by-layer self-assembly process. The opposite charges enable layer-by-layer adsorption and achieve layer-by-layer self-assembly.

[0146] Figure 2 、 Figure 3 respectively are the variation diagrams of the adsorption thickness and the adsorption mass of different self-assembly layers in the self-assembly process of Example 1. In order to quantify the adsorption amount of bioactive substances on the nanofiber membrane in the layer-by-layer self-assembly process, the quartz crystal microbalance (QCM) was used to measure the LBL n thickness and mass of the sample, and it was found that the overall thickness of the fiber membrane gradually increased with the increase of the number of self-assembly layers, which further verified the overall increase of the fiber diameter at the microscale. In the 1-time self-assembly process of Example 1, after the assembly of the two bioactive substances respectively, the thickness of the fiber membrane increased by 1.135±0.006 nm and 2.184±0.008 nm respectively. With the increase of the number of self-assembly layers, the thickness and mass of the membrane increased in turn, and until the thickness of the LBL5 fiber membrane increased by 5.118±0.035 nm and the total mass increased by 800±90 ng / cm 2 The test results show that the bioactive substances are successfully deposited on the surface of the electrospun nanofiber through electrostatic interaction.

[0147] Figure 4 is a scanning electron microscope image of the electrospun polycaprolactone (PCL) fiber membrane of Example 1, Figure 5 、 Figure 6 are scanning electron microscope images of the LBL1 and LBL5 fiber membranes of Example 1, respectively, the microstructure and diameter of the electrospun fibers at different self-assembly layers are characterized and counted by a scanning electron microscope (SEM); it can be seen from the figure that the original electrospun nanofibers have a smooth surface and good orientation, and with the increase of the number of LBL self-assembly layers and repeated soaking procedures, the nanofiber surface is slightly convex, and the fiber diameter also gradually increases. The average diameter of the PCL nanofiber in Example 1 is 460±150nm, and the fiber diameter of the LBL5 fiber membrane increases to 940±370nm. It can be seen from the SEM image that the LBL n (n=1,2,3,4,5) fiber membranes still maintain good morphology, porosity and good orientation, and covering complex structures without changing the original morphology is also an important advantage of the present application. Figure 11 and Figure 12 are scanning electron microscope images of the LBL 10 fiber of Example 2 and the LBL3 fiber membrane of Example 6, respectively, the LBL 10 fiber has an average diameter of 1130±320nm, and the average diameter of the LBL3 fiber is 680±240nm, and the nanofiber surface is convex, and the fiber membrane still maintains good morphology, porosity and good orientation.

[0148] Table 1 is an EDS analysis table of the nanofiber layer-by-layer self-assembly 1 and 5 times of Example 1;

[0149] Table 1

[0150]

[0151] Table 1 is the test result of LBL1 and LBL5 membrane samples of Example 1 by X-ray energy spectrometer (EDS), it can be seen that for the effective adsorption of collagen and chondroitin sulfate in Example 1, EDS shows the existence of negatively charged bioactive substances S element and N element in two bioactive substances on the LBL1 fiber membrane, and the content of N and S elements in LBL5 is higher than that of LBL1 fiber membrane, N element increases from 3.41% to 6.19%, S element increases from 0.21% to 0.51%, the increase ratio depends on the content of the two elements in the bioactive substance, indicating that more bioactive substances are adsorbed on the nanofiber with the increase of the number of self-assembly layers.

[0152] Figure 7 is the electrospun polycaprolactone (PCL) fiber membrane and LBL nFTIR spectra of fiber membranes (n = 1, 5) The original fiber membrane and layer-by-layer self-assembly 1 layer and 5 layers of LBL1 and LBL5 fiber membranes were characterized by infrared spectrum analysis, and the infrared spectra of the two bioactive substances were compared and analyzed. It can be seen from the figure that among the new spectral peaks of LBL1 and LBL5 fiber membranes: the peak at 3500-3100 cm -1 is mainly the stretching vibration of N-H and O-H containing amide bond in bioactive substances, 1650 cm -1 represents the stretching vibration of C=O, 1560 cm -1 is considered to be the N-H bending vibration, O-H deformation vibration or C-N stretching vibration of amide III band at 1410 cm -1 , and the weak peak at 1065 cm -1 corresponds to the sulfate group of the negatively charged bioactive substance. The results show that the bioactive substances are successfully assembled on the surface of the fiber membrane, and it is proved that the gradient design and construction of the fiber membrane can be well controlled by adjusting the deposition layer number of layer-by-layer self-assembly.

[0153] Figure 8 For the self-assembly process of Example 1, electrospun polycaprolactone (PCL) fiber membrane and LBL n The change diagram of water contact angle of fiber membranes (n = 1-5) The surface contact angle of fiber membranes with different self-assembly layers was measured by using a water contact angle measuring instrument. The pure fiber membrane has poor hydrophilicity, and the water contact angle of the electrospun polycaprolactone (PCL) fiber membrane of Example 1 without plasma treatment is 127±1.3°, while the hydrophilicity of the nanofiber membrane after plasma treatment is greatly improved, and the water contact angle is reduced to 34±1.4°. This can be attributed to the introduction of hydrophilic groups (-C-OH, -C-O- and -C=O) into the hydrophobic nanofiber. In addition, with the increase of the number of self-assembly layers, the hydrophilicity of LBL n The fiber membrane is further enhanced due to the increase of bioactive substance adsorption, and the LBL5 fiber membrane shows strong hydrophilicity, and the contact angle of the LBL5 fiber membrane in Example 1 is 13±0.3°. This also proves the successful occurrence of layer-by-layer self-assembly behavior, and the improvement of hydrophilicity is conducive to the adhesion and growth of cells.

[0154] Figure 9 For Example 1 in electrospun polycaprolactone (PCL) fiber membrane, LBL nThe proliferation results of BMSCs on the fiber membranes (n = 1-5) and blank samples after 7 days of culture. The proliferation of BMSCs on different self-assembly layer numbers of the fiber membranes was tested by CCK-8 detection. The cell proliferation rate of each group increased with time, indicating that each group had good biocompatibility. More importantly, it showed stronger cell viability with the increase of the number of self-assembly layers. In Example 1, compared with the original nanofiber membrane, the LBL5 nanofiber membrane showed very high cell viability after 7 days of incubation. This shows that the bioactive substances adsorbed by layer-by-layer self-assembly can contribute more bioactivity to the scaffold, and excellent biocompatibility ensures the enhanced interaction between cells and materials, thus showing more attractive cell viability.

[0155] Figure 10 The fiber membranes with gradient scaffolds prepared in Example 1, electrospun polycaprolactone (PCL) fiber membranes, LBL n The migration distance of cells after 3 days of cell migration of the fiber membranes (n = 1-5). The migration of bone marrow mesenchymal stem cells (BMSCs) on different self-assembly layer numbers of the fiber membranes was studied. It can be seen that the migration distance of BMSCs is longer with the increase of the number of self-assembly layers. Among them, the gradient fiber membrane shows excellent and uniform migration distance. In Example 1, the longest and average migration distances of cells on the gradient fiber membrane are longer than those on the original nanofiber membrane and samples LBL1, LBL2, LBL3 and LBL4. Although it is not as good as the LBL5 nanofiber membrane in the longest migration distance, there is no significant difference in the average migration distance. In comparison, the concentration of bioactive substances used by LBL5 uniform scaffold is much higher than that of the gradient fiber membrane. All these results show that by designing a gradient structure of bioactive components on electrospun nanofibers, cell behavior can be well regulated to promote cell migration.

[0156] The test results prove that the present application successfully develops a general method for constructing a double gradient on an electrospun fiber membrane based on layer-by-layer self-assembly technology. The tissue engineering scaffold with a double gradient of bioactive components constructed by this method can effectively promote the proliferation and migration of cells. At the same time, this simple method of constructing a double gradient of bioactive components can be easily extended to the preparation of different types of gradient scaffolds, providing deeper insights for the preparation of gradient structures simulating natural tissues and related applications.

Claims

1. A method for preparing a gradient fiber membrane, the method comprising the following steps: (1) adding a high molecular polymer into a solvent to obtain a solution after sufficient dissolution; (2) electrospinning the solution obtained in step (1) to obtain an electrospun nanofiber membrane; (3) respectively preparing a cation solution and an anion solution; the cation solution is a solution of a positively charged bioactive substance; the anion solution is a solution of a negatively charged bioactive substance; (4) placing the electrospun nanofiber membrane obtained in step (2) vertically in a height direction, first immersing it in the cation solution, then immersing it in deionized water to wash away the unabsorbed positively charged bioactive substance, and then immersing it in the anion solution, and immersing it in deionized water to wash away the unabsorbed negatively charged bioactive substance, to obtain a first self-assembled fiber membrane; the cation solution and the anion solution have the same liquid level height, and the liquid level height is lower than the height of the electrospun nanofiber membrane; (5) raising the liquid level height of the cation solution and the anion solution, still maintaining the same liquid level height, and repeating the self-assembly process of step (4) with the first self-assembled fiber membrane obtained in step (4) to obtain a second self-assembled fiber membrane; (6) repeating step (5) n-2 times until step (4) is repeated with an n-1th fiber membrane, the liquid level height reaches the electrospun nanofiber membrane, and the n th self-assembled fiber membrane is obtained to obtain the gradient fiber membrane; 3≤n≤15. 2.The method for preparing a gradient fiber membrane according to claim 1, wherein: in step (1), the high molecular polymer is at least one of a synthetic high molecular polymer and a natural high molecular polymer; and / or the solvent is at least one of trifluoroethanol, N, N'-dimethylformamide, dichloromethane, acetic acid, hexafluoroisopropanol, acetone, dimethyl sulfoxide, tetrahydrofuran, trichloromethane, methanol and deionized water. 3.The method for preparing a gradient fiber membrane according to claim 2, wherein: the high molecular polymer is at least one of polycaprolactone, polylactic acid, polyurethane, polyvinyl alcohol, poly (lactic-co-glycolic acid), and poly (lactic-co-glycolic acid-co-caprolactone) ; and the natural high molecular polymer is at least one of gelatin, silk fibroin, collagen, chitosan, starch, cellulose and elastin. 4.The method for preparing a gradient fiber membrane according to claim 1, wherein: in step (1), the mass fraction of the high molecular polymer in the solution is 5-20%. 5.The method for preparing a gradient fiber membrane according to claim 1, wherein: in step (2), the electrospinning solution feeding rate is 0.2-2.5 mL / h; the spinning voltage is 10-25 kV; the receiving distance is 10-25 cm; and / or the obtained electrospun nanofiber membrane is dried at room temperature for 12-48 h. 6.The method for preparing a gradient fiber membrane according to claim 5, wherein: the electrospinning solution feeding rate is 0.5-2.0 mL / h; and / or the receiving distance is 15-20 cm. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ After the obtained electrospun nanofiber membrane is dried at room temperature for 12-48 hours, it is soaked in a sodium hydroxide solution or treated by plasma; the concentration of the sodium hydroxide solution is 0.02-1.0 M; the soaking time is 2-48 hours.

7. The method of claim 6, wherein: the concentration of the sodium hydroxide solution is 0.05-0.1 M; and the soaking time is 12-48 hours.

8. The method of claim 1, wherein: step (3), the positively charged bioactive substance is added to deionized water or a deionized water solution with a pH adjusted to form the cation solution; the concentration of the cation solution is 0.1-5 mg / mL; the deionized water solution is a deionized water solution of acetic acid and sodium chloride; and / or the negatively charged bioactive substance is added to deionized water or a deionized water solution with a pH adjusted to form the anion solution; the concentration of the anion solution is 0.1-5 mg / mL; the deionized water solution is a deionized water solution of sodium chloride.

9. The method of claim 8, wherein: the concentration of the cation solution is 1-2 mg / mL; the concentration of the acetic acid is 0.1-1 M; the concentration of the sodium chloride solution is 0.1-1 M; the pH is adjusted to 4-10 using a sodium hydroxide solution; the concentration of the sodium hydroxide solution is 0.1-2 M; and / or the concentration of the anion solution is 1-2 mg / mL; the deionized water solution is a deionized water solution of sodium chloride; the concentration of the sodium chloride solution is 0.1-1 M; the pH is adjusted to 4-10 using an acetic acid solution; and the concentration of the acetic acid solution is 0.1-1 M.

10. The method of claim 9, wherein: the pH of the cation solution is adjusted to 4-6 using a sodium hydroxide solution; and / or the pH of the anion solution is adjusted to 4-6 using an acetic acid solution.

11. The method of claim 1, wherein: step (4), the electrospun nanofiber membrane is placed upright in a container, the cation solution is added, the liquid level is 1 / n of the upright height of the electrospun nanofiber membrane, the soaking time is 10-60 minutes, and after soaking, the electrospun nanofiber membrane is removed, washed in deionized water, ultrapure water, a sodium chloride solution, or phosphate buffered saline (PBS) for 5-30 minutes, and dried; then the electrospun nanofiber membrane is placed upright in a container, the same volume of the anion solution as the cation solution is added, the liquid level is also the same as the height of the cation solution, and after soaking for the same time, the electrospun nanofiber membrane is removed, washed in deionized water, ultrapure water, a sodium chloride solution, or phosphate buffered saline (PBS) for 5-30 minutes, and dried.

12. The method of claim 11, wherein: the concentration of the sodium chloride solution is independently selected from 0.1-1 M.

13. The method of claim 1, wherein: The positively charged bioactive substance and the negatively charged bioactive substance are independently selected from at least one of collagen, quaternary ammonium chitin, chondroitin sulfate, chitosan, phospholucin, lysozyme, ovalbumin, polyallylamine hydrochloride, silk fibroin, hydroxyapatite, beta-tricalcium phosphate, bioglass, laminin, fibronectin, gelatin, and growth factor; The growth factor is at least one of vascular endothelial growth factor, nerve growth factor, brain-derived nerve growth factor, and human acidic fibroblast growth factor.

14. The method of claim 1, wherein: Step (5), The height of the cation solution and the anion solution is 2 / n of the vertical height of the electrospun nanofiber membrane, and the remaining process is the same as step (4), to obtain a 2-time self-assembled electrospun fiber membrane.

15. The method of claim 1, wherein: Step (6), In 3-time self-assembly, the height of the cation solution and the anion solution is 3 / n of the vertical height of the electrospun nanofiber membrane; until n-time self-assembly, the cation solution and the anion solution completely submerge the vertical height of the electrospun nanofiber membrane; the process of each self-assembly is the same as step (4); and / or, 3≤n≤10。 16. A gradient fiber membrane prepared by the method of any one of claims 1-15.

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