Magnetic particle-biomacromolecule composite fiber assembly, preparation method thereof and application of magnetic particle-biomacromolecule composite fiber assembly as culture scaffold

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to a magnetic particle-biomacromolecule composite fiber assembly, a preparation method thereof and application of the magnetic particle-biomacromolecule composite fiber assembly as a culture scaffold. Silicon oxide coated magnetic microspheres, biomacromolecule substances and a buffer solution are mixed and incubated, an obtained magnetic particle-biomacromolecule composite material solution is magnetically attracted in an external magnetic field and then mixed with a coupling agent, and then assembling is conducted on the surface of a substrate in the external magnetic field; and obtaining the magnetic particle-biomacromolecule composite fiber assembly on the surface of the substrate. The phagocytic ability of BV2 treated by using the magnetic particle-biomacromolecule composite fiber assembly prepared by the invention as a scaffold is enhanced and is not excessively activated, so that not only can a potential mechanism for regulating and controlling the behaviors of microglia be disclosed, but also the interaction of microglia and dopaminergic neurons can be researched, an in-vitro model is provided for a PD microenvironment, and the application prospect is broad. And the method has important significance for developing a microglial cell treatment strategy aiming at PD.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a magnetic particle-biomacromolecule composite fiber assembly and a preparation method thereof and an application thereof as a culture support. Background Art

[0002] Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons and the formation of Lewy bodies. Microglia play an important role in the pathogenesis of PD. Under normal circumstances, microglia maintain the stability of the nervous system through immune surveillance and debris removal, but in PD, their overactivation can lead to neuronal damage, especially damage to dopaminergic neurons, exacerbating the disease process (Yildirim-Balatan C, Fenyi A, Besnault P, et al. Parkinson's disease-derived α-synuclein assemblies combined with chronic-type inflammatory cues promote a neurotoxic microglial phenotype [J]. Journal of Neuroinflammation, 2024, 21 (1)).

[0003] Due to the complex physiological environment in the brain, it is very difficult to study the specific mechanism of microglia in PD. Therefore, it is crucial to construct a simplified matrix environment in vitro to simulate and regulate the behavior of microglia and study their interaction with dopaminergic neurons to reveal the pathological mechanism of PD. In recent years, materials with micropatterns and micromorphologies have been found to induce and regulate cell behavior (Kim MJ, Lee B, Yang K, et al. BMP-2peptide-functionalized nanopatterned substrates for enhanced osteogenic differentiation of human mesenchymal stem cells [J]. Biomaterials, 2013, 34 (30): 7236-7246.), but existing materials such as PDMS and PMMA (Yang K, Jung H, Lee HR, et al. Multiscale, Hierarchically Patterned Topography for Directing Human Neural Stem Cells into Functional Neurons [J]. ACS NANO, 2014, 8(8): 7809-7822.) Although nanofibers can be generated by electrospinning, there are still problems in terms of the orderliness of highly oriented arrangement and the simplicity and repeatability of the operation process. It is difficult to effectively simulate the microenvironment of PD. In addition, during the electrospinning process, proteins may undergo physical processes such as stretching, shearing and drying, which may affect their structure and activity. Although the fiber formation process can be controlled to a certain extent by optimizing spinning parameters such as voltage, collection distance, solution concentration and composition, and the adverse effects on protein structure and activity can be reduced, it will further increase the complexity of preparation and control. Overall, the preparation method is complicated, which limits its application in in vitro research and regulation of microglial behavior in PD. Summary of the invention

[0004] The purpose of the present invention is to provide a magnetic particle-biomacromolecule composite fiber assembly and a preparation method thereof and an application as a culture scaffold. The magnetic particle-biomacromolecule composite fiber assembly provided by the present invention can effectively simulate the microenvironment of PD, regulate the behavior of microglia and its interaction with dopaminergic neurons, so that it can be used as a culture scaffold for studying and regulating the behavior of microglia in Parkinson's disease. At the same time, the preparation method of the magnetic particle-biomacromolecule composite fiber assembly is simple, and it has the characteristics of good flexibility, regular morphology, rich biomacromolecule active sites and modifiable controllable.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a magnetic particle-biomacromolecule composite fiber assembly, comprising the following steps:

[0007] The magnetic nanoparticles, silane coupling agent and solvent are mixed, and hydrolyzed and coated in an alkaline environment to obtain silicon oxide coated magnetic microspheres;

[0008] The silica-coated magnetic microspheres, biomacromolecules and a buffer solution are mixed and incubated to obtain a magnetic particle-biomacromolecule composite material solution, wherein the biomacromolecules include proteins and / or extracellular polysaccharides, and the magnetic particle-biomacromolecule composite material solution contains the magnetic particle-biomacromolecule composite material;

[0009] The magnetic particle-biomacromolecule composite material solution is subjected to magnetic attraction in an external magnetic field to obtain a magnetic particle-biomacromolecule composite pre-assembly solution;

[0010] Mixing the magnetic particle-biomacromolecule composite pre-assembly solution and a coupling agent to obtain a mixed system;

[0011] The mixed system is assembled on the surface of a substrate in an external magnetic field to obtain a magnetic particle-biomacromolecule composite fiber assembly on the surface of the substrate.

[0012] Preferably, the magnetic nanoparticles include one or more of Fe3O4 nanoparticles, γ-Fe2O3 nanoparticles, CoFe2O4 nanoparticles and Prussian blue nanoparticles;

[0013] The particle size of the magnetic nanoparticles is 1 to 300 nm;

[0014] The silane coupling agent includes ethyl orthosilicate or aminopropyltriethoxysilane.

[0015] Preferably, the biomacromolecule comprises one or more of bovine serum albumin, xanthan gum, collagen and adhesion protein;

[0016] The mass ratio of the silicon oxide coated magnetic microspheres to the biomacromolecules is 1:(0.001-5);

[0017] The incubation temperature is room temperature and the incubation time is 20 to 30 minutes.

[0018] Preferably, the magnetic attraction time is 2 to 5 minutes.

[0019] Preferably, the coupling agent includes a first coupling agent or a second coupling agent; the first coupling agent is N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2-morpholineethanesulfonic acid, and the molar ratio of N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2-morpholineethanesulfonic acid is 2:1:1; the second coupling agent is glutaraldehyde.

[0020] Preferably, the magnetic field strength of the external magnetic field used in the assembly is 0.1-1.5 mT; the temperature of the assembly is 35-37° C., and the time is 12-24 h.

[0021] The present invention provides a magnetic particle-biomacromolecule composite fiber assembly prepared by the preparation method described in the above technical solution, wherein the magnetic particle-biomacromolecule composite fiber assembly is a coating structure assembled on the surface of the substrate and formed by the spatial arrangement of the magnetic particle-biomacromolecule composite fibers.

[0022] The present invention provides the use of the magnetic particle-biomacromolecule composite fiber assembly described in the above technical solution as a culture scaffold.

[0023] Preferably, the culture scaffold is used to study the behavior of nervous system cells in vitro.

[0024] Preferably, the nervous system cells are nervous system cells associated with Parkinson's disease, and the nervous system cells include microglia (BV2 cells) and / or astrocytes;

[0025] The application comprises the following steps:

[0026] The cell suspension is inoculated into a culture medium in a culture container for culture, and the surface of the base of the culture container is assembled with the magnetic particle-biomacromolecule composite fiber assembly described in the above technical solution.

[0027] Preferably, the cell suspension includes microglia and / or astrocytes; and the inoculation amount of cells in the cell suspension during the inoculation is 30-40%.

[0028] The invention provides a method for preparing a magnetic particle-biomacromolecule composite fiber assembly, comprising the following steps: mixing magnetic nanoparticles, a silane coupling agent and a solvent, and performing hydrolysis and coating in an alkaline environment to obtain silicon oxide coated magnetic microspheres; mixing the silicon oxide coated magnetic microspheres with biomacromolecule substances and a buffer solution for incubation to obtain a magnetic particle-biomacromolecule composite material solution, wherein the biomacromolecule substances include proteins and / or extracellular polysaccharides, and the magnetic particle-biomacromolecule composite material solution contains magnetic particle-biomacromolecule composite materials; magnetically attracting the magnetic particle-biomacromolecule composite material solution in an external magnetic field to obtain a magnetic particle-biomacromolecule composite preassembly solution; mixing the magnetic particle-biomacromolecule composite preassembly solution and a coupling agent to obtain a mixed system; assembling the mixed system on a substrate surface in an external magnetic field to obtain a magnetic particle-biomacromolecule composite fiber assembly on the substrate surface. The present invention uses silicon oxide coated magnetic microspheres as the core, and through a three-step assembly method, firstly, a layer of biomacromolecules is uniformly coated on the surface of the silicon oxide coated magnetic microspheres; then, under the action of the first external magnetic field, the magnetic particle-biomacromolecule composite material in the magnetic particle-biomacromolecule composite material solution is preassembled to obtain a solution of a magnetic particle-biomacromolecule composite preassembled body; finally, a coupling agent is added to realize a coupling reaction between the magnetic particle-biomacromolecule composite preassembled body and the substrate surface, and at the same time, under the action of the second external magnetic field, the magnetic particle-biomacromolecule composite preassembled body is assembled on the substrate surface to form a single-layer magnetic composite fiber (i.e., magnetic particle-biomacromolecule composite fiber) with uniform spatial arrangement, and an organic fiber micro-pattern (coating) is formed on the substrate surface, and at the same time, the coupling reaction induced by the coupling agent can enhance the binding force between the single-layer magnetic particle-biomacromolecule composite fiber and the substrate surface. Therefore, the present invention generates fiber morphologies of different specifications on the substrate surface to form an assembly (coating structure) through the change of the external magnetic field, and the preparation method is simple and has high repeatability. The magnetic particle-biomacromolecule composite fiber assembly prepared by the present invention can achieve behavioral regulation of cells in vitro, simulating the interaction between microglia and dopaminergic neuronal cells. The magnetic particle-biomacromolecule composite fiber assembly prepared by the present invention has the following characteristics as a culture scaffold: the magnetic particle-biomacromolecule composite fiber assembly prepared by the present invention has good stability in water, PBS buffer and cell culture medium; the magnetic particle-biomacromolecule composite fiber assembly prepared by the present invention has strong anisotropy and bioactive signals, which can intervene in the regulation of cell behavior.The results of the examples show that the magnetic particle-biomacromolecule composite fiber assembly prepared by the present invention as an in vitro culture scaffold treated with PD-related nervous system cells (BV2 in the example) has enhanced phagocytic ability and is not over-activated, which can not only reveal the potential mechanism for regulating microglial behavior, but also study the interaction between microglia and dopaminergic neurons, providing an in vitro model for the PD microenvironment, which is of great significance for the development of microglial treatment strategies for PD.

[0029] The present invention provides a magnetic particle-biomacromolecule composite fiber assembly prepared by the preparation method described in the above technical solution, wherein the magnetic particle-biomacromolecule composite fiber assembly is a coating structure formed by the spatial arrangement of magnetic particle-biomacromolecule composite fibers assembled on the surface of the substrate, and the magnetic composite fibers have anisotropy and biological activity signals. The magnetic particle-biomacromolecule composite fiber assembly provided by the present invention is used as a culture scaffold to process and analyze the phagocytic ability of BV2 microglia. RNA-seq technical analysis shows that BV2 cells cultured with the magnetic particle-biomacromolecule composite fiber assembly as a culture scaffold show enhanced phagocytic ability, some inflammatory factors are downregulated, and the expression levels of genes related to apoptosis and cell adhesion are downregulated, while the expression levels of genes related to ubiquitin-mediated proteolysis are upregulated, and the cytoskeleton also changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A typical transmission electron microscope photograph of the silicon oxide-coated magnetic microspheres prepared in Example 1;

[0031] Figure 2 A typical scanning electron microscope photograph of the magnetic particle-biomacromolecule composite fiber prepared in Example 1;

[0032] Figure 3 The results of immunofluorescence staining of the magnetic particle-biomacromolecule composite fiber assembly prepared in Example 1 as a culture scaffold to promote lysosomal activation and phagocytosis enhancement;

[0033] Figure 4 The results are that microglia phagocytize PFFs;

[0034] Figure 5 This is a diagram of the cell culture and digestion effects. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing a magnetic particle-biomacromolecule composite fiber assembly, comprising the following steps:

[0036] The magnetic nanoparticles, silane coupling agent and solvent are mixed, and hydrolyzed and coated in an alkaline environment to obtain silicon oxide coated magnetic microspheres;

[0037] The silicon oxide coated magnetic microspheres, biomacromolecules and buffer solution are mixed and incubated to obtain a magnetic particle-biomacromolecule composite material solution, wherein the biomacromolecules include proteins and / or extracellular polysaccharides, and the magnetic particle-biomacromolecule composite material solution contains magnetic particle-biomacromolecule composite materials.

[0038] The magnetic particle-biomacromolecule composite material solution is subjected to magnetic attraction in an external magnetic field to obtain a magnetic particle-biomacromolecule composite pre-assembly solution;

[0039] Mixing the magnetic particle-biomacromolecule composite pre-assembly solution and a coupling agent to obtain a mixed system;

[0040] The mixed system is assembled on the surface of a substrate in an external magnetic field to obtain a magnetic particle-biomacromolecule composite fiber assembly on the surface of the substrate.

[0041] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0042] The present invention mixes magnetic nanoparticles, a silane coupling agent and a solvent, and performs hydrolysis coating in an alkaline environment to obtain silicon oxide coated magnetic microspheres. In the present invention, the magnetic nanoparticles preferably include one or more of Fe3O4 nanoparticles, γ-Fe2O3 nanoparticles, CoFe2O4 nanoparticles and Prussian blue nanoparticles. In a specific embodiment of the present invention, the magnetic nanoparticles are specifically Fe3O4 nanoparticles. The particle size of the magnetic nanoparticles is preferably 1 to 300 nm.

[0043] In a specific embodiment of the present invention, the magnetic nanoparticles are Fe3O4 nanoparticles.

[0044] In a specific embodiment of the present invention, when the magnetic nanoparticles are specifically selected as Fe3O4 nanoparticles, the preparation method of the Fe3O4 nanoparticles preferably comprises the following steps: mixing an inorganic water-soluble iron salt, polyacrylic acid, urea and an alcohol solvent for a solvothermal reaction to obtain Fe3O4 nanoparticles. The inorganic water-soluble iron salt is preferably one or more of ferric chloride, ferric nitrate and ferric sulfate. The alcohol solvent is preferably ethylene glycol. The mass ratio of the inorganic water-soluble iron salt, polyacrylic acid and urea is preferably (0.8-0.85): (0.25-0.3): (1.5-2), and can be specifically selected as 0.81: 0.29: 1.8. The present invention has no special requirements for the amount of the alcohol solvent. The mixing of the inorganic water-soluble iron salt, polyacrylic acid, urea and the alcohol solvent is preferably carried out under ultrasonic conditions, and the solvothermal reaction is preferably carried out in a stainless steel reactor with a polytetrafluoroethylene liner. The temperature of the solvothermal reaction is preferably 200-300°C. The time of the solvothermal reaction is preferably ≥ 12h. The solid product obtained by the hydrothermal reaction is preferably washed with ethanol and then with water in sequence to obtain Fe3O4 nanoparticles. The ethanol washing is preferably carried out under ultrasonic conditions. The water washing is preferably ultrapure water washing or deionized water washing, and the water washing is preferably carried out under ultrasonic conditions, and the number of water washings is preferably 3 to 5 times. The time for the ethanol washing is preferably 5 to 10 minutes, and the time for each water washing is preferably 5 to 10 minutes. In the present invention, the obtained Fe3O4 nanoparticles are preferably dispersed in deionized water and stored at 4°C.

[0045] In the present invention, the silane coupling agent preferably includes tetraethyl orthosilicate (TEOS) or aminopropyltriethoxysilane. In a specific embodiment of the present invention, the silane coupling agent is specifically TEOS. The solvent is preferably ethanol and water, and the water is preferably deionized water. In an embodiment of the present invention, the amount ratio of the magnetic nanoparticles and the silane coupling agent is specifically 150 mg: 80 μL. The more the silane coupling agent (tetraethyl orthosilicate) is used in the present invention, the thicker the silica shell of the silica-coated magnetic microspheres formed. The mixing of the magnetic nanoparticles, the silane coupling agent and the solvent preferably includes: dispersing the magnetic nanoparticles in part of the solvent to obtain a magnetic nanoparticle dispersion; adjusting the pH value of the magnetic nanoparticle dispersion to alkaline using a pH adjuster to obtain an alkaline magnetic nanoparticle dispersion; dissolving the silane coupling agent in the remaining solvent to obtain a silane coupling agent solution; and dropping the silane coupling agent solution into the alkaline magnetic nanoparticle dispersion for hydrolysis and coating. The partial solvent is preferably ethanol and water, and the volume ratio of ethanol to water is preferably 3:7. The pH regulator is preferably ammonia water, and the present invention has no special requirements on the mass concentration of the ammonia water, and the present invention has no special requirements on the amount of the pH regulator, as long as the reaction solution is in an alkaline environment during the hydrolysis coating process.

[0046] In the present invention, the dispersion is preferably carried out under ultrasonic conditions. The residual solvent is preferably ethanol. The volume ratio of the silane coupling agent to the residual solvent is preferably 0.08:1. The dripping is preferably carried out using a peristaltic pump, and the dripping speed is preferably 1 drop / 10s. In the present invention, the temperature of the hydrolysis coating is preferably room temperature, and the time of the hydrolysis coating is preferably 12 to 24h. The hydrolysis coating is preferably carried out under stirring. The time of the hydrolysis coating is calculated from the time when the silane coupling agent solution starts to be dripped.

[0047] In the present invention, the solid product obtained by the hydrolysis coating is preferably washed with ethanol and washed with water in sequence to obtain silica-coated magnetic microspheres. The ethanol washing is preferably carried out under ultrasonic conditions. The water washing is preferably ultrapure water washing or deionized water washing, and the water washing is preferably carried out under ultrasonic conditions. The number of water washings is preferably 3 to 5 times. The time for the ethanol washing is preferably 5 to 10 minutes, and the time for each water washing is preferably 5 to 10 minutes.

[0048] After obtaining the silica-coated magnetic microspheres, the present invention mixes the silica-coated magnetic microspheres, biomacromolecules and a buffer solution for incubation to obtain a magnetic particle-biomacromolecule composite material solution, wherein the biomacromolecules preferably include proteins and / or extracellular polysaccharides, and the magnetic particle-biomacromolecule composite material solution contains a magnetic particle-biomacromolecule composite material. In the present invention, the biomacromolecules more preferably include one or more of bovine serum albumin, xanthan gum, collagen and adhesion protein, and further preferably one or two of bovine serum albumin, xanthan gum, collagen and adhesion protein, and further preferably serum albumin, or xanthan gum, or a mixture of collagen and adhesion protein. The mass ratio of collagen to the adhesion protein in the mixture of collagen and adhesion protein is preferably 2:3. The mass ratio of the silica-coated magnetic microspheres and the biomacromolecules is preferably 1: (0.001-5), and can be specifically 1: 5, 1: 0.01, 1: 0.7 or 1: 1. The buffer solution is specifically preferably PBS. The mixing of the silicon oxide coated magnetic microspheres, biomacromolecules and buffer solution preferably includes: dissolving the biomacromolecules in the buffer solution to obtain a biomacromolecule solution; and mixing the biomacromolecule solution with the silicon oxide coated magnetic microspheres. The incubation temperature is preferably room temperature, and the incubation time is preferably 20 to 30 minutes.

[0049] In the present invention, the magnetic particle-biomacromolecule composite material solution contains the magnetic particle-biomacromolecule composite material. The magnetic particle-biomacromolecule composite material includes the silicon oxide coated magnetic microspheres and the biomacromolecule substances coated on the surface of the silicon oxide coated magnetic microspheres.

[0050] After obtaining the magnetic particle-biomacromolecule composite material solution, the present invention magnetically absorbs the magnetic particle-biomacromolecule composite material solution in an external magnetic field to obtain a magnetic particle-biomacromolecule composite pre-assembly solution. In the present invention, the external magnetic field is preferably provided by a solid magnet. The strength of the external magnetic field used for the magnetic absorption is preferably 0.1 to 1.5 mT, preferably 0.29 to 0.88 mT. The time of the magnetic absorption is preferably 2 to 5 min. After the magnetic absorption, the magnetic particle-biomacromolecule composite material solution forms dark brown agglomerates on the container wall. At this time, the external magnetic field is removed to obtain a magnetic composite fiber pre-assembly. The magnetic composite fiber pre-assembly is gently blown several times to allow the magnetic composite fiber pre-assembly that adheres to the wall to be resuspended in the liquid phase to obtain a magnetic particle-biomacromolecule composite pre-assembly solution.

[0051] After obtaining the magnetic particle-biomacromolecule composite preassembly solution, the present invention mixes the magnetic particle-biomacromolecule composite preassembly solution and a coupling agent to obtain a mixed system. In the present invention, the coupling agent preferably includes a first coupling agent or a second coupling agent. The first coupling agent preferably includes N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 2-morpholineethanesulfonic acid (MES), and the molar ratio of N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 2-morpholineethanesulfonic acid is preferably 2:1:1. The second coupling agent is glutaraldehyde. In the present invention, the first coupling agent promotes the crosslinking of amino and carboxyl groups. The second coupling agent promotes the crosslinking reaction of amino pre-amino groups. The present invention preferably enables the magnetic particle-biomacromolecule composite preassembly to simultaneously fix the fiber on the substrate in the subsequent assembly process by adding a coupling agent. In the present invention, the volume ratio of the coupling agent to the magnetic particle-biomacromolecule composite pre-assembly solution is preferably 1:10.

[0052] After obtaining the mixed system, the present invention assembles the mixed system on the surface of the substrate in an external magnetic field to obtain a magnetic particle-biomacromolecule composite fiber assembly on the surface of the substrate. In the present invention, the substrate is preferably a well plate substrate. The well plate is preferably a 48-well plate, a 24-well plate, a 12-well plate or a 6-well plate. The present invention preferably transfers the mixed system to a well plate for assembly. The strength of the external magnetic field used in the assembly is preferably 0.1 to 1.5 mT, preferably 0.29 to 0.88 mT. The assembly preferably places the well plate containing the mixed system in an incubator and leaves it to stand. The temperature of the assembly is preferably 35 to 37°C, and the time is preferably 12 to 24 hours.

[0053] After the assembly is completed, the present invention preferably removes the supernatant and dries to obtain a magnetic particle-biomacromolecule composite fiber assembly on the substrate surface. The drying is preferably performed in an incubator, the drying temperature is preferably 37° C., and the drying time is preferably 12 hours.

[0054] The present invention provides a magnetic particle-biomacromolecule composite fiber assembly prepared by the preparation method described in the above technical solution, wherein the magnetic particle-biomacromolecule composite fiber assembly is a coating structure assembled on the surface of the substrate and formed by the spatial arrangement of the magnetic particle-biomacromolecule composite fibers.

[0055] In the present invention, the magnetic particle-biomacromolecule composite fiber is formed by directional arrangement of the magnetic particle-biomacromolecule composite material along the direction of the magnetic field line.

[0056] In the present invention, the magnetic particle-biomacromolecule composite fiber assembly has anisotropy and biological activity signals.

[0057] The present invention provides the use of the magnetic particle-biomacromolecule composite fiber assembly described in the above technical solution as a culture scaffold. In the present invention, the culture scaffold is preferably used to study the behavior of nervous system cells in vitro. In a specific embodiment of the present invention, the culture scaffold is specifically used to study the behavior of nervous system cells related to Parkinson's disease in vitro. The nervous system cells related to Parkinson's disease preferably include microglia and / or astrocytes.

[0058] In the present invention, the application preferably comprises the following steps:

[0059] The cell suspension is inoculated into a culture medium in a culture container for culture, and the surface of the base of the culture container is assembled with the magnetic particle-biomacromolecule composite fiber assembly described in the above technical solution.

[0060] In the present invention, the cell suspension preferably includes microglia and / or astrocytes. The culture medium preferably contains: 90% DMEM, 10% FBS and 1% penicillin-streptomycin dual antibody system. The inoculation amount of cells in the cell suspension during inoculation is preferably 30-40%. In the present invention, the inoculation amount of cells in the cell suspension is an estimated value. After the cells adhere to the wall, they occupy 30-40% of the bottom area. At this time, the cells are relatively dispersed and have at least half of the growth space.

[0061] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] Example 1

[0063] (1) Accurately weigh 0.81 g of ferric chloride, 0.288 g of polyacrylic acid, and 1.8 g of urea, disperse the above reagents in 30 mL of ethylene glycol, stir ultrasonically to dissolve them fully, transfer them to a reactor and heat them in a high-temperature oven at 200 °C for 12 h to prepare a Fe3O4 superparamagnetic core by a one-pot hydrothermal synthesis method. After the reaction is completed, wait for the reactor to cool to room temperature, and ultrasonically disperse the obtained black precipitate in ethanol for 5 to 10 min. After magnetic separation, the precipitate is dispersed in deionized water again, ultrasonically dispersed for 5 to 10 min, and magnetically separated using an external magnetic field. The precipitate is washed repeatedly for 3 to 5 times and dispersed in deionized water to obtain a Fe3O4 superparamagnetic core with a particle size of 100 nm.

[0064] (2) Disperse 150 mg of the product in step (1) in a mixture of ethanol and deionized water, disperse it evenly by ultrasonication, add 2 mL of ammonia water to adjust the pH value, add 80 μL of tetraethyl orthosilicate (TEOS) pre-dissolved in 1 mL of ethanol to the reaction system using a peristaltic pump, control the drop rate to 1 drop / 10 s, and stir at room temperature for 12 hours; after the reaction, disperse the obtained product in ethanol, disperse it by ultrasonication for 5 to 10 minutes, separate it by magnetic attraction, disperse the precipitate in deionized water again, ultrasonicate for 5 to 10 minutes, separate it by magnetic attraction using an external magnetic field, wash it repeatedly for 3 to 5 times, and disperse it in deionized water to obtain silica-coated magnetic microspheres with a hydraulic diameter of about 150 nm. Typical transmission electron micrographs of silica-coated magnetic microspheres prepared in this example are shown in FIG. Figure 1 shown.

[0065] (3) Accurately weigh 5 mg of bovine serum albumin and place it in a centrifuge tube. Add 10 mL of PBS solution to prepare a protein solution (protein concentration 0.5 mg / mL). Gently invert the tube until it is completely dissolved. Accurately pipette 0.1 mg of the silica-coated magnetic microspheres from step (2) and add it to 1 mL of the protein solution. Gently invert the tube and incubate at room temperature for 30 min.

[0066] (4) The liquid phase mixed system in step (3) was magnetically attracted for 2 minutes using an external magnetic field (provided by a solid magnet with a magnetic field strength of 0.29 to 0.88 mT). After observing the formation of dark brown aggregates on the wall of the centrifuge tube, the magnetic field was removed and the aggregates were resuspended by a pipette to obtain a magnetic particle-biomacromolecule composite pre-assembly solution. The coupling agent was added at a volume ratio of 1:10 of coupling agent: magnetic particle-biomacromolecule composite pre-assembly solution. The coupling agent was NHS, EDC and MES, and the molar ratio of NHS, EDC and MES was 2:1:1. The mixture was resuspended by a pipette into a 24-well plate and the magnetic field was reapplied (magnetic field strength of 0.29 to 0.88 mT). The mixture was allowed to stand in a 37°C incubator for 12 hours to obtain a linear bovine serum albumin-magnetic composite microsphere assembly with stable morphology. The scanning electron microscope photograph of the linear bovine serum albumin-magnetic composite microsphere fiber is shown in FIG. Figure 2 shown.

[0067] (5) Carefully remove the supernatant from the system assembled by precipitation in step (4) along the tube wall, and place it back in a 37°C incubator for drying for 12 hours. If the coating is stable and does not fall off, subsequent cell culture can be carried out.

[0068] (6) BV2 culture: 55,000 to 65,000 BV2 cells were added to each well of a 24-well plate and cultured for 24 h and 48 h for observation and subsequent experimental treatment.

[0069] Figure 3 This is the immunofluorescence staining result of using the magnetic particle-biomacromolecule composite fiber assembly prepared in Example 1 as a culture scaffold to promote lysosomal activation and phagocytosis enhancement. Figure 4 The result is that microglia phagocytize PFFs (α-synuclein preformed fibrils). Figure 5 This is a diagram of the cell culture and digestion effects.

[0070] Figure 3 This is the result of laser scanning confocal microscopy. After culturing on the cell culture scaffold for 24 hours, the cells were digested on the scaffold, and the lysosomes were stained with lyso-tracker and the cell nuclei were located with hoechst. Figure 3 The results showed that compared with the blank group, the lysosome activation of cells treated with the scaffold was more significant. The digested cells were observed for 72 hours. After 72 hours, the lysosomes remained activated and the average fluorescence intensity was higher than that of the Blank group, proving that the scaffold promoted lysosomal phagocytosis and retained a memory effect on phagocytosis.

[0071] Figure 4This is a laser scanning confocal microscope (confocal) image of engulfed PFF. PFF was modified with pHrodo-red. When pHrodo-red-PFF was engulfed into lysosomes, the pHrodo-red-PFF glowed red because the lysosomes were acidic organelles. After being treated with 0.5mg / mL BSA magnetic protein fiber scaffolds, the cells engulfed more pHrodo-red-PFFs than other groups, proving that the scaffolds improved the phagocytic ability of BV2.

[0072] Figure 5 The picture was taken for optical microscopy. BV2 was cultured on a magnetic protein fiber scaffold and digested with trypsin after the culture. The cells were able to completely detach from the scaffold, and the magnetic protein fiber assembly at the bottom was not destroyed during the digestion and washing process, indicating the stability of the assembly.

[0073] Example 2

[0074] (1) A Fe3O4 superparamagnetic core with a particle size of 100 nm was prepared using the same method as in Example 1.

[0075] (2) The method for preparing silica-coated magnetic microspheres is basically the same as that in Example 1, except that the amount of TEOS is increased to 100 μL.

[0076] (3) Accurately weigh 1 mg of xanthan gum and place it in a centrifuge tube. Add 10 mL of PBS solution to make a solution (xanthan gum concentration 0.1 mg / mL). Gently invert until completely dissolved. Continue to dilute 100 times to 0.001 mg / mL. Accurately pipette 0.1 mg of the silica-coated magnetic microspheres from step (2) and add it to 1 mL of the xanthan gum solution. Gently invert the centrifuge tube and incubate at room temperature for 30 min.

[0077] Steps (4) to (6) are the same as those in Example 1.

[0078] Example 3

[0079] (1) A Fe3O4 superparamagnetic core with a particle size of 100 nm was prepared using the same method as in Example 1.

[0080] (2) Silicon oxide-coated magnetic microspheres were prepared using the same method as step (2) of Example 2.

[0081] (3) Accurately weigh 1 mg of collagen in a centrifuge tube, add 10 mL of PBS solution to prepare a protein solution (collagen concentration 0.1 mg / mL), and prepare 0.05 mg / mL of laminin in the same way. Take a total volume of 1 mL of collagen solution and laminin solution, where the volume ratio of collagen solution to laminin solution is 4:6, to obtain a mixed protein solution, accurately pipette 0.1 mg of silica-coated magnetic microspheres from step (2), add it to 1 mL of the mixed protein solution, gently flip the centrifuge tube, and incubate at room temperature for 30 minutes.

[0082] Steps (4) to (6) are the same as those in Example 1.

[0083] It can be seen from the above embodiments that the present invention uses Fe3SO4@SiO2 nanoparticles with magnetic field responsiveness as the core, and uniformly wraps a layer of protein or polysaccharide crown on the outside of the magnetic core through a three-step assembly method. Under the action of the magnetic field, these nanoparticles form a single layer of magnetic protein fibers with uniform spatial arrangement. The morphology and size of this organic fiber micropattern can be regulated according to the magnetic field, and it has the advantages of good flexibility, morphology and controllable modification. The present invention processed and analyzed the phagocytic ability of BV2 microglia by using the magnetic composite fiber culture scaffold prepared by the above method. RNA-seq technical analysis showed that BV2 cells cultured on magnetic protein fiber scaffolds showed enhanced phagocytic ability, some inflammatory factors were downregulated, and the expression levels of genes related to apoptosis and cell adhesion were downregulated, while the expression levels of genes related to ubiquitin-mediated proteolysis were upregulated, and the cytoskeleton also changed.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a magnetic particle-biomacromolecule composite fiber assembly, characterized in that: The following steps are involved: The magnetic nanoparticles, silane coupling agent and solvent are mixed, and hydrolyzed and coated in an alkaline environment to obtain silicon oxide coated magnetic microspheres; The silica-coated magnetic microspheres, biomacromolecules and a buffer solution are mixed and incubated to obtain a magnetic particle-biomacromolecule composite material solution, wherein the biomacromolecules include proteins and / or extracellular polysaccharides, and the magnetic particle-biomacromolecule composite material solution contains the magnetic particle-biomacromolecule composite material; The magnetic particle-biomacromolecule composite material solution is subjected to magnetic attraction in an external magnetic field to obtain a magnetic particle-biomacromolecule composite pre-assembly solution; Mixing the magnetic particle-biomacromolecule composite pre-assembly solution and a coupling agent to obtain a mixed system; The mixed system is assembled on the surface of a substrate in an external magnetic field to obtain a magnetic particle-biomacromolecule composite fiber assembly on the surface of the substrate.

2. The preparation method according to claim 1, characterized in that: The magnetic nanoparticles include one or more of Fe3O4 nanoparticles, γ-Fe2O3 nanoparticles, CoFe2O4 nanoparticles and Prussian blue nanoparticles; The particle size of the magnetic nanoparticles is 1 to 300 nm; The silane coupling agent includes ethyl orthosilicate or aminopropyltriethoxysilane.

3. The preparation method according to claim 1, characterized in that: The biomacromolecule substance includes one or more of bovine serum albumin, xanthan gum, collagen and adhesion protein; The mass ratio of the silicon oxide coated magnetic microspheres to the biomacromolecules is 1:(0.001-5); The incubation temperature is room temperature and the incubation time is 20 to 30 minutes.

4. The preparation method according to claim 1, characterized in that: The magnetic attraction time is 2 to 5 minutes.

5. The preparation method according to claim 1, characterized in that: The coupling agent includes a first coupling agent or a second coupling agent; the first coupling agent is N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2-morpholineethanesulfonic acid, and the molar ratio of the N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2-morpholineethanesulfonic acid is 2:1:1; the second coupling agent is glutaraldehyde.

6. The preparation method according to claim 1 or 5, characterized in that: The magnetic field strength of the external magnetic field used in the assembly is 0.1-1.5 mT; the temperature of the assembly is 25-37° C., and the time is 12-24 hours.

7. The magnetic particle-biomacromolecule composite fiber assembly prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The magnetic particle-biomacromolecule composite fiber assembly is a coating structure assembled on the surface of the substrate and formed by the spatial arrangement of the magnetic particle-biomacromolecule composite fibers.

8. Use of the magnetic particle-biomacromolecule composite fiber assembly according to claim 7 as a culture scaffold.

9. The use according to claim 8, characterized in that: The culture scaffold is used for studying the behavior of nervous system cells in vitro.

10. The use according to claim 9, characterized in that: The nervous system cells include microglia and / or astrocytes; The application comprises the following steps: Inoculating the cell suspension into a culture medium in a culture container for culturing, wherein the magnetic particle-biomacromolecule composite fiber assembly according to claim 7 is assembled on the base surface of the culture container; The cell suspension includes microglia and / or astrocytes; during the inoculation, the inoculation amount of cells in the cell suspension is 30-40%.

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