A magnetic composite microsphere self-assembly driven in series by magnetic field induction and protein electrostatic force and its preparation method

Abstract

The present invention belongs to the field of biomaterial technology for biomimetic micro-nano assemblies and provides a method for preparing a magnetic composite microsphere self-assembly, comprising: dispersing a trivalent inorganic iron source and a surfactant in ethylene glycol; preparing magnetic composite microspheres containing an Fe3O4 magnetic core via a one-pot hydrothermal synthesis method; and assembling the magnetic composite microspheres by incubating them with proteins, placing them in an external magnetic field for magnetic attraction, removing the magnetic field for redispersion, and finally allowing them to stand to form the magnetic composite microsphere self-assembly. The present invention also provides corresponding magnetic composite microspheres, magnetic composite microsphere self-assemblies, and their applications. The self-assembly preparation process of the present invention does not require replacement of the reaction system and is gentle, convenient, and reproducible. The product exhibits excellent structural and chemical stability, and the assembly process can be terminated or even disassembled within a short time window. These characteristics give the assembly a wide range of potential applications in biological testing, tissue engineering, and cell culture scaffolds.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterial technology for biomimetic micro-nano assemblies. Specifically, it relates to a micron-scale self-assembled structure with a significant aspect ratio, formed using a magnetic nanoparticle-protein complex as the assembly unit and driven in series by magnetic field induction and protein electrostatic forces, and its preparation method. This self-assembled micro-nanostructure is composed of superparamagnetic composite particles and surface-adsorbed protein molecules, thus combining multiple properties such as magnetic physical manipulation, the biological effects of protein molecules, and micro-nano interfaces. The present invention also includes applications of this self-assembled micro-nanostructure. Background Art

[0002] Magnetic field-induced self-assembly is a simple and convenient means of constructing ordered micro- and nanoscale structures using magnetic materials as monomers. By regulating the direction of the magnetic field and the distribution of magnetic field lines, various ordered linear assemblies can be easily obtained. It is applied to the regulation of material morphology in mesoscopic systems and is widely used in energy, catalysis, environment, and bioengineering.

[0003] In traditional magnetic assembly systems, when no external magnetic field is applied, single magnetic particles are dispersed in a medium, with their magnetic moments in a chaotic manner, resulting in a macroscopic magnetic moment that cancels out each other and becomes zero. Under the influence of an external magnetic field, the magnetic particles align along the easy axis of the magnetic field lines. The mutual repulsion or attraction of the magnetic dipole moments orients the magnetic particles, forming anisotropic chain-like or linear assembly structures. In this traditional single system, due to the characteristics of the magnetic dipole force, the magnetic self-assembly structure is usually controlled by parameters such as the strength of the external magnetic field, the specific saturation magnetization of the magnetic particles, and the viscosity of the dispersion (Xu Dan, Duan Haibao, Lu Chunhua, et al. Research Progress on Preparation of Ordered Structured Materials by Magnetic Field-Induced Self-Assembly [J]. Magnetic Materials and Devices, 2015, 000(004): 74-79).

[0004] In order to further expand the application scenarios of magnetic assemblies, single magnetic nanoparticle systems have been introduced into other designs for improvement. In terms of assembly driving force, in addition to the original magnetic field induction, the particles are modified with hydrophobic blocks, and clusters are assembled by the interaction of two-phase interfaces, and agglomerates or two-dimensional arrays are formed by solvent evaporation; specific covalent modification of the magnetic particle surface, such as the base complementary pairing of DNA molecules modified on the particle surface, is used to achieve anisotropic self-assembly. There are also methods such as electrospinning and template in situ synthesis (Tao Ke, Dou Hongjing, Sun Kang. Chemical preparation of iron oxide nanoparticles and their assemblies [J]. Progress in Chemistry, 2006, 18 (11).). Compared with the above systems, magnetic field-induced assembly can often more easily prepare one-dimensional, two-dimensional and three-dimensional ordered structures. However, it is difficult to maintain the original morphology and orientation when the external magnetic field driving force is removed. Therefore, the development of an assembly and its preparation method that has the advantages of magnetic induced assembly, does not rely on external magnetic field forces to maintain morphological and structural stability, and has rich protein molecules on the surface, has significant technical innovation characteristics and good application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide an assembly that can be assembled by magnetic induction and does not rely on external magnetic field force to maintain the stability of the morphology and structure, and a preparation method thereof.

[0006] Given that nanoparticles adsorb multiple layers of protein molecules in a protein solution to form a protein corona (Casals E, Pfaller T, Duschl A, et al., Time Evolution of the Nanoparticle Protein Corona [J]. Acs Nano, 2010, 4 (7): 3623-3632.), the present invention provides a method for preparing an anisotropic assembly of micrometer scale formed by nanoscale magnetic composite particles, which is driven in series by magnetic induction and protein electrostatic forces. The complex formed by the interaction of paramagnetic composite microspheres with different surface modifications and protein molecules is used as a monomer to achieve self-assembly of a three-dimensional assembly under magnetic field induction and electrostatic interaction between molecules of specific protein domains. The three-dimensional assembly is composed of superparamagnetic composite microspheres of ferroferric oxide with different surface modifications of 80-300 nm as the main body. The proteins in the complex include common components in human and animal plasma, such as fibrinogen, immunoglobulin and serum albumin. The self-assembly is a micrometer-scale product visible under a light microscope and has a significant aspect ratio. Further studies have found that supernatant extracts from various cell lines can also promote the self-assembly process.

[0007] The present invention is the first to incorporate the electrostatic force between protein molecules and the magnetic force of an external magnetic field into the magnetic nanoparticle self-assembly system, and after the first step of the magnetic field force driving, the magnetic field is removed and the force between specific protein molecules adsorbed on the surface of the magnetic composite microspheres is used to adjust the structural stability and size, aspect ratio and other parameters of the assembly. Utilizing this time-sharing series dual driving force system, on the one hand, the adjustable range of the morphology structure of the assembly can be increased, the structural stability of the assembly can be increased, and ordered assemblies can be prepared under conditions of wider protein concentrations and more types of protein molecules. The assembly process is implemented in a single liquid phase system, and an in vitro simulation method is provided to promote the formation of stable and ordered self-assemblies of nanoparticles under physiological conditions. The present invention relates to the key parameters of the assembly, the overall preparation process is gentle and convenient, has good repeatability, the product structure and chemical stability are good, and the required protein reagents have good biocompatibility. During the preparation process, by adding polyelectrolytes of different charges, the assembly process can be stopped or even disassembled within a shorter time window. These characteristics give the assembly a wide range of potential applications in biological testing, tissue engineering, and cell culture scaffolds, and provide a new approach to developing in situ self-assemblies suitable for complex physiological environments within the body. The present invention was completed based on this research.

[0008] In one aspect, the present invention provides a method for preparing a magnetic composite microsphere self-assembly, the method comprising the following steps:

[0009] S1, dispersing a trivalent inorganic iron source and a surfactant in ethylene glycol and fully dissolving them; then placing them in a reactor containing a polytetrafluoroethylene liner and heating them at a high temperature of not less than 200°C overnight to prepare a superparamagnetic core by a one-pot hydrothermal synthesis method to obtain an Fe3O4 magnetic core;

[0010] S2, placing the magnetic composite microspheres in a solution containing protein and incubating for at least 5 minutes;

[0011] S3, placing the mixture obtained in step S2 in an external magnetic field for magnetic attraction for at least 2 minutes, removing the magnetic field, redispersing the mixture, and then allowing it to stand to obtain a magnetic composite microsphere self-assembly.

[0012] Preferably, in step S1, after the superparamagnetic core is prepared by a one-pot hydrothermal synthesis method, the obtained magnetic core is repeatedly washed with ethanol and deionized water in the presence of a solid magnet.

[0013] Preferably, repeated washing means dispersing the obtained magnetic core in ethanol, ultrasonically dispersing for 5-20 minutes, dispersing in deionized water, continuing ultrasonication for 5-10 minutes, magnetically separating by applying an external magnetic field, washing for a total of 3-5 times, and dispersing in deionized water.

[0014] Preferably, in step S1, the high temperature of not less than 200°C means that the reaction temperature should be not less than 200°C, for example, heating at 200-220°C.

[0015] The trivalent inorganic iron source is a trivalent inorganic iron salt selected from ferric chloride (FeCl), ferric sulfate (Fe(SO)), and ferric nitrate (Fe(NO)). For example, in a preferred embodiment of the present invention, ferric chloride hexahydrate is used.

[0016] Preferably, in step S2, more ethanol is added to the mixture of ethanol and deionized water than water, for example, the volume ratio of the added ethanol to deionized water is (2-5):1.

[0017] Preferably, the surfactant in step S1 includes but is not limited to one or more of the following: polyacrylic acid, polyglutamic acid, hyaluronic acid, polyvinyl pyrrolidone or sodium acetate.

[0018] Preferably, a pH regulator is added to the liquid in step S1. For example, in a preferred embodiment of the present invention, urea is used as the pH regulator.

[0019] Preferably, in step S2, the protein content in the protein-containing solution is 0-10 mg / mL. The protein content in the solution can be relatively low, but not zero.

[0020] Preferably, the protein is derived from:

[0021] Serum proteins of various components extracted from animal plasma;

[0022] Or the culture supernatant of the cell line.

[0023] Preferably, in step S2, the incubation time is usually not less than 5 minutes, which can be adjusted according to actual conditions, for example, 5-15 minutes, so as to achieve the binding of the magnetic composite microspheres with the protein or polypeptide.

[0024] Preferably, in step S3, the assembly is driven in series by magnetic field induction and protein electrostatic force. For example, the magnetic field is first applied for several minutes, the magnetic field is removed, and the protein is resuspended and dispersed before the assembly is driven by protein force.

[0025] Preferably, the standing time in step S3 is usually not less than 10 minutes, such as 15, 20, 30, 60, 120 minutes or longer. In a preferred embodiment of the present invention, the standing time is 180 minutes.

[0026] Preferably, resuspending and dispersing means resuspending the precipitate by pipetting or re-dispersing it by slight ultrasound.

[0027] Preferably, the method for preparing the self-assembly further comprises the step of modifying the Fe3O4 magnetic core;

[0028] The steps of modifying the Fe3O4 magnetic core are selected from:

[0029] Modification with negatively charged surfactants to obtain magnetic composite microspheres containing a Fe3O4 magnetic core and a surface stabilized by negatively charged surfactants; or

[0030] The surface of the magnetic core particles is coated with one or more layers of silicon oxide shell to obtain magnetic composite microspheres containing a Fe3O4 magnetic core and a negatively charged silicon oxide surface;

[0031] The magnetic core particles coated with one or more layers of silicon oxide shell are positively charged to obtain magnetic composite microspheres containing Fe3O4 magnetic cores and positively charged surfaces and coated with silicon oxide;

[0032] The magnetic core particles coated with one or more layers of silicon oxide shell are carboxylated to obtain magnetic composite microspheres containing Fe3O4 magnetic cores and negatively charged carboxyl groups on the surface and coated with silicon oxide.

[0033] Preferably, when the surface of the magnetic core particles is coated with one or more layers of silicon oxide shell,

[0034] The Fe3O4 magnetic core is dispersed uniformly in a mixture of ethanol and deionized water, and a silicon source pre-dissolved in ethanol is added to the mixture under weak alkaline conditions to obtain silicon oxide-coated magnetic composite microspheres.

[0035] Wherein, in the mixture of ethanol and deionized water, the volume of the added ethanol is greater than the volume of the added deionized water.

[0036] Preferably, in the mixture of ethanol and deionized water, the volume ratio of ethanol to deionized water is (2-5):1.

[0037] Among them, weak alkalinity can adopt pH=8.5-10.5.

[0038] Preferably, the silicon source used includes a silane coupling agent, wherein the silane coupling agent is selected from: ethyl orthosilicate or aminopropyltriethoxysilane.

[0039] Preferably, the surface of the magnetic composite microspheres obtained in step S2 is modified by dispersing the magnetic composite microspheres in methanol, adding branched polyethyleneimine, and washing after ultrasonic stirring for 2-4 hours.

[0040] Preferably, the silica magnetic composite microspheres can be modified with PEI. Alternatively, the composite microspheres can be modified with other polymers containing positively charged amino groups, such as polylysine or PEG derivatives containing amino groups, to obtain positively charged microspheres. In a preferred embodiment of the present invention, 20 mg of silica magnetic composite microspheres can be modified with 20 mg of PEI. The amount of PEI used can be between 15 and 50 mg.

[0041] The washing after ultrasonic stirring for 2-4 hours means washing the superparamagnetic composite microspheres obtained after the reaction with methanol and deionized water for 3-5 times respectively.

[0042] Preferably, the molecular weight of the branched polyethyleneimine is between 2 and 25 k.

[0043] Preferably, in step S2, the solution containing protein can be a PBS solution containing protein, and the protein content in PBS is 0.1-10 mg / mL, or the protein content is close to 0 but not equal to 0.

[0044] Preferably, the protein is selected from:

[0045] Serum proteins of various components extracted from animal plasma;

[0046] Or culture medium supernatant of various cell lines.

[0047] The cells of the present invention are not limited to tumor cell lines, but may also be normal cells such as fibroblasts and leukocytes.

[0048] Preferably, the magnetic composite microsphere self-assembly is prepared by driving in series with magnetic field induction and protein electrostatic force.

[0049] Preferably, in step S3, the duration of applying the external magnetic field may be 5-15 minutes, for example, 7, 8, 10, 12, 14 minutes or longer.

[0050] Preferably, the external magnetic field is selected from: a static magnetic field or a dynamic magnetic field, a uniform or non-uniform magnetic field.

[0051] Preferably, in step S3, after the external magnetic field is removed, the protein-bound magnetic composite microspheres are redispersed and then allowed to stand for at least 10 minutes, for example, 20, 30, 40, 50, 60 minutes or longer. Of course, in practice, the standing time can be appropriately shortened to balance the duration of action and the effect.

[0052] Preferably, a gentle method is used for redispersion, because a too drastic method can easily destroy the interaction between the protein and the magnetic composite microspheres, and even destroy the structure and stability of the protein and other substances in the reaction system.

[0053] Preferably, the magnetic composite microsphere self-assembly is formed on a suitable substrate, which is selected from mica sheets, silicon sheets, glass sheets or plastic well plates.

[0054] Specifically, the self-assembled magnetic composite microspheres obtained by the preparation method of the present invention are obtained by time-sharing serial drive of magnetic induction and protein electrostatic forces. In a preferred embodiment of the present invention, the preparation method comprises the following steps:

[0055] (1) Using ferric chloride as the iron source, polyacrylic acid or sodium acetate as the surfactant, and urea to adjust the pH, the reagents are dispersed in ethylene glycol solvent, and after ultrasonic stirring and dissolution, they are transferred to a stainless steel reactor with a polytetrafluoroethylene liner and heated in a high-temperature oven at 200°C for 12-24 hours to prepare a superparamagnetic core by a solvothermal method; after the reaction is completed, the reactor is cooled to room temperature, and the reaction product is washed three times with ethanol and deionized water respectively with the help of a solid magnet to obtain a Fe3O4 nanoparticle core;

[0056] (2) The product of step (1) is coated with silica, and a certain amount of core particles are dispersed in a mixture of ethanol and deionized water (volume ratio 3:7), uniformly dispersed by ultrasonication, and ammonia water is added to adjust the pH value. Tetraethyl orthosilicate (TEOS) pre-dissolved in ethanol is added to the mixed system using a peristaltic pump, and the appropriate dropwise addition speed is controlled, and the mixture is stirred at room temperature for 12 hours. After the reaction is completed, the obtained superparamagnetic composite microspheres are washed three times with ethanol and deionized water respectively, and dispersed in deionized water to obtain magnetic composite microspheres with negative charges on the surface, which are set aside.

[0057] (3) The product in step (2) can be modified with positive surface charge according to the application purpose, dispersed in methanol, added with an appropriate amount of branched polyethyleneimine (molecular weight between 2-25k), and ultrasonically stirred for 2-4 hours. After the reaction is completed, the obtained superparamagnetic composite microspheres are washed three times with methanol and deionized water respectively to obtain magnetic composite microspheres with positive surface charge, which are dispersed in deionized water and set aside;

[0058] (4) Prepare protein PBS solution, take appropriate amounts of different types of proteins and dissolve them in PBS, with a concentration range of 0.1-10 mg / mL; add 1 mL of protein solution to a centrifuge tube, add appropriate amounts of superparamagnetic composite microsphere aqueous dispersion with different surface modifications, gently flip the centrifuge tube, and incubate at room temperature for 10-15 minutes; to prepare magnetic beads coated with hard protein coronas, add the magnetically separated precipitate to an appropriate amount of PBS and resuspend it; magnetic beads coated with soft protein coronas can be obtained without magnetic separation and resuspending. Both types of magnetic beads coated with protein coronas are dispersed in PBS and set aside;

[0059] (5) Pre-assembly of magnetic microsphere-protein complexes driven by external magnetic field and self-assembly driven by electrostatic interaction between protein molecules after removing the magnetic field. The PBS mixed system of magnetic microsphere-protein complexes obtained in step (4) is magnetically attracted by a solid magnet with a high magnetic field strength for 2-5 minutes. Dark brown aggregates can be observed to form on the wall of the centrifuge tube. The magnetic field is removed to obtain a pre-assembly of magnetic composite microspheres. Then, a 1 mL pipette is used to gently blow several times to allow the adherent aggregates to be resuspended in the PBS liquid phase. The centrifuge tube is allowed to stand for at least 10 minutes to obtain a micrometer-scale linear stable magnetic composite microsphere-protein complex assembly.

[0060] In the present invention, the superparamagnetic composite microspheres in step (4) and step (5) include: Fe3O4 magnetic particles without any secondary modification, with a strong negative charge on the surface; or magnetic composite microspheres coated with a silica shell, the silica shell thickness is adjustable, and the composite microsphere particle size is between 80-300 nm; or the silica magnetic beads are further surface-modified with cationic polyelectrolytes to obtain magnetic composite microspheres with obvious surface positive charge;

[0061] In the present invention, the protein solution can be a solution using PBS as a medium. The protein can be selected from, but not limited to, serum proteins of various components extracted from animal plasma, such as fibrinogen, serum albumin, or immunoglobulins, or PBS supernatant solutions obtained after culturing various cells for a period of time. The various cells can be selected from, but not limited to, HeLa (human cervical cancer cells), 4T1 (mouse breast cancer cells), FB (fibroblasts), leukocytes, and the like.

[0062] PBS is phosphate buffered saline. In a preferred embodiment of the present invention, the formula of the PBS used is:

[0063] Potassium dihydrogen phosphate (KH2PO4): 0.27g, disodium hydrogen phosphate (Na2HPO4): 1.42g, sodium chloride (NaCl): 8g, potassium chloride (KCl) 0.2g, add about 800mL of deionized water and stir thoroughly to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.2-7.4, and finally adjust the volume to 1L.

[0064] In the present invention, the assembly can be formed on a suitable substrate, and the subsequent treatment of the assembly can be a single formation in situ, or a secondary formation after physical disturbance and static formation, or a third formation after physical disturbance and magnetic attraction. The corresponding treatment method is selected according to the application purpose and the surface modification of the particles; the magnetic field induction and the protein electrostatic force are driven in series in a time-sharing manner; the suitable substrate is selected from mica sheets, silicon sheets, glass sheets or plastic well plates.

[0065] In the present invention, the above preparation method can be used to obtain a micron-scale self-assembly with a significant aspect ratio, which is formed by using magnetic nanoparticle-protein complexes as assembly units and driven in series by magnetic field induction and protein electrostatic forces.

[0066] On the other hand, the present invention provides a magnetic composite microsphere self-assembly, which is a three-dimensional assembly that is self-assembled under the induction of a magnetic field and the electrostatic interaction between molecules in specific structural domains of the protein, using the complex formed by the interaction of ferroferric oxide superparamagnetic composite microspheres and protein molecules as a monomer.

[0067] In another aspect, the present invention provides an application of the magnetic composite microsphere self-assembly, wherein the magnetic composite microsphere self-assembly is applied to biological detection, tissue engineering or preparation of cell culture scaffolds.

[0068] Preferably, the magnetic composite microsphere self-assembly can recognize or bind to a target protein or peptide. Alternatively, proteins adsorbed on the surface of magnetic nanoparticles can be used as bridging molecules for the particles, allowing them to align along magnetic lines of force under magnetic induction. This approach combines the rapid response assembly characteristics of magnetic drive with the advantages of protein bridging in maintaining the structural stability of the assembly. Through time-sharing tandem drive using magnetic induction and protein electrostatic forces, a gentle, convenient, rapid, morphology-controllable, and biocompatible micron-scale assembly can be developed across a wide range of protein concentrations and pH media. This approach will provide new insights into applications such as biomonitoring, tissue engineering, and cell culture scaffolds, while also providing an in vitro model system for the in situ self-assembly of nanoparticles in vivo.

[0069] Due to the adoption of the above technical solution, the beneficial effects obtained by the present invention include: the self-assembly obtained by the present invention has more cell binding sites than spherical materials, which can increase the affinity with cells and the specificity of targeting; when used in tissue engineering, one-dimensional materials with a longer aspect ratio can be woven and arranged to form a 3D scaffold material with a long aspect ratio, which is more efficient, more controllable, and more uniform.

[0070] The self-assembly conditions of the present invention are mild, rapid and convenient, the reagents and equipment used are relatively common materials and methods, the operation is simple and the cost is low.

[0071] The protein reagents used in the present invention are all commonly available components in organisms, with universal applicability and good biosafety. Starting from protein binding, the present invention can be completed in a single system, eliminating the need for separation and purification steps, which not only reduces the probability of contamination but also better simulates the actual physiological environment.

[0072] The protein concentration range selected in the present invention covers high concentration to low concentration, which can reach 10 mg / mL or even more, which is closer to the multi-component, high-concentration protein environment under physiological conditions in vivo.

[0073] The self-assembling particles used in the present invention are composite magnetic nanoparticles with superparamagnetism that adsorb and form a complex of soft or hard protein coronas. Under the induction of an external magnetic field, the easy magnetization axis of the magnetic particle core is arranged along the magnetic field lines. During the pre-assembly process, needle-shaped aggregates with a scale of several microns are first formed. In the subsequent assembly process after the magnetic field is removed, the needle-shaped aggregates are first blown and dispersed, and then the nanoparticles are bridged through the intermolecular electrostatic effect of protein molecules adsorbed on the particle surface, thereby ensuring that after the external magnetic field is removed, the pre-assembly forms a linear assembly with a larger size and a larger aspect ratio under the action of liquid microfluid flow. The assembly has good stability under the short-range force of the protein molecules, can resist the interference of fetal bovine serum such as FBS, and cannot be blown apart by a pipette. After the magnetic field is removed, the self-assembly process, once linear assemblies have formed, can be rapidly terminated by adding positively or negatively charged small molecules or polyelectrolytes, such as polystyrene sulfonic acid (PSS), polycyclic aromatic hydrocarbons (PAH), and polyethyleneimine (PEI). Redirecting the microfluidic flow can also lead to disassembly, restoring the micron-sized linear aggregates to nanoparticle-protein complexes. The assembly and disassembly processes exhibit a short response time window and excellent reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0075] Figure 1 is a typical transmission electron microscope image of the magnetic composite microspheres;

[0076] Figure 2 is the hysteresis loop of the magnetic composite microsphere;

[0077] Figure 3 The TGA thermal analysis diagrams of the assembly of the magnetic composite microspheres with bovine fibrinogen and the assembly with bovine serum albumin;

[0078] Figure 4 Representative optical micrographs of the assemblies with added bovine fibrinogen;

[0079] Figure 5 A typical optical micrograph of the assembly with the addition of bovine serum albumin;

[0080] Figure 6 A typical transmission electron micrograph of the assembly is shown in Figure 2. DETAILED DESCRIPTION

[0081] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0082] Example 1

[0083] (1) Accurately weigh 0.081 g of ferric chloride, 0.288 g of polyacrylic acid, and 1.8 g of urea, and disperse the above reagents in 30 ml of ethylene glycol. After ultrasonic stirring and sufficient dissolution, transfer the mixture to a reactor and heat the reaction in a high-temperature oven at 200°C for 12 h. Prepare a 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 5-10 min. After magnetic separation, disperse the precipitate in deionized water, ultrasonically disperse it for 5-10 min, and magnetically separate it using an external magnetic field. Repeatedly wash 3-5 times and disperse it in deionized water to obtain a Fe3O4 superparamagnetic core with a particle size of 100 nm.

[0084] (2) The product of step (1) was dispersed in 120 ml of a mixture of ethanol and deionized water, and ultrasonically dispersed uniformly. 2 ml of ammonia water was added to adjust the pH value. 80 μl of tetraethyl orthosilicate (TEOS) pre-dissolved in 1 ml of ethanol was added to the reaction system using a peristaltic pump. The dropwise addition rate was controlled to be 1 d / 10 s, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the obtained product was dispersed in ethanol, and the mixture was repeatedly washed with ethanol and deionized water for 3-5 times according to the above steps, and then dispersed in deionized water. Silica magnetic composite microspheres with a hydraulic diameter of about 150 nm were obtained.

[0085] (3) Accurately weigh 1 mg of bovine fibrinogen and place it in a centrifuge tube. Add 1 ml of PBS solution to prepare a protein solution with a protein concentration of 1 mg / ml. Gently invert the tube until the protein is completely dissolved. Pipette the aqueous dispersion containing 0.1 mg of the silica magnetic composite microspheres prepared in step (2) and add it to the protein solution. Gently invert the centrifuge tube and incubate at room temperature for 10 min. The product does not need to be washed and is set aside.

[0086] (4) The liquid phase system in step (3) was gently blown with a pipette tip to resuspend and then transferred to a well plate with a mica sheet on the bottom (the mica sheet was placed to facilitate the subsequent direct characterization of the assembly morphology). A solid magnet was applied to the bottom of the well plate for 5 minutes with a magnetic field strength of about 3000 Gauss. After dark brown aggregates were formed on the wall of the vessel, the external magnetic field was removed; a 200μl pipette was used to gently blow and resuspend the aggregates in the liquid phase. The reaction was allowed to stand for 10 minutes, and the bovine fibrinogen-magnetic composite microsphere self-assembly was obtained on the surface of the mica sheet. The mica sheet at the bottom was slowly removed with tweezers, and the morphology and size of the assembly could be directly observed under an optical microscope. Under typical conditions, the assembly exhibited a linear structure with a diameter of 2.0μm and an aspect ratio of about 30.

[0087] Example 2

[0088] (1) Using the same method for synthesizing the superparamagnetic core as in Example 1, the amount of polyacrylic acid was reduced to 0.23 g, while other experimental conditions remained unchanged, to prepare superparamagnetic core particles with a particle size of 150 nm. In the subsequent silica coating process, the amount of TEOS was increased to 100 μl, and 220 nm magnetic silica composite microspheres were prepared.

[0089] (2) Accurately weigh 1 mg of bovine serum albumin and place it in a centrifuge tube. Add 10 ml of PBS solution to prepare a protein solution with a protein concentration of 0.1 mg / ml. Gently invert the tube until it is completely dissolved. Accurately pipette 0.1 mg of the superparamagnetic composite microspheres prepared in step (1) and add it to 1 ml of the protein solution. Gently invert the tube and incubate at room temperature for 10 min.

[0090] (3) The liquid phase mixed system in step (2) was magnetically attracted for 2 minutes using an external magnetic field. After observing the formation of dark brown aggregates on the wall of the centrifuge tube, the magnetic field was removed and the aggregates were aspirated and resuspended in the well plate using a pipette and allowed to stand for 180 minutes to obtain a linear bovine serum albumin-magnetic composite microsphere self-assembly with a stable morphology, having a diameter in the range of 1.5-2.0 μm and an aspect ratio of 20.

[0091] The assembly obtained by the above method is as follows Figure 5 shown.

[0092] Example 3

[0093] (1) Using the same method for synthesizing a superparamagnetic core as in Example 1, except that polyacrylic acid was replaced with sodium acetate and the reagent dosage was 0.56 g, a magnetic core with a particle size of 250 nm was prepared. A similar silica coating process was then performed to obtain silica magnetic composite microspheres with a diameter of approximately 320 nm.

[0094] (2) Disperse 500,000 HeLa cells in 6 ml of PBS, wash with a pipette for 30 seconds, centrifuge at 1200 rpm for 3 minutes, repeat the wash twice, resuspend the cells in 1.5 ml of PBS, let stand at room temperature for 10 minutes, centrifuge at 1500 rpm for 5 minutes, aspirate 1 ml of supernatant, including PBS solution containing cell secretory factors, into a centrifuge tube, accurately aspirate 0.1 mg of the silica magnetic composite microspheres obtained in step (1), add them to 1 ml of the protein solution, gently flip the centrifuge tube, and incubate at room temperature for 10 minutes;

[0095] The liquid phase mixed system in step (2) was magnetically attracted for 2 minutes using an external magnetic field. After observing the formation of dark brown agglomerates on the wall of the centrifuge tube, the magnetic field was removed and the agglomerates were blown and resuspended in the well plate using a pipette and allowed to stand for 10 minutes to obtain a linear self-assembly of magnetic composite microspheres adsorbed with HeLa cell secretory factors with a stable morphology, with a diameter of about 2.5 μm and an aspect ratio of 30.

[0096] Example 4

[0097] The same superparamagnetic core synthesis method as in Example 1 was used, except that 0.578 g of ferric chloride was used, polyacrylic acid was replaced with polyglutamic acid (molecular weight 100-170 w), and the reagent dosage was 0.214 g to prepare a magnetic core with a particle size of 125 nm. In the subsequent silica coating process, the amount of TEOS used was 100 μl, and magnetic composite microspheres with a diameter of approximately 215 nm were obtained.

[0098] In 30 ml of anhydrous methanol, 10 mg of the above-mentioned silica magnetic composite microspheres were added and ultrasonically dispersed for 1 min. Then, 15 mg of branched polyethyleneimine was added and mechanically stirred for reaction at room temperature for 2 h. The microspheres were washed with anhydrous ethanol and deionized water three times each to obtain silica magnetic composite microspheres with positive surface charge.

[0099] Accurately weigh 0.1 mg of growth factor EGF protein and place it in a centrifuge tube. Add 1 ml of PBS solution to prepare a protein solution. Accurately pipette 0.1 mg of superparamagnetic composite microspheres and add them to 1 ml of protein solution. Gently flip the centrifuge tube and incubate at room temperature for 15 minutes.

[0100] The cells were magnetically attracted for 5 minutes using an external magnetic field. After observing the formation of dark brown aggregates on the wall of the centrifuge tube, the magnetic field was removed and the aggregates were aspirated and resuspended in the well plate using a pipette and allowed to stand for 60 minutes to obtain morphologically stable linear bovine serum albumin-magnetic composite microsphere self-assemblies with a diameter in the range of 2.5 μm and an aspect ratio of 15.

[0101] Example 5

[0102] The same method for synthesizing a superparamagnetic core as in Example 1 was used, except that polyacrylic acid was replaced with hyaluronic acid HA and polyethyleneimine for co-stabilization. The reagent dosage was 0.08 g of HA and 0.3 g of PEI to prepare a magnetic core with a particle size of 120 nm. After silica coating, the surface was modified with positive charge to obtain silica magnetic composite microspheres with a diameter of about 200 nm.

[0103] Accurately weigh 15 mg of serum albumin, 10 mg of growth factor TGF, and 15 mg of immunoglobulin protein powder and place them in a centrifuge tube. Mix well and add 10 ml of PBS solution to prepare a protein solution. Accurately pipette 0.1 mg of superparamagnetic composite microspheres and add them to 1 ml of protein solution. Gently invert the centrifuge tube and incubate at room temperature for 10 min.

[0104] The cells were magnetically attracted for 8 minutes using an external magnetic field. After observing the formation of dark brown aggregates on the wall of the centrifuge tube, the magnetic field was removed and the aggregates were aspirated and resuspended in the well plate using a pipette and allowed to stand for 20 minutes to obtain a linear magnetic composite microsphere self-assembly with stable morphology.

[0105] The above descriptions and embodiments are provided to facilitate understanding and application of the present invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these contents and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above descriptions and embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for preparing a magnetic composite microsphere self-assembly, characterized in that: The preparation method comprises the following steps in sequence: S1, dispersing a trivalent inorganic iron salt and a surfactant in a liquid containing ethylene glycol and fully dissolving them; then heating them in a reactor containing a polytetrafluoroethylene liner overnight at a temperature not lower than 200°C to prepare a superparamagnetic core by a one-pot hydrothermal synthesis method, and preparing magnetic composite microspheres containing an Fe3O4 magnetic core based on the superparamagnetic core; S2, placing the magnetic composite microspheres in a solution containing protein and incubating for at least 5 minutes; S3, placing the mixture obtained in step S2 in an external magnetic field for magnetic attraction for at least 2 minutes, removing the magnetic field, redispersing the mixture, and allowing it to stand for more than 10 minutes to obtain a magnetic composite microsphere self-assembly.

2. The preparation method according to claim 1, characterized in that In step S1, after the superparamagnetic core is prepared by a one-pot hydrothermal synthesis method, the obtained magnetic core is repeatedly washed with ethanol and deionized water in the presence of a solid magnet.

3. The preparation method according to claim 2, characterized in that Repeated washing means dispersing the obtained magnetic core in ethanol, ultrasonically dispersing for 5-20 minutes, dispersing in deionized water, continuing ultrasonication for 5-10 minutes, magnetically separating by applying an external magnetic field, washing for a total of 3-5 times, and dispersing in deionized water.

4. The preparation method according to claim 1, characterized in that The trivalent inorganic iron salt in step S1 is selected from: ferric chloride, ferric sulfate, and ferric nitrate.

5. The preparation method according to claim 1, characterized in that The surfactant in step S1 includes but is not limited to one or more of the following: Polyacrylic acid, polyglutamic acid, hyaluronic acid, polyvinylpyrrolidone or sodium acetate.

6. The preparation method according to claim 1, characterized in that In step S1, a pH regulator is further added to the liquid containing ethylene glycol.

7. The preparation method according to claim 6, characterized in that In step S1, urea is used as a pH regulator.

8. The preparation method according to claim 1, characterized in that In step S2, the protein content in the protein-containing solution is 0.05-10 mg / mL.

9. The preparation method according to claim 1, characterized in that The protein is derived from: Serum proteins of various fractions extracted from animal plasma; or Cell-secreted factors in the culture supernatant of cell lines.

10. The preparation method according to claim 1, characterized in that In step S2, the incubation time is 5-15 minutes.

11. The preparation method according to claim 1, characterized in that In step S3, the magnetic field induction and the electrostatic force of the protein are driven in series: the magnetic field is first applied for several minutes, the magnetic field is removed, and then the particles are resuspended and dispersed before the assembly is driven by the protein force.

12. The preparation method according to claim 11, characterized in that Resuspension and dispersion means resuspending the precipitate by pipetting or re-dispersing it by slight ultrasound.

13. The preparation method according to claim 1, characterized in that The external magnetic field in step S3 is selected from: a static magnetic field or a dynamic magnetic field, a uniform or non-uniform magnetic field.

14. The preparation method according to claim 1, characterized in that The magnetic composite microspheres are self-assembled and formed on a suitable substrate; The suitable substrate is selected from mica sheet, silicon sheet, glass sheet or plastic well plate.

15. The preparation method according to any one of claims 1 to 14, characterized in that: It also includes the step of modifying the Fe3O4 magnetic core; The steps of modifying the Fe3O4 magnetic core are selected from: Modification with negatively charged surfactants to obtain magnetic nanoparticles containing a Fe3O4 magnetic core and a negatively charged surfactant-stabilized surface; or The surface of the magnetic core particles is coated with one or more layers of silicon oxide shell to obtain magnetic composite microspheres containing a Fe3O4 magnetic core and a negatively charged silicon oxide surface; The magnetic core particles coated with one or more layers of silicon oxide shell are positively charged to obtain magnetic composite microspheres containing Fe3O4 magnetic core, silicon oxide coating and positively charged surface; The magnetic core particles coated with one or more layers of silicon oxide shell are carboxylated to obtain magnetic composite microspheres containing Fe3O4 magnetic core, silicon oxide coating and negatively charged carboxyl groups on the surface.

16. The preparation method according to claim 15, characterized in that When the surface of the magnetic core particles is coated with one or more layers of silicon oxide shell, The Fe3O4 magnetic core is dispersed uniformly in a mixture of ethanol and deionized water, and a silicon source pre-dissolved in ethanol is added to the mixture under weak alkaline conditions to obtain silicon oxide-coated magnetic composite microspheres. Wherein, in the mixture of ethanol and deionized water, the volume of the added ethanol is greater than the volume of the added deionized water.

17. The preparation method according to claim 16, characterized in that In the mixture of ethanol and deionized water, the volume ratio of ethanol to deionized water is (2-5):

1.

18. The preparation method according to claim 16, characterized in that Weak alkalinity refers to pH = 8.5-10.

5.

19. The preparation method according to claim 16, characterized in that The silicon source used includes a silane coupling agent; The silane coupling agent is selected from: ethyl orthosilicate or aminopropyltriethoxysilane.

20. The preparation method according to claim 15, characterized in that When the magnetic core particles coated with one or more layers of silicon oxide shell are positively charged, The Fe3O4 magnetic core particles coated with one or more layers of silicon oxide shell are dispersed in methanol, and branched polyethyleneimine or polylysine or PEG derivatives with terminal amino groups are added, and ultrasonic stirring is performed for 2-4 hours and then washed.

21. The preparation method according to claim 20, characterized in that Washing after ultrasonic stirring for 2-4 hours means washing the superparamagnetic composite microspheres obtained after the reaction with methanol and deionized water for 3-5 times respectively.

22. The method for preparing a magnetic composite microsphere self-assembly according to claim 20, characterized in that: The molecular weight of the branched polyethyleneimine is between 2 and 25k.

23. The use of the preparation method according to claims 1-14, characterized in that: A micrometer-scale self-assembly with a significant aspect ratio is prepared, using magnetic nanoparticle-protein complexes as assembly units and driven in series by magnetic field induction and protein electrostatic forces.

24. A magnetic composite microsphere self-assembly, characterized in that: The magnetic composite microsphere self-assembly uses the complex formed by the interaction of surface-modified ferroferric oxide superparamagnetic composite microspheres and protein molecules as a monomer, and realizes self-assembly into a three-dimensional assembly under the serial time-sharing drive of magnetic field induction and electrostatic interaction between specific protein domain molecules.

25. The magnetic composite microsphere self-assembly according to claim 24, characterized in that: The particle size of the magnetic composite microsphere self-assembly is rod-shaped or wire-shaped, with a diameter of 1-3 microns and an aspect ratio of 15-30.

26. The magnetic composite microsphere self-assembly according to claim 24, characterized in that: The magnetic composite microsphere self-assembly is prepared by the preparation method described in any one of claims 1-14.

27. Use of the magnetic composite microsphere self-assembly according to any one of claims 24 to 26, characterized in that: The magnetic composite microsphere self-assembly is used in biological detection, tissue engineering or preparation of cell culture scaffolds; or the magnetic composite microsphere self-assembly is placed in an in vitro cell growth environment to detect, promote or improve cell growth status.

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