CN109507418B

CN109507418B - Magnetic nanoparticle with cell-like structure, immunomagnetic nanoparticle, and preparation method and application thereof

Info

Publication number: CN109507418B
Application number: CN201811260412.3A
Authority: CN
Inventors: 吴尧; 周小熙; 易强英; 张宇佳; 康珂
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-10-26
Filing date: 2018-10-26
Publication date: 2020-01-10
Anticipated expiration: 2038-10-26
Also published as: CN109507418A

Abstract

The invention discloses magnetic nanoparticles with a cell-like structure, immunomagnetic nanoparticles, and a preparation method and application thereof. The magnetic nano-particles with the cell-like structure are made of superparamagnetic Fe₃O₄The magnetic nanoparticle is used as an inner core, and the surface of the magnetic nanoparticle is coated with a leukocyte membrane, so that the magnetic nanoparticle not only shows excellent magnetic response performance, but also has homology between the leukocyte membrane coated on the surface of the magnetic nanoparticle and leukocytes, and can effectively weaken non-specific adsorption to the leukocytes; the antibody is mediated and modified on a leukocyte membrane coated on the surface of the magnetic nanoparticles through lipid molecules-polyethylene glycol-biotin molecules and avidin to obtain the immunomagnetic nanoparticles with simulated cell structures, so that the high-efficiency and high-purity enrichment and separation of CTCs can be realized.

Description

Magnetic nanoparticle with cell-like structure, immunomagnetic nanoparticle, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of bionic immune magnetic nano materials, and relates to a magnetic material with a cell-like structure, a preparation method and application of the magnetic material in the aspect of circulating tumor cell enrichment.

Background

Circulating Tumor Cells (CTCs) refer to tumor cells that have been shed from solid tumors into the blood circulation, and the CTCs spread to other tissues or organs of the body through the circulation system, playing an important role in tumor metastasis and recurrence. By detecting the CTCs, indicative information can be provided for cancer treatment, cancer metastasis, cancer prognosis and the like. Therefore, the detection of circulating tumor cells in peripheral blood has important clinical application value.

However, the extremely low concentration of CTCs in blood (1mL of blood contains only one to several tens of CTCs), seriously hampers the counting of CTCs and further intensive studies. At present, the CTCs enrichment and separation technology can be divided into two categories according to its separation principle: (1) based on the differences of the physical properties of the CTCs and the blood cells, including density, size, deformability, etc., such as a bich density gradient centrifugation solution (FicollHypaque density centrifugation) designed based on the separation principle, a microporous Chip (microporous Chip), etc.; however, because the density, size, deformability and the like of the CTCs and blood cells are greatly overlapped, the CTCs obtained by enrichment based on the difference of physical properties have low purity; (2) based on that the CTCs and blood cells have different antigens on the surfaces, the CTCs are enriched and separated from the blood by modifying antibodies or aptamers corresponding to the antigens on a separation matrix, such as an immunomagnetic separation platform (e.g., CellSearch system), a CTC chip, a MagSweeper device and the like designed based on the separation principle. Among them, the immunomagnetic separation technology has attracted much attention because of its advantages of convenient modification of functional components, simple operation, high capture efficiency, etc. Although the immunomagnetic separation technology has a good prospect, the purity of CTC in an enriched sample is low due to the poor leukocyte absorption resistance of the immunomagnetic separation technology, and the downstream research of CTCs is greatly limited. For example, in the enriched sample of the CellSearch system, which is the only approved CTCs separation platform by FDA, thousands of leukocytes still exist, and the purity of the CTCs is only 0.1-1.4%.

In order to improve the leukocyte absorption resistance without affecting the trapping efficiency, nonionic hydrophilic polyethylene glycol and an artificial lipid bilayer similar to the structure of a cell membrane are modified on a CTCs separation platform. However, the results were not as satisfactory as expected, and when only a small number of CTCs were added to the blood sample, there were still tens to thousands of leukocytes in the sample after enrichment. Subsequently, it was gradually recognized that a natural cell membrane, which is composed mainly of protein and phospholipid bilayers and protects cells from the external environment, may be an ideal material to disguise the material as a homogenous cell. For the platform after leucocyte membrane camouflage, it may be possible to reject the leucocytes (about 10 per ml of blood) present in the blood in large amounts and affecting the targeting of CTCs⁷And so as to create an environment conducive to the separation of high purity CTCs. For example, Xiong et al functionalize cell membranes with azide, extract the cell membranes, and coat the cell membranes with Fe₃O₄On the nanoclusters, dibenzooctyl-modified antibodies were finally attached by click chemistry (Xiong et cl. advanced materials 2016,28,7929-7935.DOI: 10.1002/adma.201601643). Rao et al andthe leukocyte membrane and the platelet membrane are fused, the mixed membrane is coated on magnetic beads in a coextrusion mode, and finally, antibodies capable of specifically targeting CTCs are modified (Rao et cl. advanced functional materials.2018,1803531.DOI: 10.1002/adfm.201803531). Although both of these CTCs separation platforms achieve good anti-leukocyte performance, in order to extract the membrane fraction of interest, it is necessary to combine the steps of low permeability, mechanical disruption and a series of gradient centrifugation to remove the non-disrupted cells and cell contents. Furthermore, the natural membrane components must be integrated with the magnetic particles by means of ultrasound and/or multiple mechanical pressing. In conclusion, the traditional processes of extracting cell membranes and integrating the cell membranes and the magnetic nano platforms involve complex processes and long periods, and special extruders and other equipment are required. Therefore, there is a need to develop a method for conveniently extracting cell membranes and integrating the cell membranes with magnetic nanoparticles.

Disclosure of Invention

The present invention aims to solve the above problems in the prior art, and provide a magnetic nanoparticle with a cell-like structure, which enhances the leukocyte adsorption resistance of the magnetic nanoparticle through the leukocyte membrane coated on the surface of the magnetic nanoparticle.

Another object of the present invention is to provide a method for preparing the magnetic nanoparticles with cell-like structures.

The third purpose of the invention is to provide an immunomagnetic nanoparticle with a cell-like structure, which realizes the enrichment and separation of circulating tumor cells with high efficiency and high purity.

The fourth purpose of the invention is to provide a preparation method of the immunomagnetic nanoparticles with the cell-like structure.

The fifth purpose of the invention is to provide the application of the immunomagnetic nanoparticles with the cell-like structure in enriching the circulating tumor cells.

The magnetic nano-particle with the cell-like structure provided by the invention is made of Fe₃O₄Magnetic nanoparticles sequentially coated on Fe₃O₄Polymer polymer on surface of magnetic nanoparticleThe compound layer, the graphene layer and the leukocyte membrane. The magnetic nano-particles with the cell-like structure are Fe with ultrahigh saturation magnetization₃O₄The nanoparticles are inner core (57.21emu g)^-1) So that the overall saturation intensity of the magnetic nanoparticles reaches the final saturation intensity (50.27emu g)^-1) Has higher saturation magnetization intensity, thereby having good magnetic response performance to an external magnetic field (all magnetic nano particles can be almost completely absorbed by a magnet within 2 min). Fe₃O₄The high molecular polymer layer coated outside the magnetic nano particles is a transition layer, and the high molecular polymer adopted in the invention is polyacrylamide hydrochloride or polyethyleneimine. The high molecular polymer is positively charged and can be used for adsorbing graphene. The graphene coated outside the high polymer layer is carboxylated graphene, is of a nanosheet structure, and can be obtained by market outsourcing; the graphene can be inserted into leukocyte membranes, and a large number of phospholipid molecules are scraped, so that the leukocyte membranes are adsorbed to Fe₃O₄On the magnetic nanoparticles. On one hand, the magnetic nano-particles coated by the leukocyte membrane are repelled by the leukocyte due to the homology with the leukocyte, and the nonspecific leukocyte adsorption is obviously inhibited; on the other hand, the biological compatibility is good, and the biological activity can exist in blood relatively stably. Therefore, the modified corresponding antibody or aptamer and the like of the magnetic nanoparticle with the cell-like structure can be used for targeting, enriching and separating target cells, exosomes and the like in complex samples (such as whole blood, serum, artificial whole blood and the like).

The invention further provides a preparation method of the magnetic nano-particle with the cell-like structure, and the technical idea is that the magnetic nano-particle is prepared by using superparamagnetic Fe₃O₄Graphene nanosheets capable of inserting and extracting a large amount of phospholipids from cell membranes are modified on the surfaces of the nanoparticles (MNs), so that magnetic nanoparticles coated by leukocyte membranes, namely magnetic nanoparticles (BMNs for short) with simulated cell structures, are rapidly obtained. Specifically, first, Fe₃O₄The surfaces of the magnetic nano particles are sequentially coated with a high polymer Layer and a graphene Layer by a Layer-by-Layer self-assembly technology (LbL); as the graphene is negatively charged,therefore, the invention uses the polyacrylamide hydrochloride or polyethyleneimine with positive charge as a high polymer layer, and the graphene is coated on the Fe through the electrostatic effect₃O₄On the magnetic nanoparticles. And then incubating the magnetic nanoparticles obtained by self-assembly with the white blood cells to obtain the magnetic nanoparticles coated by the white blood cells. In a specific implementation mode, the preparation method of the magnetic nanoparticles with the cell-like structure comprises the following steps:

(1) by electrostatic interaction at Fe₃O₄Sequentially coating a high molecular polymer layer and a graphene layer on the surfaces of the magnetic nanoparticles to obtain a product L1;

(2) uniformly mixing the product L1 with a culture medium in which cells are uniformly dispersed, incubating for 1.5-2 h at 37 ℃ and at a carbon dioxide volume concentration of 5%, then carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product to obtain the magnetic nanoparticles of the leukocyte membrane-coated product L1, namely the magnetic nanoparticles with the cell-like structure.

The preparation method of the magnetic nanoparticles with the simulated cell structure comprises the step (1) of performing electrostatic adsorption on Fe₃O₄The surface of the magnetic nanoparticle is sequentially coated with a high polymer layer and a graphene layer, and the specific implementation mode comprises the following steps:

(11) will be uniformly dispersed with Fe₃O₄Mixing the suspension of the magnetic nanoparticles and the aqueous solution of the high-molecular polymer to form a reaction system, reacting for 1-3 hours under an oscillation condition, then carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product to obtain Fe coated with a high-molecular polymer layer₃O₄Magnetic nanoparticles, denoted product L11; fe in the reaction system₃O₄The mass ratio of the magnetic nano particles to the high molecular polymer is 1 (0.08-0.4);

(12) mixing the suspension liquid in which the product L11 is uniformly dispersed with the suspension liquid in which the graphene is uniformly dispersed to form a reaction system, reacting for 1-3 h under the oscillation condition, and reacting the obtained productPerforming magnetic separation on the reaction solution, collecting the separated solid product, and washing the solid product to obtain Fe₃O₄Sequentially coating a high molecular polymer layer and a graphene layer on the surfaces of the magnetic nanoparticles to obtain a product L1; the mass ratio of the product L11 to the graphene in the reaction system is 1 (0.1-0.11).

The high molecular polymer is polyacrylamide hydrochloride (PAH) or Polyethyleneimine (PEI), and the graphene is carboxylated graphene and can be obtained by market outsourcing. Said uniform dispersion of Fe in steps (11) and (12)₃O₄Fe in suspension of magnetic nanoparticles₃O₄The concentration of the graphene is 1-1.25 mg/mL, the concentration of the high molecular polymer in the water solution of the high molecular polymer is 1-2 mg/mL, the concentration of the product L11 in the suspension liquid in which the product L11 is uniformly dispersed is 0.25-0.33 mg/mL, and the concentration of the graphene in the suspension liquid in which the graphene is dispersed is 1-1.1 mg/mL. The washing in steps (11) and (12) aims to remove the high molecular polymer and graphene raw materials which are not coated on the surface of the magnetic nanoparticles, and the obtained solid product is washed by deionized water.

In the preparation method of the magnetic nanoparticles with the cell-like structure, the purpose of the step (2) is to coat the outer surface of the product L1 with leukocyte membranes, and the product L1 is obtained by incubating with cells. The culture medium containing uniformly dispersed cells is obtained by uniformly dispersing cells derived from mouse mononuclear macrophage or human T lymphocyte leukemia cell (Jurkat cell) in a serum-free culture medium. The mass-to-volume ratio of the product L1 to the culture medium is 1 (2-3), the mass is mg, the volume is mL, the number of cells in each 1mL of culture medium is at least 1 x 10⁵And (4) respectively. The preparation process of the cell comprises the following steps: culturing the cells at 37 ℃ and 5% of carbon dioxide volume concentration, digesting the cells to obtain cells such as mouse mononuclear macrophage cells or human T lymphocyte leukemia cells (Jurkat cells) when the cell fusion rate reaches more than 90%, washing the obtained cells for 2-3 times by using PBS buffer solution, and finally dispersing the cells in serum-free high-glucose DMEM (dulbecco's modified eaglemedium) culture medium. The purpose of the washing in step (2) was to remove the leukocyte membrane coated on the surface of the product L1And other impurities can be directly washed by deionized water for 8-20 times.

In the preparation method of the magnetic nanoparticles with the cell-like structure, the reactions in the steps (1) to (2) are carried out under the oscillation condition, and the rotation speed of an oscillator adopted by the oscillation operation is 300-450 r/min.

The invention further provides an immune magnetic nanoparticle with a cell-like structure, which is obtained by inserting lipid molecules-polyethylene glycol-biotin molecules into a leukocyte membrane of the magnetic nanoparticle with the cell-like structure, modifying avidin and finally modifying an antibody. The lipid molecule-polyethylene glycol-biotin molecule of the invention is: monoacyl lipid (C18) -polyethylene glycol-biotin (C18-PEG)₂₀₀₀-Biotin), cholesterol-polyethylene glycol-Biotin (cholestrol-PEG)₂₀₀₀-Biotin) or distearoylphosphatidylethanolamine acylphosphatidylethanolamine-polyethylene glycol-Biotin (DSPE-PEG)₂₀₀₀-Biotin); the Avidin is streptavidin or Avidin (Avidin); the antibody is a Biotin-modified anti-epithelial cell adhesion molecule antibody (anti-EpCAM-Biotin). The immunomagnetic nanoparticles with the simulated cell structure are modified with anti-EpCAM-Biotin antibodies, so that specific targeting on circulating tumor cells can be realized, and leukocyte membranes can inhibit nonspecific adsorption on the leukocytes, thereby greatly improving the purity of enriched circulating tumor cells.

The invention further provides a preparation method of the immunomagnetic nanoparticles with the simulated cell structure, which is used for simply, quickly and efficiently introducing the lipid molecules-polyethylene glycol-biotin molecules and the antibodies, and firstly based on the similarity between the lipid molecules in the lipid molecules-polyethylene glycol-biotin molecules and the phospholipid bilayer in the leukocyte membrane, the lipid molecules-polyethylene glycol-biotin molecules are inserted into the leukocyte membrane by utilizing the hydrophobic effect, so that the magnetic nanoparticles are successfully modified with polyethylene glycol with anti-nonspecific adsorption, and biotin for subsequent introduction of the antibodies. And then avidin is combined on biotin, so that subsequent antibody modification capable of specifically targeting CTCs is facilitated, and immune magnetic nanoparticles (BIMNs) with simulated cell structures are obtained. In a specific implementation mode, the preparation method of the immunomagnetic nanoparticles with the cell-like structure comprises the following steps:

(31) mixing the magnetic nanoparticles with the cell-like structure with a PBS buffer solution containing lipid molecules, polyethylene glycol and biotin molecules to form a reaction system, reacting for 2-5 hours under an oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting a separated solid product, and washing the solid product to obtain a product L31; the mass ratio of the magnetic nanoparticles with the cell-like structure to the lipid molecules-polyethylene glycol-biotin molecules in the reaction system is 5000: (1-3);

(32) mixing the product L31 with a PBS buffer solution containing avidin to form a reaction system, reacting for 2-5 h under the oscillation condition, performing magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product to obtain a product L32; the mass ratio of the product L31 to the avidin in the reaction system is 5000: (1-3);

(33) mixing the product L32 with a PBS buffer solution containing an antibody to form a reaction system, reacting for 6-12 h at 4-7 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product to obtain the immunomagnetic nanoparticles with the simulated cell structure; the mass ratio of the product L32 to the antibody in the reaction system is 1000: (0.5 to 1.5).

In the preparation method of the immunomagnetic nanoparticles with the cell-like structure, the lipid molecules-polyethylene glycol-biotin molecules are: monoacyl lipid (C18) -polyethylene glycol-biotin (C18-PEG)₂₀₀₀-Biotin), cholesterol-polyethylene glycol-Biotin (cholestrol-PEG)₂₀₀₀-Biotin) or distearoylphosphatidylethanolamine-polyethylene glycol-Biotin (DSPE-PEG)₂₀₀₀-Biotin); the concentration of the lipid molecules-polyethylene glycol-biotin molecules in the PBS buffer solution containing the lipid molecules-polyethylene glycol-biotin molecules is 10-30 mu g/mL; the Avidin is streptavidin or Avidin (Avidin) and the like, and the concentration of the Avidin in the PBS buffer solution containing the Avidin is 10-30 mu g/mL; the antibody is biotin-modified anti-epithelial cell adhesion molecule antibody(Biotin-modified anti-EpCAM-Biotin), wherein the concentration of the antibody in the PBS buffer solution containing the antibody is 0.25-0.75 mu g/mu L. And (4) washing in the steps (31) to (33) for removing the biotin, avidin and antibody modified on the surface of the magnetic nanoparticle coated with the leukocyte membrane, and washing the obtained solid product by using deionized water.

In order to reduce non-specific adsorption to cells other than target cells, the preparation method of the immunomagnetic nanoparticles with the cell-like structure comprises the step of co-incubating the immunomagnetic nanoparticles and bovine serum albumin in a PBS buffer solution to reduce the non-specific adsorption. The specific implementation mode is as follows: in the step (33), the obtained immunomagnetic nanoparticles with the cell-like structure are subjected to the following post-treatment: dispersing the obtained immunomagnetic nanoparticles (BIMNs) with the cell-like structure in PBS buffer solution with the bovine serum albumin mass concentration of 0.5% -1.0%, reacting for 20-30 min at 4-7 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product to finish the post-treatment of the immunomagnetic nanoparticles with the cell-like structure. The purpose of washing the solid product is to remove the redundant bovine serum albumin on the surface of the obtained product, and the obtained solid product is washed by deionized water. And (4) resuspending the treated BIMNs in a PBS buffer solution, and storing at 4-7 ℃ for later use.

In the preparation method of the immunomagnetic nanoparticles with the cell-like structure, the reactions in the steps (31) to (33) are carried out under the oscillation condition, and the rotation speed of an oscillator adopted in the oscillation operation is 300-450 r/min.

The immunomagnetic nanoparticles with the cell-like structure can be applied to the high-efficiency and high-purity enrichment and separation of circulating tumor cells. Epithelial cell adhesion molecule (EpCAM) and cytokeratin family members (CK8, CK18, and CK19) are present on epithelial tumor cells but not on blood cells; thus, epithelial cell adhesion molecule (EpCAM) and cytokeratin family members (CK8, CK18, and CK19) have become "gold standards" for the detection of CTCs in patients with epithelial phenotypes; therefore, the immunomagnetic nanoparticles modified with anti-EpCAM antibodies can specifically target and combine with CTCs. The leukocyte membrane coated on the surface of the magnetic nanoparticles can inhibit the nonspecific adsorption of leukocytes, thereby greatly improving the enrichment purity of circulating tumor cells.

Compared with the prior art, the invention has the following beneficial effects:

1. the magnetic nano-particle with the cell-like structure provided by the invention adopts superparamagnetic Fe₃O₄The magnetic nanoparticle is used as an inner core, and the surface of the magnetic nanoparticle is coated with a leukocyte membrane, so that the magnetic nanoparticle not only shows excellent magnetic response performance, but also has homology between the leukocyte membrane coated on the surface of the magnetic nanoparticle and leukocytes, so that nonspecific adsorption to the leukocytes can be effectively weakened, and the magnetic nanoparticle can be suitable for targeting, enriching and separating target cells, exosomes and the like in complex samples (such as whole blood, serum, artificial whole blood and the like).

2. In the preparation process of the magnetic nano-particles with the cell-like structure, the coating of Fe is utilized₃O₄The dispersion interaction between the graphene on the surface of the magnetic nanoparticle and the phospholipid can realize the rapid extraction of the leukocyte membrane, and the magnetic nanoparticle coated with the leukocyte membrane is obtained.

3. The immune magnetic nanoparticles with the cell-like structure provided by the invention modify antibodies on leukocyte membranes coated on the surfaces of the magnetic nanoparticles through the mediation of lipid molecules-polyethylene glycol-biotin molecules and avidin, the immune magnetic nanoparticles not only can have higher enrichment efficiency on CTCs, but also greatly improve the purity (the purity is up to more than 96%) of the enriched CTCs due to the effective inhibition of the leukocyte membranes on the nonspecific adsorption of homologous leukocytes, and have very important significance on the downstream research of CTCs, such as in-vitro culture, amplification and extraction of high-purity DNA, RNA and protein.

4. The immunomagnetic nanoparticles with the simulated cell structures provided by the invention can rapidly modify lipid molecules-polyethylene glycol-biotin to the surfaces of the magnetic nanoparticles coating leukocyte membranes by utilizing the mutual hydrophobic interaction of the lipid molecules and the leukocyte membranes in the lipid molecules-polyethylene glycol-biotin molecules, the polyethylene glycol has the function of nonspecific adsorption resistance, and the biotin lays a foundation for the rapid modification of subsequent antibodies of specific targeting CTCs.

5. The immunomagnetic nanoparticles with the cell-like structure provided by the invention can complete the enrichment and separation of CTCs in a short time (about 5min), thereby ensuring that the enriched CTCs have higher activity and being beneficial to the downstream research of CTCs.

6. The magnetic nanoparticles with the cell-like structure and the preparation method of the immunomagnetic nanoparticles provided by the invention have the advantages of simple process and mild reaction conditions, have great potential values in clinical tests and application aspects, and are suitable for popularization and application in the field of biological medicines.

Drawings

FIG. 1 is a morphology characterization chart of the magnetic nanoparticles prepared in example 1 of the present invention obtained by transmission electron microscopy; wherein B, D, F, H are TEM images (scale: 100 μm) of the PEI coated magnetic nanoparticles (MNs @ PEI), graphene coated magnetic nanoparticles (MNs @ PEI @ G), leukocyte membrane coated magnetic nanoparticles (BMNs) and leukocyte membrane coated immunomagnetic nanoparticles (BIMNs) prepared in example 1, and A, C, E, G are corresponding magnified images (scale: 20 μm), respectively.

FIG. 2 is a graph showing the surface potential change of particles during the preparation of magnetic nanoparticles having a cell-like structure according to example 1 of the present invention.

FIG. 3 is a confocal view and an SDS-PAGE (10 μm scale) view of the magnetic nanoparticles prepared in example 1 under 488nm laser excitation; wherein A is confocal drawing of material obtained by incubating MNs @ PEI @ G and fluorescent dye DiO-prestained J774A.1 cells under 488nm laser excitation, B is SDS-PAGE (Coomassie blue staining drawing) of Cell protein (Cell), Cell membrane protein (M) and leukocyte membrane protein contained in magnetic nanoparticles (BMNs) with Cell-like structure, and C is DSPE-PEG modified staining drawing₂₀₀₀-the confocal image of the magnetic nanoparticles obtained after the co-incubation of the magnetic nanoparticles of Biotin and the fluorescent-labeled streptavidin of isothiocyanic acid (FITC-SA) under the excitation of 488nm laser, D is the imitated fine particleAnd (3) incubating immune magnetic nanoparticles (BIMNs) with cellular structures with donkey anti-goat antibodies marked by fluorescein isothiocyanate to obtain a confocal image of the magnetic nanoparticles under the excitation of 488nm laser.

FIG. 4 is a graph showing the magnetic properties of the magnetic nanoparticles prepared in example 1 of the present invention; wherein A is Fe₃O₄Magnetic nanoparticles and biomagnetic nanoparticles (BIMNs) with cell-like structures prepared in example 1 were measured at room temperature for a hysteresis loop, B is an ultraviolet absorption spectrum of aqueous solutions of the BIMNs at different concentrations at 600nm, and C is a time-efficiency graph of permanent magnet enriched BIMNs.

FIG. 5 is a graph showing the effect of the biomagnetic nanoparticles (BIMNs) with cell-like structures prepared in example 1 on the capture of CTCs in application example 1; wherein, A is a histogram of the capture efficiency of BIMNs to CTCs cells along with the change of the concentration of the BIMNs, B is a histogram of the capture efficiency of BIMNs to CTCs cells along with the change of incubation time, and C is the capture efficiency of BIMNs and the BIMNs of unmodified antibodies to different types of cells under the optimal concentration and the optimal incubation time.

FIG. 6 is a schematic diagram showing the detection limits and the average detection efficiency of the biomagnetic nanoparticles (BIMNs) with the simulated cell structures prepared in example 1 in application example 2 in different systems.

FIG. 7 is a graph showing the effect of anti-leukocyte adsorption capacity of IMMUNOMAGNETIC NANOPARTICLES (BIMNs) having a cell-imitated structure prepared in application example 3 using the method of example 1 and America-Tian-whirly commercial magnetic beads on a sample consisting of MCF-7 cells and Jurkat cells (number ratio: 1:1, MCF-7 cells stained red in advance, and Jurkat cells stained green); wherein A is a confocal superposition map (scale is 50 μm) of a product obtained after capture by American, Tian, whirly and commercial magnetic beads under laser excitation, B is a confocal superposition map (scale is 50 μm) of a product obtained after capture by BIMNs under laser excitation, and C is a purity histogram before and after capture of MCF-7 cells.

FIG. 8 is a trichromatic immunostaining confocal image (10 μm scale) of typical MCF-7 cells and non-specifically captured leukocytes enriched from MCF-7 cell-dispersed whole blood using the biomagnetic nanoparticles (BIMNs) of the mock cell structure prepared in example 1 in application example 4.

FIG. 9 is a graph showing the effect of using the biomagnetic nanoparticles (BIMNs) with simulated cell structures prepared in example 1 and the American day and whirlwind commercial magnetic beads in application example 5 on the leukocyte adsorption capacity of the erythrocyte-removed blood containing MCF-7 cells and the whole blood simulant sample containing MCF-7 cells; a, D is a confocal overlay (50 μm scale) of products obtained by capturing two blood samples with American-day-whirling commercial magnetic beads, B, E is a confocal overlay (50 μm scale) of products obtained by capturing two blood samples with BIMNs under laser excitation, and C, F is a histogram of capture efficiency and capture purity of MCF-7 cells with American-day-whirling commercial magnetic beads and BIMNs.

FIG. 10 is a confocal map (with a scale of 100 μm) of the cell death and survival staining of MCF-7 in application example 6, wherein A is the confocal map of the cell death and survival staining of MCF captured by the cell-like immunomagnetic nanoparticles (BIMNs) prepared in example 1 in the experimental group in an enrichment manner, and B is the confocal map of the cell death and survival staining of MCF in the control group without the enrichment and capture process.

FIG. 11 is a diagram of the in vitro culture of MCF-7 cells enriched in the experimental group of FIG. 10, wherein A, B, C is a diagram of the culture effect of the first generation cell culture stage for 4h, 24h and 48h, and D, E, F is a diagram of the culture effect when the fusion rate reaches about 90% in the first, second and third generation stages, respectively (the scale is 100 μm).

Detailed Description

The technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the technical solutions provided by the present invention clearer, the following provides a more detailed description and explanation with reference to the embodiments.

Fe used in the following examples₃O₄Magnetic nanoparticlesThe specific preparation process comprises the following steps: adding 1.157g of ferric chloride hexahydrate, 3.303g of ammonium acetate and 0.4g of sodium citrate into a reaction kettle containing 60mL of ethylene glycol, magnetically stirring for 1-3 h to uniformly mix the raw materials, removing a stirrer, putting the reaction kettle into a heating furnace, heating to 200 ℃, reacting for 16 h, turning off the power supply of the heating furnace, preserving the heat of the reaction kettle in the heating furnace for 1h, and taking out the reaction kettle. After the reaction kettle is cooled to room temperature, carrying out magnetic separation on the reaction liquid for more than 1h and collecting the separated solid product; then, the solid product is repeatedly washed for about 5 times by using ethanol and deionized water in sequence, the supernatant is completely clear and transparent, and the collected black magnetic nanoparticles are Fe₃O₄And (3) placing the magnetic nanoparticles in a refrigerator at 4-7 ℃ for later use.

Fe obtained by the above method₃O₄The magnetic nano-particles can be well dispersed in water to form stable superparamagnetic nano-particle suspension. By analysis, Fe₃O₄The particle size of the magnetic nanoparticles is about 300 nm.

In the following examples, the oscillation is performed in a conventional oscillator, and the rotation speed of the oscillator is 300-450 rpm.

The antibody used in the following examples is a Biotin-modified anti-epithelial cell adhesion molecule antibody (anti-EpCAM-Biotin), which is commercially available from R & D Systems, under the trade designation BAF 960.

The graphene adopted in the following examples is carboxylated graphene, which is purchased from outside the market, and is manufactured by Nanjing Ginko nanotechnology Co., Ltd, with the product number of JCG-1-50n-COOH, the diameter of the carboxylated graphene nanosheet is about 50nm, the thickness of the carboxylated graphene nanosheet is about 0.8-1.2 nm, and about 8% of carbon atoms are carboxylated.

Example 1

The preparation of the immunomagnetic nanoparticles with the cell-like structure of the embodiment comprises the following steps:

(1) preparation of graphene and PEI coated Fe₃O₄The magnetic nanoparticles (denoted as product L1, MNs @ PEI @ G) of (1), comprising the sub-steps of:

(11) mixing Fe₃O₄Uniformly dispersing magnetic nano particles (MNs) into deionized water to obtain Fe₃O₄Dissolving Polyethyleneimine (PEI) into deionized water to obtain a PEI aqueous solution with the concentration of 1.5mg/mL by using a suspension with the magnetic nanoparticle concentration of 1 mg/mL; 5mL of a solution in which Fe was uniformly dispersed₃O₄Mixing the suspension of the magnetic nanoparticles with 0.5mL of PEI (polyetherimide) aqueous solution, reacting for 2h under the oscillation condition, carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain PEI-coated Fe₃O₄The magnetic nanoparticles (MNs @ PEI) of (a), denoted as product L11;

(12) uniformly dispersing the product L11 into deionized water to obtain a suspension with the concentration of the product L11 being 0.33mg/mL, and uniformly dispersing graphene into the deionized water to obtain a suspension with the concentration of the graphene being 1 mg/mL; mixing 15mL of suspension in which the product L11 is uniformly dispersed with 0.5mL of suspension in which graphene is uniformly dispersed, reacting for 2h under the oscillation condition, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain magnetic nanoparticles of the graphene-coated product L11, namely a product L1(MNs @ PEI @ G);

(2) preparing magnetic nanoparticles (BMNs) with a cell-like structure, comprising the following substeps:

(21) fusing the mouse mononuclear macrophage (J774.1, about 2-3 × 10)⁶One) was scraped with a cell scraper, and the scraped cells were washed 3 times with PBS buffer and uniformly dispersed in 10mL of high-sugar serum-free culture medium DMEM (Gibco | Life Technologies (Grand Island, USA)) to obtain a culture medium in which the cells were uniformly dispersed;

(22) adding 5mg of the product L1 into a culture medium in which cells are uniformly dispersed, uniformly mixing, incubating for 2h at 37 ℃ and under the condition that the volume concentration of carbon dioxide is 5%, then carrying out magnetic separation on the obtained reaction, collecting the separated solid product, and washing the solid product for 10 times by using deionized water to obtain magnetic nanoparticles of a leukocyte membrane coated product L1, namely the magnetic nanoparticles (BMNs) with a cell-like structure;

(3) preparation of immunomagnetic nanoparticles (BIMNs) with a cell-like structure comprising the steps of:

(31) mixing DSPE-PEG₂₀₀₀-Biotin (Daorhifang) dissolved in PBS buffer to obtain DSPE-PEG₂₀₀₀-PBS buffer with Biotin concentration of 20 μ g/mL; adding 0.1mLDSPE-PEG into 5mg of magnetic nano particle BMNs with the cell-like structure₂₀₀₀-Biotin in PBS buffer, reacting for 3h under shaking conditions, then performing magnetic separation on the obtained reaction and collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain a product L31;

(32) dissolving streptavidin (Aladdin, CAS number: 9013-20-1, product number S103034) in PBS buffer solution to obtain PBS buffer solution with streptavidin concentration of 20 μ g/mL; adding 0.1mL of streptavidin PBS buffer solution into 5mg of the product L31, reacting for 3h under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain a product L32;

(33) dispersing Biotin-modified anti-epithelial cell adhesion molecule antibody (Biotin-modified anti-EpCAM-Biotin) into PBS buffer solution to obtain PBS buffer solution with the concentration of the anti-EpCAM-Biotin being 0.5 mu g/mu L; adding 10 mu of Lanti-EpCAM-Biotin PBS buffer solution into 5mg of the product L32, reacting for 10h at 4 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution and collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain the immunomagnetic nanoparticles with the imitated cell structure;

(34) dispersing the obtained immune magnetic nanoparticles with the cell-like structure in PBS buffer solution with bovine serum albumin volume concentration of 0.5%, reacting for 30min at 4 ℃ under the condition of oscillation, then carrying out magnetic separation on the obtained reaction solution and collecting separated solid products, washing the solid products for 3 times by using deionized water to obtain post-treated immune magnetic nanoparticles (BIMNs) with the cell-like structure, and suspending the particles in the PBS buffer solution to be placed in a refrigerator at 4 ℃ for later use.

The product obtained in this example was analyzed for morphology, microstructure and magnetic properties:

1. topography analysis

The transmission electron microscope is adopted to analyze the shapes of the magnetic nanoparticles MNs @ PEI, MNs @ PEI @ G, BMNs and BIMNs, and the obtained TEM shape graph is shown in FIGS. 1A-H. Comparing fig. 1A and C, it can be seen that, in fig. 1C, substances with lower contrast and in a flake shape are added on the surface of the magnetic nanoparticle, which indicates that the graphene nanosheet has been successfully modified on the surface of the magnetic nanoparticle MNs @ PEI. As can be seen from fig. 1E and F, after the magnetic nanoparticles MNs @ PEI @ G are incubated with j774a.1 cells, a layer of halo with relatively low contrast is covered on the surface of the magnetic nanoparticles, which indicates that cell membranes are scraped by graphene, and magnetic nanoparticles BMNs coated by target cell membranes are obtained. As shown in FIGS. 1G and H, after biotin-modified anti-epithelial cell adhesion molecule antibodies (anti-EpCAM) capable of specifically targeting CTCs are further modified on the magnetic nanoparticle BMNs, the obtained magnetic nanoparticle BIMNs have a thicker halo with a shallower surface contrast, which indicates that the biomagnetic nanoparticle BIMNs with a cell-like structure is successfully constructed.

2. Microstructure analysis

The surface potentials of the magnetic nanoparticles MNs, MNs @ PEI @ G, BMNs are tested by adopting a Zetasizer Nano ZS90 type particle size analyzer, the analysis result is shown in figure 2, and the surface potential of the material can be seen to change along with the layer-by-layer self-assembly process, so that Fe and Fe are obtained₃O₄The surface potential of the magnetic nanoparticles is-20 mV, after the magnetic nanoparticles are sequentially coated with the PEI with positive electricity and the graphene with negative electricity, the surface potential of the magnetic nanoparticles is deflected to be +37mV and-10.5 mV respectively, and further the PEI is successfully coated on the surface of the magnetic nanoparticles. After the magnetic nanoparticles MNs @ PEI @ G and J774A.1 cells are incubated together, the surface charge of the magnetic nanoparticles is changed to-27 mV, which indicates that a negatively charged cell membrane is successfully scraped off and coated on the surfaces of the magnetic nanoparticles, and the magnetic nanoparticles BMNs with the cell-like structure are obtained.

When the J774A.1 cells washed in the step (21) are pre-stained by a cell membrane green fluorescent probe (DiO) (a lipophilic dye which is combined with a phospholipid bilayer of a cell membrane and emits green fluorescence under 488nm laser excitation), the pre-stained cells and the magnetic nanoparticles MNs PEI @ G are incubated together to obtain the magnetic nanoparticles BMNs with the simulated cell structure, and the BMNs emit green fluorescence under 488nm laser excitation, as shown in FIG. 3A, the lipid molecules on the cell membrane are successfully scraped to the surfaces of the magnetic nanoparticles MNs @ PEI @ G.

The total protein of J774A.1 cells, the protein of J774A.1 cell membranes and the protein contained in the BMNs of the magnetic nanoparticles with the cell-imitated structure are compared by an SDS-PAGE Coomassie blue staining method, as shown in figure 3B, the protein contained in the BMNs of the magnetic nanoparticles with the cell-imitated structure is similar to the protein of the cell membranes, which indicates that the magnetic nanoparticles coated with the cell membranes prepared by the method have greater similarity with the white blood cells, so the BMNs of the magnetic nanoparticles with the cell-imitated structure are repelled by the white blood cells due to the homology, and the nonspecific adsorption of the white blood cells can be obviously inhibited.

When the streptavidin adopted in the step (32) is marked by fluorescein isothiocyanate, the streptavidin is modified by DSPE-PEG₂₀₀₀The magnetic nanoparticles (product L32) obtained after the incubation of the magnetic nanoparticles of Biotin (product L31) with the dyed streptavidin emit green fluorescence under the excitation of 488nm laser, as shown in FIG. 3C, which illustrates that DSPE-PEG emits green fluorescence₂₀₀₀Biotin molecules and streptavidin have been successfully modified on magnetic nanoparticle BMNs with a cell-like structure.

When the immunomagnetic nanoparticles BIMNs with the cell-like structure and the donkey anti-goat antibody marked by fluorescein isothiocyanate are incubated for 2h under the oscillation condition, the incubated magnetic nanoparticles emit green fluorescence under the excitation of 488nm laser, which indicates that the anti-EpCAM-Biotin is successfully modified on the immunomagnetic nanoparticles with the cell-like structure.

3. Detection of magnetic Properties

Model BHV-525 Vibration Sample Magnetometer (VSM) is adopted to respectively detect Fe₃O₄The magnetic hysteresis loops of the Magnetic Nanoparticles (MNs) and the immunomagnetic nanoparticles (BIMNs) with cell structures in the range of-18000 Oe to 18000Oe are shown in FIG. 4A, and the results are shown in FIG. 4A, and it can be seen from the graph that the hysteresis loops of all the samples pass through the origin, have no remanence and coercivity, and show that MNs and BIMNs have no remanence and coercivity and have super-cisMagnetic property and high saturation magnetization of 57.21emu/g and 50.27 emu/g.

In order to further test the magnetic response time of the BIMNs, the absorption intensity of the BIMNs aqueous solutions with different concentrations at 600nm is detected, and the standard curve of the concentration of the BIMNs-the ultraviolet absorption intensity is shown in FIG. 4B. It can be seen that the uv absorption intensity of BIMNs at 600nm increases linearly with increasing concentration. Adsorbing 100 μ g/mL BIMNs water solution with a permanent magnet, measuring the absorbance values of APMNs adsorbed by the permanent magnet at 600nm at different adsorption times, calculating the percentage of BIMNs adsorbed by the permanent magnet at different adsorption times according to a BIMNs concentration-ultraviolet absorption intensity standard curve, and obtaining a change curve of the percentage of BIMNs adsorbed with adsorption time as shown in FIG. 4C. As can be seen from FIG. 4C, about 95% of BIMNs were separated by magnetic separation for 30 s. The fact that the magnetic response capability of the BIMNs is very fast shows that the BIMNs can be rapidly separated after being captured by the BIMNs in the subsequent CTCs capturing process.

Example 2

(1) preparation of graphene and PAH-coated Fe₃O₄The magnetic nanoparticles (denoted as product L1, MNs @ PAH @ G) of (a), comprising the sub-steps of:

(11) mixing Fe₃O₄Uniformly dispersing magnetic nano particles (MNs) into deionized water to obtain Fe₃O₄Dissolving polyacrylamide hydrochloride (PAH) into deionized water to obtain a PEI aqueous solution with the concentration of 1mg/mL, wherein the magnetic nano particle concentration of the suspension is 1.25 mg/mL; 5mL of a solution in which Fe was uniformly dispersed₃O₄Mixing the suspension of the magnetic nanoparticles with 0.5mL of PAH aqueous solution, reacting for 1h under oscillation, carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain PAH-coated Fe₃O₄The magnetic nanoparticles (MNs @ PAH) of (1), denoted as product L11;

(12) uniformly dispersing the product L11 into deionized water to obtain a suspension with the concentration of the product L11 of 0.33mg/mL, and uniformly dispersing graphene into the deionized water to obtain a suspension with the concentration of 1.1 mg/mL; mixing 15mL of suspension in which the product L11 is uniformly dispersed with 0.5mL of suspension in which graphene is uniformly dispersed, reacting for 1h under the oscillation condition, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain magnetic nanoparticles of the graphene-coated product L11, namely a product L1(MNs @ PAH @ G);

(21) human T lymphocyte leukemia cells (Jurkat, about 2-3 × 10) with fusion rate of 90%⁶One) was scraped with a cell scraper, and the scraped cells were washed 2 times with PBS buffer and uniformly dispersed in 15mL of high-sugar serum-free medium DMEM (Gibco | Life Technologies (Grand Island, USA)) to obtain a medium in which the cells were uniformly dispersed;

(22) adding 5mg of the product L1 into a culture medium in which cells are uniformly dispersed, uniformly mixing, incubating for 1.5h at 37 ℃ and under the condition that the volume concentration of carbon dioxide is 5%, then carrying out magnetic separation on the obtained reaction, collecting the separated solid product, and washing the solid product with deionized water for 8 times to obtain magnetic nanoparticles of a leukocyte membrane-coated product L1, namely magnetic nanoparticles (BMNs) with a cell-like structure;

(31) mixing C18-PEG₂₀₀₀-Biotin (Peng) was dissolved in PBS buffer to obtain C18(C18) -PEG₂₀₀₀-PBS buffer with Biotin concentration of 10 μ g/mL; adding 0.1mLC18-PEG into 5mg of magnetic nano-particle BMNs with imitated cell structures₂₀₀₀-Biotin in PBS buffer, reacting for 2h under shaking conditions, then performing magnetic separation on the obtained reaction and collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain a product L31;

(32) avidin (Sigma, CAS No.: 1405-69-2, product No. 215-783-6) was dissolved in PBS buffer to obtain PBS buffer with Avidin concentration of 10. mu.g/mL; adding 0.1ml of PBS buffer solution of avidin into 5mg of the product L31, reacting for 2 hours under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product for 3 times by using deionized water to obtain a product L32;

(33) dispersing Biotin-modified anti-EpCAM-Biotin into PBS buffer solution to obtain PBS buffer solution with the concentration of the anti-EpCAM-Biotin being 0.25 mu g/mu L; adding 10 mu L of anti-EpCAM-Biotin PBS buffer solution into 5mg of the product L32, reacting for 6h at 7 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution and collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain the immunomagnetic nanoparticles with the imitated cell structure;

(34) dispersing the obtained immune magnetic nanoparticles with the cell-like structure in PBS buffer solution with bovine serum albumin volume concentration of 1%, reacting for 20min at 7 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution and collecting separated solid products, washing the solid products for 3 times by deionized water to obtain post-treated immune magnetic nanoparticles (BIMNs) with the cell-like structure, and re-suspending the particles in the PBS buffer solution and placing the particles in a refrigerator at 7 ℃ for later use.

Example 3

(11) mixing Fe₃O₄Uniformly dispersing magnetic nano particles (MNs) into deionized water to obtain Fe₃O₄Dissolving Polyethyleneimine (PEI) into deionized water to obtain a PEI aqueous solution with the concentration of 2mg/mL by using suspension with the magnetic nanoparticle concentration of 1.25 mg/mL; 2mL of a solution in which Fe was uniformly dispersed₃O₄Mixing the suspension of the magnetic nanoparticles with 0.5mL of PEI (polyetherimide) aqueous solution, reacting for 3h under the oscillation condition, carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain PEI-coated Fe₃O₄The magnetic nanoparticles (MNs @ PEI) of (a), denoted as product L11;

(12) uniformly dispersing the product L11 into deionized water to obtain a suspension with the concentration of the product L11 being 0.25mg/mL, and uniformly dispersing graphene into the deionized water to obtain a suspension with the concentration of the graphene being 1 mg/mL; mixing 20mL of suspension in which the product L11 is uniformly dispersed with 0.5mL of suspension in which graphene is uniformly dispersed, reacting for 3h under the oscillation condition, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain magnetic nanoparticles of the graphene-coated product L11, namely a product L1(MNs @ PAH @ G);

(21) fusing the mouse mononuclear macrophage (J774.1, about 2-3 × 10)⁶One) was scraped with a cell scraper, and the scraped cells were washed 2 times with PBS buffer and uniformly dispersed in 10mL of high-sugar serum-free culture medium DMEM (Gibco | Life Technologies (Grand Island, USA)) to obtain a culture medium in which the cells were uniformly dispersed;

(22) adding 5mg of the product L1 into a culture medium in which cells are uniformly dispersed, uniformly mixing, incubating for 2h at 37 ℃ and under the condition that the volume concentration of carbon dioxide is 5%, then carrying out magnetic separation on the obtained reaction, collecting the separated solid product, and washing the solid product for 20 times by using deionized water to obtain magnetic nanoparticles of a leukocyte membrane coated product L1, namely the magnetic nanoparticles (BMNs) with a cell-like structure;

(31) mixing cholestrol-PEG₂₀₀₀-Biotin (Peng Shuo biol) dissolved in PBS buffer to obtain cholestrol-PEG₂₀₀₀-PBS buffer with Biotin concentration of 30 μ g/mL; adding 0.1mLcholesterol-PEG into 5mg of magnetic nano particle BMNs with simulated cell structures₂₀₀₀Biotin in PBS buffer, reacting for 5h under shaking, magnetically separating the reaction and collecting the separated solid product, washing the solid product with deionized water for 3 times to obtainTo product L31;

(32) dissolving streptavidin (Aladdin, CAS number: 9013-20-1, product number S103034) in PBS buffer solution to obtain PBS buffer solution with streptavidin concentration of 30 μ g/mL; adding 0.1mL of streptavidin PBS buffer solution into 5mg of the product L31, reacting for 5h under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain a product L32;

(33) dispersing Biotin-modified anti-EpCAM-Biotin into PBS buffer solution to obtain PBS buffer solution with the concentration of the anti-EpCAM-Biotin being 0.75 mu g/mu L; adding 10 mu L of anti-EpCAM-Biotin PBS buffer solution into 5mg of the product L32, reacting for 12h at 4 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution and collecting the separated solid product, and washing the solid product with deionized water for 3 times to obtain the immunomagnetic nanoparticles with the imitated cell structure;

(34) dispersing the obtained immune magnetic nanoparticles with the cell-like structure in PBS buffer solution with bovine serum albumin volume concentration of 1.0%, reacting for 30min at 4 ℃ under the condition of oscillation, then carrying out magnetic separation on the obtained reaction solution and collecting separated solid products, washing the solid products for 3 times by using deionized water to obtain post-treated immune magnetic nanoparticles (BIMNs) with the cell-like structure, and suspending the particles in the PBS buffer solution to be placed in a refrigerator at 4 ℃ for later use.

Application example

The application examples below illustrate the enrichment of CTCs with the cellular-structured immunomagnetic nanoparticles BIMNs prepared in example 1, using MCF-7 or GFP-MCF-7 cells as examples.

Human breast cancer cells (MCF-7), human liver cancer cells (HepG2), T-lymphocyte leukemia cells (Jurkat), and mouse mononuclear macrophages (J774A.1) used in the following application examples were purchased from Shanghai Baeyer.

Application example 1

The purpose of this application example is to examine the influence of the concentration and incubation time of the immunomagnetic nanoparticles BIMNs with cell structures on the enrichment efficiency of the BIMNs-enriched CTCs cells. In the embodiment, human breast cancer cells (MCF-7) and human liver cancer cells (HepG2) with high epithelial cell adhesion molecule expression are taken as model cells of circulating tumor cells, and T lymphocyte leukemia cells (Jurkat) and mouse mononuclear macrophages (J774A.1) with or without epithelial cell adhesion molecule expression are taken as control group cells.

In six 1mL portions of PBS buffer containing MCF-7 cells (MCF-7 cells in a number of 2X 10)⁵Respectively adding different masses of BIMNs prepared in example 1 (the final concentration of the BIMNs is shown in figure 5A), incubating for 15min at room temperature, carrying out magnetic separation on the incubated product after the incubation is finished, collecting supernatant, counting cells in the supernatant by using a cell counter, measuring each sample for three times, and taking the average value as the number of the cells in the supernatant, thereby obtaining the cell concentration of the supernatant. According to the total cell number of the non-enriched cells (the cell concentration of the supernatant multiplied by the volume of the supernatant), the number of the non-enriched MCF-7 cells can be obtained, and according to the enrichment efficiency (1-the total cell number of the non-enriched cells/the total cell number of the input multiplied by 100%), the enrichment efficiency of the BIMNs on the MCF-7 cells can be obtained, and the test result is shown in FIG. 5A, and the capture efficiency of the BIMNs on the MCF-7 cells is increased from 95.4% to 98.5% along with the increase of the concentration of the BIMNs (75 mu g/m to 125 mu g/mL). However, when the concentration of BIMNs is further increased to 250 mu g/mL, the capture efficiency is still maintained at about 98.5%, which indicates that 2X 10⁵The optimum dose of the MCF-7 cells and BIMNs is about 125 mug.

In six 1mL portions of PBS buffer containing MCF-7 cells (MCF-7 cells in a number of 2X 10)⁵Respectively adding 125 μ g of the BIMNs prepared in example 1, incubating for different times at room temperature (the incubation time is shown in figure 5B), carrying out magnetic separation on the incubated product after the incubation is finished, collecting the supernatant, counting the cells in the supernatant by using a cell counter, measuring each sample for three times, and taking the average value to be recorded as the number of the cells in the supernatant, thereby obtaining the cell concentration of the supernatant. According to the total cell number of the non-enriched cells (cell concentration of the supernatant multiplied by the volume of the supernatant), the number of the non-enriched MCF-7 cells can be obtained, and according to the enrichment efficiency (1-total cell number of the non-enriched cells/total cell number of the input multiplied by 100%), the enrichment efficiency of BIMNs on the MCF-7 cells can be obtained, and the test result is shown in figure 5B, wherein the test result is that BIMNs on the MCF-7 cellsThe capture efficiency of the method can reach 96.8 percent when the capture time is 90 s; further extension of incubation time maintained the efficiency of capture of the BIMNs on MCF-7 cells at around 96.5%, indicating that for 2X 10⁵The optimal incubation time for MCF-7 cells and BIMNs was approximately 90 s.

PBS buffer in 1mL MCF-7 cells (MCF-7 cell number 4X 10)⁵One), 1mL of PBS buffer solution of HepG2 cells (4X 10 number of HepG2 cells)⁵One), 1mL of PBS buffer solution of Jurkat cells (the number of Jurkat cells is 4X 10)⁵One), 1mL of PBS buffer solution of J774A.1 cells (J774A.1 cells in number of 4X 10)⁵Respectively adding 250 μ g of BIMNs prepared in example 1, and incubating at room temperature for 90 s; while in 1mL of MCF-7 cell PBS buffer (MCF-7 cell count 4X 10)⁵One), 1mL of PBS buffer solution of HepG2 cells (number of HepG2 cells is 2X 10)⁵One), 1mL of PBS buffer solution of Jurkat cells (the number of Jurkat cells is 4X 10)⁵One), 1mL of PBS buffer solution of J774A.1 cells (J774A.1 cells in number of 4X 10)⁵Respectively adding 250 μ g of the antibody-free BIMNs prepared in example 1 (the product obtained in step (32)) and incubating at room temperature for 90 s; and after the incubation is finished, carrying out magnetic separation on the incubation product, collecting the supernatant, counting the cells in the supernatant by using a cell counter, measuring each sample for three times, and recording the average value as the number of the cells in the supernatant so as to obtain the cell concentration of the supernatant. The number of cells in which each cell was not enriched was obtained from the total number of cells in the supernatant cell concentration × the volume of the supernatant, and the enrichment efficiency of BIMNs for each cell was obtained from the enrichment efficiency (1-total number of cells not enriched/total number of cells put) × 100%, and the results of the non-test are shown in fig. 5C. As can be seen from the figure, the capture efficiency of the magnetic nanoparticles without the anti-EpCAM-Biotin antibody on four cells is very low (about 5%), while the capture efficiency of BIMNs with the anti-EpCAM-Biotin antibody on MCF-7 cells and HepG2 cells is increased to 95%, and the non-specific capture efficiency on Jurkat and J774A.1 cells is still maintained at about 5%. Shows that the immunomagnetic nanoparticles BIMNs with the cell structure have higher capture efficiency on the cells with high expression of the epithelial cell adhesion molecules and have no/low expression of the epithelial cell adhesion moleculesThe nonspecific adsorption of the cells is low, and the cell adsorption has strong nonspecific coating cell adsorption resistance, thereby laying a foundation for the subsequent application in complex samples (such as whole blood).

Application example 2

To investigate the detection limit of BIMNs on target cells in different environments, the application example was performed in 1mL of PBS buffer (pH 7.4) and 10⁶1-150 human breast cancer cells (GFP-MCF-7) with green fluorescent protein are respectively placed in Jurkat T cells and healthy human blood to obtain three groups of artificial samples. After adding 125 μ g of each of the BIMNs prepared in example 1 to each of the three groups of artificial samples, incubating the mixture for 90 seconds under a room temperature shaking condition (200 to 300 rpm), magnetically separating the incubated products, collecting the magnetically separated products, re-dispersing the magnetically separated products in 0.5 to 1.2mL of PBS buffer (pH 7.4), adding the products to a 96-well plate, and counting green fluorescent cells one by one under a fluorescence microscope (GFP-MCF-7 which is regarded as the captured green fluorescent protein), wherein the statistical result is shown in fig. 6.

As can be seen, BIMNs were in PBS buffer, 10⁶The detection limits of 1, 3 and 3 in each of Jurkat T cells and complex whole blood were found, and BIMNs were found to be present in PBS buffer and 10⁶The detection limit was very low in individual Jurkat T cells and complex whole blood. BIMNs in PBS buffer, 10⁶The average enrichment efficiency in individual Jurkat T cells and complex whole blood reached 98.7%, 92.7% and 86.7%, respectively (slope of the fitted curve). The immune magnetic nano particle BIMNs with the simulated cell structure prepared by the invention can efficiently capture CTCs in complex blood samples with low detection limit and has great potential for separating CTCs from whole blood samples.

Application example 3

The application example aims to examine the nonspecific adsorption resistance of the immunomagnetic nanoparticles BIMNs with the cell structures, which are prepared by the method, in the mixed cell simulation sample.

The application example comprises the following steps:

(1) 2 x 10 to⁵MCF-7 cells [ manufactured by cell tracker Deep Red (manufacturer Invitrogen, trade Mark.)C7025) Prestained, red at 633nm laser excitation, 2X 10⁵Jurkat cells [ cell tracker CMFDA (manufacturer Invitrogen, brand C34565) pre-stained, 488nm laser excitation under green color) were mixed to prepare two sets of mixed cell samples, all dispersed in 1mL PBS buffer.

(2) To the first mixed cells, 100uL of american and whirly commercial magnetic beads were added, incubated at 4 ℃ for 30 minutes, and then the resulting incubations were separated with a corresponding magnetic separation column, and fractions containing the target MCF-7 cells after enrichment were collected and dispersed in PBS buffer (pH 7.4), and placed on a glass plate, and then the corresponding confocal overlay was obtained under excitation with 488nm and 633nm lasers, as shown in fig. 7A.

(3) To the second mixed cell, 125 μ g of BIMNs prepared in example 1 was added, and after incubation for 90s, the product obtained by incubation was subjected to magnetic separation, and the product obtained by magnetic separation was collected, and then re-dispersed in PBS buffer (pH 7.4), and placed on a glass plate, and the corresponding confocal overlay was obtained under excitation of 488nm and 633nm lasers, as shown in fig. 7B.

The MCF-7 cell number in the mixed cell system before capture, after capture by BIMNs and after capture by American, whirly and commercial magnetic beads was counted to obtain the MCF-7 cell purity before and after capture, as shown in FIG. 7C. It can be seen that the purity of MCF-7 cells obtained by using BIMNs for capture is as high as 99.4%, and Jurkat cells do not exist in the whole visual field. While MCF-7 obtained by adopting the capture of the American and whirly commercial magnetic beads has lower purity which can only reach 77.5 percent, and the enriched product contains more Jurkat cells. Therefore, the immunomagnetic nanoparticles BIMNs with the cell structure provided by the invention can effectively inhibit the adsorption of leukocytes in mixed cell simulation samples, greatly increase the purity of MCF-7 cells in enriched samples, and lay a good foundation for subsequent CTCs downstream research.

Application example 4

The application example adopts a three-color cell immunization method to identify the number of CTCs enriched in whole blood of a patient with epithelial cell carcinoma and a healthy normal volunteer.

To whole blood (1.5mL) of a cancer patient (patient information shown in Table 1) was added 37.5. mu.g of the kit prepared in example 1And (3) incubating the immunomagnetic nanoparticles BIMNs with the cell-like structure for 5 minutes, carrying out magnetic separation on the incubated product, and collecting the product obtained by magnetic separation. Adding 25 mu g of immunomagnetic nanoparticles BIMNs with a cell-like structure prepared in example 1 into whole blood (1mL) of healthy volunteers (the information of the healthy volunteers is shown in Table 2), incubating for 5 minutes under the condition of room temperature oscillation (200-300 rpm), performing magnetic separation on the incubated product, and collecting the product obtained by the magnetic separation. Fixing the collected cells with 4% (mass to volume, mass in mg, volume in mL) of paraformaldehyde (manufacturer Solebao Co., Ltd., trade name P1110) in PBS buffer for 10 minutes, and washing with PBS buffer for three times after fixing; then, the cells were incubated for 10 minutes with PBS buffer solution of polyethylene glycol octyl phenyl ether (CAS: 9002-93-1, cat: T8200, Co., Ltd.) at a volume concentration of 0.1%, and after the incubation was completed, the cells were washed three times with PBS buffer solution; then, the mixture was incubated with 1% (mass to volume, mass in mg, volume in mL) of bovine serum albumin (Wuhan Google Bio Inc., cat: G5001, Lot: 160862) in PBS buffer for 30 minutes, and after the incubation, the mixture was washed three times with PBS buffer; then 30. mu.g mL of^-1Alexa Fluor 647-CK18，30μg mL^-1Alexa Fluor 488-CD45, 20mM Hochestt 33324, and the solid product after the trichromatic cell immunostaining is obtained by staining for 12 hours at 4 ℃ and washing three times by using PBS buffer solution after the staining is finished. After the staining process is finished, the stained cells are re-dispersed in PBS buffer solution, placed on a glass plate, and excited by laser at 405nm, 488nm and 633nm to obtain a corresponding confocal image and a confocal superposition image (as shown in FIG. 8). The above experiment was repeated three times, and the number of CTCs detected in each time in the whole blood sample was counted, as shown in tables 1 and 2.

As can be seen from FIG. 8, the cells with blue fluorescence signals and red fluorescence signals are considered to be CTCs, and the cells with blue fluorescence signals and green fluorescence signals are considered to be leukocytes, which indicates that the differentiation between CTCs and leukocytes can be achieved by using the trichrome staining method, and the method can be used in subsequent experiments to distinguish circulating tumor cells from leukocytes.

TABLE 1 basic information Table for cancer patients and statistical Table for the number of CTCs in whole blood of cancer patients

^aThe average RSD is 8.7 +/-5.6 percent

TABLE 2 statistical table of basic information and circulating tumor cell number of healthy volunteers

The statistics of the number of CTCs found in the blood samples of cancer patients and healthy volunteers are shown in Table 1 and Table 2, 2 to 48 circulating tumor cells in 1.5ml of blood of 8 cancer patients are detected, and no circulating tumor cells are found in 5 healthy samples, which shows that the magnetic nanoparticle BIMNs with the cell-like structure prepared by the invention can be successfully applied to real clinical blood samples. In addition, the results of three counts of circulating tumor cells in the same sample are not very different, and the good reproducibility (the average relative standard deviation is 8.7 +/-5.6%) is shown, so that the magnetic nanoparticle BIMNs with the simulated cell structure, which is prepared by the method, has good reproducibility and reliability, and has a great prospect in clinical application.

Application example 5

The application example aims to examine the nonspecific adsorption resistance of the immunomagnetic nanoparticles BIMNs with the cell structures, which are prepared by the invention, in a whole blood simulation sample.

The application example comprises the following steps:

(1) first, 1X 10 of the total volume of 100uL of whole blood/red-removed whole blood was added⁴MCF-7 cells to obtain two whole blood simulation samples, respectively adding 125 mu g of BIMNs prepared in the example 1 into the two whole blood simulation samples, incubating for 90s under the condition of room temperature oscillation (100-300 r/min), carrying out magnetic separation on the incubated product, and collecting the product obtained by magnetic separation.

(2) First, 1X 10 of the total volume of 100uL of whole blood/red-removed whole blood was added⁴And obtaining two whole blood simulation samples after MCF-7 cells, respectively adding 100uL of American and whirlwind magnetic beads into the two whole blood simulation samples, incubating at 4 ℃ for 30 minutes, separating the obtained incubation liquid by using a corresponding magnetic separation column, and collecting the separated solid product.

(3) After the solid product obtained from the magnetic separation of the red-removed whole blood mock sample in the steps (1) and (2) is subjected to trichrome cellular immunostaining, the obtained stained solid product is dispersed in a PBS buffer solution (pH 7.4), placed on a glass plate, and excited by laser at 405nm, 488nm and 633nm, so as to obtain a corresponding confocal superposition map, as shown in FIGS. 9A-B.

(4) After the solid products obtained from the magnetic separation of the whole blood mock sample in the steps (1) and (2) were subjected to trichrome cellular immunostaining, the obtained stained solid products were dispersed in PBS buffer (pH 7.4), placed on a glass plate, and excited by laser light at 405nm, 488nm, and 633nm, to obtain the corresponding confocal overlay images, as shown in fig. 9D-E.

The three-color cell immunostaining operation is as follows: standard trichromatic ICC methods typically include probes that selectively bind to epithelial cells (Alexa Fluor 647 labeled anti-cytokeratin 18 monoclonal antibody, Alexa Fluor 647-CK18, 633nm laser-excitation, red), leukocytes (Alexa Fluor 488 labeled anti-leukocyte common antigen, Alexa Fluor 488-CD45 (manufacturer abcam, brand ab197730), 405nm laser-excitation, green), and nuclear dyes (Hochest 33324 (manufacturer abcam, brand ab206269), 405nm laser-excitation, blue). The trichromatic cell immunostaining step is as follows: fixing the cells collected from the whole blood sample in the step (1) by 4% (mass to volume ratio, mass in mg and volume in mL) of paraformaldehyde (manufacturer Solebao Co., Ltd., brand P1110) in PBS buffer for 10 minutes, and washing the cells with PBS buffer for three times after the fixation is finished; then, the cells were incubated for 10 minutes with PBS buffer solution containing 0.1% by volume of octyl phenyl ether polyethylene glycol (CAS: 9002-93-1, cat: T8200, from Solebao Co., Ltd.) and washed three times with PBS buffer solution after the incubation; then 1% (mass to volume, mass in mg, volume in mL) of bovine serum albumin (Wuhan google biology ltd, cat: g5001, Lot: 160862) for 30 minutes in PBS buffer, and washing three times with PBS buffer after the incubation is finished; then 30. mu.g mL of^-1Alexa Fluor 647-CK18，30μg mL^-1Alexa Fluor 488-CD45, 20mM biochest 33324, and the solid product after the trichromatic cell immunostaining is obtained by staining for 12 hours at 4 ℃ and washing three times by PBS buffer solution after the staining is finished. The capture efficiency and capture purity of the MCF-7 cells captured from the red-removed whole blood mock sample and the whole blood mock sample by the trichrome cell immunostaining method in the steps (1) and (2) were statistically calculated by using commercial magnetic beads and BIMNs, as shown in FIG. 8C and FIG. 8F. The capture efficiency was obtained from the number of MCF-7 cells captured compared to the number of MCF-7 cells placed in the whole blood sample. The capture purity was obtained from the number of MCF-cells captured compared to the total number of cells captured.

FIG. 9A shows the addition of 1X 10 to 100uL of red-removed whole blood⁴MCF-7 cells, using commercial magnetic beads to capture the cells, and obtaining a laser confocal superposition image after trichromatic cell immunostaining. FIG. 9B shows the addition of 1X 10 to 100uL of red-removed whole blood⁴After MCF-7 cells, BIMNs are used for capturing the cells, and after trichromatic cell immunostaining, a laser confocal superposition graph is obtained. FIG. 9C is a statistical plot of the capture efficiency and purity of the MCF-7 cells in a de-reddened blood sample containing MCF-7 cells using commercial magnetic beads and BIMNs. FIG. 9D shows the addition of 1X 10 to 100uL of whole blood⁴MCF-7 cells, using commercial magnetic beads to capture cells, after three-color cell immunostaining, the laser confocal overlay. FIG. 9E shows the addition of 1X 10 to 100uL of whole blood⁴After MCF-7 cells, BIMNs are used for capturing the cells, and after trichromatic cell immunostaining, the laser confocal superposition map is obtained. FIG. 9F is a statistical plot of the capture efficiency and purity of MCF-7 cells in whole blood samples containing MCF-7 cells using commercial magnetic beads and BIMNs.

As can be seen from fig. 9, the capture efficiencies of the BIMNs in the whole blood sample and the de-reddened blood sample are 81.2% and 84.2%, respectively, and the capture purities are 96.7% and 96.9%, respectively, which indicates that the immunomagnetic nanoparticles with the cell-like structure can still achieve high-efficiency and high-purity CTCs capture in extremely complex blood. However, the capture efficiency of the commercial magnetic beads in the whole blood sample and the red blood removed sample is 10.7%, 69.5%, and the capture purity is 44.3%, 72.5%, respectively. It is clear that the large number of red blood cells in whole blood greatly impairs the capture efficiency and capture purity of commercial magnetic beads. The above results confirm the superior performance of immunomagnetic nanoparticles BIMNs with cellular structure, in particular the ability of whole blood to resist leukocytes. Thus, the direct use of BIMNs in whole blood may provide more accurate results than commercial magnetic beads.

Application example 6

The application example aims to investigate the cell activity of the CTCs enriched by the immunomagnetic nanoparticles BIMNs with the cell structure prepared by the invention.

The application example comprises the following steps:

(A) to 1X 10⁶625 mu g of immunomagnetic nanoparticles BIMNs with the cell-like structure prepared in the embodiment 1 are added into MCF-7 cells, and after incubation for 90s under the condition of room temperature oscillation (200-300 r/min), the product obtained by incubation is subjected to magnetic separation, and the product obtained by magnetic separation is collected. 1/5 magnetic separation solid products are taken and dispersed in 1mL PBS buffer (pH 7.4), AO/PI cell dead and live staining solution (Solebao Co., Ltd., AO cat No. CA1143, PI cat No. P8080) is added, after 5 minutes of incubation, the solid products are washed once by PBS buffer, the solid products are collected by centrifugation at 1200 r/min, the collected solid products are dispersed in 1mL PBS buffer again and placed on a glass plate, and under the excitation of 488nm and 543nm laser, the corresponding confocal superposition map is obtained, as shown in FIG. 10A.

(B) 2 x 10 to⁵MCF-7 cells are dispersed in 1mL PBS buffer solution, AO/PI cell dead and live staining solution is added, after 5 minutes of incubation, the cells are washed once by PBS buffer solution, solid products are collected by centrifugation at 1200 rpm, the collected solid products are dispersed in 1mL PBS buffer solution again, the mixture is placed on a glass bottom dish, and under the excitation of 488nm laser at 543nm, a corresponding confocal superposition map is obtained, as shown in FIG. 10B.

(C) To 1X 10⁶625 mu g of BIMNs with the cell-imitated structure prepared in example 1 are added into MCF-7 cells, after incubation for 90s, the product obtained by incubation is subjected to magnetic separation, and the product obtained by magnetic separation is collected. Will magnetically divideThe isolated solid product was dispersed in 2mL of complete medium (DMEM, high sugar), placed in a 6-well plate, cultured at 37 ℃ in an incubator with 5% carbon dioxide, and passaged when the cell fusion rate reached 85% or more. The whole culture and passage process is kept sterile. The bright field cell state diagrams obtained at different stages of cell passaging are shown in FIG. 11.

Acridine Orange (AO) is a membrane-permeable fluorescent dye that can bind to DNA/RNA and fluoresce yellow-green under excitation of 488nm laser. And Propidium Iodide (PI) can be combined with DNA, and emits red fluorescence under the excitation of 543nm laser. However, the dye is not membrane permeable and only the DNA of dead cells can bind to it. As can be seen from FIGS. 10A-B, the activity of the MCF-7 cells is equivalent to that of the control group (about 98%) through the whole enrichment and separation process, which indicates that the effect of the incubation and separation process on the activity of the MCF-7 cells is small, and this indicates that the biomagnetic nanoparticles BIMNs with the cell-like structure can be used for obtaining high-activity CTCs.

As can be seen from the enriched cell in vitro culture diagram of FIG. 11, after the incubation and separation processes, the adherence ability and proliferation ability of MCF-7 cells are not affected and there is no obvious morphological change. The influence of the incubation and separation processes on the proliferation function and the morphology of the MCF-7 cells is small, which shows that the circulating tumor cells enriched by the biomins with the immune magnetic nanoparticles with the cell-like structure can be directly cultured in vitro.

Claims

1. Magnetic nanoparticles with a cell-like structure, characterized in that they consist of Fe₃O₄Magnetic nanoparticles sequentially coated on Fe₃O₄The magnetic nanoparticle consists of a high molecular polymer layer on the surface of the magnetic nanoparticle, a graphene layer and a leukocyte membrane layer.

2. The magnetic nanoparticle with cell-like structure of claim 1, wherein the high molecular polymer is polyacrylamide hydrochloride or polyethyleneimine; the graphene is carboxylated graphene.

3. The method for preparing magnetic nanoparticles with cell-like structure of claim 1, characterized by the following steps:

(2) uniformly mixing the product L1 with a culture medium in which cells are uniformly dispersed, incubating for 1.5-2 h at 37 ℃ and under the condition that the volume concentration of carbon dioxide is 5%, then carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product to obtain the magnetic nanoparticles of the leukocyte membrane coated product L1, namely the magnetic nanoparticles with the cell-like structure.

4. The method for preparing magnetic nanoparticles with cell-like structure according to claim 3, wherein the sub-steps of the step (1) are as follows:

(12) mixing the suspension liquid in which the product L11 is uniformly dispersed with the suspension liquid in which the graphene is uniformly dispersed to form a reaction system, reacting for 1-3 h under an oscillation condition, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, and washing the solid product to obtain the Fe-Fe alloy material₃O₄The surfaces of the magnetic nanoparticles are sequentially coated with a high polymer layer and a product L1 of a graphene layer; the mass ratio of the product L11 to the graphene in the reaction system is 1 (0.1-0.11).

5. The method for preparing magnetic nanoparticles with cell-like structure according to claim 3 or 4, wherein the high molecular polymer is polyacrylamide hydrochloride or polyethyleneimine; the graphene is carboxylated graphene; the cells in the culture medium with uniformly dispersed cells are derived from mouse mononuclear macrophages or human T lymphocyte leukemia cells.

6. An immunomagnetic nanoparticle with a cell-like structure, which is obtained by sequentially modifying the leukocyte membrane of the magnetic nanoparticle with a cell-like structure of claim 1 or 2 with a lipid molecule-polyethylene glycol-biotin molecule, avidin, and an antibody.

7. The immunomagnetic nanoparticle with cell-like structure according to claim 6, wherein the lipid molecule-PEG-biotin molecule is C18-PEG₂₀₀₀-Biotin、cholesterol-PEG₂₀₀₀-Biotin or DSPE-PEG₂₀₀₀-Biotin; the avidin is streptavidin or avidin; the antibody is a biotin-modified anti-epithelial cell adhesion molecule antibody.

8. The method for preparing the immunomagnetic nanoparticles with the cell-like structure of claim 6, is characterized by comprising the following steps:

(31) mixing the magnetic nanoparticles with the cell-like structure of claim 1 or 2 with a PBS buffer solution containing lipid molecules-polyethylene glycol-biotin molecules to form a reaction system, reacting for 2-5 h under oscillation, performing magnetic separation on the obtained reaction solution, collecting a separated solid product, and washing the solid product to obtain a product L31; the mass ratio of the magnetic nanoparticles with the cell-like structure to the lipid molecules-polyethylene glycol-biotin molecules in the reaction system is 5000: (1-3);

9. The method for preparing immunomagnetic nanoparticles with cell-like structure according to claim 8, wherein the lipid molecule-polyethylene glycol-biotin molecule is C18-PEG₂₀₀₀-Biotin、cholesterol-PEG₂₀₀₀-Biotin or DSPE-PEG₂₀₀₀-Biotin; the avidin is streptavidin or avidin; the antibody is a biotin-modified anti-epithelial cell adhesion molecule antibody;

carrying out the following post-treatment on the immunomagnetic nanoparticles with the simulated cell structures obtained in the step (33): dispersing the immune magnetic nanoparticles with the cell-like structure in PBS buffer solution with the mass concentration of bovine serum albumin of 0.5-1.0%, reacting for 20-30 min at 4-7 ℃ under the oscillation condition, then carrying out magnetic separation on the obtained reaction solution, collecting the separated solid product, and then washing the solid product.

10. Use of the immunomagnetic nanoparticles with a cell-like structure according to claim 6 or 7 for enriching circulating tumor cells.