CN114487391A - Bionic immune magnetic nanoparticle, preparation method and application thereof - Google Patents

Bionic immune magnetic nanoparticle, preparation method and application thereof Download PDF

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CN114487391A
CN114487391A CN202210339436.8A CN202210339436A CN114487391A CN 114487391 A CN114487391 A CN 114487391A CN 202210339436 A CN202210339436 A CN 202210339436A CN 114487391 A CN114487391 A CN 114487391A
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magnetic nanoparticles
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nanoparticle
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饶浪
吴显佳
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Shenzhen Bay Laboratory
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Abstract

The invention discloses a bionic immune magnetic nanoparticle, a preparation method and application thereof. The bionic immune magnetic nanoparticle comprises: the magnetic nanoparticles and the neutrophilic granulocyte membrane vesicles are coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles. The bionic magnetic nano-particles are connected with a targeting antibody subsequently and can be used for separating biological components. The bionic immune magnetic nanoparticle at least has the following advantages: the lipid bilayer of Neu-vesicles reduces the adsorption of non-specific proteins and maintains the targeting ability of IMNs in the blood; the interaction between CTCs and neutrophils is enhanced, and the separation efficiency of the IMNs on the CTCs is improved; the interaction between the leukocytes and the neutrophils is reduced, the interference of WBCs in the separation process is reduced, and the purity of the separated CTC is improved; the soft interaction between Neu-vesicles and CTCs enhances the viability of isolated CTCs.

Description

Bionic immune magnetic nanoparticle, preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to bionic immune magnetic nanoparticles and a preparation method and application thereof.
Background
The separation of Circulating Tumor Cells (CTC) from clinical peripheral blood samples belongs to non-invasive liquid biopsy and has wide prospect in the early detection and diagnosis of cancers. To date, a variety of emerging methods have been explored to separate CTCs from peripheral blood, such as physical property-based enrichment methods, biological feature-based enrichment methods, and differential nucleated cell-free enrichment methods, among others. Among them, the most widely used strategy is based on immunomagnetic nanoparticles (IMNs), where magnetic nanoparticles are used as separation media and specific antibodies are used as capture ligands. Due to the scarcity of CTCs in peripheral blood (only a few to tens of CTCs per ml of blood), it is a significant challenge to isolate CTCs with native biological activity in high efficiency and purity for subsequent cellular analysis.
The current methods for isolating circulating tumor cells still have certain technical problems, such as: Qian-Fan Meng, et al, biomedical immunological Nanoparticles with Minimal Non-Specific biomolecular Adsorption for Enhanced Isolation of Circulating cells ACS Applied Materials & Interfaces,2019,11,28732-28739 discloses that modification of the magnetic Nanoparticles obtained with targeting antibodies to isolate CTCs can improve the efficiency of isolating CTCs to some extent, but the purity of the isolated CTCs is not high. For another example: lang Rao, et al, plant let-Leucocyte Hybrid Membrane-Coated immunological Beads for high efficiency and Specific Isolation of Circulating Tumor cells, advanced Functional materials 2018,28,1803531 discloses a mixed Membrane of platelets and leukocytes Coated on MBs, and then the obtained magnetic nanoparticles are modified with a targeting antibody for separating CTC, which can improve the separation purity of CTC to some extent. But the stability is poor, the procedure is complex and the encapsulation efficiency is not high.
Disclosure of Invention
The invention aims to provide a bionic immune magnetic nanoparticle, a preparation method and application thereof, and aims to provide a novel bionic immune magnetic nanoparticle which can improve the separation efficiency and purity of biological components.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a biomimetic immunomagnetic nanoparticle. The bionic immune magnetic nanoparticle comprises: the magnetic nanoparticles and the neutrophilic granulocyte membrane vesicles are coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles.
Further, the magnetic nanoparticles are Fe3O4Magnetic nanoparticles.
According to another aspect of the present invention, there is provided a biomimetic immunomagnetic nanoparticle for isolation. The bionic immune magnetic nanoparticle comprises: the bionic immune magnetic nanoparticle and the targeting antibody are physically and/or covalently connected with the bionic magnetic nanoparticle.
Further, the targeting antibody is a CTC targeting antibody.
Further, the CTC targeting antibody is an anti-epithelial cell adhesion molecule antibody.
Further, the physical linkage and/or covalent linkage is a conjugate linkage or a click linkage.
According to another aspect of the invention, a preparation method of the bionic immune magnetic nanoparticle is provided. The preparation method comprises the following steps: s1, preparing a neutrophil membrane vesicle; and S2, coating the magnetic nanoparticles with the neutrophil membrane vesicles to obtain the bionic magnetic nanoparticles.
Further, S1 includes: neutrophil membrane vesicles are prepared by isolating neutrophils from peripheral blood.
Further, a gradient density centrifugation method was used to separate neutrophils from peripheral blood.
Further, the separated neutrophils are destroyed by a hypotonic lysis buffer solution and a repeated freeze-thaw method, cell membranes are collected by ultracentrifugation, and then the cells are extruded by a micro extruder to prepare the neutrophil membrane vesicles.
Further, a micro-extruder extrudes through 800 and 400nm polycarbonate porous membranes to obtain neutrophil membrane vesicles.
Further, S2 includes: injecting the neutrophile granulocyte membrane vesicle and the magnetic nanoparticles into the microfluidic photo-perforation chip, and fusing the neutrophile granulocyte membrane vesicle and the magnetic nanoparticles in a photo-perforation area to enable the neutrophile granulocyte membrane vesicle to be coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles.
Further, when the mixture passes through the light perforated region at the optimal flow rate of 2 ml/h, the mixture is irradiated by using pulse laser with the laser energy density of 0.12 joule/square centimeter, and the laser pulse can effectively promote the magnetic nanoparticles to enter the neutral particle cell membrane vesicles to form the bionic magnetic nanoparticles.
Further, the neutrophil membrane vesicles are associated with magnetic nanoparticles (1:1) - (1: 10).
Further, S2 also includes other common methods such as mechanical pushing and ultrasonic mixing, which illustrate the advantages of the photo-perforation method over the mechanical pushing and ultrasonic mixing method. The mechanical extrusion method is to mix the neutrophilic granulocyte membrane vesicles with the magnetic nanoparticles, and then repeatedly extrude the obtained mixture through a miniature extruder by adopting 400nm holes for many times, so that the neutrophilic granulocyte membrane vesicles are coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles. The ultrasonic fusion is to mix the neutrophilic granulocyte membrane vesicles with the magnetic nanoparticles, and then ultrasonically mix for 3 minutes to coat the neutrophilic granulocyte membrane vesicles on the magnetic nanoparticles to form the bionic magnetic nanoparticles.
According to still another aspect of the present invention, there is provided a method for preparing biomimetic immunomagnetic nanoparticles for separation. The preparation method comprises the following steps: preparing the bionic immune magnetic nanoparticles according to the preparation method, S3, and obtaining the bionic immune magnetic nanoparticles by physically and/or covalently connecting the targeting antibody and the bionic magnetic nanoparticles.
Further, the targeting antibody is a CTC targeting antibody.
Further, the CTC targeting antibody is an anti-epithelial cell adhesion molecule antibody.
Further, S3 includes: and (2) treating the bionic magnetic nanoparticles prepared by S2 with carboxyl-polyethylene glycol-phospholipid, adding NHS/EDC for activation, crosslinking streptavidin, and combining a biotinylated CTC targeted antibody with the streptavidin to obtain the bionic immune magnetic nanoparticles.
According to another aspect of the invention, the application of the bionic immune magnetic nanoparticles for separation in preparing products for enriching, separating or detecting circulating tumor cells is provided.
According to still another aspect of the present invention, there is provided a use of the isolated biomimetic immunomagnetic nanoparticles in the preparation of a medicament for treating a tumor metastasis associated disease.
Further, the tumor metastasis-associated diseases are tumors associated with high expression of epithelial cell adhesion factor, preferably colorectal cancer, breast cancer and gastric cancer.
By applying the technical scheme of the invention, the bionic magnetic nanoparticles are connected with the target antibody subsequently, can be used for separating biological components, and are wrapped by neutral particle cell membrane derived vesicles (Neu-vesicles) to obtain the bionic immune magnetic nanoparticles (Neu-IMNs). The bionic immune magnetic nanoparticle has at least the following advantages: 1) the lipid bilayer of Neu-vesicles reduces the adsorption of non-specific proteins and maintains the targeting ability of IMNs in the blood; 2) the interaction between CTCs and neutrophils is enhanced, and the separation efficiency of the IMNs on the CTCs is improved; 3) the interaction between White Blood Cells (WBCs) and neutrophils is reduced, the interference of WBCs in the separation process is reduced, and the purity of the separated CTC is improved; 4) the soft interaction between Neu-vesicles and CTCs enhances the viability of isolated CTCs, making subsequent cell analysis possible. In addition, the bionic magnetic nanoparticle of the neutrophilic granulocyte membrane vesicle prepared by the microfluidic photo-perforation method has the following two outstanding advantages: 1) compared with other common preparation methods (such as mechanical extrusion and ultrasonic mixing), the applied photo-perforation preparation method is optimal, the encapsulation efficiency is highest, the required raw materials are least, and the capture efficiency of CTC is highest, so that the method has the greatest application and transformation prospects; 2) compared with bionic magnetic nanoparticles wrapped by other hybrid membranes, the bionic magnetic nanoparticles of the neutral-particle cell membrane vesicles have better encapsulation efficiency and stability, and can realize higher CTC capture efficiency.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
fig. 1 shows a schematic diagram of preparation of a neutrophil membrane vesicle biomimetic magnetic nanoparticle by using a microfluidic photo-perforation chip.
FIG. 2 shows a schematic of (a) a scheme for preparing Neu-IMNs to (b) enhance CTC isolation.
Fig. 3 shows a physical diagram of a micro-fluidic photo-perforated chip.
FIG. 4 shows a schematic of the pathway of Neu-MNs modifying anti-EpCAM antibodies.
FIG. 5 shows a bright field image of neutrophils isolated from a peripheral blood sample in example 1. Scale bar, 100 μm.
FIG. 6 shows Fe in example 13O4XRD spectra of MNs.
Fig. 7 shows the photo-perforation envelope efficiency at different laser energy densities, with an optimal laser energy density of 0.12 joules per square centimeter.
Fig. 8 shows the photo-perforation encapsulation efficiency at different flow rates in the microfluidic chip, with an optimal flow rate of 2 ml/h.
Figure 9 shows the encapsulation efficiency at different neutrophil membrane vesicle and magnetic nanoparticle ratios (1:1, 1:2, 1:5 and 1:10) using different preparation methods (photo-perforation, micro-extruder extrusion, ultrasonic mixing).
Fig. 10 shows the encapsulation efficiency of a single neutrophil membrane and hybrid membrane 1 (neutrophil and macrophage hybrid membrane), hybrid membrane 2 (leukocyte and erythrocyte hybrid membrane) and magnetic nanoparticles at different ratios (1:1, 1:2, 1:5 and 1: 10).
Figure 11 shows the particle size change of single neutrophil membrane and hybrid membrane 1 (neutrophil and macrophage hybrid membrane), hybrid membrane 2 (leukocyte and erythrocyte hybrid membrane) biomimetic nanoparticles within 2 weeks, single neutrophil membrane biomimetic nanoparticles show better stability.
Fig. 12 shows fourier infrared characterization results of single neutrophil membrane and hybrid membrane 1 (neutrophil and macrophage hybrid membrane), hybrid membrane 2 (leukocyte and erythrocyte hybrid membrane) biomimetic nanoparticles 2 weeks after preparation, the neutrophil membrane biomimetic nanoparticles exhibiting C-H bonds of the cell membrane showing better encapsulation stability compared to hybrid membrane 1 (neutrophil and macrophage hybrid membrane), hybrid membrane 2 (leukocyte and erythrocyte hybrid membrane) biomimetic nanoparticles.
FIG. 13 shows the characterization of Neu-IMNs: (a) average diameter and zeta potential of MN and Neu-MN. (b) TEM images of MNs and (c) Neu-MNs. Scale bar, 100 nm. (d) Neu-visiles and Neu-MNs were protein identified by SDS-PAGE. (e) Confocal images of biotin-FITC labeled Neu-MNs. Scale bar, 20 μm. (f) SEM images of single MCF-7 cells captured by Neu-IMNs. Scale bar, 10 μm and 2.5 μm (magnified). Arrows indicate Neu-IMN. (g) The efficiency of Neu-MN or Neu-IMN separation of MCF-7 and HeLa cells in PBS. All data are expressed as mean ± standard deviation (n ═ 5).
A in FIG. 14 shows a hysteresis curve of Neu-IMNs; b shows the photographs of the aqueous solution of Neu-IMNs before and after the treatment with the external magnetic field.
Figure 15 shows the dimensional changes of IMNs before and after incubation with 10% plasma.
FIG. 16 shows that Neu-IMNs show reduced protein adsorption and enhanced interaction with cancer cells. (a) Protein content on IMN after co-incubation with 10% plasma. (b) Size change of Neu-IMN incubated in 10% plasma. (c) After incubation in 10% plasma, the heatmap shows the abundance changes of some of the most common proteins on the Neu-IMN surface. (d) ICP-AES tested the interaction between MCF-7 cells and various MNs. (e) Capture efficiency of various MNs against different cancer cell lines. All data are expressed as mean ± standard deviation (n-5).
FIG. 17 shows the interaction of ICP-AES assay MCF-7 cells co-incubated with different nanoparticles at a concentration of 100. mu.g/ml for different times.
Figure 18 shows the MCF-7 cell capture efficiency of neutrophil biomimetic immunomagnetic nanoparticles prepared by different preparation methods (photoperforation, micro-extruder extrusion, ultrasound mixing).
Figure 19 shows MCF-7 cell capture efficiency of single neutrophil membrane and hybrid membrane 1 (neutrophil and macrophage hybrid membrane), hybrid membrane 2 (leukocyte and erythrocyte hybrid membrane) biomimetic immunomagnetic nanoparticles.
Figure 20 shows the cell capture efficiency of different nanoparticles in PBS.
Figure 21 shows Neu-IMN shows reduced WBC interference and enhanced viability of isolated cells: (a) the interaction between WBCs and various MNs was measured by ICP-AES. (b) Capture efficiency of different MNs with different numbers of MCF-7 cells added to whole blood. (c) Isolation purity of MCF-7 cells by various MNs. (d) MCF-7 cell viability after separation of IMNs and Neu-IMNs. All data are expressed as mean ± standard deviation (n ═ 5).
FIG. 22 shows the interaction of ICP-AES assay for white blood cells co-incubated with different nanoparticles at a concentration of 100. mu.g/ml for different times.
Figure 23 shows the number of CTCs captured per ml of blood sample by neutrophil biomimetic immunomagnetic nanoparticles prepared by different preparation methods (photoperforation, micro-extruder extrusion, ultrasound mixing).
Figure 24 shows the number of CTCs captured per ml of blood sample by a single neutrophil membrane and hybrid membrane 1 (neutrophil and macrophage hybrid membrane), hybrid membrane 2 (leukocyte and erythrocyte hybrid membrane) biomimetic immunomagnetic nanoparticle.
Figure 25 shows identification, enumeration and analysis of CTCs. (a) Confocal images of CTC and WBC isolated from Neu-IMNs. Scale bar, 15 μm. (b) Count and purity of CTCs isolated from blood samples of healthy or breast cancer patients. (c) SEM images of individual CTCs after Neu-IMNs isolation and two rounds of incubation. Scale bar, 5 μm. (d) Sanger sequencing of CTCs after isolation of Neu-IMNs.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
Noun interpretation
CTC: circulating the tumor cells.
MNs: magnetic nanoparticles.
IMN: immunomagnetic nanoparticles.
Neu-visicles: neutrophil membrane-derived vesicles, otherwise known as neutrophil membrane vesicles.
Neu-IMNs: the bionic immune magnetic nanoparticle is formed by wrapping magnetic nanoparticles with neutral granulocyte cell membrane-derived vesicles, and a targeting antibody is connected to the bionic immune magnetic nanoparticle, and a CTC targeting antibody is connected to the bionic immune magnetic nanoparticle.
Neu-MNs: the bionic immune magnetic nanoparticle is formed by wrapping magnetic nanoparticles by utilizing neutral particle cell membrane derived vesicles.
WBCs: white blood cells.
anti-EpCAM: anti-epithelial cell adhesion molecules.
PBS: phosphate buffered saline.
The detection and analysis of rare Circulating Tumor Cells (CTCs) with immunomagnetic nanoparticles (IMNs) has shown promising promise in noninvasive cancer diagnosis. However, the inventors of the present application found that after entering biological fluids, the IMNs adsorb non-specific proteins, and the formed protein corona covers the surface targeting ligand, limiting the detection efficiency of the IMNs. In addition, the surface-targeting ligands have strong interactions with White Blood Cells (WBCs) in the blood, limiting the purity of CTCs isolated from IMN. In response, the present application proposes surface functional modification of IMNs with neutrophil membrane, which can significantly reduce non-specific protein adsorption, enhance the interaction with CTCs, reduce interference with WBCs, and improve the activity of isolated CTCs.
According to an exemplary embodiment of the present application, there is provided a biomimetic immunomagnetic nanoparticle for isolating CTCs. Referring to a in fig. 2, the biomimetic immunomagnetic nanoparticle for isolating CTCs comprises: the magnetic nanoparticles and the neutrophilic granulocyte membrane vesicles are coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles.
After the bionic magnetic nanoparticles are connected with the targeting antibody, the bionic magnetic nanoparticles can be used for separating biological components, for example, the targeting antibody can be a CTC targeting antibody, and other targeting antibodies can be connected according to the requirements of specific separation targets.
Referring to b in fig. 2, the biomimetic immunomagnetic nanoparticle has at least the following advantages: 1) the lipid bilayer of Neu-vesicles reduces the adsorption of non-specific proteins and maintains the targeting ability of IMNs in the blood; 2) the interaction between CTCs and neutrophils is enhanced, and the separation efficiency of the IMNs on the CTCs is improved; 3) the interaction between White Blood Cells (WBCs) and neutrophils is reduced, the interference of WBCs in the separation process is reduced, and the purity of the separated CTC is improved; 4) the soft interaction between Neu-vesicles and CTCs enhances the viability of isolated CTCs, making subsequent cell analysis possible.
In a typical embodiment of the present application, the CTC targeting antibody is an anti-epithelial cell adhesion molecule antibody.
In a typical embodiment of the present application, the magnetic nanoparticles are Fe3O4Magnetic nanoparticles.
According to an exemplary embodiment of the present application, a method for preparing the biomimetic immunomagnetic nanoparticles is provided. The preparation method comprises the following steps: s1, preparing a neutrophil membrane vesicle; and S2, coating the magnetic nanoparticles with the neutrophilic granulocyte membrane vesicles to obtain the bionic magnetic nanoparticles. Correspondingly, the preparation method of the bionic immune magnetic nanoparticle for separation comprises the preparation method of the bionic immune magnetic nanoparticle and S3, wherein the CTC targeting antibody is physically and/or covalently connected with the bionic magnetic nanoparticle, and the physical connection and/or covalent connection is conjugate connection or chemical click connection.
In a typical embodiment of the present application, the CTC targeting antibody is an anti-epithelial cell adhesion molecule antibody.
In an embodiment of the present application, S1 includes: separating neutrophils from peripheral blood to prepare neutrophil membrane vesicles; preferably, the method comprises separating neutrophils from peripheral blood by gradient density centrifugation; of course, the isolation of neutrophils from tissue is also possible.
Preferably, the separated neutrophils are destroyed by a hypotonic lysis buffer solution and a repeated freeze thawing method, cell membranes are collected centrifugally and then extruded by a miniature extruder to prepare the neutrophil membrane vesicles; in an exemplary embodiment of the present application, the micro-extruder extrudes through the 800nm and 400nm polycarbonate porous membranes to obtain the neutrophilic granulocyte membrane vesicles, although porous membranes with other pore sizes may be selected according to actual needs.
Common methods for coating the nanoparticles with the cell membrane vesicles comprise mechanical extrusion and ultrasonic mixing, and the methods have the defects of low encapsulation efficiency, unstable encapsulation and the like. In order to improve the defects, the bionic magnetic nanoparticles are prepared by a photo-perforation method, referring to fig. 1, a neutrophilic granulocyte membrane vesicle and magnetic nanoparticles are injected into a micro-fluidic photo-perforation chip, and when the neutrophilic granulocyte membrane vesicle and the magnetic nanoparticles enter a photo-perforation area, laser pulses promote the magnetic nanoparticles to enter the neutrophilic granulocyte membrane vesicle to form the bionic magnetic nanoparticles. The method can effectively improve encapsulation efficiency and stability, and can improve the capture efficiency of subsequent CTCs, as shown in FIGS. 9, 18 and 23.
According to the application, the magnetic nanoparticles are coated by the single neutrophilic granulocyte membrane vesicle to prepare the bionic magnetic nanoparticles, compared with the magnetic nanoparticles coated by other hybrid membrane vesicles, the magnetic nanoparticles are better in encapsulation efficiency and stability, and higher CTC capture efficiency can be realized, as shown in fig. 10, fig. 11, fig. 12, fig. 19 and fig. 24.
In an embodiment of the present application, S2 includes: injecting the neutrophile granulocyte membrane vesicle and the magnetic nanoparticles into the microfluidic photo-perforation chip, and fusing the neutrophile granulocyte membrane vesicle and the magnetic nanoparticles in a photo-perforation area to enable the neutrophile granulocyte membrane vesicle to be coated on the magnetic nanoparticles to form bionic magnetic nanoparticles; in one embodiment of the application, when the mixture passes through the photoperforated region, the laser pulse can effectively promote the magnetic nanoparticles to enter the neutral granulocyte cell membrane vesicles to form bionic magnetic nanoparticles; preferably, the mole ratio of the neutrophil membrane vesicles to the magnetic nanoparticles is (1:1) to (1:10), for example, 1:1, 1:2, 1:5 or 1:10, and the coating can be performed well.
S2 also includes other common methods such as mechanical pushing and ultrasonic mixing, which illustrate the advantages of the photo-perforation method over the mechanical pushing and ultrasonic mixing methods. The mechanical extrusion method is to mix the neutrophilic granulocyte membrane vesicle with magnetic nanoparticles, and then repeatedly extrude the obtained mixture through a miniature extruder by adopting 400nm holes for many times, so that the neutrophilic granulocyte membrane vesicle is coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles. And the ultrasonic fusion is to mix the neutrophilic granulocyte membrane vesicles with the magnetic nanoparticles, and then ultrasonically mix for 3 minutes to coat the neutrophilic granulocyte membrane vesicles on the magnetic nanoparticles to form the bionic magnetic nanoparticles.
According to an exemplary embodiment of the present application, S3 includes: and (2) treating the bionic magnetic nanoparticles prepared by S2 with carboxyl-polyethylene glycol-phospholipid, adding NHS/EDC for activation, crosslinking streptavidin, and combining a biotinylated CTC targeted antibody with the streptavidin to obtain the bionic immune magnetic nanoparticles.
In another exemplary embodiment of the present application, S4 is further included, and the reduction of protein crown adsorption of Neu-IMNs is verified by liquid chromatography tandem mass spectrometry and Sanger sequencing is adopted to identify circulating tumor cells separated by Neu-IMNs.
According to an exemplary embodiment of the present application, there is provided a use of biomimetic immunomagnetic nanoparticles for isolating CTCs in the preparation of a product for enriching, isolating or detecting circulating tumor cells.
According to an exemplary embodiment of the present application, there is provided a use of biomimetic immunomagnetic nanoparticles for isolating CTCs in the preparation of a medicament for treating a tumor metastasis associated disease. The tumor metastasis related diseases are tumors related to high expression of epithelial cell adhesion factors, and preferably colorectal cancer, breast cancer and gastric cancer.
The advantageous effects of the present invention will be further explained below in conjunction with the test data. The steps or reagents described in the following examples, if not described in detail, can be performed using methods or reagents conventional in the art.
Example 1
The preparation and application steps of the immunomagnetic nanoparticles (Neu-IMNs) coated by the neutrophil membrane vesicles are as follows: (1) preparing a micro-fluidic photo-perforating chip; (2) separating neutrophils from clinical peripheral blood to prepare Neu-vesicles; (3) coating Magnetic Nanoparticles (MNs) with the prepared Neu-visiles to obtain bionic magnetic nanoparticles (Neu-MNs); and (4) bioconjugate linking an anti-epithelial cell adhesion molecule (anti-EpCAM) antibody to the Neu-MNs. (5) And (3) verifying the reduction of protein crown adsorption of the Neu-IMNs by using a liquid chromatography tandem mass spectrometry and identifying the circulating tumor cells separated by the Neu-IMNs by using Sanger sequencing.
The method comprises the following specific steps:
(1) preparing a micro-fluidic photo-perforated chip:
glue homogenizing: and (3) putting the cleaned silicon wafer on a spin coater, spin-coating a layer of SU-8-2050 photoresist, rotating at 600rpm, operating for 30s, adjusting the rotation speed to 1000rpm, and operating for 40 s. Pre-baking: heating on a drying table at 65 ℃ for 5min, transferring to a drying table at 95 ℃ for heating for 12min, and then cooling for 10 min. Exposure: and (4) placing the silicon chip on a photoetching machine, covering a mask plate, and exposing for 35 s. Post-baking: and (3) heating the exposed silicon wafer on a 65 ℃ baking table for 2.5min, heating the silicon wafer on a 95 ℃ baking table for 8.5min, and then cooling the silicon wafer for 10 min. Developing and hardening: immersing the silicon wafer into a developing solution, developing for 9min for 30s, taking out with tweezers, washing off the redundant developing solution, and drying with a nitrogen gun; then the mixture is placed on a baking table at 120 ℃ and heated for 30 min. Pouring PDMS: according to A, B, glue 30: 3, preparing 33g of PDMS; pouring PDMS onto a silicon chip, standing for 30min, and blowing off large bubbles by using a blowing balloon; vacuumizing twice by using a vacuum machine for three minutes each time; then put into an oven to be baked for one hour at 80 ℃. Punching and bonding: taking PDMS from the silicon chip, punching and cutting, putting the PDMS and cleaned glass into an ion cleaning machine for 3min, taking out the glass to adhere with the PDMS, completing the sealing of the chip channel, and putting the chip channel on an oven to be dried for one day. Thus, the micro-fluidic photo-punching chip is completed (fig. 3).
(2) Preparation of neutrophil membrane vesicles (Neu-vesicles):
neutrophils were first isolated from fresh clinical peripheral blood by gradient density centrifugation (figure 5, bright field picture of neutrophils isolated from peripheral blood samples, scale bar, 100 μm). To obtain neutrophil membrane vesicles, isolated neutrophils are disrupted by hypotonic lysis buffer and repeated freeze-thaw methods. The collected cells were washed with cold PBS and then suspended in hypotonic solution (Gibico brand PBS and deionized water mixed at 1:4 volumes). Thereafter, neutrophils were disrupted 3 times using a repeated freeze-thaw method, and then the solution was centrifuged at 2000g for 30 minutes at 4 ℃, and then the enriched supernatant was further ultracentrifuged at 100000g for 40 minutes to collect cell membranes. The collected cell membranes were resuspended in PBS and gradually extruded through 800 and 400nm polycarbonate porous membranes by an Avanti mini extruder to produce neutrophil membrane vesicles.
(3) Magnetic nanoparticle synthesis and coating of magnetic nanoparticles with neutrophile cell membrane vesicles:
hydrothermal method for synthesizing Fe3O4Magnetic Nanoparticles (MNs). First 1.35g FeCl3·6H2O was dissolved in 40ml of ethylene glycol and 3.6g of NaAc was added. The mixed solution was vigorously stirred for 1h, and then sealed in a 50ml stainless steel autoclave lined with Teflon, and reacted at 200 ℃ for 8 h. The reaction product was washed several times with ethanol and deionized water. The obtained MNs were then characterized by means of an X-ray diffractometer (XRD; D8 Advance, AXS Instruments, Germany) and the results are shown in FIG. 6 (Fe)3O4XRD spectrum of MNs). In order to obtain magnetic nanoparticles (Neu-MNs) coated with cell vesicles, 50 μ g of magnetic nanoparticles and neutrophilic granulocyte membrane vesicles were injected into a microfluidic photoperforating chip, neutrophilic granulocyte membraneThe vesicle and magnetic nanoparticle mixture was irradiated with a pulsed laser with an optimal laser energy density of 0.12 joules per square centimeter while passing through the photoperforated region at an optimal flow rate of 2 ml/hr, the laser pulses promoting the magnetic nanoparticles to enter the neutrophile cell membrane vesicles to form biomimetic magnetic nanoparticles (fig. 7, fig. 8). The mechanical extrusion method is to mix the neutrophilic granulocyte membrane vesicles with the magnetic nanoparticles, and then repeatedly extrude the obtained mixture through a miniature extruder by adopting 400nm holes for many times, so that the neutrophilic granulocyte membrane vesicles are coated on the magnetic nanoparticles to form the bionic magnetic nanoparticles. And the ultrasonic fusion is to mix the neutrophilic granulocyte membrane vesicles with the magnetic nanoparticles, and then ultrasonically mix for 3 minutes to coat the neutrophilic granulocyte membrane vesicles on the magnetic nanoparticles to form the bionic magnetic nanoparticles. Experimental data show that at different neutrophil membrane vesicle and magnetic nanoparticle ratios (1:1, 1:2, 1:5 and 1:10), photo-perforation all showed higher encapsulation efficiency compared to mechanical push and ultrasonic mixing (figure 9). Dynamic diameters and zeta potentials of MNs and Neu-MNs were measured by Dynamic Light Scattering (DLS), and morphologies of MNs and Neu-MNs were characterized by transmission electron microscopy. The magnetism of Neu-MNs was measured by a physical property measuring system (PPMS-9Quantum Design). SDS-PAGE was used to test and analyze the protein content of Neu-visiles and Neu-MNs. The proteins of Neu-visiles and Neu-MNs were denatured at 100 ℃ for 10 minutes, and each sample with the same protein content of 5. mu.g was loaded into parallel wells of a 10% SDS-polyacrylamide gel (EpiZyme Biotechnology, China). Subsequently, the electrophoresis tank was run at 110V for 2h, and the resulting polyacrylamide gel was stained with Coomassie Brilliant blue solution for 30min and destained overnight. And then exposed to image with a chemiluminescent gel imaging system.
Compared with other magnetic nanoparticles coated by hybrid membrane vesicles, the bionic magnetic nanoparticles prepared by coating the magnetic nanoparticles with single neutrophil membrane vesicles have higher encapsulation efficiency (figure 10). The Neu-MNs particle size was more stable over a 2-week time range than that of the hybrid membrane biomimetic nanoparticles as determined by dynamic light scattering (fig. 11). Fourier infrared spectroscopy confirmed that Neu-MNs still have C-H bonds of cell membranes after being placed for 2 weeks, and the surface functional groups are more stable (FIG. 12). DLS results indicate that the mean diameter of Neu-MNs is about 19nm for MN alone (a in FIG. 13). By TEM observation and recording of the morphological features of MNs and Neu-MNs, the images showed that the film-coated nanoparticles were about 9nm thicker than the bare cores (b, c in fig. 13), and Neu-MNs exhibited a distinct core-shell structure. Subsequently, SDS-PAGE was used to identify the membrane protein component in Neu-MNs, showing that Neu-MNs fully inherit the Neu-visicles membrane protein component (d of FIG. 13).
(4) Anti-epithelial cell adhesion molecule (anti-EpCAM) antibodies are bioconjugated to Neu-MNs:
the prepared Neu-MNs are treated by DSPE-PEG-COOH (carboxyl-polyethylene glycol-phospholipid), then NHS/EDC (N-hydroxysuccinimide/(1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)) is added for activation for 1 hour, Streptavidin (Streptavidin) is added, Streptavidin-FITC (biotin-coupled fluorescent marker) is used for marking Streptavidin-linked Neu-MNs, and then confocal imaging is used for verifying whether the Neu-MNs can be successfully bioconjugated with anti-epithelial cell adhesion molecules (anti-EpCAM). After successful validation, anti-EpCAM antibodies were mixed with Neu-MNs for bioconjugate ligation. The pathway for Neu-MNs to modify the anti-EpCAM antibody is shown in FIG. 4. Streptavidin-linked Neu-MNs were labeled with biotin-FITC and confocal images showed significant FITC fluorescence on the nanoparticles (e in FIG. 13), indicating that Neu-MNs were able to bioconjugate to anti-EpCAM. Furthermore, the inventors have also demonstrated that Neu-IMN has excellent paramagnetism (fig. 14), which ensures successful isolation of CTCs in complex biological media.
(5) And (3) verifying the reduction of protein crown adsorption of the Neu-IMNs by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identifying circulating tumor cells separated by the Neu-IMNs by Sanger sequencing:
the protein component of the protein corona was analyzed by LC-MS/MS using BCA kit (KeygEN BioTECH, China). All samples were added to 400. mu.l of a 1M solution of urea and 25mM ammonium bicarbonate and then mixed with 5. mu.l of 1M solution of dithiothreitol. Then, the mixed solution was reacted at 200 ℃ for 40 minutes, followed by cooling to room temperature. The mixture was then added with a final molar concentration of 15mM iodoacetamide solution. Then, aqueous dithiothreitol solution was added to the mixture to quench the iodoacetamide and shaken at room temperature for 10 minutes. The sample was then digested with proteomic grade trypsin at 37 ℃ for 16 hours. Excess solvent was removed using a Speedvac (Thermo Scientific, USA) before analysis using LC-MS/MS (Thermo Scientific, USA). Samples with the same protein content were analyzed by LC-MS/MS, supplemented with a mass spectrometer connected to ultra-high performance LC. The most abundant peaks were detected by the mass spectrometer and the spectra were searched using bioworks browse 3.3.1sp1 software. Normalization of the spectral counts was achieved using the following equation:
Figure BDA0003578360910000101
isolated circulating tumor cells were identified by sanger sequencing, first using a micromanipulator to obtain isolated cells. gDNA was extracted and amplified separately for each CTC using a Whole Genome Amplification (WGA) kit. Thereafter, the PIK3CA gene was PCR amplified using exon 9 and exon 20 specific primers. gDNA was amplified on exon 9 and exon 20 of PIK3CA gene. Finally, Sanger sequencing was performed in the Qingke Biotech Ltd using the above primers to detect the PIK3CA mutation. In addition, WBCs were also sequenced as controls.
Wherein, PIK3 CA-exon 9 forward primer (SEQ ID NO: 1): 5'-GGGAAAAATATGACAAAGAAAGC-3', respectively;
PIK3 CA-exon 9 reverse primer (SEQ ID NO: 2): 5'-CTGAGATCAGCCAAATTCAGTT-3', respectively;
PIK3 CA-exon 9 sequence (SEQ ID NO: 3): 5'-TAGCTAGAGACAATGAATTAAGGGAAA-3', respectively;
PIK3 CA-exon 20 forward primer (SEQ ID NO: 4): 5'-CTCAATGATGCTTGGCTCTG-3', respectively;
PIK3 CA-exon 20-reverse primer (SEQ ID NO: 5): 5'-TGGAATCCAGAGTGAGCTTTC-3', respectively;
PIK3 CA-exon 20 sequence (SEQ ID NO: 6): 5'-TTGATGACATTGCATACATTCG-3' are provided.
The capture performance of cancer cells was verified by co-incubation of Neu-IMNs with EpCAM positive MCF-7 cells. SEM images showed that a large number of Neu-IMNs were attached to the MCF-7 cell surface (f of FIG. 13), indicating that there was strong binding between Neu-IMNs and cancer cells. In addition, the inventors found that under the conditions of optimal incubation concentration and time (100. mu.g/ml, 60min), the capture efficiency of Neu-IMNs on MCF-7 cells is about 95% (g in FIG. 13), while the value of Neu-MNs is lower than 20%, which effectively proves that the capture efficiency is greatly improved after anti-EpCAM modification.
Example 2
Once the nanoparticles enter a biological environment (physiological fluid), protein crowns are formed on the surfaces of the nanoparticles quickly, which changes the interface structure and functions of the nanoparticles and has a vital influence on the fate of the nanoparticles in biological fluid. After confirming that the neutrophil membrane coating is successful, the inventors found that the neutrophil membrane can effectively reduce the formation of protein corona:
IMNs (procedures for preparing IMNs are the same as those for coupling Neu-MNs to antibodies) and Neu-IMNs are co-incubated with 10% human plasma (100. mu.g/ml, 60min), respectively, and subsequently centrifuged and washed with PBS to remove excess biomolecules. After treatment, the size of the IMNs increased significantly (fig. 15), due to the production of protein corona on the surface of the IMNs. The LC-MS/MS results indicate that certain biomolecules have aggregated onto the IMN after a period of incubation (a in fig. 16). In sharp contrast, the sizes of the Neu-IMNs were not significantly changed (b in fig. 16), indicating that neutrophil membranes are effective in inhibiting the adsorption of non-specific biomolecules. The protein content remained essentially unchanged before and after treatment (c in FIG. 16 and Table 1), indicating that neutrophil membrane coating was effective in reducing the aggregation of non-specific biomolecules on the surface of Neu-IMN.
TABLE 1 comprehensive list of normalized percentages of change before and after each protein on Neu-IMN identified by liquid chromatography tandem mass spectrometry after 4 hours incubation in 10% plasma. NSpC: the spectral counts were normalized.
Figure BDA0003578360910000111
Figure BDA0003578360910000121
Figure BDA0003578360910000131
Figure BDA0003578360910000141
Figure BDA0003578360910000151
Figure BDA0003578360910000161
Figure BDA0003578360910000171
Figure BDA0003578360910000181
Example 3
During tumor metastasis, neutrophils are able to differentiate and communicate with CTCs, and in this application the inventors found that Neu-IMNs can potentiate the interaction with CTCs:
various MNs were co-cultured with MCF-7 cells (concentration 100. mu.g/ml, time 60min) and tested for binding capacity by ICP-AES, and the results showed that the neutrophil membrane coating could effectively enhance the interaction between MNs and cancer cells (d in FIG. 16 and FIG. 17). Neu-IMNs prepared using the photo-perforation method showed higher MCF-7 cell capture efficiency compared to the mechanical push and ultrasonic mixing method (fig. 18). Neu-IMNs showed higher MCF-7 cell capture efficiency in PBS than the hybrid membrane biomimetic nanoparticles (fig. 19). Neu-IMNs show significantly improved separation efficiency in PBS over IMNs (FIG. 20), which is due to the enhanced interaction between Neu-IMNs and cancer cells. In addition, the inventors added MCF-7, PC-3 and HeLa cells to 10% of plasma and tested the cell separation efficiency. Neu-IMN showed superior capture efficiency of EpCAM positive cells (i.e., MCF-7 and PC-3 cells) compared to other IMNs (e in fig. 16). For EpCAM negative cells (i.e., HeLa cells), the capture efficiency of Neu-IMNs was almost 2 times higher than that of IMNs, indicating that neutrophil membrane coating alone can significantly improve cell separation efficiency, which benefits from neutrophil-CTC interaction. Furthermore, the cell capture efficiency of IMN in 10% plasma drops dramatically to about 60% compared to about 90% capture efficiency in PBS (e in fig. 16 and fig. 20), which is likely caused by the formation of protein corona. In sharp contrast, the Neu-IMNs maintain the excellent separation efficiency of MCF-7 cells in a complex biological environment, and further proves that the cell membrane coating can effectively reduce the blockage of anti-EpCAM.
Example 4
The homologous leukocytes in circulation do not form cell clusters, and the neutrophils are one kind of leukocytes, so that the invention discovers that the Neu-IMN inherits the characteristic of WBC:
large amounts of MNs were incubated with WBCs isolated from fresh human peripheral blood samples (concentration 100. mu.g/ml, time 60min) and then detected by ICP-AES. The results showed that Neu-IMNs accumulated less in WBCs (a in FIG. 21 and FIG. 22), indicating that neutrophil membranes can successfully limit the interaction between Neu-IMNs and WBCs. Furthermore, to better simulate the complex physiological environment in vivo, MCF-7 cells were spiked into fresh healthy blood samples and the same experiment was performed. The efficiency of separation of cancer cells by Neu-IMNs was superior to that of other IMNs (b in FIG. 21), and it was further confirmed that Neu-IMNs could effectively improve the efficiency of cancer cell capture even in whole blood. More importantly, Neu-IMN showed better isolation purity (c in fig. 21) than other IMNs when there were a large number of WBCs, because interference by WBCs was reduced. Notably, the number of CTCs in human blood samples was much lower than in human samples. Thus, the effect of background leukocytes is dramatically amplified during the isolation process. Recent advances in chemistry and materials science have shown that soft interactions between the biofilm interface and the cells provide better viability of the isolated cells. The inventors tested the effect of Neu-IMNs on the viability of isolated cells and found that Neu-IMNs indeed have better biocompatibility than IMNs (d in FIG. 21), ensuring the feasibility of subsequent cell analysis.
Example 5
Based on clinical blood samples, the inventors compared Neu-IMN with
Figure BDA0003578360910000191
Capture efficiency of CTCs by IMN. Isolated CTCs were identified by immunofluorescent staining with DAPI, PE-anti-CK and FITC-anti-CD45 used to differentiate nuclei, CTCs and WBCs, respectively. Fluorescence information was used to distinguish CTCs, i.e. DAPI +, CK +, CD45-, and WBCs, i.e. DAPI +, CK-, CD45+ (a in fig. 25) as observed by confocal microscopy. For blood samples from healthy donors, no CTC signal was detected by IMN or Neu-IMN (b in fig. 25); for breast cancer blood samples, the following are used
Figure BDA0003578360910000192
The separation efficiency and purity of Neu-IMN were significantly higher compared to IMN (b in fig. 25). Of the 20 blood samples donated by breast cancer patients, 19 samples successfully isolated CTCs by Neu-IMN. It is worth mentioning that the purity of the CTCs captured by Neu-IMNs is higher than that of IMNs. Furthermore, the inventors compared the capture efficiency of CTC by Neu-IMN prepared by different methods, and Neu-IMN prepared by the photo-perforation method had the highest capture efficiency of CTC (FIG. 23). Neu-IMN also exhibited higher CTC capture efficiency compared to other hybrid membrane biomimetic immunomagnetic nanoparticles (fig. 24).
Using the excellent biocompatibility of Neu-IMNs, CTCs isolated from Neu-IMNs were successfully cultured and observed under SEM (c in FIG. 25). More importantly, the inventors tested the frequently mutated PIK3CA gene of captured CTCs, taking into account the different mutation points of CTCs. Polymerase Chain Reaction (PCR) and Sanger sequencing were used for gene mutation analysis. The results showed that cancer metastasis was positively correlated with 3140A/G mutation (d in fig. 25). In contrast, PIK3CA had no mutations for isolated WBCs. The result shows that the CTCs captured by the Neu-IMNs have high purity and excellent biological activity and can be directly used for CTC sequencing.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:
1) compared with Qian-Fan Meng, et al, biomedical immunological Nanoparticles with Minimal Non-Specific biomolecular Adsorption for Enhanced Isolation of Circulating cells ACS Applied Materials & Interfaces,2019,11,28732-28739, the application improves the separation efficiency and purity of CTC by enhancing the interaction with CTC and reducing the interference to leukocytes, so that the separated CTC has good bioactivity and is convenient for subsequent analysis;
2) compared with Lang Rao, et al, plant let-Leukocyte Hybrid Membrane-Coated immunological Beads for high efficiency and Specific Isolation of Circulating Tumor cells, 2018,28,1803531, the preparation of the neutrophile cell Membrane vesicle of the present application does not need a complicated process, is more convenient in design, and has better stability of the encapsulation rate and better encapsulation rate due to the single cell Membrane. Based on the advantages, the technology can realize efficient and high-purity separation of CTC with low cost, and the separated CTC has good biological activity and is convenient for subsequent analysis.
3) Experimental data show that the preparation of the immunomagnetic nanoparticles (Neu-IMNs) coated with the neutrophilic granulocyte membrane vesicles by using the photo-perforation method is optimal compared with other common preparation methods (such as mechanical extrusion and ultrasonic mixing), the encapsulation efficiency is highest, the required raw materials are the least, the capture efficiency of the CTC is the highest, and therefore the application and transformation prospects are the most.
4) Experimental data show that compared with bionic magnetic nanoparticles coated by other hybrid membrane vesicles (neutrophil and macrophage hybrid membranes and erythrocyte and leukocyte hybrid membranes), the bionic magnetic nanoparticles of the neutrophil membrane vesicles developed based on the photoporation method have better encapsulation rate and stability, and can realize higher CTC capture efficiency.
5) Experimental data show that the immunomagnetic nanoparticles (Neu-IMNs) coated by the neutrophilic granulocyte membrane vesicles can simultaneously reduce nonspecific protein adsorption, enhance the interaction with CTC, reduce the interference of WBC and improve the activity of separated CTC. Compared with magnetic nanoparticles coated with erythrocyte membrane vesicles, the Neu-IMNs can improve the purity of the captured CTCs while improving the capture efficiency of the CTCs, and the two purposes are achieved. In clinical practice of separating CTC from peripheral blood samples, the Neu-IMNs designed by the method show excellent performance, and can lay a good foundation for wide application of the Neu-IMNs in noninvasive early diagnosis.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (21)

1. A biomimetic immunomagnetic nanoparticle, comprising:
magnetic nanoparticles, and
and the neutrophil membrane vesicles are coated on the magnetic nanoparticles to form bionic magnetic nanoparticles.
2. The biomimic immunomagnetic nanoparticle of claim 1, wherein the magnetic nanoparticle is Fe3O4Magnetic nanoparticles.
3. A biomimetic immunomagnetic nanoparticle for isolation, comprising:
the biomimetic immunomagnetic nanoparticle as recited in claim 1 or 2, and
a targeting antibody physically and/or covalently linked to the biomimetic magnetic nanoparticle.
4. The biomimic immunomagnetic nanoparticle of claim 3, wherein the targeting antibody is a CTC targeting antibody.
5. The biomimic immunomagnetic nanoparticle according to claim 4, wherein the CTC targeting antibody is an anti-epithelial cell adhesion molecule antibody.
6. The biomimic immunomagnetic nanoparticle according to claim 3, wherein the physical linkage and/or covalent linkage is a conjugate linkage or a click linkage.
7. A method for preparing biomimetic immunomagnetic nanoparticles according to claim 1 or 2, comprising the steps of:
s1, preparing a neutrophil membrane vesicle; and
and S2, coating the magnetic nanoparticles with the neutrophil membrane vesicles to obtain the bionic magnetic nanoparticles.
8. The method according to claim 7, wherein the S1 includes: the neutrophil membrane vesicles are prepared by isolating neutrophils from peripheral blood.
9. The method of claim 8, wherein the neutrophils are separated from the peripheral blood using gradient density centrifugation.
10. The method of claim 8, wherein the isolated neutrophils are disrupted by hypotonic lysis buffer and repeated freezing and thawing, ultracentrifugation to collect cell membranes, and then extrusion through a micro extruder to produce the neutrophils membrane vesicles.
11. The production method according to claim 10, wherein the micro-extruder extrudes through 800nm and 400nm polycarbonate porous membranes to obtain the neutrophil membrane vesicles.
12. The method according to claim 7, wherein the S2 includes: injecting the neutrophile granulocyte membrane vesicle and the magnetic nanoparticles into a micro-fluidic photo-perforation chip, and fusing the neutrophile granulocyte membrane vesicle and the magnetic nanoparticles in a photo-perforation area to coat the neutrophile granulocyte membrane vesicle on the magnetic nanoparticles to form the bionic magnetic nanoparticles.
13. The preparation method according to claim 12, wherein the flow rate of the mixture of the neutrophil membrane vesicles and the magnetic nanoparticles passing through the light-perforated region of the micro-fluidic light-perforated chip is 2 ml/h, and the bionic magnetic nanoparticles are obtained by promoting the magnetic nanoparticles to enter the neutrophil membrane vesicles by irradiating with a pulsed laser having a laser energy density of 0.12 joules/cm.
14. The method according to claim 12, wherein the molar ratio of the neutrophil membrane vesicles to the magnetic nanoparticles is (1:1) to (1: 10).
15. A method for preparing biomimetic immunomagnetic nanoparticles for isolation according to any of claims 3 to 6, comprising the steps of:
the preparation method according to any one of claims 7 to 14,
s3, the bionic immune magnetic nanoparticle is obtained by physically and/or covalently connecting the targeting antibody with the bionic magnetic nanoparticle.
16. The method of making of claim 15, wherein the targeting antibody is a CTC targeting antibody.
17. The method of making of claim 16, wherein the CTC targeting antibody is an anti-epithelial cell adhesion molecule antibody.
18. The method according to claim 17, wherein the S3 includes: and treating the bionic magnetic nanoparticles prepared by the S2 with carboxyl-polyethylene glycol-phospholipid, adding NHS/EDC for activation, crosslinking streptavidin, and combining a biotinylated CTC targeting antibody with the streptavidin to obtain the bionic immune magnetic nanoparticles.
19. Use of the biomimetic immunomagnetic nanoparticles according to any of claims 3-6 for isolation in the preparation of a product for enriching, isolating or detecting circulating tumor cells.
20. Use of the isolated biomimetic immunomagnetic nanoparticle of any of claims 3-6 in the preparation of a medicament for treating a tumor metastasis associated disease.
21. The use according to claim 20, wherein the tumor metastasis associated disease is a tumor associated with high expression of epithelial cell adhesion factor, preferably colorectal cancer, breast cancer and gastric cancer.
CN202210339436.8A 2022-04-01 2022-04-01 Bionic immune magnetic nanoparticle, preparation method and application thereof Pending CN114487391A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115058344A (en) * 2022-08-05 2022-09-16 深圳湾实验室 Bait micro-robot for removing SARS-CoV-2 and its variant strain in waste water, its preparation method and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115058344A (en) * 2022-08-05 2022-09-16 深圳湾实验室 Bait micro-robot for removing SARS-CoV-2 and its variant strain in waste water, its preparation method and application
CN115058344B (en) * 2022-08-05 2022-11-18 深圳湾实验室 Bait micro-robot for removing SARS-CoV-2 and its variant strain in waste water, its preparation method and application

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