A magnetic nanobead for cell sorting and its preparation method

Abstract

This invention discloses a method for preparing magnetic nanobeads for cell sorting. Using hemicellulose, arabinogalactan, FeCl3, FeCl2, and Na2CO3 as raw materials and DMSO as solvent, monodisperse hemicellulose / arabinogalactan@Fe3O4 magnetic nanobeads are generated by a solvothermal reaction. Subsequently, the nanobeads are further activated by 4-fluorobenzenesulfonyl chloride to introduce fluorobenzenesulfonyl groups. The fluorobenzenesulfonyl groups are then coupled with cell sorting antibody CD3. The coupled antibody magnetic beads are then used to purify and sort cells.

Description

Technical Field

[0001] This invention belongs to the field of antibody-magnetic bead conjugation, specifically relating to a nanomagnetic bead for cell sorting and its preparation method, and more particularly to a biodegradable hemicellulose matrix nanomagnetic bead for cell sorting and its preparation method. Background Technology

[0002] Immunomagnetic beads, or simply magnetic beads, possess excellent superparamagnetism, are simple and convenient to handle, and have a large specific surface area, making them a good separation material. As a solid material, magnetic beads are modified to have chemical groups on their surface, allowing them to covalently couple with protein molecules. Furthermore, due to their large specific surface area, they can bind more proteins, making them suitable for cell sorting.

[0003] Currently, the most effective and popular choice on the market is the magnetic bead for cell sorting using a sorting column developed by Medtronic. Little is publicly available information about the coupling technology of these magnetic beads, and such beads are also scarce, being trade secrets. Most products on the market use carboxyl-coupled antibodies, but this method is unsuitable for Medtronic cell sorting; the magnetic beads cannot remain in the buffer for extended periods without settling and cannot pass through Medtronic's sorting column. Other conventional magnetic beads have too large a particle size and poor hydrophilicity, easily clogging the sorting column. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings and deficiencies of the prior art by providing a nanomagnetic bead for cell sorting and its preparation method.

[0005] This invention involves coating Fe3O4 nanoparticles with hemicellulose / arabinogalactan. Hemicellulose / arabinogalactan is rich in hydroxyl groups, which, after coating the magnetic beads, make them more hydrophilic and better dispersed in water. The beads do not sink in the buffer for extended periods and can pass through the sorting column of a micro-sorptive agent. The hydroxyl groups also serve for subsequent modification with fluorobenzenesulfonyl groups. Compared to other coating materials, such as PVA, which is a high-molecular-weight polymer obtained through chemical synthesis and has some toxicity and is not biodegradable, this invention is suitable for cell sorting, as the cells obtained are generally used in clinical CAR-T cell therapy for cancer. Therefore, the magnetic beads used for cell sorting should ideally be non-toxic and biodegradable. Hemicellulose and arabinogalactan meet these requirements. Hemicellulose primarily provides hydroxyl groups for subsequent modification, while arabinogalactan, as a surfactant, mainly acts as a surfactant for the magnetic beads, making them easier to disperse in water.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a magnetic nanoparticle for cell sorting, wherein the magnetic nanoparticle has a core-shell structure; the outer shell is antibody-functionalized hemicellulose / arabinogalactan, and the core is Fe3O4 nanoparticles;

[0008] Antibody-functionalized hemicellulose / arabinogalactan is produced by activating the hydroxyl groups on hemicellulose and arabinogalactan with 4-fluorobenzenesulfonyl chloride and triethylamine to obtain magnetic nanobeads coated with fluorobenzenesulfonyl groups. These nanobeads are then coupled with antibodies to obtain antibody-coated hemicellulose and arabinogalactan, i.e., antibody-functionalized hemicellulose / arabinogalactan.

[0009] Preferably, the nanomagnetic beads are monodisperse particles with a transmission electron microscope (TEM) diameter of 8-12 nanometers. The nanomagnetic beads obtained by this invention are the best monodisperse, smallest in size, and non-sinking magnetic beads currently available in the technology. This solves the problem of precipitation after long-term storage following conjugation of magnetic beads and antibodies, and allows the conjugated antibody magnetic beads to bind more fully to the target biomolecules, thus improving the binding efficiency.

[0010] The Fe3O4 nanoparticles of the present invention are used to obtain fluorobenzenesulfonyl group modified magnetic nanobeads by activating the hydroxyl groups on hemicellulose / arabinogalactan with 4-fluorobenzenesulfonyl chloride and triethylamine. The fluorobenzenesulfonyl groups on the surface of the obtained magnetic nanobeads are coupled with the T cell recognition antibody CD3 to obtain CD3 antibody-coated magnetic nanobeads. Then, the CD3 antibody-coated magnetic nanobeads are used to enrich CD3-containing immune cells.

[0011] Secondly, the present invention relates to a method for preparing biodegradable hemicellulose matrix nanobeads for cell sorting, the method comprising the following steps:

[0012] S1. Using hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate as raw materials, these are added to a solvent and reacted via a solvothermal method to generate monodisperse, core-shell structured hemicellulose / arabinogalactan@Fe3O4 nanoparticles.

[0013] S2. Activation reaction of hemicellulose / arabinogalactan@Fe3O4 nanoparticles was carried out using 4-fluorobenzenesulfonyl chloride and triethylamine to obtain fluorobenzenesulfonyl group modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles.

[0014] S3. The obtained fluorobenzenesulfonyl group modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles are coupled with antibodies to obtain antibody-coated hemicellulose / arabinogalactan@Fe3O4 nanoparticles, which are the nanomagnetic beads used for cell sorting.

[0015] Preferably, in step S1, the ratio of hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate is 0.2–0.8 g : 0.1–0.4 g : 0.02–0.62 g : 0.012–0.25 g : 0.4–0.65 g. The molar ratio of ferric chloride or its hydrate and ferrous chloride or its hydrate is preferably a fixed 2:1.

[0016] Preferably, in step S1, the ratio of solvent, hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate is 0.1–2 ml: 0.2–0.8 g: 0.1–0.4 g: 0.02–0.62 g: 0.012–0.25 g: 0.4–0.65 g.

[0017] Preferably, in step S1, ferric chloride is added in the form of ferric chloride or its hydrate, ferrous chloride is added in the form of ferrous chloride or its hydrate, and sodium carbonate is added in the form of sodium carbonate or its hydrate. The preferred ferric chloride hydrate is FeCl3·6H2O, the preferred ferrous chloride hydrate is FeCl2·4H2O, and the preferred sodium carbonate hydrate is Na2CO3·10H2O.

[0018] Preferably, in step S1, the solvent is DMSO.

[0019] Preferably, in step S1, the reaction temperature of the solvothermal reaction is 120–220°C, and the reaction time is 2–48 hours.

[0020] Preferably, in step S1, the hemicellulose / arabinogalactan@Fe3O4 nanoparticles are hemicellulose / arabinogalactan-coated Fe3O4 nanoparticles.

[0021] Preferably, in step S2, the activation reaction involves adding hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine to a solvent and reacting at 25–80°C for 2–8 hours to obtain fluorobenzenesulfonyl-modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles. The solvent is DMSO. Activation involves reacting the hydroxyl groups on the hemicellulose / arabinogalactan with 4-fluorobenzenesulfonyl chloride and triethylamine to obtain fluorobenzenesulfonyl-coated magnetic nanobeads.

[0022] This invention uses a fluorobenzenesulfonyl group to conjugate antibodies, which results in a shorter antibody conjugation time and higher conjugation efficiency compared to other conjugation methods.

[0023] Preferably, in step S2, the ratio of hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine is 1–5 g : 0.5–5 ml : 0.1–0.4 ml. Triethylamine acts as an organic base catalyzer.

[0024] Preferably, in step S3, the antibody conjugation is achieved by reacting fluorobenzenesulfonyl-modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles with the antibody in a sodium carbonate-sodium bicarbonate solution (pH 9.5) at 4–50°C for 1–3 hours.

[0025] Preferably, in step S3, the mass ratio of fluorobenzenesulfonyl group modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles to antibody is 0.1-5:0.1-5.

[0026] Preferably, in step S3, the antibody is an antibody against CD3 protein, which recognizes CD3 protein on the surface of T cells.

[0027] Thirdly, the present invention also relates to the application of the aforementioned magnetic nanobeads in the purification and sorting of cells.

[0028] Preferably, the application is the use of nanomagnetic beads in the sorting column of Medtronic to purify and sort cells.

[0029] Preferably, the application includes the following steps:

[0030] A1. Prepare cell samples, resuspend cell particles in buffer solution, add magnetic nanobeads and mix well.

[0031] A2. Incubate the well-mixed cell sample, wash the cells with buffer, then centrifuge, completely aspirate the supernatant, and resuspend in buffer.

[0032] A3. Perform magnetic cell separation on the resuspended cell sample.

[0033] In step A1, the buffer is 1X PBS pH 7.2-7.4 including 2mM EDTA and 0.5% HSA buffer (Ca-free). 2 + ).

[0034] In step A1, before using the cell sample, the cell suspension is centrifuged at 300g for 10 minutes, and the supernatant is completely aspirated.

[0035] In step A1, the cell particles are resuspended in buffer solution at a ratio of 1 x 10⁻⁶. 7Resuspend the cell particles in 80 μL of buffer (you can remove clumped or large cells beforehand using a 30 μm cell filter), 1 x 10 7 Add 20 μL of magnetic nanoparticle dispersion to each cell (the amount of magnetic beads is 5 x 10). 10 indivual).

[0036] Each 1x10 7 Each cell was resuspended in 60 μL of buffer solution, and 20 μL of magnetic nanoparticle suspension corresponding to CD3 protein was added.

[0037] In step A2, the incubation time is 15 minutes, and the temperature is 2-8℃. If fluorescent labeling is to be performed, 10 μL of fluorescent antibody can be added after incubation, and the mixture can be incubated in the dark for 5 minutes.

[0038] In step A2, cells are washed with buffer solution, 1 x 10 7 Wash cells with 1-2 mL of buffer. Centrifuge at 300 g for 10 minutes.

[0039] In step A2, during resuspension, the number of cells resuspended in 500 μL of buffer should not exceed 1 x 102. 8 The number of cells does not exceed 1 x 103. 7 When using this method, the buffer volume should be no less than 50 μL. For higher cell numbers, increase the buffer volume accordingly.

[0040] In step A3, magnetic cell separation is performed using Miltenyi MS or LS columns.

[0041] In step A3, the magnetic cell separation step is as follows:

[0042] (1) Magnetic separation was performed using METI MS or LS columns;

[0043] (2) Place the separation column in the separation magnet of the MACS Separator;

[0044] (3) Rinse the column with buffer solution (MS: 500 μL, LS: 3 mL);

[0045] (4) Drop the cell suspension onto the separation column and collect the eluent containing unlabeled cells;

[0046] (5) Rinse the separation column with buffer (MS: 3 x 500 μL, LS: 3 x 3 mL), collect the unlabeled cells that have passed through, and mix with the effluent from step (3);

[0047] (6) Remove the separation column from the separation magnet, place it in the collection tube, transfer the buffer to the separation column, push the plunger into the separation column and flush out the magnetically labeled cells (MS: 1 mL, LS: 5 mL);

[0048] (7) Finally, flow cytometry analysis was performed.

[0049] In step (5), once the separation column reservoir is empty, an equal amount of buffer solution is immediately added for a cleaning step.

[0050] The hemicellulose used in this invention is a type of natural polysaccharide widely found in plant cell walls, forming a major component of plant cell walls along with cellulose and lignin. Unlike the linear structure of cellulose, hemicellulose has a branched structure, a smaller molecular weight, and diverse structures, often composed of various sugar units such as xylose, mannose, glucose, galactose, and arabinose. This invention uses hemicellulose to coat magnetic beads, which is easily degraded by microorganisms during use, does not accumulate in the environment over a long period, and as a natural polysaccharide, is harmless to the human body.

[0051] The nanomagnetic beads of this invention have higher sorting efficiency, are simpler, produce purer cells, cause less damage to cells, and have a higher recovery rate.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The magnetic nanoparticles obtained by this invention are the best monodisperse, have the smallest particle size, and do not sink in water. This solves the problem of precipitation after long-term storage of magnetic beads and antibodies. At the same time, it allows the magnetic beads with conjugated antibodies to bind more fully with the target biomolecules, thus improving the binding efficiency.

[0054] (2) The sorting efficiency is higher, the recovery rate is higher, the process is simple, and the operation is convenient. Attached Figure Description

[0055] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0056] Figure 1 This is a transmission electron microscope (TEM) image (10 nm) of the magnetic nanobeads used for cell sorting obtained in Example 1 of the present invention.

[0057] Figure 2 This is a transmission electron microscope (TEM) image (10 nm) of the magnetic nanobeads used for cell sorting obtained in Example 1 of the present invention.

[0058] Figure 3 The CD3 antibody cells obtained in Example 1 of this invention are used to sort PBMC cells using magnetic beads; where a is the purity of CD3 cells in PBMCs before cell sorting (69.77%), and b is the purity after enrichment with magnetic beads (97.34%).

[0059] Figure 4 This is a scanning electron microscope image of the failed example magnetic bead obtained in Comparative Example 1 of the present invention.

[0060] Figure 5 This is a scanning electron microscope image of the failed example magnetic bead obtained in Comparative Example 1 of the present invention.

[0061] Figure 6 This is a scanning electron microscope image of the failed example magnetic bead obtained in Comparative Example 2 of this invention.

[0062] Figure 7 This is a scanning electron microscope image of the failed example magnetic bead obtained in Comparative Example 2 of this invention.

[0063] Figure 8 This is a scanning electron microscope image of the failed example magnetic bead obtained in Comparative Example 3 of the present invention.

[0064] Figure 9 This is a scanning electron microscope image of the failed example magnetic bead obtained in Comparative Example 3 of the present invention. Detailed Implementation

[0065] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0066] The hemicellulose used in this invention has a molecular weight of 7500 g / mol, and its composition, by mass fraction, mainly includes 17% glucose, 65% mannose, and 15% galactose.

[0067] Example 1

[0068] This embodiment relates to a novel method for preparing and applying magnetic nanobeads for cell sorting, as detailed below:

[0069] I. The preparation method of magnetic beads includes the following steps:

[0070] In the first step, 0.5 g of hemicellulose, 0.25 g of (+)-arabinogalactan, 0.5 g of FeCl3·6H2O, 0.368 g of FeCl2·4H2O, and 1.5 g of Na2CO3·10H2O were mixed in 20 mL of DMSO solvent and mechanically stirred at 300 rpm until completely dissolved. The solution was then transferred to a 50 mL reaction vessel and reacted at 195 °C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 mL centrifuge tube, washed with deionized water, collected with a magnet, and vacuum dried for storage to obtain black Fe3O4 nanoparticles.

[0071] The second step involves surface modification of the magnetic beads to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads: 3 g of hemicellulose-coated magnetic nanobeads, 0.4 mL of triethylamine and 2 mL of 4-fluorobenzenesulfonyl chloride were reacted in 30 mL of DMSO solvent at 50 °C for 3 hours, followed by washing with anhydrous ethanol and deionized water multiple times to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads.

[0072] The third step is to introduce CD3 antibody onto the surface of the magnetic beads: 2 mg of fluorobenzenesulfonyl group modified magnetic nanobeads and 1 mg of CD3 antibody are reacted in 8 mL of 0.5 M sodium bicarbonate solution at 37 °C for 16 hours. Then, the mixture is washed multiple times with deionized water to obtain CD3 antibody modified magnetic nanobeads.

[0073] II. The CD3 antibody nanoparticles prepared in this embodiment are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0074] Sample preparation:

[0075] Peripheral blood cells (PBMCs), buffer 1X PBS pH 7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca. 2+ ;

[0076] 1. Determine the cell number.

[0077] 2. Centrifuge the cell suspension at 300g for 10 minutes and completely aspirate the supernatant.

[0078] 3. According to each 1x10 7 Resuspend the cell particles in 80 μL of buffer (you can remove clumped or large cells beforehand using a 30 μm cell filter), 1 x 10 7 Add 20 μL of CD3 nanoparticle magnetic bead dispersion to each cell (the amount of magnetic beads is 5 x 10). 10 (One), used for CD3+ cell isolation.

[0079] 4. Mix well and incubate in the refrigerator for 15 minutes (2-8℃).

[0080] 5. Each 1x10 7 Wash the cells with 1-2 mL of buffer, then centrifuge at 300 g for 10 minutes and completely aspirate the supernatant.

[0081] 6. Resuspend in 500 μL of buffer at a maximum of 1 x 10⁻⁶. 8 1 x 10 cells 7Alternatively, resuspend fewer cells in 50 μL of buffer; for higher cell numbers, increase the buffer volume accordingly.

[0082] 7. Perform magnetic separation.

[0083] Magnetic cell separation:

[0084] 1. Use Miltenyi MS or LS columns for magnetic separation.

[0085] 2. Place the separation column in the separation magnet of a suitable MACS Separator.

[0086] 3. Rinse the column with an appropriate amount of buffer solution: MS: 500 μL, LS: 3 mL.

[0087] 4. Drop the cell suspension onto the separation column and collect the eluent containing unlabeled cells.

[0088] 5. Rinse the separation column with an appropriate amount of buffer, collect the passed unlabeled cells, and mix with the effluent from step 3. MS: 3 x 500 μL, LS: 3 x 3 mL.

[0089] • Note: Once the separation column reservoir is empty, immediately add an equal volume of buffer solution for the washing step.

[0090] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Transfer an appropriate amount of buffer solution onto the separation column. Push the plunger into the separation column to flush out the magnetically labeled cells. MS: 1 mL, LS: 5 mL.

[0091] Then flow cytometry analysis was performed.

[0092] Transmission electron microscopy (10 nm) image of the magnetic nanobeads obtained in Example 1 for cell sorting. Figure 1 , 2 As shown. CD3 antibody cell sorting magnetic beads sorting PBMC cells, as shown. Figure 3 As shown; where a is the purity of CD3 cells in PBMC before cell sorting (69.77%), and b is the purity after enrichment with sorting magnetic beads (97.34%).

[0093] Comparative Example 1

[0094] This embodiment relates to a novel method for preparing and applying magnetic nanobeads for cell sorting, as detailed below:

[0095] The preparation method of magnetic beads includes the following steps:

[0096] In the first step, 0.5 g of hemicellulose, 0.25 g of (+)-arabinogalactan, 0.5 g of FeCl3·6H2O, 0.368 g of FeCl2·4H2O, and 2 g of Na2CO3·10H2O were mixed in 20 mL of DMSO solvent and mechanically stirred at 300 rpm until completely dissolved. The solution was then transferred to a 50 mL reactor and reacted at 195 °C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 mL centrifuge tube, washed with deionized water, collected with a magnet, and vacuum dried for storage to obtain black Fe3O4 nanoparticles.

[0097] The second step involves surface modification of the magnetic beads to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads: 3 g of hemicellulose-coated magnetic nanobeads, 0.4 mL of triethylamine and 2 mL of 4-fluorobenzenesulfonyl chloride were reacted in 30 mL of DMSO solvent at 50 °C for 3 hours, followed by washing with anhydrous ethanol and deionized water multiple times to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads.

[0098] The third step is to introduce CD3 antibody onto the surface of the magnetic beads: 2 mg of fluorobenzenesulfonyl group modified magnetic nanobeads and 1 mg of CD3 antibody are reacted in 8 mL of 0.5 M sodium bicarbonate solution at 37 °C for 16 hours. Then, the mixture is washed multiple times with deionized water to obtain CD3 antibody modified magnetic nanobeads.

[0099] The CD3 antibody nanoparticles prepared in this embodiment are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0100] A1, Buffer Solution:

[0101] Sample preparation:

[0102] Peripheral blood cells (PBMCs), 1X PBS pH 7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca2+. 2+ ;

[0103] 1. Determine the cell number.

[0104] 2. Centrifuge the cell suspension at 300g for 10 minutes and completely aspirate the supernatant.

[0105] 3. According to each 1x10 7 Resuspend the cell particles in 80 μL of buffer (you can remove clumped or large cells beforehand using a 30 μm cell filter), 1 x 10 7 Add 20 μL of CD3 nanoparticle magnetic bead dispersion to each cell (the amount of magnetic beads is 5 x 10). 10(One), used for CD3+ cell isolation.

[0106] 4. Mix well and incubate in the refrigerator for 15 minutes (2-8℃).

[0107] 5. Each 1x10 7 Wash the cells with 1-2 mL of buffer, then centrifuge at 300 g for 10 minutes and completely aspirate the supernatant.

[0108] 6. Resuspend in 500 μL of buffer at a maximum of 1 x 10⁻⁶. 8 1 x 10 cells 7 Alternatively, resuspend fewer cells in 50 μL of buffer; for higher cell numbers, increase the buffer volume accordingly.

[0109] 7. Perform magnetic separation.

[0110] Magnetic cell separation

[0111] 1. Use Miltenyi MS or LS columns for magnetic separation.

[0112] 2. Place the separation column in the separation magnet of a suitable MACS Separator.

[0113] 3. Rinse the column with an appropriate amount of buffer solution: MS: 500 μL, LS: 3 mL.

[0114] 4. Drop the cell suspension onto the separation column and collect the eluent containing unlabeled cells.

[0115] 5. Rinse the separation column with an appropriate amount of buffer, collect the passed unlabeled cells, and mix with the effluent from step 3. MS: 3 x 500 μL, LS: 3 x 3 mL.

[0116] • Note: Once the separation column reservoir is empty, immediately add an equal volume of buffer solution for the washing step.

[0117] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Transfer an appropriate amount of buffer to the separation column, push the plunger into the separation column to flush out the magnetically labeled cells. MS: 1 mL, LS: 5 mL.

[0118] Scanning electron microscope (SEM) images of the failed magnetic beads obtained in Example 2 are shown below. Figure 4 , 5 As shown, the magnetic beads agglomerated. This agglomeration caused blockage in the sorting column, preventing the plunger from ejecting the magnetically labeled cells.

[0119] Comparative Example 2

[0120] This embodiment relates to a novel method for preparing and applying magnetic nanobeads for cell sorting, as detailed below:

[0121] The preparation method of magnetic beads includes the following steps:

[0122] In the first step, 1 g of hemicellulose, 0.5 g of (+)-arabinogalactan, 0.5 g of FeCl3·6H2O, 0.368 g of FeCl2·4H2O, and 2 g of Na2CO3·10H2O were mixed in 20 mL of DMSO solvent and mechanically stirred at 300 rpm until completely dissolved. The solution was then transferred to a 50 mL reaction vessel and reacted at 195 °C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 mL centrifuge tube, washed with deionized water, collected with a magnet, and vacuum dried for storage to obtain black Fe3O4 nanoparticles.

[0123] The second step involves surface modification of the magnetic beads to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads: 3 g of hemicellulose-coated magnetic nanobeads, 0.4 mL of triethylamine and 2 mL of 4-fluorobenzenesulfonyl chloride were reacted in 30 mL of DMSO solvent at 50 °C for 3 hours, followed by washing with anhydrous ethanol and deionized water multiple times to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads.

[0124] The third step is to introduce CD3 antibody onto the surface of the magnetic beads: 2 mg of fluorobenzenesulfonyl group modified magnetic nanobeads and 1 mg of CD3 antibody are reacted in 8 mL of 0.5 M sodium bicarbonate solution at 37 °C for 16 hours. Then, the mixture is washed multiple times with deionized water to obtain CD3 antibody modified magnetic nanobeads.

[0125] The CD3 antibody nanoparticles prepared in this embodiment are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0126] A1, Buffer Solution:

[0127] Sample preparation:

[0128] Peripheral blood cells (PBMCs), 1X PBS pH 7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca2+. 2+ ;

[0129] 1. Determine the cell number.

[0130] 2. Centrifuge the cell suspension at 300g for 10 minutes and completely aspirate the supernatant.

[0131] 3. According to each 1x10 7Resuspend the cell particles in 80 μL of buffer (you can remove clumped or large cells beforehand using a 30 μm cell filter), 1 x 10 7 Add 20 μL of CD3 nanoparticle magnetic bead dispersion to each cell (the amount of magnetic beads is 5 x 10). 10 (One), used for CD3+ cell isolation.

[0132] 4. Mix thoroughly and incubate in a refrigerator for 15 minutes (+2 to +8°C). If fluorescent labeling is to be performed, add 10 μL of fluorescent antibody after 15 minutes and incubate in the dark for 5 minutes.

[0133] 5. Each 1x10 7 Wash the cells with 1-2 mL of buffer, then centrifuge at 300 g for 10 minutes and completely aspirate the supernatant.

[0134] 6. Resuspend in 500 μL of buffer at a maximum of 1 x 10⁻⁶. 8 1 x 10 cells 7 Alternatively, resuspend fewer cells in 50 μL of buffer; for higher cell numbers, increase the buffer volume accordingly.

[0135] 7. Perform magnetic separation.

[0136] Magnetic cell separation

[0137] 1. Use Miltenyi MS or LS columns for magnetic separation.

[0138] 2. Place the separation column in the separation magnet of a suitable MACS Separator.

[0139] 3. Rinse the column with an appropriate amount of buffer solution: MS: 500 μL, LS: 3 mL.

[0140] 4. Drop the cell suspension onto the separation column and collect the eluent containing unlabeled cells.

[0141] 5. Rinse the separation column with an appropriate amount of buffer, collect the passed unlabeled cells, and mix with the effluent from step 3. MS: 3 x 500 μL, LS: 3 x 3 mL.

[0142] • Note: Once the separation column reservoir is empty, immediately add an equal volume of buffer solution for the washing step.

[0143] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Transfer an appropriate amount of buffer to the separation column, push the plunger into the separation column to flush out the magnetically labeled cells. MS: 1 mL, LS: 5 mL.

[0144] Scanning electron microscope (SEM) images of the failed magnetic beads obtained in Example 3 are shown below. Figure 6, 7 The magnetic beads shown have agglomerated. This agglomeration causes blockage in the sorting column, preventing the plunger from ejecting the magnetically labeled cells.

[0145] Comparative Example 3

[0146] This embodiment relates to a novel method for preparing and applying magnetic nanobeads for cell sorting, as detailed below:

[0147] The preparation method of magnetic beads includes the following steps:

[0148] In the first step, 1 g of hemicellulose, 0.25 g of (+)-arabinogalactan, 0.5 g of FeCl3·6H2O, 0.368 g of FeCl2·4H2O, and 2 g of Na2CO3·10H2O were mixed in 20 mL of DMSO solvent and mechanically stirred at 300 rpm until completely dissolved. The solution was then transferred to a 50 mL reactor and reacted at 195 °C for 25 hours. After cooling to room temperature, the reaction product was transferred to a 50 mL centrifuge tube, washed with deionized water, collected with a magnet, and vacuum dried for storage to obtain black Fe3O4 nanoparticles.

[0149] The second step involves surface modification of the magnetic beads to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads: 3 g of hemicellulose-coated magnetic nanobeads, 0.4 mL of triethylamine and 2 mL of 4-fluorobenzenesulfonyl chloride were reacted in 30 mL of DMSO solvent at 50 °C for 3 hours, followed by washing with anhydrous ethanol and deionized water multiple times to obtain fluorobenzenesulfonyl group-modified magnetic nanobeads.

[0150] The third step is to introduce CD3 antibody onto the surface of the magnetic beads: 2 mg of fluorobenzenesulfonyl group modified magnetic nanobeads and 1 mg of CD3 antibody are reacted in 8 mL of 0.5 M sodium bicarbonate solution at 37 °C for 16 hours. Then, the mixture is washed multiple times with deionized water to obtain CD3 antibody modified magnetic nanobeads.

[0151] Step 5

[0152] The CD3 antibody nanoparticles prepared in this embodiment are used to purify and sort CD3 immune cells. The purification method includes the following steps:

[0153] A1, Buffer Solution:

[0154] Sample preparation:

[0155] Peripheral blood cells (PBMCs), 1X PBS pH 7.2-7.4 containing 2mM EDTA, 0.5% HSA buffer without Ca2+. 2+ ;

[0156] 1. Determine the cell number.

[0157] 2. Centrifuge the cell suspension at 300g for 10 minutes and completely aspirate the supernatant.

[0158] 3. According to each 1x10 7 Resuspend the cell particles in 80 μL of buffer (you can remove clumped or large cells beforehand using a 30 μm cell filter), 1 x 10 7 Add 20 μL of CD3 nanoparticle magnetic bead dispersion to each cell (the amount of magnetic beads is 5 x 10). 10 (One) is used for CD3+ cell isolation.

[0159] 4. Mix well and incubate in the refrigerator for 15 minutes (2-8℃).

[0160] 5. Each 1x10 7 Wash the cells with 1-2 mL of buffer, then centrifuge at 300 g for 10 minutes and completely aspirate the supernatant.

[0161] 6. Resuspend in 500 μL of buffer at a maximum of 1 x 10⁻⁶. 8 1 x 10 cells 7 Alternatively, resuspend fewer cells in 50 μL of buffer; for higher cell numbers, increase the buffer volume accordingly.

[0162] 7. Perform magnetic separation.

[0163] Magnetic cell separation

[0164] 1. Use Miltenyi MS or LS columns for magnetic separation.

[0165] 2. Place the separation column in the separation magnet of a suitable MACS Separator.

[0166] 3. Rinse the column with an appropriate amount of buffer solution: MS: 500 μL, LS: 3 mL.

[0167] 4. Drop the cell suspension onto the separation column and collect the eluent containing unlabeled cells.

[0168] 5. Rinse the separation column with an appropriate amount of buffer, collect the passed unlabeled cells, and mix with the effluent from step 3. MS: 3 x 500 μL, LS: 3 x 3 mL.

[0169] • Note: Once the separation column reservoir is empty, immediately add an equal volume of buffer solution for the washing step.

[0170] 6. Remove the separation column from the separation magnet and place it in a suitable collection tube. Transfer an appropriate amount of buffer to the separation column, push the plunger into the separation column to flush out the magnetically labeled cells. MS: 1 mL, LS: 5 mL.

[0171] The scanning electron microscope image of the failed magnetic bead obtained in Example 4 is as follows: Figure 8 , 9 As shown, the magnetic beads agglomerated. This agglomeration caused blockage in the sorting column, preventing the plunger from ejecting the magnetically labeled cells.

[0172] Comparative Example 4

[0173] This comparative example relates to a novel method for preparing and applying magnetic nanobeads for cell sorting. The steps are basically the same as in Example 1, except that 0.5 g of hemicellulose and 0.25 g of (+)-arabinogalactan are replaced with 0.75 g of hemicellulose. The magnetic beads agglomerated, causing blockage of the sorting column.

[0174] Comparative Example 5

[0175] This comparative example relates to a novel method for preparing and applying magnetic nanobeads for cell sorting. The steps are basically the same as in Example 1, except that 0.5 g of hemicellulose and 0.25 g of (+)-arabinogalactan are replaced with 0.75 g of (+)-arabinogalactan. The magnetic beads agglomerated, causing blockage of the sorting column.

[0176] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A type of magnetic nanobead for cell sorting, characterized in that, The magnetic nanobeads have a core-shell structure; the outer shell is antibody-functionalized hemicellulose / arabinogalactan, and the core is Fe3O4 nanoparticles. Antibody-functionalized hemicellulose / arabinogalactan is produced by activating the hydroxyl groups on hemicellulose and arabinogalactan with 4-fluorobenzenesulfonyl chloride and triethylamine to obtain magnetic nanobeads coated with fluorobenzenesulfonyl groups. These nanobeads are then coupled with antibodies to obtain antibody-coated hemicellulose and arabinogalactan, i.e., antibody-functionalized hemicellulose / arabinogalactan.

2. The magnetic nanobeads for cell sorting according to claim 1, characterized in that, The magnetic nanobeads are monodisperse particles with a particle size of 8-12 nanometers.

3. A method for preparing magnetic nanobeads for cell sorting, characterized in that, The preparation method includes the following steps: S1. Using hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate as raw materials, they are added to a solvent and reacted by a solvothermal method to generate monodisperse, core-shell structured hemicellulose / arabinogalactan@Fe3O4 nanoparticles. S2. Activation reaction of hemicellulose / arabinogalactan@Fe3O4 nanoparticles was carried out using 4-fluorobenzenesulfonyl chloride and triethylamine to obtain fluorobenzenesulfonyl group modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles. S3. The obtained fluorobenzenesulfonyl group modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles are coupled with antibodies to obtain antibody-coated hemicellulose / arabinogalactan@Fe3O4 nanoparticles, which are the nanomagnetic beads used for cell sorting.

4. The method for preparing nanomagnetic beads according to claim 3, characterized in that, In step S1, the ratio of hemicellulose, arabinogalactan, ferric chloride, ferrous chloride, and sodium carbonate is 0.2–0.8 g: 0.1–0.4 g: 0.02–0.62 g: 0.012–0.25 g: 0.4–0.65 g.

5. The method for preparing nanomagnetic beads according to claim 3, characterized in that, In step S1, the solvent includes DMSO.

6. The method for preparing nanomagnetic beads according to claim 3, characterized in that, In step S1, the reaction temperature of the solvothermal reaction is 120–220°C, and the reaction time is 2–48 hours.

7. The method for preparing nanomagnetic beads according to claim 3, characterized in that, In step S2, the activation reaction involves adding hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine to a solvent and reacting at 25–80°C for 2–8 hours to obtain fluorobenzenesulfonyl group modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles. And / or, the ratio of hemicellulose / arabinogalactan@Fe3O4 nanoparticles, 4-fluorobenzenesulfonyl chloride, and triethylamine is 1–5 g : 0.5–5 ml : 0.1–0.4 ml.

8. The method for preparing nanomagnetic beads according to claim 3, characterized in that, In step S3, the antibody conjugation is achieved by reacting fluorobenzenesulfonyl-modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles with the antibody in a sodium carbonate-sodium bicarbonate solution at 4–50°C for 1–3 hours. And / or, the mass ratio of fluorobenzenesulfonyl-modified hemicellulose / arabinogalactan@Fe3O4 nanoparticles to antibody is 0.1–5:0.1–5; And / or, the antibody is an antibody against the CD3 protein that recognizes the CD3 protein on the surface of T cells.

9. The application of the magnetic nanobeads as described in claim 1 or 2 in the purification and sorting of cells.

10. The application according to claim 9, characterized in that, The application includes the following steps: A1. Prepare cell samples, resuspend cell particles in buffer solution, add magnetic nanobeads and mix well. A2. Incubate the well-mixed cell sample, wash the cells with buffer, then centrifuge, completely aspirate the supernatant, and resuspend in buffer. A3. Perform magnetic cell separation on the resuspended cell sample.

Images