Sorting method of T lymphocytes
By preparing magnetic nanobeads with a median particle size of 50–200 nm and good uniformity, and coating their surface with carboxyglucan and conjugated antibodies, the problems of unsuitable particle size and poor uniformity of magnetic nanobeads in the prior art have been solved. This has enabled efficient separation of T lymphocytes without significant impact on cell function, and is applicable to the biomedical field.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-07
- Publication Date
- 2026-04-03
AI Technical Summary
The existing magnetic nanobeads have an unsuitable particle size range and poor uniformity, resulting in low cell sorting efficiency and affecting cell function and biocompatibility.
Aqueous solutions of ferric and ferrous iron were prepared under ice bath conditions, the pH was adjusted to 10–12, and the reaction was carried out at 65–90 °C to prepare magnetic nanobeads with a median particle size of 50–200 nm and good particle size uniformity. Subsequently, carboxyglucan was coated on the surface and coupled with antibodies.
It achieves efficient separation of T lymphocytes, ensuring sorting results while having minimal impact on cell function, good biocompatibility, and is suitable for large-scale industrial production.
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Figure CN121780431A_ABST
Abstract
Description
[0001] This application is a divisional application of application number 2023108300022, filed on July 7, 2023, entitled "A Nanomagnetic Bead for Cell Sorting and a Method for Preparing the Same". Technical Field
[0002] This invention belongs to the field of cell separation technology, specifically relating to a method for sorting T lymphocytes. Background Technology
[0003] Immunomagnetic bead cell sorting is based on the property that cell surface antigens can bind to specific antibodies linked to magnetic beads. In an external magnetic field, cells linked to the magnetic beads by antibodies are adsorbed and retained in the field, while cells lacking the specific antibody cannot bind to the beads and are therefore non-magnetic, thus avoiding the magnetic field and achieving cell separation. Magnetic bead cell sorting requires simple equipment, does not demand highly skilled technicians, and yields cells with high sensitivity, good purity, and good cell viability and recovery rate. It has minimal impact on downstream applications and has broad application prospects.
[0004] When choosing magnetic beads for cell sorting, it's crucial to consider the cell sorting efficiency, the impact of the beads on target cells, and potential problems in later applications. Larger particle sizes, while allowing for the loading of more labeled cell receptor sites, can lead to slower separation speeds and, in biomedical applications, difficulty in penetrating biological tissues, resulting in poor therapeutic efficacy. Smaller particle sizes may result in weaker magnetism and easier endocytosis, making it difficult to sort cells using magnetic separation. More importantly, achieving better cell capture requires attaching more magnetic particles to the cell surface, leading to the occupation of more functional epitopes, affecting subsequent detection and cell function. Furthermore, the uniformity of magnetic bead size plays a critical role in sorting efficiency. Magnetic beads with high uniformity and suitable size range exhibit greater stability in cell sorting systems, significantly reducing the probability of aggregation and effectively controlling the number of labeled groups on the bead surface, thus ensuring effective antibody conjugation and achieving more efficient cell sorting.
[0005] Methods for preparing magnetic nanobeads include physical and chemical methods. Physical methods are primarily represented by mechanical ball milling, which involves grinding micron or submicron particles for an extended period before dispersing them in an oil-based medium. This method yields particles with a relatively wide size distribution but is also time-consuming. Chemical methods include coprecipitation, high-temperature decomposition, microemulsion, sol-gel, ultrasonic chemistry, laser decomposition, and electrochemical deposition. Coprecipitation is the most widely studied method due to its simple reaction principle, relatively inexpensive equipment and raw materials, and suitability for mass production. However, to date, no method has been found that can effectively control the particle size range and uniformity of the magnetic beads. Summary of the Invention
[0006] The purpose of this invention is to provide a method for sorting T lymphocytes. PBMCs isolated from peripheral blood samples are mixed with magnetic nanobeads for sorting T lymphocytes and incubated, followed by separation of T lymphocytes using a sorting column. The method for preparing the magnetic nanobeads for sorting T lymphocytes is as follows:
[0007] (1) Prepare an aqueous solution containing ferric and ferrous iron under ice bath conditions and nitrogen protection;
[0008] (2) Under nitrogen protection, the pH of the aqueous solution containing ferric and ferrous iron is adjusted to 10-12 using a NaOH solution with a concentration of 6-10 mol / L. The NaOH solution is deoxygenated by nitrogen before feeding.
[0009] (3) React at 65-90℃, and wash with deionized water after the reaction is completed to obtain nano-magnetic beads;
[0010] (4) The surface of the nanomagnetic beads in step (3) is coated with carboxyglucan and then coupled with an antibody that can specifically bind to T lymphocytes to obtain the nanomagnetic beads used for sorting T lymphocytes.
[0011] Preferably, the incubation conditions are 4°C in the dark.
[0012] Preferably, the antibody is a CD4 / CD8 antibody.
[0013] Preferably, the sorting column is a METI Ms sorting column.
[0014] Preferably, in step (1), the molar ratio of ferric iron to ferrous iron is (1-2):1.
[0015] Preferably, in step (1), the ferric iron comes from FeCl3·6H2O and the ferrous iron comes from FeCl2·4H2O.
[0016] Preferably, the reaction temperature in step (3) is 70–85°C.
[0017] Preferably, step (3) is carried out under stirring at 300-500 rpm.
[0018] Preferably, the reaction time in step (3) is 1 to 3 hours.
[0019] Preferably, step (4) specifically involves: mixing carboxyglucan and the magnetic nanoparticles from step (3) at a mass ratio of 1:(3-10), reacting at 40-60°C, separating the magnetic nanoparticles coated with carboxyglucan after the reaction, resuspending them in MES buffer, activating them with EDC and NHS, and then reacting them with the antibody at 36-38°C. After the reaction, blocking is performed using a blocking solution, and the resulting solid is the magnetic nanoparticles used for sorting T lymphocytes, which are stored in a preservation solution.
[0020] More preferably, the blocking solution is a PBS solution containing BSA, and the preservation solution is a PBS solution containing Tween 20.
[0021] Preferably, the carboxylated dextran is obtained by reacting dextran and succinic anhydride in the presence of 4-dimethylaminopyridine and dimethyl sulfoxide under nitrogen protection at 50–70°C.
[0022] Preferably, the median particle size of the nanomagnetic beads is 50–200 nm.
[0023] Another object of the present invention is to provide a method for preparing magnetic nanobeads for cell sorting, wherein the median particle size of the magnetic nanobeads prepared by this method is controlled within the range of 50–200 nm, and the particle size uniformity is good. A further object of the present invention is to provide magnetic nanobeads prepared by the above method, which are suitable for cell sorting, and can minimize the impact on target cell function and exhibit good biocompatibility while ensuring sorting effectiveness.
[0024] Another objective of this invention is to provide magnetic nanobeads for sorting T lymphocytes, which can efficiently separate T lymphocytes and have virtually no adverse effects on subsequent T lymphocyte detection and applications.
[0025] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0026] A method for preparing magnetic nanobeads includes the following steps:
[0027] (1) Prepare an aqueous solution containing ferric and ferrous iron under ice bath conditions;
[0028] (2) The pH of the aqueous solution containing ferric and ferrous iron is adjusted to 10-12 using an alkaline substance;
[0029] (3) The reaction is carried out at 65-90℃ to obtain an aqueous solution containing the nanomagnetic beads.
[0030] Preferably, both steps (1) and (2) are carried out under nitrogen protection.
[0031] Preferably, in step (1), the molar ratio of ferric iron to ferrous iron is (1-2):1, for example 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1.
[0032] Preferably, in step (1), the ferric iron comes from FeCl3·6H2O and the ferrous iron comes from FeCl2·4H2O.
[0033] Preferably, in step (2), the alkaline substance is NaOH or ammonia; and / or, the alkaline substance is fed in the form of an aqueous solution and is deoxygenated by nitrogen before feeding.
[0034] Preferably, in step (2), the alkaline substance is fed in the form of a NaOH solution with a concentration of 6 to 10 mol / L, and more preferably, the concentration of the NaOH solution is 7 to 9 mol / L.
[0035] Preferably, the reaction temperature in step (3) is 70 to 85°C, for example, 70°C, 75°C, 80°C, or 85°C.
[0036] Preferably, step (3) is carried out under stirring at 300-500 rpm, and more preferably under stirring at 350-450 rpm.
[0037] Preferably, the reaction time in step (3) is 1 to 3 hours, and more preferably 1.5 to 2.5 hours.
[0038] Preferably, the preparation method further includes a post-processing step, wherein the post-processing step involves washing the aqueous solution containing the nanomagnetic beads with water, and the solid obtained after washing is the nanomagnetic beads, and the nanomagnetic beads are stored in deionized water.
[0039] The present invention also provides a magnetic nanoparticle for cell sorting, wherein the magnetic nanoparticle for cell sorting is prepared by the above preparation method, and the median particle size of the magnetic nanoparticle for cell sorting is 50-200 nm.
[0040] The present invention also provides a magnetic nanobead for sorting T lymphocytes, wherein the magnetic nanobead for sorting T lymphocytes comprises the magnetic nanobead for cell sorting, and the magnetic nanobead for cell sorting is coupled with an antibody capable of specifically binding to T lymphocytes.
[0041] Preferably, the surface of the magnetic nanobeads used for cell sorting is coated with carboxyglucan, and the carboxyglucan is coupled to the antibody.
[0042] The present invention also provides a method for preparing the aforementioned magnetic nanobeads for sorting T lymphocytes. Carboxyglucan and the aforementioned magnetic nanobeads for cell sorting are mixed at a mass ratio of 1:(3-10) and reacted at 40-60°C. After the reaction, the magnetic nanobeads coated with carboxyglucan are separated, resuspended in MES buffer, activated by EDC and NHS, and then reacted with the antibody at 36-38°C. After the reaction, the nanobeads are blocked with a blocking solution, and the resulting solid is the aforementioned magnetic nanobeads for sorting T lymphocytes, which are stored in a preservation solution.
[0043] Preferably, the carboxylated dextran is obtained by reacting dextran and succinic anhydride in the presence of 4-dimethylaminopyridine and dimethyl sulfoxide under nitrogen protection at 50–70°C.
[0044] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:
[0045] The preparation method of this invention can control the median particle size of the magnetic nanobeads to 50–200 nm with good particle size uniformity. While ensuring cell sorting efficiency, it has minimal impact on the function of target cells and good biocompatibility, showing great promise for application in the biomedical field. Furthermore, the preparation method of this invention is simple and easy to operate, suitable for large-scale industrial production. Attached Figure Description
[0046] Figure 1 The particle size distribution diagram of the nanomagnetic beads (bare magnetic beads) prepared in Example 1;
[0047] Figure 2 The particle size distribution of the nanomagnetic beads (bare magnetic beads) prepared in Comparative Example 1 is shown.
[0048] Figure 3 The particle size distribution of the nanomagnetic beads (bare magnetic beads) prepared in Comparative Example 2 is shown.
[0049] Figure 4 The particle size distribution of the nanomagnetic beads prepared in Example 1 after being coated with carboxydextrin (the mass ratio of carboxydextrin to nanomagnetic beads is 1:5).
[0050] Figure 5 The particle size distribution of the nanomagnetic beads prepared in Example 1 after being coated with carboxydextrin (the mass ratio of carboxydextrin to nanomagnetic beads is 1:10).
[0051] Figure 6This is a comparison of the sorting effects of the magnetic beads prepared from the magnetic beads of Example 1 and Comparative Example 1 on human lymphocyte T cells. Detailed Implementation
[0052] In a series of preliminary experimental studies, the inventors discovered that magnetic beads with a median particle size of 50–200 nm can effectively balance the sorting effect of human lymphocyte T cells and the function of sorted human lymphocyte T cells. While ensuring the sorting efficiency of human lymphocyte T cells, they have little impact on the function of human lymphocyte T cells, good biocompatibility, and have great application prospects in the field of biomedicine.
[0053] When the median particle size is greater than 200 nm, although the magnetic bead surface can load more specific binding epitopes for target cells to recognize, excessive binding often leads to overstimulation of the target cells, causing depletion and affecting their biological function. Furthermore, in biomedical applications, there is a risk that the magnetic beads may not be able to penetrate biological tissues, causing embolism and thus affecting subsequent treatment outcomes. When the median particle size is less than 50 nm, the magnetism may be weak, and the beads are easily endocytosed by cells, making it difficult to sort cells using magnetic separation. More importantly, to achieve better cell capture, more magnetic particles need to be attached to the cell surface, which leads to more cellular functional epitopes being occupied, affecting both subsequent detection and cell function.
[0054] While there are many existing processes for preparing magnetic nanobeads, the particle size range and uniformity of the prepared beads are not ideal. For example, the magnetic beads prepared in patent CN101554574B have a particle size of no more than 20 nm, exhibit poor stability in the system, are prone to aggregation, and are easily endocytosed by cells during cell sorting, making magnetic separation difficult. To achieve better cell capture, more magnetic particles need to be attached to the cell surface, resulting in the occupation of more cellular functional epitopes, which affects subsequent detection and has a potential impact on cell function.
[0055] To obtain nanomagnetic beads (bare magnetic beads) with suitable particle size (ideally with a median particle size controlled between 50 and 200 nm) and good uniformity, the inventors conducted extensive research and experimental verification, summarizing the technical solution of this invention. Specifically, this invention involves two steps: preparing a magnetite core and coating magnetic beads with carboxyglucan. In preparing the magnetite core, an aqueous solution containing ferric and ferrous iron is first prepared in an ice bath. The pH is then adjusted to 10–12, and the reaction is carried out at 65–90°C. This yields magnetite cores with superparamagnetism, a median particle size controlled between 50 and 200 nm, and uniform particle size—the bare magnetic beads. Subsequently, when carboxyglucan is added to the surface of these bare magnetic beads, the amount of carboxyl groups on the surface can be controlled, thus enabling the production of uniformly high-quality nanomagnetic beads after antibody conjugation. Experiments have shown that the magnetic nanobeads prepared using this method for sorting T lymphocytes have a good sorting effect on human T lymphocytes and have virtually no effect on human T lymphocyte function. The magnetite core has good biocompatibility and does not need to be removed during subsequent in vivo use.
[0056] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0057] The reagents and other materials used in this invention are all commercially available.
[0058] In this invention, room temperature refers to 25±5℃.
[0059] In this invention, bare magnetic beads refer to magnetic beads that have not been marked, coated, or coupled with any substance.
[0060] In this invention, the particle size distribution was tested using a Malvern Nano ZS90 nanoparticle size analyzer.
[0061] Example 1
[0062] 1. Weigh 3.2245 g (0.012 mol) of FeCl3·6H2O using an analytical balance and place it in a 50 mL three-necked flask. Add 17 mL of deionized water to the flask and stir until the ferric chloride dissolves to obtain a ferric chloride solution.
[0063] 2. In an ice-water bath (temperature maintained at 0-4℃), continuously pass N2 into the three-necked flask to remove oxygen for about 30 minutes.
[0064] 3. Weigh 1.5905g (0.008mol) of FeCl2·4H2O using an analytical balance and dissolve it in 3mL of deionized water to obtain a ferrous solution. While in an ice-water bath (temperature maintained at 0-4℃) and with N2 continuously flowing through, use a syringe to inject the above ferrous solution into the ferric chloride solution from step 1. Continue stirring for 30min to obtain a mixture of ferric and ferrous iron.
[0065] 4. Prepare an 8 mol / L NaOH solution, purge with N2 to remove oxygen, and after approximately 15 minutes, use a peristaltic pump to add the NaOH solution to the mixed solution of ferric and ferrous iron from step 3 at a flow rate of 8 mL / min until the pH of the solution reaches 11. Continue stirring for 30 minutes, then raise the temperature to 80℃ and react at 400 rpm for 2 hours. After stopping the reaction, wash with deionized water to obtain nano-magnetic beads. Store the nano-magnetic beads in deionized water; their particle size distribution is shown in [Figure showing particle size distribution]. Figure 1 The nanomagnetic beads in this embodiment have a particle size range of 40–120 nm, a median particle size (D50) of approximately 80 nm, and good uniformity.
[0066] Example 2
[0067] 1. Weigh 3.2245 g (0.012 mol) of FeCl3·6H2O using an analytical balance and place it in a 50 mL three-necked flask. Add 17 mL of deionized water to the flask and stir until the ferric chloride dissolves to obtain a ferric chloride solution.
[0068] 2. In an ice-water bath (temperature maintained at 0-4℃), continuously pass N2 into the three-necked flask to remove oxygen for about 30 minutes.
[0069] 3. Weigh 2.1112 g (0.0107 mol) of FeCl2·4H2O using an analytical balance and dissolve it in 3 mL of deionized water to obtain a ferrous solution. While in an ice-water bath (temperature maintained at 0–4 °C) and with N2 continuously flowing through, use a syringe to inject the above ferrous solution into the ferric chloride solution from step 1. Continue stirring for 30 min to obtain a mixture of ferric and ferrous iron.
[0070] 4. Prepare an 8 mol / L NaOH solution, purge with N2 to remove oxygen, and after approximately 15 minutes, use a peristaltic pump to add the NaOH solution to the ferric and ferrous iron mixture from step 3 at a flow rate of 8 mL / min until the pH of the solution reaches 11. Continue stirring for 30 minutes, then raise the temperature to 80°C and react at 400 rpm for 2 hours. After the reaction is complete, wash with deionized water to obtain the magnetic nanobeads, which are then stored in deionized water. The median particle size (D50) of the magnetic nanobeads in this embodiment is approximately 149 nm, exhibiting good uniformity.
[0071] Example 3
[0072] 1. Weigh 32.245 g (0.12 mol) of FeCl3·6H2O using an analytical balance and place it in a 500 mL three-necked flask. Add 200 mL of deionized water to the three-necked flask and stir until the ferric chloride dissolves to obtain a ferric chloride solution.
[0073] 2. In an ice-water bath (temperature maintained at 0-4℃), continuously pass N2 into the three-necked flask to remove oxygen for about 30 minutes.
[0074] 3. Weigh 15.905 g (0.08 mol) of FeCl2·4H2O using an analytical balance and dissolve it in 30 mL of deionized water to obtain a ferrous solution. While in an ice-water bath (temperature maintained at 0–4 °C) and with N2 continuously flowing through, use a syringe to inject the above ferrous solution into the ferric chloride solution from step 1. Continue stirring for 30 min to obtain a mixture of ferric and ferrous iron.
[0075] 4. Prepare an 8 mol / L NaOH solution, purge with N2 to remove oxygen, and after approximately 15 minutes, use a peristaltic pump to add the NaOH solution to the ferric and ferrous iron mixture from step 3 at a flow rate of 8 mL / min until the pH of the solution reaches 11. Continue stirring for 30 minutes, then raise the temperature to 80°C and react at 400 rpm for 2 hours. After the reaction is complete, wash with deionized water to obtain the magnetic nanoparticles, which are then stored in deionized water. The magnetic nanoparticles in this embodiment have a particle size range of 70–140 nm, a median particle size (D50) of approximately 82 nm, and good uniformity.
[0076] Comparative Example 1
[0077] 1. Weigh 3.2245 g (0.012 mol) of FeCl3·6H2O using an analytical balance and place it in a 50 mL three-necked flask. Add 17 mL of deionized water to the flask and stir until the ferric chloride dissolves to obtain a ferric chloride solution.
[0078] 2. At room temperature, continuously pass N2 into the three-necked flask to remove oxygen for about 30 minutes.
[0079] 3. Weigh 1.5905 g (0.008 mol) of FeCl2·4H2O using an analytical balance and dissolve it in 3 mL of deionized water to obtain a ferrous solution. At room temperature and while continuously passing N2 through the solution, inject the ferrous solution into the ferric chloride solution from step 1 using a syringe. Continue stirring for 30 min to obtain a mixture of ferric and ferrous iron.
[0080] 4. Prepare an 8 mol / L NaOH solution, purge with N2 to remove oxygen, and after approximately 15 minutes, use a peristaltic pump to add the NaOH solution to the mixed solution of ferric and ferrous iron from step 3 at a flow rate of 8 mL / min until the pH of the solution reaches 11. Raise the temperature to 80℃ and react at 400 rpm for 2 hours. After the reaction is complete, wash with deionized water to obtain nanomagnetic beads. Store the nanomagnetic beads in deionized water. Their particle size distribution is shown in [Figure showing...]. Figure 2 .like Figure 2As shown, the uniformity of the nanomagnetic beads in this comparative example is poor, with a median particle size (D50) of 244.9 nm and a high proportion of large-sized magnetic beads exceeding 130 nm.
[0081] Comparative Example 2
[0082] 1. Weigh 3.2245 g (0.012 mol) of FeCl3·6H2O using an analytical balance and place it in a 50 mL three-necked flask. Add 17 mL of deionized water to the flask and stir until the ferric chloride dissolves to obtain a ferric chloride solution.
[0083] 2. In an ice-water bath (temperature maintained at 0-4℃), continuously pass N2 into the three-necked flask to remove oxygen for about 30 minutes.
[0084] 3. Weigh 1.5905g (0.008mol) of FeCl2·4H2O using an analytical balance and dissolve it in 3mL of deionized water to obtain a ferrous solution. While in an ice-water bath (temperature maintained at 0-4℃) and with N2 continuously flowing through, use a syringe to inject the above ferrous solution into the ferric chloride solution from step 1. Continue stirring for 30min to obtain a mixture of ferric and ferrous iron.
[0085] 4. Prepare an 8 mol / L NaOH solution, purge with N2 to remove oxygen, and after approximately 15 minutes, use a peristaltic pump to add the NaOH solution to the mixed solution of ferric and ferrous iron from step 3 at a flow rate of 8 mL / min until the pH of the solution reaches 11. Raise the temperature to 60℃ and react at 400 rpm for 2 hours. After the reaction is complete, wash with deionized water to obtain nanomagnetic beads. The particle size distribution is shown in the figure. Figure 3 .like Figure 3 As shown, the magnetic beads in this comparative example have poor uniformity, with a high proportion of large-sized magnetic beads (some magnetic beads have a particle size exceeding 1μm). The magnetic beads in this comparative example are not suitable for cell sorting.
[0086] Magnetic nanobeads for sorting human lymphocytes were prepared using magnetic beads from Example 1 and Comparative Example 1, respectively. The preparation methods are as follows:
[0087] (1) Preparation of carboxyglucan: 4g of dextran and 8g of succinic anhydride were dissolved in 10mL of anhydrous dimethyl sulfoxide (DMSO). After purging with N2 for 15min to remove oxygen, 1mL of DMSO solution containing 10mg of 4-dimethylaminopyridine (DMAP) was added as a catalyst. The liquid was heated to 60℃ and stirred for 24h. After the reaction was completed, the liquid was dropped into ice-cold ethanol to precipitate, and the solid was obtained by filtration. The solid was dissolved in deionized water and added to a dialysis bag for dialysis for 48h (with water changed every six hours during the process). The solid was then freeze-dried to obtain carboxyglucan.
[0088] (2) Coating magnetic beads with carboxyglucan: Carboxyglucan aqueous solution and magnetic beads were added to a flask at a mass ratio of 1:5 or 1:10, stirred and heated at 50°C for 4 hours, and magnetic beads coated with carboxyglucan were obtained by magnetic separation and screening.
[0089] (3) Conjugation of CD4 / CD8 antibody (Baiying Biotechnology): The carboxyglucan-coated magnetic beads were washed with 25mM pH 5.0 MES buffer using magnetic separation, and the washed magnetic beads were resuspended to 1mL with 25mM pH 5.0 MES buffer. 10μL (10mg / mL) EDC and 20μL (10mg / mL) NHS were added, and the carboxyl groups were activated at 37℃ for 0.5h. The supernatant was removed by magnetic separation, and the beads were resuspended to 1mL with 25mM pH 5.0 MES buffer. 60μg of CD4 / CD8 antibody was added, and the mixture was incubated at 37℃ using a rotary incubator for 6h. After magnetic separation, blocking buffer (PBS solution containing BSA) was added, and the beads were washed with preservation buffer (PBS solution containing Tween 20) and stored in the preservation buffer. When the mass ratio of carboxyglucan to the magnetic nanobeads of Example 1 was 1:5, the particle size distribution of the magnetic beads coated with carboxyglucan was as follows. Figure 4 As shown, the magnetic beads are uniformly distributed, with a median particle size of approximately 84 nm and a potential of approximately -42 mV. When the dextran-coated magnetic beads were placed at room temperature for a period of time, the particle size showed no significant change (Table 1), indicating that the dextran-coated magnetic beads have good stability.
[0090] Table 1
[0091] Time (day) 1 7 30 60 Size (nm) 89.2 85.4 91.5 87.2
[0092] When the mass ratio of carboxyglucan to the magnetic nanobeads of Example 1 is 1:10, the particle size distribution of the magnetic beads coated with carboxyglucan is as follows: Figure 5 As shown, the particles are uniformly distributed with a median particle size of approximately 100 nm and a potential of approximately -44 mV. The magnetic beads prepared by mass ratios of carboxyglucan to the magnetic beads from Example 1 of 1:5 and 1:10 exhibited particle sizes and potentials within the target range. Different mass ratios were selected depending on the conjugated antibody.
[0093] The magnetic beads used in Example 1 and Comparative Example 1 were used to prepare magnetic nanobeads for sorting human lymphocytes (the mass ratio of carboxyglucan to the magnetic nanobeads of Example 1 was 1:5), and human lymphocyte sorting experiments were performed respectively:
[0094] Take 5 mL of apheresis blood, dilute it 5-fold, add 15 mL of human peripheral blood lymphocyte separation medium (Ficoll), centrifuge at 800 g for 30 min, aspirate the white membrane layer, wash three times with PBS, and obtain PBMCs. Perform cell counting, and take 2 x 10... 7Each cell was divided into two aliquots, and 10 μL of sorting magnetic beads prepared from the magnetic beads of Example 1 and Comparative Example 1 were added to each aliquot. After incubation at 4°C in the dark for 30 min, the cells were separated using a Miltenyi Ms sorting column to obtain two aliquots, A and B. Flow cytometry analysis was performed on these aliquots, and the results are as follows: Figure 6 As shown. Figure 6 Figure A shows the sorting effect of the magnetic beads prepared from Example 1 on human lymphocyte T cells, specifically the CD4 target cells. + CD8 - CD4 - CD8 + CD4 + CD8 + The percentage of cells in Figure B is 98%; Figure B shows the sorting effect of the magnetic beads prepared from the magnetic beads in Comparative Example 1 on human lymphocyte T cells, and the target cells are CD4. + CD8 - CD4 - CD8 + CD4 + CD8 + The percentage of cells was 91%. The sorting magnetic beads prepared from the magnetic beads of Example 1 showed significantly better sorting effect on human lymphocyte T cells than those prepared from the magnetic beads of Comparative Example 1. The magnetic beads of Comparative Example 1 had a significantly larger particle size than those of Example 1, and their excessive surface loading of specific binding epitopes would affect the biological function of the target cells. Furthermore, the excessively large magnetic beads were not easily able to penetrate biological tissues, posing a risk of embolism.
[0095] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for sorting T lymphocytes, characterized in that: PBMCs isolated from peripheral blood samples were mixed and incubated with magnetic nanobeads for sorting T lymphocytes, and then T lymphocytes were separated using a sorting column. The method for preparing the magnetic nanobeads for sorting T lymphocytes is as follows: (1) Prepare an aqueous solution containing ferric and ferrous iron under ice bath conditions and nitrogen protection; (2) Under nitrogen protection, the pH of the aqueous solution containing ferric and ferrous iron is adjusted to 10-12 using a NaOH solution with a concentration of 6-10 mol / L. The NaOH solution is deoxygenated by nitrogen before feeding. (3) React at 65-90℃, and wash with deionized water after the reaction is completed to obtain nano-magnetic beads; (4) The surface of the nanomagnetic beads in step (3) is coated with carboxyglucan and then coupled with an antibody that can specifically bind to T lymphocytes to obtain the nanomagnetic beads used for sorting T lymphocytes.
2. The method for sorting T lymphocytes according to claim 1, characterized in that: The incubation conditions were 4°C and protected from light.
3. The method for sorting T lymphocytes according to claim 1, characterized in that: The antibody is a CD4 / CD8 antibody.
4. The method for sorting T lymphocytes according to claim 1, characterized in that: The sorting column is a METI Ms sorting column.
5. The method for sorting T lymphocytes according to claim 1, characterized in that: In step (1), the molar ratio of ferric iron to ferrous iron is (1-2):1; And / or, in step (1), ferric iron comes from FeCl3·6H2O and ferrous iron comes from FeCl2·4H2O.
6. The method for sorting T lymphocytes according to claim 1, characterized in that: The reaction temperature in step (3) is 70–85°C; And / or, step (3) is carried out with stirring at 300–500 rpm; And / or, the reaction time for step (3) is 1 to 3 hours.
7. The method for sorting lymphocytes according to claim 1, characterized in that: The specific steps of step (4) are as follows: carboxyglucan and the magnetic nanoparticles from step (3) are mixed at a mass ratio of 1:(3-10) and reacted at 40-60°C. After the reaction is completed, the magnetic nanoparticles coated with carboxyglucan are separated, resuspended in MES buffer, activated by EDC and NHS, and then reacted with the antibody at 36-38°C. After the reaction is completed, the nanoparticles are blocked with blocking solution, and the resulting solid is the magnetic nanoparticles used for sorting T lymphocytes, which are stored in a preservation solution.
8. The method for sorting lymphocytes according to claim 6, characterized in that: The blocking solution is a PBS solution containing BSA, and the preservation solution is a PBS solution containing Tween 20.
9. The method for sorting lymphocytes according to claim 1, characterized in that: The carboxylated dextran is obtained by reacting dextran and succinic anhydride in the presence of 4-dimethylaminopyridine and dimethyl sulfoxide under nitrogen protection at 50–70 °C.
10. The method for sorting lymphocytes according to claim 1, characterized in that: The median particle size of the nanomagnetic beads is 50–200 nm.
Citation Information
Patent Citations
Superparamagnetism nano particle jointly modified by aminosilane coupling agent and hyskon and preparation method thereof
CN101554574B