A one-step method for preparing nanocluster magnetic beads and its application

Abstract

This invention discloses a method for preparing and applying nanocluster magnetic beads, comprising the following steps: adding a salt solution containing ferrous and ferric salts to an aqueous solution, dissolving it uniformly, then adding a dispersant to obtain a mixed solution, stirring evenly, and heating; adding ammonia water to the heated mixed solution, stirring the reaction to obtain nanocluster magnetic beads. This invention directly synthesizes nanocluster magnetic beads using an innovative one-step method, eliminating the need for multi-step synthesis and subsequent separation steps, and the synthesized nanocluster magnetic beads can be used for cell separation and MRI imaging.

Description

Technical Field

[0001] This invention belongs to the field of inorganic nanomaterial synthesis technology, specifically relating to a one-step method for preparing nanocluster magnetic beads and its application. Background Technology

[0002] Magnetic nanobeads have wide applications in in vitro diagnostics, analytical chemistry, biochemistry, biomedicine, and clinical diagnostics. Their main physicochemical properties include nanoscale size, superparamagnetism, MRI imaging capabilities, and magnetothermal therapy. Designing suitable magnetic bead products using different synthesis methods for different application scenarios can significantly enhance their effectiveness. Currently, the main synthesis methods for magnetic nanobeads include chemical coprecipitation, high-temperature pyrolysis, biomineralization, and ball milling. Among these, chemical coprecipitation is widely used due to its simple operation, mild reaction conditions, and green reagents.

[0003] Magnetic beads synthesized using the coprecipitation method typically have very small particle sizes, around 10 nm. Due to their extremely small size, they can exhibit toxicity to biological cells in applications such as cell biology. Larger magnetic beads, such as those larger than 500 nm, have extremely poor dispersibility, are prone to precipitation, and are not easily stable in systems, leading to instability in some biological applications. Their size also limits their application in cells and animals. For example, in cell separation, very small magnetic beads are easily endocytosed and enter the cell, and the cytotoxicity of extremely small beads can damage cell viability, thus adversely affecting the cell. Larger magnetic beads bind to the cell surface, requiring further elution steps after cell separation, which is not only cumbersome and complex but also potentially harmful to the cells. Therefore, magnetic beads of suitable size are extremely important for their application.

[0004] Patent CN107416910A discloses a simple method for controlling the morphology of Fe3O4 nanoclusters. This method involves first preparing a precursor solution, and then preparing Fe3O4 nanoparticles from the precursor solution. However, it suffers from drawbacks such as high reaction temperature, long reaction time, and high energy and time consumption.

[0005] The invention patent with publication number CN115607693A discloses polyvinylpyrrolidone modified ferric oxide magnetic clusters, their preparation method and application. It adopts a synthesis step of first synthesizing nanomagnetic beads, further assembling them into nanoclusters, and finally separating large-sized particles to form PVP dispersed nanoclusters.

[0006] A search revealed no patent reports on the innovative one-step synthesis method for synthesizing nanocluster magnetic beads and its applications. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the purpose of this invention is to provide a one-step method for preparing nanocluster magnetic beads and its application.

[0008] This invention employs an innovative one-step method to synthesize a novel type of nanocluster magnetic beads and demonstrates their applicability for cell separation and MRI imaging. The synthesized nanocluster magnetic beads are formed by the self-assembly of smaller Fe3O4 nanoparticles into larger clusters, exhibiting superparamagnetism. Furthermore, in the presence of a stabilizer, the surface of the nanocluster magnetic beads possesses a large number of functional groups, which can be further used for the modification of functional molecules for applications in other fields such as biology, analytical chemistry, and MRI imaging.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing nanocluster magnetic beads, comprising the following steps:

[0011] Add ferrous salt and ferric salt to water and dissolve them evenly. Then add a dispersant to obtain a mixed solution. Stir evenly and heat to 50-95℃. Add ammonia water to the heated mixed solution and stir for 10-120 minutes to obtain nano-cluster magnetic beads.

[0012] This invention employs a co-precipitation method, a one-step synthesis that is safer, with a lower reaction temperature and shorter reaction time. Furthermore, this reaction utilizes Fe... 3+ and Fe 2+ Two ions are used, eliminating the need for ethylene glycol, thus avoiding the toxicity of ethylene glycol to the human body and the pollution of the raw materials to the environment during synthesis.

[0013] Preferably, the ratio of the ferrous salt, ferric salt, water and dispersant is (2-4)g:(5.4-10.8)g:(100-200)ml:(5-12)g.

[0014] Preferably, the volume ratio of water to ammonia is (100-200):(8-10).

[0015] Preferably, the ferrous salt includes one or more of ferrous chloride, ferrous nitrate, ferrous acetate, and ferrous sulfate, and the ferric salt includes one or more of ferric chloride, ferric nitrate, ferric acetate, and ferric sulfate.

[0016] Preferably, the dispersant comprises one or more of the following: dextran, carboxylated dextran, aminolated dextran, polyethylene glycol, carboxylated polyethylene glycol, aminolated polyethylene glycol, polyacrylic acid, 2,3-dimercaptosuccinic acid, glutathione, cystine, and citric acid.

[0017] The dispersant used in this invention has a large number of functional groups on its surface, which can modify the surface of the nanocluster magnetic beads with biofunctional molecules, such as cell-targeting antibodies and cell-targeting biomolecules, for cell separation and MRI targeted imaging. The functional groups include, but are not limited to, carboxylated, aminated, thiolated, hydroxylated, epoxy-based, anhydride-based, and azide-based dispersants. Furthermore, the high molecular weight of the dispersant results in more stable nanoclusters with a longer shelf life.

[0018] In a second aspect, the present invention provides nanocluster magnetic beads prepared by the above-described preparation method.

[0019] A third aspect of the present invention provides the application of the above-mentioned nanocluster magnetic beads in cell separation.

[0020] Preferably, the nanocluster magnetic beads are modified with targeted antibodies; cell separation includes, but is not limited to, magnetic separation of stem cells, magnetic separation of T cells, magnetic separation of NK cells, magnetic separation of macrophages, magnetic separation of nerve cells and / or separation of exosomes.

[0021] Preferably, the targeting antibody includes, but is not limited to, CD3 antibody, CD56 antibody and / or CD44 antibody.

[0022] A fourth aspect of the present invention provides the application of the above-described nanocluster magnetic beads in cellular and animal MRI imaging.

[0023] The beneficial effects of this invention are:

[0024] (1) This invention directly synthesizes nanocluster magnetic beads using an innovative one-step method, without the need for multi-step synthesis and subsequent separation steps. The yield is high, and the synthesized nanocluster magnetic beads can be used for cell separation and MRI imaging.

[0025] (2) Compared with other magnetic bead products, the magnetic beads synthesized in this invention are formed by assembling nano-sized Fe3O4 particles into nano clusters. Their size is moderate, in the range of 20-100nm. They are non-toxic to cells and biological tissues, have good biocompatibility, and can be used to develop various biomagnetic products.

[0026] (3) This method uses a one-step method to synthesize nano-clusters of magnetic beads. Compared with other methods for synthesizing magnetic beads, this method only requires one step to complete the synthesis. It does not require a multi-step synthesis process. The synthesis method is simple, the synthesis process is green and pollution-free, and the synthesized product has reliable quality.

[0027] (4) Under the action of the dispersant, the nanocluster magnetic beads, after binding with the targeting antibody molecules, can specifically target and bind to various cells, thereby achieving magnetic targeting and magnetic separation of multiple cells. Furthermore, the magnetic beads synthesized in this invention exhibit excellent T2 mode MRI imaging performance and can be used for MRI imaging of cells, tissues, and animals. Attached Figure Description

[0028] Figure 1 Transmission electron microscope (TEM) image of synthesized nanocluster magnetic beads.

[0029] Figure 2 High-resolution transmission electron microscope (HRTEM) image of synthesized nanocluster magnetic beads.

[0030] Figure 3 Figure a shows the X-ray diffraction (XRD) analysis pattern of the synthesized nanocluster magnetic beads; Figure b shows the X-ray photoelectron diffraction (XPS) energy spectrum of the synthesized nanocluster magnetic beads; Figure c shows the resonant sample magnetometer pattern of the nanocluster magnetic beads; and Figure d shows the laser dynamic light scattering (DLS) particle size analysis pattern of the nanocluster magnetic beads.

[0031] Figure 4 Scanning electron microscope (SEM) image of CD3 antibody-modified nanoclusters of magnetic beads specifically binding to T cells.

[0032] Figure 5 : Magnetic separation diagram of CD3 antibody-modified nanocluster magnetic beads after specific binding to T cells under the action of a magnetic field.

[0033] Figure 6 Scanning electron microscope (SEM) image of CD56 antibody-modified nanocluster magnetic beads specifically binding to NK cells.

[0034] Figure 7 : Magnetic targeting separation of CD56 antibody-modified nanocluster magnetic beads after specific binding to NK cells under the action of a magnetic field.

[0035] Figure 8 Bright-field microscopy image of CD44 antibody-modified nanocluster magnetic beads specifically binding to mesenchymal stem cells.

[0036] Figure 9 T2-mode MRI imaging of RGD-modified nanocluster magnetic beads after binding with tumor cells.

[0037] Figure 10 T2-mode MRI imaging of tumor tissue by RGD-modified nanocluster magnetic beads, where Fe3O4 represents nanocluster magnetic beads and Fe3O4-RGD represents RGD-modified nanocluster magnetic beads. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] The one-step synthesis of nanocluster magnetic beads described in this invention employs a one-step co-precipitation method, utilizing the dispersing effect of a stabilizer to synthesize nanoclusters with good dispersibility, superparamagnetism, and suitable size. Its key feature is the use of a one-step co-precipitation method, eliminating the need for the complex steps of first synthesizing magnetic nanoparticles followed by assembly and purification; only a single synthesis step is required to prepare uniformly sized nanocluster magnetic beads. The synthesized nanocluster magnetic beads have a suitable size, ranging from 20-100 nm in diameter, and are assembled from multiple small Fe3O4 nanoparticles. The synthesized nanocluster magnetic beads exhibit good superparamagnetism and can be used for cell magnetic separation after grafting cell-targeting molecules. Simultaneously, the synthesized nanocluster magnetic beads possess excellent T2-mode MRI imaging properties and can be used for MRI imaging of cells and animals.

[0040] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0041] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0042] In the following examples, the polyacrylic acid, carboxylated dextran, and 2,3-dimercaptosuccinic acid used were all purchased from McLean Company.

[0043] Example 1:

[0044] 1. One-step coprecipitation method for synthesizing nanocluster magnetic beads:

[0045] Add 2g of FeCl2·4H2O and 5.4g of FeCl3·6H2O to 100mL of water and stir thoroughly to dissolve. Add 10g of carboxylated dextran with a molecular weight of 5000 and stir until fully dissolved to obtain a mixed solution. Heat the mixed solution to 80℃, quickly add 10mL of ammonia water, and continue stirring for 30min to generate carboxylated dextran-modified nanocluster magnetic beads.

[0046] 2. Material and chemical characterization of nanocluster magnetic beads:

[0047] The morphology of the synthesized nanocluster magnetic beads was characterized by transmission electron microscopy (TEM), and their crystal properties were characterized by high-resolution transmission electron microscopy (HETEM). The crystal structure type of the nanocluster magnetic beads was analyzed by X-ray diffraction (XRD). The magnetic intensity was measured using a resonant sample magnetometer. The elemental valence states of the materials were analyzed by X-ray photoelectron spectroscopy (XPS). The hydrated particle size of the nanoclusters was analyzed and detected by laser particle size analyzer (DLS).

[0048] The material properties of the synthesized nanocluster magnetic beads were characterized under the above conditions. For example... Figure 1 As shown, TEM testing was performed on the synthesized nanocluster magnetic beads. The results showed that the synthesized clusters had uniform morphology, and the nanoclusters were assembled from several small nanoparticles with a cluster size of approximately 20-50 nm. Figure 2 As shown, HRTEM results indicate that the synthesized nanoclusters are assembled from small particles, and each nanoparticle exhibits good crystallinity. The synthesized nanoclusters were further characterized using various tests. Figure 3 As shown, the synthesized nanocluster magnetic beads were tested by XRD, and the results are as follows. Figure 3 As shown in Figure a, the nanocluster magnetic beads have a pure Fe3O4 phase crystal structure, meaning the synthesized products are Fe3O4 nanocluster magnetic beads. The nanocluster magnetic beads were tested using XPS, and the results are as follows... Figure 3 As shown in b, Fe appeared in the nanocluster magnetic beads. 2+ and Fe 3+ The peak values ​​are consistent with the XRD test results. The hysteresis loop test results of the synthesized nanocluster magnetic beads are as follows: Figure 3 As shown in c, the synthesized nanocluster magnetic beads have a magnetic strength of 50 emu / g, which is high and can be used for the research and development of magnetic separation products. The hydrated particle size of the synthesized nanocluster magnetic beads is as follows: Figure 3 As shown in d, the results show that the hydrated particle size of the clusters is in the range of 20-100 nm, with the main size being 50 nm.

[0049] Example 2: Synthesis of nanocluster magnetic beads by one-step coprecipitation method.

[0050] Add 4g of FeCl2·4H2O and 10.8g of FeCl3·6H2O to 200mL of water and stir thoroughly to dissolve. Add 20g of carboxylated dextran with a molecular weight of 5000 and stir until fully dissolved. Heat the mixture to 80℃, quickly add 10mL of ammonia water, and continue stirring for 30min to generate carboxylated dextran-modified nanocluster magnetic beads.

[0051] Example 3: Synthesis of nanocluster magnetic beads by one-step coprecipitation method.

[0052] Add 3.6 g of Fe(NO3)2 and 9.6 g of Fe(NO3)3 to 200 mL of water and stir thoroughly to dissolve. Add 12 g of polyacrylic acid with a molecular weight of 2000 and stir until fully dissolved to obtain a mixed solution. Heat the mixed solution to 90 °C, quickly add 8 mL of ammonia water, and continue stirring for 40 min to generate polyacrylic acid-modified nanocluster magnetic beads.

[0053] Example 4: Synthesis of nanocluster magnetic beads by one-step coprecipitation method.

[0054] 3.48 g of ferrous acetate and 7.76 g of ferric acetate were added to 150 mL of water and stirred thoroughly to dissolve. 5 g of 2,3-dimercaptosuccinic acid was added and stirred until fully dissolved to obtain a mixed solution. The mixed solution was heated to 85 °C, and 10 mL of ammonia water was quickly added. After stirring and reacting for 60 min, 2,3-dimercaptosuccinic acid-modified nanocluster magnetic beads were generated.

[0055] Application Example 1:

[0056] (1) The dispersant-stabilized magnetic nanoclusters synthesized in Example 1 were coupled with a T-cell-specific antibody, CD3, to form CD3 antibody-modified magnetic nanoclusters. These biomagnetic beads can be used for magnetic targeting and magnetic separation of T cells. The synthesis steps are as follows:

[0057] Take 2 mg of the nanocluster magnetic beads synthesized in Example 1, add 0.1 mM EDC and 0.1 mM NHS solution, activate for 30 min, add 0.1 mg of CD3 antibody, incubate overnight at 4 °C, and collect the reaction product by centrifugation to obtain CD3 antibody modified nanocluster magnetic beads.

[0058] (2) CD3 antibody-modified nanocluster magnetic beads for magnetic labeling and magnetic separation of T cells:

[0059] Take 20 μL of 2 mg / mL CD3 nanocluster magnetic beads and add it to 1 mL of 1×10 5 A T cell suspension of 1 / mL can achieve highly efficient magnetic labeling of T cells.

[0060] T cells labeled with magnetic nanoclusters were characterized and magnetically separated under the above conditions. The magnetically labeled T cells were characterized using SEM. Figure 4 As shown, after CD3 antibody-labeled magnetic beads bind to the CD3 antigen on the surface of T cells, the nanoclusters of magnetic beads are uniformly distributed on the T cell surface, thereby achieving highly efficient magnetic labeling of T cells. Magnetically labeled T cells can respond rapidly to external magnetic fields, such as... Figure 5 As shown, under the attraction of a magnet, T cells labeled with nanocluster magnetic beads can be quickly attracted by the magnet, achieving magnetic separation and enrichment of T cells.

[0061] Application Example 2:

[0062] (1) The dispersant-stabilized magnetic nanoclusters synthesized in Example 2 were coupled with the NK cell-specific antibody CD56 to form CD56 antibody-modified magnetic nanoclusters. These biomagnetic beads can be used for magnetic targeting and magnetic separation of NK cells. The synthesis steps are as follows:

[0063] Take 5 mg of the nanocluster magnetic beads synthesized in Example 2, add 0.1 mM EDC and 0.1 mM NHS solution, activate for 30 min, add 0.2 mg of CD56 antibody, incubate overnight at 4 °C, and collect the reaction product by centrifugation to obtain CD56 antibody modified nanocluster magnetic beads.

[0064] (2) CD56 antibody-modified nanocluster magnetic beads for magnetic labeling and magnetic separation of NK cells:

[0065] Take 20 μL of 2 mg / mL CD56 nanocluster magnetic beads and add it to 1 mL of 1×10 5 A NK cell suspension of 1 / mL can achieve highly efficient magnetic labeling of NK cells.

[0066] Under the above conditions, NK cells labeled with nanocluster magnetic beads were characterized and magnetically separated. The magnetically labeled NK cells were characterized using SEM. Figure 6 As shown, after CD56 antibody-labeled magnetic beads bind to the CD56 antigen on the surface of NK cells, the nanoclusters of magnetic beads are uniformly distributed on the surface of NK cells, thereby achieving highly efficient magnetic labeling of NK cells. Magnetically labeled NK cells can respond rapidly to external magnetic fields, such as... Figure 7 As shown, under the attraction of a magnet, NK cells labeled with nanocluster magnetic beads can be quickly attracted by the magnet, achieving magnetic separation and enrichment of NK cells.

[0067] Application Example 3:

[0068] (1) The dispersant-stabilized magnetic nanoclusters synthesized in Example 1 were coupled with a mesenchymal stem cell-specific antibody, CD44, to form CD44 antibody-modified magnetic nanoclusters. These biomagnetic beads can be used for magnetic targeting and magnetic separation of mesenchymal stem cells. The synthesis steps are as follows:

[0069] Take 2 mg of the nanocluster magnetic beads synthesized in Example 1, add 0.1 mM EDC and 0.1 mM NHS solution, activate for 30 min, add 0.1 mg of CD44 antibody, incubate overnight at 4 °C, and collect the reaction product by centrifugation to obtain CD44 antibody modified nanocluster magnetic beads.

[0070] (2) D44 antibody-modified nanocluster magnetic beads were used for magnetic labeling and magnetic separation of bone marrow mesenchymal stem cells:

[0071] Take 20 μL of 2 mg / mL CD44 nanocluster magnetic beads and add it to 1 mL of 1×10 5 A suspension of bone marrow mesenchymal stem cells at a concentration of 1000 mL can achieve highly efficient magnetic labeling of bone marrow mesenchymal stem cells.

[0072] Bone marrow mesenchymal stem cells labeled with nanocluster magnetic beads were characterized and magnetically separated under the above conditions. The magnetically labeled bone marrow mesenchymal stem cells were characterized using an inverted phase-contrast microscope. Figure 8 As shown, after CD44 antibody-labeled magnetic beads bind to the CD44 antigen on the surface of bone marrow mesenchymal stem cells, the nanoclusters of magnetic beads are evenly distributed on the surface of bone marrow mesenchymal stem cells, thereby achieving efficient magnetic labeling of bone marrow mesenchymal stem cells.

[0073] Application Example 4:

[0074] (1) The dispersant-stabilized magnetic nanoclusters synthesized in Example 1 were coupled with tumor cell-targeting RGD peptides to form RGD peptide-modified magnetic nanoclusters. These biomagnetic beads can be used for tumor cell-targeted MRI imaging. The synthesis steps are as follows:

[0075] Take 4 mg of the nanocluster magnetic beads synthesized in Example 1, add 0.2 mM EDC and 0.2 mM NHS solution, activate for 30 min, add 1 mg of RGD peptide, incubate overnight at 4 °C, and collect the reaction product by centrifugation to obtain RGD peptide-modified nanocluster magnetic beads.

[0076] (2) RGD peptide-modified nanocluster magnetic beads for magnetic labeling of tumor cells and MRI imaging:

[0077] Take 20 μL of the 2 mg / mL nanocluster magnetic beads prepared in Example 1 and the RGD peptide-modified nanocluster magnetic beads synthesized in the above steps, and add them to 1 mL of 1×10 5 Highly efficient magnetic labeling of tumor cells can be achieved by incubating a tumor cell suspension at a concentration of 1000 mL / mL for 0, 0.5, 1, 2, and 4 hours, respectively. The magnetically labeled tumor cells are then imaged using a clinical 3T MRI scanner.

[0078] (3) RGD peptide-modified nanocluster magnetic beads for in vivo MRI imaging of animal tumors:

[0079] RGD peptide-modified nanocluster magnetic beads were injected into mice at a concentration of 5 mg / mL in 100 μL. MRI imaging of tumor sites in mice was achieved by clinical 3T MRI scanning.

[0080] Under the above conditions, tumor cells labeled with nanocluster magnetic beads were magnetically labeled and subjected to MRI imaging, and MRI imaging of tumors could be achieved in vivo in animals. The MRI imaging results of tumor cells are as follows: Figure 9 As shown, the nanocluster magnetic beads can perform T2 mode MRI imaging of 4T1 tumor cells, and the signal intensity in the T2 mode increases with increasing incubation time. After binding with the RGD tumor-targeting peptide, the MRI signal intensity shows a significant enhancement, demonstrating the tumor-targeting effect of the RGD peptide. The results after injecting the nanoclusters into 4T1 tumor-bearing mice are as follows... Figure 10 As shown, both pure nanocluster magnetic beads and RGD-modified nanocluster magnetic beads exhibited good T2-mode MRI imaging effects on tumor sites. With increasing post-injection time, the best MRI enhancement was observed at 4 hours post-injection, after which the signal gradually weakened. Labeling with RGD molecules significantly enhanced the MRI signal. Therefore, RGD-modified nanocluster magnetic beads can achieve enhanced MRI imaging.

[0081] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A nanocluster magnetic bead, characterized in that, The preparation method of the nanocluster magnetic beads comprises the following steps: adding ferrous salt and iron salt into water, uniformly dissolving, then adding dispersant to obtain a mixed solution, uniformly stirring, and heating to 50-95℃; adding ammonia water into the heated mixed solution, and stirring for 10-120min to obtain the nanocluster magnetic beads; the ferrous salt comprises one or more of ferrous chloride, ferrous nitrate, ferrous acetate and ferrous sulfate, and the iron salt comprises one or more of ferric chloride, ferric nitrate, ferric acetate and ferric sulfate; the dispersant comprises one or more of carboxylated dextran, polyacrylic acid and 2,3-dimercaptosuccinic acid; the use amount ratio of the ferrous salt, iron salt, water and dispersant is (2-4)g:(5.4-10.8)g:(100-200)ml:(5-12)g; the volume ratio of the water and ammonia water is (100-200):(8-10).

2. The application of the nanocluster magnetic beads in claim 1 in the preparation of a material for enhancing T2 signal in cell and animal MRI imaging.

3. Use according to claim 2, characterized in that, The nanocluster magnetic beads are modified by RGD polypeptide.

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