A cell membrane chitosan ferroferric oxide microsphere and its preparation method and application

Abstract

The invention discloses a cell membrane chitosan ferroferric oxide microsphere, a preparation method and an application thereof, and the preparation methods include: (1) preparation of a cell membrane suspension; (2) preparation of a ferroferric oxide suspension; (3) preparation of chitosan ferroferric oxide microspheres; and (4) preparation of cell membrane chitosan ferroferric oxide microspheres. The invention uses magnetic microspheres as carriers and uses external magnetic force for separation, thereby avoiding the influence of factors within the system on the analysis work. It does not require steps such as column loading and column balancing required for traditional cell membrane chromatography, simplifies the active component analysis process, and shortens the analysis cycle. Compared with traditional cell membrane chromatography columns, the amount of cell membrane used is reduced. It does not require column loading and column balancing, thereby reducing the amount of buffer used. Chitosan has biocompatibility, stabilizes magnetic microspheres, avoids aggregation in aqueous media, and has low toxicity, good biodegradability and adhesion. The magnetic microspheres coated with chitosan can be bonded to a variety of ligand proteins for affinity analysis.

Description

Technical Field

[0001] The invention belongs to the field of affinity chromatography screening of active chemical components. It specifically relates to a stable cell membrane chitosan ferric oxide microsphere and its preparation method and application. This technology can achieve the purpose of targeted rapid screening of biologically active substances contained in natural products. Background Art

[0002] Natural products are the main source of active compound discovery. For example, artemisinin was isolated and discovered from the traditional Chinese medicine Artemisia annua. Currently, this compound and its derivative synthetics have been widely used in clinical practice as effective antimalarial drugs. However, screening candidate drugs from natural products often follows the method of chemical separation first, analytical identification, and then combining efficacy experiments to determine the active ingredients. However, such a chemical component separation, structural identification and activity evaluation system is limited to the separation and purification capabilities of existing chromatographic technology, and can only separate stable monomer organic chemical components; water-soluble components, trace components and unstable components are more difficult to obtain. Therefore, how to quickly identify active compounds in natural products remains a problem that needs to be solved.

[0003] Since the main target of more than 50% of the drugs on the market is membrane proteins, Professor He Langchong and others have developed a series of cell membrane-based chromatography methods. These methods are used to study the interaction of membrane proteins and screen complex mixtures of active compounds. At present, a variety of cell membrane chromatography models have been used to screen effective ingredients of traditional Chinese medicine, and have achieved good results. However, although cell membrane chromatography is feasible and has broad application prospects, this type of method requires the preparation of stationary phases and filling of chromatographic columns, and the chromatographic system needs to be balanced during use. These operating processes not only require complex procedures, but also require high-pressure pumps to fill the chromatographic columns. This process limits the further widespread application of cell membrane chromatography.

[0004] Therefore, Professor Zhu Quan and others improved the cell membrane chromatography and directly used the red blood cell membrane to fish for the potential active ingredients in angelica. Combining high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance technology, they identified potentially active compounds such as ligustilide in the red blood cell membrane dissociation solution. This proves that the use of cell membranes to "fish" for active ingredients in natural products can be applied to predict the biological activity of ingredients in traditional Chinese medicine. However, this technology still requires repeated ultrafiltration and centrifugation steps to separate the cell membranes bonded to the active ingredients from the extract, and the active ingredients need to be re-analyzed. The whole process is also time-consuming.

[0005] Ligand fishing was originally used to extract target biomolecules from complex cell matrix proteins and enzymes and immobilize them on micro- or nanospheres to screen active compounds. The use of magnetic microspheres for fishing mainly relies on the magnetic force outside the system for separation, and can avoid the influence of factors within the system (for example, pH, ionic strength, or surface charge) on the analysis. In 2007, Moaddel et al. first used magnetic microspheres in ligand fishing experiments. In the work, the researchers immobilized human serum albumin on the surface of magnetic microspheres, and the fishing mixture contained known ligands and non-ligands. Since then, more and more proteins have been immobilized on the surface of magnetic microspheres to detect active compounds. However, there are no reports of cell membranes being immobilized on chitosan Fe3O4 magnetic microspheres. Summary of the invention

[0006] The purpose of the invention is to overcome the deficiencies of the prior art and provide a cell membrane chitosan ferroferric oxide microsphere.

[0007] The second object of the present invention is to provide a method for preparing cell membrane chitosan ferroferric oxide microspheres.

[0008] The third object of the present invention is to provide a use of cell membrane chitosan ferroferric oxide microspheres for analyzing active substances in natural products.

[0009] The technical solution of the present invention is summarized as follows:

[0010] A method for preparing cell membrane chitosan ferroferric oxide microspheres comprises the following steps:

[0011] (1) Add phenylmethylsulfonyl fluoride at a final concentration of 0.1-1 mM to the cell suspension and mix well; use a cell ultrasonic disruptor at 4-20°C for 5-10 min, centrifuge at 200-1000×g for 5-10 min at 4-20°C, and obtain the supernatant; then centrifuge at 10000-20000×g for 15-60 min, discard the supernatant, obtain the precipitate, wash it with water or 5-50 mM phosphate buffer with a pH of 7.2-7.4, add water or the buffer to the precipitate, and adjust the concentration to obtain a cell membrane suspension with a concentration of 1-5 mg / mL;

[0012] (2) Weigh 216 mg of FeCl3·6H2O and 80 mg of FeCl2·4H2O, add 2 mL of pure water to dissolve; pass N2 for 10 min, add 1 mL of 8M ammonia water, heat to 80 °C and react for 1 h. Fe3O4 solid is generated, wash with water until neutral; add water to obtain a suspension;

[0013] (3) adjusting the concentration of the suspension obtained in step (2) to 5 mg / mL, taking 1 mL and adding 5 mL of 0.5 mg / mL sodium tripolyphosphate aqueous solution, mixing well, and then mixing with 0.8 mL of 0.5 mg / mL chitosan solution, ultrasonicating for 20 min, discarding the supernatant, rinsing the precipitate with water, and obtaining chitosan ferrous oxide microspheres; the solvent of the chitosan solution is 1%-10% by volume acetic acid aqueous solution;

[0014] (4) Under oscillation conditions, the cell membrane suspension with a concentration of 1-5 mg / mL obtained in step (1) is adsorbed onto the surface of chitosan ferric oxide microspheres by a reduced pressure method, and the mixture is oscillated at a constant temperature of 4-10° C. for 2-8 hours to obtain cell membrane chitosan ferric oxide microspheres.

[0015] Preferably, the molecular weight of chitosan is in the range of 200 kda-500 kda, and the degree of deacetylation is 85%.

[0016] The cell membrane chitosan ferroferric oxide microspheres are prepared by the above-mentioned preparation method.

[0017] The above-mentioned cell membrane chitosan ferroferric oxide microspheres are used in analyzing active substances in natural products.

[0018] Preferably: the above application includes the following steps:

[0019] At 37°C, cell membrane chitosan ferric oxide microspheres were added to a natural product alcohol extract solution, the solvent of which was a 50 mM phosphate buffer solution at pH = 7.4, and incubated for 30 minutes; the cell membrane chitosan ferric oxide microspheres bound to the active ingredients of the natural product were sucked out by a magnetic field force, and the unbound components were washed away with a 5 mM phosphate buffer solution at pH = 7.4, and then a 20% volume concentration of acetic acid aqueous solution was added as a dissociation solution to dissociate the active ingredients of the natural product in the natural product alcohol extract from the cell membrane chitosan ferric oxide microspheres, and analyzed by chromatography-mass spectrometry.

[0020] Advantages of the present invention:

[0021] (1) Magnetic microspheres are used as carriers, and separation is mainly based on the magnetic force outside the system, thus avoiding the influence of factors within the system (such as pH value, ionic strength, or surface charge) on the analysis.

[0022] There is no need for necessary steps of traditional cell membrane chromatography such as column packing and column equilibration, which simplifies the active ingredient analysis process and shortens the analysis cycle.

[0023] Compared with traditional cell membrane chromatography columns, the amount of cell membrane used is significantly reduced.

[0024] Since there is no need to pack or balance the chromatographic column, this method reduces the amount of buffer used.

[0025] The present invention uses chitosan, a biocompatible polymer, to stabilize magnetic microspheres, avoid aggregation in aqueous media, and has low toxicity and good biodegradability and adhesion. The magnetic microspheres coated with chitosan can be bonded to a variety of ligand proteins for affinity analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a fluorescence confocal microscope image of cell membrane chitosan ferric oxide microspheres:

[0027] DiI-labeled erythrocyte membrane A1 under visible light;

[0028] DiI-labeled chitosan Fe3O4 microspheres B1;

[0029] DiI-labeled cell membrane chitosan Fe3O4 microspheres C1;

[0030] DiI-labeled erythrocyte membrane A2 under fluorescence;

[0031] DiI-labeled chitosan Fe3O4 microspheres B2;

[0032] DiI-labeled cell membrane chitosan Fe3O4 microspheres C2.

[0033] Figure 2 This is the chromatogram of the alcohol extract solution of Cnidium monnieri.

[0034] Figure 3 This is the chromatogram of the alcohol extract solution of Cnidium monnieri treated with cell membrane chitosan ferric oxide microspheres. DETAILED DESCRIPTION

[0035] The cell suspension is obtained by adding rat blood cells, rabbit blood cells, or cultured cells to physiological saline, DMEM culture medium, or phosphate buffer with a pH of 7.2-7.4 and a concentration of 50 mM.

[0036] The following examples are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the following examples. Inventions and creations completed by ordinary technicians in the relevant technical field according to the above disclosure of the present invention and the ranges of various parameters all belong to the protection scope of the present invention. Example

[0037] A method for preparing cell membrane chitosan ferroferric oxide microspheres comprises the following steps:

[0038] (1) Preparation of cell membrane suspension

[0039] Red blood cells were obtained from rabbit blood and prepared into cell suspension in 50 mM phosphate buffer at pH 7.4;

[0040] Add phenylmethylsulfonyl fluoride with a final concentration of 0.1 mM to the cell suspension and mix well; use a cell ultrasonic disruptor to disrupt for 5 minutes at 4°C, centrifuge at 1000×g for 5 minutes at 4°C, and take the supernatant; centrifuge at 20000×g for 30 minutes, discard the supernatant, take the precipitate, wash it three times with 5 mM phosphate buffer at pH=7.4, add the buffer to the precipitate, and adjust the concentration to obtain a cell membrane suspension;

[0041] The cell membrane protein content in the cell membrane suspension was determined to be 2 mg / mL using the total protein quantitative test kit from Nanjing Jiancheng Bioengineering Institute;

[0042] (2) Weigh 216 mg of FeCl3·6H2O and 80 mg of FeCl2·4H2O, add 2 mL of pure water to dissolve; pass N2 for 10 min, add 1 mL of 8M ammonia water, heat to 80 °C and react for 1 h. Fe3O4 solid is generated, wash with water until neutral; add water to obtain a suspension;

[0043] (3) The concentration of the suspension obtained in step (2) was adjusted to 5 mg / mL, and 1 mL was added to 5 mL of 0.5 mg / mL sodium tripolyphosphate aqueous solution, mixed well, and then mixed with 0.8 mL of 0.5 mg / mL chitosan solution, ultrasonicated for 20 min, the supernatant was discarded, and the precipitate was rinsed with water for 3 times to obtain chitosan ferrous oxide microspheres; the solvent of the chitosan solution was 5% by volume acetic acid aqueous solution;

[0044] The molecular weight of chitosan is 300 kda and the degree of deacetylation is 85%.

[0045] (4) Under oscillation conditions, the cell membrane suspension with a concentration of 2 mg / mL obtained in step (1) is adsorbed onto the surface of chitosan ferric oxide microspheres by a reduced pressure method, and the mixture is oscillated at a constant temperature of 4° C. for 8 h to obtain cell membrane chitosan ferric oxide microspheres.

[0046] Using DiI to label cell membrane chitosan ferric oxide microspheres:

[0047] Take 10 μL of 1 mg / mL DiI solution (solvent dimethyl sulfoxide) and add 1 mL of red blood cell membrane suspension, 1 mL of chitosan ferric oxide microspheres water suspension, and 1 mL of cell membrane chitosan ferric oxide microspheres water suspension, respectively, and store at 4°C in the dark for 8 h. Rinse with 5 mM PBS to remove excess DiI. Observe with fluorescence confocal microscopy, the results show that the cell membrane has been fixed on the surface of chitosan ferric oxide microspheres (see Figure 1 ). Example

[0048] 1. Preparation of Cell Membrane Suspension

[0049] Red blood cells were obtained from rabbit blood and prepared into cell suspension in 50 mM phosphate buffer at pH 7.4;

[0050] Add phenylmethylsulfonyl fluoride with a final concentration of 0.5 mM to the cell suspension and mix well; use a cell ultrasonic disruptor at 6°C for 8 min, centrifuge at 500×g for 8 min at 4°C, and take the supernatant; centrifuge at 15000×g for 15 min, discard the supernatant, take the precipitate, wash it 3 times with 5 mM phosphate buffer at pH=7.4, add the buffer to the precipitate, and adjust the concentration to obtain a cell membrane suspension; use the total protein quantitative test kit of Nanjing Jiancheng Bioengineering Institute to determine the cell membrane protein content in the cell membrane suspension to be 1 mg / mL;

[0051] (2) Weigh 216 mg of FeCl3·6H2O and 80 mg of FeCl2·4H2O, add 2 mL of pure water to dissolve; pass N2 for 10 min, add 1 mL of 8M ammonia water, heat to 80 °C and react for 1 h. Fe3O4 solid is generated, wash with water until neutral; add water to obtain a suspension;

[0052] (3) The concentration of the suspension obtained in step (2) was adjusted to 5 mg / mL, and 1 mL was added to 5 mL of 0.5 mg / mL sodium tripolyphosphate aqueous solution, mixed and then mixed with 0.8 mL of 0.5 mg / mL chitosan solution, ultrasonicated for 20 min, the supernatant was discarded, and the precipitate was rinsed with water twice to obtain chitosan ferrous oxide microspheres; the solvent of the chitosan solution was 1% by volume acetic acid aqueous solution;

[0053] The molecular weight of chitosan is 200 kDa and the degree of deacetylation is 85%.

[0054] (4) Under oscillation conditions, the cell membrane suspension with a concentration of 1 mg / mL obtained in step (1) is adsorbed onto the surface of chitosan ferric oxide microspheres by a reduced pressure method, and the mixture is oscillated at a constant temperature of 8° C. for 5 h to obtain cell membrane chitosan ferric oxide microspheres. Example

[0055] 1. Preparation of Cell Membrane Suspension

[0056] Red blood cells were obtained from rabbit blood and prepared into cell suspension in 50 mM phosphate buffer at pH 7.4;

[0057] The corresponding cell suspension can be obtained by replacing the red blood cells obtained from the rabbit blood of this embodiment with rat red blood cells or cultured cells, and the other conditions are the same as this embodiment; the corresponding cell suspension can be obtained by replacing the 50mM phosphate buffer with pH=7.4 of this embodiment with physiological saline, DMEM culture medium or 50mM phosphate buffer with pH=7.2, and the other conditions are the same as this embodiment.

[0058] Add phenylmethylsulfonyl fluoride with a final concentration of 1 mM to the cell suspension and mix well; use a cell ultrasonic disruptor at 20°C for 10 min, centrifuge at 200×g for 10 min at 20°C, and take the supernatant; centrifuge at 10000×g for 60 min, discard the supernatant, take the precipitate, wash it three times with 50 mM phosphate buffer at pH=7.2, add the buffer to the precipitate to obtain a cell membrane suspension, and adjust the concentration; use the total protein quantitative test kit of Nanjing Jiancheng Bioengineering Institute to determine the cell membrane protein content in the cell membrane suspension to be 5 mg / mL;

[0059] (2) Weigh 216 mg of FeCl3·6H2O and 80 mg of FeCl2·4H2O, add 2 mL of pure water to dissolve; pass N2 for 10 min, add 1 mL of 8M ammonia water, heat to 80 °C and react for 1 h. Fe3O4 solid is generated, wash with water until neutral; add water to obtain a suspension;

[0060] (3) The concentration of the suspension obtained in step (2) was adjusted to 5 mg / mL, and 1 mL was added to 5 mL of 0.5 mg / mL sodium tripolyphosphate aqueous solution, mixed and then mixed with 0.8 mL of 0.5 mg / mL chitosan solution, ultrasonicated for 20 min, the supernatant was discarded, and the precipitate was rinsed with water for 4 times to obtain chitosan ferrous oxide microspheres; the solvent of the chitosan solution was 10% by volume acetic acid aqueous solution;

[0061] The molecular weight of chitosan is 500 kDa and the degree of deacetylation is 85%.

[0062] (4) Under oscillation conditions, the cell membrane suspension with a concentration of 5 mg / mL obtained in step (1) is adsorbed onto the surface of chitosan ferric oxide microspheres by a reduced pressure method, and the mixture is oscillated at a constant temperature of 10° C. for 2 h to obtain cell membrane chitosan ferric oxide microspheres. Example

[0063] The application of cell membrane chitosan ferric oxide microspheres in the analysis of active substances in natural products (taking Cnidium monnieri as an example) includes the following steps:

[0064] Preparation of natural product (Cnidium monnieri) alcohol extract solution:

[0065] Take 5 g of natural product powder and use 50 mL of 70% ethanol aqueous solution for hot reflux extraction for 1 h, filter, and freeze-dry the filter to obtain the natural product alcohol extract, reconstitute it with 50 mM phosphate buffer at pH = 7.4, filter it with a 0.22 μm filter membrane, and obtain the natural product alcohol extract solution, which is stored at 4°C for later use.

[0066] At 37°C, the cell membrane chitosan ferric oxide microspheres prepared in Example 1 were added to the natural product alcohol extract solution and incubated for 30 min. The cell membrane chitosan ferric oxide microspheres bound to the active ingredients of the natural product were sucked out by magnetic field force, and the unbound components were washed away with 5 mM phosphate buffer at pH = 7.4. Then, a 20% volume concentration of acetic acid aqueous solution was added as a dissociation solution to dissociate the natural product active ingredients in the natural product alcohol extract from the cell membrane chitosan ferric oxide microspheres, and the results were analyzed by chromatography-mass spectrometry (see Figure 2 , Figure 3 ).

[0067] from Figure 2 , Figure 3 It can be seen that the chromatogram of the Cnidium monnieri alcohol extract solution treated with cell membrane chitosan ferric oxide microspheres ( Figure 3 ) is significantly higher than the chromatographic peaks of the ethanol extract of Cnidium monnieri ( Figure 2 ) decreased. One of the chromatographic peaks is imperatorin, which is the active ingredient of Cnidium monnieri.

[0068] The cell membrane chitosan ferric oxide microspheres prepared in Examples 2 and 3 were used to replace the cell membrane chitosan ferric oxide microspheres in this example. The other aspects were the same as in this example. The results obtained by chromatography-mass spectrometry analysis were similar to those in Example 1.

Claims

1. A method for preparing cell membrane chitosan ferroferric oxide microspheres, characterized in that The steps include: (1) Add phenylmethylsulfonyl fluoride at a final concentration of 0.1-1 mM to the cell suspension and mix well; use a cell ultrasonic disruptor at 4-20°C for 5-10 min, centrifuge at 200-1000×g for 5-10 min at 4-20°C, and obtain the supernatant; then centrifuge at 10000-20000×g for 15-60 min, discard the supernatant, obtain the precipitate, wash it with water or 5-50 mM phosphate buffer with a pH of 7.2-7.4, add water or the buffer to the precipitate, and adjust the concentration to obtain a cell membrane suspension with a concentration of 1-5 mg / mL; (2) Weigh 216 mg of FeCl3·6H2O and 80 mg of FeCl2·4H2O, add 2 mL of pure water to dissolve; pass N2 for 10 min, add 1 mL of 8M ammonia water, heat to 80 °C and react for 1 h. Fe3O4 solid is generated, wash with water until neutral; add water to obtain a suspension; (3) adjusting the concentration of the suspension obtained in step (2) to 5 mg / mL, taking 1 mL and adding 5 mL of 0.5 mg / mL sodium tripolyphosphate aqueous solution, mixing well, and then mixing with 0.8 mL of 0.5 mg / mL chitosan solution, ultrasonicating for 20 min, discarding the supernatant, rinsing the precipitate with water, and obtaining chitosan ferrous oxide microspheres; the solvent of the chitosan solution is 1%-10% by volume acetic acid aqueous solution; (4) Under oscillation conditions, the cell membrane suspension with a concentration of 1-5 mg / mL obtained in step (1) is adsorbed onto the surface of chitosan ferric oxide microspheres by a reduced pressure method, and the mixture is oscillated at a constant temperature of 4-10° C. for 2-8 hours to obtain cell membrane chitosan ferric oxide microspheres; The molecular weight of the chitosan is in the range of 200 kda to 500 kda, and the degree of deacetylation is 85%.

2. Cell membrane chitosan ferroferric oxide microspheres prepared by the preparation method described in claim 1.

3. Use of the cell membrane chitosan ferroferric oxide microspheres according to claim 2 in analyzing active substances in natural products.

4. The use according to claim 3, characterized in that The steps include: At 37°C, the cell membrane chitosan ferric oxide microspheres are added to a natural product alcohol extract solution, the solvent of the natural product alcohol extract solution is a 50mM phosphate buffer solution with a pH of 7.4, and incubated for 30 minutes; the cell membrane chitosan ferric oxide microspheres combined with the active ingredients of the natural product are sucked out by a magnetic field force, and the unbound components are washed away with a 5mM phosphate buffer solution with a pH of 7.4, and then a 20% volume concentration of acetic acid aqueous solution is added as a dissociation solution to dissociate the active ingredients of the natural product in the natural product alcohol extract from the cell membrane chitosan ferric oxide microspheres, and the chromatography-mass spectrometry analysis is performed.

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