Cell membrane gold magnetic screening medium as well as application and method of cell membrane gold magnetic screening medium in screening of anti-breast tumor active components

Abstract

The invention provides a cell membrane gold magnetic screening medium as well as application and a method thereof in screening of anti-breast tumor active ingredients, the cell membrane gold magnetic screening medium of a bioactive ligand comprises gold magnetic particles and an SKBR3 cell membrane coating the surfaces of the gold magnetic particles, the gold magnetic particles comprise a Fe3O4 (at) SiO2 carrier, polyethyleneimine modified on the surface of the Fe3O4 (at) SiO2 carrier, gold nanoparticles and PEG-SH (Polyethylene Glycol-Sulfonate). Through electrostatic force and metal coordination force, efficient and stable coating of the SKBR3 cell membrane on the surfaces of the gold magnetic particles is achieved, and the sensitivity and accuracy of screening of traditional Chinese medicine active ingredients are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of functional materials and active drug component screening, and in particular relates to a cell membrane gold magnetic screening medium and its application and method in screening anti-breast tumor active components. Background Art

[0002] Most of the active ingredients in natural products can strongly interact with target proteins, thereby producing biological effects on organisms. According to statistics, more than 50% of the small molecule targeted drugs approved by the FDA interact with the cell membrane protein receptors of their target cells (Yin H., Flynn A. Annu Rev Biomed Eng 2016, 18, 51-76). Due to the superparamagnetism of magnetic materials, the material and liquid can be quickly separated by applying an external magnetic field. The bionic platform combining magnetic materials with cell membranes can conveniently, quickly and efficiently screen out active ligands in natural products.

[0003] However, the preparation technology of cell membrane-coated magnetic materials is not easy. At present, the binding of cell membrane and magnetic materials is mainly achieved by covalent bonds or ultrasonic adsorption. Covalent binding requires cumbersome chemical modification of cell membrane and magnetic materials, including steps such as activation of linking groups, which is not only time-consuming, but may also affect the biological activity of cell membrane (Fu J., Jia Q., Liang P., et al., Anal Chem 2021, 93, 11719-11728; Hu Q., Jia L., Zhang X., et al., Acta Pharm Sin B 2022, 12, 394-405; Bu Y., Wu D., Zhao Y., et al., ACS Appl Mater Interfaces 2023, 15, 52150-52161). In addition, covalent binding may also destroy the integrity and biological activity of cell membranes, reducing their screening effect. Although ultrasonic adsorption is easy to operate, the strong ultrasonic effect can easily damage the cell membrane, affecting its stability and screening accuracy. At the same time, the cell membrane adsorbed on the surface of the magnetic material is easy to fall off, resulting in reduced screening efficiency. Therefore, how to overcome the problems of long cell membrane coating time, cumbersome steps and poor stability has become the main challenge facing the current preparation technology of cell membrane-coated magnetic materials.

[0004] Breast cancer has become the most common cancer in the world, seriously endangering women's physical and mental health (CA Cancer JClin 2021, 71, 209-249). Given that human epidermal growth factor 2 (HER2)-positive breast cancer is more likely to recur and metastasize, screening drugs targeting HER2 breast cancer has important clinical significance. Ancient Chinese medical books record that heat-clearing and detoxifying drugs can be used to treat tumors. One of the representative drugs is Scutellaria baicalensis, which has been found to inhibit the proliferation and metastasis of various tumor cells. Therefore, further exploring the anti-breast cancer active ingredients in Scutellaria baicalensis is of great significance to the study of its anti-tumor mechanism and the development of new products based on Scutellaria baicalensis Chinese medicine. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a cell membrane gold magnetic screening medium and its application and method in the screening of anti-breast tumor active ingredients. Through electrostatic force and metal coordination force, efficient and stable coating of SKBR3 cell membrane on the surface of gold magnetic particles is achieved, thereby improving the sensitivity of screening of active ingredients of traditional Chinese medicine.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a cell membrane gold magnetic screening medium for biologically active ligands, comprising gold magnetic particles and SKBR3 cell membranes coated on the surface of the gold magnetic particles, wherein the gold magnetic particles comprise Fe3O4@SiO2 carriers, polyethyleneimine modified on the surface of the Fe3O4@SiO2 carriers, gold nanoparticles and PEG-SH.

[0007] Furthermore, 1 mg of gold magnetic particles was added to every 400 μg to 700 μg of cell membrane.

[0008] Furthermore, the Fe3O4@SiO2 carrier includes Fe3O4 bare magnet and SiO2 coating wrapped on the surface of Fe3O4 bare magnet, the particle size of Fe3O4 bare magnet is 150nm~500nm, and the thickness of SiO2 coating is 10nm~30nm.

[0009] The present invention also provides a method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand, the specific steps of which are as follows: The Fe3O4@SiO2 carrier dispersion liquid is mixed with the polyethyleneimine solution, and after ultrasonication and washing, a first reaction liquid is obtained; The gold nanoparticle solution and the first reaction solution are mixed, ultrasonicated and washed, and then the PEG-SH solution is added, and the reaction is followed by drying to obtain gold magnetic particles; The gold magnetic particles are added to the PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, mixed and combined, and then washed to obtain the gold magnetic particles coated with SKBR3 cell membranes, i.e., the cell membrane gold magnetic screening medium; The composition of PBS is 2.7mM KCl, 2.0mM KH2PO4, 137mM NaCl, 10mM Na2HPO4, and the pH value is 7.3~7.5@25ºC.

[0010] Further, in the step of mixing the Fe3O4@SiO2 dispersion with the polyethyleneimine solution, ultrasonicating and washing to obtain the first reaction solution: The concentration of the Fe3O4@SiO2 dispersion is 0.2mg / mL~0.8mg / mL; the final concentration of the Fe3O4@SiO2 dispersion after mixing with the polyethyleneimine solution is 0.8mg / mL~2mg / mL.

[0011] Further, the gold nanoparticle solution and the first reaction solution are mixed, ultrasonicated and washed, and then a PEG-SH solution is added, and the reaction is followed by drying to obtain the gold magnetic particles: The preparation of the gold nanoparticle solution is specifically as follows: a 2mmol / L~7mmol / L chloroauric acid aqueous solution is mixed with 2 times the volume of a 2mmol / L~7mmol / L sodium citrate solution, sodium borohydride solution is added dropwise thereto, and the mixture is reacted to obtain a gold nanoparticle solution with a particle size of less than 10nm.

[0012] Further, the gold nanoparticle solution and the first reaction solution are mixed, ultrasonicated and washed, and then a PEG-SH solution is added, and the reaction is followed by drying to obtain the gold magnetic particles: Mixing equal volumes of the first reaction solution and the gold nanoparticle solution; The concentration of PEG-SH solution is 1mmol / L~2.5mmol / L.

[0013] Further, the gold magnetic particles are added to the PBS suspension of the HER2-positive breast tumor SKBR3 cell membrane, mixed and combined, and then washed to obtain the gold magnetic particles coated with the SKBR3 cell membrane: The gold magnetic particles were added to the PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, vortexed to mix, and allowed to bind at room temperature for 1 h to 2 h. Unbound cell membranes were washed away with PBS to obtain gold magnetic particles coated with SKBR3 cell membranes.

[0014] The present invention also provides a method for extracting anti-HER2 breast tumor active components, which is carried out using the cell membrane gold magnetic screening medium of the above-mentioned biologically active ligand, and the specific steps are as follows: Adding the cell membrane gold magnetic screening medium obtained by magnetic separation and removing the supernatant to the substrate to be screened for incubation to obtain an incubation mixture; The incubation mixture is subjected to magnetic separation and the supernatant is discarded. After washing, an eluent is added for incubation, and the supernatant is collected to obtain the anti-HER2 breast tumor active component in the substrate to be screened.

[0015] Furthermore, the substrate to be screened is Scutellaria baicalensis extract.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention discloses a cell membrane gold magnetic screening medium of a bioactive ligand, comprising gold magnetic particles and SKBR3 cell membrane coated on the surface of the gold magnetic particles, wherein the gold magnetic particles include Fe3O4@SiO2 carriers, polyethyleneimine modified on the surface of the Fe3O4@SiO2 carriers, gold nanoparticles and PEG-SH, which realizes efficient and stable coating of SKBR3 cell membrane on the surface of the gold magnetic particles, avoiding the problem of easy detachment of the cell membrane in the traditional method; the cell membrane gold magnetic screening medium is prepared by self-assembly combining the positively charged polymer on the surface of the magnetic material and the gold nanoparticles with the cell membrane through electrostatic force and metal coordination force, thereby improving the binding stability of the cell membrane and ensuring the sensitivity and accuracy in the screening process. The prepared SKBR3 / CMAuM magnetic screening medium has excellent magnetism, can be quickly separated under an external magnetic field, and is convenient for subsequent operations. The screening medium can not only be used for the screening of anti-HER2 breast tumor active ingredients, but can also be expanded to the screening field of other tumor active ingredients, providing new ideas and methods for new drug research and development.

[0017] The present invention discloses a method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand, in which the cell membrane is incubated in the PBS suspension provided by the present invention, and the cell membrane is combined with the magnetic particles through electrostatic force and coordination force, the steps are simple, no ultrasound or activation is required, and the cell membrane binding stability is high. The method of the present invention realizes efficient and stable coating of the cell membrane on the surface of gold magnetic particles through electrostatic force and metal coordination force, and improves the sensitivity and stability of screening active ingredients of traditional Chinese medicine. The SKBR3 / CMAuM magnetic screening medium prepared by the present invention has excellent magnetism and cell membrane binding stability, can be quickly separated under an external magnetic field, and the cell membrane is not easy to fall off.

[0018] The invention discloses a method for extracting active ingredients against HER2 breast tumors, which can conveniently, quickly and efficiently screen out ingredients with anti-HER2 breast cancer activity in scutellaria baicalensis using a bionic screening platform combining magnetic materials with cell membranes. The prepared cell membrane gold magnetic particles are used to fish for potential active ingredients against HER2 breast tumors in scutellaria baicalensis, achieving the purpose of discovering anti-HER2 breast cancer drug lead in scutellaria baicalensis, and will also provide useful reference and reference for anti-tumor research of other traditional Chinese medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The AuM particle size and transmission electron microscope characterization diagram obtained in the embodiment of the present invention; Figure 2AuM and CMAuM hysteresis regression lines obtained in the embodiments of the present invention; Figure 3 The CMAuM scanning electron microscope and fluorescence confocal images obtained in the embodiments of the present invention; Figure 4 This is a test diagram of the performance of AuM-loaded cell membrane obtained in an embodiment of the present invention; Figure 5 This is a diagram showing the feasibility results of SKBR3 / CMAuM screening for the positive drug lapatinib. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] The present invention provides a method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand, based on the combination of positively charged polymers and gold nanoparticles on the surface of magnetic materials with cell membranes through electrostatic force and metal coordination force, and specifically comprises the following steps: S1 Preparation of gold magnetic particles 1) Preparation of AuM microparticle carriers: At 200ºC, ethylene glycol is used as a reducing agent in a hydrothermal reactor. The size and magnetism of the bare magnet are controlled by changing the feed ratio of ferric chloride, sodium citrate and sodium acetate and the reaction time. The bare magnet particle size is controlled within the range of 150nm~500nm to give it a high specific surface area to increase the subsequent loading rate on the cell membrane. SiO2 is coated on the surface of the bare magnet, and the concentration of ammonia water and tetraethyl orthosilicate is changed to adjust the thickness of the SiO2 coating within 10nm~30nm to ensure that the water solubility, stability and biocompatibility of the magnetic particles are enhanced with less magnetic influence. The Fe3O4@SiO2 carrier is dispersed in pure water, added into a polyethyleneimine (PEI, Mw: 1800-25000) aqueous solution, ultrasonicated for 1-2 hours, and washed with pure water for multiple times to obtain a first reaction solution; The concentration range of Fe3O4@SiO2 carrier dispersed in pure water is 0.2~0.8mg / mL; the final concentration after PEI is added is 0.8~2mg / mL.

[0022] 2) Preparation of gold magnetic particles; The gold nanoparticle (Au NPs) solution and the first reaction solution were mixed in equal volumes, ultrasonicated for 1h~2h, washed with pure water for several times, and then a PEG-SH (polyethylene glycol thiol) aqueous solution with a concentration of 1~2.5 mmol / L was added, stirred at room temperature for 4~6h, and dried to obtain gold magnetic microparticles (AuM).

[0023] The gold nanoparticle solution is obtained by dropwise adding a freshly prepared sodium borohydride solution to a 2-7 mmol / L aqueous solution of chloroauric acid and a 2-fold amount of a 2-7 mmol / L sodium citrate solution, and stirring at room temperature for 2-5 minutes. The gold nanoparticle solution is pink and has a particle size of less than 10 nm.

[0024] S2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium) The cells were disrupted by cell disruptor and centrifuged to collect the HER2-positive breast tumor SKBR3 cell membranes, which were then resuspended in PBS; Add gold magnetic particles to the cell membrane suspension, vortex mix, place on a rotary mixer, bind for 1h~2h at room temperature, and wash away the unbound cell membrane with PBS to obtain SKBR3 cell membrane-coated magnetic particles (SKBR3 / CMAuM magnetic screening medium).

[0025] Preferably, the PBS used has a composition of 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4, and a pH value of 7.3-7.5 @ 25°C.

[0026] Preferably, the ratio of gold magnetic particles to cell membrane is 400 μg to 700 μg of cell membrane per 1 mg AuM.

[0027] The present invention uses the cell membrane gold magnetic screening medium for screening anti-HER2 breast tumor active ingredients, specifically comprising the following steps: The prepared SKBR3 / CMAuM magnetic screening medium is magnetically separated, and after removing the supernatant, the scutellaria baicalensis extract to be screened is added thereto, vortexed, and then placed on a rotary mixer for incubation; After the incubation, discard the supernatant by magnetic separation, wash 2-3 times with PBS solution, add the eluent prepared with a volume ratio of DMSO:H2O=1:1 to the magnetic particles, vortex, incubate on a rotary mixer for 10-20 minutes, and collect the supernatant; The supernatant was analyzed by liquid chromatography-mass spectrometry to obtain the anti-HER2 breast tumor active components in the Scutellaria baicalensis extract.

[0028] The present invention combines positively charged polymers and gold nanoparticles on the surface of magnetic materials with cell membranes through electrostatic force and metal coordination force to prepare cell membrane gold magnetic screening media. Compared with the existing cell membrane fixation technology, it has the advantages of being more efficient, convenient and rapid, and can overcome the problems of long cell membrane fixation time and complicated steps. Compared with the existing cell membrane fixation technology, the cell membrane gold magnetic screening medium prepared by the present invention has a higher cell membrane coverage rate and cell membrane fixation stability, thereby improving the efficiency and accuracy of drug active ingredient screening; The preparation method of the cell membrane gold magnetic screening medium provided by the present invention is simple to operate and low in cost, and can be widely used in the screening field of other tumor active components. Using the cell membrane gold magnetic screening medium to screen the scutellaria extract can efficiently discover new anti-HER2 breast tumor active components with targeting and selectivity, providing new drug options for the clinical treatment of HER2-positive breast cancer.

[0029] Example 1 A method for preparing a cell membrane gold magnetic screening medium for a biologically active ligand comprises the following steps: Step 1: Preparation of AuM microparticle carrier Step 101, bare magnet synthesis: Weigh 1.00g of ferric chloride hexahydrate into a beaker, then add 25ml of ethylene glycol, 0.20g of disodium citrate, and 1.30g of anhydrous sodium acetate in sequence, and heat and stir until completely dissolved. Transfer the mixed solution to a hydrothermal reactor and react in a 200℃ oven for 10 hours. Turn off the oven power and take out the reactor after it cools to room temperature. Wash alternately with anhydrous ethanol and pure water 3 times, disperse in a small amount of anhydrous ethanol, dry in a 37℃ oven, and collect in a centrifuge tube.

[0030] Step 102, silicon dioxide coating modification: Weigh 0.1g of ferroferric oxide magnetic particles in a three-necked flask, add 80ml of ethanol and 20ml of pure water, stir to mix, and then add 2.5ml of 25% ammonia solution. Add 0.2ml of tetraethyl orthosilicate dropwise under stirring, start timing from the addition of tetraethyl orthosilicate, and stir ultrasonically for 4 hours. Wash with anhydrous ethanol and pure water alternately for 3 times, disperse in a small amount of anhydrous ethanol, and dry in an oven at 37°C to obtain a Fe3O4@SiO2 carrier.

[0031] Step 103: Preparation of AuM particles: Take a Fe3O4@SiO2 carrier solution with a concentration of 0.8 mg / mL, add 1 ml of 2 mg / ml polyethyleneimine aqueous solution dropwise under stirring, stir ultrasonically for 1 hour, wash with pure water 5 times, and disperse in 20 ml of pure water.

[0032] Take another 10 ml of 2 mmol / L chloroauric acid aqueous solution and 2 times the volume of 2 mmol / L sodium citrate solution in a flask, and add 50 mmol / L freshly prepared sodium borohydride solution dropwise under magnetic stirring to obtain a pink small-size gold nanoparticle solution. The gold nanoparticle solution and the polyethyleneimine-modified Fe3O4@SiO2 solution were mixed in equal volumes, ultrasonically stirred for 1 hour, washed with ultrapure water 5 times, and then dispersed in 10 ml of pure water. Then 10 ml of 1.25 mmol / L PEG was added. 5000 -SH solution was stirred at room temperature for 4 hours, washed with pure water 5 times, dispersed in a small amount of pure water, and dried at 37°C to obtain AuM microparticles.

[0033] Step 2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium) Step 201, SKBR3 cells were cultured in a complete medium consisting of 10% fetal bovine serum, McCoy's 5A medium, and 1% double antibody in an environment of 5% carbon dioxide and 37°C. Cells in the exponential growth phase were collected by trypsin digestion, washed three times by centrifugation at 4°C, 800g, and 5 minutes using phosphate buffer, and resuspended in phosphate buffer. Cells were disrupted using a cell ultrasonic disruptor under ice bath conditions, with the parameters set to ultrasonic 4 seconds, stop 6 seconds, cycle 6 times, and power 40%. After centrifugation at 4°C, 4000g for 10 minutes, the precipitate was discarded; the supernatant was centrifuged at 4°C, 18000g for 30 minutes, the supernatant was discarded, the precipitate was washed twice with phosphate buffer and resuspended in phosphate buffer.

[0034] Step 202, take 200 μL of 10 mg / ml AuM solution and add it to 2 ml of 400 μg / ml cell membrane solution, vortex mix, place it on a rotary mixer and incubate it for 1.5 hours, wash it twice with phosphate buffer, and disperse it in 200 μL of phosphate buffer to obtain SKBR3 cell membrane-coated magnetic particles (SKBR3 / CMAuM magnetic screening medium).

[0035] Embodiment 2: A method for preparing a cell membrane gold magnetic screening medium for a biologically active ligand comprises the following steps: Step 1: Preparation of AuM microparticle carrier Step 101, bare magnet synthesis: Weigh 1.5g of ferric chloride hexahydrate, add 30ml of ethylene glycol, 0.3g of disodium citrate, and 1.8g of anhydrous sodium acetate, heat to 80℃ and stir to dissolve. Transfer the mixed solution to a hydrothermal reactor and react in an oven at 180℃ for 8 hours. Turn off the oven power and take out the reactor after it cools naturally to room temperature. Wash alternately with anhydrous ethanol and pure water 4 times, disperse in an appropriate amount of anhydrous ethanol, dry in an oven at 40℃, and collect in a centrifuge tube.

[0036] Step 102, silicon dioxide coating modification: Weigh 0.15g of ferroferric oxide magnetic particles in a three-necked flask, add 90ml of ethanol and 30ml of pure water, stir to mix, and then add 3ml of 25% ammonia solution. Slowly drop 0.3ml of tetraethyl orthosilicate under stirring, start timing from the addition of tetraethyl orthosilicate, and stir ultrasonically for 5 hours. Wash with anhydrous ethanol and pure water alternately for 4 times, disperse in an appropriate amount of anhydrous ethanol, and dry in an oven at 40℃ to obtain Fe3O4@SiO2 carrier.

[0037] Step 103: Preparation of AuM particles: Take a Fe3O4@SiO2 carrier solution with a concentration of 0.2 mg / mL, slowly add 1.2 ml of 0.8 mg / ml polyethyleneimine aqueous solution under stirring, ultrasonically stir for 1.5 hours, wash with pure water 6 times, and disperse in 25 ml of pure water.

[0038] Take another 12 ml of 7 mmol / L chloroauric acid aqueous solution and 2 times the volume of 7 mmol / L sodium citrate solution in a flask, and slowly add 60 mmol / L freshly prepared sodium borohydride solution under magnetic stirring to obtain a pink small-size gold nanoparticle solution.

[0039] The gold nanoparticle solution and the polyethyleneimine-modified Fe3O4@SiO2 solution were mixed in equal volumes, stirred ultrasonically for 1.5 hours, washed with ultrapure water for 6 times, and then dispersed in 12 ml of pure water. Then 12 ml of 1 mmol / L PEG was added. 3400 -SH solution was stirred at room temperature for 5 hours, washed with pure water for 6 times, dispersed in an appropriate amount of pure water, and dried at 40°C to obtain AuM particles.

[0040] Step 2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium) Step 201, MCF-7 cells are cultured in a complete medium consisting of 15% fetal bovine serum, RPMI-1640 medium, and 1% double antibody in an environment of 5% carbon dioxide and 37°C. Cells in the logarithmic growth phase are collected by trypsin-EDTA digestion, washed three times by centrifugation at 4°C, 1000g, and 8 minutes using phosphate buffer, and resuspended in phosphate buffer. Cells are disrupted using a cell ultrasonic disruptor under ice bath conditions, with the parameters set to ultrasonic 5 seconds, stop 8 seconds, cycle 8 times, and power 45%. After centrifugation at 4°C, 4000g for 15 minutes, the precipitate is discarded; the supernatant is centrifuged at 4°C, 20000g for 40 minutes, the supernatant is discarded, the precipitate is washed three times with phosphate buffer and resuspended in phosphate buffer.

[0041] Step 202, take 200 μL of 10 mg / ml AuM solution and add it to 2 ml of 500 μg / ml cell membrane solution, vortex mix, place it on a rotary mixer and incubate it for 2 hours, wash it three times with phosphate buffer, and disperse it in 300 μL of phosphate buffer to obtain SKBR3 cell membrane-coated magnetic particles (SKBR3 / CMAuM magnetic screening medium).

[0042] Embodiment 3: A method for preparing a cell membrane gold magnetic screening medium for a biologically active ligand comprises the following steps: Step 1: Preparation of AuM microparticle carrier Step 101, bare magnet synthesis: Weigh 0.8g of ferric chloride hexahydrate, add 20ml of ethylene glycol, 0.15g of disodium citrate, and 1g of anhydrous sodium acetate, heat to 70℃ and stir to dissolve. Transfer the mixed solution to a hydrothermal reactor and react in an oven at 180℃ for 8 hours. Turn off the oven power and take out the reactor after it cools naturally to room temperature. Wash alternately with anhydrous ethanol and pure water 4 times, disperse in an appropriate amount of anhydrous ethanol, dry in an oven at 40℃, and collect in a centrifuge tube.

[0043] Step 102, silicon dioxide coating modification: Weigh 0.12g of ferroferric oxide magnetic particles in a three-necked flask, add 75ml of ethanol and 25ml of pure water, stir to mix, and then add 2ml of 25% ammonia solution. Slowly drop 0.25ml of tetraethyl orthosilicate under stirring, start timing from the addition of tetraethyl orthosilicate, and stir ultrasonically for 4.5 hours. Wash with anhydrous ethanol and pure water alternately for 5 times, disperse in an appropriate amount of anhydrous ethanol, and dry in an oven at 35℃ to obtain Fe3O4@SiO2 carrier.

[0044] Step 103: Preparation of AuM particles: Take a Fe3O4@SiO2 carrier solution with a concentration of 0.5 mg / mL, slowly add 1 ml of 1.5 mg / ml polyethyleneimine aqueous solution under stirring, ultrasonically stir for 1.5 hours, wash with pure water 6 times, and disperse in 22 ml of pure water.

[0045] Take another 14 ml of 2 mmol / L chloroauric acid aqueous solution and 2 times the volume of 2 mmol / L sodium citrate solution in a flask, and slowly add 55 mmol / L freshly prepared sodium borohydride solution under magnetic stirring to obtain a pink small-size gold nanoparticle solution.

[0046] The gold nanoparticle solution and the polyethyleneimine-modified Fe3O4@SiO2 solution were mixed in equal volumes, ultrasonically stirred for 2 hours, washed with ultrapure water 7 times, and then dispersed in 15 ml of pure water. Then 15 ml of 2.5 mmol / L PEG was added.4500 -SH solution was stirred at room temperature for 6 hours, washed with pure water 6 times, dispersed in an appropriate amount of pure water, and dried at 40°C to obtain AuM particles.

[0047] Step 2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium) Step 201, MCF-7 cells are cultured in a complete medium consisting of 15% fetal bovine serum, RPMI-1640 medium, and 1% double antibody in an environment of 5% carbon dioxide and 37°C. The cells in the logarithmic growth phase are collected by trypsin-EDTA digestion, washed three times by centrifugation at 4°C, 1000g, and 8 minutes using phosphate buffer, and resuspended in phosphate buffer. The cells are disrupted with a cell ultrasonic disruptor under ice bath conditions, and the parameters are set to ultrasonic 5 seconds, stop 8 seconds, cycle 8 times, and power 45%. After centrifugation at 3500g for 15 minutes at 4°C, the precipitate is discarded; the supernatant is centrifuged at 20000g for 40 minutes at 4°C, the supernatant is discarded, the precipitate is washed three times with phosphate buffer and resuspended in phosphate buffer.

[0048] Step 202, take 200 μL of 10 mg / ml AuM solution and add it to 2 ml of 700 μg / ml cell membrane solution, vortex mix, place it on a rotary mixer and incubate it for 1 hour, wash it three times with phosphate buffer, and disperse it in 300 μL of phosphate buffer to obtain SKBR3 cell membrane-coated magnetic particles (SKBR3 / CMAuM magnetic screening medium).

[0049] The magnetic materials were characterized by dynamic light scattering particle size analyzer, transmission electron microscope (TEM), vibrating sample magnetometer (VSM) and laser confocal microscope: hydrated particle size and transmission electron microscope image ( Figure 1 ) shows that the size of AuM particles is in the submicron range (100nm~1000nm), about 200nm, indicating that AuM particles have a high specific surface area and can achieve efficient cell membrane loading. Transmission electron microscopy images show that gold nanoparticles (about 4nm) are adsorbed on the surface of magnetic particles at a very high density, indicating that AuM particles have the ability to coordinate and bind to cell membranes.

[0050] The magnetic properties of AuM and CMAuM were studied using a vibrating sample magnetometer (VSM). Figure 2 ) The magnetization intensity of CMAuM particles coated with SKBR3 is slightly lower than that of AuM particles, but it is still greater than 30emu / g. CMAuM has a strong magnetization intensity and can quickly separate the material from the solution under an external magnetic field.

[0051] The surface morphology of AuM and CMAuM was observed by SEM. The results showed that compared with AuM, CMAuM had a layer of cell membrane adsorbed on its surface ( Figure 3 a, Figure 3 b); Dil (Ex / Em=549 / 565nm) was used to stain the cell membrane. Under a laser confocal microscope, CMAuM emitted red fluorescence ( Figure 3 c). This demonstrates that the cell membrane is fixed on the AuM.

[0052] In order to investigate the efficiency and stability of gold magnetic particles adsorbing cell membranes, the amount of protein before and after adsorption by magnetic particles was measured by BCA protein detection kit to obtain the amount of protein adsorbed on magnetic particles. The protein amount was used instead of the cell membrane mass, and the membrane adsorption rate and membrane shedding rate were calculated according to the following formula. The results showed that the adsorption efficiency of SKBR3 cell membranes in 1×PBS was higher than that in 0.5×PBS ( Figure 4 a), indicating that 1×PBS is a better buffer environment for cell membrane coating; it can also be seen that the adsorption of Fe3O4@SiO2 and AuM surfaces reached saturation at 90 min, with adsorption rates of 149.5 μg / mg and 186.6 μg / mg, respectively ( Figure 4 b), indicating that the AuM prepared by the present invention has a higher adsorption efficiency on the cell membrane. In addition, by detecting the change of cell membrane shedding rate over the days, it was found that after the 8th day, the cell membrane shedding rate on the Fe3O4@SiO2 surface gradually increased, while the cell membrane shedding rate on the AuM surface was still very low ( Figure 4 c), indicating that the cell membrane binding stability of the AuM surface prepared by the present invention is better than that of the traditional Fe3O4@SiO2.

[0053] Film adsorption rate (%) = (mass of gold magnetic particle surface film / mass of gold magnetic particle) × 100%; Membrane shedding rate (%) = (mass of cell membrane in supernatant / mass of initial membrane on the surface of gold magnetic particles) × 100%.

[0054] The present invention uses the SKBR3 cell membrane-coated magnetic particles prepared above to screen effective ingredients against HER2 breast cancer, specifically comprising the following steps: Step 1: CMAuM screening performance investigation Take 100μL, 1mg / mL SKBR3 / CMAuM magnetic screening medium prepared above, magnetically separate, remove the supernatant, add 200μL 0.1mg / mL positive drug lapatinib solution, vortex for 10s, incubate on a rotary mixer for 0-7min, magnetically separate after incubation, collect the adsorbed supernatant for HPLC test, and analyze the effect of drug screening time. The chromatographic conditions used are ODS column, acetonitrile-0.005mol / L ammonium acetate solution (55:45) mobile phase, 260nm detection wavelength and 1.0 mL / min flow rate. From the results, it can be seen that at 5min, the adsorption of lapatinib by SKBR3 / CMAuM magnetic screening medium reached saturation ( Figure 5 a, b).

[0055] The SKBR3 / CMAuM after adsorption of lapatinib was washed twice with PBS solution, and 100 μL of eluent prepared with a volume ratio of DMSO:H2O=1:1 was added to the magnetic particles, vortexed for 10 seconds, incubated on a rotary mixer for 15 minutes, and the eluent was collected for HPLC analysis. Figure 5 c shows an obvious chromatographic peak of lapatinib. The above results indicate that SKBR3 / CMAuM has the ability to screen the positive drug lapatinib.

[0056] Step 2: The above SKBR3 / CMAuM magnetic screening medium is used to screen the anti-HER2 tumor components in the root of Scutellaria baicalensis. The specific steps are as follows: (1) After the root of Scutellaria baicalensis was crushed and sieved, 1.0 g of the powder was taken into a three-necked flask, and 100 mL of 70% ethanol was added. The mixture was refluxed at 80°C for 3 h, filtered, and concentrated to about 5 mL using a vacuum rotary concentrator. The root extract of Scutellaria baicalensis was diluted 20 times with 1× PBS and filtered through a 0.22 μm filter membrane for screening.

[0057] (2) Take 1 mL of the above-mentioned Scutellaria baicalensis root screening solution and add it to 1 mg of SKBR3 / CMAuM magnetic screening medium for 5 min. Discard the supernatant after magnetic separation, wash twice with PBS, add 500 μL of elution solution prepared with a volume ratio of DMSO:H2O=1:1, elute for 15 min, and collect the fishing solution.

[0058] The active ingredients of the fishing liquid were identified by Shimadzu LCMS-8045 triple quadrupole liquid-mass spectrometer. The chromatographic column used was an ODS column; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; gradient elution program: 0-8min, 10%-60% B, 8-10min, 60%-90% B, 10-15min, 90%-100% B; 15-20min, 100% B; flow rate: 0.3mL / min; the mass spectrometry system used full scan mode, 10-1000 m / z. After component analysis, the results of active ingredients are shown in Table 1. There are three potential anti-HER2 tumor components in the root of Scutellaria baicalensis obtained by fishing, namely baicalin, wogonin and baicalein.

[0059] Table 1 Compound information

[0060] The present invention provides a high coverage, stable and rapid strategy for fixing magnetic carriers of cell membranes, and uses the cell membrane magnetic screening medium for fishing active ingredients in traditional Chinese medicine. At room temperature, in PBS with a pH value of 7.3 to 7.5, the cell membrane is self-assembled for 1 to 2 hours and then fixed on the magnetic carrier. Such an environment can improve the coverage and stability of the cell membrane, and improve the sensitivity and accuracy of the screening medium for traditional Chinese medicine screening.

[0061] The above is a preferred embodiment of the present invention. For ordinary technicians in this technical field, various equivalent modifications to the present invention without departing from the principle of the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A cell membrane gold magnetic screening medium for biologically active ligands, characterized in that: The invention comprises gold magnetic particles and SKBR3 cell membranes coated on the surface of the gold magnetic particles. The gold magnetic particles comprise Fe3O4@SiO2 carriers, polyethyleneimine modified on the surface of the Fe3O4@SiO2 carriers, gold nanoparticles and PEG-SH.

2. The cell membrane gold magnetic screening medium of a biologically active ligand according to claim 1, characterized in that: Add 1 mg of gold magnetic particles to every 400 μg~700 μg of cell membrane.

3. The cell membrane gold magnetic screening medium of a biologically active ligand according to claim 1, characterized in that: The Fe3O4@SiO2 carrier includes Fe3O4 bare magnet and SiO2 coating wrapped on the surface of Fe3O4 bare magnet. The particle size of Fe3O4 bare magnet is 150nm~500nm, and the thickness of SiO2 coating is 10nm~30nm.

4. The method for preparing a cell membrane gold magnetic screening medium of a bioactive ligand according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: The Fe3O4@SiO2 carrier dispersion liquid is mixed with the polyethyleneimine solution, and after ultrasonication and washing, a first reaction liquid is obtained; The gold nanoparticle solution and the first reaction solution are mixed, ultrasonicated and washed, and then the PEG-SH solution is added, and the reaction is followed by drying to obtain gold magnetic particles; The gold magnetic particles are added to the PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, mixed and combined, and then washed to obtain the gold magnetic particles coated with SKBR3 cell membranes, i.e., the cell membrane gold magnetic screening medium; The composition of PBS is 2.7mM KCl, 2.0mM KH2PO4, 137mM NaCl, 10mM Na2HPO4, and the pH value is 7.3~7.5@25ºC.

5. The method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand according to claim 4, characterized in that: In the step of mixing the Fe3O4@SiO2 dispersion with the polyethyleneimine solution, ultrasonicating and washing, to obtain the first reaction solution: The concentration of the Fe3O4@SiO2 dispersion is 0.2mg / mL~0.8mg / mL; the final concentration of the Fe3O4@SiO2 dispersion after mixing with the polyethyleneimine solution is 0.8mg / mL~2mg / mL.

6. The method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand according to claim 4, characterized in that: The step of mixing the gold nanoparticle solution and the first reaction solution, ultrasonicating and washing, adding the PEG-SH solution, and drying after the reaction to obtain the gold magnetic particles: The preparation of the gold nanoparticle solution is specifically as follows: a 2mmol / L~7mmol / L chloroauric acid aqueous solution is mixed with 2 times the volume of a 2mmol / L~7mmol / L sodium citrate solution, sodium borohydride solution is added dropwise thereto, and the mixture is reacted to obtain a gold nanoparticle solution with a particle size of less than 10nm.

7. The method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand according to claim 6, characterized in that: The step of mixing the gold nanoparticle solution and the first reaction solution, ultrasonicating and washing, adding the PEG-SH solution, and drying after the reaction to obtain the gold magnetic particles: Mixing equal volumes of the first reaction solution and the gold nanoparticle solution; The concentration of PEG-SH solution is 1 mmol / L~2.5 mmol / L.

8. The method for preparing a cell membrane gold magnetic screening medium of a biologically active ligand according to claim 4, characterized in that: The step of adding gold magnetic particles to a PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, mixing and combining, and then washing to obtain gold magnetic particles coated with SKBR3 cell membranes: The gold magnetic particles were added to the PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, vortexed to mix, and allowed to bind at room temperature for 1 h to 2 h. Unbound cell membranes were washed away with PBS to obtain gold magnetic particles coated with SKBR3 cell membranes.

9. A method for extracting an anti-HER2 breast tumor active ingredient, characterized in that: The method is carried out using a cell membrane gold magnetic screening medium of a bioactive ligand according to any one of claims 1 to 3, and the specific steps are as follows: Adding the cell membrane gold magnetic screening medium obtained by magnetic separation and removing the supernatant to the substrate to be screened for incubation to obtain an incubation mixture; The incubation mixture is subjected to magnetic separation and the supernatant is discarded. After washing, an eluent is added for incubation, and the supernatant is collected to obtain the anti-HER2 breast tumor active component in the substrate to be screened.

10. The method for extracting the anti-HER2 breast tumor active ingredient in a traditional Chinese medicine according to claim 9, characterized in that: The substrate to be screened is Scutellaria baicalensis extract.

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