Cell membrane gold magnetic screening medium and its application and method in screening of anti-breast tumor active ingredients

Abstract

The application provides a cell membrane gold magnetic screening medium and an application and method thereof in screening of anti-breast tumor active ingredients, the cell membrane gold magnetic screening medium of a bioactive ligand comprises gold magnetic microparticles and SKBR3 cell membranes coated on surfaces of the gold magnetic microparticles, the gold magnetic microparticles comprise Fe3O4@SiO2 carriers, polyethyleneimine modified on surfaces of the Fe3O4@SiO2 carriers, gold nanoparticles and PEG-SH. Through electrostatic force and metal coordination force, the application realizes efficient and stable coating of the SKBR3 cell membranes on surfaces of the gold magnetic microparticles, and improves sensitivity and accuracy of screening of traditional Chinese medicine active ingredients.

Description

Technical Field

[0001] This invention belongs to the field of functional materials and drug active ingredient screening technology, specifically relating to a cell membrane gold magnetic screening medium and its application and method in screening active ingredients against breast tumors. Background Technology

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

[0003] However, the preparation technology of cell membrane-coated magnetic materials is not easy. Currently, the binding of cell membranes to magnetic materials is mainly achieved through covalent bonding or ultrasonic adsorption. Covalent bonding requires cumbersome chemical modification of both the cell membrane and the magnetic material, including the activation of linking groups, which is not only time-consuming but may also affect the biological activity of the 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 bonding may also damage the integrity and biological activity of the cell membrane, reducing its screening effect. While ultrasonic adsorption is simple to operate, the strong ultrasonic action can easily damage cell membranes, affecting their stability and screening accuracy. Furthermore, cell membranes adsorbed on the surface of magnetic materials are prone to detachment, leading to reduced screening efficiency. Therefore, overcoming the problems of long cell membrane coating time, cumbersome procedures, and poor stability has become the main challenge facing current cell membrane-coated magnetic material preparation technology.

[0004] Breast cancer has become the most common cancer worldwide, 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 prone to recurrence and metastasis, screening drugs targeting HER2-positive breast cancer is of significant clinical importance. Ancient Chinese medicine texts record that heat-clearing and detoxifying herbs can be used to treat tumors, with Scutellaria baicalensis (Huang Qin) being one of the representative herbs, and it has been found to inhibit the proliferation and metastasis of various tumor cells. Therefore, further exploration of the anti-breast cancer active components in Scutellaria baicalensis is of great significance for research on the anti-tumor mechanism of Scutellaria baicalensis and the development of new traditional Chinese medicine products based on Scutellaria baicalensis. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a cell membrane gold magnetic screening medium and its application and method in screening active ingredients for anti-breast tumors. Through electrostatic forces and metal coordination forces, efficient and stable coating of SKBR3 cell membranes on the surface of gold magnetic microparticles is achieved, thereby improving the sensitivity of screening active ingredients of traditional Chinese medicine.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a cell membrane gold magnetic screening medium for bioactive ligands, comprising gold magnetic microparticles and an SKBR3 cell membrane coated on the surface of the gold magnetic microparticles, wherein the gold magnetic microparticles include a Fe3O4@SiO2 carrier, polyethyleneimine modified on the surface of the Fe3O4@SiO2 carrier, gold nanoparticles, and PEG-SH.

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

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

[0009] This invention also provides a method for preparing a cell membrane gold magnetic screening medium for bioactive ligands, the specific steps of which are as follows:

[0010] The Fe3O4@SiO2 carrier dispersion was mixed with a polyethyleneimine solution, and after sonication and washing, the first reaction solution was obtained.

[0011] The gold nanoparticle solution and the first reaction solution were mixed, sonicated, washed, and then PEG-SH solution was added. After the reaction, the mixture was dried to obtain gold magnetic microparticles.

[0012] Gold magnetic microparticles were added to a PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, mixed and bound, and then washed to obtain gold magnetic microparticles coated with SKBR3 cell membranes, i.e., cell membrane gold magnetic screening medium.

[0013] The PBS composition is 2.7mM KCl, 2.0mM KH2PO4, 137mM NaCl, 10mM Na2HPO4, with a pH of 7.3~7.5 at 25ºC.

[0014] Further, in the step of mixing the Fe3O4@SiO2 dispersion with a polyethyleneimine solution, followed by sonication and washing to obtain the first reaction solution:

[0015] The concentration of the Fe3O4@SiO2 dispersion is 0.2 mg / mL to 0.8 mg / mL; the final concentration of the Fe3O4@SiO2 dispersion after mixing with the polyethyleneimine solution is 0.8 mg / mL to 2 mg / mL.

[0016] Further, in the step of mixing the gold nanoparticle solution and the first reaction solution, sonicating and washing, adding PEG-SH solution, reacting and drying to obtain gold magnetic microparticles:

[0017] The preparation of the gold nanoparticle solution is as follows: a chloroauric acid aqueous solution with a concentration of 2 mmol / L to 7 mmol / L is mixed with two volumes of sodium citrate solution with a concentration of 2 mmol / L to 7 mmol / L, and sodium borohydride solution is added dropwise to the mixture. The mixture reacts to obtain a gold nanoparticle solution with a particle size of less than 10 nm.

[0018] Further, in the step of mixing the gold nanoparticle solution and the first reaction solution, sonicating and washing, adding PEG-SH solution, reacting and drying to obtain gold magnetic microparticles:

[0019] Mix the first reaction solution and the gold nanoparticle solution in equal volumes;

[0020] The concentration of the PEG-SH solution is 1 mmol / L to 2.5 mmol / L.

[0021] Further, in the step of adding gold magnetic microparticles to a PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, mixing and binding, and then washing to obtain gold magnetic microparticles coated on SKBR3 cell membranes:

[0022] Gold magnetic microparticles were added to a PBS suspension of HER2-positive breast tumor SKBR3 cell membranes, vortexed and mixed, and bound at room temperature for 1-2 hours. Unbound cell membranes were washed away with PBS to obtain gold magnetic microparticles coated with SKBR3 cell membranes.

[0023] This invention also provides a method for extracting an active ingredient against HER2 breast tumors, using a cell membrane gold magnetic screening medium containing one of the aforementioned bioactive ligands. The specific steps are as follows:

[0024] The cell membrane gold magnetic screening medium, after magnetic separation and supernatant removal, was added to the substrate to be screened and incubated to obtain an incubation mixture.

[0025] The supernatant of the incubation mixture was magnetically separated and discarded. After washing, elution buffer was added and incubated. The supernatant was then collected to obtain the anti-HER2 breast tumor active ingredient in the substrate to be screened.

[0026] Furthermore, the substrate to be screened is Scutellaria baicalensis extract.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] This invention discloses a gold magnetic screening medium for cell membranes containing bioactive ligands, comprising gold magnetic microparticles and SKBR3 cell membranes coated on the surface of the gold magnetic microparticles. The gold magnetic microparticles include a Fe3O4@SiO2 carrier, polyethyleneimine modified on the surface of the Fe3O4@SiO2 carrier, gold nanoparticles, and PEG-SH. This achieves efficient and stable coating of the SKBR3 cell membrane onto the surface of the gold magnetic microparticles, avoiding the problem of easy cell membrane detachment in traditional methods. The cell membrane gold magnetic screening medium is prepared by self-assembling the positively charged polymer on the surface of the magnetic material and the gold nanoparticles with the cell membrane through electrostatic and metal coordination forces, improving the binding stability of the cell membrane and ensuring the sensitivity and accuracy of the screening process. The prepared SKBR3 / CMAuM magnetic screening medium exhibits excellent magnetism, enabling rapid separation under an external magnetic field, facilitating subsequent operations. This screening medium can not only be used for screening active ingredients against HER2 breast tumors but can also be extended to the screening of other tumor active ingredients, providing new ideas and methods for new drug development.

[0029] This invention discloses a method for preparing a cell membrane gold magnetic screening medium containing bioactive ligands. The method involves incubation in a PBS suspension provided by this invention. The cell membrane binds to the magnetic particles through electrostatic and coordination forces. The procedure is simple, requires no sonication or activation, and exhibits high cell membrane binding stability. This method achieves efficient and stable coating of the cell membrane onto the surface of gold magnetic microparticles through electrostatic and metal coordination forces, improving the sensitivity and stability of screening active ingredients in traditional Chinese medicine. The SKBR3 / CMAuM magnetic screening medium prepared by this invention exhibits excellent magnetic and cell membrane binding stability, can rapidly separate under an external magnetic field, and the cell membrane is not easily detached.

[0030] This invention discloses a method for extracting active ingredients against HER2-positive breast cancer. Utilizing a biomimetic screening platform combining magnetic materials with cell membranes, it can conveniently, rapidly, and efficiently screen for components in Scutellaria baicalensis with anti-HER2 breast cancer activity. The prepared cell membrane gold magnetic particles are then used to target potential active ingredients in Scutellaria baicalensis against HER2-positive breast cancer, achieving the goal of discovering lead compounds for anti-HER2 breast cancer drugs from Scutellaria baicalensis. This also provides valuable reference and guidance for anti-tumor research on other traditional Chinese medicines. Attached Figure Description

[0031] Figure 1 These are AuM particle size and transmission electron microscopy characterization images obtained in the embodiments of the present invention;

[0032] Figure 2 The AuM and CMAuM hysteresis regression lines obtained in the embodiments of the present invention;

[0033] Figure 3 These are CMAuM scanning electron microscope and fluorescence confocal images obtained in embodiments of the present invention;

[0034] Figure 4 The graph shows the performance test results of AuM-loaded cell membranes obtained in an embodiment of the present invention.

[0035] Figure 5 Figure showing the feasibility results of screening the positive drug lapatinib for SKBR3 / CMAuM. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0037] This invention provides a method for preparing a cell membrane gold magnetic screening medium containing bioactive ligands. The method is based on the binding of positively charged polymers and gold nanoparticles on the surface of magnetic materials to the cell membrane via electrostatic and metal coordination forces. Specifically, the method includes the following steps:

[0038] S1 Preparation of Gold Magnetic Particles

[0039] 1) Preparation of AuM microparticle carriers:

[0040] At 200ºC, ethylene glycol was used as a reducing agent in a hydrothermal reactor. The size and magnetism of the bare magnets were controlled by changing the feed ratio of ferric chloride, sodium citrate, and sodium acetate, as well as the reaction time. The particle size of the bare magnets was controlled within the range of 150nm to 500nm, giving them a high specific surface area to improve the subsequent loading rate on the cell membrane. SiO2 was coated on the surface of the bare magnets. The concentration of ammonia and tetraethyl orthosilicate was changed to adjust the SiO2 coating thickness between 10nm and 30nm, so as to enhance the water solubility, stability, and biocompatibility of the magnetic particles while minimizing the influence of magnetism.

[0041] The above Fe3O4@SiO2 support was dispersed in pure water, and then added to an aqueous solution of polyethyleneimine (PEI, Mw: 1800~25000). The mixture was sonicated for 1~2 hours and washed several times with pure water to obtain the first reaction solution.

[0042] The concentration range of Fe3O4@SiO2 carrier dispersed in pure water is 0.2~0.8 mg / mL; the final concentration after adding PEI is 0.8~2 mg / mL.

[0043] 2) Preparation of gold magnetic microparticles;

[0044] The gold nanoparticle (Au NPs) solution and the first reaction solution were mixed in equal volumes, sonicated for 1-2 hours, washed several times with pure water, and then a PEG-SH (polyethylene glycol mercapto) aqueous solution with a concentration of 1-2.5 mmol / L was added. The mixture was stirred at room temperature for 4-6 hours and then dried to obtain gold magnetic microparticles (AuM).

[0045] The above-mentioned gold nanoparticle solution was obtained by adding freshly prepared sodium borohydride solution dropwise to a 2-7 mmol / L aqueous chloroauric acid solution and twice the amount of 2-7 mmol / L sodium citrate solution, and stirring at room temperature for 2-5 min. The gold nanoparticle solution was pink and the particle size was less than 10 nm.

[0046] S2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium)

[0047] Cells were disrupted using a cell disruptor and centrifuged to collect HER2-positive breast tumor SKBR3 cell membranes, which were then resuspended in PBS.

[0048] The gold magnetic microparticles were added to the cell membrane suspension, vortexed and mixed, and placed on a rotary mixer to bind at room temperature for 1-2 hours. Unbound cell membranes were washed away with PBS to obtain SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium).

[0049] Preferably, the PBS used consists of 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, and 10 mM Na2HPO4, with a pH of 7.3~7.5 at 25ºC.

[0050] Preferably, the ratio of gold magnetic microparticles to cell membrane is 400μg~700μg of cell membrane per 1mg AuM.

[0051] This invention utilizes the aforementioned cell membrane gold magnetic screening medium for screening active ingredients against HER2 breast tumors, specifically including the following steps:

[0052] Take the prepared SKBR3 / CMAuM magnetic screening medium, separate it magnetically, remove the supernatant, add the Scutellaria baicalensis extract to be screened, vortex it, and then place it on a rotary mixer for incubation.

[0053] After incubation, the supernatant was magnetically discarded, and the particles were magnetically washed 2-3 times with PBS solution. Eluent prepared with a volume ratio of DMSO:H2O = 1:1 was added to the magnetic particles, and the particles were vortexed and incubated on a rotary mixer for 10-20 minutes. The supernatant was then collected.

[0054] Liquid chromatography-mass spectrometry (LC-MS) analysis of the supernatant yielded the anti-HER2 breast tumor active ingredient in Scutellaria baicalensis extract.

[0055] This invention utilizes the self-assembly of positively charged polymers and gold nanoparticles on the surface of magnetic materials with cell membranes through electrostatic and metal coordination forces to prepare gold magnetic sieving media for cell membranes. Compared with existing cell membrane fixation techniques, this method offers advantages such as higher efficiency, convenience, and speed, overcoming the problems of long fixation times and cumbersome procedures.

[0056] The cell membrane gold magnetic screening medium prepared by this invention has a higher cell membrane coverage and cell membrane fixation stability compared with existing cell membrane fixation technologies, thereby improving the efficiency and accuracy of drug active ingredient screening.

[0057] The method for preparing cell membrane gold magnetic screening media provided by this invention is simple to operate, low in cost, and can be widely applied to the screening of other tumor active ingredients. Using cell membrane gold magnetic screening media to screen Scutellaria baicalensis extract can efficiently discover novel anti-HER2 breast tumor active ingredients with targeting and selectivity, providing new drug options for the clinical treatment of HER2-positive breast cancer.

[0058] Example 1

[0059] A method for preparing a cell membrane gold magnetic screening medium for a bioactive ligand includes the following steps:

[0060] Step 1: Preparation of AuM microparticle carriers

[0061] Step 101, Bare Magnet Synthesis:

[0062] Weigh 1.00 g of ferric chloride hexahydrate into a beaker, then add 25 ml of ethylene glycol, 0.20 g of disodium citrate, and 1.30 g of anhydrous sodium acetate in sequence. Heat and stir until completely dissolved. Transfer the mixture to a hydrothermal reactor and react in an oven at 200 °C for 10 hours. Turn off the oven power and remove the reactor after it has cooled to room temperature. Wash the reactor three times alternately with anhydrous ethanol and pure water, disperse it in a small amount of anhydrous ethanol, dry it in an oven at 37 °C, and collect it in a centrifuge tube.

[0063] Step 102, Silica coating modification:

[0064] Weigh 0.1 g of Fe3O4 magnetic microparticles into a three-necked flask, add 80 ml of ethanol and 20 ml of pure water, stir to mix well, and then add 2.5 ml of 25% ammonia solution. While stirring, add 0.2 ml of tetraethyl orthosilicate dropwise, and start timing from the addition of tetraethyl orthosilicate. Sonicate for 4 hours. Wash three times alternately with anhydrous ethanol and pure water, disperse in a small amount of anhydrous ethanol, and dry in an oven at 37°C to obtain the Fe3O4@SiO2 support.

[0065] Step 103, AuM microparticle preparation:

[0066] 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 while stirring, sonicate for 1 hour, wash 5 times with pure water, and disperse in 20 mL of pure water.

[0067] Take 10 ml of 2 mmol / L chloroauric acid aqueous solution and twice 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 solution of small-diameter gold nanoparticles.

[0068] The above gold nanoparticle solution was mixed with an equal volume of polyethyleneimine-modified Fe3O4@SiO2 solution, ultrasonically stirred for 1 hour, washed 5 times with ultrapure water, and then dispersed in 10 ml of pure water. Then, 10 ml of 1.25 mmol / L PEG was added. 5000 The -SH solution was stirred at room temperature for 4 hours, washed 5 times with pure water, dispersed in a small amount of pure water, and dried at 37°C to obtain AuM microparticles.

[0069] Step 2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium)

[0070] Step 201: SKBR3 cells were cultured in a complete medium consisting of 10% fetal bovine serum, McCoy's 5A medium, and 1% penicillin-dextrose antibody at 37°C and 5% CO2. Cells in the exponential growth phase were collected by trypsin digestion and washed three times by centrifugation at 800g for 5 minutes at 4°C using phosphate buffer. Cells were then resuspended in phosphate buffer. Cells were disrupted using a cell sonicator on ice with the following parameters: sonication for 4 seconds, pause for 6 seconds, 6 cycles, and 40% power. After centrifugation at 4000g for 10 minutes at 4°C, the precipitate was discarded. The supernatant was centrifuged at 18000g for 30 minutes at 4°C, the supernatant was discarded, and the precipitate was washed twice with phosphate buffer and resuspended in phosphate buffer.

[0071] Step 202: Add 200 μL of 10 mg / ml AuM solution to 2 ml of 400 μg / ml cell membrane solution, vortex to mix, incubate on a rotary mixer for 1.5 hours, wash twice with phosphate buffer, disperse in 200 μL of phosphate buffer to obtain SKBR3 cell membrane coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium).

[0072] Example 2:

[0073] A method for preparing a cell membrane gold magnetic screening medium for a bioactive ligand includes the following steps:

[0074] Step 1: Preparation of AuM microparticle carriers

[0075] Step 101, Bare Magnet Synthesis:

[0076] 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 until dissolved. Transfer the mixture to a hydrothermal reactor and react in an oven at 180℃ for 8 hours. Turn off the oven and allow the reactor to cool naturally to room temperature. Wash the reactor four times alternately with anhydrous ethanol and pure water, disperse in an appropriate amount of anhydrous ethanol, dry in an oven at 40℃, and collect in centrifuge tubes.

[0077] Step 102, Silica coating modification:

[0078] Weigh 0.15 g of Fe3O4 magnetic microparticles into a three-necked flask, add 90 ml of ethanol and 30 ml of pure water, stir to mix thoroughly, and then add 3 ml of 25% ammonia solution. While stirring, slowly add 0.3 ml of tetraethyl orthosilicate, and start timing from the addition of tetraethyl orthosilicate. Sonicate for 5 hours. Wash four times alternately with anhydrous ethanol and pure water, disperse in an appropriate amount of anhydrous ethanol, and dry in an oven at 40 °C to obtain the Fe3O4@SiO2 support.

[0079] Step 103, AuM microparticle preparation:

[0080] 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 while stirring, sonicate for 1.5 hours, wash 6 times with pure water, and disperse in 25 mL of pure water.

[0081] Take 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 dropwise under magnetic stirring to obtain a pink small-diameter gold nanoparticle solution.

[0082] The above gold nanoparticle solution was mixed with an equal volume of polyethyleneimine-modified Fe3O4@SiO2 solution, ultrasonically stirred for 1.5 hours, washed 6 times with ultrapure water, and then dispersed in 12 ml of pure water. Then, 12 ml of 1 mmol / L PEG was added. 3400 -SH solution, stirred at room temperature for 5 hours, washed 6 times with pure water, dispersed in an appropriate amount of pure water, and dried at 40℃ to obtain AuM microparticles.

[0083] Step 2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium)

[0084] Step 201: MCF-7 cells were cultured in a complete medium consisting of 15% fetal bovine serum, RPMI-1640 medium, and 1% penicillin-dextrose antibody at 37°C and 5% CO2. Cells in the logarithmic growth phase were collected by trypsin-EDTA digestion, washed three times by centrifugation at 1000g for 8 minutes at 4°C with phosphate buffer, and resuspended in phosphate buffer. Cells were then disrupted using a cell sonicator on ice with parameters set as follows: sonication for 5 seconds, pause for 8 seconds, 8 cycles, and 45% power. After centrifugation at 4000g for 15 minutes at 4°C, the precipitate was discarded; the supernatant was centrifuged at 20000g for 40 minutes at 4°C, the supernatant was discarded, and the precipitate was washed three times with phosphate buffer and resuspended in phosphate buffer.

[0085] Step 202: Add 200 μL of 10 mg / ml AuM solution to 2 ml of 500 μg / ml cell membrane solution, vortex to mix, incubate on a rotary mixer for 2 hours, wash three times with phosphate buffer, disperse in 300 μL of phosphate buffer to obtain SKBR3 cell membrane coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium).

[0086] Example 3:

[0087] A method for preparing a cell membrane gold magnetic screening medium for a bioactive ligand includes the following steps:

[0088] Step 1: Preparation of AuM microparticle carriers

[0089] Step 101, Bare Magnet Synthesis:

[0090] Weigh 0.8 g of ferric chloride hexahydrate, add 20 ml of ethylene glycol, 0.15 g of disodium citrate, and 1 g of anhydrous sodium acetate. Heat to 70°C and stir until dissolved. Transfer the mixture to a hydrothermal reactor and react in an oven at 180°C for 8 hours. Turn off the oven and allow the reactor to cool naturally to room temperature. Wash the reactor four times alternately with anhydrous ethanol and pure water, disperse in an appropriate amount of anhydrous ethanol, dry in an oven at 40°C, and collect in centrifuge tubes.

[0091] Step 102, Silica coating modification:

[0092] Weigh 0.12 g of Fe3O4 magnetic microparticles into a three-necked flask, add 75 ml of ethanol and 25 ml of pure water, stir to mix thoroughly, and then add 2 ml of 25% ammonia solution. While stirring, slowly add 0.25 ml of tetraethyl orthosilicate, and start timing from the addition of tetraethyl orthosilicate. Sonicate for 4.5 hours. Wash five times alternately with anhydrous ethanol and pure water, disperse in an appropriate amount of anhydrous ethanol, and dry in an oven at 35°C to obtain the Fe3O4@SiO2 support.

[0093] Step 103, AuM microparticle preparation:

[0094] 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 while stirring, sonicate for 1.5 hours, wash 6 times with pure water, and disperse in 22 mL of pure water.

[0095] Take 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 dropwise under magnetic stirring to obtain a pink small-diameter gold nanoparticle solution.

[0096] The above gold nanoparticle solution was mixed with an equal volume of polyethyleneimine-modified Fe3O4@SiO2 solution, ultrasonically stirred for 2 hours, washed 7 times with ultrapure water, and then dispersed in 15 ml of pure water. Then, 15 ml of 2.5 mmol / L PEG was added. 4500 The -SH solution was stirred at room temperature for 6 hours, washed 6 times with pure water, dispersed in an appropriate amount of pure water, and dried at 40°C to obtain AuM microparticles.

[0097] Step 2: Preparation of SKBR3 cell membrane-coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium)

[0098] Step 201: MCF-7 cells were cultured in a complete medium consisting of 15% fetal bovine serum, RPMI-1640 medium, and 1% penicillin-dextrose antibody at 37°C and 5% CO2. Cells in the logarithmic growth phase were collected by trypsin-EDTA digestion and washed three times by centrifugation at 1000g for 8 minutes at 4°C using phosphate buffer. Cells were then resuspended in phosphate buffer. Cells were disrupted using a cell sonicator on ice with the following parameters: sonication for 5 seconds, pause for 8 seconds, 8 cycles, and 45% power. After centrifugation at 3500g for 15 minutes at 4°C, the precipitate was discarded. The supernatant was centrifuged at 20000g for 40 minutes at 4°C, the supernatant was discarded, and the precipitate was washed three times with phosphate buffer and resuspended in phosphate buffer.

[0099] Step 202: Add 200 μL of 10 mg / ml AuM solution to 2 ml of 700 μg / ml cell membrane solution, vortex to mix, incubate on a rotary mixer for 1 hour, wash three times with phosphate buffer, disperse in 300 μL of phosphate buffer to obtain SKBR3 cell membrane coated magnetic microparticles (SKBR3 / CMAuM magnetic screening medium).

[0100] The above magnetic materials were characterized using a dynamic light scattering particle size analyzer, transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), and laser confocal microscopy: hydrated particle size and TEM images (…). Figure 1 The images show that the AuM particle size is in the submicron range (100nm~1000nm), approximately 200nm, indicating that the AuM particles have a high specific surface area and can achieve efficient cell membrane loading. Transmission electron microscopy images show that gold nanoparticles (approximately 4nm) are adsorbed at extremely high density on the surface of magnetic particles, indicating that the AuM particles have the ability to coordinate and bind to cell membranes.

[0101] The magnetic properties of AuM and CMAuM materials were studied using a vibrating sample magnetometer (VSM). It can be seen that ( Figure 2 The magnetization of CMAuM particles coated with SKBR3 is slightly lower than that of AuM particles, but it is still greater than 30 emu / g. CMAuM has a strong magnetization, which can realize the rapid separation of materials from solution under an external magnetic field.

[0102] 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); The cell membrane was stained with Dil (Ex / Em=549 / 565nm), and under a laser confocal microscope, all CMAuM cells emitted red fluorescence ( Figure 3 c). This demonstrates that the cell membrane is fixed to AuM.

[0103] To investigate the efficiency and stability of gold magnetic particle adsorption on cell membranes, the amount of protein before and after adsorption was measured using a BCA protein assay kit to obtain the amount of protein adsorbed on the magnetic particles. The protein mass was used to replace the cell membrane mass, and the membrane adsorption rate and membrane detachment rate were calculated using the following formulas. 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) indicates that 1×PBS is a superior cell membrane coating buffer environment; it can also be seen that the adsorption on the 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) This indicates that the AuM prepared in this invention has a higher adsorption efficiency for cell membranes. Furthermore, by detecting the change in cell membrane shedding rate over time, it was found that after day 8, the cell membrane shedding rate on the Fe3O4@SiO2 surface gradually increased, while the cell membrane shedding rate on the AuM surface remained very low. Figure 4 c) indicates that the AuM surface cell membrane binding stability prepared by this invention is better than that of the traditional Fe3O4@SiO2.

[0104] Film adsorption rate (%) = (mass of film on gold magnetic particle surface / mass of gold magnetic particles) × 100%;

[0105] Membrane shedding rate (%) = (mass of cell membrane in supernatant / initial membrane mass on the surface of gold magnetic particles) × 100%.

[0106] This invention utilizes the aforementioned SKBR3 cell membrane-coated magnetic microparticles for screening effective components against HER2 breast cancer, specifically including the following steps:

[0107] Step 1: Performance Evaluation of CMAuM Screening

[0108] 100 μL of the SKBR3 / CMAuM magnetic screening medium (1 mg / mL) prepared earlier was magnetically separated. After removing the supernatant, 200 μL of a 0.1 mg / mL lapatinib positive drug solution was added. The mixture was vortexed for 10 s and incubated on a rotary mixer for 0-7 min. After incubation, the supernatant was magnetically separated, and the adsorbed supernatant was collected for HPLC analysis to analyze the effect of drug screening time. The chromatographic conditions used were: ODS column, acetonitrile-0.005 mol / L ammonium acetate solution (55:45) mobile phase, 260 nm detection wavelength, and 1.0 mL / min flow rate. The results showed that the adsorption of lapatinib by the SKBR3 / CMAuM magnetic screening medium reached saturation at 5 min. Figure 5 a,b).

[0109] The SKBR3 / CMAuM particles after lapatinide adsorption were washed twice with PBS solution. 100 μL of eluent prepared with a DMSO:H2O ratio of 1:1 (v / v) was added to the magnetic particles. The mixture was vortexed for 10 s, incubated on a rotary mixer for 15 min, and the eluent was collected for HPLC analysis. Figure 5 c shows a clear lapatinib chromatographic peak. These results demonstrate that SKBR3 / CMAuM has the ability to screen for the positive drug lapatinib.

[0110] Step 2: The above-mentioned SKBR3 / CMAuM magnetic screening medium was used to screen for anti-HER2 tumor components in Scutellaria baicalensis root. The specific steps are as follows:

[0111] (1) After pulverizing and sieving the Scutellaria baicalensis root, 1.0 g of powder was placed in a three-necked flask, 100 mL of 70% ethanol was added, and the mixture was refluxed at 80ºC for 3 h. After filtration, the mixture was concentrated to about 5 mL using a vacuum rotary concentrator. The Scutellaria baicalensis root extract was diluted 20 times with 1×PBS, filtered through a 0.22 μm filter membrane, and then used for screening.

[0112] (2) Take 1 mL of the above Scutellaria baicalensis root screening solution and add it to 1 mg of SKBR3 / CMAuM magnetic screening medium and fish for 5 min. Discard the supernatant after magnetic separation, wash twice with PBS, add 500 μL of elution buffer with a volume ratio of DMSO:H2O=1:1 and elute for 15 min. Collect the fishing solution.

[0113] The active ingredients in the fishing liquid were identified using a Shimadzu LCMS-8045 triple quadrupole LC-MS / MS. An ODS column was used; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; gradient elution program: 0-8 min, 10%-60% B, 8-10 min, 60%-90% B, 10-15 min, 90%-100% B; 15-20 min, 100% B; flow rate: 0.3 mL / min; mass spectrometry system used full scan mode, 10-1000 m / z. The results of component analysis are shown in Table 1. Three potential anti-HER2 tumor components were found in Scutellaria baicalensis root: baicalin, wogonin, and baicalein.

[0114] Table 1 Compound Information Table

[0115]

[0116] This invention provides a strategy for high-coverage, stable, and rapid immobilization of cell membranes onto magnetic carriers, and applies this cell membrane magnetic screening medium to the detection of active ingredients in traditional Chinese medicine. In PBS at room temperature and pH 7.3-7.5, the cell membrane self-assembles and is immobilized on the magnetic carrier after 1-2 hours. This environment improves the cell membrane coverage and stability, thereby enhancing the sensitivity and accuracy of the screening medium for traditional Chinese medicine.

[0117] The above description represents the preferred embodiments of the present invention. For those skilled in the art, any equivalent modifications to the present invention without departing from the principles thereof are within the scope of protection of the appended claims.

Claims

1. A cell membrane gold magnetic screening medium for bioactive ligands, characterized in that, It includes gold magnetic microparticles and HER2-positive breast tumor SKBR3 cell membranes coated on the surface of the gold magnetic microparticles. The gold magnetic microparticles include Fe3O4@SiO2 carrier, polyethyleneimine modified on the surface of Fe3O4@SiO2 carrier, gold nanoparticles, and PEG-SH. Add 1 mg of gold magnetic microparticles to every 400 μg~700 μg of cell membrane; The Fe3O4@SiO2 support includes bare Fe3O4 magnets and a SiO2 coating on the surface of the bare Fe3O4 magnets. The particle size of the bare Fe3O4 magnets is 150nm~500nm, and the thickness of the SiO2 coating is 10nm~30nm. The specific steps for preparing the cell membrane gold magnetic screening medium for the bioactive ligand are as follows: The Fe3O4@SiO2 carrier dispersion was mixed with a polyethyleneimine solution, and after sonication and washing, the first reaction solution was obtained. The concentration of the Fe3O4@SiO2 dispersion is 0.2 mg / mL to 0.8 mg / mL; the final concentration of the Fe3O4@SiO2 dispersion after mixing with the polyethyleneimine solution is 0.8 mg / mL to 2 mg / mL. The gold nanoparticle solution and the first reaction solution were mixed, sonicated, washed, and then PEG-SH solution was added. After the reaction, the mixture was dried to obtain gold magnetic microparticles. The specific preparation method of the gold nanoparticle solution is as follows: a chloroauric acid aqueous solution with a concentration of 2 mmol / L to 7 mmol / L is mixed with two volumes of sodium citrate solution with a concentration of 2 mmol / L to 7 mmol / L, and sodium borohydride solution is added dropwise to the mixture to obtain a gold nanoparticle solution with a particle size of less than 10 nm. The first reaction solution and the gold nanoparticle solution were mixed in equal volumes; the concentration of the PEG-SH solution was 1 mmol / L to 2.5 mmol / L. Gold magnetic microparticles were added to a PBS suspension of SKBR3 cell membranes in HER2-positive breast tumors, vortexed and mixed, and bound at room temperature for 1-2 hours. Unbound cell membranes were washed away with PBS to obtain gold magnetic microparticles coated with SKBR3 cell membranes, i.e., 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.

2. A method for extracting an active ingredient against HER2-positive breast tumors, characterized in that, The cell membrane gold magnetic screening medium using the bioactive ligand described in claim 1 is used, and the specific steps are as follows: The cell membrane gold magnetic screening medium, after magnetic separation and supernatant removal, was added to the substrate to be screened and incubated to obtain an incubation mixture; The supernatant of the incubation mixture was magnetically separated and discarded. After washing, elution buffer was added and incubated. The supernatant was then collected to obtain the anti-HER2 breast tumor active ingredient in the substrate to be screened.

3. The method for extracting an active ingredient against HER2 breast tumors as described in claim 2, characterized in that, The substrate to be screened is Scutellaria baicalensis extract.

Images