Method for coating magnetic beads with traditional Chinese medicine active ingredients based on electroporation and application thereof

Abstract

The application discloses a method for fishing traditional Chinese medicine active ingredients by using cell membrane coated magnetic beads based on electroporation, which comprises the following steps: 1, synthesizing Fe3O4@BSA-RGD magnetic nanoparticles by coating and modification; 2, preparing nano magnetic beads coated with complete macrophage membranes by using electroporation; 3, fishing traditional Chinese medicine active ingredients; and the application also discloses application of the method to fishing active ingredients of aconite. The application synthesizes Fe3O4@BSA-RGD magnetic nanoparticles which are good in biocompatibility and have membrane targeting, and then the magnetic nanoparticles are co-cultured with target cells macrophages and electroporation is carried out, so that a nano magnetic bead system coated with complete macrophage membranes is constructed, the structural integrity of the cell membranes is ensured and the outer surface of the cell membranes faces outward, the traditional coating method does not distinguish the membrane outer / inner surface orientation, the number of target points on the surface of the fishing carrier is increased, and the accuracy and sensitivity of screening of traditional Chinese medicine active ingredients are improved; and the application realizes fishing of anti-inflammatory active ingredients in aconite.

Description

Technical Field

[0001] This invention belongs to the field of screening technology for active ingredients of traditional Chinese medicine, specifically relating to a method for fishing for active ingredients of traditional Chinese medicine based on cell membrane-coated magnetic beads using electroporation and its application. Background Technology

[0002] Traditional research on the active ingredients of traditional Chinese medicine (TCM) typically employs a combination of bioeffect-guided systematic separation and pharmacological experiments. However, TCM components are complex, and the content of active ingredients is often low. Therefore, the separation and analysis procedures are cumbersome, labor-intensive, time-consuming, and have a low success rate. Furthermore, this approach neglects the holistic effect of TCM as a "multi-component, multi-target" system.

[0003] Finding rapid screening methods and technologies for active ingredients has always been a focus for pharmaceutical companies both domestically and internationally. To date, over 500 drug targets have been identified, with more than 50% of these drugs targeting membrane receptors. Therefore, direct drug screening centered on membrane receptor targets has become an effective method for discovering lead compounds. In recent years, using magnetic nanospheres as carriers and cell membrane vesicles as functional coatings, cell membrane-functionalized magnetic nanospheres have been prepared. Under the directional control of an external magnetic field, their magnetic responsiveness and high selectivity of membrane surface affinity groups allow for the extraction of active substances from complex biological systems. This technology has been successfully applied to the screening of cell membrane-bound active ingredients in traditional Chinese medicine. However, the preparation of membrane receptor-coated magnetic beads remains a bottleneck restricting the development of this technology.

[0004] Traditional methods for coating cell membranes with magnetic nanospheres require two steps: first, ultrasonic disruption of cells to prepare cell membrane fragments; second, thorough mixing of the magnetic nanospheres and cell membrane fragments under mechanical stirring or ultrasonic treatment, followed by static settling, allows the magnetic nanospheres to physically adsorb the phospholipid bilayer of the cell membrane, thus forming a uniform cell membrane layer on its surface. While this traditional method effectively coats the cell membrane onto the surface of magnetic nanospheres, it still faces two main challenges: first, ultrasonic treatment / mechanical force damages the integrity of the cell membrane, reducing its function and potentially leading to conformational changes or even denaturation of membrane proteins, thus decreasing the accuracy of drug screening results; second, this physical adsorption process cannot distinguish between the inner and outer surfaces of the cell membrane, resulting in unclear drug screening sites. Furthermore, the coexistence of receptors on both the inner and outer surfaces reduces the number of drug screening sites per unit area, lowering the sensitivity of related screening methods. These problems limit the further development and application of cell membrane-coated magnetic bead fishing methods. Since the outer surface of the cell membrane contains a large number of ligand-binding domains for drug targets, it is of great significance to establish a method for fishing for active ingredients of traditional Chinese medicine by coating nano-magnetic beads with the outer surface of the intact cell membrane facing outward.

[0005] Electroporation is a physical process that creates temporary micropores in the cell membrane under the influence of a short-term applied electric field, increasing its permeability. This process allows molecules that would normally be unable to cross the cell membrane to freely enter and exit the cell. Once the electric field is removed, the micropores close without affecting the cell. This characteristic has led to the widespread use of electroporation in gene transfection and the construction of membrane-masked nanomedicine delivery systems. However, its application in the research of cell membrane-based active ingredient extraction from traditional Chinese medicine is not yet widespread.

[0006] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by erosive arthritis and involving multiple target cells. Macrophages play a central role in the immune system of RA due to their multifaceted functions, including secreting pro-inflammatory factors, chemokines, and tissue-degrading enzymes. Macrophages have become important target cells in RA treatment, and their membrane receptors are key targets for the exertion of anti-RA substances. Aconitum carmichaelii (Fuzi) can warm and tonify kidney yang, dispel wind and cold, and possesses anti-inflammatory and analgesic pharmacological activities, making it clinically used to treat arthritis; however, its anti-inflammatory active components remain unclear. Summary of the Invention

[0007] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for electroporation-based cell membrane-coated magnetic beads for screening active ingredients in traditional Chinese medicine. This method prepares biocompatible Fe3O4@BSA-RGD magnetic nanoparticles with membrane targeting capabilities, which are then co-cultured with target macrophages and subjected to electroporation to construct a complete macrophage membrane-coated magnetic bead system for screening active ingredients in traditional Chinese medicine. This method ensures the structural integrity of the cell membrane and the outward orientation of the outer surface, unlike traditional coating methods that do not distinguish between the outer and inner surface orientations. It also increases the number of target points on the surface of the screening carrier, improving the accuracy and sensitivity of screening for active ingredients in traditional Chinese medicine.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a method for fishing for active ingredients of traditional Chinese medicine using cell membrane-coated magnetic beads based on electroporation, characterized in that the method includes the following steps:

[0009] Step 1: Synthesis of Fe3O4@BSA-RDG magnetic nanoparticles

[0010] Step 101, Synthesis of Fe3O4 magnetic nanoparticles: Under nitrogen protection throughout the process, acetylacetone iron was first dissolved in a mixed solution of oleylamine and oleic acid under magnetic stirring to obtain acetylacetone iron solution. Then, N-methylpyrrolidone was heated to reflux under mechanical stirring. The acetylacetone iron solution was then added dropwise to the heated and refluxed N-methylpyrrolidone to carry out the reaction. After cooling to room temperature, the mixture was magnetically separated and washed to obtain Fe3O4 magnetic nanoparticles.

[0011] Step 102, Synthesis of Fe3O4@BSA magnetic nanoparticles: Bovine serum albumin and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were stirred at room temperature to obtain a BSA solution. Then, the Fe3O4 magnetic nanoparticles obtained in step 101 were added to the BSA solution and the reaction was continued by stirring. After magnetic separation, washing and freeze-drying, bovine serum albumin-coated Fe3O4 magnetic nanoparticles, i.e., Fe3O4@BSA magnetic nanoparticles, were obtained.

[0012] Step 103, Synthesis of Fe3O4@BSA-RGD magnetic nanoparticles: The Fe3O4@BSA magnetic nanoparticles obtained in step 102 were dispersed in deionized water and PBS was added for ultrasonic dispersion to obtain a Fe3O4@BSA magnetic nanoparticle dispersion. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to the Fe3O4@BSA magnetic nanoparticle dispersion and stirred under light-protected conditions. N-hydroxysuccinimide was added and stirred under light-protected conditions. Then, RGD was added and stirred at room temperature under light-protected conditions to carry out the reaction. After magnetic separation and washing, RGD-modified Fe3O4@BSA magnetic nanoparticles, namely Fe3O4@BSA-RGD magnetic nanoparticles, were obtained.

[0013] Step 2: Preparation of nanomagnetic beads coated with intact macrophage cell membranes

[0014] Step 201: Seed macrophages in DMEM medium and culture them in a constant temperature incubator until the cell density reaches 70%~80%. After discarding the culture medium, add the macrophages to a culture medium containing Fe3O4@BSA-RGD magnetic nanoparticles from step 1 for co-culture to obtain a co-culture medium.

[0015] Step 202: The co-culture medium obtained in step 201 was digested with trypsin. Cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles were collected by magnetic separation. The cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles were resuspended in a culture medium containing Fe3O4@BSA-RGD magnetic nanoparticles to obtain a cell suspension. The cell suspension was then aspirated into an electroporation cuvette and incubated on ice. Electroporation was then performed using an electroporator, and the cells were immediately transferred to an ice bath. After adding preheated culture medium, the cells were placed in an incubator for culture. After washing with DMEM culture medium, macrophages loaded with Fe3O4@BSA-RGD magnetic nanoparticles were obtained, i.e., complete macrophage membranes coated with RAW-Fe3O4@BSA-RGD magnetic nanoparticles.

[0016] Step 3: Active ingredients of traditional Chinese medicine for fishing

[0017] Step 301: Prepare the traditional Chinese medicine extract;

[0018] Step 302: The macrophage cell membrane prepared in step 2 is coated with magnetic nanoparticles and suspended to obtain a suspension. Then, the traditional Chinese medicine extract prepared in step 301 is added to the suspension and placed in an incubator for culture. The culture medium is then separated by magnetic solid-liquid separation to obtain a precipitate. The precipitate is washed with buffer solution and then eluted by shaking with an organic solvent. The fishing liquid containing the active ingredients of traditional Chinese medicine is obtained by magnetic solid-liquid separation.

[0019] This invention first prepares Fe3O4 magnetic nanoparticles, then coats them with bovine serum albumin (BSA) to obtain Fe3O4@BSA magnetic nanoparticles. Next, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added sequentially with stirring. RGD is then added to modify the Fe3O4@BSA magnetic nanoparticles. Utilizing the good biocompatibility of BSA and the easy binding of RGD to receptors on cell membranes, biocompatible and membrane-targeting Fe3O4@BSA-RGD magnetic nanoparticles are obtained. Then, target cells, namely macrophages, are co-cultured with a culture medium containing Fe3O4@BSA-RGD magnetic nanoparticles. The phagocytic function of macrophages allows the Fe3O4@BSA-RGD magnetic nanoparticles to enter the macrophages. After trypsin digestion to disperse the cells and magnetic separation, cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles are obtained and resuspended in a culture medium containing Fe3O4@BSA. Cell suspensions were obtained in a culture medium containing A-RGD magnetic nanoparticles and then incubated on ice to maintain cell viability. Electroporation was then performed using an electroporator, which, under the influence of electrical pulses, created micropores in the cell membrane and increased its permeability, allowing the Fe3O4@BSA-RGD magnetic nanoparticles in the cell suspension to be transferred into the cells. After cultivation, Fe3O4@BSA-RGD magnetic nanoparticles were obtained, thus constructing a complete macrophage membrane-coated magnetic bead system. This system eliminates the need for pre-disruption of cells, ensuring the structural integrity of the cell membrane and preventing changes or even denaturation of cell membrane protein conformations. It also preserves the structure and activity of cell membrane surface receptors and ensures that the outer surface of the cell membrane faces outwards. Compared to traditional coating methods that do not distinguish between the outer and inner surfaces, this method increases the number of target sites on the carrier surface. When cultured with traditional Chinese medicine extracts, the selectivity of the cell membrane for active ingredients in this system allows for the extraction of active ingredients from complex traditional Chinese medicine biological systems, improving the accuracy and sensitivity of screening for active ingredients in traditional Chinese medicine.

[0020] In this invention, RGD is Arg-Gly-Asp (arginyl-glycyl-aspartic acid) tripeptide.

[0021] The above-mentioned method for fishing for active ingredients of traditional Chinese medicine by electroporation-based cell membrane coating magnetic beads is characterized in that the heating reflux in step 101 uses a rotation speed of 600 rpm, a temperature of 200℃, and a time of 1 h.

[0022] The above-mentioned method for fishing for active ingredients of traditional Chinese medicine by electroporation-based cell membrane coating magnetic beads is characterized in that, in step 103, the stirring speed after adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 600 rpm for 30 min, the stirring speed after adding N-hydroxysuccinimide is 1000 rpm for 1 h, and the stirring speed after adding RGD is 1000 rpm for 24 h.

[0023] The above-mentioned method for fishing for active ingredients of traditional Chinese medicine by encapsulating cell membranes with magnetic beads based on electroporation is characterized in that the culture conditions in step 201 are: 37℃, 5% CO2 constant temperature incubator; the co-culture conditions are: 37℃, 5% CO2 constant temperature incubator, for 6 hours.

[0024] The above-mentioned method for fishing for active ingredients of traditional Chinese medicine by encapsulating cell membranes with magnetic beads based on electroporation is characterized in that the electroporation parameters in step 202 are: pulse voltage 300V, pulse time 20ms, pulse interval time 5s, and 4 consecutive pulses.

[0025] In addition, the present invention also discloses an application of the above-described method for fishing for active ingredients of traditional Chinese medicine using cell membrane-coated magnetic beads based on electroporation, characterized in that the method is applied to fishing for active ingredients of Aconitum carmichaelii.

[0026] The above application is characterized by the following steps:

[0027] Step 1: Soak Aconitum carmichaelii in purified water and then decoct it. While still hot, filter it through gauze once to obtain a primary filtrate and a primary residue. Decoction the primary residue in purified water and filter it through gauze a second time while still hot to obtain a secondary filtrate and a secondary residue. Combine the primary and secondary filtrates and concentrate them under reduced pressure to obtain a concentrated Aconitum carmichaelii extract. Dilute the concentrated Aconitum carmichaelii extract and filter it through a 0.22μm microporous membrane to remove bacteria to obtain an Aconitum carmichaelii extract solution. Analyze the chemical components in the Aconitum carmichaelii extract solution.

[0028] Step 2: The Aconitum carmichaelii extract solution obtained in Step 1 is added to the suspension prepared by suspending macrophage membrane-coated magnetic nanoparticles, and then placed in an incubator for incubation. The culture medium is then subjected to magnetic solid-liquid separation to obtain a precipitate. The precipitate is eluted with PBS buffer and then eluted with methanol by shaking. The fishing liquid containing Aconitum carmichaelii chemical components is obtained by magnetic solid-liquid separation. The chemical components in the fishing liquid are analyzed, and ion extraction analysis is performed based on the chemical components in the Aconitum carmichaelii extract solution obtained in Step 1 to obtain the active components of Aconitum carmichaelii.

[0029] This invention uses macrophages, an important target cell in the treatment of rheumatoid arthritis, as the cell membrane raw material to prepare intact macrophage membrane-coated magnetic beads. These beads are then cultured with a solution of Aconitum carmichaelii extract, a traditional Chinese medicine used to treat arthritis. This allows the anti-inflammatory active components in the Aconitum carmichaelii extract to selectively and specifically bind to receptor target sites on the surface of the macrophage membrane in the intact macrophage membrane-coated magnetic beads (a process known as "fishing"). After rinsing with PBS buffer solution, the beads are eluted with methanol and then removed by magnetic solid-liquid separation to obtain a fishing solution containing Aconitum carmichaelii chemical components, ultimately yielding the active components of Aconitum carmichaelii. This method utilizes the integrity of the macrophage membrane on the surface of the magnetic nanobeads to enhance the adsorption performance of the receptor targets on the macrophage membrane surface for the active components in Aconitum carmichaelii, thereby improving the accuracy of the screening results. Furthermore, since the outer surface of the macrophage membrane on the surface of the magnetic nanobeads is facing outwards, unlike traditional coating methods that do not distinguish between the outer and inner surface orientations, this method relatively increases the number of target points on the carrier surface, improving the sensitivity of the screening and enabling the identification of anti-inflammatory active components in Aconitum carmichaelii.

[0030] The above application is characterized by using ultra-high performance liquid chromatography-mass spectrometry to analyze the chemical components in the Aconitum carmichaelii extract solution in step one and the chemical components in the fishing liquid in step two, obtaining ion chromatograms of the chemical components in the Aconitum carmichaelii extract solution and the fishing liquid, respectively, and then performing ion extraction analysis to obtain the active components of Aconitum carmichaelii.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1. This invention employs a coating and modification method to prepare biocompatible Fe3O4@BSA-RGD magnetic nanoparticles with membrane targeting capabilities. These nanoparticles are then co-cultured with target macrophages and resuspended for electroporation, thereby constructing a complete macrophage membrane-coated magnetic nanoparticle system for screening active ingredients of traditional Chinese medicine. The electroporation process enhances cell membrane permeability, allowing the Fe3O4@BSA-RGD magnetic nanoparticles to enter macrophages, ensuring the structural integrity of the cell membrane within the system. This, in turn, preserves the structure and activity of receptors on the cell membrane surface. Furthermore, the outer surface of the cell membrane faces outwards, unlike traditional coating methods that do not differentiate between outer and inner surface orientations. This increases the number of target sites on the carrier surface, improving the accuracy and sensitivity of screening active ingredients of traditional Chinese medicine.

[0033] 2. This invention utilizes the characteristics of cell membrane electroporation combined with magnetic bead fishing to construct a method for fishing for active ingredients of traditional Chinese medicine by encapsulating nano-magnetic beads with intact cell membranes. It has high specificity, good selectivity, low workload, short cycle, and high success rate, and is suitable for screening active ingredients of traditional Chinese medicine.

[0034] 3. This invention adds an electroporation process to the preparation of magnetic nanoparticles, eliminating the need for ultrasonic cell disruption and mechanical stirring to promote the mixing and adsorption of magnetic nanoparticles and cell membrane fragments, thus saving corresponding process steps and avoiding damage to the macrophage cell membrane-coated magnetic bead system.

[0035] 4. This invention applies the constructed complete macrophage membrane-coated nanomagnetic bead system to the screening of active ingredients of Aconitum carmichaelii, a traditional Chinese medicine for treating arthritis. By using receptors on the macrophage membrane as recognition carriers for active ingredients in arthritis treatment, the anti-inflammatory active ingredients in Aconitum carmichaelii are screened, thereby identifying the anti-inflammatory active ingredients in Aconitum carmichaelii.

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0037] Figure 1 This is a transmission electron microscope (TEM) characterization image of the Fe3O4@BSA magnetic nanoparticles obtained in Example 1 of this invention.

[0038] Figure 2 The magnetic hysteresis loop is the magnetic regression line of the intact macrophage membrane coated with nano-magnetic beads obtained in Example 1 of the present invention.

[0039] Figure 3 This is a fluorescence confocal image obtained in Example 1 of the present invention.

[0040] Figure 4 The chromatograms are obtained by ultra-high performance liquid chromatography-mass spectrometry analysis of Aconitum carmichaelii extract solution A0, blank control solution A4, and fishing solution A5 obtained in Example 1 of this invention. Detailed Implementation

[0041] Example 1

[0042] This embodiment includes the following steps:

[0043] Step 1: Synthesis of Fe3O4@BSA-RDG magnetic nanoparticles

[0044] Step 101, Synthesis of Fe3O4 magnetic nanoparticles: Under nitrogen protection throughout the process, 1.0 g of acetylacetone iron was first dissolved in 20 mL of a mixed solution prepared by equal volumes of oleylamine and oleic acid under magnetic stirring to obtain acetylacetone iron solution. Then, 90 mL of N-methylpyrrolidone was added to a 250 mL three-necked flask and heated to 200 °C and refluxed for 1 h under mechanical stirring at 600 rpm. Then, the acetylacetone iron solution was added dropwise to the heated and refluxed N-methylpyrrolidone using a syringe to react for 1 h. After cooling to room temperature, Fe3O4 magnetic nanoparticles were obtained by magnetic separation and washing.

[0045] Step 102, Synthesis of Fe3O4@BSA magnetic nanoparticles: 120 mg bovine serum albumin and 70 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to a round-bottom flask containing 50 mL of PBS buffer solution. The mixture was stirred at room temperature for 15 min to obtain a BSA solution. Then, 100 mg of the Fe3O4 magnetic nanoparticles obtained in step 101 were added to the BSA solution and the reaction was continued for 24 h. After magnetic separation, washing and freeze-drying, bovine serum albumin-coated Fe3O4 magnetic nanoparticles, i.e., Fe3O4@BSA magnetic nanoparticles, were obtained.

[0046] Step 103, Synthesis of Fe3O4@BSA-RGD magnetic nanoparticles: 40 mg of Fe3O4@BSA magnetic nanoparticles obtained in step 102 were dispersed in 48 mL of deionized water, and 2 mL of pH 7.4 PBS was added and ultrasonically dispersed for 10 min to obtain Fe3O4@BSA magnetic nanoparticle dispersion. Then, 230 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to the Fe3O4@BSA magnetic nanoparticle dispersion and stirred at 600 rpm for 30 min under light-protected conditions. 50 mg of N-hydroxysuccinimide was added and stirred at 1000 rpm for 1 h under light-protected conditions. Then, 15 mL of 1 mg / mL RGD solution was added and the reaction was carried out at 1000 rpm for 24 h under light-protected conditions at room temperature. After magnetic separation and washing, RGD-modified Fe3O4@BSA magnetic nanoparticles, i.e., Fe3O4@BSA-RGD magnetic nanoparticles, were obtained.

[0047] Step 2: Preparation of nanomagnetic beads coated with intact macrophage cell membranes

[0048] Step 201: Macrophages (RAW264.7) were processed at a rate of 4 × 10⁻⁶. 5 Ten dishes were seeded at a density of 100 mm in diameter and filled with DMEM medium. The dishes were then incubated at 37°C and 5% CO2 until the cell density reached 70%–80%. After discarding the culture medium, the cells were added at a seeding rate of 150 μg / mL to DMEM medium containing Fe3O4@BSA-RGD magnetic nanoparticles from step one. The mixture was then co-cultured at 37°C and 5% CO2 for 6 hours to obtain the co-culture medium.

[0049] Step 202: The co-culture medium obtained in step 201 was digested with trypsin, and cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles were collected by magnetic separation. 1×10⁻⁶ cells were then added. 7Cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles were resuspended at a concentration of 300 μg / mL in a culture medium containing Fe3O4@BSA-RGD magnetic nanoparticles to obtain a cell suspension. The cell suspension was then transferred to an electroporation cuvette and incubated on ice for 10 min. Electroporation was then performed using a Biorad GenePulser Xcell with a pulse voltage of 300 V, a pulse duration of 20 ms, a pulse interval of 5 s, and four consecutive pulses. The cells were immediately transferred to an ice bath, preheated culture medium was added, and the cells were transferred to a 150 mm diameter culture dish and incubated for 1 h. After washing three times with DMEM culture medium, unbound Fe3O4@BSA-RGD magnetic nanoparticles were removed to obtain macrophages loaded with Fe3O4@BSA-RGD magnetic nanoparticles, i.e., macrophages with intact macrophage membranes coated with RAW-Fe3O4@BSA-RDG magnetic nanoparticles.

[0050] Step 3: Active ingredients of Aconitum carmichaelii (a traditional Chinese medicine) used in fishing.

[0051] Step 301: Preparation of Aconitum carmichaelii extract: Weigh 20g of Aconitum carmichaelii, soak it in 200mL of purified water for 1 hour, and then decoct it for 2 hours. While hot, filter it through four layers of gauze to obtain a primary filtrate and a primary residue. Decoction the primary residue in 200mL of purified water for 1 hour, and then filter it through four layers of gauze to obtain a secondary filtrate and a secondary residue. Combine the primary and secondary filtrates and concentrate them under reduced pressure to obtain a 1g / mL Aconitum carmichaelii extract concentrate. Dilute the Aconitum carmichaelii extract concentrate with sterile water for injection and filter it through a 0.22μm microporous membrane to remove bacteria, obtaining a 1mg / mL Aconitum carmichaelii extract solution, denoted as A0.

[0052] The chemical components in Aconitum carmichaelii extract solution A0 were analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). Specific analytical conditions were as follows: the liquid chromatography system was an Agilent 1290 Infinity HPLC system, the column temperature was 30℃, and the chromatographic column used was an Acquity UPLCHSS T3 C column. 18A 2.1 mm × 100 mm, 1.7 μm column was used. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile. Gradient elution was employed with the following gradient settings: 0-2 min, 5%-5% B; 2-13 min, 5%-95% B; 13-15 min, 95%-95% B; column equilibration for 5 min; flow rate 400 μL / min; injection volume 4 μL. The mass spectrometer was an Agilent 6530 Accurate-Mass Q-TOFMS tandem quadrupole-time-of-flight mass spectrometer equipped with an electrospray ionization (ESI) source. Detection was performed in positive ion mode. Detection parameters included: capillary voltage 3500 V, drying gas flow rate 11 L / min, drying gas temperature 350 °C, spray pressure 45 psig, fragmentation voltage 120 V, Skimmer voltage 60 V, and data acquisition range. m / z 50~1000; The chromatogram of the aconite extract solution A0 obtained by analysis is shown in the ultra-high performance liquid chromatography-mass spectrometry analysis chromatogram. Figure 4 As shown in Table 1, the analytical results of the chemical components in the obtained Aconitum carmichaelii extract solution A0 are presented.

[0053] Table 1

[0054]

[0055] As shown in Table 1, there are 18 chemical components in Aconitum carmichaelii extract solution A0;

[0056] Step 302: Add 1 mL of the Aconitum carmichaelii extract solution A0 obtained in Step 1 to 9 mL of the suspension prepared in Step 2, which consists of intact macrophage membrane-coated magnetic nanoparticles. Incubate at 37°C for 2 hours. Then, use magnetic solid-liquid separation to obtain a precipitate. Collect the supernatant and label it A1. Wash the precipitate with 1 mL of PBS buffer and collect the supernatant, labeling it A2. Repeat the elution process twice, collecting the supernatants A3 and A4 respectively. Since the last wash removes non-specific adsorption... After elution, the supernatant contained virtually no impurities. Supernatant A4 was used as the blank control solution A4. Finally, 1 mL of methanol was added and the mixture was gently shaken for 10 min. Magnetic solid-liquid separation was performed to obtain a fishing solution containing Aconitum carmichaelii chemical components, denoted as A5. Blank control solution A4 and fishing solution A5 were centrifuged at 12000 rpm. The resulting supernatants were analyzed according to the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) conditions described in step 301. The UHPLC-MS / MS chromatograms of blank control solution A4 and fishing solution A5 are shown below. Figure 4As shown, the extraction ion chromatography (EIC) function of Agilent Mass Hunter software was used to perform ion analysis on the ion chromatograms of the 18 chemical components of the Aconitum carmichaelii extract solution A0 in step 301 in the blank control solution A4 and the fishing solution A5. The mass window was ±20 ppm, and the active components of Aconitum carmichaelii were obtained.

[0057] Figure 1 The image shows the transmission electron microscopy (TEM) characterization of the Fe3O4@BSA magnetic nanoparticles obtained in this embodiment. Figure 1 It can be seen that the average particle size of the Fe3O4@BSA magnetic nanoparticles is 41nm±3nm.

[0058] Figure 2 The hysteresis loop of the intact macrophage membrane coated with nano-magnetic beads obtained in this embodiment was measured by a vibrating sample magnetometer (VSM) at room temperature. Figure 2 It is known that the intact macrophage membrane-coated magnetic nanobeads exhibit excellent superparamagnetism, with coercivity and remanence both approaching zero. Their saturation magnetization is approximately 45.7 emu / g. Therefore, these superparamagnetic intact macrophage membrane-coated magnetic nanobeads will be magnetized under an external magnetic field, generating strong magnetism. Once the external magnetic field is removed, there is no remanence. This characteristic makes the intact macrophage membrane-coated magnetic nanobeads highly valuable for separating and targeting active components acting on macrophage membranes.

[0059] The Fe3O4@BSA-RGD magnetic nanoparticles obtained in this embodiment were treated with Rhodamine B fluorescent dye to load them with Rhodamine B fluorescent dye, resulting in Fe3O4@BSA-RGD magnetic nanoparticles loaded with Rhodamine B fluorescent dye. Then, the macrophage membrane-coated magnetic nanoparticles preparation process in step two was performed to obtain complete macrophage membrane-coated Rhodamine B-loaded magnetic nanoparticles. The complete macrophage membrane-coated Rhodamine B-loaded magnetic nanoparticles were seeded in laser confocal microscopy culture dishes and cultured overnight. Samples were then fixed with 4% paraformaldehyde, and the cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI). Cell membrane staining was performed using a DIO cell membrane green fluorescent probe, and the staining was performed using a laser confocal microscope (LeicaTCS). SP8) recorded images, in which a 405nm laser was used to excite DAPI-stained cell nuclei, a 488nm laser was used to excite DIO-stained cell membranes, and a 552nm laser was used to excite Rhodamine B fluorescent dye loaded in Fe3O4@BSA-RGD magnetic nanoparticles. Fluorescence was collected for the corresponding wavelength ranges, and the resulting fluorescence confocal images are shown below. Figure 3 As shown.

[0060] Figure 3 This is a fluorescence confocal image obtained in this embodiment. All scale bars in the image are 10 μm. Figure 3 As can be seen, the cell nucleus stained samples corresponding to DAPI showed blue fluorescence after excitation, the cell membrane stained samples corresponding to DIO showed green fluorescence after excitation, the Rhodamine B fluorescent dye loaded in Fe3O4@BSA-RGD magnetic nanoparticles showed red fluorescence after excitation, and the intact macrophage membrane coated with Rhodamine B magnetic beads (Merge image in the figure) showed green fluorescence on the outside and blue fluorescence in the core after excitation, with red fluorescence interspersed in the core. This indicates that the Fe3O4@BSA-RGD magnetic nanoparticles in the intact macrophage membrane coated with magnetic beads prepared by electroporation in this embodiment have entered the interior of the macrophage membrane, and the macrophage membrane structure is intact with the outer surface of the membrane facing outward, thereby ensuring the structure and activity of the cell membrane surface receptors, increasing the ligand binding domain of the target sites on the outer surface of the cell membrane, which is beneficial to improving the accuracy and sensitivity of screening active ingredients of traditional Chinese medicine.

[0061] Figure 4 The chromatograms for ultra-high performance liquid chromatography-mass spectrometry analysis of Aconitum carmichaelii extract solution A0, blank control solution A4, and fishing solution A5 obtained in this embodiment are shown below. Figure 4 It can be seen that the Aconitum carmichaelii extract solution A0 corresponds to 18 chromatographic peaks (peak 1 to peak 18), the blank control solution A4 does not have any chromatographic peaks related to Aconitum carmichaelii chemical components, while the fishing solution A5 corresponds to 3 obvious chromatographic peaks (peak 7, peak 15, peak 16). By comparing with the results in Table 1, it can be seen that peak 7, peak 15, and peak 16 represent Songguoling, benzoyl aconitine, and benzoyl hypoaconitine, which are the active components in the chemical components of Aconitum carmichaelii.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for fishing traditional Chinese medicine active ingredients based on electroporation of cell membrane-coated magnetic beads, characterized in that, The method comprises the following steps: Step one, synthesis of Fe3O4@BSA-RGD magnetic nanoparticles Step 101, synthesis of Fe3O4 magnetic nanoparticles: under the protection of nitrogen gas, first, dissolve iron acetylacetonate in the mixed solution of oleylamine and oleic acid under magnetic stirring to obtain an iron acetylacetonate solution, then heat N-methylpyrrolidone under mechanical stirring to reflux, and then add the iron acetylacetonate solution dropwise into the refluxed N-methylpyrrolidone for reaction, and after cooling to room temperature, magnetic separation, washing, and drying, Fe3O4 magnetic nanoparticles are obtained; Step 102, synthesis of Fe3O4@BSA magnetic nanoparticles: stir bovine serum albumin and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at room temperature to obtain a BSA solution, then add the Fe3O4 magnetic nanoparticles obtained in step 101 into the BSA solution for further stirring and reaction, and then sequentially perform magnetic separation, washing, and freeze-drying to obtain bovine serum albumin-coated Fe3O4 magnetic nanoparticles, namely Fe3O4@BSA magnetic nanoparticles; Step 103, synthesis of Fe3O4@BSA-RGD magnetic nanoparticles: disperse the Fe3O4@BSA magnetic nanoparticles obtained in step 102 in deionized water, and add PBS for ultrasonic dispersion to obtain a Fe3O4@BSA magnetic nanoparticle dispersion, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride into the Fe3O4@BSA magnetic nanoparticle dispersion for stirring under light shielding, continue to add N-hydroxysuccinimide for stirring under light shielding, and then add RGD for stirring under light shielding at room temperature to perform reaction, and then perform magnetic separation, washing, and drying to obtain RGD-modified Fe3O4@BSA magnetic nanoparticles, namely Fe3O4@BSA-RGD magnetic nanoparticles; Step two, preparation of nanometer magnetic beads coated with complete macrophage membranes Step 201, inoculate macrophages into DMEM culture medium, and place into a constant-temperature incubator for culture until the cell density is 70% to 80%, then discard the culture medium, and add into the culture medium containing the Fe3O4@BSA-RGD magnetic nanoparticles in step one for co-culture to obtain a co-culture solution; Step 202, trypsin digestion of the co-culture solution obtained in step 201, collect the cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles by magnetic separation, resuspend the cells loaded with Fe3O4@BSA-RGD magnetic nanoparticles in the culture medium containing Fe3O4@BSA-RGD magnetic nanoparticles to obtain a cell suspension, then suck the cell suspension into an electroporation cuvette and incubate on ice, then perform electroporation using an electroporator, immediately transfer into an ice bath, add preheated culture medium and place in an incubator for culture, wash with DMEM culture medium, and obtain macrophages loaded with Fe3O4@BSA-RGD magnetic nanoparticles, i.e. intact macrophage membrane-coated nanomagnetic beads RAW-Fe3O4@BSA-RDG; the parameters for electroporation are: pulse voltage 300V, pulse time 20ms, pulse interval time 5s, and 4 continuous pulses; Step three, fishing active ingredients of traditional Chinese medicine Step 301, prepare a traditional Chinese medicine extract; Step 302, suspend RAW-Fe3O4@BSA-RDG prepared in step two to obtain a suspension, then add the traditional Chinese medicine extract prepared in step 301 to the suspension and place in an incubator for culture, then perform magnetic solid-liquid separation on the culture solution to obtain a precipitate, wash the precipitate with a buffer solution, then perform organic solvent shaking elution, and perform magnetic solid-liquid separation to obtain a fishing solution containing active ingredients of traditional Chinese medicine.

2. The method according to claim 1, wherein the method is characterized in that, The rotation speed used in the heating reflux in step 101 is 600rpm, the temperature is 200℃, and the time is 1h.

3. The method according to claim 1, wherein the method is characterized in that, The rotation speed used in the stirring after adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in step 103 is 600rpm, the time is 30min, the rotation speed used in the stirring after adding N-hydroxysuccinimide is 1000rpm, the time is 1h, the rotation speed used in the stirring after adding RGD is 1000rpm, and the time is 24h.

4. The method according to claim 1, wherein the method is characterized in that, The culture conditions in step 201 are: 37℃, 5% CO2 constant temperature incubator; the co-culture conditions are: 37℃, 5% CO2 constant temperature incubator, and the time is 6h.

5. The use of the method for coating magnetic beads with traditional Chinese medicine active ingredients based on electroporation of cell membranes according to any one of claims 1-4, characterized in that, Application of the method in fishing active ingredients of aconite.

6. Use according to claim 5, characterized in that, The method comprises the following steps: Step one, soak aconite in purified water and decoct, filter once while hot through gauze to obtain a first filtrate and a first residue, decoct the first residue with purified water, filter twice while hot through gauze to obtain a second filtrate and a second residue, combine the first filtrate and the second filtrate and perform vacuum concentration to obtain an aconite extract concentrate, dilute the aconite extract concentrate and filter through a 0.22μm microporous filter membrane to obtain an aconite extract solution, and analyze the chemical components in the aconite extract solution; Step two, the solution of the extract of aconite obtained in step one is added to the suspension prepared by RAW-Fe3O4@BSA-RDG suspension, and is placed in an incubator for incubation, and then the culture solution is subjected to magnetic solid-liquid separation to obtain a precipitate, the precipitate is eluted with PBS buffer, and then is eluted by shaking with solvent methanol, and then is subjected to magnetic solid-liquid separation to obtain a fishing solution containing the chemical components of aconite, the chemical components in the fishing solution are analyzed, and ion extraction analysis is performed according to the chemical components in the solution of the extract of aconite obtained in step one, to obtain the active components of aconite.

7. Use according to claim 6, characterized in that, The chemical components in the solution of the extract of aconite in step one and the chemical components in the fishing solution in step two are analyzed by using ultra-high performance liquid chromatography-mass spectrometry, ion chromatograms of the chemical components of the solution of the extract of aconite and the fishing solution are obtained respectively, and then ion extraction analysis is performed, to obtain the active components of aconite.

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