Magnetic hydrothermal carbon material reversely wrapped by unmarked cell membrane as well as preparation method and application of magnetic hydrothermal carbon material

Abstract

The invention discloses a magnetic hydrothermal carbon material reversely wrapped by an unmarked cell membrane as well as a preparation method and application of the magnetic hydrothermal carbon material, magnetic carbon nanospheres are prepared by a hydrothermal method, and then carboxyl groups on the surfaces of the magnetic carbon nanospheres and residual amino groups of lectin such as concanavalin A are subjected to amide condensation, so that lectinized hydrothermal magnetic carbon nanospheres are obtained. Finally, agglutination reaction can be carried out on agglutinin and carbohydrate chains on the outer surface of the cell membrane, so that the cell membrane is wrapped from inside to outside. The biomimetic material can screen drugs acting on the inner side of a cell membrane, does not need to label the cell membrane, can reduce damage to membrane protein, provides a new technical means for deeply exploring lead compounds in natural products, and promotes development of traditional Chinese medicine anti-osteoporosis drugs. According to the invention, two novel potential anti-osteoporosis components targeting the inner surface of an osteoblast cell membrane in herba epimedii are successfully found.

Description

Technical Field

[0001] This invention belongs to the field of nanobiotechnology, specifically relating to a label-free cell membrane reverse-encapsulation magnetic hydrothermal carbon material based on agglutination reaction, its preparation method, and its application in the discovery of active substances in traditional Chinese medicine. Background Technology

[0002] Discovering the pharmacodynamic material basis of traditional Chinese medicine (TCM) is an important aspect of TCM modernization. However, the TCM system is complex, and traditional solvent extraction-based screening of TCM active ingredients is often time-consuming, labor-intensive, and difficult, and easily misses trace and ultra-trace active ingredients. In recent years, cell membrane fishing technology has become a hot topic in screening TCM active ingredients (Trac-Trend. Anal. Chem., 2024, 180, 117983). Researchers used cell membrane composite materials as solid-phase extraction materials, utilizing the specific binding of cell membrane receptors to ligand compounds in TCM to enrich, elute, and identify active ingredients. This method of "fishing" for active drugs from TCM provides a meaningful direction for the discovery and identification of active ingredients (especially trace and ultra-trace components) in TCM.

[0003] Cell membranes are asymmetric, and proteins or their domains on the inner and outer surfaces of the cell membrane often have different functions, which greatly affects the performance of cell membrane biocomposites. The cell membrane targets of some drugs have been confirmed to be the intracellular domains of transmembrane receptors on the cell membrane, such as receptor tyrosine kinases. To better screen such candidate drugs, it is necessary to perform reverse orientation fixation of the cell membrane "inside out". At present, the orientation fixation method of cell membrane requires prior chemical or biological labeling of the cell membrane through covalent modification, metabolism and genetic engineering. The steps are numerous and can easily change the natural properties of the cell membrane. For example, covalent modification reagents such as activated esters and hydrazines can achieve random or directional binding of the cell membrane, but they can interfere with the biological activity of biomolecules such as membrane enzymes and proteins on the cell membrane, resulting in false positive screening results (Theranostics, 2016, 6 (7): 1012-1022; Mater. Design, 2025, 255, 114211). While genetic and metabolic biosynthetic pathways can introduce additional ligands onto cell membranes, they suffer from limitations such as gene contamination, low integration efficiency, high cost, and poor versatility and scalability (Angew. Chem. Int. Ed. 2021, 60: 6320-6325). Achieving targeted immobilization of cell membranes using a simple, label-free strategy remains a challenge, aiming to avoid altering cell membrane activity and properties and to screen for active substances in traditional Chinese medicine more efficiently and accurately.

[0004] Magnetic hydrochar nanomaterials (MHNs) are nanoscale carbon-containing products formed by the high-temperature closed reaction of biomass (such as glucose, cellulose, etc.) in aqueous solution. They have the characteristics of high hydrophilicity, superparamagnetism, low biotoxicity and good biocompatibility, and can be used as a good carrier material for cell membranes (A cell membrane bonded magnetic carbon ball composite material and its preparation method and application, ZL202211010609.8[P], 2024-07-16; Chem. Eng. J., 2023, 454, 140238).

[0005] Osteoporosis (OP) is a common skeletal disease characterized by decreased bone density and deterioration of bone microstructure, leading to increased bone fragility and a higher risk of fractures. Current treatments for osteoporosis include sclerosing protein inhibitors, bisphosphonates, parathyroid hormone, selective estrogen receptor modulators, and calcium supplements; however, single-component chemical drugs have some adverse reactions. In traditional Chinese medicine (TCM) theory, osteoporosis falls under the categories of "bone fullness," "bone soreness," or "lower back pain." Based on the TCM theory that the kidneys govern bones, the selection of Chinese herbal medicines primarily focuses on tonifying the kidneys. Epimedium (Hypericum striatum) enters the liver and kidney meridians and has the effects of dispelling wind and dampness, tonifying the kidneys and strengthening yang. It is an important component of clinical formulas for tonifying the kidneys and strengthening bones, such as Xianling Gubao capsules and Erxian Tang. The main active ingredient of Epimedium, such as icariin, has been proven to promote bone formation and combat osteoporosis; however, research on other pharmacodynamic substances is lacking, hindering the in-depth development of Epimedium's medicinal value in combating osteoporosis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a label-free magnetic hydrothermal carbon material with reverse cell membrane wrapping, its preparation method and application, in order to address the shortcomings of the prior art.

[0007] This invention discovers that lectins possess the ability to recognize and specifically bind to specific glycan structures. For example, concanavalin A (ConA), a typical lectin, can specifically bind to sugars such as mannose and glucose. ConA can also specifically and non-covalently bind to glycoprotein glycans on the outer surface of cell membranes, potentially enabling label-free reverse coating of cell membranes on magnetic hydrothermal carbon materials. Currently, concanavalin A has wide applications in ligand-receptor interaction studies, primarily for the enrichment and purification of glycoproteins; however, its application in cell membrane lectin technology and the screening of active ingredients in traditional Chinese medicine is yet to be observed. Based on this, the present invention uses lectin (ConA as an example) to prepare lectin-bonded magnetic hydrothermal carbon materials (Lectin-bonded MHNs, L-MHNs) with good biocompatibility. Then, it binds to the glycan chains on the outer surface of the cell membrane, thereby achieving directional immobilization of the cell membrane on the MHNs with the "inside facing out" without any labeling or modification of the cell membrane. This ensures the bioactivity of cell membrane proteins in the system. Compared with traditional coating methods that do not distinguish between the outer / inner surface orientation, this method can expose the inner membrane proteins, improving the comprehensiveness of screening for active ingredients of drugs, especially traditional Chinese medicine.

[0008] The technical solution adopted in this invention is as follows:

[0009] In a first aspect, the present invention provides a label-free magnetic hydrothermal carbon material with reverse cell membrane encapsulation, wherein the label-free magnetic hydrothermal carbon material with reverse cell membrane encapsulation is prepared by agglutination reaction of glycoprotein glycan chains on the inner surface of the cell membrane with lectinized magnetic hydrothermal carbon material to achieve reverse cell membrane encapsulation.

[0010] In one specific implementation scheme, the condensed magnetic hydrothermal carbon material is obtained by the following preparation method:

[0011] Activated magnetic hydrothermal carbon material is obtained by activating the carboxyl groups on the surface of hydrothermal magnetic carbon nanospheres with a hydroxyl activating agent.

[0012] A mixed activated magnetic hydrothermal carbon material and agglutinin are used to form an amide condensation between the amino groups of the activated magnetic hydrothermal carbon material and the agglutinin to obtain agglutinated magnetic hydrothermal carbon material.

[0013] In one specific implementation, the ratio of cell membrane to lectinized magnetic hydrothermal carbon material is 6-12 million / 10 mg.

[0014] In one specific implementation, the agglutination reaction is carried out at 4°C with shaking at 50-150 r / min for 6-18 hours.

[0015] In one specific implementation scheme, lectins include, but are not limited to, concanavalin A, wheat germ protein, peanut lectin, red kidney bean lectin, mannose-binding lectin, and Amanita fulva. Glycoproteins refer to glycoproteins or sugar-bound proteins purified from plants or animals.

[0016] In one specific implementation, the carboxyl groups on the surface of the magnetic hydrothermal carbon bind to the amino groups of lectin and bind to the sugar chains on the outer surface of the cell membrane in a natural, unmodified state, wherein the cell membrane is any single cell membrane or a mixture of multiple cell membranes.

[0017] In one specific implementation, the hydrothermal magnetic carbon nanospheres are prepared by a hydrothermal method.

[0018] In a more specific implementation, the preparation steps of the hydrothermal magnetic carbon nanospheres are as follows:

[0019] Hydrothermal synthesis of Fe3O4 magnetic nanoparticles;

[0020] Hydrothermal magnetic carbon nanospheres were prepared by hydrothermal reaction of Fe3O4 magnetic nanoparticles and glucose.

[0021] In a more specific implementation, the hydrothermal synthesis of Fe3O4 magnetic nanoparticles specifically involves:

[0022] Ferric chloride hexahydrate and sodium citrate were dissolved in ethylene glycol to form a solution. Sodium acetate was then added, and the mixture was stirred magnetically at a constant temperature of 50°C for 30 min until fully dissolved. The solution was then transferred to a hydrothermal reactor and reacted at a constant temperature. After the reaction was complete, Fe3O4 magnetic nanoparticles were obtained by washing. The mass ratio of ferric chloride hexahydrate, sodium citrate, and sodium acetate was (1-5):(0.2-1.2):(1.3-2.4); the concentration of ferric chloride hexahydrate in ethylene glycol was 0.036-0.06 g / mL.

[0023] In one specific implementation plan, the isothermal reaction is carried out at a temperature of 200°C for 12 hours.

[0024] In a more specific implementation, the mass ratio of Fe3O4 magnetic nanoparticles to glucose is (1-5):(10-50).

[0025] In a more specific implementation, the Fe3O4 magnetic nanoparticles and glucose are subjected to a hydrothermal reaction at 200-220°C for 10-14 h.

[0026] In one specific embodiment, the carboxyl activating agent is at least one of EDC and NHS, and the mass ratio of magnetic carbon spheres, EDC and NHS is (1-5):(2-12):(4-24).

[0027] In a second aspect, the present invention provides a method for preparing a label-free magnetic hydrothermal carbon material with reverse cell membrane encapsulation, comprising:

[0028] Activated magnetic hydrothermal carbon material obtained by activating the carboxyl groups on the surface of hydrothermal magnetic carbon nanospheres with a hydroxyl activating agent;

[0029] A mixture of activated magnetic hydrothermal carbon material and lectin was used to form an amide condensation between the amino groups of the activated magnetic hydrothermal carbon material and the lectin to obtain a lectinized magnetic hydrothermal carbon material.

[0030] Cell membranes were added to lectinized magnetic hydrothermal carbon materials, and the cell membranes were reverse-encapsulated through a lectinization reaction to obtain label-free magnetic hydrothermal carbon materials with reverse-encapsulated cell membranes.

[0031] In one specific embodiment, the carboxyl activating agent is at least one of EDC and NHS, and the mass ratio of magnetic carbon spheres, EDC and NHS is (1-5):(2-12):(4-24).

[0032] The activated magnetic hydrothermal carbon material is amide-condensed with the amino group of lectin to obtain a lectinized magnetic hydrothermal carbon material, specifically:

[0033] Concanavalin A was activated in a binding buffer containing MnCl2 and CaCl2 in a molar ratio of 1:1 at pH 7.2, and then activated magnetic hydrothermal carbon material was added and incubated at a constant temperature to obtain lectinized magnetic hydrothermal carbon material.

[0034] In one specific implementation, 1-100 mg of activated magnetic hydrothermal carbon material is added to 0.8-80 mg / mL of concanavalin A.

[0035] In one specific implementation, the specific formulation of the binding buffer containing MnCl2 and CaCl2 in a molar ratio of (1:1) at pH 7.2 is: 20 mM Tris-HCl, 1 mM MnCl2, 1 mM Cacl2, pH = 7.2.

[0036] In one specific implementation, the concentration of concanavalin A in the binding buffer is 1.6 mg / mL.

[0037] In one specific implementation, the constant temperature incubation conditions are: incubation at 37°C and 50-300 rpm for 4-12 hours.

[0038] In one specific implementation scheme, the synthesis steps of the condensed magnetic hydrothermal carbon material L-MHNs are as follows:

[0039] Step 1-1: Synthesis of Fe3O4 Magnetic Nanoparticles: Ferric chloride hexahydrate and sodium citrate were dissolved in ethylene glycol to form a solution. Sodium acetate was then added, and the mixture was stirred magnetically at a constant temperature of 50°C for 30 min until fully dissolved. The solution was then transferred to a Teflon-lined stainless steel hydrothermal reactor and reacted at a constant temperature. After the reaction was complete, the hydrothermal reaction product was cooled to room temperature and transferred to a 30 mL centrifuge tube. Impurities were removed by magnetic separation, and the liquid was discarded. This process was repeated 5 times. Ultrapure water was added back to the centrifuge tube, and the mixture was sonicated, then separated magnetically again, and the liquid was discarded. This process was repeated 5 times. After washing, anhydrous ethanol was used as the reagent, and the washing process was repeated five times. Finally, Fe3O4 magnetic nanospheres were obtained and stored in ultrapure water.

[0040] Steps 1-2: Synthesis of MHNs: The Fe3O4 magnetic nanospheres obtained in Step 1-1 were ultrasonically dispersed in ultrapure water. Glucose was added, and the mixture was mechanically stirred at room temperature until the glucose was completely dissolved. Next, the mixture was treated at a constant temperature, and the resulting hydrothermal magnetic carbon nanospheres were collected. Finally, the nanospheres were washed five times each with ultrapure water and anhydrous ethanol, and stored in ultrapure water for subsequent use.

[0041] Steps 1-3: Synthesis of L-MHNs magnetic nanoparticles: The Fe3O4 magnetic hydrothermal carbon nanoparticles from Step 1-2 were dissolved in 0.1 M 4-morpholinoethanesulfonic acid (MES) buffer. Then, the carboxyl activators 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added stepwise. The mixture was sonicated and oscillated to complete activation. After activation, the material was washed three times with MES buffer to remove unreacted reagents. Subsequently, concanavalin A (ConA) was added to a binding buffer containing MnCl2 and CaCl2 at pH 7.2, followed by sonication. Finally, the material was incubated at a constant temperature. After magnetic separation and washing, ConA-modified Fe3O4 magnetic hydrothermal carbon nanoparticles, i.e., L-MHNs magnetic nanoparticles, were obtained.

[0042] In one specific implementation, the constant temperature heating in step 1-1 is 200°C for 12 hours.

[0043] In one specific implementation, the constant temperature heating in steps 1-2 is 200-220℃ for 10-14 hours.

[0044] In one specific implementation, the stirring in steps 1-3 after adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is performed at a speed of 150 rpm, a temperature of 37°C, and a time of 30 min.

[0045] In one specific implementation, EDC and NHS are used to activate the carboxyl groups, converting them into active ester groups, with the mass ratio of hydrothermal carbon balls, EDC, and NHS being 10:80:120.

[0046] In one specific implementation, the addition of ConA in steps 1-3 is 8 mg / mL, the temperature is 37°C, and the time is 6 h.

[0047] In one specific implementation scheme, the preparation steps of osteoblast membrane reverse coating hydrothermal magnetic carbon nanospheres are as follows:

[0048] Step 2-1, Osteoblast culture: Select MC3T3-E1 cells in good growth condition and seed them in α-MEM complete culture medium containing sodium β-glycerophosphate, dexamethasone, and ascorbic acid. Mature osteoblasts are obtained after 5 days of culture.

[0049] Step 2-2: Obtaining osteoblast membranes: Cells were counted using a hemocytometer. When the cell count reached the predetermined range, they were collected in centrifuge tubes and centrifuged for 5 minutes. The supernatant was then aspirated, and the cells were resuspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution (HEPES). Subsequently, the cells were disrupted using an ultrasonic disruptor under ice-water bath conditions. After disruption, the cell suspension was transferred to a high-speed refrigerated centrifuge for low-temperature centrifugation to remove organelles. After centrifugation, the supernatant containing cell membrane fragments was carefully removed and centrifuged again at low temperature. The supernatant was discarded after centrifugation, pre-cooled HEPES solution was added, and the cells were disrupted again using an ultrasonic disruptor to fully disperse the cell membrane fragments, finally obtaining a cell membrane suspension, which was stored at 4°C for later use.

[0050] Step 2-3: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres: The L-MHNs magnetic nanoparticles obtained in Step 1-3 were added to the cell membrane suspension obtained in Step 2-2, and ultrasonically dispersed under ice-water bath conditions. After binding, the nanospheres were separated and washed with a magnet to obtain osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres, i.e., OMR@MHNs.

[0051] In one specific implementation, the inoculation density in step 2-1 is 15 million per dish, and the culture conditions are: 37°C, 5% CO2 constant temperature incubator.

[0052] In one specific implementation, the amount of cell membrane used in steps 2-3 is 60 million, and the amount of L-MHNs used is 10 mg.

[0053] A third aspect of the present invention also provides an application of the aforementioned label-free cell membrane reverse-wrapped magnetic hydrothermal carbon material in the field of pharmacological discovery.

[0054] In one specific implementation plan, the active ingredient of traditional Chinese medicine for fishing is applied, and the specific steps are as follows:

[0055] Step 3-1: Prepare the Chinese herbal extract;

[0056] Step 3-2: After suspending the hydrothermal magnetic carbon nanospheres with reverse-coated osteoblast membranes prepared in Step 2, a suspension is obtained. Then, the traditional Chinese medicine extract prepared in Step 3-1 is added to the suspension and incubated at 37°C for 30 min. The mixture is then separated by magnets, washed, and eluted with an elution buffer to finally obtain a fishing liquid containing active ingredients of traditional Chinese medicine.

[0057] In one specific implementation method, the application of the active ingredient of Epimedium brevicornu for fishing includes the following steps:

[0058] Step 1: After soaking Epimedium in ethanol and water, decoct the mixture and filter it once to obtain a primary filtrate and a primary residue. Then, decoct the primary residue in ethanol and water again while hot and filter it a second time 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 Epimedium extract. Dilute the concentrated Epimedium extract with water and filter it through a 0.22 μm microporous membrane for sterilization to obtain an Epimedium extract solution. Analyze the chemical components in the Epimedium extract solution.

[0059] Step 2: The Epimedium extract solution obtained in Step 1 is added to the suspension of hydrothermal magnetic carbon nanospheres with reverse-coated osteoblast membranes. The culture medium is then subjected to magnetic solid-liquid separation to obtain a precipitate. The precipitate is washed with ultrapure water and eluted with 6% acetic acid. The fishing solution containing Epimedium chemical components is obtained by magnetic solid-liquid separation. The chemical components in the fishing solution are analyzed, and the active ingredients are obtained based on the chemical components in the Epimedium extract solution obtained in Step 1.

[0060] In one specific implementation, the fishing solution containing the active ingredients of traditional Chinese medicine is obtained by eluting with 3 mL of 6% acetic acid aqueous solution for 3 min.

[0061] This invention is particularly applicable to the screening and identification of potential active components acting on specific receptors in complex systems.

[0062] This invention relates to a method for targeting active ingredients in traditional Chinese medicine (TCM) using osteoblast membranes with an "inside-out" reverse coating of hydrothermal magnetic carbon nanospheres based on agglutination reactions, belonging to the field of pharmaceutical analysis. This method improves the comprehensiveness and accuracy of screening active ingredients in TCM by constructing a cell membrane reverse-coated hydrothermal magnetic carbon nanosphere system with a specific membrane orientation. First, Fe3O4 magnetic nanoparticles are prepared using a hydrothermal synthesis method: ferric chloride hexahydrate and sodium citrate are used as precursors, ethylene glycol as solvent, and sodium acetate is added. The reaction is carried out at 200°C for 12 hours, followed by magnetic separation and ultrasonic cleaning to obtain Fe3O4 magnetic nanospheres. Subsequently, hydrothermal magnetic carbon nanospheres (MHNs) are prepared through glucose-assisted carbonization treatment. After activation with the carboxyl activator EDC / NHS, they are then placed in Mn... 2+ / Ca 2+ Biocompatible L-MHNs were constructed by conjugating concanavalin A in a buffer solution. Osteoblast cell membranes were then extracted using sonication combined with gradient centrifugation to obtain a cell membrane suspension. The L-MHNs and cell membrane suspension were ultrasonically dispersed in an ice bath and bound at low speed (4°C) for 6 hours to form osteoblast membranes that "face outward" in reverse coating of MHNs (OMR@MHNs). These were then cultured with traditional Chinese medicine extracts. Utilizing the selectivity of the cell membrane for active ingredients in this system, active components were extracted from the complex biological system of traditional Chinese medicine, improving the comprehensiveness of the screening for active ingredients in traditional Chinese medicine.

[0063] This invention utilizes osteoblasts—key target cells in osteoporosis treatment—as cell membrane raw materials to prepare hydrothermal magnetic carbon nanospheres with reverse-coated cell membranes. These nanospheres are incubated with a solution of Epimedium extract, a traditional Chinese medicine used in osteoporosis treatment. This allows the active components in the Epimedium extract to selectively and specifically bind to receptors on the osteoblast membrane surface of the reverse-coated hydrothermal magnetic carbon nanospheres (a process known as "fishing"). After washing with ultrapure water and eluting with 6% acetic acid, the reverse-coated hydrothermal magnetic carbon nanospheres are removed by magnetic solid-liquid separation, yielding a fishing solution containing Epimedium chemical components. Ultimately, the active components of Epimedium are obtained. This method utilizes the aggregation reaction between the glycan chains of the cell membrane and ConA, achieving an "inside-out" cell membrane orientation, thus improving the comprehensiveness of the fishing screening results and enabling the targeting of anti-inflammatory active components in Epimedium, thereby identifying the active components in Epimedium that combat osteoporosis.

[0064] Compared with the prior art, the present invention has the following advantages:

[0065] 1. This invention uses a coating and modification method to prepare biocompatible Fe3O4@lectin magnetic nanoparticles with membrane targeting properties. These nanoparticles are then combined with osteoblast membranes to construct a hydrothermal magnetic carbon nanosphere system with reverse coating of osteoblast membranes. This system is used for the screening of active ingredients in traditional Chinese medicine. By utilizing the aggregation reaction between the glycan chains of the cell membrane and lectins, the cell membrane is oriented "inside to outside" on the surface of the hydrothermal magnetic carbon nanospheres, thus improving the comprehensiveness of the screening results.

[0066] 2. In the process of preparing magnetic nanoparticles, the present invention uses lectins for modification, which eliminates the need for additional modification of the cell membrane, avoids damage to the membrane proteins on the cell membrane, and also saves corresponding process steps.

[0067] 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

[0068] Figure 1 This is a schematic diagram of a preparation process for a cell membrane reverse-coated magnetic hydrothermal nano-carbon biomimetic material in this invention.

[0069] Figure 2 The hysteresis regression lines (a) and infrared spectra (b) of MHNs, L-MHNs, and OMR@MHNs obtained in Example 1 of this invention are shown.

[0070] Figure 3 The images show the laser confocal images of MHNs, L-MHNs, and OMR@MHNs obtained in Example 1 of this invention.

[0071] Figure 4 The images are transmission electron microscope (TEM) images of hydrothermal magnetic carbon nanospheres (MHNs), L-MHNs, and OMR@MHNs obtained in Example 1 of this invention after immunochromatographic gold staining.

[0072] Figure 5 Figure 1 shows the adsorption selectivity test results of OMR@MHNs, cell membrane randomly bonded MHNs (OM-MHNs), L-MHNs and MHNs for the positive drug icariin II and the negative drug methotrexate.

[0073] Figure 6 This is an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis chromatogram of Epimedium extract EMB and MHNs, L-MHNs and OMR@MHNs in Epimedium extract EMB.

[0074] Figure 7Figure 1 shows the effects of the two newly discovered active ingredients, diphylloside A and sagittatoside C, on osteoblast growth (a, b), alkaline phosphatase activity (c, d), and articular mineralization level (f). n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal group (Ctrl).

[0075] Figure 8 Figures show the results of molecular docking (a), cell membrane thermal transfer, and Western blot analysis of the two novel active ingredients with osteoporosis-related membrane receptors (b)-e). n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal group. Detailed Implementation

[0076] The technical solution of the present invention will be further described in detail below through specific embodiments and with reference to the accompanying drawings. In the following embodiments, % refers to volume percentage.

[0077] This invention provides a label-free magnetic hydrothermal carbon material with reverse-coated cell membranes, its preparation method, and its applications. Magnetic hydrothermal carbon nanospheres are prepared and activated through a hydrothermal reaction of Fe3O4 magnetic spheres and glucose, and then bonded to lectins such as concanavalin A to obtain lectinized magnetic hydrothermal carbon nanospheres. Simultaneously, adherent cells meeting technical requirements are digested, disrupted, and centrifuged to obtain cell membranes. The lectinized magnetic hydrothermal carbon nanospheres are thoroughly mixed with the cell membranes. Utilizing the specific binding of immobilized concanavalin A to the glycosyl groups on the outer surface of the cell membrane, the cell membrane is reverse-coated "inside out" onto the surface of the magnetic hydrothermal carbon nanospheres, fully exposing the outer surface of the cell membrane. This allows for efficient and highly active screening of active components targeting the inner surface of the cell membrane. The reverse-coated magnetic hydrothermal carbon nanospheres of this invention eliminate the need for additional labeling or modification of the cell membrane, maximizing the preservation of the original structure and activity of membrane proteins, saving time and costs. This provides a new technical means for drug development targeting the inner surface of cell membranes and is also beneficial for the comprehensive exploration of the active substances in traditional Chinese medicine.

[0078] Example 1

[0079] The preparation method of label-free cell membrane reverse-coated magnetic hydrothermal carbon nanospheres described in this invention is as follows: Figure 1 As shown, it includes the following operations:

[0080] Step 1: Synthesis of OMR@MHNs biomimetic materials

[0081] Step 1-1, Synthesis of Fe3O4 Magnetic Particles: 1.8 g of ferric chloride hexahydrate and 0.36 g of sodium citrate were dissolved in 50 mL of ethylene glycol. Then, 2.4 g of sodium acetate was added, and the mixture was stirred magnetically at a constant temperature of 50°C for 30 min until fully dissolved. The solution was then transferred to a 100 mL Teflon-lined stainless steel hydrothermal reactor and reacted at 200°C for 12 h. After the reaction was complete, the hydrothermal reaction product was cooled to room temperature and transferred to a 30 mL centrifuge tube. Impurities were removed by magnetic separation, and the liquid was discarded. This process was repeated 5 times. Ultrapure water was added back to the centrifuge tube, followed by sonication and magnetic separation, and the liquid was discarded. This process was repeated 5 times. After washing, anhydrous ethanol was used as the reagent, and the washing process was repeated five times. Finally, Fe3O4 magnetic nanospheres were obtained and stored in ultrapure water.

[0082] Steps 1-2: Synthesis of Magnetic Hydrothermal Carbon Nanoparticles: 0.3 g of the Fe3O4 magnetic nanospheres obtained in Step 1-1 were ultrasonically dispersed in ultrapure water. 1.2 g of glucose was added, and the mixture was mechanically stirred at room temperature for 30 min until the glucose was completely dissolved. Next, the mixture was treated at 200℃ for 12 h, and the resulting hydrothermal magnetic carbon nanospheres were collected. Finally, the nanospheres were washed five times each with ultrapure water and anhydrous ethanol to obtain MHNs, which were then stored in ultrapure water for subsequent use.

[0083] Steps 1-3, Synthesis of L-MHNs: 10–20 mg of the hydrothermal magnetic carbon nanospheres from Step 1-2 were dissolved in 5–10 mL of 0.1–0.2 M 4-morpholinoethanesulfonic acid (MES) buffer. Then, 40–80 mg of the carboxyl activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 60–120 mg of N-hydroxysuccinimide (NHS) were gradually added. The mixture was sonicated and oscillated to complete activation. After activation, the material was washed three times with MES buffer to remove unreacted reagents. Subsequently, 16 mg of concanavalin A (ConA) was added to 10 mL of a binding buffer containing MnCl2 and CaCl2 (pH = 7.2) and sonicated. Finally, 20 mg of magnetic carbon spheres were added and incubated at a constant temperature of 37 °C for 6 h. After magnetic separation and washing, ConA-modified Fe3O4 magnetic hydrothermal carbon nanoparticles, namely L-MHNs magnetic nanoparticles, were obtained. Simultaneously, activated hydrothermal magnetic carbon nanoparticles were directly reacted with cell membranes to prepare randomly bonded cell membrane hydrothermal magnetic carbon nanoparticles, denoted as OM-MHNs (see CN115389659B).

[0084] Step 2: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres

[0085] Step 2-1, Osteoblast culture: MC3T3-E1 cells were used as the source of osteoblasts and cultured in α-MEM medium at 37°C in humid air containing 5% CO2. The cells were cultured with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin. A large number of osteoblasts were obtained after 5 days of culture.

[0086] The cell count is approximately 6 × 10⁶ 7 Remove the culture medium, wash several times with PBS, digest with 0.25% trypsin, collect cells by centrifugation at 4°C, resuspend in PBS to a concentration of approximately 3–12 × 10⁻⁶ cells. 7 / mL.

[0087] Step 2-2, Obtaining osteoblast membranes: Cells are counted using a hemocytometer. When the cell count reaches the predetermined range (2 × 10⁻⁶), the cell count is determined. 7 -1.2×10 8 Cells were collected in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was then aspirated, and the cells were resuspended in 3 mL of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution (HEPES). Subsequently, the cells were disrupted by sonication (550 W, 2-second sonication interval, 5-second cooling, total 10 minutes) under ice-water bath conditions. After disruption, the cell suspension was transferred to a high-speed refrigerated centrifuge and centrifuged at low temperature (4°C, 1500×g, 20 min) to remove organelles. After centrifugation, the supernatant containing cell membrane fragments was carefully removed and centrifuged again at low temperature (4°C, 15000×g, 20 min). After centrifugation, the supernatant was discarded, 3 mL of pre-cooled HEPES solution was added, and the cell membrane fragments were fully dispersed by sonication (550 W, sonication interval 2s, cooling 5s, 5 min) to obtain a cell membrane suspension, which was then stored at 4℃ for later use.

[0088] Steps 2-3: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres: All L-MHNs magnetic nanoparticles obtained in Steps 1-3 were added to 3 mL of cell membrane suspension obtained in Step 2-2 and ultrasonically dispersed under ice-water bath conditions. Subsequently, they were incubated on a shaker at 4℃, 80 rpm for 6 h to bind. After magnetic separation and washing, osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres, i.e., OMR@MHNs, were obtained.

[0089] Figure 1This is a schematic diagram illustrating the implementation of the present invention. Fe3O4 undergoes a hydrothermal reaction to generate MHNs, which are then activated by EDCI and NHS. After binding with ConA, they further bind to the glycan chains on the outer surface of the MC3T3-E1 cell membrane. This exposes the inner surface of the cell membrane to the solution, enabling efficient and precise screening of medicinal substances targeting the inner surface of the MC3T3-E1 cell membrane.

[0090] Figure 2 The images show the hysteresis regression lines and infrared spectra of MHNs, L-MHNs, and OMR@MHNs obtained in Example 1 of this invention. The cell membrane-coated hydrothermal magnetic carbon nanospheres, as measured by a vibrating sample magnetometer (VSM) at room temperature, exhibit good superparamagnetism, with coercivity and remanence approximately zero, and a saturation magnetization of approximately 39.89 emu / g. Figure 2 a). From Figure 2 As shown in b, the L-MHNs magnetic nanoparticles contain amide bonds, which is due to the amide condensation reaction between concanavalin A and the carboxyl group of hydrothermal carbon. Furthermore, the CH bonds in OMR@MHNs are strengthened, while the Fe-O bonds are weakened; these results all indicate the successful incorporation of the cell membrane.

[0091] Figure 3 The images show laser confocal images of MHNs, L-MHNs, and OMR@MHNs obtained in Example 1 of this invention. OMR@MHNs hydrothermal magnetic carbon nanospheres were seeded in laser confocal culture dishes, and the cell membranes were stained with a DiI red fluorescent probe. Images were recorded using a laser confocal microscope (LSM880), where 549 nm and 488 nm lasers were used to excite the DiI and FITC-ConA stained cell membrane samples. Red fluorescence was present in the OMR@MHNs hydrothermal magnetic carbon nanospheres, but green fluorescence disappeared, indicating that the OMR@MHNs hydrothermal magnetic carbon nanospheres achieved an inside-out orientation of the cell membrane.

[0092] Figure 4 The images show transmission electron microscopy (TEM) images of hydrothermal magnetic carbon nanospheres (MHNs), L-MHNs, and OMR@MHNs obtained in Example 1 of this invention after immunochromatographic gold staining. The OMR@MHNs magnetic nanoparticles showed significant gold enrichment under C-terminal antibody labeling, while no obvious gold particles were observed in the N-terminal antibody-labeled group. This difference confirms that the cell membrane successfully achieved an "inside-out" effect.

[0093] Figure 5This study presents the adsorption selectivity of OMR@MHNs, randomly bonded cell membrane MHNs (OM-MHNs), L-MHNs, and MHNs for the positive drug icariin II (5 μg / mL) and the negative drug methotrexate (450 μg / mL). At room temperature, mobile phase A consisted of acetonitrile (containing 0.1% formic acid), and mobile phase B consisted of 0.1% formic acid in water, with a mobile phase ratio of 80% A: 20% B. Isocratic elution was performed using a Waters UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). Comparison of recoveries showed that only OMR@MHNs exhibited good enrichment of the positive drug icariin II, whose domain of action is on the inner side of the cell membrane. OM-MHNs, L-MHNs, and MHNs all showed weak selective enrichment of icariin II. This confirms that the cell membrane successfully achieves an "inside-out" effect.

[0094] Step 3: Active ingredient steps of the traditional Chinese medicine Epimedium in fishing.

[0095] 3-1. Preparation of Epimedium extract: Weigh 5 g of Epimedium and reflux it with 100 mL of 70% ethanol aqueous solution for 2 h. Filter it while hot to obtain a primary filtrate and a primary residue. Reflux the primary residue with 100 mL of 70% ethanol aqueous solution for another 2 h. Filter it while 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 Epimedium extract. Dissolve the concentrated Epimedium extract in 10 mL of ultrapure water for later use. The Epimedium extract solution, denoted as A0, was obtained by filtration through a 0.22 μm microporous membrane for sterilization. The eluent was analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) under the following conditions: Waters Empower HPLC system, column temperature 25℃, Waters UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm), mobile phase A: acetonitrile (containing 0.1% formic acid), mobile phase B: 0.1% formic acid in water. Gradient elution was used, with the gradient settings as follows: 0-5 min, 5%-12% A; 5-10 min, 12%-14% A; 10-15 min, 14%-20% A; 15-20 min, 20-25% A; 20-25 min, 25%-55% A; 25-30 min, 55%-85% A; 30-32 min, 85%-100% A; 2-33.5 min, 100% A. The flow rate was 0.2 mL / min. ‒1The injection volume was 2 μL. The mass spectrometer used was a Waters (SYNAPT G2-Si) tandem quadrupole-time-of-flight mass spectrometer equipped with an electrospray ionization (ESI) source, employing both positive and negative ion mass spectrometry modes. The capillary voltage was set to 2.5 kV; the sample cone voltage to 40 V; the source bias voltage to 80 V; the source temperature to 120 °C; the cone gas flow rate to 50 L / h; the desolvation gas flow rate to 800 L / h; and the nebulizer operating pressure to 6.0 Bar.

[0096] Step 3-2: Add 3 mL of the Epimedium extract solution obtained in Step 3-1 to 10 mg of the osteoblast membrane "inside-out" coated hydrothermal magnetic carbon nanospheres prepared in Step 2, and incubate at 37℃ for 30 min. Then, use magnetic solid-liquid separation to obtain a precipitate. Wash the precipitate with ultrapure water until the supernatant is colorless. Elute the precipitate with 3 mL of 6% acetic acid, repeating the elution twice, and collect the eluent. Simultaneously, randomly coat Epimedium with the osteoblast membrane obtained in Step 1-3 and fish for it under the same conditions, collecting the eluent. Fish for Epimedium with hydrothermal magnetic carbon nanospheres, and collect the eluent in the same manner. Concentrate the above eluent under reduced pressure and then redissolve it with 0.5 mL of methanol. Analyze the obtained supernatant according to the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) conditions in Step 3-1. The UHPLC-MS chromatograms are shown below. Figure 6 As shown, mass spectrometry analysis was performed using unifi software.

[0097] like Figure 6 As shown, the osteoblast membrane "inside-out" reverse-encapsulated hydrothermal magnetic carbon nanospheres prepared in this invention screened four highly specific representative components from Epimedium extract. These components were identified by ultra-high performance liquid chromatography and mass spectrometry as diphylloside A (Peak 1), sagittatoside C (Peak 2), epimedoside B (Peak 3), and icariin II (Peak 4). Among these, diphylloside A (Peak 1) and sagittatoside C (Peak 2) are previously unreported potential active components in Epimedium. Further analysis of these two components was conducted.

[0098] Step 4: Activity Study of Potential Components

[0099] Step 4-1, CCK-8 assay for osteoblast viability: MC3T3-E1 cells were cultured at 1×10⁻⁶ cells per well. 4The cells were seeded at a density of 100% in 96-well plates and cultured for 12 hours in a 37°C incubator containing 5% CO2 to ensure full cell adhesion.

[0100] Step 4-2, Cell Grouping and Drug Administration: MC3T3-E1 cells were seeded in culture dishes. After cell attachment, the experimental groups were divided into a control group and low, medium, and high-dose drug administration groups. The control group received a drug with a concentration of 10... -2 M β-glycerophosphate sodium, 10 -8 Cells were cultured in α-MEM complete medium containing dexamethasone and 50 μg / mL ascorbic acid. The drug-treated groups received different concentrations of the test drugs (screened potential active ingredients (bis-podoside A and strophanthidin C)) added to the above medium and were treated for 48 hours. The medium was changed every other day in all groups. Subsequently, all medium was removed from each well. CCK-8 working solution was prepared by mixing α-MEM medium and CCK-8 solution at a volume ratio of 10:1, and 100 μL was added to each well. The 96-well plates were incubated at 37°C for 30 minutes, and then the absorbance was measured at 450 nm using a microplate reader. Figure 7 a, b, and bisposide A (1-10 μM) and bisposide C (10-100 nM) can all promote osteoblast proliferation in a dose-dependent manner.

[0101] Step 4-3, Osteoblast alkaline phosphatase staining: MC3T3-E1 cells in logarithmic growth phase were stained with 1×10⁻⁶ cells. 4 Cells were seeded at a density of [number] cells / well and incubated in a 37°C, 5% CO2 incubator. Following the experimental protocol described in step 4-2, cell groups and drug treatments were completed. After 5 days of intervention, the culture medium was discarded, and cells were washed twice with PBS buffer. Subsequently, 50 μL of 4% paraformaldehyde solution was added to each well for 30 min for fixation, followed by washing with PBS to remove the fixative. The staining procedure was completed according to the alkaline phosphatase staining kit protocol. Finally, observation and recording were performed under a microscope. Figure 7 As shown in e.

[0102] Step 4-4, Osteoblast alkaline phosphatase activity assay: MC3T3-E1 cells in logarithmic growth phase were subjected to a 1×10⁻⁶ thiocyanate incubation. 4The cells were seeded at a density of cells / well and incubated in a 37°C, 5% CO2 incubator. After the cells were fully adhered, they were treated for 5 days using the grouping and drug concentration described in step 4-2. The culture medium was then removed, and the cells were washed three times with PBS buffer. Cells were lysed on ice for 30 min using Western blotting and IP cell lysis buffer, followed by centrifugation at 12000 rpm for 5 min to obtain the supernatant. Different treatment groups, including control and test samples, were set up in 96-well plates. Detection buffer, chromogenic agent, test sample, and working concentration standard solution were added according to the operating procedure. The plate was incubated at 37°C for 20 min, followed by the addition of 160 μL of reaction stop solution per well to terminate the reaction. The absorbance at 405 nm was then measured to calculate the alkaline phosphatase activity in the sample. Figure 7 As shown in c-7d.

[0103] It can be seen that bis(t)-glucoside A (1-10 μM) and bis(t)-glucoside C (10-100 nM) can significantly increase the activity (c, d) and production (e) of osteoblast alkaline phosphatase, and the same dose-dependent relationship exists.

[0104] Steps 4-5: Osteoblast bone nodule formation assay: MC3T3-E1 at 1× 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated at 37°C in a 5% CO2 incubator. After the cells had fully adhered, they were treated for 21 days using the grouping and drug concentration described in step 4-2, followed by washing three times with PBS buffer. The cells were then fixed with 4% paraformaldehyde for 20 min, and washed again with PBS to remove the fixative. The staining procedure was completed according to the Alizarin Red staining kit, and the results were observed and recorded under a microscope. Figure 7 f shows the results of alizarin red staining of osteoblasts by bis(tibaridin) A and bis(tibaridin) C. Both active compounds can induce osteoblast bone nodule formation in a dose-dependent manner at certain concentrations.

[0105] Figure 8 This study analyzed the molecular docking, cell membrane thermal transfer, and Western blot analysis of two novel active ingredients with osteoporosis-related membrane receptors. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal group. Figure 8 a shows the docking results of biposide A and succinoside C with osteoblast domains on the membrane. Among them, biposide A has the lowest molecular binding energy with EPHB4 and succinoside C has the lowest molecular binding energy with IGF1R, indicating that the two compounds have a strong affinity for these two proteins. Figure 8b and c are cell heat transfer experiments of EPHB4 and IGF1R with the two compounds. Bismoposide A and strophoside C can enhance the thermal stability of EPHB4 and IGF1R, respectively, further demonstrating that the two have a strong affinity. Figure 8 Images d and e show that bisporamin A and strophanthidin C can promote the expression of EPHB4 and IGF1R in osteoblasts, respectively. These results indicate that EPHB4 and IGF1R are potential targets of bisporamin A and strophanthidin C, respectively.

[0106] Example 2

[0107] The preparation method of label-free cell membrane reverse-coated magnetic hydrothermal carbon nanospheres described in this invention is as follows: Figure 1 As shown, it includes the following operations:

[0108] Step 1: Synthesis of OMR@MHNs biomimetic materials

[0109] Step 1-1: Synthesis of Fe3O4 magnetic particles: 3 g of ferric chloride hexahydrate and 3.6 g of sodium citrate were dissolved in 50 mL of ethylene glycol. Then, 7.2 g of sodium acetate was added, and the mixture was stirred magnetically at a constant temperature of 50 °C for 30 min until fully dissolved. The solution was then transferred to a 100 mL Teflon-lined stainless steel hydrothermal reactor and reacted at 200 °C for 12 h. After the reaction was complete, the hydrothermal reaction product was cooled to room temperature and transferred to a 30 mL centrifuge tube. Impurities were removed by magnetic separation, and the liquid was discarded. This process was repeated 5 times. Ultrapure water was added back to the centrifuge tube, followed by sonication and magnetic separation, and the liquid was discarded. This process was repeated 5 times. After washing, anhydrous ethanol was used as the reagent, and the washing process was repeated five times. Finally, Fe3O4 magnetic nanospheres were obtained and stored in ultrapure water.

[0110] Steps 1-2: Synthesis of Magnetic Hydrothermal Carbon Nanoparticles: 0.3 g of the Fe3O4 magnetic nanospheres obtained in Step 1-1 were ultrasonically dispersed in ultrapure water. 15 g of glucose was added, and the mixture was mechanically stirred at room temperature for 30 min until the glucose was completely dissolved. Next, the mixture was treated at 220℃ for 14 h, and the resulting hydrothermal magnetic carbon nanospheres were collected. Finally, the nanospheres were washed five times each with ultrapure water and anhydrous ethanol to obtain MHNs, which were then stored in ultrapure water for subsequent use.

[0111] Steps 1-3, Synthesis of L-MHNs: 10-20 mg of the hydrothermal magnetic carbon nanospheres from Step 1-2 were dissolved in 5-10 mL of 0.1-0.2 M 4-morpholinoethanesulfonic acid (MES) buffer. Then, 40-80 mg of the carboxyl activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 60-120 mg of N-hydroxysuccinimide (NHS) were gradually added. The mixture was sonicated and oscillated to complete activation. After activation, the material was washed three times with MES buffer to remove unreacted reagents. Subsequently, 80 mg of concanavalin A (ConA) was added to 10 mL of a binding buffer containing MnCl2 and CaCl2 (pH = 7.2) and sonicated. Finally, 100 mg of magnetic carbon spheres were added and incubated at a constant temperature of 37 °C for 6 h. After magnetic separation and washing, ConA-modified Fe3O4 magnetic hydrothermal carbon nanoparticles, namely L-MHNs magnetic nanoparticles, were obtained. Simultaneously, activated hydrothermal magnetic carbon nanoparticles were directly reacted with cell membranes to prepare randomly bonded cell membrane hydrothermal magnetic carbon nanoparticles, denoted as OM-MHNs (see CN115389659B).

[0112] Step 2: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres

[0113] Step 2-1, Osteoblast culture: MC3T3-E1 cells were used as the source of osteoblasts and cultured in α-MEM medium at 37°C in humid air containing 5% CO2. The cells were cultured with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin. A large number of osteoblasts were obtained after 5 days of culture.

[0114] The cell count is approximately 6 × 10⁶ 7 Remove the culture medium, wash several times with PBS, digest with 0.25% trypsin, collect cells by centrifugation at 4°C, resuspend in PBS to a concentration of approximately 3–12 × 10⁻⁶ cells. 7 / mL.

[0115] Step 2-2, Obtaining osteoblast membranes: Cells are counted using a hemocytometer. When the cell count reaches the predetermined range (2 × 10⁻⁶), the cell count is determined. 7 -1.2×10 8Cells were collected in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was then aspirated, and the cells were resuspended in 3 mL of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution (HEPES). Subsequently, the cells were disrupted by sonication (550 W, 2-second sonication interval, 5-second cooling, total 10 minutes) under ice-water bath conditions. After disruption, the cell suspension was transferred to a high-speed refrigerated centrifuge and centrifuged at low temperature (4°C, 1500×g, 20 min) to remove organelles. After centrifugation, the supernatant containing cell membrane fragments was carefully removed and centrifuged again at low temperature (4°C, 15000×g, 20 min). After centrifugation, the supernatant was discarded, 3 mL of pre-cooled HEPES solution was added, and the cell membrane fragments were fully dispersed by sonication (550 W, sonication interval 2s, cooling 5s, 5 min) to obtain a cell membrane suspension, which was then stored at 4℃ for later use.

[0116] Steps 2-3: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres: 10 mg of L-MHNs magnetic nanoparticles obtained in Step 1-3 were added to 3 mL of cell membrane suspension obtained in Step 2-2, and ultrasonically dispersed under ice-water bath conditions. Subsequently, the mixture was incubated on a shaker at 4℃, 80 rpm for 6 h to bind. After magnetic separation and washing, osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres, i.e., OMR@MHNs, were obtained.

[0117] Tests have shown that the osteoblast membrane-coated hydrothermal magnetic carbon nanospheres exhibit excellent screening effects for fishing.

[0118] Example 3

[0119] The preparation method of label-free cell membrane reverse-coated magnetic hydrothermal carbon nanospheres described in this invention is as follows: Figure 1 As shown, it includes the following operations:

[0120] Step 1: Synthesis of OMR@MHNs biomimetic materials

[0121] Step 1-1, Synthesis of Fe3O4 Magnetic Particles: 1.8 g of ferric chloride hexahydrate and 0.072 g of sodium citrate were dissolved in 50 mL of ethylene glycol. Then, 0.468 g of sodium acetate was added, and the mixture was stirred magnetically at a constant temperature of 50°C for 30 min until fully dissolved. The solution was then transferred to a 100 mL Teflon-lined stainless steel hydrothermal reactor and reacted at 200°C for 12 h. After the reaction was complete, the hydrothermal reaction product was cooled to room temperature and transferred to a 30 mL centrifuge tube. Impurities were removed by magnetic separation, and the liquid was discarded. This process was repeated 5 times. Ultrapure water was added back to the centrifuge tube, followed by sonication and magnetic separation, and the liquid was discarded. This process was repeated 5 times. After washing, anhydrous ethanol was used as the reagent, and the washing process was repeated five times. Finally, Fe3O4 magnetic nanospheres were obtained and stored in ultrapure water.

[0122] Steps 1-2: Synthesis of Magnetic Hydrothermal Carbon Nanoparticles: 0.3 g of the Fe3O4 magnetic nanospheres obtained in Step 1-1 were ultrasonically dispersed in ultrapure water. 0.6 g of glucose was added, and the mixture was mechanically stirred at room temperature for 30 min until the glucose was completely dissolved. Next, the mixture was treated at 210℃ for 10 h, and the resulting hydrothermal magnetic carbon nanospheres were collected. Finally, the nanospheres were washed five times each with ultrapure water and anhydrous ethanol to obtain MHNs, which were then stored in ultrapure water for subsequent use.

[0123] Steps 1-3, Synthesis of L-MHNs: 10-20 mg of the hydrothermal magnetic carbon nanospheres from Step 1-2 were dissolved in 5-10 mL of 0.1-0.2 M 4-morpholinoethanesulfonic acid (MES) buffer. Then, 40-80 mg of the carboxyl activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 60-120 mg of N-hydroxysuccinimide (NHS) were gradually added. The mixture was sonicated and oscillated to complete activation. After activation, the material was washed three times with MES buffer to remove unreacted reagents. Subsequently, 0.8 mg of concanavalin A (ConA) was added to 10 mL of a binding buffer containing MnCl2 and CaCl2 (pH = 7.2) and sonicated. Finally, 1 mg of magnetic carbon spheres were added and incubated at a constant temperature of 37°C for 6 h. After magnetic separation and washing, ConA-modified Fe3O4 magnetic hydrothermal carbon nanoparticles, namely L-MHNs magnetic nanoparticles, were obtained. Simultaneously, activated hydrothermal magnetic carbon nanoparticles were directly reacted with cell membranes to prepare randomly bonded cell membrane hydrothermal magnetic carbon nanoparticles, denoted as OM-MHNs (see CN115389659B).

[0124] Step 2: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres

[0125] Step 2-1, Osteoblast culture: MC3T3-E1 cells were used as the source of osteoblasts and cultured in α-MEM medium at 37°C in humid air containing 5% CO2. The cells were cultured with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin. A large number of osteoblasts were obtained after 5 days of culture.

[0126] The cell count is approximately 6 × 10⁶ 7 Remove the culture medium, wash several times with PBS, digest with 0.25% trypsin, collect cells by centrifugation at 4°C, resuspend in PBS to a concentration of approximately 3–12 × 10⁻⁶ cells. 7 / mL.

[0127] Step 2-2, Obtaining osteoblast membranes: Cells are counted using a hemocytometer. When the cell count reaches the predetermined range (2 × 10⁻⁶), the cell count is determined. 7 -1.2×10 8 Cells were collected in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was then aspirated, and the cells were resuspended in 3 mL of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution (HEPES). Subsequently, the cells were disrupted by sonication (550 W, 2-second sonication interval, 5-second cooling, total 10 minutes) under ice-water bath conditions. After disruption, the cell suspension was transferred to a high-speed refrigerated centrifuge and centrifuged at low temperature (4°C, 1500×g, 20 min) to remove organelles. After centrifugation, the supernatant containing cell membrane fragments was carefully removed and centrifuged again at low temperature (4°C, 15000×g, 20 min). After centrifugation, the supernatant was discarded, 3 mL of pre-cooled HEPES solution was added, and the cell membrane fragments were fully dispersed by sonication (550 W, sonication interval 2s, cooling 5s, 5 min) to obtain a cell membrane suspension, which was then stored at 4℃ for later use.

[0128] Steps 2-3: Preparation of osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres: 10 mg of L-MHNs magnetic nanoparticles obtained in Step 1-3 were added to 3 mL of cell membrane suspension obtained in Step 2-2, and ultrasonically dispersed under ice-water bath conditions. Subsequently, the mixture was incubated on a shaker at 4℃, 80 rpm for 6 h to bind. After magnetic separation and washing, osteoblast membrane reverse-coated hydrothermal magnetic carbon nanospheres, i.e., OMR@MHNs, were obtained.

[0129] Tests have shown that the osteoblast membrane-coated hydrothermal magnetic carbon nanospheres exhibit excellent screening effects for fishing.

[0130] 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 label-free cell membrane reverse wrapped magnetic hydrothermal carbon material, characterized in that, The label-free cell membrane reverse-wrapped magnetic hydrothermal carbon material is prepared by reverse wrapping of cell membranes through agglutination of glycoprotein sugar chains on the inner surface of the cell membranes and the lectinized magnetic hydrothermal carbon material.

2. The label-free cell membrane reverse wrapped magnetic hydrothermal carbon material of claim 1, wherein, The lectinized magnetic hydrothermal carbon material is obtained by the following preparation method: The activated magnetic hydrothermal carbon material is obtained by activating the carboxyl groups on the surface of the hydrothermal magnetic carbon nanospheres with a hydroxyl group activating agent. The lectinized magnetic hydrothermal carbon material is obtained by mixing the activated magnetic hydrothermal carbon material with the lectin to form amide condensation between the amino groups of the activated magnetic hydrothermal carbon material and the lectin.

3. The label-free cell membrane reverse wrapped magnetic hydrothermal carbon material of claim 2, wherein, The lectin includes one or more of concanavalin A, wheat germ agglutinin, peanut agglutinin, red kidney bean agglutinin, mannose-binding agglutinin, and orange cap tricholoma.

4. The label-free cell membrane reverse wrapped magnetic hydrothermal carbon material of claim 1, wherein, The cell membrane is any single cell membrane or a mixture of multiple cell membranes.

5. A method of preparing the label-free cell membrane reverse-wrapped magnetic hydrothermal carbon material of claim 1, characterized in that, The method comprises: The activated magnetic hydrothermal carbon material is obtained by activating the carboxyl groups on the surface of the hydrothermal magnetic carbon nanospheres with a hydroxyl group activating agent. The lectinized magnetic hydrothermal carbon material is obtained by mixing the activated magnetic hydrothermal carbon material with the lectin to form amide condensation between the amino groups of the activated magnetic hydrothermal carbon material and the lectin. The label-free cell membrane reverse-wrapped magnetic hydrothermal carbon material is prepared by reverse wrapping of cell membranes through agglutination of glycoprotein sugar chains on the inner surface of the cell membranes and the lectinized magnetic hydrothermal carbon material.

6. The production method according to claim 5, wherein The carboxyl activating agent is at least one of EDC and NHS, and the mass ratio of the hydrothermal magnetic carbon nanospheres, EDC, and NHS is (1-5):(2-12):(4-24).

7. The preparation method according to claim 5, characterized in that, The lectinized magnetic hydrothermal carbon material is obtained by mixing the activated magnetic hydrothermal carbon material with the lectin to form amide condensation between the amino groups of the activated magnetic hydrothermal carbon material and the lectin. Concanavalin A is activated in a combined buffer containing MnCl2 and CaCl2 at a molar concentration ratio of 1:1 at a pH value of 7.2, and then the activated magnetic hydrothermal carbon material is added for constant temperature incubation to obtain the lectinized magnetic hydrothermal carbon material.

8. The preparation method according to claim 7, characterized in that, 0.8-80 mg / mL of concanavalin A is added per 1-100 mg of activated magnetic hydrothermal carbon material.

9. The preparation method according to claim 7, characterized in that, The constant temperature incubation conditions are: incubation at 37°C at 50-300 rpm for 4-12 h.

10. Use of the label-free cell membrane reverse-wrapped magnetic hydrothermal carbon material of claim 1 in the field of drug efficacy substance discovery.

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