Preparation method for and use of scopoletin-functionalized magnetic nanoprobe

Abstract

A preparation method for and use of a scopoletin-functionalized magnetic nanoprobe. The preparation method comprises steps such as preparation of amino-terminal modified nanoparticles Fe3O4@SiO2-NH2, preparation of a photoaffinity linker-scopoletin conjugate, preparation of the scopoletin-functionalized magnetic nanoprobe, etc. The method features simple and rapid operation, and eliminates the need for prior clarification of the structure-activity relationship of the active compounds, without compromising all-round contact between the active compounds and target proteins. The prepared scopoletin-functionalized magnetic nanoprobe is used for target fishing in cellular and animal models of rheumatoid arthritis. After the captured target proteins are isolated and subjected to mass spectrometry identification, a western blot experiment successfully verifies vimentin as the target protein of scopoletin on HFLS-RA cell membranes, thereby providing a novel therapeutic target for rheumatoid arthritis.

Description

Preparation method and application of scopolamine functionalized magnetic nanoprobe Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation method and application of a scopoletin functionalized magnetic nanoprobe. Background Art

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by synovial hyperplasia and bone destruction, leading to significant declines in patient function and quality of life, creating a significant social burden. The global prevalence of RA is approximately 1%, affecting all age groups. RA is complex, with a long and recurrent course and a high incidence of complications. Currently, there is no cure for RA. Existing treatments include glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and disease-modifying antirheumatic drugs (DMARDs); these medications only alleviate symptoms but cannot control disease progression, and they are associated with unavoidable adverse effects. In recent years, highly targeted biologics (such as TNF inhibitors) have been increasingly used in the treatment of RA. Recently, the emergence of small molecule targeted drugs (such as JAK inhibitors) has further overcome the limitations of protein-based biologics. Therefore, the development of small molecule targeted drugs has become a new direction in RA drug research and a key to achieving personalized precision medicine.

[0003] Currently, chemically synthesized targeted drugs (such as the small molecule JAK inhibitors tofacitinib and baricitinib) face serious clinical safety issues. To date, attempts to modify the chemical structure of existing RA targeted drugs to mitigate toxic side effects have not been successful. Compared to chemically synthesized compounds, natural products possess complex and diverse skeletons and rich functional groups, possessing unique biological activities and making them suitable starting points for new drug screening.

[0004] Studies have shown that scopoletin is a safe and effective treatment for RA, with a target protein identified in RA models. In animal models, oral administration of the compound to rats with adjuvant-induced arthritis (AIA) significantly alleviated clinical symptoms (redness and swelling of the hind paws, tail ulceration), modulated inflammatory cytokine responses (selectively inhibiting COX-2 expression and regulating levels of the cytokines IL-10 and IL-6 in ankle synovial tissue), and improved joint pathology (soft tissue swelling, joint cavity narrowing, cartilage erosion, cellular infiltration, and pannus formation) without significant cardiovascular toxicity. In the cell membrane model, the compound effectively inhibited inflammatory synoviocyte migration and invasion, as well as NF-κB signaling, in fibroblast-like synoviocytes from RA patients (HFLS-RA).

[0005] Target identification of natural active compounds is a key step in new drug development. It not only helps discover promising compound structures, but also facilitates the safety and efficacy assessment of compounds, and can even discover new therapeutic targets for complex diseases outside of classical pharmacological signaling pathways. Target identification methods for active compounds can be divided into two categories: indirect and direct. Indirect methods include phenotypic screening, genetic screening, and virtual screening, but are limited in that they cannot rule out false negative results. In contrast, direct methods are widely used, and based on "drug-target" affinity, active compounds are chemically linked to solid supports for target protein enrichment and identification.

[0006] Due to their unique properties, magnetic nanoparticles (MNPs) are suitable for preparing compound-functionalized magnetic nanoprobes for use as solid supports for target fishing. Currently, these emerging probes are often synthesized by structurally modifying compounds and then linking them to MNPs. However, structural modification of compounds is not conducive to efficient target fishing because: 1) before the structure-activity relationship of the active compound is clearly defined, structural changes hinder its comprehensive access to the target protein; and 2) natural active compounds have diverse structures. If the connection to MNPs must be individually designed based on molecular structural characteristics, it is difficult to create probes in a unified and convenient manner. Summary of the Invention

[0007] To solve the above problems, the primary purpose of the present invention is to provide a method for preparing scopolamine-functionalized magnetic nanoprobes. This method uses photocrosslinking technology, is simple and rapid to operate, does not require prior clarification of the structure-activity relationship of the active compound, and does not affect its all-round contact with the target protein.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing a scopoletin-functionalized magnetic nanoprobe comprises the following steps:

[0010] (1) Ferric chloride hexahydrate, polyvinyl pyrrolidone and sodium acetate were dispersed in ethylene glycol to react and obtain Fe3O4 nanoparticles; Fe3O4 nanoparticles were ultrasonically reacted with ethyl orthosilicate to obtain Fe3O4@SiO2 nanoparticles;

[0011] (2) Using ethanol as solvent, aminopropyltriethoxysilane was added to Fe3O4@SiO2 nanoparticles and refluxed to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2;

[0012] (3) Mix the photoaffinity linker and scopoletin and fully dissolve them in an organic solvent, irradiate with 365 nm ultraviolet light at room temperature, and after the reaction is completed, spin dry to remove the solvent to obtain a photoaffinity linker-scopoletin coupling product;

[0013] (4) The photoaffinity linker-scopoletin coupling product was redissolved in an organic solvent, and triethylamine was added dropwise. The amino-terminus-modified nanoparticles Fe3O4@SiO2-NH2 were added, mixed and shaken in the dark overnight. After the reaction was completed, the product was separated by magnet and washed with methanol to obtain a scopoletin-functionalized magnetic nanoprobe.

[0014] Preferably, in step (3), the photoaffinity linker is 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid

[0015] (TDBA), 3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diazirine (SDAD) or 3-methyl-3H-diazirine-3-propionic acid (MPDA), more preferably 4-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzoic acid.

[0016] Preferably, in step (3), the molar ratio of the photoaffinity linker to scopoletin is 1-4:1.

[0017] Preferably, in step (3), the organic solvent is one or more of anhydrous methanol, dichloromethane or carbon tetrachloride.

[0018] Anhydrous methanol is more preferred.

[0019] Preferably, in step (3), the ultraviolet light irradiation time at room temperature is 15-60 minutes.

[0020] Preferably, in step (4), the organic solvent is one or more of a mixed solution of isopropyl alcohol and dimethylformamide in a volume ratio of 1:1, dimethylformamide or 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate, preferably a mixed solution of isopropyl alcohol and dimethylformamide in a volume ratio of 1:1.

[0021] The present invention also provides a scopolamine functionalized magnetic nanoprobe, which is prepared by the above-mentioned preparation method.

[0022] The present invention also provides the use of the aforementioned scopoletin-functionalized magnetic nanoprobe for target protein fishing and / or identification. Scopoletin-functionalized magnetic nanoprobes were used to target protein fishing in a rheumatoid arthritis model. The fished target proteins were separated by gel electrophoresis and identified by liquid chromatography-mass spectrometry. Western blotting confirmed that scopoletin's target protein on the HFLS-RA cell membrane was vimentin, and that the specific binding between vimentin and scopoletin involved covalent and hydrogen bonding.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses photocrosslinking reaction technology, in which photoaffinity linkers are irradiated with ultraviolet light to generate carbenes, which cross-link various active compounds on the solid surface in a non-selective manner. This method is suitable for various natural active compound structures and does not require individual design based on molecular structural characteristics. The operation is simple and rapid, and there is no need to clarify the structure-activity relationship of the active compound in advance, which does not affect its all-round contact with the target protein.

[0025] The present invention uses the prepared scopoletin-functionalized magnetic nanoprobe to perform target fishing in cells and animal models of rheumatoid arthritis. After separation and mass spectrometry identification of the target protein, the protein immunoblotting experiment was successfully conducted, verifying that the target protein of scopoletin on the HFLS-RA cell membrane is vimentin, providing a new therapeutic target for rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flow chart for the preparation of amino-terminally modified Fe3O4@SiO2-NH2 nanoparticles;

[0027] FIG2 is a flow chart of the preparation of scopolamine-functionalized magnetic nanoprobes;

[0028] Figure 3 shows the optimization results of the sequence of photocrosslinking reaction and dehydration condensation reaction;

[0029] Figure 4 shows the optimization results of the photocrosslinking reaction (A: photoaffinity linker selection; B: photoaffinity linker dosage; C: physical state of the reaction system; D: solvent selection of the reaction system; E: reaction time);

[0030] Figure 5 shows the optimization results of the dehydration condensation reaction (A: physical state of the reaction system; B: solvent selection for the reaction system);

[0031] Figure 6 shows the validation results of the probe preparation method: functionalized magnetic nanoprobes with different compounds (A: Rhodamine B; B: Coumarin 6);

[0032] Figure 7 shows the results of probe target fishing verification: Celecoxib functionalized magnetic nanoprobes (Cel-MNPs) fishing and verification

[0033] Target protein (silver staining and western blotting results: 1, cell membrane protein solution; 2, blank control group; 3, normal fishing group; 4, competition control group. The arrow indicates the protein COX-2 that is specifically and covalently bound to celecoxib);

[0034] Figure 8 shows the silver staining results of Sco-MNPs fishing and separating target proteins from the RA cell model protein group (1, cell membrane protein solution; 4, blank control group; 5, normal fishing group; 6, competition control group. Arrows indicate the main protein bands that are specifically and covalently bound to scopoletin);

[0035] Figure 9 shows the silver staining results of Sco-MNPs fishing and isolating target proteins from the intestinal bacterial proteome of RA animal models (1, intestinal bacterial protein solution; 2, blank control group; 3, normal fishing group; 4, competition control group. Arrows and boxes indicate the main protein bands that are specifically and covalently bound to scopoletin);

[0036] Figure 10 shows the immunoblotting results of the target proteins fished by Sco-MNPs from the RA cell model protein group: 1, cell membrane protein solution; 2, blank control group; 3, normal fishing group; 4, competition control group). DETAILED DESCRIPTION

[0037] The present invention will be further described below by way of specific embodiments. The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.

[0038] 20 mmol of ferric chloride hexahydrate (FeCl3·6H2O), 0.05 mmol of polyvinylpyrrolidone (PVP) and 40 mmol of sodium acetate (NaOAc) were dispersed in 20 mL of ethylene glycol (EG) and sealed in a polytetrafluoroethylene-lined stainless steel autoclave for reaction at 200 °C for 10 h to obtain Fe3O4 nanoparticles (Fe3O4MNPs);

[0039] 50 mg of Fe3O4MNPs were dispersed in 50 mL of ethanol / water / ammonia solution (45:5:1, v / v), 0.1 mmol of tetraethyl orthosilicate (TEOS) was added, and ultrasonic reaction was carried out for 1 h to obtain Fe3O4@SiO2 nanoparticles (Fe3O4@SiO2MNPs);

[0040] Using 45 mL of ethanol as the solvent, 5 mL of aminopropyltriethoxysilane (APTES) was added to 50 mg of nanoparticles, and the mixture was refluxed for 2 h. The product was separated by a magnet, washed with deionized water, and dried to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2. The preparation process is shown in Figure 1.

[0041] 1 µmol of 4-[3-(trifluoromethyl)-3H-bis(aziridin-3-yl)benzoic acid (TDBA) and 0.25 µmol of scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm ultraviolet light at a distance of 1 cm for 15 minutes at room temperature. After the reaction was completed, the solvent was removed by rotary drying to obtain TDBA-scopoletin conjugate (TDBA-Sco).

[0042] TDBA-Sco was redissolved in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio), and 1.6 µL of triethylamine was added dropwise. 10 mg of NH2-MNPs was added, mixed, and shaken overnight in the dark. After the reaction was completed, the product was separated by magnet and washed with methanol to obtain scopoletin-functionalized magnetic nanoprobes (Sco-MNPs). The preparation process is shown in Figure 2.

[0043] Example 2: Optimization of process conditions for preparing scopolamine functionalized magnetic nanoprobes

[0044] The preparation of scopoletin-functionalized magnetic nanoprobes involves two main steps: coupling the photoaffinity linker to scopoletin via photocrosslinking, and coupling the amino groups at the terminals of the MNPs to the carboxyl groups of the photoaffinity linker via a dehydration condensation reaction. Many factors influence the coupling efficiency of scopoletin on the probe surface, and thus the fishing performance of the final probe. These factors primarily include: 1) the order of the two coupling reactions; 2) the choice and dosage of the photoaffinity linker during the photocrosslinking reaction, the physical conditions of the reaction system, the choice of solvent, and the reaction time; and 3) the physical conditions of the reaction system and the choice of solvent during the dehydration condensation reaction.

[0045] The present invention optimizes and screens the above factors, and the specific process is as follows:

[0046] (1) Optimization of the order of two coupled reactions

[0047] Sample 1: First do the photocrosslinking reaction, then do the dehydration condensation reaction

[0048] 1 µmol of 4-[3-(trifluoromethyl)-3H-bis(aziridin-3-yl)benzoic acid (TDBA) and 0.25 µmol of scopoletin were mixed and thoroughly dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. After completion of the reaction, the solvent was removed by rotary drying to obtain the TDBA-scopoletin conjugate (TDBA-Sco). TDBA-Sco was reconstituted in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio), and 1.6 µL of triethylamine was added dropwise. 10 mg of NH2-MNPs was added, mixed, and shaken overnight in the dark. After completion of the reaction, the product was separated using a magnet and washed with methanol to obtain scopoletin-functionalized magnetic nanoprobes (Sco-MNPs).

[0049] Sample 2: Dehydration condensation reaction first, then photocrosslinking reaction

[0050] 4-[3-(Trifluoromethyl)-3H-bis(aziridin-3-yl)benzoic acid (TDBA) was dissolved in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio), and 1.6 µL of triethylamine was added dropwise. 10 mg of NH2-MNPs was added, mixed, and shaken overnight in the dark. After the reaction, the product was separated by magnetism and washed with methanol to obtain a blank probe (TDBA-MNPs). The blank probe containing 1 µmol of TDBA and 0.25 µmol of scopoletin were mixed and thoroughly dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. After the reaction, the product was separated by magnetism and washed with methanol to obtain scopoletin-functionalized magnetic nanoprobes (Sco-MNPs).

[0051] For Samples 1 and 2, after the second reaction was complete, the concentration of scopoletin in the supernatant was measured by liquid chromatography-mass spectrometry. This concentration was compared with the initial scopoletin concentration to determine the coupling efficiency of scopoletin on the probe surface. As shown in Figure 3, the coupling efficiency of scopoletin in Sample 1 was significantly higher than that in Sample 2, suggesting that the photocrosslinking reaction should be performed first, followed by the dehydration condensation reaction.

[0052] (2) Optimization of photocrosslinking reaction

[0053] For each sample in this step, the ion peak area of ​​scopoletin in the supernatant before and after the reaction was determined by liquid chromatography-mass spectrometry; the two peak areas were subtracted and divided by the peak area before the reaction to obtain the coupling rate of scopoletin and the photoaffinity linker.

[0054] 2.1 Optimizing the selection of photoaffinity linkers:

[0055] Three photoaffinity linkers (1 µmol each) were mixed with 0.25 µmol of scopoletin and thoroughly dissolved in 1 mL of anhydrous methanol. The three photoaffinity linkers were 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid (TDBA), 3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diaziridin (SDAD), and 3-methyl-3H-diaziridin-3-propionic acid (MPDA). The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. The results are shown in Figure 4A. The coupling efficiency of the three photoaffinity linkers with scopoletin varied, with the order from highest to lowest being TDBA > SDAD > MPDA. Therefore, TDBA was the optimal photoaffinity linker.

[0056] 2.2 Optimize the amount of photoaffinity linker:

[0057] 0.25, 1, and 2.5 µmol of TDBA and 0.25 µmol of scopoletin were mixed and thoroughly dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. As shown in Figure 4B, when the photoaffinity linker dosage was 1 µmol or less, the coupling efficiency of scopoletin increased with increasing dosage. However, when the dosage exceeded 1 µmol, the adsorption of brucein D by MIPs decreased significantly. Therefore, the optimal photoaffinity linker dosage was 1 µmol.

[0058] 2.3 Optimize the physical state of the reaction system:

[0059] 1 µmol of TDBA and 0.25 µmol of scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol. The mixture was then spread evenly in a sealed glass container and dried under vacuum, nitrogen purge, or not dried in the solvent. The mixture was then irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. The results are shown in Figure 4, C. The coupling efficiency of the photoaffinity linker to scopoletin varied with the drying method, with the order from highest to lowest being not dried in the solvent > nitrogen purge > vacuum drying. Therefore, the optimal physical state of the reaction system is preferably dissolved in solution.

[0060] 2.4 Optimize the solvent selection of the reaction system:

[0061] 1 µmol of TDBA and 0.25 µmol of scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, dichloromethane, and carbon tetrachloride, respectively. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. The results are shown in Figure 4, D. The coupling efficiency of the photoaffinity linker to scopoletin varied with different solvents, with the order from highest to lowest being anhydrous methanol > carbon tetrachloride > dichloromethane. Therefore, anhydrous methanol was the optimal solvent for the reaction system.

[0062] 2.5 Optimize reaction time:

[0063] 1 µmol of TDBA and 0.25 µmol of scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15, 40, and 60 minutes at room temperature. As shown in Figure 4, E, after 15 minutes, the coupling rate between the photoaffinity linker and scopoletin did not increase significantly with time. Therefore, the optimal reaction time is 15 minutes.

[0064] (3) Optimization of dehydration condensation reaction

[0065] For each sample in this step, the ion peak area of ​​the TDBA-scopoletin coupling product in the supernatant before and after the reaction was determined by liquid chromatography-mass spectrometry; the two peak areas were subtracted and divided by the peak area before the reaction to obtain the coupling rate between the coupling product and NH2-MNPs.

[0066] 3.1 Optimize the physical state of the reaction system:

[0067] 1 µmol of TDBA and 0.25 µmol of scopoletin were mixed and thoroughly dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. After completion of the reaction, the solvent was removed by rotary drying to obtain the TDBA-scopoletin conjugate (TDBA-Sco). TDBA-Sco was reconstituted in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio) or dried under vacuum. 1.6 µL of triethylamine was then added dropwise, and 10 mg of NH2-MNPs were added. The mixture was then shaken overnight in the dark. As shown in Figure 5A, the TDBA-Sco coupling efficiency of the sample in solvent was significantly higher than that of the dried sample. Therefore, the optimal physical state of the reaction system is preferably dissolved in solution.

[0068] 3.2 Optimize the solvent selection of the reaction system:

[0069] 1 µmol of TDBA and 0.25 µmol of scopoletin were mixed and thoroughly dissolved in 1 mL of anhydrous methanol. The mixture was spread evenly in a sealed glass container and irradiated with 365 nm UV light at a distance of 1 cm for 15 minutes at room temperature. After the reaction, the solvent was removed by rotary drying to obtain the TDBA-scopoletin conjugate (TDBA-Sco). TDBA-Sco was reconstituted with 1 mL of isopropanol / dimethylformamide (IPA / DMF; 1:1 volume ratio), dimethylformamide (DMF), and 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), respectively. 1.6 µL of triethylamine was added dropwise, and 10 mg of NH2-MNPs was added. The mixture was mixed thoroughly and incubated in the dark overnight with shaking. The results are shown in Figure 5B. Using different solvents, the coupling rates of TDBA-Sco and NH2-MNPs are different, from high to low, in the order of IPA / DMF>DMF>HATU. Therefore, the optimal solvent for the reaction system is IPA / DMF (1:1 volume ratio).

[0070] The preparation method was the same as that in Example 1, except that scopoletin was replaced with rhodamine B (red fluorescent dye) and coumarin 6 (green fluorescent dye) to prepare rhodamine B and coumarin 6 functionalized magnetic nanoprobes (Rho-MNPs and Cou-MNPs), respectively.

[0071] Microscopic observations of Rho-MNPs and Cou-MNPs are shown in Figure 6. The products are quasi-spherical, relatively uniform in size, and well dispersed, demonstrating that the physical properties of the probe products are satisfactory. Under fluorescence, the nanoparticles exhibit uniform red and green light, respectively, indicating that rhodamine B and coumarin 6 are uniformly coupled to the surface of the magnetic nanoprobes, and both compounds have high coupling rates. Using a unified operating procedure, functionalized magnetic nanoprobes with different compounds were prepared, and significant coupling effects were achieved, verifying the feasibility of the probe preparation method of the present invention.

[0072] Prepare fishing buffer: add 50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 to deionized water. 2­ .

[0073] Prepare elution buffer: add 0.3% TritonX-100 to the fishing buffer.

[0074] Prepare electrophoresis buffer: 1 mL deionized water, 1 mL 0.5 M Tris-Cl (pH 6.8), 1 mL glycerol, 1 mL 10% SDS, 0.1 mL mercaptoethanol, and 0.2 mL 0.05% bromophenol blue.

[0075] (1) Preparation of celecoxib-functionalized magnetic nanoprobes

[0076] The preparation method was the same as that in Example 1, except that scopoletin was replaced with celecoxib (a COX-2 selective inhibitor) to prepare celecoxib functionalized magnetic nanoprobes (Cel-MNPs).

[0077] (2) Preparation of cell membrane protein solution:

[0078] HFLS-RA cells were taken, cell membrane proteins were extracted, and the concentration was adjusted to 3 mg / mL with fishing buffer.

[0079] (3) Prepare blank control group samples:

[0080] To 250 µL of cell membrane protein solution, 0.5 µL of DMSO was added and incubated at 4°C for 1 hour. Then, 2.5 mg of TDBA-MNPs (10 mg / mL, suspended in fishing buffer) was added and incubated at 4°C overnight with shaking. After incubation, the probes were collected by magnetic separation, washed three times with elution buffer, and 100 µL of electrophoresis buffer was added. The samples were boiled at 100°C for 5 minutes and then cooled to room temperature.

[0081] (4) Make a normal fishing group sample:

[0082] 0.5 µL of DMSO was added to 250 µL of the cell membrane protein solution and incubated at 4°C for 1 hour. Then, 2.5 mg of Cel-MNPs (10 mg / mL, suspended in fishing buffer) was added and incubated at 4°C with shaking overnight. After incubation, the probes were collected by magnetic separation, washed three times with elution buffer, and 100 µL of electrophoresis buffer was added. The samples were boiled at 100°C for 5 minutes and then cooled to room temperature.

[0083] (5) Preparation of competitive control group samples:

[0084] To 250 µL of the cell membrane protein solution, 0.5 µL of a 10 mM celecoxib solution in DMSO was added and incubated at 4°C for 1 hour. Then, 2.5 mg of Sco-MNPs (10 mg / mL, suspended in fishing buffer) were added and incubated overnight at 4°C with shaking. After incubation, the probes were collected by magnetic separation, washed three times with elution buffer, and 100 µL of electrophoresis buffer was added. The samples were boiled at 100°C for 5 minutes and then cooled to room temperature.

[0085] (6) Target protein separation, identification, and verification:

[0086] After boiling and cooling, each sample was separated by gel electrophoresis to observe the band differences. Differential protein bands were excised, decolorized, dehydrated, alkylated, and enzymatically digested to extract peptides. After purification, the peptides were analyzed by liquid chromatography-mass spectrometry. Protein structures were inferred through database comparison. Proteins identified in the bands were verified by western blotting.

[0087] The fishing and validation results are shown in Figure 7. The cell membrane protein solution contains numerous proteins (lane 1). No distinct bands were detected in the blank control group, indicating no significant nonspecific binding between the blank probe and the protein (lane 2). Comparing the normal fishing group with the blank control group, the normal fishing group showed significantly more bands and darker colors, indicating significant specific binding of the coupled celecoxib on the probe to the protein, including hydrogen bonding and covalent binding (lane 3). In the competition control group, since some target proteins were already stably covalently bound to free celecoxib prior to fishing, the remaining target proteins were only able to be fished out through hydrogen bonding with the coupled celecoxib on the probe (lane 4). Comparing the normal fishing group with the competition control group, a distinct differential protein band at approximately 74 kDa was observed in the normal fishing group, indicating covalent binding of the protein to celecoxib. Western blotting confirmed that the protein was COX-2, the selective inhibitory target of celecoxib. The above results indicate that the method of fishing for target proteins using compound-functionalized probes is feasible.

[0088] Example 5: Scopoletin-functionalized magnetic nanoprobes (Sco-MNPs) fishing and isolation of target proteins from the proteome of RA cell models

[0089] (1) Prepare blank control group, normal fishing group, and competition control group samples:

[0090] The methods for preparing cell membrane protein solutions and each group of samples were the same as in Example 4.

[0091] (2) Sample protein separation:

[0092] After boiling and cooling, the samples of each group were separated by gel electrophoresis and the band differences were observed.

[0093] The results are shown in Figure 8. The cell membrane protein solution contained numerous proteins (lane 1), while the blank control group exhibited no distinct bands (lane 4). Comparing the normal fishing group with the blank control group, the normal fishing group exhibited distinct bands, indicating that scopoletin specifically binds to the protein group (lane 5). Comparing the normal fishing group with the competition control group (lane 6), distinct differential protein bands at approximately 57, 50, and 40 kDa were observed in the normal fishing group, with the band at approximately 57 kDa being the most prominent, indicating that these proteins are targets of scopoletin and can covalently bind to it.

[0094] Example 6: Scopoletin-functionalized magnetic nanoprobes (Sco-MNPs) fishing and isolation of target proteins from the proteome of RA animal models

[0095] (1) Preparation of intestinal bacterial protein solution:

[0096] Male Sprague-Dawley rats were subcutaneously injected with 0.1 mL of complete Freund's adjuvant at the base of the tail to induce adjuvant arthritis. Twenty-one days after modeling, the rats were sacrificed and fresh feces collected. 10 g of feces was thoroughly homogenized in 40 mL of PBS, centrifuged at 700 g for 1 min, and the supernatant was collected. The pellet was then centrifuged again at 6000 g for 3 min, washed twice with 10 mL of PBS, added to 250 mL of anaerobic medium, and incubated in an anaerobic bag at 37°C for 12 h. After incubation, the pellet was centrifuged at 4000 g for 15 min and suspended in 1 mL of PBS to obtain the intestinal bacterial culture medium. The intestinal bacterial culture medium was then homogenized in HEPES lysis buffer and centrifuged at 700 g for 5 min at 4°C. The supernatant was collected and ultrafiltrated, and washed with lysis buffer to obtain the intestinal bacterial protein solution.

[0097] (2) Prepare blank control group, normal fishing group, and competition control group samples:

[0098] The method is the same as that of Example 4, except that the cell membrane protein solution is replaced with an intestinal bacteria protein solution.

[0099] (3) Sample protein separation:

[0100] After boiling and cooling, the samples of each group were separated by gel electrophoresis and the band differences were observed.

[0101] The results are shown in Figure 9. The cell membrane protein solution contained numerous proteins (lane 1), while the blank control group showed no distinct bands (lane 2). Comparing the normal fishing group with the blank control group, the normal fishing group showed distinct bands, indicating that scopoletin specifically binds to the proteins (lane 3). Comparing the normal fishing group with the competition control group (lane 4), the normal fishing group showed distinct differential protein bands at approximately 66 and 50–20 kDa, with the band at approximately 57 kDa being the most prominent, indicating that these proteins are targets of scopoletin and can covalently bind to it.

[0102] Example 7: Identification of target proteins angling from the RA cell model proteome by Sco-MNPs

[0103] Based on the results of Example 5, a differential protein band at approximately 57 kDa was excised. The excised protein band was sequentially decolorized, dehydrated, alkylated, and enzymatically digested to extract peptides. After purification, the peptides were detected by liquid chromatography-mass spectrometry. After database search and comparison, the protein was hypothesized to be vimentin.

[0104] Western blotting results are shown in Figure 10. The band at approximately 57 kDa was confirmed to be vimentin. Vimentin was observed in the cell membrane protein solution (lane 1), the normal fishing group (lane 3), and the competitive control group (lane 4), while no vimentin was detected in the blank fishing group (lane 2). The band in the normal fishing group (lane 3) was significantly denser and thicker than that in the competitive control group (lane 4), indicating that vimentin specifically binds to scopoletin, through both hydrogen bonding and covalent bonding.

Claims

1. A preparation method of scopoletin-functionalized magnetic nanoprobes, characterized in that, It includes the following steps: (1) React ferric chloride hexahydrate, polyvinylpyrrolidone and sodium acetate by dispersing them in ethylene glycol to obtain Fe3O4 nanoparticles; ultrasonically react the Fe3O4 nanoparticles and tetraethyl orthosilicate to obtain Fe3O4@SiO2 nanoparticles; (2) Using ethanol as a solvent, add 3-aminopropyltriethoxysilane to the Fe3O4@SiO2 nanoparticles and reflux to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2; (3) Mix the photoaffinity linker and scopolactone and fully dissolve them in an organic solvent, irradiate with 365 nm ultraviolet light at room temperature, after the reaction is completed, remove the solvent by rotary evaporation to obtain the photoaffinity linker-scopolactone conjugate; (4) Redissolve the photoaffinity linker-scopolactone conjugate in an organic solvent, dropwise add triethylamine, add the amino-terminally modified nanoparticles Fe3O4@SiO2-NH2, mix well and shake overnight in the dark. After the reaction is completed, separate the product with a magnet and wash with methanol to obtain the scopolactone-functionalized magnetic nanoprobe.

2. The preparation method of the scopoletin-functionalized magnetic nanoprobe according to claim 1, characterized in that: In step (3), the photoaffinity linker is one or more of 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid, 3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diazirin or 3-methyl-3H-diaziridin-3-propionic acid, preferably 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid.

3. The preparation method of the scopoletin-functionalized magnetic nanoprobe according to claim 1, wherein: In step (3), the molar ratio of the photoaffinity linker to scopolactone is 1-4:

1.

4. The preparation method of the scopoletin-functionalized magnetic nanoprobe according to claim 1, characterized in that: In step (3), the organic solvent is one or more of anhydrous methanol, dichloromethane or carbon tetrachloride, preferably anhydrous methanol.

5. The preparation method of the scopoletin-functionalized magnetic nanoprobe according to claim 1, characterized in that: In step (3), the irradiation time of the ultraviolet light at room temperature is 15-60 min.

6. The preparation method of the scopoletin-functionalized magnetic nanoprobe according to claim 1, characterized in that: In step (4), the organic solvent is one or more of a mixed solution of isopropanol and dimethylformamide in a volume ratio of 1:1, dimethylformamide or 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, preferably a mixed solution of isopropanol and dimethylformamide in a volume ratio of 1:

1.

7. A scopolactone-functionalized magnetic nanoprobe prepared by the preparation method according to any one of claims 1-6.

8. Use of the scopoletin-functionalized magnetic nanoprobe according to claim 7 in fishing and / or recognizing a target protein, characterized in that, Use the scopolactone-functionalized magnetic nanoprobe to perform target fishing on the proteome of a rheumatoid arthritis model.

9. The application according to claim 8, wherein The target protein of scopolactone is vimentin, and the specific binding of vimentin to scopolactone includes covalent binding and hydrogen bond binding.

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