Bile acid action target specifically enriched photoaffinity magnetic nano-microspheres as well as construction method and application thereof

Abstract

The invention discloses photoaffinity magnetic nano-microspheres specifically enriched with bile acid acting targets as well as a construction method and application of the photoaffinity magnetic nano-microspheres. The microsphere is sequentially composed of Fe3O4 magnetic nanoparticles, a TAD photoaffinity link layer and a bile acid molecular layer from inside to outside, and is called BAs microsphere for short. Bile acid molecules are fixed on the surfaces of the BAs microspheres through a photoactivation coupling technology to serve as molecular bait, potential targets in a biological sample are captured by the bile acid molecules fixed on the surfaces of the microspheres on the basis of specific interaction characteristics, and different targets are enriched on the surfaces of solid-phase microspheres. The preparation method comprises the following steps: firstly, preparing a target spot, stripping the target spot from the surface of the microsphere through specific elution, and accurately identifying and analyzing by adopting a high-resolution mass spectrum, so as to obtain a direct acting target spot of a specific bile acid component, and further explaining the pharmacological mechanism of bile acid from the source of the target spot.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a photoaffinity magnetic nanosphere specifically enriched for bile acid targets, its construction method, and its application. Background Technology

[0002] Bile acids are molecules with a steroidal skeleton (cyclopentanoperhydrophenanthrene), whose structure is ingeniously designed with a hydrophilic α-side and a hydrophobic β-side. Their core features are as follows: The steroidal core consists of three six-membered rings (A, B, and C rings) and one five-membered ring (D ring). This core is hydrophobic. Hydroxyl groups are attached to specific positions on the core (most commonly C-3, C-7, and C-12). These hydroxyl groups are hydrophilic, and their configuration is typically α-oriented. At the C-17 position of the D ring, a saturated 5-carbon side chain is attached, ending with a carboxyl group. This carboxyl group is usually deprotonated and negatively charged at physiological pH, making it a strongly hydrophilic group. Based on their source and synthetic pathway, bile acids are mainly classified into primary and secondary bile acids, such as cholic acid and chenodeoxycholic acid (primary bile acids) and deoxycholic acid and lithocholic acid (secondary bile acids). According to the modification of their chemical structure, they are mainly classified into free and conjugated bile acids, such as cholic acid and ursodeoxycholic acid (free bile acids). In the liver, the carboxyl side chain of free bile acids can combine with glycine or taurine via an amide bond to form conjugated bile acids. Bile acids are efficiently utilized through enterohepatic circulation and are an important pathway for cholesterol excretion.

[0003] Bile acids, as important signaling molecules in the body, not only play a key role in lipid absorption and metabolism, but also participate extensively in regulating physiological and pathological processes such as glucose homeostasis, energy metabolism and immune inflammation by activating a variety of nuclear and membrane receptors such as farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5). Research on their mechanism of action has become a cutting-edge hot topic in the development of drugs for metabolic diseases.

[0004] However, our current understanding of the mechanisms of action of bile acids in vivo is still limited to a few known receptors, and their direct and efficient target network in complex biological systems has not yet been fully revealed. This has become a key scientific problem restricting the development of this field. This cognitive bottleneck mainly stems from the limitations of existing target discovery technologies: traditional biochemical methods usually target a single pre-set target, making it difficult to achieve unbiased global target discovery; and the interactions between bile acids and their potential targets are often transient, have weak affinity, and depend on the cellular microenvironment, posing significant challenges to their capture and identification.

[0005] "Target fishing" technology provides a powerful research tool for overcoming this challenge. The core of this technology lies in immobilizing bile acids as "molecular bait" on the surface of a solid support, thereby specifically "fishing out" and enriching the proteins bound to them from complex biological samples (such as cell lysates and tissue homogenates), and finally identifying them using high-resolution mass spectrometry. However, the key to the success of this technology lies in how to efficiently, stably, and while maintaining the native active conformation of small bile acid molecules, immobilize them on the support. Existing immobilization strategies, especially those relying on specific chemical reactions such as carboxyl-amino coupling, usually have significant limitations: they require tedious pre-modification of the cross-linked molecules, a process that not only introduces uncertainty but may also lead to a loss or deviation in the binding ability to the true target due to changes in the three-dimensional conformation of the molecule after immobilization.

[0006] In recent years, photoaffinity labeling technology has opened up new avenues for target fishing techniques due to its unique mechanism of "locking in" transient, weak-affinity interactions. This technology generates active intermediates through ultraviolet light excitation of photosensitive groups (such as benzophenone and TAD), thereby causing irreversible covalent cross-linking with neighboring proteins. For example, Professor Zhang Ao's research group developed agarose microspheres with a surface-bonded bidentate ortho-dicarbonyl groups, which were successfully used to immobilize active targets in unpurified cell lysates. Professor Tu Pengfei's research group developed a polymeric solid-phase material with a surface-bonded benzophenone, which was successfully used to immobilize traditional Chinese medicine components and fish for targets. Based on existing research, we recognize that an ideal solid-phase carrier must simultaneously solve two core challenges: first, steric hindrance, ensuring that immobilized small molecules are fully exposed and accessible to target proteins; and second, characterizing immobilization efficiency, which requires accurate quantification of the amount of molecules immobilized on the carrier surface. While traditional photosensitive group materials have advantages, they still have inherent limitations: due to the direct connection of small molecules, the accessibility of target proteins may be low. At the same time, the immobilized small molecule-carrier complex is difficult to dissociate, making it difficult to quantitatively evaluate the immobilization efficiency.

[0007] To precisely overcome the technical bottleneck of bile acid target identification, this study designed and constructed a novel bile acid-guided photoaffinity magnetic microsphere. This microsphere possesses a distinct "core-shell" structure: an inner layer of Fe3O4 magnetic nanoparticles as the core, a middle layer modified with a photoaffinity linking layer, and an outer layer covalently immobilized with bile acid molecules; hereinafter referred to as "BAs microspheres". The innovation of this design is mainly reflected in four aspects: 1) Utilizing the excellent photocrosslinking efficiency and steric hindrance sensitivity of the TAD group, efficient immobilization of bile acid molecules is achieved under mild conditions, maximizing the preservation of their native conformation and biological activity; 2) The magnetic core makes solid-liquid separation during the "fishing" process extremely simple and rapid, significantly improving operational efficiency and making it suitable for high-throughput screening; 3) The entire system is specifically designed for single and well-defined bile acid "molecular baits," enabling comprehensive and unbiased capture of interacting proteins from the complex "molecular ocean" of biological samples, thereby accurately constructing a bile acid-target interaction map. 4) It can efficiently dissociate the immobilized small molecule-carrier complex and accurately quantify the molecular immobilization efficiency, providing key evidence for the reliability and reproducibility of the method. Summary of the Invention

[0008] This invention aims to provide photoaffinity magnetic nanospheres (denoted as BAs microspheres) specifically enriched for bile acid targets, along with their construction method and applications. Using these BAs microspheres, the interaction between bile acid systems and target proteins can be studied, and target proteins specifically binding to bile acid systems can be identified.

[0009] The technical solution of the present invention is as follows: A photoaffinity magnetic nanosphere specifically enriched for bile acid targets, abbreviated as BAs microspheres, is composed of Fe3O4 magnetic nanoparticles, a TAD photoaffinity linking layer, and a bile acid molecular layer from the inside out; wherein, TAD represents trifluoromethyl aryldiazirines. In the “BAs microspheres,” Fe3O4 magnetic nanoparticles are prepared by co-precipitation of FeCl3·6H2O and FeCl2·4H2O. The TAD photoaffinity linking layer and the bile acid molecular layer are the core layers of the “BAs microspheres.” As a photoaffinity group, TAD can covalently link small molecules with different structures to the immobilized matrix through the photoaffinity group. The binding of TAD to small molecules does not depend on specific functional groups. This compound generates highly reactive carbene (nitrobenzene) under ultraviolet 365nm irradiation and can insert into various chemical bonds, such as CH, OH, NH, etc., to form novel functionalized photoaffinity magnetic “BAs microspheres,” which can realize the self-assembly and immobilization of bile acid molecules and selective dissociation. In this invention, the microsphere system of each layer is characterized by scanning electron microscopy, XRD, and infrared spectroscopy. At the same time, the construction of the core layer of the “BAs microspheres”—the bile acid active ingredient layer—is analyzed by liquid chromatography. The "BAs microspheres" of this invention have the structure shown in formula (I), formula (II), or formula (III): (I) In formula (I), X1 is H or hydroxyl, and X2 is H or hydroxyl; the carbene undergoes a photocrosslinking reaction with the carboxyhydroxyl group to form a high-energy, zwitterionic intermediate. This intermediate rapidly undergoes proton migration and electron rearrangement, eventually generating a stable N-acyloxy derivative. (II) In formula (II), X1 is H or hydroxyl, X2 is H or hydroxyl, and Y1-Y8 are all CH3; the generated carbene attacks the CH2 group on the steroid nucleus or side chain of bile acid, inserts into the CH bond, and directly "stitches" itself onto the carbon skeleton of bile acid; (III) In formula (III), X1 is H or hydroxyl, X2 is H or hydroxyl, and Y1 is carboxyl or sulfonic acid group; the generated carbene preferentially attacks the NH bond of the amide or amine in the bile acid side chain and inserts into the NH bond to form a new NN bond.

[0010] The method for preparing BAs microspheres of the present invention includes the following steps: (1) Preparation of Fe3O4 magnetic nanoparticles FeCl2·4H2O, FeCl3·6H2O and ultrapure water were mixed and heated to 50~60℃. Ammonia water was added under N2 protection and stirred (450~500rpm) for 30min. After that, the mixture was allowed to stand and magnetically separated to discard the supernatant. The solid product was washed and dried to obtain Fe3O4 magnetic nanoparticles. The preferred molar ratio of FeCl2·4H2O and FeCl3·6H2O is 1:1.5~3; (2) Preparation of microspheres loaded with TAD photoaffinity linking layer The Fe3O4 magnetic nanoparticles obtained in step (1) were dispersed in ultrapure water, heated to 50-60℃, and oleic acid was added. The mixture was stirred (350-500 rpm) under N2 protection for 1-3 hours. The supernatant was removed by magnetic separation. The resulting black oily precipitate was washed, dried by N2, and dispersed in n-hexane. An aqueous solution of sodium periodate was added dropwise under stirring. The mixture was reacted at room temperature for 2-4 hours. The supernatant was removed by magnetic separation. NHS (N-hydroxysuccinimide) and EDC·HCl (1-ethyl-3-(3-dimethylamino)) were added to the resulting solid. A solution of N,N-dimethylformamide (N,N-dimethylformamide) carbodiimide hydrochloride was prepared and reacted at room temperature with stirring (500-600 rpm) for 2-4 hours. The supernatant was discarded by magnetic separation. The solid was then mixed with DIPEA (N,N-diisopropylethylamine) and TAD (trifluoromethylphenylbisacrylamide) in DMF and reacted at room temperature with stirring (500 rpm) in the dark for 8-12 hours. After magnetic separation and washing, the mixture was placed in a blocking solution and stirred at room temperature in the dark for 1-2 hours. The mixture was then washed and vacuum dried to obtain microspheres loaded with a TAD photoaffinity linking layer. The preferred feeding ratio of Fe3O4 magnetic nanoparticles, oleic acid, and sodium periodate is 1g:8mL:3.76g; The preferred feeding ratio of Fe3O4 magnetic nanoparticles, NHS, and EDC·HCl is 1.8g:8.29g:13.81g; The preferred feeding ratio of Fe3O4 magnetic nanoparticles, DIPEA, and TAD is 1.8g:369μL:180mg; The blocking solution was a 1 mol / L ethanolamine DMF solution; The resulting microspheres loaded with TAD photoaffinity linking layers have the structure shown in formula (IV): (IV) (3) Preparation of BAs microspheres The microspheres loaded with TAD photoaffinity linking layer obtained in step (2) were mixed with bile acid molecule solution, the solvent was evaporated under vacuum at 45°C, and then irradiated under 365nm ultraviolet light for 3~5h. After washing and vacuum drying, BAs microspheres were obtained (stored in a -20°C refrigerator for later use). The solvent for dissolving bile acid molecules is methanol or PBS; preferably, the concentration of the bile acid molecule solution is 25 mg / mL. The preferred feeding ratio of bile acid molecular solution to microspheres loaded with TAD photoaffinity linker layer is 5~8mL:600mg; In microspheres (IV) loaded with TAD photoaffinity linking layers, biacpropidine can generate highly reactive intermediate carbene (mainly nitrogen bene in the microsphere) after ultraviolet light irradiation. It rapidly undergoes photocrosslinking reaction with the NH, OH, and CH of bile acid molecules, forming covalent bonds through insertion reaction, thereby fixing bile acid molecules to the surface of the microsphere. Bile acid molecules, for example: cholic acid Chenodeoxycholic acid Ursodeoxycholic acid Deoxycholic acid Lithocholic acid Glycine deoxycholic acid Tauroursodeoxycholic acid.

[0011] The BAs microspheres of this invention can be used for bile acid interacting group target fishing.

[0012] The specific application methods are as follows: (1) Preparation of protein lysis buffer: (Sources of protein lysis buffer include, but are not limited to, cell lysis buffer, tissue lysis buffer, and commercially available lysis buffer) a. Obtaining cell protein lysis buffer: Aspirate the cell culture supernatant and wash the cells with pre-chilled PBS; add cell lysis buffer (NP-40:PMSF=100:1), spread evenly at the bottom, and place on ice for 10 min with shaking; scrape cells from the cell culture tube into an EP tube; place on ice for 20 min, vortexing once every 3 minutes; place the EP tube in a pre-chilled centrifuge at 4°C, 14000 rpm, for 12 min, until cell debris settles to the bottom and proteins dissolve in the NP-40 buffer; transfer the supernatant to a new pre-chilled EP tube for storage; determine protein concentration using the BCA assay kit according to the manufacturer's instructions; b. Obtaining tissue protein lysis buffer: Add 1 mL of NP-40 lysis buffer and 10 μg of PMSF per 100 mg of tissue; add grinding beads and homogenize the tissue for 1 min using a homogenizer at 4 °C; after complete lysis, centrifuge at 12000 rpm for 15 min at 4 °C, take the supernatant, and use the BCA assay kit to determine the protein concentration according to the manufacturer's instructions. (2) Co-incubation of “BAs microspheres” with protein lysis buffer: The experimental groups included a blank group, a binding group, and a competition group, with a third parallel experiment in each group. All experiments were conducted on ice. ① Blank group: Blank photoaffinity magnetic nanospheres without bile acid molecules were incubated with tissue lysis buffer at 4°C for 8-12 h; ② Binding group: "BAs microspheres" were incubated with tissue lysis buffer at 4°C for 8-12 h; ③ Competition group: Tissue lysis buffer was pre-incubated with bile acid molecule solution for 2 h, then "BAs microspheres" were added and incubated at 4°C for 8-12 h. After incubation, non-specifically adsorbed proteins were washed away with pre-cooled PBS and NP-40. After magnetically removing the supernatant, each group was boiled for 8-10 min with an appropriate amount of diluted loading buffer (6×), followed by boiling at 12000 rpm. -1 Centrifuge for 5 minutes, collect the supernatant, and store at -80℃ for later use. (3) Protein spectroscopy identification: The supernatant was subjected to SDS-PAGE gel electrophoresis. After enzymatic digestion of the obtained protein bands, each peptide was identified using the Q-Exactive liquid chromatography-mass spectrometry system, thereby accurately determining the properties and identity of each target protein.

[0013] This is an innovative solution specifically designed for the molecular characteristics of bile acids, mainly reflected in: 1. The magnetic carrier design enables rapid separation and microsphere reuse, which is crucial for capturing low-abundance bile acid-binding proteins; 2. Photoaffinity coupling technology can selectively immobilize the carboxyl, hydroxyl, and other groups of bile acids, thus preserving their natural conformation and binding activity to the greatest extent.

[0014] 3. The specially designed three sets of controls (blank / binding / competitive) can effectively distinguish between specific binding and non-specific adsorption, ensuring the reliability of the identification results.

[0015] The integration of the above three points has specifically overcome the technical challenge of "efficiently and specifically enriching low-abundance, weakly interacting bile acid targets in near-natural conditions," achieving both high efficiency and versatility.

[0016] Compared with the prior art, the present invention has the following significant advantages: The "BAs microspheres" used in this invention can be rapidly separated from tissue lysis fluid using a magnetic scaffold, eliminating the need for repeated high-speed centrifugation, greatly simplifying the operation process and significantly improving the efficiency of target enrichment experiments. This method supports magnetic recovery, enabling the reuse of the microspheres.

[0017] These "BAs microspheres" do not rely on specific functional groups of small molecules, enabling rapid and universal immobilization of small molecules. Through photoactivated coupling technology, bile acid molecules can be covalently bonded to the surface of solid-phase magnetic microspheres, constructing a stable probe system. The bile acid-modified magnetic microspheres are co-incubated with cell or tissue lysates. Utilizing the specific interactions between the bile acid molecules immobilized on the microsphere surface and target proteins, different target sites are efficiently enriched on the surface of the solid-phase microspheres. Subsequently, specific elution removes the target proteins from the microsphere surface for high-resolution mass spectrometry identification and analysis, ultimately obtaining a direct target group interacting with bile acids. This strategy helps elucidate the pharmacological mechanism of action of bile acids from the source of the target. Attached Figure Description

[0018] Figure 1 Schematic diagram of the "BAs microspheres" structure.

[0019] Figure 2 Scanning electron microscope images and particle size comparison diagrams of Fe3O4 magnetic nanolayer microspheres and "BAs microspheres" in Example 1 of this invention.

[0020] Figure 3 XRD diagram of the three-layer structure of "BAs microspheres" in Embodiment 1 of the present invention.

[0021] Figure 4 Infrared characterization image of the three-layer structure of "BAs microspheres" in Embodiment 1 of the present invention.

[0022] Figure 5 The following is a liquid phase detection image of the solution of the "BAs microspheres" before and after crosslinking with microspheres loaded with photoaffinity groups in Example 1 of this invention.

[0023] Figure 6 : Gel electrophoresis diagram of the enriched protein in Example 2 of the present invention.

[0024] Figure 7 : Volcano diagram of target proteins in Embodiment 2 of the present invention.

[0025] Figure 8 KEGG analysis diagram in Embodiment 2 of the present invention.

[0026] Figure 9 : GO analysis diagram in Embodiment 2 of the present invention. Detailed Implementation

[0027] To illustrate the technical solution of the present invention in detail, the following description is provided in conjunction with the accompanying drawings and embodiments. The provided embodiments are merely some examples of the present invention and do not constitute any limitation on the scope of protection of the claims.

[0028] Example 1: This example provides a method for synthesizing and characterizing "BAs microspheres" based on tauroursodeoxycholic acid. The specific reaction formula is as follows:

[0029] In tauroursodeoxycholic acid, the NH bond is a polar bond, and the oxygen and nitrogen atoms have high electron cloud densities. Nitrogen atoms are preferentially attracted to these electron-rich sites, resulting in a lower reaction energy barrier.

[0030] The C-3 and C-7 hydroxyl groups and the CH bonds at the C-1, C-2, C-4, and C-18 positions of tauroursodeoxycholic acid are all potential sites for photocrosslinking reactions, and azirone can also undergo covalent insertion at the same time.

[0031] 1. Construction of “BAs microspheres”: (1) Fe3O4 magnetic nanolayer microspheres: FeCl3·6H2O (11.2mM, final concentration, the same below) and FeCl2·4H2O (5.6mM) were added to 750mL of ultrapure water, transferred to a three-necked flask and heated to 50℃ in an oil bath. Under the condition of 50℃ and nitrogen gas, 62.5mL of ammonia water was quickly added, and the mixture was stirred at 500rpm for 30min. After washing with deoxygenated water, Fe3O4 magnetic nanolayer microspheres were obtained.

[0032] (2) Photoaffinity linking layer microspheres: 1.8g of Fe3O4 magnetic nanolayer microspheres obtained in step (1) were dispersed in 750mL of ultrapure water. After the temperature in the flask was raised to 50℃, 8mL of oleic acid was added. Under N2 protection, the mixture was stirred at 400rpm for 1h. The supernatant was separated by magnetic adsorption, and a black oily precipitate was obtained. The precipitate was washed three times with ethanol and the residual solvent on the surface of the magnetic beads was dried by N2. The obtained magnetic nanoparticles were dispersed in 90 mL of n-hexane, and sodium periodate (3.76 g, 137 mL) aqueous solution was added dropwise under stirring. The reaction was carried out at room temperature for 2 h, and the supernatant was discarded after magnetic separation. The obtained product was washed three times with ethanol and pure water, and dried under vacuum. 288 mL of DMF solution containing NHS (8.29 g, 200 mmol / L) and EDC·HCl (13.81 g, 200 mmol / L) was added to the solid material, and the reaction was carried out at room temperature for 2 h with stirring speed of 550 rpm. The activated and washed Fe3O4 magnetic nanospheres with long-chain carboxylic acids were added with DIPEA (369 μL, 50 mmol / L) and TAD (180 mg, 10 mmol / L) and DMF to a final volume of 45 mL. The reaction was carried out overnight at room temperature in the dark with stirring at 500 rpm. Magnetic separation was performed, followed by vortex washing with DMF. The washed carboxyl magnetic beads were placed in a round-bottom flask containing a blocking solution (1 mol / L ethanolamine, 225 mL) and stirred at room temperature in the dark for 1 h. After washing with ultrapure water and anhydrous ethanol, the beads were vacuum dried to obtain photoaffinity linking layer microspheres.

[0033] (3) Tauroursodeoxycholic acid molecular layer microspheres: Prepare a 25 mg / mL tauroursodeoxycholic acid (TUDCA) methanol solution. Accurately weigh 600 mg of TAD photoaffinity-linked microspheres (enough for one target fishing attempt) into a weighing bottle, add 5 mL of the above TUDCA solution, and gently shake to evenly spread the microspheres on the bottom of the bottle. Then, place the weighing bottle in a vacuum drying oven at 45°C to dry the solvent for 1–2 hours to completely remove the solvent. Transfer the dried microspheres to 365 nm ultraviolet light for 4 hours to induce a photocrosslinking reaction between the TAD groups and bile acid molecules. After the reaction, wash the microspheres three times with methanol to remove uncovalently bonded molecules, finally obtaining bile acid molecular layer microspheres, i.e., "BAs microspheres". Vacuum dry and store at -20°C for later use.

[0034] In the above construction method, Fe3O4 magnetic nanolayers were prepared by co-precipitation, controlling the molar ratio of Fe... 3+ :Fe 2+ The ratio of Fe3O4 magnetic nanospheres was 1.5:1 to 3:1. Subsequently, sodium periodate was used to break the C=C bonds of the Fe3O4 magnetic nanospheres and convert them into carboxyl groups, thereby obtaining Fe3O4 magnetic nanospheres with long-chain carboxyl group modification. Using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as dual activators, carboxyl magnetic beads were activated in equal volumes at a molar ratio of 1:1. Then, benzyltrifluorobisacrylidine was covalently linked to the surface of the activated magnetic beads to form a photoaffinity linking layer. A certain amount of microspheres loaded with photoaffinity groups was weighed and placed in a weighing bottle. Tauroursodeoxycholic acid was dissolved in methanol to prepare a 25 mg / mL solution, and 5 mL of this solution was added to the weighing bottle. The bottle was gently shaken to evenly spread the microspheres at the bottom. The sample was placed in a vacuum drying oven at 40–50 °C for 1–2 hours to remove excess solvent. Afterward, the dried magnetic beads were irradiated under a UV instrument at 365 nm for 3–5 hours to allow the photoaffinity groups to covalently cross-link with bile acids, forming an active small molecule layer. Finally, the microspheres were washed three times with methanol to remove unreacted molecules. The washed microspheres were then dried in a vacuum drying oven to obtain "BAs microspheres". A 5% sodium hydroxide solution was prepared as the dissociation solution. 100–150 mg of "BAs microspheres" was weighed and added to 5–10 ml of the dissociation solution. After ultrasonic treatment to ensure thorough dispersion, the mixture was transferred to a round-bottom flask and refluxed in a 90°C water bath for 2 hours. After the reaction, the pH of the dissociation solution was adjusted to 6–7 with 2M hydrochloric acid. The supernatant was collected by magnetic absorption, and then the liquid was evaporated to dryness using a rotary evaporator. It was then reconstituted with methanol and stored at 4°C for later use.

[0035] Each layer of the "BAs microspheres" construct was characterized by scanning electron microscopy, particle size distribution, XRD, and infrared spectroscopy. The results are shown in the figures below. Figure 2 , Figure 3 , Figure 4 .

[0036] Figure 2 This is a 100,000x scanning electron microscope image of Fe3O4 magnetic nanospheres. The Fe3O4 magnetic nanospheres have a relatively regular morphology, exhibiting a near-spherical shape, with an average particle size of 11-12 nm. The Fe3O4-TUDCA composite microspheres show a certain degree of aggregation due to the presence of the drug layer on the surface. Their average particle size increases to 18-20 nm, and this trend of increasing particle size from the inside out proves the successful preparation of "BAs microspheres".

[0037] Figure 3 The XRD patterns of the three-layer structure of "BAs microspheres" are Fe3O4, Fe3O4-TAD, and Fe3O4-TUDCA. Obvious diffraction peaks can be observed at 2θ = 18.2°, 30.1°, 35.4°, 43.1°, 53.4°, 56.9°, 62.5°, 70.9°, and 73.9°, respectively. These peaks correspond to the 111, 220, 311, 400, 422, 333, 440, 620, and 533 crystal planes of Fe3O4, consistent with the characteristics of the Fe3O4 peaks in the standard card PDF#74-0748. The positions of the diffraction peaks before and after coating are basically consistent, indicating that the crystal structure of the Fe3O4 core remains unchanged after sequential modification with the photoaffinity linking layer and the TUDCA molecular layer, and it still occupies the main component in the composite material. Furthermore, the diffraction peaks of the Fe3O4-TUDCA composite are sharper and have higher intensity, which may be attributed to the highly ordered crystal structure of TUDCA itself. After it is connected to the surface of the microspheres, it induces the formation of a more ordered assembly, and may even construct a new crystal structure, thereby enhancing the overall crystallinity and diffraction signal.

[0038] Figure 4 The infrared spectrum of the three-layer structure of "BAs microspheres" is shown, consisting of Fe3O4, Fe3O4-TAD, and Fe3O4-TUDCA, at 583 cm⁻¹. -1 The characteristic absorption peak at 3420 cm⁻¹ indicates the successful preparation of Fe₃O₄ magnetic nanolayers via the coprecipitation method. The Fe₃O₄ spectrum shows a peak at 3420 cm⁻¹. -1 The nearby absorption peak is caused by the stretching vibration of the -OH group, which is due to the addition of ammonia during the preparation process. At 1650 cm⁻¹... -1 The absorption peak at 2924 cm⁻¹ is the C=O peak. -1 and 2850cm -1The absorption peaks at these locations represent the asymmetric and symmetric stretching vibrations of CH3, respectively. These characteristic peaks collectively indicate the presence of organic molecules with carboxylic acid groups and alkyl chains on the surface of the nanoparticles. In the Fe3O4-TAD spectrum, the peak at 1618 cm⁻¹ may be the N=N stretching vibration peak, and the peak at 1350 cm⁻¹... -1 The absorption peak at 1050 cm⁻¹ is likely due to the stretching vibration of -CF₃, indicating that the photoaffinity linking layer has been successfully modified onto the microsphere surface. In the Fe₃O₄-TUDCA spectrum, the peak at 1050 cm⁻¹ is also present. -1 A characteristic absorption peak, possibly attributable to the CO stretching vibration in the TUDCA molecule, was observed nearby at 900 cm⁻¹. -1 Nearby, an NN stretching peak was observed after TUDCA reacted with the photoaffinity linker layer. The NN stretching peak is usually weak, indicating that the tauroursodeoxycholic acid small molecule layer has been successfully constructed.

[0039] Figure 5 This is a liquid chromatography (LC) analysis of the bile acid component layer, the core layer of the "BAs microspheres". The construction of the bile acid component layer of the "BAs microspheres" was performed using LC. A methanol solution of TUDCA was used as the pre-crosslinking sample. The washing solution was evaporated to dryness and then reconstituted with methanol to prepare the post-crosslinking sample. The dissociation solution of the crosslinked TUDCA "BAs microspheres" was evaporated to dryness and then reconstituted with methanol to prepare the post-dissociation sample. The peak area difference of the three groups of samples was determined by high performance liquid chromatography. The liquid chromatography detection was performed on an Aglient Eclipse C18 column (4.6 mm × 250 mm, 5.0 μm). The chromatographic conditions were as follows: the mobile phase was acetonitrile (A) - 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.5 with 80% phosphoric acid) (B) as the mobile phase for elution; elution conditions: 0-8 min, 20% A; 8-30 min, 45% A; 30 min, 20% A; flow rate 1 mL / min; detection wavelength 205 nm; injection volume 10 μL; column temperature 30℃. Under the above chromatographic conditions, the theoretical plate number calculated based on tauroursodeoxycholic acid shall not be less than 6000, and the tailing factor shall not exceed 2.0.

[0040]

[0041] The high-performance liquid chromatography (HPLC) method is as follows: The above sample is filtered through a 0.22 μm microporous membrane, and the filtrate is analyzed by liquid chromatography. (See figure...) Figure 5 Record the peak areas of the chromatograms and compare the differences among the three groups of samples, as shown in the table below:

[0042] Differential analysis of chromatographic peak areas confirmed the successful fixation and dissociation of TUDCA. a. The difference in peak area between the samples before and after crosslinking indicates that tauroursodeoxycholic acid has been successfully immobilized in the microspheres.

[0043] b. A significant TUDCA chromatographic peak was detected again in the sample after dissociation, confirming its effective recovery. Based on the peak areas before and after crosslinking, the fixation efficiency was calculated to be 60.91%. c. Based on the plotted TUDCA standard curve, the concentration of TUDCA in the dissociation solution was quantified, and the loading amount of TUDCA on the microspheres was calculated to be 152.48 μg / mg according to the loading formula.

[0044] Example 2

[0045] This embodiment provides a method for directly targeting specific sites in the liver tissue of diabetic nephropathy mice using tauroursodeoxycholic acid photoaffinity magnetic beads, which is carried out according to the following steps: (1) Preparation of liver tissue protein lysis buffer: All reagents and consumables required must be pre-cooled, and all operations must be performed on ice or at 4°C. Mouse liver tissue was taken from samples frozen at -80°C. Partially thawed on ice or at 4°C, maintaining the low temperature. The tissue was rinsed with pre-cooled PBS buffer or physiological saline to remove blood and impurities. Excess water was blotted with filter paper, and the tissue was cut into 1-2 mm³ pieces using surgical scissors or a blade. The shredded tissue was transferred to EP tubes. 100g of tissue was weighed and added to an appropriate amount of pre-cooled lysis buffer (RIPA:PMSF=100:1), along with three grinding beads. The sample was then homogenized in a low-temperature homogenizer at 60 Hz for 120 s until the tissue was completely lysed. The homogenate was placed in a pre-cooled centrifuge at 4°C and centrifuged at 12000 rpm for 5 min. The tissue fragments settled to the bottom, and the supernatant was collected as the total tissue protein lysis buffer, which was then transferred to new pre-cooled EP tubes for storage.

[0046] Protein quantification was performed using the BCA method: Protein standards were added to the standard wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, and then brought to a final volume of 20 μL with PBS. These concentrations corresponded to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively, and a standard curve was constructed.

[0047] Add an appropriate volume of sample to the wells of a 96-well plate. If the sample volume is less than 20 μL, add standard diluent PBS to bring it to 20 μL. Prepare an appropriate amount of BCA working solution by mixing 50 volumes of BCA reagent A with 1 volume of BCA reagent B (50:1), according to the sample quantity. Mix thoroughly, add 200 μL of BCA working solution to each well, and incubate at 37°C for 20-30 minutes. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and sample volume, and quantify using 500 μg / ml as the standard.

[0048] (2) Co-incubation of “BAs microspheres” with protein lysis buffer: The liver tissue lysate was divided into 9 aliquots, each containing 500 μg of protein. Three replicates were set up for each group: a blank control, a binding control, and a competition control. The blank control group was incubated with 100 mg of unbonded TUDCA magnetic nanospheres at 4°C for 12 h. The binding control group was incubated with 100 mg of TUDCA "BAs microspheres" at 4°C for 12 h. The competition control group was pre-incubated with tauroursodeoxycholic acid solution for 2 h, followed by incubation with 100 mg of tauroursodeoxycholic acid "BAs microspheres" at 4°C for 12 h. After incubation, each group was washed three times with 1 mL of pre-cooled PBS solution and then three times with 1 mL of elution buffer, each time for 3 min at a shaking frequency of 140 rpm. All washing was performed on ice. Loading buffer (1×) was added to each group of magnetic nanospheres after washing, and the mixture was boiled for 8-10 min. The supernatant was collected by magnetic absorption. Centrifuge the supernatant at 12,000 rpm for 5 min, collect the supernatant after centrifugation, and store it at -80℃ for later analysis.

[0049] (3) Identification of target proteins based on high-resolution mass spectrometry: SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining: Prepare an 8% separating gel and a 5% stacking gel according to the instructions. Add the prepared supernatant protein sample sequentially to each well, with a sample volume of 15 μL per well. Set the initial voltage to 50V to begin electrophoresis. When the bromophenol blue band just leaves the stacking gel and enters the separating gel, adjust the power supply voltage to 110V. Stop electrophoresis when the target band reaches the optimal viewing area on the separating gel. After electrophoresis, place the gel in Coomassie Brilliant Blue R-250 staining solution and shake for 1 hour. After staining, place the gel in destaining solution and shake for 3–4 hours. Remove the destaining solution, add an appropriate amount of ultrapure water, and shake overnight until the protein bands are clearly visible. See the results below. Figure 6 .

[0050] Protein bands in the gel were enzymatically digested, and the resulting peptides were analyzed and identified using a Q-Exactive liquid chromatography-mass spectrometry (LC-MS) system. Chromatographic separation was performed on a Thermo Acclaim PepMap C18 column (75 μm × 250 mm); chromatographic conditions: mobile phase: 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B); elution conditions: 0–45 min, 0%–45% B; 45–55 min, 45%–80% B; 55–60 min, 80% B; flow rate: 350 nL / min; injection volume: 1 μL. Mass spectrometry conditions: electrospray ionization voltage: 1.8 kV; ion source: ESI; ion source temperature: 320 °C; full scan range: m / z 350–1800. Finally, Proteome Discoverer software was used to match and search for peptides to identify target proteins.

[0051] (4) Target group identification and analysis: Based on mass spectrometry identification results, proteins with a (binding group mass spectrometry intensity - blank group mass spectrometry intensity) / (competitive group mass spectrometry intensity - blank group mass spectrometry intensity) greater than 1.5 and P < 0.05 were selected as differentially expressed proteins. The results are shown in […]. Figure 7 KEGG enrichment analysis was performed on the 719 screened proteins, ultimately identifying 11 pathways closely related to bile acids and the pathology of diabetes, including pathways regulating bile acids, lipid metabolism and inflammation, fibrosis, and reactive oxygen species. Among these, the pathways regulating bile acids mainly involve bile secretion and primary bile acid biosynthesis. The results are shown in [Figure number missing]. Figure 8 This study screened TUDCA targets using a pecking protein assay and then performed GO enrichment analysis on differentially expressed proteins (P < 0.01) to reveal the functional localization and mechanism of action of proteins in cells from three dimensions: biological processes (BP), cellular composition (CC), and molecular function (MF). TUDCA may be associated with pathological processes through the following biological pathways: TUDCA can affect biological processes such as cytoplasmic translation, fatty acid oxidation, protein folding, bile acid biosynthesis, mitochondrial ATP synthesis, and regulation of substance transport; differentially expressed proteins are mainly concentrated in secretion-related structures (such as cytoplasm, ribosomes, and peroxisomes), energy metabolism hubs (such as mitochondria), and dynamic cell membrane structures (such as focal adhesion); key molecular functions involve protein-protein interaction networks (such as ATP binding, RNA binding, and enzyme binding). The results are shown in [Figure number missing]. Figure 9 .

Claims

1. A photoaffinity magnetic nanosphere specifically enriched at the target site of bile acids, abbreviated as BAs microspheres, is composed of Fe3O4 magnetic nanoparticles, a TAD photoaffinity linking layer, and a bile acid molecular layer from the inside out; wherein, TAD represents trifluoromethylphenyl bisacrylidine.

2. The BAs microspheres as described in claim 1, characterized in that, It has the structure shown in Equation (I), Equation (II), or Equation (III): （I） In formula (I), X1 is H or a hydroxyl group, and X2 is H or a hydroxyl group; (II) In formula (II), X1 is H or hydroxyl, X2 is H or hydroxyl, and Y1-Y8 are all CH3; (III) In formula (III), X1 is H or hydroxyl, X2 is H or hydroxyl, and Y1 is carboxyl or sulfonic acid group.

3. The method for preparing BAs microspheres as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of Fe3O4 magnetic nanoparticles FeCl2·4H2O, FeCl3·6H2O and ultrapure water were mixed and heated to 50~60℃. Ammonia water was added under N2 protection and stirred for 30 min. After standing, the supernatant was discarded by magnetic adsorption separation. The solid product was washed and dried to obtain Fe3O4 magnetic nanoparticles. (2) Preparation of microspheres loaded with TAD photoaffinity linking layer The Fe3O4 magnetic nanoparticles obtained in step (1) were dispersed in ultrapure water, heated to 50-60℃, oleic acid was added, and the reaction was stirred for 1-3 hours under N2 protection. The supernatant was removed by magnetic separation. The resulting black oily precipitate was washed, dried by N2, and dispersed in n-hexane. An aqueous solution of sodium periodate was added dropwise under stirring. The reaction was carried out at room temperature for 2-4 hours. The supernatant was removed by magnetic separation. A DMF solution of NHS and EDC·HCl was added to the obtained solid material. The reaction was carried out at room temperature for 2-4 hours. The supernatant was removed by magnetic separation. The solid material was mixed with DIPEA and TAD in DMF and stirred at room temperature in the dark for 8-12 hours. After magnetic separation and washing, the mixture was placed in a blocking liquid and stirred at room temperature in the dark for 1-2 hours. The mixture was washed and vacuum dried to obtain microspheres loaded with a TAD photoaffinity linking layer. The blocking solution is a DMF solution of ethanolamine; The resulting microspheres loaded with TAD photoaffinity linking layers have the structure shown in formula (IV): (IV) (3) Preparation of BAs microspheres The microspheres loaded with TAD photoaffinity linking layer obtained in step (2) were mixed with bile acid molecular solution, the solvent was evaporated under vacuum at 45°C, and then irradiated under 365nm ultraviolet light for 3-5 hours. After washing and vacuum drying, BAs microspheres were obtained.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of FeCl2·4H2O and FeCl3·6H2O is 1:1.5~3.

5. The preparation method according to claim 3, characterized in that, In step (2), the feeding ratio of Fe3O4 magnetic nanoparticles, oleic acid, and sodium periodate is 1g:8mL:3.76g.

6. The preparation method according to claim 3, characterized in that, In step (2), the feeding ratio of Fe3O4 magnetic nanoparticles, NHS, and EDC·HCl is 1.8g: 8.29g: 13.81g.

7. The preparation method according to claim 3, characterized in that, In step (2), the feeding ratio of Fe3O4 magnetic nanoparticles, DIPEA, and TAD is 1.8g: 369μL: 180mg.

8. The preparation method according to claim 3, characterized in that, In step (3), the solvent for dissolving bile acid molecules is methanol or PBS, and the concentration of the bile acid molecule solution is 25 mg / mL; the feeding ratio of bile acid molecule solution to microspheres loaded with TAD photoaffinity linking layer is 5~8 mL: 600 mg.

9. The preparation method according to claim 3, characterized in that, In step (3), bile acid molecules are selected from: cholic acid Goose-extracted biliary acid Ursodeoxycholic acid Deoxycholic acid Lithocholic acid Glycine deoxycholic acid Tauroursodeoxycholic acid.

10. The application of BAs microspheres as described in claim 1 in bile acid interacting group target fishing.

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