A nano-drug for recurrent spontaneous abortion and a preparation method and use thereof

Abstract

The application belongs to the technical field of targeted drug delivery, and particularly relates to a nano-drug for recurrent abortion and a preparation method and application thereof. The nano-drug is prepared by loading baicalin into a transferrin-baitao exosome conjugate, the preparation method is simple, and the nano-drug shows good immunoregulation and anti-embryo resorption effects in an in-vitro immune inflammation model and an in-vivo recurrent abortion model. Safety testing shows that the nano-drug has good compatibility.

Description

Technical Field

[0001] This invention belongs to the field of targeted drug delivery technology, specifically relating to a nanomedicine for recurrent miscarriage, its preparation method, and its uses. Background Technology

[0002] Recurrent spontaneous abortion (RSA) refers to the loss of three or more pregnancies with the same partner. It is a major reproductive health challenge affecting approximately 1% to 5% of couples of reproductive age worldwide. Its etiology is complex, placing a heavy physical and psychological burden on patients. Endometrial homeostasis is a prerequisite for the establishment and maintenance of pregnancy, a process closely dependent on the precise dynamic balance of the local immune microenvironment. This balance is maintained by a complex regulatory network composed of epithelial cells, stromal cells, and immune cells, primarily uterine natural killer cells (uNK cells) and macrophages. Within this network, the functional polarization of decidual macrophages is particularly crucial; the balance between their classic pro-inflammatory M1 phenotype and the alternative anti-inflammatory M2 phenotype is central to establishing and maintaining maternal-fetal immune tolerance. Numerous studies have shown that the endometrium of patients with recurrent spontaneous abortion (RSA) exhibits significant immune dysregulation, characterized by abnormal overactivation of M1 macrophages. This leads to the massive release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), creating a persistent inflammatory microenvironment unfavorable to embryo implantation and development, ultimately resulting in pregnancy failure. However, current clinical immunomodulatory strategies for RSA, such as glucocorticoids or intravenous immunoglobulins, often suffer from inconsistent efficacy, lack of tissue targeting, and potential systemic side effects. Therefore, the development of novel targeted therapies capable of precisely regulating local immune homeostasis in the endometrium is urgently needed.

[0003] Scutellaria baicalensis and Atractylodes macrocephala are commonly used herbal pairs in compound formulas for preventing miscarriage (e.g., CN1305840A). Baicalin, a compound known to regulate the maternal-fetal immune microenvironment and thus improve miscarriage (Ma Yannan, Yang Xiaoqi. Effects of baicalin on the maternal-fetal immune microenvironment in LPS-induced miscarriage mice [J]. Journal of Changchun University of Traditional Chinese Medicine, 2018, 34(2):4.), is considered the main active ingredient in Scutellaria baicalensis for preventing miscarriage. Meanwhile, Atractylodes macrocephala polysaccharide is considered the main active ingredient in Atractylodes macrocephala for preventing miscarriage (CN111000860A).

[0004] Exosomes are extracellular vesicles with a diameter of approximately 30-200 nm, secreted by cells, and carrying various substances such as proteins, lipids, DNA, and RNA glycoconjugates. Research on Atractylodes macrocephala exosomes is currently limited; published literature indicates that exosomes containing Atractylodes macrocephala exosomes can alleviate ovarian cancer (CN120605312A). Previous research by the inventors found that Atractylodes macrocephala exosomes have significant immunomodulatory effects (the pharmacodynamic material basis and mechanism are still unclear), but as nanoscale complex structures, exosomes are extremely difficult to reach the maternal-fetal interface or endometrial region, thus limiting their ability to regulate miscarriage; similarly, baicalin has low bioavailability.

[0005] To date, there are no reports on the use of Atractylodes macrocephala exosomes for abortion or on technical means to overcome the difficulty of Atractylodes macrocephala exosomes and baicalin in targeting the maternal-fetal interface. Summary of the Invention

[0006] To address the above problems, the purpose of this invention is to provide a nanomedicine for recurrent miscarriage, its preparation method, and its uses. The technical solution of this invention is as follows:

[0007] A nanomedicine for recurrent miscarriage, wherein the nanomedicine is a transferrin-Atractylodes macrocephala exosome covalent conjugate loaded with baicalin.

[0008] Preferably, the mass ratio of transferrin-Atractylodes macrocephala exosome covalent conjugate to baicalin in the nanomedicine is (0.3~3):10.

[0009] Preferably, the mass ratio of transferrin to Atractylodes macrocephala exosomes in the nanomedicine is (0.03~0.3):2.

[0010] Preferably, the nanomedicine exhibits a spherical structure under a scanning electron microscope; the diameter of the spherical structure of the drug is 30 nm to 150 nm.

[0011] Preferably, the nanomedicine has a carbon content of 6%~7%, a nitrogen content of 54%~55%, an oxygen content of 28%~29%, a sulfur content of 1%~2%, and a phosphorus content of 8%~9%.

[0012] Preferably, the zeta potential of the nanomedicine is 1.5mV to 5mV.

[0013] Preferably, the ultraviolet-visible absorption spectrum of the nanomedicine has an absorption peak at 270 nm.

[0014] The aforementioned method for preparing nanomedicines for recurrent miscarriage includes the following steps:

[0015] S2 Atractylodes macrocephala exosome activation: Atractylodes macrocephala exosomes and activator are mixed in solvent and reacted in the dark; after the reaction is completed, the supernatant is discarded by centrifugation, the residual activator is washed away and then resuspended to obtain activated Atractylodes macrocephala exosomes;

[0016] S3 Transferrin Coupling: Activated Atractylodes macrocephala exosomes were added to a transferrin solution and mixed to react for coupling. After the reaction was completed, the unreacted activation sites were blocked, the supernatant was discarded by centrifugation, and the uncoupled transferrin was removed to obtain the transferrin-Atractylodes macrocephala exosome covalent conjugate.

[0017] S4-loaded baicalin: Transferrin-Atractylodes macrocephala exosome covalent conjugate and baicalin were mixed in a solvent, and baicalin was loaded using electroporation and incubated to obtain baicalin-loaded transferrin-Atractylodes macrocephala exosome covalent conjugate.

[0018] Preferably, in step S2, the activator is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and bis(N-hydroxysuccinimide) polyethylene glycol ester. Bis(N-hydroxysuccinimide) polyethylene glycol ester, also known as NHS-PEG-NHS, has been shown to be useful for coupling protein peptides to the surface of exosomes, as detailed in patent document CN119548642A.

[0019] Preferably, the preparation method further includes the following steps: S1 Preparation of Atractylodes macrocephala exosomes: Atractylodes macrocephala is hydrolyzed by cellulase and pectinase, the supernatant is collected by centrifugation, the filtrate is collected by filtration through a filter membrane, the supernatant is removed by ultracentrifugation, and Atractylodes macrocephala exosomes are prepared.

[0020] The aforementioned nanomedicines for recurrent miscarriage are used in the preparation of drugs for the treatment of recurrent miscarriage.

[0021] Beneficial effects:

[0022] The inventors of this invention discovered in their research that Atractylodes macrocephala exosomes have good immunomodulatory effects. However, due to their physical properties, Atractylodes macrocephala exosomes and baicalin are difficult to effectively target the endometrium or the maternal-fetal interface, and thus cannot effectively regulate the immune balance of the endometrium or the maternal-fetal interface, which limits their application in recurrent miscarriage.

[0023] The inventors constructed a complex drug based on transferrin covalently coupled to Atractylodes macrocephala exosomes and electroporated to load baicalin. Studies have shown that this drug can effectively target the endometrium or maternal-fetal interface, thereby improving targeted treatment for recurrent miscarriage. In addition, Atractylodes macrocephala exosomes and baicalin exhibited an unexpected synergistic effect, and the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin unexpectedly showed uniform dispersion and was not prone to aggregation, making it easier to control drug release and tissue distribution. It also facilitates pH-dependent drug release at the target site (rapid release in pathological acidic environments and slower release in physiological pH environments), showing a superior effect on inflammation and embryonic resorption at the target site. Therefore, it has better application prospects in recurrent miscarriage. Attached Figure Description

[0024] Figure 1 Scanning electron microscope images of the transferrin-Atractylodes macrocephala exosome conjugate sample (A) and Atractylodes macrocephala exosomes (B) loaded with baicalin in Example 1;

[0025] Figure 2 The following are the elemental mass percentage statistics of the transferrin-Atractylodes macrocephala exosome conjugate sample loaded with baicalin in Example 1: (A) elemental distribution map of O element and (C) elemental distribution map of P, N, C, and S elemental distribution map.

[0026] Figure 3 (A) Comparison of the hydrodynamic size distribution of Atractylodes macrocephala exosomes, transferrin-Atractylodes macrocephala exosome conjugates, and transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin; (B) 14-day particle size change of transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin; (C) Hydrodynamic size distribution and 14-day particle size change of Atractylodes macrocephala exosomes.

[0027] Figure 4 The diagrams show the zeta potentials of (A) Atractylodes macrocephala exosomes, transferrin-Atractylodes macrocephala exosome conjugates, and transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin; (B) the 14-day zeta potential changes of transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin; and (C) the 14-day zeta potential changes of Atractylodes macrocephala exosomes.

[0028] Figure 5 Comparison of UV-Vis absorption spectra of Atractylodes macrocephala exosomes, transferrin-Atractylodes macrocephala exosome conjugates and transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin on day 1 (A) and day 15 (B);

[0029] Figure 6XPS spectra of the transferrin-Atractylodes macrocephala exosome conjugate sample loaded with baicalin in Example 1: (A) total spectrum; (B) P 2p spectrum; (C) S 2p spectrum; (D) N 1s spectrum; (E) C 1s spectrum and (F) O 1s spectrum;

[0030] Figure 7 The image shows a comparison of Fourier transform infrared spectra of (A) baicalin, transferrin, transferrin-Atractylodes macrocephala exosome conjugate and transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin, and (B) immunoblotting analysis of specific labeled proteins of Atractylodes macrocephala exosome and transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin.

[0031] Figure 8 (A) UV absorption spectra of baicalin solutions at different concentrations; (B) standard curves and (C) drug release curves of transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin at different pH values;

[0032] Figure 9 Comparison of (A) interleukin-10, (B) tumor necrosis factor-α and (C) interleukin-6 concentrations in RAW 264.7 cells treated with different samples in Example 2;

[0033] Figure 10 This is a graph showing the statistical results of embryo absorption rates in each group of animals in Example 3;

[0034] Figure 11 These are HE staining pathological images of animal organs from each group in Example 3;

[0035] Figure 12 For the in vitro biosafety assessment of Example 4, (A) images of the supernatant of different samples at 12 h; (B) images of the supernatant of different samples at 24 h; (C) relative hemolysis rate of different samples at 12 h; and (D) relative hemolysis rate of different samples at 24 h.

[0036] Figure 13 The statistical results of (A) in vivo anti-embryonic absorption of transferrin-Atractylodes macrocephala exosome conjugates loaded with different compounds and the statistical results of their regulatory effects on (B) interleukin-10, (C) tumor necrosis factor-α and (D) interleukin-6 are shown in the figure.

[0037] Figure 14 The statistical results of (A) in vivo anti-embryonic absorption of different exosome-transferrin conjugates loaded with baicalin and the statistical results of their regulatory effects on (B) interleukin-10, (C) tumor necrosis factor-α and (D) interleukin-6 are shown in the figure. Detailed Implementation

[0038] The present invention and its effects are further illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are merely illustrative of the implementation methods and effects of the present invention and are not intended to limit the scope of the invention. After reading the contents of this invention, those skilled in the art can make various simple modifications or alterations to the invention without creative effort, and these equivalent / identical transformations also fall within the protection scope of this invention.

[0039] Example 1: Preparation, analysis, and characterization of transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin.

[0040] 1. Preparation of transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin

[0041] (1) Extraction of exosomes by ultracentrifugation: Pulverized dried rhizome slices of Atractylodes macrocephala (dried rhizomes dried at constant temperature to a moisture content <12%) were added to a PBS buffer containing cellulase and pectinase (the concentrations of cellulase and pectinase in the PBS buffer were 2 mg / L and 5 mg / L, respectively; 20 mL of PBS buffer was used per gram of Atractylodes macrocephala sample). The mixture was then centrifuged to remove residues to obtain the Atractylodes macrocephala enzymatic hydrolysate, which was stored at -80°C. The hydrolysate was then rapidly thawed at 37°C. The sample was transferred to a new centrifuge tube and centrifuged at 2000 × g, 4°C for 30 min. The supernatant was transferred to a new centrifuge tube and centrifuged again at 10,000 × g, 4°C for 45 min to remove larger vesicles. The supernatant was collected and filtered through a 0.45 μm filter membrane. Transfer the filtrate to a new centrifuge tube and centrifuge at 100,000 × g for 70 min at 4 ℃. Remove the supernatant, resuspend in 10 mL of pre-chilled PBS (phosphate-buffered saline, pH=7.4), and centrifuge again at 100,000 × g for 70 min at 4 ℃. Remove the supernatant and resuspend in 5200 μL of pre-chilled PBS (pH=7.4) to obtain purified Atractylodes macrocephala exosomes. 20 μL was used for electron microscopy, 10 μL for particle size analysis, and 10 μL for protein extraction. 100 μL was added to 700 μL of RNA lysis buffer, and the remaining exosomes were stored at -80 ℃. The purified exosomes were characterized by morphology observation under transmission electron microscopy, particle size distribution analysis using hydrodynamics, and particle size variation analysis.

[0042] (2) Exosome activation: The purified Atractylodes macrocephala exosomes were diluted to 1 mg / mL with PBS (pH=7.4). 2 mL of the diluted solution was transferred to a 10 mL centrifuge tube, and 2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide were added. The mixture was gently vortexed and incubated at room temperature in the dark for 30 min. After the reaction, the mixture was ultracentrifuged at 100,000 × g for 10 min, and the supernatant was discarded. The mixture was washed with 2 mL of PBS (pH=7.4) and ultracentrifuged at 100,000 × g for 10 min to thoroughly remove any residual activating agents. Finally, the activated Atractylodes macrocephala exosomes were resuspended in 1 mL of 2-morpholinoethanesulfonic acid (MES) buffer.

[0043] (3) Transferrin conjugation: Add 100 µL of transferrin aqueous solution (1 mg / mL) to the activated Atractylodes macrocephala exosomes and mix gently. Place on a rotary mixer and slowly rotate overnight at room temperature. That is, the mass ratio of transferrin to Atractylodes macrocephala exosomes is approximately 0.1:2. After the conjugation reaction is complete, add 2 mL of tris(hydroxymethyl)aminomethane buffer (50 mM, pH 8.5) and slowly rotate at room temperature for 2 h to block unreacted activation sites. After blocking, centrifuge at 100,000 × g for 10 min and discard the supernatant to remove unconjugated transferrin. Then wash with PBS (pH=7.4) and centrifuge again at 100,000 × g for 10 min. Resuspend the precipitate in PBS (pH=7.4), collect the sample, and store the transferrin-Atractylodes macrocephala exosome conjugate at 4 ℃ for later use.

[0044] (4) Loading baicalin: Accurately weigh 10 mg of baicalin, add 10 mL of PBS (pH=7.4), vortex until completely dissolved, and prepare a drug solution of 1 mg / mL. Take 200 μL of transferrin-Atractylodes macrocephala exosome conjugate (0.5 mg / mL) suspension into a 5 mL centrifuge tube, and add 1 mL of the above baicalin drug solution (1 mg / mL). Loading was performed using an electroporator (voltage: 100 V; capacitance: 100 μF; number of pulses: 1; pulse duration: 3 ms; buffer: PBS pH=7.4; electroporator: Bio-Rad MicroPulser). The mass ratio of transferrin-Atractylodes macrocephala exosome conjugate to baicalin was approximately 1:10. Then, the mixture was placed on a constant temperature shaker and incubated at 4 °C for 4 h. Then, it was transferred to a 37 °C shaker and incubated for 30 min. After incubation, the supernatant was discarded by ultracentrifugation to remove unencapsulated drug. Subsequently, the sample was washed once with PBS (pH=7.4) under the same conditions to obtain the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin.

[0045] 2. Analysis and characterization of intermediate products and transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin

[0046] The intermediate products and the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin from the preparation process in Part 1 of this embodiment were characterized and analyzed as follows:

[0047] 2.1 Morphological, dimensional, and elemental analysis

[0048] (1) Scanning electron microscopy (SEM) analysis: Take an appropriate amount of sample, dilute it with PBS (pH=7.4), and add 5~10 µL to the surface of a special silicon wafer. Let it stand at room temperature for 5 min to allow the vesicles to adsorb. Then add an equal volume of 4% paraformaldehyde solution and fix it at room temperature for 30 min. After fixation, carefully absorb the excess liquid from the edge with filter paper. Add 20 µL of 2% (w / v) sodium phosphotungstenate solution (pH 7.0) for negative staining, and remove the stain after 1~2 min. Place the sample in a clean environment to air dry. After the silicon wafer is completely dry, place it in the sample holder of the scanning electron microscope and observe the morphology, structure and size distribution of exosomes under a suitable accelerating voltage.

[0049] The results are as follows Figure 1 As shown. The transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin exhibits a classic round or nearly round spherical structure, with uniform size and a diameter of approximately 80 nm. Figure 1 The A in the figure is consistent with the classic morphology of exosomes reported in most literature. Furthermore, its clear edges and complete structure are consistent with the morphology of unmodified Atractylodes macrocephala exosomes. Figure 1 (See section B) This indicates that the coupling modification of transferrin on the surface of Atractylodes macrocephala exosomes successfully preserves the morphologically intact natural nanovesicle structure. Furthermore, compared to Atractylodes macrocephala exosomes, the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin exhibits relatively uniform dispersion and no obvious aggregation. In contrast, Atractylodes macrocephala exosomes themselves are highly prone to aggregation (see section B). Figure 1 The aggregate in the upper right corner of B in the formula is one of the reasons why it is not easily absorbed or difficult to target the endometrium or the maternal-fetal interface.

[0050] In addition, SEM images of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin were analyzed on day 15. The nanoparticles exhibited a spherical morphology, with the scale bar indicating a size of approximately 80 nm. The particles were also relatively uniformly distributed with no obvious aggregation. This result reflects, from the perspective of material morphology, that the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin could still maintain good structural integrity and dispersibility after 15 days, demonstrating its excellent stability.

[0051] (2) Elemental Analysis: To further investigate and compare the elemental composition of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin, EDS (energy-dispersive X-ray spectroscopy) was used to analyze its elements, and the mass percentage of each element was quantitatively calculated. The results are as follows: Figure 2 As shown. Figure 2 In the figure, A represents the final elemental mass percentage statistical result of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin. Figure 2 B and C in the figure are elemental distribution maps of different elements in the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin. It can be seen that the C element content in the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin is 6.85%, which mainly comes from phospholipids and cholesterol in the exosome lipid bilayer membrane, as well as the backbone of biomacromolecules such as proteins and nucleic acids carried inside, the surface-modified transferrin, and the internally loaded baicalin; the N element content is as high as 54.39%, which strongly points to the rich protein components in Atractylodes macrocephala exosomes, including the membrane-modified transferrin and various functional proteins in the lumen; the O element content is 28.75%, which is related to carbohydrates (such as polysaccharides on the surface of exosomes), proteins, loaded drugs, and oxygen-containing functional groups in lipids. Figure 2 The elemental distribution mapping of B and C visualizes the quantitative composition in space. The green (O element distribution), cyan (N element distribution), and purple (C element distribution) signal points are uniformly distributed across the outline regions of single or multiple exosome particles. This not only confirms the coexistence of carbon, nitrogen, and oxygen—the three core elements—in the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin, but more importantly, it demonstrates the uniformity of their distribution, rather than aggregation or bias towards a particular area. This indicates that the chemical composition of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin is integrated within its structure, with its protein (nitrogen signal), lipid / carbohydrate (carbon and oxygen signals), and other components collectively forming a complete and homogeneous nanoparticle. Meanwhile, the sulfur content is 1.35%, possibly originating from sulfur-containing amino acids such as cysteine ​​and methionine in transferrin. The explicit presence of this signal is one of the direct chemical evidences of the successful covalent coupling of transferrin to the surface of Atractylodes macrocephala exosomes. The detected 8.66% of the phosphorus (P) mainly comes from the phospholipid bilayer in the exosome biomembrane structure. In addition, the nucleic acids (such as mRNA and miRNA) naturally carried inside the exosome, whose backbones are linked by phosphodiester bonds, also contribute the P signal.

[0052] In summary, the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin constructed in Part 1 of Example 1 has standard exosome nanosize and morphology, is rich in organic components such as proteins and lipids, and has a uniform elemental distribution.

[0053] (3) Dynamic light scattering (DLS) analysis and stability test: Five 2 mL samples were aliquoted into 5 mL centrifuge tubes and stored at 4 °C as total stock solution. The hydrodynamic dimensions and distribution of the samples were measured and analyzed at 1, 3, 5, 7 and 14 days after the start of storage using a dynamic light scattering instrument.

[0054] Figure 3 A in the figure is a comparison of the hydrodynamic size distribution of Atractylodes macrocephala exosomes, transferrin-Atractylodes macrocephala exosome conjugates, and transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin. Figure 3 B in the figure represents the 14-day particle size change of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin. Figure 3 Figure C shows the hydrodynamic size distribution and 14-day particle size variation of Atractylodes macrocephala exosomes, respectively. The hydrodynamic size distribution reveals that the hydrated particle size of the baicalin-loaded transferrin-Atractylodes macrocephala exosome conjugate is approximately 84.56 nm, showing a gradual increasing trend compared to unmodified Atractylodes macrocephala exosomes (78.29 nm) and the unloaded transferrin-Atractylodes macrocephala exosome conjugate (80.23 nm). This particle size variation is attributed to the combined effects of surface-modified transferrin molecules (biomacromolecule conjugation) and loaded baicalin (drug encapsulation / adsorption). Notably, although the particle size increases slightly with modification and drug loading, the particle size distribution of the baicalin-loaded transferrin-Atractylodes macrocephala exosome conjugate sample exhibits a sharp peak shape (narrow half-peak width), indicating excellent homogeneity. This homogeneity can reduce in vivo pharmacokinetic fluctuations caused by size heterogeneity and improve delivery efficiency.

[0055] The 14-day particle size variation diagram shows that the particle size of Atractylodes macrocephala exosomes and the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin has small variation within 14 days, fluctuating in the range of 80-90 nm and 70-90 nm, respectively. The particle size fluctuation range of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin is particularly small, indicating that it is more stable than Atractylodes macrocephala exosomes.

[0056] (4) Zeta potential and stability monitoring: Five 2 mL samples were aliquoted into 5 mL centrifuge tubes and stored at 4 °C as total stock solution. The surface potential of the samples was measured at 1, 3, 5, 7 and 14 days after the start of storage using a Zeta potential analyzer.

[0057] Figure 4 In the diagram, A represents the Zeta potential of Atractylodes macrocephala exosomes, transferrin-Atractylodes macrocephala exosome conjugates, and transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin. Figure 4 B in the figure represents the 14-day zeta potential change of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin; Figure 4C in the figure represents the 14-day Zeta potential change of Atractylodes macrocephala exosomes.

[0058] The zeta potential of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin was near neutral (2.34±0.4 mV), a significant shift compared to the Atractylodes macrocephala exosomes (-7.82±1.5 mV). This change is mainly attributed to the isoelectric point characteristics of transferrin molecules (pI≈5.9), which carry a weak positive charge under physiological pH conditions, neutralizing the negative charge on the substrate surface. The slight adsorption of baicalin further fine-tuned the potential value. The near-neutral zeta potential effectively reduced the non-specific adsorption of plasma proteins, prolonging blood circulation time while preserving the active targeting function of transferrin. Within 14 days, the zeta potential of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin remained stable in the 0-2 mV range. This stability is attributed to the synergistic effect of the inherent lipid bilayer membrane structure of exosomes and the engineered transferrin modification; the covalent coupling of transferrin enhanced membrane rigidity, while the loading of baicalin did not significantly disrupt vesicle integrity.

[0059] 2.2 UV-Vis absorption spectroscopy analysis: The transferrin-Atractylodes macrocephala exosome conjugate dispersion loaded with baicalin was diluted to a suitable concentration with buffer solution. Using the corresponding buffer solution as a reference, a full spectrum scan was performed using a UV-Vis spectrophotometer to record its absorption spectrum and determine the characteristic absorption peaks. The changes in UV-Vis absorption spectra at different time points (days 1 and 15) were also recorded.

[0060] Figure 5 A in the figure is a comparison of the UV-Vis absorption spectra of Atractylodes macrocephala exosomes, transferrin-Atractylodes macrocephala exosome conjugates, and transferrin-Atractylodes macrocephala exosome conjugates loaded with baicalin on day 1. Figure 5 B in the figure is a comparison of the UV-Vis absorption spectra of the sample on day 15.

[0061] In the UV-Vis absorption spectrum of the transferrin-Atractylodes macrocephala exosome conjugate, an absorption peak appeared at 260 nm, which was contributed by the benzene ring in the phenylalanine residue of transferrin. After loading baicalin, a strong absorption peak was generated at 270 nm, which was mainly generated by the conjugated aromatic system and specific chromophores in its molecular structure through "π-π* electronic transition". This result directly confirms that the functionalization modification of transferrin and the loading of baicalin have been successfully achieved. On day 15, the position, shape, and absorbance attenuation of the absorption peaks of each sample showed little difference. This indicates that the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin maintained good stability within 15 days, without significant degradation, aggregation, or component loss that would interfere with its performance.

[0062] 2.3 X-ray photoelectron spectroscopy (XPS) analysis: To further verify the successful engineering modification of Atractylodes macrocephala exosomes, XPS spectroscopy was used to reveal their chemical composition and surface modification characteristics through multi-element synergistic analysis. The transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin was subjected to full-spectrum and high-resolution narrow-spectrum scanning of the sample surface using a monochromatic Al Kα beam as the excitation source. The binding energy was calibrated using the C 1s peak (284.8 eV), and peak fitting of characteristic elements was performed to analyze the surface elemental composition and chemical valence state.

[0063] Figure 6 The XPS spectra of the transferrin-Atractylodes macrocephala exosome conjugate sample loaded with baicalin in Example 1 are shown in the overall and partial spectra. The overall spectrum shows that the sample contains five elements: C, O, N, S, and P. This indicates that the transferrin-Atractylodes macrocephala exosome-loaded baicalin is an integration of multiple components: the Atractylodes macrocephala exosome base (containing C, O, and N), transferrin modification (introducing S and P), and baicalin loading (containing O and N). The P 2p spectrum shows a bimodal structure, with the main peak located in the higher binding energy region (approximately 133 eV), corresponding to phosphorus in the phosphate group, suggesting the presence of phosphorylation modification in the transferrin molecule; while the secondary peak may originate from the phosphate ester bond introduced during the modification process. The bimodal distribution of the S 2p spectrum (approximately 163 eV and 164 eV) reveals that sulfur exists in two chemical states: the low binding energy peak corresponds to the disulfide bond (-SS-) between cysteine ​​residues, reflecting the integrity of the native transferrin structure; the high binding energy peak may originate from the covalent coupling between the thiol group and the carrier surface during modification. The fine splitting peaks of the N 1s spectrum (399.5 eV and 401.2 eV) correspond to the amide nitrogen (-NH-CO-) and amino group (-NH2) in the amide bond, respectively. The former originates from the transferrin protein backbone, while the latter may originate from the primary amine group introduced during targeted modification. All three elemental spectral analyses confirm the successful modification of transferrin.

[0064] Furthermore, the chemical environment of carbon was differentiated using C 1s spectra: aromatic ring / alkyl carbon (CC), ether bond / ester group carbon (COC), and carboxylic acid group (OC=O). The appearance of the carboxylic acid peak is directly related to the phenolic hydroxyl / carboxylic acid functional group of baicalin. The bimodal structure of the O 1s spectrum (530.2 eV, 533.8 eV) was assigned to the phenolic hydroxyl oxygen (COH) and carboxylic acid oxygen (O=C-OH), respectively, further confirming that the active group of baicalin participates in the construction of the carrier. At the same time, the inherent sugar chain components of exosomes contribute to the COC peak.

[0065] Synergistic analysis of elemental spectra showed that the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin had been successfully constructed, providing key evidence for the synergistic mechanism of the "targeting ligand-carrier-drug" ternary system.

[0066] 2.4 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0067] Prepare four groups of 200 μL liquid samples, namely baicalin, transferrin, transferrin-Atractylodes macrocephala exosome conjugate, and transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin. The protein concentration of the protein-containing samples is ≥ 1 mg / mL.

[0068] The above sample was pre-frozen at -80 ℃ for 4 h, then transferred to a freeze dryer and freeze-dried at -50 ℃ and a vacuum degree of <0.1 mbar for 24 h to obtain a dry powdery solid.

[0069] In a dry agate mortar, add 1–2 mg of the above-mentioned freeze-dried powdered solid and mix with 150–200 mg of dry spectral-grade potassium bromide powder. Grind rapidly and thoroughly under an infrared lamp for 5–10 min until the mixture is homogeneous, fine, and free of noticeable particles. Finally, transfer the mixed powder to a tableting mold and press under 10–15 MPa pressure for 1 min to form a transparent, uniform thin sheet. Simultaneously, press a pure KBr thin sheet for background spectrum acquisition. Analyze the sample using a Fourier transform infrared spectrometer.

[0070] like Figure 7 As shown in A, at 3400 cm -1 The broad peaks in the vicinity are generally attributed to the stretching vibrations of OH and NH. The peak intensity of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin in this region is significantly lower than that of baicalin and transferrin. This strongly suggests that a new hydrogen bond network has been formed in the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin. Intermolecular interactions have occurred between the phenolic hydroxyl groups of baicalin, the hydroxyl / amino groups of the exosome membrane components, and the amino acid residues of transferrin, resulting in an increase in hydrogen-bonded hydroxyl / amino groups, which in turn leads to a decrease in peak intensity. (That is, the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin actually contains not only the Atractylodes macrocephala exosome-transferrin conjugate, but also the non-covalent conjugate of baicalin and the transferrin-Atractylodes macrocephala exosome conjugate, and the three form a "complex".)

[0071] It is worth noting that at 1600 cm -1 Near the surface, both transferrin and the transferrin-Atractylodes macrocephala exosome conjugate showed characteristic peaks generated by amide I and amide II bands, indicating that transferrin had been successfully modified onto the surface of Atractylodes macrocephala exosomes. However, when baicalin was loaded, these characteristic peaks weakened. The core reason for this may be that the aromatic ring of baicalin underwent π-π stacking with transferrin, leading to a weakening of the characteristic peaks generated by amide I and amide II bands. Baicalin, at 1100 cm⁻¹... -1The characteristic peaks appearing nearby are mainly attributed to the COC ether bond and C-OH stretching vibrations on the glucuronic acid units in its molecular structure. These characteristic peaks also persisted after loading with the transferrin-Atractylodes macrocephala exosome conjugate, indicating that baicalin was successfully loaded onto Atractylodes macrocephala exosomes-transferrin. However, the decrease in peak intensity may be due to the drug being encapsulated within a hydrophobic cavity, resulting in a microenvironment shielding effect.

[0072] 2.5 Immunoblotting detection

[0073] Characteristic proteins of Atractylodes macrocephala exosomes and transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin were detected using conventional Western blotting. Except for Atractylodes macrocephala exosomes and the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin, all reagents and materials used were commercially available and commonly used in this method.

[0074] (1) Sample lysis: Transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin and Atractylodes macrocephala exosomes (control group) were added to protein lysis buffer and incubated on ice for 15 min. After centrifugation at 12000 rpm for 5 min, the supernatant was retained and the protein concentration was detected. Then, 5× loading buffer (protein volume: loading buffer volume = 4:1) was added, and finally the sample was boiled in boiling water for 15 min.

[0075] (2) SDS-polyacrylamide gel electrophoresis: Install the pre-made gel into the electrophoresis tank and add sufficient electrophoresis buffer. Add the pre-stained protein marker and the processed protein samples of each group to the corresponding lanes in sequence. Set the initial voltage to 80 V. After the sample enters the separating gel and the bromophenol blue band is pressed into a line, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the gel.

[0076] (3) Transfer: After electrophoresis, cut out the target separating gel region and immerse it together with the pre-cut PVDF membrane (pre-activated in methanol for 15 seconds) in the transfer solution. Prepare a "sandwich" structure in the order of "cathode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode" to ensure no air bubbles. Place it in the transfer tank and add pre-cooled transfer solution. Transfer the membrane at a constant current of 300 mA for 90 min under ice bath conditions.

[0077] (4) Primary antibody incubation: After transfer, immerse the PVDF membrane in TBST solution containing 5% skim milk and slowly block it on a shaker for 1 hour at room temperature. After completion, briefly wash the membrane with TBST. Cut membrane strips according to the molecular weight of the target protein and place them in the corresponding primary antibody dilution solution. Finally, incubate overnight at 4 °C with gentle shaking.

[0078] (5) Secondary antibody incubation: Remove the membrane strip from the primary antibody and wash it three times with TBST for 10 minutes each time. Immerse the membrane strip in a secondary antibody dilution solution (1:5000) corresponding to the species of the primary antibody and incubate at room temperature for 1 hour. After completion, wash it three more times with TBST for 10 minutes each time.

[0079] (6) Image acquisition: Mix equal volumes of solutions A and B of the ECL luminescent reagent. Use filter paper to absorb excess liquid from the membrane strip, and place the membrane face up into the imaging instrument. Evenly drop the mixed ECL working solution onto the membrane, ensuring complete coverage. Acquire images using the chemiluminescence imaging system.

[0080] The results are as follows Figure 7 As shown in Figure B, both the Atractylodes macrocephala exosome group and the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin clearly show bands of three exosome marker proteins: apoptosis linker gene 2 interacting protein X (Alix) (approximately 100 kDa), differentiation cluster 63 protein (CD63) (approximately 55 kDa), and tumor susceptibility gene 101 protein (TSG101) (approximately 44 kDa). This indicates that during the process of transferrin modification and baicalin loading of Atractylodes macrocephala exosomes, the specific marker proteins of Atractylodes macrocephala exosomes were not destroyed, and their origin characteristics and structural integrity were preserved.

[0081] 2.6 Investigation of in vitro drug release characteristics

[0082] Standard curve plotting: Baicalin drug solutions of different concentration gradients were prepared using PBS (pH=7.4) to obtain standard solutions with concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 μg / mL. The absorbance of different drug concentrations (276 nm) was measured using a UV spectrophotometer, and a standard curve was plotted.

[0083] (1) Take 2 mL of transferrin-Atractylodes macrocephala exosome conjugate sample loaded with baicalin (1 mg / mL), place it in a 10 mL centrifuge tube, and make up to 10 mL with PBS (pH=7.4). Mix well and use it as a sample for drug release test.

[0084] (2) Transfer the above sample solution to a pretreated dialysis bag (molecular weight cutoff: 3 kDa). Place the dialysis bag in a 50 mL beaker containing 25 mL of PBS release medium (pH 5.5, 7.5), ensuring the dialysis bag is completely submerged. Place the beaker in a constant temperature shaker and perform dialysis at room temperature and 100 rpm.

[0085] (3) At time points of 1, 2, 6, 12, 24 and 48 h respectively, accurately measure 3 mL of dialysis fluid (release medium) from the beaker and place it into a 10 mL centrifuge tube, and immediately add 3 mL of fresh PBS buffer at the same temperature and volume to the beaker to maintain the leak conditions.

[0086] (4) Using a UV-Vis spectrophotometer, the absorbance of the dialysate samples taken at each time point was measured at the characteristic absorption wavelength of baicalin. The cumulative drug concentration was calculated based on the pre-established standard curve, and the release kinetic curve was plotted.

[0087] Figure 8 In the figure, A represents the ultraviolet absorption spectra of baicalin solutions at different concentrations; Figure 8 In this context, B represents the standard curve, and the curve parameters are: ;

[0088] Figure 8 C in the figure represents the drug release curves of the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin at different pH levels. Over time, the drug release rate gradually increased in both pH environments, with the release rate in the pH=5.5 group being significantly faster than that in the pH=7.5 group. This provides a promising precision treatment strategy for addressing the clinical challenge of recurrent miscarriage. In pathological conditions, the endometrium experiences immune homeostasis imbalance and excessive secretion of pro-inflammatory factors. Simultaneously, the synergistic effect of increased vascular permeability and local microcirculatory disturbances at the site of inflammation jointly promotes a decrease in interstitial pH, thus forming a unique local acidic microenvironment. This indicates that the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin is more likely to release the drug rapidly in the acidic environment of the target site, while achieving sustained release at the normal physiological pH (7.5), which helps improve the targeting and safety of drug delivery and reduce toxic side effects on normal tissues. The reason for this result may be that the drug delivery system undergoes protonation under acidic conditions, resulting in charge repulsion, which increases the pore size of the carrier, improves membrane permeability, and accelerates the outward diffusion of the drug.

[0089] In summary, this embodiment prepared a transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin, and through a large number of characterization experiments, its physicochemical properties were systematically and comprehensively evaluated from multiple dimensions, including morphology, size, potential, stability, drug loading characteristics, characteristic proteins, responsive release and chemical structure.

[0090] Example 2: Culture and Effect Evaluation of Mouse Mononuclear Macrophage Leukemia Cells RAW 264.7

[0091] Cell Culture: RAW 264.7 cells (mouse mononuclear macrophage leukemia cells) were retrieved from liquid nitrogen cryovials and rapidly thawed by shaking in a 37°C water bath. The cell suspension was transferred to centrifuge tubes containing 5 mL of DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in complete medium and seeded into culture flasks. The cells were then cultured in a 37°C, 5% CO2 incubator. Cells were passaged every 2–3 days, gently scraping off cells with a cell scraper to avoid over-digestion. When cells reached 70%–80% confluence, the medium was replaced with complete medium containing 100 ng / mL lipopolysaccharide (LPS) and 20 ng / mL interferon-γ to induce M1 polarization. After 24 h of culture, the cell supernatant was collected for subsequent assays.

[0092] Cell grouping: M1 polarized cells were randomly divided into model group, transferrin group, baicalin group, Atractylodes macrocephala exosome group, Atractylodes macrocephala polysaccharide-baicalin mixture group (purified Atractylodes macrocephala polysaccharide was extracted using the literature method BZJP: Yang Ying. Isolation, purification and structural analysis of Atractylodes macrocephala polysaccharide and its protective effect on acute alcoholic liver injury [D]. Nanjing University of Chinese Medicine, 2021; Atractylodes macrocephala polysaccharide + baicalin compound was prepared according to the ratio of CN111000860A), transferrin-Atractylodes macrocephala exosome conjugate group (prepared according to steps (1) to (3) of Part 1 of Example 1), Atractylodes macrocephala exosome loaded with baicalin group (prepared according to steps (1) and (4) of Part 1 of Example 1, i.e., no activation of Atractylodes macrocephala exosomes and transferrin conjugate were performed, and other steps and parameters were the same), and transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin (prepared according to Part 1 of Example 1). Except for the model group, all test samples (liquid samples were first centrifuged to obtain a precipitate) were uniformly adjusted to a concentration of 10 mg / mL using PBS (pH 7.4).

[0093] Enzyme-linked immunosorbent assay (ELISA): Cell culture supernatant was collected after induction in each group, centrifuged at 300×g for 5 min at 4℃ to remove cell debris and dead cells, and the supernatant was aliquoted and stored at -80℃ for later use. Mouse TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), and IL-10 (interleukin-10) ELISA kits (Byabscience) were used for detection according to the kit instructions (all wells were set to 3 replicates).

[0094] The results are as follows Figure 9 As shown. Figure 9The symbols in the table represent the following: Compared with the model group: *p<0.05, **p<0.01, ***p<0.001. Compared with the transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin: &p<0.05, &&p<0.01, &&&p<0.001. Compared with the Atractylodes macrocephala exosome group loaded with baicalin: #p<0.05. ns indicates no statistically significant difference between the two groups.

[0095] Except for the transferrin group, all other drug groups inhibited pro-inflammatory factors (TNF-α, IL-6) and increased anti-inflammatory factors (IL-10) in model cells. However, further inter-group comparisons showed that, at the same total dose (total concentration of the mixture group, concentration of the conjugate / complex group, and concentration of the single compound group), the anti-inflammatory activity of the transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin was significantly higher than that of the other groups (p<0.05), and the difference was extremely significant compared with the model group (p<0.001). Compared with the baicalin group, Atractylodes macrocephala exosome group, Atractylodes macrocephala polysaccharide-baicalin mixture group, transferrin-Atractylodes macrocephala exosome conjugate group, and Atractylodes macrocephala exosome loaded with baicalin, the baicalin group was significantly higher. The differences were significant (p<0.05); except for the transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin and the transferrin group, the differences between the other groups and the model group were also significant (p<0.01). Among them, the anti-inflammatory effect of the Atractylodes macrocephala polysaccharide-baicalin mixture was significantly lower than that of the Atractylodes macrocephala exosome loaded with baicalin group (p<0.05), suggesting that the RNA or protein components in Atractylodes macrocephala exosomes have an important contribution to its activity; the anti-inflammatory effect of the Atractylodes macrocephala exosome loaded with baicalin group was significantly higher than that of the Atractylodes macrocephala exosome group (p<0.05) and the baicalin group (p<0.05).

[0096] The same model was further used to investigate the immunomodulatory effects of baicalin, Atractylodes macrocephala exosomes, and Atractylodes macrocephala exosomes loaded with baicalin on model cells (including the regulation of pro-inflammatory and anti-inflammatory factors). In the experiment, five equivalent concentration gradients (0.1, 0.5, 1, 5, 10 μg / mL) were set based on a fixed mass ratio of baicalin loaded with Atractylodes macrocephala exosomes. The concentration of the single-drug control group was set to the equivalent concentration of the corresponding component in the complex. The obtained test data were input into CompuSyn software, and the combination index was calculated according to the Chou-Talalay intermediate-effect principle. The results showed that at the 50% effect level (ED50), the CI for TNF-α was 0.56 ± 0.05, the CI for IL-6 was 0.54 ± 0.06, and the CI for IL-10 was 0.61 ± 0.07 (n = 3). According to the Chou-Talalay combination drug index evaluation criteria (CI < 0.7 indicates a strong synergistic effect), the CI of all the above factors was < 0.7, indicating that baicalin and Atractylodes macrocephala exosomes have significant strong synergistic effects in inhibiting TNF-α and IL-6 secretion and promoting IL-10 secretion. That is, the immunomodulatory effect of Atractylodes macrocephala exosomes loaded with baicalin is significantly better than the simple addition of the two drugs used alone.

[0097] Example 3: Construction and Effect Evaluation of a Mouse Model for Recurrent Abortion (RSA)

[0098] An RSA mouse model and a normal pregnancy control group were established using the literature method (Zhao X, Zhao Y, Jiang Y, et al. HK2-driven histone H3K18lactylation promotes stromal cell senescence and decidualization deficiency in URSA via CUX1-mediated SASP factor transcription. Cell Mol Biol Lett. 2026;31(1):31).

[0099] Eight-week-old female CBA / J mice were paired with eight-week-old male DBA / 2 mice to establish a recurrent spontaneous abortion (RSA) model, simulating human recurrent miscarriage. A control group consisting of eight-week-old female CBA / J mice and eight-week-old male BALB / c mice served as the normal pregnancy control group, exhibiting a lower embryo resorption rate. Mice were purchased from Huafukang Biotechnology Co., Ltd. in China and housed under standard laboratory conditions. Male and female mice were housed at a 2:1 ratio to establish the RSA group and the normal pregnancy group, respectively. Pregnancy time was determined daily by examining vaginal plugs (the day a vaginal plug was found was recorded as day 1 of pregnancy).

[0100] Animal experimental grouping: The CBA / J × BALB / c mating combination served as the control group, and RSA was used. Mice were randomly divided into: model group, transferrin group, baicalin group, Atractylodes macrocephala exosome group, Atractylodes macrocephala polysaccharide-baicalin mixture group (purified Atractylodes macrocephala polysaccharide was extracted using the literature method BZJP: Yang Ying. Isolation, purification and structural analysis of Atractylodes macrocephala polysaccharide and its protective effect on acute alcoholic liver injury [D]. Nanjing University of Chinese Medicine, 2021; Atractylodes macrocephala polysaccharide + baicalin compound was prepared according to the ratio of CN111000860A), transferrin-Atractylodes macrocephala exosome conjugate group (prepared according to steps (1) to (3) of Part 1 of Example 1), Atractylodes macrocephala exosome loaded with baicalin group (prepared according to steps (1) and (4) of Part 1 of Example 1, i.e., without activation of Atractylodes macrocephala exosomes and transferrin conjugate, other steps and parameters are the same), and transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin (prepared according to Part 1 of Example 1). Ten animals were included in each group (seven animals per group for both the RSA group and the normal pregnancy group). Except for the model group, all test substances (liquid samples were first centrifuged to obtain a precipitate) were uniformly adjusted to a concentration of 100 mg / kg using PBS (pH 7.4). On day 0.5 of pregnancy (pregnancy confirmed by vaginal suppository examination; the day the suppository was found was recorded as day 0.5 of pregnancy), each group was administered intravenously at a dose of 100 μL (equal volume administration) (equal total dose, equal volume administration; for mixture groups, the total concentration of the mixture was used; for conjugates / complexes, the concentration of the conjugate / complex was used; for single-compound groups, the concentration of the single compound was used). Administration was once daily for a total of 14 doses. Mice were sacrificed 24 hours after the last administration. The uterus, heart, liver, spleen, lungs, kidneys, and placenta were dissected. Routine pathological sections of the heart, liver, spleen, lungs, kidneys, and placenta were prepared and stained with hematoxylin and eosin (HE) for pathological observation. Embryo resorption and the number of embryos were observed, recorded, and analyzed. Embryo resorption rate (R) is calculated using the following formula: R = Re / (Re + F), where Re represents the number of resorbed embryos and F represents the number of surviving embryos.

[0101] Statistical results of embryo absorption rate are as follows Figure 10 As shown. Figure 10 The symbols in the table represent the following: Compared with the model group: *p<0.05, **p<0.01, ***p<0.001. Compared with the transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin: &p<0.05, &&&p<0.001. Compared with the Atractylodes macrocephala exosome group loaded with baicalin: #p<0.05. ns indicates no statistically significant difference between the two groups.

[0102] Depend on Figure 10As can be seen, the embryo resorption rate in the RSA model group was approximately 39%, which was significantly higher than that in the normal pregnancy group (p<0.001); there was no statistically significant difference in embryo resorption rate between the transferrin group and the model group; the embryo resorption rates in all other groups were significantly or extremely significantly lower than those in the RSA model group (p<0.05, 0.01, or 0.001). Further intergroup comparisons showed that, at the same total dose, the embryonic absorption rate of the transferrin-Atractylodes macrocephala exosome conjugate group loaded with baicalin was significantly lower than that of other groups (p<0.05 or 0.001), and the difference was extremely significant compared with the model group (p<0.001). The difference was also significant compared with the baicalin group, Atractylodes macrocephala exosome group, Atractylodes macrocephala polysaccharide-baicalin mixture group, transferrin-Atractylodes macrocephala exosome conjugate group, and Atractylodes macrocephala exosome loaded with baicalin (p<0.05 or 0.001). The embryonic absorption rate of the Atractylodes macrocephala exosome loaded with baicalin group was significantly lower than that of the Atractylodes macrocephala polysaccharide-baicalin mixture group (p<0.05), suggesting that the RNA or protein components in Atractylodes macrocephala exosomes play an important role in inhibiting embryonic absorption.

[0103] No obvious pathological damage was found in the organ pathological examinations of any group of animals (see...). Figure 11 ).

[0104] Example 4 Biosafety assessment of transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin

[0105] Fresh rat red blood cells were washed three times with PBS buffer (pH=7.4) and then resuspended in PBS buffer to a final concentration of 4% (v / v). The 4% red blood cell suspension was incubated with samples of different concentrations (3.125, 6.25, 12.5, 25, 50, 100 μg / mL; concentration calculations were the same as in Examples 2 and 3: for mixture groups, the total concentration of the mixture was used; for conjugates / complexes, the concentration of the conjugate / complex was used; for single-compound groups, the concentration of the single compound was used) at 37°C for 24 h. At each time point (12 h, 24 h), samples were centrifuged at 5000 rpm for 5 min, and the supernatant was collected. The absorbance at 540 nm was measured using a microplate reader. A positive control was achieved using 0.1% (v / v) Triton X-100, and a negative control was achieved using PBS. The hemolysis rate was calculated as: Hemolysis rate (%) = (OD sample - OD negative) / (OD positive - OD negative) × 100%. Wherein, OD sample, OD negative, and OD positive represent the absorbance of the sample and the negative and positive controls, respectively. The sample groups include transferrin (Tf), baicalin, Atractylodes macrocephala exosomes (AMV), transferrin-Atractylodes macrocephala exosome conjugate (Tf-AMV), and transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin (Tf-AMV@Baicalin). The sample preparation and source are the same as in Examples 2 and 3.

[0106] The results are as follows Figure 12As shown, at 12 h ( Figure 12 In sample A), the supernatant of each sample group was generally lighter in color, which contrasted sharply with the deep red of the positive control (+) representing complete hemolysis, and was close to the pale color of the negative control (-) representing no hemolysis; even after 24 hours ( Figure 12 (B) The color of the supernatant in each sample group did not show a significant deepening or large-area reddening, maintaining a low hemolysis state similar to the negative control. From Figure 12 C in Figure 12 As can be seen from D, even when the concentration of each component reached 100 μg / mL, the hemolysis rate was still far below the international safety standard (<5%). These results confirm that the transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin possesses excellent blood compatibility and demonstrates good potential for clinical translation.

[0107] Example 5: Investigation of different exosome-transferrin conjugates loaded with different drugs

[0108] This example is a comparative example (exploratory example) examined during the research process, and its in vivo and in vitro effects were investigated.

[0109] Transferrin-Atractylodes macrocephala exosome conjugates loaded with curcumin or resveratrol were prepared using the method described in Example 1 (except that baicalin was replaced with an equal amount of curcumin or resveratrol; otherwise, the preparation method was the same as in Part 1 of Example 1). In vitro and in vivo anti-inflammatory and anti-RSA effects were investigated using the same methods as in Examples 2 and 3 (including dosage, administration route, and frequency).

[0110] Curcuma and ginseng exosomes were prepared using the method described in Example 1 (the only difference from step (1) in Part 1 of Example 1 was that the raw materials were replaced with curcuma (dried curcuma rhizome slices, pulverized to 40-60 mesh, the dried rhizome was dried at a constant temperature to a moisture content of <12%) and ginseng (dried ginseng root slices, pulverized to 40-60 mesh, the dried root was naturally sun-dried to a moisture content of <12%), with other parameters remaining unchanged). Curcuma exosome-transferrin conjugates loaded with baicalin and ginseng exosome-transferrin conjugates loaded with baicalin were prepared using the method described in Part 1 of Example 1 (preparation method is the same as in Part 1 of Example 1). In vitro and in vivo anti-inflammatory and anti-RSA effects were investigated using the same methods as in Examples 2 and 3 (including dosage, administration method, and frequency). Results are shown in […]. Figure 13 and 14 .

[0111] Depend on Figure 13 and Figure 14It is evident that, at the same dosage, the in vivo inhibitory effect of transferrin-Atractylodes macrocephala exosome conjugate loaded with baicalin on embryonic absorption and immunomodulation was significantly better than that of the model group, and significantly better than that of transferrin-Atractylodes macrocephala exosome conjugate loaded with curcumin or resveratrol, as well as that of curcumin exosome-transferrin conjugate loaded with baicalin or ginseng exosome-transferrin conjugate.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nanomedicine for recurrent miscarriage, characterized in that, The nanomedicine is a transferrin-Atractylodes macrocephala exosome covalently coupled with baicalin; the mass ratio of the transferrin-Atractylodes macrocephala exosome covalently coupled with baicalin in the nanomedicine is (0.3~3):10; the nanomedicine for recurrent miscarriage is characterized in that the mass ratio of transferrin to Atractylodes macrocephala exosomes in the nanomedicine is (0.03~0.3):2; the preparation method of the nanomedicine includes the following steps: Preparation of S1 Atractylodes macrocephala exosomes: Atractylodes macrocephala was hydrolyzed by cellulase and pectinase, the supernatant was collected by centrifugation, the filtrate was collected by membrane filtration, the supernatant was removed by ultracentrifugation, and Atractylodes macrocephala exosomes were prepared. S2 Atractylodes macrocephala exosome activation: Atractylodes macrocephala exosomes and activator are mixed in solvent and reacted in the dark; after the reaction is completed, the supernatant is discarded by centrifugation, the residual activator is washed away and then resuspended to obtain activated Atractylodes macrocephala exosomes; S3 Transferrin Coupling: Activated Atractylodes macrocephala exosomes were added to a transferrin solution and mixed to react for coupling. After the reaction was completed, the unreacted activation sites were blocked, the supernatant was discarded by centrifugation, and the uncoupled transferrin was removed to obtain the transferrin-Atractylodes macrocephala exosome covalent conjugate. S4-loaded baicalin: The transferrin-Atractylodes macrocephala exosome covalent conjugate and baicalin were mixed in a solvent, and baicalin was loaded and incubated by electroporation to obtain the transferrin-Atractylodes macrocephala exosome covalent conjugate loaded with baicalin. In step S2, the activator is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and bis(N-hydroxysuccinimide) polyethylene glycol ester.

2. The drug for recurrent miscarriage according to claim 1, characterized in that, The nanomedicine appears as a spherical structure under a scanning electron microscope.

3. The drug for recurrent miscarriage according to claim 2, characterized in that, The spherical structure of the nanomedicine has a diameter of 30nm to 150nm.

4. The nanomedicine for recurrent miscarriage according to claim 1, characterized in that, The nanomedicine contains 6%~7% C, 54%~55% N, 28%~29% O, 1%~2% S, and 8%~9% P.

5. Use of the nanomedicine for recurrent miscarriage as described in claim 1 in the preparation of a drug for treating recurrent miscarriage.

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