Natural exosome-like nanovesicles from tetrastigma hemsleyanum and anti-tumor application thereof

Abstract

The present application relates to a kind of natural exosome-like nanovesicle of origin of three-leafed ginseng and its antitumor application;The raw material of natural exosome-like nanovesicle of origin of three-leafed ginseng provided by the present application is dried three-leafed ginseng traditional Chinese medicine finished product, not fresh three-leafed ginseng and other finished products;The present application provides a kind of simple, economical extraction method of natural exosome-like nanovesicle of origin of three-leafed ginseng, ensures that raw material can be sustainable supply, stability and low cost, opens new road for commercial production of natural exosome-like nanovesicle of origin of three-leafed ginseng.The present application applies natural exosome-like nanovesicle of origin of three-leafed ginseng to breast cancer treatment, provides a kind of safe, efficient new idea for the treatment of clinical breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biology and medicine, specifically relating to a natural exosome-like nanovesicle derived from *Tripterygium wilfordii* and its anti-tumor applications. Background Technology

[0002] Triple-negative breast cancer (TNC) is one of the most aggressive and difficult-to-treat subtypes of breast cancer, characterized by high recurrence rates, early metastasis, and poor clinical prognosis. Notably, this subtype is considered a typical "cold immune" tumor, characterized by low levels of tumor-infiltrating lymphocytes, impaired antigen-presenting function, and a dominant microenvironment of immunosuppressive cells (such as regulatory T cells and M2 macrophages). These characteristics severely limit the responsiveness of TNC to immune checkpoint blockade and other immunotherapies. Therefore, transforming it from a "cold" immune state to a "warm," immune-active microenvironment has become a key scientific issue in the current treatment of TNC.

[0003] In recent years, the gut-tumor immune axis has received widespread attention as an important mechanism for systemic regulation of anti-tumor immunity. The intestinal mucosa contains a rich network of immune cells that not only participate in local immune responses but also regulate the distant tumor microenvironment through immune cell migration, cytokine release, and antigen cross-presentation. Studies have shown that reshaping the intestinal immune landscape by modulating gut microbiota balance or enhancing mucosal immune responses can effectively improve the sensitivity of solid tumors to immunotherapy. However, ideal interventions that can simultaneously regulate both gut and tumor immunity and can be delivered non-invasively are still lacking.

[0004] Traditional Chinese medicine (TCM) has a long history in cancer treatment, and some herbs have been proven to possess anti-proliferative, anti-inflammatory, and immunomodulatory activities. Among them, *Tripterygium wilfordii* is commonly used in traditional medicine for clearing heat and detoxifying, and inhibiting tumors. Preclinical studies have shown that extracts from this plant can inhibit tumor cell proliferation and regulate inflammatory responses; however, the specific mechanisms by which it regulates systemic immunity and the tumor immune microenvironment remain unclear, especially its potential impact on the gut-tumor immune axis, which requires further elucidation. Although the immunomodulatory effects of TCM are increasingly valued, related research is mostly at the empirical level, lacking a systematic connection with modern immunobiological mechanisms. Furthermore, its clinical application faces challenges such as low oral bioavailability, poor targeting, and potential systemic toxicity. Therefore, there is an urgent need to develop novel delivery systems and conduct mechanistic studies to elucidate its ability to reshape the gut-tumor immune axis.

[0005] Building upon existing technologies, this study extracted exosome-like nanoparticles (TNPs) from the abundant traditional Chinese medicine *Tetrastigma hemsleyanum* Diels & Gilg. By concentrating the active components and removing excess impurities, the study enhanced its antitumor efficacy while ensuring biosafety. This medicinal herb is widely cultivated in Zhejiang and other regions of my country, with stable yields, making industrial-scale production feasible. Furthermore, as a traditional medicinal plant, it enjoys high public acceptance, facilitating clinical translation. By developing an efficient extraction process that maximizes the preservation of bioactivity, this study aims to advance the application of this nanocarrier in the intervention of diseases such as breast cancer and provide methodological references for the extraction and development of exosomes from other plant sources. Summary of the Invention

[0006] The purpose of this invention is to provide a natural exosome-like nanovesicle derived from *Tripterygium wilfordii*, its preparation method, and its anti-tumor application, which solves the problems of low oral bioavailability, poor targeting, and low standardization of traditional *Tripterygium wilfordii* drugs. At the same time, it fills the technological gap in the immunotherapy of triple-negative breast cancer by lacking non-invasive and modulating intervention methods for the gut-tumor immune axis.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A preparation of Tripterygium wilfordii exovesicles, comprising Tripterygium wilfordii exovesicles.

[0008] Preferably, the concentration of *Trifolium repens* exovesicles is 1×10⁻⁶. 12 ~1×10 14 Particles / mL.

[0009] Preferably, the particle size of the exovesicles of *Trifolium repens* is 100~300 nm.

[0010] Preferably, the outer vesicles of *Trifolium repens* are derived from the traditional Chinese medicine *Trifolium repens*.

[0011] This invention also discloses a method for preparing a *Trifolium repens* exovesicle formulation, comprising, The dried Chinese herbal medicine *Trifolium repens* was ground into a fine powder to obtain *Trifolium repens* dry powder. The *Trifolium repens* dry powder was added to PBS solution for soaking and filtered to obtain crude juice. The crude juice was centrifuged at low speed, and the supernatant was collected and extracted twice. Then, it was centrifuged at high speed, and the supernatant was discarded to obtain *Trifolium repens* exovesicles. The outer vesicles of *Tripterygium wilfordii* were resuspended in PBS solution to obtain a resuspended solution of *Tripterygium wilfordii*; the resuspended solution of *Tripterygium wilfordii* was then filtered through multiple stages to obtain the outer vesicle preparation of *Tripterygium wilfordii*.

[0012] Preferably, the mass-to-volume ratio of *Trifolium repens* powder to PBS solution is 1 g: 5~50 mL.

[0013] Preferably, the low-speed centrifugation speed is 9000~10000×g and the duration is 40~80min.

[0014] Preferably, the high-speed centrifugation speed is 130,000~170,000×g, and the duration is 40~80min.

[0015] Preferably, the *Trifolium repens* suspension includes a sisal saponin dispersion.

[0016] Preferably, the sisal saponin dispersion comprises sisal saponin and PBS solution, wherein the mass-to-volume ratio of sisal saponin to PBS solution is 1 mg: 5~20 mL.

[0017] Preferably, the volume ratio of *Trifolium repens* exovesicles to sisal saponin dispersion is 2-6:1.

[0018] Sisalin can significantly improve the in vitro stability of Tripterygium wilfordii exovesicle preparations, and also synergistically enhances the effect of Tripterygium wilfordii exovesicles, effectively increasing the tumor cell proliferation inhibition rate and enhancing the overall antitumor activity of the preparation. This effect may be due to the fact that sisal saponin, relying on its amphiphilic structure, forms a steric barrier that can effectively shield the mutual attraction between exovesicle particles, reduce particle aggregation, and thus significantly reduce the aggregation rate of the preparation under 4°C refrigeration conditions. At the same time, it can synergize with the natural antitumor activity of Tripterygium wilfordii exovesicles, further strengthening the inhibitory effect on tumor cell proliferation.

[0019] Preferably, the sisal saponin dispersion includes apigenin-7-glucoside, and the mass ratio of apigenin-7-glucoside to sisal saponin is 1:2~8.

[0020] Apigenin-7-glucoside can further enhance the in vitro storage stability of *Tripterygium wilfordii* vesicle preparations and amplify the synergistic antitumor activity of *Tripterygium wilfordii* vesicles and sisal saponins, significantly improving the inhibitory effect on tumor cell proliferation. The glycosyl group of this component can form hydrogen bonds with the vesicle membrane components, enhancing the hydrophilicity of the vesicle interface layer and forming a dual protective system with sisal saponins. Compared with the addition of sisal saponins alone, it further reduces the aggregation rate of the preparation. Moreover, this component is mainly distributed in the aqueous phase, which can further enhance the inhibitory effect on tumor cell proliferation.

[0021] This invention also discloses the application of *Trifolium repens* exovesicle preparations in the preparation of antitumor drugs.

[0022] Preferably, the tumor includes triple-negative breast cancer.

[0023] More preferably, the sisal saponin dispersion further includes 4',5,7-trihydroxyflavanone, wherein the mass ratio of 4',5,7-trihydroxyflavanone to sisal saponin is 1:5~15.

[0024] 4',5,7-Trihydroxyflavanone can optimize the in vitro stability of *Trifolium repens* exovesicle formulations, while maximizing the inhibition rate of tumor cell proliferation, achieving the best tumor suppression effect, and enhancing the therapeutic effect on "immune cold" tumors such as triple-negative breast cancer. This flavonoid compound also has antioxidant activity and can synergistically work with sisal saponins and apigenin-7-glucoside to further optimize the exovesicle interface properties, minimize the aggregation rate of the formulation, and amplify the antitumor activity of each component.

[0025] This invention also provides a method for preparing a Tripterygium wilfordii exovesicular formulation, comprising the steps of preparing Tripterygium wilfordii exovesiculars and the steps of preparing a Tripterygium wilfordii exovesicular formulation: Step 1: Preparation of Tripterygium wilfordii exovesicles: Grind Tripterygium wilfordii into a fine powder to obtain dried Tripterygium wilfordii powder; add the dried Tripterygium wilfordii powder to PBS solution and soak for 10-15 hours, then filter through a filter with a pore size of 80-120µm to obtain crude Tripterygium wilfordii juice; centrifuge the crude Tripterygium wilfordii juice at 9000-10000×g for 40-80 minutes, take the supernatant and repeat the extraction twice, centrifuge at 130000-170000×g for 40-80 minutes, discard the supernatant, and obtain Tripterygium wilfordii exovesicles.

[0026] Preferably, the mass-to-volume ratio of *Trifolium repens* powder to PBS solution is 1 g: 5~50 mL.

[0027] Preferably, the particle size of the exovesicles of *Trifolium repens* is 100~300 nm.

[0028] Step 2: Preparation of Tripterygium wilfordii external vesicle formulation: Resuspend Tripterygium wilfordii external vesicles in PBS solution to obtain Tripterygium wilfordii resuspension; filter Tripterygium wilfordii resuspension through a 0.3~0.5μm filter, and then filter through a 0.2~0.25μm filter to obtain Tripterygium wilfordii external vesicle formulation.

[0029] Preferably, in the *Tripterygium wilfordii* exovesicle formulation, the concentration of *Tripterygium wilfordii* exovesicles is 1×10⁻⁶. 12 ~1×10 14 Particles / mL.

[0030] More preferably, the preparation of the *Tripterygium wilfordii* exovesicle formulation includes: dispersing sisal saponin in PBS solution and stirring for 30 min to obtain a sisal saponin dispersion; resuspending the *Tripterygium wilfordii* exovesicles in PBS solution, adding the sisal saponin dispersion, and incubating at room temperature for 20 min to obtain a *Tripterygium wilfordii* resuspension; filtering the *Tripterygium wilfordii* resuspension through a 0.4 μm filter and then through a 0.22 μm filter to obtain the *Tripterygium wilfordii* exovesicle formulation.

[0031] More preferably, in the sisal saponin dispersion, the mass-to-volume ratio of sisal saponin to PBS solution is 1 mg: 5~20 mL.

[0032] More preferably, in the *Tripterygium wilfordii* resuspension, the volume ratio of *Tripterygium wilfordii* exovesicles to sisal saponin dispersion is 2-6:1.

[0033] More preferably, in the *Tripterygium wilfordii* exovesicle formulation, the concentration of *Tripterygium wilfordii* exovesicles is 1×10⁻⁶. 12 ~1×10 14 Particles / mL.

[0034] More preferably, the sisal saponin dispersion includes apigenin-7-glucoside, and the mass ratio of apigenin-7-glucoside to sisal saponin is 1:2~8.

[0035] More preferably, the sisal saponin dispersion further includes 4',5,7-trihydroxyflavanone, and the mass ratio of 4',5,7-trihydroxyflavanone to sisal saponin is 1:5~15.

[0036] This invention provides an exosome preparation derived from the traditional Chinese medicine *Trifolium repens*. Compared with the traditional method of directly administering *Trifolium repens* decoction orally, the exosome extraction technology offers several significant improvements: (1) Significantly improved ease of administration: Trifoliate exosomes are ready-to-take preparations that do not require pre-processing steps such as decoction; while traditional decoctions require complex decoction and often need to be combined with other Chinese medicines to follow the principle of "principal, assistant, adjuvant and guide" formulation, which is cumbersome and has a low degree of standardization. (2) Enrichment and precise delivery of active ingredients: Through extraction and concentration processes, exosomes can efficiently enrich the functional ingredients with anti-tumor activity in Tripterygium wilfordii, while removing most irrelevant impurities, thereby achieving a more significant anti-tumor effect while reducing the intake dose and helping to reduce potential adverse reactions. (3) Standardization and industrialization prospects of preparation process: The extraction method established by this invention has readily available raw materials, stable process and controllable cost, and is easy to achieve standardized production, which provides a solid technical foundation for the large-scale preparation of Tripterygium wilfordii exosomes and subsequent clinical transformation and commercial promotion.

[0037] Therefore, this invention provides a natural exosome-like nanovesicle and its formulation, which uses dried Trifolium repens as raw material, has a simple and economical preparation process, and is industrializable. The nanovesicles have controllable particle size, good biocompatibility, and can effectively inhibit triple-negative breast cancer without significant in vivo or in vitro toxicity. This invention provides a safe and efficient new solution for the clinical treatment of breast cancer and also provides a methodological reference for the development of plant exosomes. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the transmission electron microscope image of the outer vesicles of *Tripterygium wilfordii*.

[0039] Figure 2 This is a schematic diagram showing the size distribution of the external vesicles of *Tripterygium wilfordii*.

[0040] Figure 3 This is a schematic diagram of the drug administration regimen and grouping for the experimental animals in Experiment Example 2.

[0041] Figure 4 This is a schematic diagram showing the changes in tumor volume in each group of mice in Experiment Example 2.

[0042] Figure 5 This is a schematic diagram showing the tumor weight statistics of mice in each group in Experiment Example 2.

[0043] Figure 6 This is a schematic diagram showing the weight statistics of mice in each group in Experiment Example 2.

[0044] Figure 7 This is a schematic diagram showing the changes in tumor volume in each group of mice in Experiment 3.

[0045] Figure 8 This is a schematic diagram showing the tumor weight statistics of mice in each group in Experiment 3.

[0046] Figure 9 This is a schematic diagram showing the weight statistics of mice in each group in Experiment 3.

[0047] Figure 10 This is a schematic diagram illustrating the cytotoxicity evaluation of the outer vesicles of *Tripterygium wilfordii* on endothelial cells.

[0048] Figure 11 This is a schematic diagram illustrating the cytotoxicity evaluation of *Tripterygium wilfordii* external vesicles on macrophages.

[0049] Figure 12 This is a schematic diagram illustrating the immunomodulatory effect of the outer vesicles of *Tripterygium wilfordii*. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0052] Example 1: This example provides a method for preparing a Tripterygium wilfordii vesicle preparation. The preparation steps mainly include the preparation steps of Tripterygium wilfordii vesicles and the preparation steps of Tripterygium wilfordii vesicle preparation, as detailed below.

[0053] Step 1: Preparation of *Trifolium repens* exovesicles: Grind *Trifolium repens* into a fine powder to obtain dried *Trifolium repens* powder; add the dried *Trifolium repens* powder to PBS solution and soak for 12 hours; filter through a 100µm pore size filter to obtain crude *Trifolium repens* juice; centrifuge the crude *Trifolium repens* juice at 9600×g for 60 min, collect the supernatant and repeat the extraction twice, centrifuge at 150000×g for 60 min, discard the supernatant, and obtain *Trifolium repens* exovesicles. The mass-to-volume ratio of dried *Trifolium repens* powder to PBS solution is 1 g: 10 mL.

[0054] Step 2: Preparation of *Tripterygium wilfordii* exovesicle formulation: *Tripterygium wilfordii* exovesicles were resuspended in PBS solution to obtain a *Tripterygium wilfordii* resuspension. The resuspension was filtered through a 0.4 μm filter, followed by filtration through a 0.22 μm filter to obtain the *Tripterygium wilfordii* exovesicle formulation. Nanoparticle tracking analysis showed that the concentration of *Tripterygium wilfordii* exovesicles in the formulation was 1 × 10⁻⁶. 13 Particles / mL.

[0055] Example 2: Compared with Example 1, the only difference in this example is the preparation step of the *Tripterygium wilfordii* exovesicle formulation. In Example 2, the concentration of *Tripterygium wilfordii* exovesicles is increased from 1×10⁻⁶. 13 Particles / mL adjusted to 1×10 12 Particles / mL.

[0056] Example 3: This example differs from Example 1 only in the preparation steps of the *Tripterygium wilfordii* external vesicle formulation, as detailed below: Step 2: Preparation of *Tripterygium wilfordii* exovesicle formulation: Sisal saponin was dispersed in PBS solution and stirred for 30 min to obtain a sisal saponin dispersion. *Tripterygium wilfordii* exovesicles were resuspended in PBS solution, and the sisal saponin dispersion was added. The mixture was incubated at room temperature for 20 min to obtain a *Tripterygium wilfordii* resuspension. The *Tripterygium wilfordii* resuspension was filtered through a 0.4 μm filter, and then through a 0.22 μm filter to obtain the *Tripterygium wilfordii* exovesicle formulation. In the sisal saponin dispersion, the CAS number of sisal saponin was 77-60-1, and the mass-to-volume ratio of sisal saponin to PBS solution was 1 mg:10 mL. In the *Tripterygium wilfordii* resuspension, the volume ratio of *Tripterygium wilfordii* exovesicles to sisal saponin dispersion was 4:1. In the *Tripterygium wilfordii* exovesicle formulation, the concentration of *Tripterygium wilfordii* exovesicles was 1 × 10⁻⁶. 13 Particles / mL.

[0057] Example 4: The only difference between this example and Example 3 is that in the preparation steps of the *Trifolium repens* vesicle preparation, the volume ratio of *Trifolium repens* vesicles to sisal saponin dispersion in Example 4 is adjusted from 4:1 to 3:1.

[0058] Example 5: The only difference between this example and Example 4 is in the preparation steps of the *Trifolium repens* exovesicle preparation. The sisal saponin dispersion includes apigenin-7-glucoside. Sisal saponin and apigenin-7-glucoside are dispersed in PBS solution and stirred for 30 min to obtain the sisal saponin dispersion. The CAS number of apigenin-7-glucoside is 578-74-5, and the mass ratio of apigenin-7-glucoside to sisal saponin is 1:5.

[0059] Example 6: Compared with Example 5, the only difference in this example is that in the preparation steps of the *Trifolium repens* exovesicle formulation, the sisal saponin dispersion also includes 4',5,7-trihydroxyflavanone; sisal saponin, apigenin-7-glucoside and 4',5,7-trihydroxyflavanone are dispersed in PBS solution and stirred for 30 min to obtain the sisal saponin dispersion; the CAS number of 4',5,7-trihydroxyflavanone is 480-41-1, and the mass ratio of 4',5,7-trihydroxyflavanone to sisal saponin is 1:10.

[0060] Comparative Example 1: The only difference between this comparative example and Example 3 is that in the preparation steps of the *Tripterygium wilfordii* vesicle formulation, the volume ratio of *Tripterygium wilfordii* vesicles to sisal saponin dispersion in Comparative Example 1 was adjusted from 4:1 to 1:1.

[0061] Comparative Example 2: The only difference between this comparative example and Example 4 is that in the preparation steps of the *Trifolium repens* exovesicle formulation, the sisal saponin dispersion includes rutin; sisal saponin and rutin are dispersed in PBS solution and stirred for 30 min to obtain the sisal saponin dispersion; the CAS number of rutin is 949926-49-2, and the mass ratio of rutin to sisal saponin is 1:5.

[0062] Comparative Example 3: The only difference between this comparative example and Example 5 is that in the preparation steps of the *Trifolium repens* exovesicle formulation, the sisal saponin dispersion also includes dihydroisoflavone; sisal saponin, apigenin-7-glucoside and dihydroisoflavone are dispersed in PBS solution and stirred for 30 min to obtain the sisal saponin dispersion; the CAS number of dihydroisoflavone is 21554-71-2, and the mass ratio of dihydroisoflavone to sisal saponin is 1:10.

[0063] Experimental Example 1: Microstructural characterization of the outer vesicles of *Tripterygium wilfordii*.

[0064] Test sample: Trifoliate vesicles prepared in Example 1.

[0065] Test methods: The *Tripterygium wilfordii* exovesicles prepared in Example 1 were fixed with 2% paraformaldehyde and stained with 1.9% methylcellulose and 0.3% uranium acetate for negative contrast staining. The micrographs were observed and collected using a HITACHI HT-7700 electron microscope. The size distribution of *Tripterygium wilfordii*-derived exosomes was analyzed using a Zetasizer Nano ZSE (Malvern Instrument, UK) with dynamic light scattering.

[0066] A transmission electron microscope schematic diagram of the outer vesicles of *Tripterygium wilfordii* is shown below. Figure 1 As shown in the diagram, the size distribution of the external vesicles of *Trifolium repens* is as follows: Figure 2 As shown in the figure, the results indicate that the exosomes exhibit a round or cup-shaped appearance under transmission electron microscopy, with a typical disc-shaped and concave morphology, highly similar to the structure of exosomes derived from mammalian cells. This demonstrates that the extraction process of this invention can completely preserve the natural structure of *Tripterygium wilfordii* exovesicles, avoiding structural damage caused by the extraction process. Dynamic light scattering experiments and electron microscopy observations corroborate each other, showing that the particle size of *Tripterygium wilfordii* exovesicles is concentrated between 100 and 300 nm. This size range matches the core particle size characteristics of exosome-like nanovesicles. Furthermore, this particle size lays the structural foundation for its subsequent delivery, transmembrane transport, and targeting effects in vivo. It also proves that the extraction method of this invention has good specificity, effectively separating the target nanovesicle components and eliminating interference from large-particle impurities.

[0067] Experimental Example 2: Antitumor Application Test of Tripterygium wilfordii External Vesicles.

[0068] Test samples: Trifoliate vesicle preparations of Trifoliate Bark prepared in Examples 1 and 2.

[0069] Test method: Take well-grown 4T1 cells. After the cells reach confluence, collect the cells and resuspend them with an appropriate amount of PBS buffer to adjust the cell concentration to 1×10⁻⁶. 6Cells were administered at a concentration of 50 μL and placed on ice for later use. Fifteen 5-week-old female Balb / c mice were acclimatized for one week, then anesthetized with isoflurane. The skin of the 4th and 5th mammary glands was prepared (treated with depilatory cream), and the mice were fixed in a supine position. 50 μL of 4T1 cell suspension was drawn using a 1 mL insulin syringe and injected into the fat pad of the 4th pair of left mammary glands to establish an orthotopic breast cancer mouse model. Starting from the 5th day after model establishment, mice were treated orally once a day and divided into three groups: a blank control group, Example 1 group, and Example 2 group. The drug concentration was 1 mL / Kg, with 5 mice in each group. The mice were given the drug a total of 7 times. During the drug administration period, the weight and tumor size of the mice were recorded. On the 13th day, the mice were sacrificed and the size and weight of the tumors were recorded. The tumor volume was measured by calipers on the day of tumor formation and on days 1, 2, 3, 4, 5, 6, 7, and 9 after tumor formation.

[0070] The dosing regimen and grouping diagram for experimental animals in Experiment Example 2 are shown below. Figure 3 As shown in the diagram, the changes in tumor volume in each group of mice in Experiment 2 are illustrated below. Figure 4 As shown in the diagram, the tumor weight statistics of mice in each group in Experiment 2 are illustrated below. Figure 5 As shown in the diagram, the weight statistics of mice in each group in Experiment 2 are illustrated below. Figure 6 As shown in the figure, the results indicate that this experiment used an orthotopic breast cancer mouse model constructed with 4T1 cells as the research subject to explore the in vivo antitumor effects and biosafety of different concentrations of *Tripterygium wilfordii* vesicle preparations. The results clearly showed that high-concentration *Tripterygium wilfordii* vesicles had a significant inhibitory effect on breast cancer, and the preparation had no obvious in vivo toxicity. Compared with the blank control group, the high-concentration *Tripterygium wilfordii* vesicle preparation prepared in Example 1 significantly reduced the growth rate of mouse tumor volume, and the final tumor weight also showed a significant decreasing trend. The low-concentration preparation in Example 2 did not show a significant antitumor effect. This result indicates that the antitumor effect of *Tripterygium wilfordii* vesicles is significantly concentration-dependent; only at a certain effective concentration can it exert a significant tumor-inhibiting effect in vivo. Meanwhile, the body weight of mice in each treatment group was not significantly different from that in the blank control group, indicating that under the experimentally set concentration and administration method, the *Tripterygium wilfordii* vesicle preparation did not cause significant systemic toxicity in mice and did not affect the normal physiological state of the mice. This confirms the preliminary biosafety of the preparation in vivo and provides important in vivo safety data to support its subsequent clinical translation.

[0071] Experimental Example 3: Test on the auxiliary effect of intestinal flora on the outer vesicles of Clematis armandii.

[0072] Test sample: Trifoliate vesicles prepared in Example 1.

[0073] Test method: The same as in Experiment 2, a 4T1 cell orthotopic breast cancer mouse model was established. The model mice were divided into a group without intestinal flora and a group with normal intestinal flora. The group without intestinal flora was fed drinking water containing combined antibiotics (ampicillin 0.3g / L + metronidazole 0.3g / L + vancomycin 0.15g / L + neomycin 0.3g / L) from the second day after the model was established. The group with normal intestinal flora was fed ordinary drinking water. Both groups had free access to food and water throughout the process. All model mice were divided into 4 groups of 5 mice each. The specific grouping and administration methods are as follows: Blank control group: normal intestinal flora, orally administered with an equal volume of PBS buffer at a concentration of 1 mL / Kg daily from day 5 of model construction; Example 1 group: normal intestinal flora, orally administered with the Tripterygium wilfordii vesicle preparation prepared in Example 1 at a concentration of 1 mL / Kg daily from day 5 of model construction; Example 2 group: normal intestinal flora, orally administered with the Tripterygium wilfordii vesicle preparation prepared in Example 2 at a concentration of 1 mL / Kg daily from day 5 of model construction; Antibiotic group: no intestinal flora (treated with antibiotics throughout the process), orally administered with the Tripterygium wilfordii vesicle preparation prepared in Example 1 at a concentration of 1 mL / Kg daily from day 5 of model construction. All groups were administered the drug a total of 7 times. During the administration period, the weight of the mice was recorded daily. On the day of tumor formation and on days 1, 2, 3, 4, 5, 6, 7 and 9 after tumor formation, the longest and shortest diameters of the tumor were measured with calipers, the tumor volume was calculated and the changes were recorded. On day 13 of model construction, the mice were sacrificed, the tumor tissue was dissected and weighed, and the tumor weight of each group of mice was recorded.

[0074] The diagram below shows the changes in tumor volume in each group of mice in Experiment 3. Figure 7 As shown, the tumor volume growth rate in the mice in Example 1 group was significantly slower than that in the antibiotic group, and the tumor suppression effect was better than that in Example 2 group; the tumor weight statistics of each group of mice in Experiment 3 are shown in the figure below. Figure 8 As shown, the final tumor weight of mice in Example 1 was significantly lower than that in the antibiotic group and still lower than that in Example 2. These results indicate that the antitumor effect of *Tripterygium wilfordii* vesicles depends on the assistance of gut microbiota. The presence of gut microbiota significantly enhances the inhibitory effect of high-concentration *Tripterygium wilfordii* vesicle preparations on triple-negative breast cancer, while the absence of gut microbiota significantly weakens its antitumor activity. This further verifies that the antitumor effect of *Tripterygium wilfordii* vesicle preparations is concentration-dependent; only high-concentration preparations can exert a significant tumor-suppressing effect in vivo. The weight statistics of mice in each group in Experiment 3 are shown in the figure below. Figure 9As shown, the body weight of mice in the Example 1 group, Example 2 group, and antibiotic group was not significantly different from that in the blank control group. This indicates that the treatment of clearing intestinal flora with combined antibiotics and the oral administration of different concentrations of Tripterygium wilfordii vesicle preparations did not cause significant systemic toxicity in mice. This confirms the biosafety of the preparation administration and related treatment methods in this experiment, and also provides direct experimental evidence for the anti-tumor effect of Tripterygium wilfordii vesicle preparations by regulating the gut-tumor immune axis. It suggests that its tumor-suppressing mechanism may be closely related to regulating the structure of intestinal flora, reshaping the intestinal immune landscape, and further regulating the distal tumor microenvironment.

[0075] Experimental Example 4: Cytotoxicity Evaluation Test of Tripterygium wilfordii Exovesicles.

[0076] Test sample: Trifoliate vesicles prepared in Example 1.

[0077] Assay Method: Endothelial cells (HUVECs) and macrophages (RAW264.7) were incubated with PBS (control group), low concentration (10 µg / mL), and high concentration (20 µg / mL) of *Trifolium repens* exovesicles for 24 hours, respectively. After treatment, the cells were washed with PBS to remove residual nanoparticles, collected, and stained according to the Annexin V-FITC apoptosis detection kit instructions. Subsequently, the proportion of apoptotic cells was quantitatively analyzed by flow cytometry.

[0078] A schematic diagram illustrating the cytotoxicity evaluation of *Trifolium repens* exovesicles on endothelial cells is shown below. Figure 10 As shown in the diagram, the evaluation of the cytotoxicity of Trifolium repens external vesicles on macrophages is as follows: Figure 11 As shown, the results confirmed that *Tripterygium wilfordii* exovesicles had no significant in vitro toxicity to normal somatic cells and exhibited good biocompatibility. Endothelial cells, as the core components of the blood vessel wall, and macrophages, as important cells of the body's immune system, are both key normal cells that exosomes may encounter when exerting their effects in vivo. Under the influence of low concentrations (10 µg / mL) and high concentrations (20 µg / mL) of *Tripterygium wilfordii* exovesicles, the apoptosis rates of both cell types were not significantly different compared to the PBS control group. This indicates that neither low nor high concentrations of *Tripterygium wilfordii* exovesicles induced apoptosis in normal endothelial cells and macrophages, nor did they disrupt their normal physiological state.

[0079] Experimental Example 5: Evaluation Test of the Immunomodulatory Effect of Tripterygium wilfordii External Vesicles.

[0080] Test sample: Trifoliate vesicles prepared in Example 1.

[0081] Test method: Healthy 8-week-old female C57BL / 6 mice were selected and euthanized by cervical dislocation. The femur and tibia of the mice were removed, and the ends of the bones were cut off. Bone marrow cells were blown out with pre-cooled PBS buffer, and the bone marrow cells were homogenized. The cell suspension was filtered through a 70µm filter and centrifuged at 500×g for 5 min at room temperature. The cell pellet was collected, and an appropriate amount of erythrocyte lysis buffer was added. The cells were lysed at room temperature for 2 min. The lysis was terminated by adding an appropriate amount of PBS buffer and centrifuged at 500×g for 5 min. The resulting cell pellet was resuspended in an appropriate amount of mouse lymphocyte culture medium (10% FBS + 1% penicillin antibody) containing GM-CSF (20 ng / mL) and cultured in a 37℃ cell culture incubator. After 6 days of culture, the cells were basically completely induced into dendritic cells. The cells were collected and seeded into 6-well plates to continue inducing dendritic cell maturation. After the induction of differentiation was completed, 10 µg / mL and 20 µg / mL of natural exosome-like nanovesicles derived from Trifolium repens were added respectively. The cells were divided into three groups, including a blank control group, Example 1 group and Example 2 group, with a drug concentration of 1 mL / Kg.

[0082] A schematic diagram illustrating the evaluation of the immunomodulatory effects of *Trifolium repens* external vesicles is shown below. Figure 12 As shown, the results confirm that *Tripterygium wilfordii* external vesicles have a significant immune-activating effect, effectively promoting the maturation of dendritic cells and providing a core mechanism for reshaping the tumor immune microenvironment and achieving anti-tumor immunity. Dendritic cells, as the body's "professional antigen-presenting cells," directly determine the efficiency of the body's anti-tumor immune response, and are a key link in transforming "immunely cold" tumors into "immunely hot" tumors. Compared with the blank control group, different concentrations of *Tripterygium wilfordii* external vesicles prepared in Examples 1 and 2 significantly increased the proportion of mature dendritic cells, indicating that *Tripterygium wilfordii* external vesicles can effectively activate the maturation process of dendritic cells, and this immunomodulatory effect can be exerted within the experimentally set concentration range. This result reveals one of the core immune mechanisms of *Tripterygium wilfordii* external vesicles in anti-tumor treatment: by promoting dendritic cell maturation and enhancing antigen-presenting capacity, it activates subsequent T-cell immune responses and improves the local immunosuppressive microenvironment of the tumor. This provides a key mechanistic explanation for its application in the treatment of "immunely cold" tumors such as triple-negative breast cancer, and also provides experimental evidence for its combined application with other immunotherapy strategies.

[0083] Experimental Example 6: In vitro stability test of Tripterygium wilfordii vesicle formulation.

[0084] Test samples: Trifoliate vesicles prepared in each example and comparative example.

[0085] Test method: The *Tripterygium wilfordii* vesicle preparations prepared in each example and comparative example were sealed and stored at 4°C. Samples were taken on days 0, 7, 14, and 21 of storage. The particle size distribution of *Tripterygium wilfordii* vesicles in the samples at each time point was determined by nanoparticle tracking analysis technology, and the average particle size was recorded. The aggregation rate (%) was calculated according to the formula: Agglomeration rate (%) = (average particle size at test time point - initial average particle size) / initial average particle size × 100%.

[0086] The in vitro stability test results of the *Trifolium repens* vesicle formulation are shown in Table 1.

[0087] Table 1. Results of in vitro stability tests on *Trifolium repens* vesicle formulations.

[0088] According to the results in Table 1, compared with Example 1, Examples 3 and 4, which added sisal saponin to the basic formulation, significantly reduced the aggregation rate and effectively improved the stability by utilizing the steric barrier formed by the amphiphilic structure of sisal saponin to shield the attractive forces between particles. Example 5 added apigenin-7-glucoside to sisal saponin. This component binds to the outer vesicle membrane through hydrogen bonding, further enhancing the interfacial layer density and hydrophilicity, achieving dual protection, further reducing the aggregation rate and continuously improving the stability. Example 6 introduced 4',5,7-trihydroxyflavanone to construct a multi-component composite interfacial modification system, reducing the aggregation rate to the lowest level and achieving optimal in vitro stability. Comparative Example 1 introduced an excessive amount of sisal saponin. The surfactant coverage was uneven, and local bridging and aggregation were easy, with an aggregation rate higher than that of Examples 3 and 4, and the stability was weaker than that of this type of optimized formulation. Comparative Example 2 replaced the added ingredient with rutin, but the interfacial barrier strength and density formed were insufficient, and the stability was lower than that of Example 5. Comparative Example 3 replaced the added ingredient with dihydroisoflavone, which lacked interfacial regulation with other components, and the interfacial layer structure was loose, with an aggregation rate higher than that of Example 6. The above results indicate that the addition of sisal saponin, apigenin-7-glucoside and 4',5,7-trihydroxyflavanone to the *Trifolium repens* exovesicle formulation can effectively reduce the aggregation rate of the formulation and significantly improve its in vitro stability under 4°C refrigeration through interfacial steric hindrance regulation and multi-level intermolecular interactions.

[0089] Experimental Example 7: Tumor cell inhibition rate test of Tripterygium wilfordii external vesicle preparation.

[0090] Test samples: Trifoliate vesicles prepared in each example and comparative example.

[0091] Test methods: Cell culture: MDA-MB-231 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics, and incubated at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for the experiment; Cell plating: The concentration of digested MDA-MB-231 cells was adjusted to 5 × 10⁻⁶ cells / mL. 3 Cells were seeded per well in a 96-well cell culture plate with 100 μL of culture medium per well and incubated for 24 h. Treatment: The original culture medium was discarded. 100 μL of the corresponding sample diluent was added to each well of the experimental group, and diluted with serum-free medium to a final concentration equivalent to the clinically effective oral concentration of the formulation. 100 μL of serum-free medium was added to the negative control group. Each group had 6 replicates, and the cells were incubated for another 48 h. Absorbance was measured: 10 μL of LCK-8 reagent was added to each well, and after 2 h of incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Proliferation inhibition rate was calculated as follows: Proliferation inhibition rate (%) = (OD value of negative control group - OD value of experimental group) / average OD value of negative control group × 100%.

[0092] The tumor cell inhibition rate test results of the *Trifolium repens* vesicle preparation are shown in Table 2.

[0093] Table 2. Results of tumor cell inhibition rate test of *Tripterygium wilfordii* vesicle preparation.

[0094] According to the results in Table 2, the basic *Tripterygium wilfordii* vesicle preparation in Example 1 can directly inhibit the proliferation of tumor cells, demonstrating the natural antitumor activity of *Tripterygium wilfordii* vesicles. In Examples 3 and 4, the introduction of sisal saponins into the basic preparations significantly improved the tumor cell proliferation inhibition rate and greatly enhanced the inhibitory effect, achieving a synergistic effect between sisal saponins and *Tripterygium wilfordii* vesicles' antitumor activity. In Example 5, the introduction of apigenin-7-glucoside into sisal saponins further improved the inhibition rate and enhanced the antitumor effect of the active ingredients. In Example 6, the introduction of 4',5,7-trihydroxyflavanone to construct a multi-active ingredient system slightly increased the inhibition rate to its highest level, achieving the optimal tumor cell inhibition effect. This invention achieves the synergistic and amplified antitumor effects of multiple active ingredients. Comparative Example 1, due to a significant increase in the amount of sisal saponin, broke the optimal ratio of this invention, resulting in a much lower tumor cell proliferation inhibition rate than Examples 3 and 4. Excessive addition weakened the antitumor effect between the components. Comparative Example 2, by replacing the added ingredient with rutin, showed a significantly lower inhibition rate than Example 5, which combined apigenin-7-glucoside, demonstrating the suitability and superiority of the selected apigenin-7-glucoside in enhancing antitumor activity. Comparative Example 3, by replacing the added ingredient with dihydroisoflavone, still showed a lower inhibition rate than Example 6, highlighting the key role of the selected 4',5,7-trihydroxyflavanone in enhancing the antitumor effect in a multi-component system. This indicates that in the *Trifolium repens* vesicle preparation, by adding sisal saponin, apigenin-7-glucoside, and 4',5,7-trihydroxyflavanone, the antitumor activity of each component and the *Trifolium repens* vesicle itself can be synergistically enhanced, thereby improving the inhibitory effect on tumor cell proliferation.

[0095] Experimental Example 8: Cell compatibility test of Tripterygium wilfordii exovesicle preparation.

[0096] Test samples: Trifoliate vesicles prepared in each embodiment.

[0097] Test methods: Cell culture: NCM460 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics, and incubated at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for the experiment; Cell plating and drug administration: The procedure was the same as for MDA-MB-231 cells, and the cell concentration was adjusted to 4×10⁶ cells / mL. 3 Cells / well, the experimental group was added with the corresponding sample diluent of the same concentration as the breast cancer cell test, and the negative control group was added with serum-free culture medium; absorbance was detected and cell viability was calculated: the operation was the same as in Experiment 7, cell viability (%) = (average OD value of experimental group / average OD value of negative control group) × 100%, and cell viability ≥ 80% was judged as good cell compatibility.

[0098] The *Tripterygium wilfordii* vesicle formulations prepared in each embodiment showed good compatibility with normal human intestinal mucosal epithelial NCM460 cells and no significant in vitro toxicity. This result aligns with the application scenario of oral administration, demonstrating that after oral administration, the formulation does not cause significant damage to intestinal mucosal epithelial cells, avoiding potential side effects such as intestinal irritation caused by oral administration. This provides important in vitro safety data to support the oral clinical application of the formulation.

[0099] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0100] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing a Trifoliate Oryza sativa vesicle formulation, characterized in that: include, The dried Chinese herbal medicine *Trifolium repens* was ground into a fine powder to obtain *Trifolium repens* dry powder. The *Trifolium repens* dry powder was added to PBS solution for soaking and then filtered to obtain crude juice. Centrifuge the crude juice at low speed, take the supernatant and repeat the extraction twice, then centrifuge at high speed, discard the supernatant to obtain the three-leaf cyanophyll exovesicles; The outer vesicles of *Trifolium repens* were resuspended in PBS solution, and sisal saponin dispersion was added. The mixture was incubated at room temperature for 20 min to obtain a resuspended solution of *Trifolium repens*. The resuspended solution of *Trifolium repens* was filtered through multiple stages to obtain the outer vesicle preparation of *Trifolium repens*. In the *Tripterygium wilfordii* resuspension, the volume ratio of *Tripterygium wilfordii* exovesicles to sisal saponin dispersion is 2-6:

1. The sisal saponin dispersion comprises sisal saponin and PBS solution, wherein the mass-to-volume ratio of sisal saponin to PBS solution is 1 mg: 5~20 mL. The low-speed centrifugation speed is 9000~10000×g, and the duration is 40~80min; The high-speed centrifugation speed is 130,000~170,000×g, and the duration is 40~80min; The concentration of the *Trifolium repens* vesicles was 1×10⁻⁶. 13 ~1×10 14 Particles / mL, the particle size of the *Trifolium repens* exovesicles is 100~300 nm.

2. The preparation method according to claim 1, characterized in that: In the crude juice, the mass-to-volume ratio of dried *Trifolium repens* powder to PBS solution is 1 g: 5-50 mL.

3. The preparation method according to claim 1, characterized in that: The soaking time for the dried *Trifolium repens* powder after adding it to PBS solution is 10-15 hours.

4. The preparation method according to claim 1, characterized in that: The sisal saponin dispersion also includes apigenin-7-glucoside, wherein the mass ratio of apigenin-7-glucoside to sisal saponin is 1:2~8.

5. The *Trifolium repens* exovesicle formulation obtained by any one of the preparation methods according to claims 1-4, characterized in that: The *Trifolium repens* external vesicle preparation includes *Trifolium repens* external vesicles, which are derived from the traditional Chinese medicine *Trifolium repens*.

6. The use of the *Trifolium repens* exovesicle formulation according to claim 5 in the preparation of antitumor drugs, characterized in that: The tumor is a triple-negative breast cancer.

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