Membrane vesicles derived from parabacteroides coumarini as well as preparation method and application of membrane vesicles

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a parabacteroides coumarini-sourced membrane vesicle as well as a preparation method and application thereof. The membrane vesicle is prepared through a gradient centrifugation method, the average particle size of the membrane vesicle is 115 nm, the Zeta potential is-12 mV, and the concentration is 2.69 * 10 < 11 > + / -1.22 * 10 < 9 > particles / mL; in-vitro experiments prove that the parabacteroides coumarini-sourced membrane vesicles can significantly inhibit the proliferation activity of breast cancer cells; in-vivo experiments show that the parabacteroides coumarini-sourced membrane vesicles can significantly inhibit tumor growth of breast cancer mice, and have good biological safety; mechanism discussion shows that the parabacteroides coumarini-sourced membrane vesicles mainly play an anti-tumor role by promoting the infiltration level of CD8 + T cells in a tumor microenvironment; in conclusion, the parabacteroides coumarini-sourced membrane vesicles have good development potential and application prospects in breast cancer immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine and microbiological technology, and particularly relates to application of a Parabacteroides goldsteinii-derived membrane vesicle in preparation of a product for preventing, treating or relieving breast cancer. BACKGROUND

[0002] Breast cancer is one of the malignant tumors with the highest incidence in the global population and the Chinese female population, and it has brought a heavy disease burden to the society and the patient's family. According to the estimation data of GLOBOCAN 2022, about 2.3 million new cases of breast cancer occur globally each year, which has replaced lung cancer and ranked first in the global cancer incidence spectrum. In China, the incidence rate continues to grow rapidly and shows a trend of youth, and it has become a major public health problem for women in both urban and rural areas.

[0003] Triple-negative breast cancer lacks estrogen receptor, progesterone receptor and HER 2 expression, resulting in ineffective targeted therapy and endocrine therapy. At present, chemotherapy is still the main systemic treatment, but it has the problems of limited efficacy, great side effects and easy drug resistance, and the tumor has strong invasiveness and is prone to metastasis and recurrence, resulting in poor overall prognosis of patients. In recent years, tumor immunotherapy has made a breakthrough, but how to effectively activate the anti-tumor immune response in the tumor microenvironment is still a core challenge. The use of bacterial components for tumor immunotherapy has attracted attention due to its unique and efficient way of activating the human immune system to precisely fight tumors. This concept can be traced back to the late nineteenth century, when Dr. William Coley successfully induced tumor regression in some patients by injecting pyogenic streptococci and later standardized inactivated bacterial mixtures (i.e., "Coley toxin"). This was regarded as the beginning of cancer immunotherapy. However, due to the potential toxicity, inconsistent efficacy and unknown mechanism of the therapy, its use is limited.

[0004] With the in-depth development of microbiology, bacterial membrane vesicles (MVs) have attracted widespread attention as a new biological treatment platform. MVs are naturally released by bacteria during growth, with a particle size of about 20-250 nm, and they contain biological active substances such as proteins, nucleic acids and peptidoglycan from the parent bacteria. As a natural delivery system, MVs can efficiently deliver these immunogenic components to host cells, activate innate immunity through pattern recognition receptors, and promote the maturation and antigen cross-presentation of dendritic cells, thereby initiating antigen-specific CD8 + T cell immune response. Compared with live bacteria, MVs have a clear nanostructure, good biological safety and precisely controllable drug dosage, and show great application potential in the field of tumor immunotherapy.

[0005] Currently, some studies have reported that MVs from certain pathogenic bacteria (such as Escherichia coli and Salmonella) can induce anti-tumor effects in breast cancer models by carrying toxins or antigens, however, these pathogen-derived MVs usually have inherent toxicity such as high endotoxin activity, and have a high risk of clinical transformation, therefore, the development of MVs derived from human symbiotic probiotics with clear mechanism of action and high safety has become a new trend in this field; Parabacteroides goldsteinii is a common symbiotic bacterium in the human intestine, which has been confirmed to have anti-inflammatory and immunoregulatory functions, and recent studies have shown that Parabacteroides goldsteinii or its specific components can play a beneficial role in metabolic diseases and colorectal cancer liver metastasis models by regulating the body's metabolic and immune microenvironment.

[0006] Based on the above background, the present application aims to provide a membrane vesicle derived from the probiotic Parabacteroides goldsteinii, in order to provide a new, safe and efficient microbial-derived nanodrug candidate for the clinical treatment of breast cancer. SUMMARY

[0007] In order to solve the problems in the prior art, the present application provides a Parabacteroides goldsteinii-derived membrane vesicle preparation method and its application in related products for preventing, treating or relieving breast cancer.

[0008] A Parabacteroides goldsteinii-derived membrane vesicle, wherein the membrane vesicle is derived from Parabacteroides goldsteinii.

[0009] Preferably, the average particle size of the membrane vesicle is 115 nm, and the Zeta potential is -12 mV.

[0010] A preparation method of a Parabacteroides goldsteinii-derived membrane vesicle is as follows: Parabacteroides goldsteinii is cultured to the late logarithmic growth phase, and then the culture supernatant is collected, gradient centrifugation and filtration are performed on the culture supernatant to remove bacteria and debris, the obtained filtrate is ultrafiltrated and concentrated, and then the concentrated solution is subjected to ultracentrifugation, the precipitate is collected and resuspended with a phosphate buffer to obtain the membrane vesicle.

[0011] Application of a Parabacteroides goldsteinii-derived membrane vesicle in the preparation of a drug for preventing or treating breast cancer.

[0012] Preferably, the drug can play a role by activating the body's anti-tumor immune response, specifically by significantly increasing the infiltration level of CD8 + T cells in tumor tissues, thereby inhibiting tumor growth.

[0013] Preferably, the drug comprises a therapeutically effective amount of Parabacteroides goldsteinii-derived membrane vesicles and a pharmaceutically acceptable excipient, wherein the excipient comprises one or more of a diluent, a stabilizer, a buffer, a preservative or a carrier, and the concentration of the membrane vesicles in the drug or pharmaceutical composition is not less than 2.69 x 1011 ±1.22 x 10 9 particles ml.

[0014] A key benefit of the present application is to provide a preparation method of Parabacteroides goldsteinii-derived membrane vesicles and its application in breast cancer prevention and treatment. The method is stable and easy to scale up. By gradient centrifugation combined with 60,000 x g ultracentrifugation, structurally intact membrane vesicles can be efficiently obtained from culture supernatants. Experiments have shown that the membrane vesicles can significantly inhibit the proliferation of MDA-MB-231 breast cancer cells in vitro, effectively inhibit tumor growth in vivo, and have no significant toxicity to major organs, with excellent biological safety. In summary, Parabacteroides goldsteinii-derived membrane vesicles have both direct anti-tumor activity and immunomodulatory function, with a clear preparation process, high safety, and important application value in the development of breast cancer treatment drugs.

[0015] Other features of the present application, and their advantages, will become apparent in the non-limiting detailed description of exemplary embodiments of the application which follows, with reference to the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0017] Figure 1 is a preparation scheme of Parabacteroides goldsteinii-derived membrane vesicles provided by the embodiments of the present application.

[0018] Figure 2 is a transmission electron microscope image of Parabacteroides goldsteinii-derived membrane vesicles provided by the embodiments of the present application.

[0019] Figure 3 is a particle size distribution graph of Parabacteroides goldsteinii-derived membrane vesicles provided by the embodiments of the present application.

[0020] Figure 4 is a SDS-PAGE and WB graph of Parabacteroides goldsteinii-derived membrane vesicles provided by the embodiments of the present application.

[0021] Figure 5 is a CCK-8 detection result graph of the proliferation activity of Parabacteroides goldsteinii-derived membrane vesicles on breast cancer cells provided by the embodiments of the present application.

[0022] Figure 6 is a Parabacteroides goldsteinii-derived membrane vesicle in vitro hemolysis experiment result graph provided by the embodiments of the present application.

[0023] Figure 7 is a Parabacteroides goldsteinii-derived membrane vesicle in vivo efficacy result graph in a breast cancer orthotopic model provided by the embodiments of the present application.

[0024] Figure 8 FIG. 7 is a graph of the pharmacodynamic evaluation results of the Parabacteroides goldsteinii-derived membrane vesicles in a mouse breast cancer prevention model provided by the embodiments of the present application.

[0025] Figure 9 FIG. 8 is a graph of the regulation of the immune microenvironment of breast cancer tumors by the Parabacteroides goldsteinii-derived membrane vesicles provided by the embodiments of the present application.

[0026] Figure 10 FIG. 9 is a graph of the HE staining results of the main organs of the breast cancer mice provided by the embodiments of the present application. DETAILED DESCRIPTION

[0027] In order to make the purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be described in detail below in combination with specific embodiments. It should be understood that the embodiments described in the present specification are only for the purpose of explaining the present application and are not intended to limit the present application.

[0028] For the sake of simplicity, only some numerical ranges are explicitly disclosed herein, however, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, likewise any upper limit can be combined with any other upper limit to form a range not explicitly recited; and further, although not explicitly recited, every point or individual number within the range is included in the range; thus every point or individual number can serve as its own lower limit or upper limit to form a range not explicitly recited.

[0029] In the description herein, it should be noted that, unless otherwise specified, "above", "below" include the number itself, and "several" in "one or several" means two or more.

[0030] The above summary of the present application is not intended to describe each disclosed embodiment or implementation in the present application; the exemplary embodiments are illustrated in more detail in the following description; in various places throughout the application, a series of embodiments are provided for guidance, which can be used in various combinations, and in each instance, the enumeration is only representative of a group, and should not be interpreted as exhaustive.

[0031] The application discloses a preparation method of membrane vesicles of Parabacteroides goldsteinii and application of the membrane vesicles in prevention and treatment of breast cancer. The method adopts a two-step process of "gradient centrifugation + 60000xg ultracentrifugation", can stably and efficiently obtain nano-scale vesicles with complete structure from supernatant of bacterial culture, and is easy to scale up. In vitro experiments show that the obtained vesicles can significantly inhibit proliferation of MDA-MB-231 breast cancer cells. A mouse model further proves that the membrane vesicles can effectively inhibit tumor growth, have no obvious toxicity to main organs such as heart, liver, spleen, lung and kidney, and have good biological safety. In summary, the membrane vesicles from Parabacteroides goldsteinii have dual functions of direct anti-tumor and immune regulation, the preparation process is clear, the quality is controllable, and the membrane vesicles have important application prospects in development of breast cancer treatment drugs and immunological preparations.

[0032] Example 1: Preparation and characterization of P-MVs Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): the technology separates proteins in polyacrylamide gel according to the difference in protein molecular weight, and is currently widely used in expression level, distribution and purity analysis in the process of expression and purification of exogenous proteins.

[0033] Western Blot (WB): the technology separates proteins according to molecular weight by SDS-PAGE, transfers the proteins to a solid phase membrane, and then detects the target protein by specific reaction of antigen and antibody.

[0034] 1. Experimental method 1.1 Extraction of Parabacteroides goldsteinii membrane vesicles Parabacteroides goldsteinii is purchased from the Guangdong Microbial Culture Collection Center, and the GDMCC number is 1.2815. GAM culture medium is used to configure liquid culture medium according to the instruction manual. After Parabacteroides goldsteinii grows to the late logarithmic growth phase, the culture supernatant is collected in a sterile tube (OD600=0.8-2.0). The bacterial cells and cell fragments are removed by gradient centrifugation at 1000xg, 3000xg, 5000xg and 8000xg, respectively. Then, the supernatant is further filtered by a 0.22 μm filter. Then, the supernatant is further concentrated by using a 100KDa ultrafiltration membrane. Then, the supernatant is ultracentrifuged at 60000g for 30 min at 4°C, washed by phosphate buffered saline (PBS), ultracentrifuged at 120000g for 2h at 4°C, washed by PBS once, resuspended by adding appropriate PBS, filtered by a 0.22 μm pore size filter, removed of bacterial cells or cell fragments (note: reduce loss), collected P-MVs, and stored at -80°C until use (not more than 3 months).

[0035] 1.2 Transmission electron microscopy and NTA are used to detect the morphology and particle size distribution of the nano-vesicles, respectively. Take 50 μL P-MVs suspension drop on copper mesh, after standing, use forceps to hold the copper mesh in the culture dish placed in PBS, then, after glutaraldehyde fixation for 5 minutes, wash with DEPC water for 1 minute, then acetaldehyde uranyl staining for 5 minutes, after staining, place the copper mesh on a stainless steel ring, use filter paper to absorb excess water, dry at room temperature for 10 minutes, then observe the morphology of P-MVs under transmission electron microscope.

[0036] Take another P-MVs sample, resuspend with 1 mL PBS, add P-MVs into a colorimetric cup or syringe to measure particle size and potential using particle size and zeta potential analyzer, follow the standard process of the instrument for specific operation.

[0037] 1.3 SDS-PAGE 1.3.1 Gel preparation and sample loading: Install the precast gel plate in the electrophoresis clamp, add the electrophoresis buffer and remove the comb, use the microsyringe to add the protein molecular weight marker (Marker) and the sample to be tested vertically in turn.

[0038] 1.3.2 Electrophoresis: Place the electrophoresis clamp in the electrophoresis tank, add enough buffer, set the initial voltage to 80V, after the sample enters the separation gel, adjust the voltage to 120V, continue electrophoresis until the bromophenol blue indicator is about 0.5 cm from the bottom of the gel.

[0039] 1.3.3 Staining and destaining: After electrophoresis, take out the gel, dye with Coomassie brilliant blue staining solution for more than 10 minutes, then use destaining solution for destaining until the background is clear and the bands are obvious, save in double distilled water and image.

[0040] 1.4 Western Blot 1.4.1 Membrane transfer: After electrophoresis, cut the corresponding gel area according to the molecular weight range of the target protein, prepare the membrane transfer "sandwich" structure in the membrane transfer buffer, in the order of "negative electrode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-positive electrode", ensure that there is no air bubble between each layer, transfer the membrane at 300 mA constant current for 45-60 minutes in ice bath.

[0041] 1.4.2 Blocking: After membrane transfer, immerse the PVDF membrane in 5% skim milk blocking solution, shake at room temperature for 2 hours.

[0042] 1.4.3 Antibody incubation: After blocking, incubate the membrane with specific primary antibody at 4°C overnight, then wash with PBST buffer for 3 times, 10 minutes each time, after washing, incubate with the corresponding secondary antibody at room temperature for 2 hours, wash with PBST buffer for 3 times again, 10 minutes each time.

[0043] 1.4.4 Development: After mixing equal volume of ECL chemiluminescence reagent A and B, evenly cover on the membrane, use chemiluminescence imaging system for detection and image acquisition.

[0044] 2. Experimental results Reference Figure 1 , Figure 1 Figure for the preparation of membrane vesicles from Parabacteroides goldsteinii.

[0045] Reference Figure 2 (Transmission electron microscopy image, TEM), from Figure 2 It can be seen that the membrane vesicles secreted by Parabacteroides goldsteinii are spherical, double-membrane structure, with typical characteristics of bacterial membrane vesicles.

[0046] Reference Figure 3 (Particle size distribution diagram), from Figure 3 It can be seen that the average diameter of P-MVs is about 115.0 nm, the Zeta potential is-12 mV, and the concentration is 2.69x10 11 ±1.22x10 9 particles / mL.

[0047] Reference Figure 4 A (SDS-PAGE result diagram), from Figure 4 A It can be seen that the membrane vesicles from Parabacteroides goldsteinii can observe the characteristic proteins of MVs such as flagellin virulence protein B (FliB) and various outer membrane proteins (OMPs) usually expressed on the outer membrane of bacteria.

[0048] Reference Figure 4 B (Western Blot result diagram), from Figure 4 B It can be seen that the membrane vesicles from Parabacteroides goldsteinii can detect the expression of outer membrane protein ompA, suggesting the source of MVs (from Parabacteroides goldsteinii).

[0049] Example 2: CCK8 experiment CCK-8 (Cell Counting Kit-8) reagent can be used for efficient and accurate detection of cell proliferation and toxicity; its core principle is that: water-soluble tetrazolium salt WST-8 in the reagent, in the presence of electron carrier 1-methoxy PMS, can be reduced by dehydrogenase in living cells to water-soluble yellow formazan, under certain conditions, the amount of generated formazan is proportional to the number of living cells, and the cell viability can be quantitatively analyzed by detecting OD value.

[0050] 1. Experimental method MDA-MB-231 cells (purchased from American Type Culture Collection, ATCC) in logarithmic growth phase were seeded in 96-well plates at an appropriate density, and after adhesion, fresh medium containing different concentrations of empty P-MVs (0, 50, 100, 200, 400, 600 μg / mL) was replaced, and the cells were treated for 6 hours, then CCK-8 solution was added to each well, and the cells were incubated in the cell incubator for an appropriate time (usually 0.5-3 hours, determined according to the pre-experiment), finally, the absorbance (OD value) of each well was determined at 450 nm wavelength using a microplate reader.

[0051] 2. Experimental results See Figure 5 (CCK-8 result graph), from Figure 5 It can be seen that P-MVs at a concentration of 400 μg / mL can achieve a significant inhibitory effect on the proliferation of breast cancer cells.

[0052] Example Three: In vitro hemolysis experiment In vitro hemolysis test is an important means to evaluate the blood compatibility and safety of biomedical materials or drug preparations. By observing the hemolytic reaction of red blood cells to drug preparations, the safety of drug preparations in vivo can be evaluated.

[0053] 1. Experimental method Take the orbital blood of healthy BALB / c mice into an anticoagulant centrifuge tube, gently invert to fully anticoagulate, centrifuge at 3500 rpm for 10 minutes, discard the supernatant, and reserve the red blood cell precipitate. Wash the red blood cells with physiological saline at 4°C and 1000 rpm for 5 times until the supernatant is colorless. Take 100 μL of purified red blood cells into a 1.5 mL centrifuge tube, add 100 μL of different concentrations of P-MVs (5 μg / mL, 10 μg / mL, 25 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) solution in the experimental group, use physiological saline as the negative control and deionized water as the positive control. Incubate the mixture at 37°C for 4 hours, centrifuge at 3000 rpm for 5 minutes, and take 150 μL of supernatant to a 96-well plate. Measure the absorbance value (OD value) at 570 nm. The calculation formula of hemolysis rate is as follows: Hemolysis (%) = (sample OD - negative control OD) / (positive control OD - negative control OD) x 100% 2. Experimental results Reference Figure 6 (in vitro hemolysis experiment result graph), from Figure 6 It can be seen that even at a higher concentration (200 μg / mL), P-MVs were not observed to have significant hemolysis (<5%).

[0054] Example Four: Mouse experimental breast cancer orthotopic treatment model 1. Experimental animals and ethics: All animal experimental protocols were approved by the Animal Ethics Committee of Ningxia Medical University (Approval No. IACUC-NYLAC-2024-064). SPF grade, 6-8 week old female BALB / c mice were purchased from and raised in the SPF barrier facility of the Experimental Animal Center of Ningxia Medical University.

[0055] 2. Cells and strains: Human breast cancer MDA-MB-231 cells were purchased from the American Type Culture Collection; Parabacteroides distasonii strains were purchased from the Guangdong Microbial Culture Collection Center.

[0056] 3. Establishment of animal models, grouping and intervention: As shown in Figure 7 A, breast cancer orthotopic xenograft models were established by subcutaneous injection of MDA-MB-231 cells into the mammary fat pad of mice. When the tumor volume grew to about 100 mm 3 , the tumor-bearing mice were randomly divided into two groups (n=5): Control group: injection of equal volume of PBS.

[0057] P-MVs treatment group: intratumoral injection of P-MVs (15 μg / time, once every 3 days, a total of 4 times).

[0058] 4. Observation index and sample collection: The intervention lasted for 3 weeks. During this period, the body weight of the mice was measured and recorded every two days. At the end of the experiment, all mice were sacrificed by cervical dislocation, and the tumor tissue was separated and measured with a vernier caliper to calculate the tumor volume.

[0059] 5. Experimental results Reference Figure 7 A, Figure 7 A is a schematic diagram of the mouse experimental breast cancer orthotopic treatment model.

[0060] Reference Figure 7 B, from Figure 7 B can be seen that there is no statistical difference in the body weight of the mice in the two groups during the entire experiment, indicating that P-MVs do not cause systemic toxicity at an effective dose and have good biological safety.

[0061] Reference Figure 7 C, D, from Figure 7 C and D can be seen that the antitumor effect in mice shows that the tumor volume of the P-MVs treatment group is significantly reduced compared with the control group, indicating that it has significant antitumor efficacy.

[0062] Example Five: Mouse experimental breast cancer prevention model Experimental animals and ethics and cells and strains are exactly the same as in Example Four, which will not be repeated here.

[0063] 1. Animal model establishment, grouping and intervention: According to the scheme shown in Figure 8 A, a breast cancer prevention model was established: MDA-MB-231 cells were injected into the mammary fat pad of mice on day 0, and the tumor-bearing mice were randomly divided into two groups (n=5): control group; P-MVs intervention group: P-MVs (5x10 7 CFU / 200 μL) were injected via the tail vein once every 7 days for a total of 4 times of intervention.

[0064] 2. Observation index and sample collection: The mice were observed for 3 weeks, during which their body weight and survival status were recorded every two days. At the end of the experiment (day 21), all mice were sacrificed by cervical dislocation, and the tumor tissue was isolated and its volume was measured.

[0065] 3. Experimental results Reference Figure 8 A, Figure 8 A is a schematic diagram of a mouse experimental breast cancer prevention model.

[0066] Reference Figure 8 B, C, from Figure 8 As can be seen from B and C, the average tumor volume of mice in the P-MVs intervention group was significantly smaller than that of the control group, indicating that P-MVs had a significant tumor inhibition effect.

[0067] Reference Figure 8 D, from Figure 8 As can be seen from D, there was no significant difference in body weight between the two groups of mice during the entire experiment, indicating that the intervention scheme did not cause systemic toxicity and had good biological safety.

[0068] The above results show that P-MVs effectively inhibit the progression of breast cancer while exhibiting good treatment safety.

[0069] Example Six: FCM detection of changes in infiltrating immune cells in tumor tissue after treatment The tumor immune microenvironment is composed of tumor cells, immune cells, stromal cells, and signal molecules, and the level of immune cell infiltration directly affects the strength of the anti-tumor immune response and the therapeutic effect. To explore the remodeling effect of P-MVs on the immune microenvironment of breast cancer, this study quantitatively analyzed the proportion changes of key immune cell subsets in the spleen, inguinal lymph nodes, and tumor tissue of tumor-bearing mice by flow cytometry.

[0070] 1. Experimental method 1.1 Sample preparation: The spleen, inguinal lymph nodes, and tumor tissue of mice in each group were collected at the end of the experiment to prepare single-cell suspensions.

[0071] 1.2 Fluorescent antibody staining: To distinguish different immune cell subsets, the single-cell suspension was labeled and stained with multi-color fluorescent antibodies.

[0072] 1.2.1 Cell surface staining: Add specific fluorescent antibodies against surface antigens, incubate at 4°C for 30 minutes in the dark, centrifuge after washing with Buffer, and discard the supernatant.

[0073] 1.2.2 Intracellular staining: For samples that need to detect intracellular cytokines (such as γ-IFN), after completing surface staining, use a membrane-breaking agent to permeabilize the cells, then add intracellular factor-specific antibodies, incubate at 4°C for 35 minutes in the dark, and wash again with Buffer.

[0074] 1.3 Detection and analysis: Resuspend the stained cells in an appropriate amount of Buffer, filter through a 200-mesh sieve, and use a flow cytometer for detection. Use FlowJo software (v10.8.1) to analyze γ-IFN + CD8 + T cells, CD86 + CD11c + The percentage of cells such as CD8

[0075] 2. Experimental results Reference Figure 9 A, by Figure 9 A can be seen that compared with the control group, the proportion of CD8 + T cells and their subpopulation secreting γ-IFN in the tumor tissue of P-MVs treated mice increased significantly.

[0076] Reference Figure 9 B, by Figure 9 B can be seen that the central memory T cells (Tcm) and effector memory T cells (Tem) in the tumor also increased significantly, indicating that the immune response has the potential for persistence and effect.

[0077] Reference Figure 9 C, by Figure 9 C can be seen that the proportion of mature dendritic cells and macrophages in the P-MVs group increased, suggesting that the antigen presentation and immune activation ability were enhanced.

[0078] The above results show that P-MVs can effectively remodel the tumor immune microenvironment, promote the activation and infiltration of T cell subpopulations with killing and memory functions, and enhance the activation and infiltration of antigen-presenting cells, which may be an important immunological mechanism for its anti-breast cancer effect.

[0079] Example Seven: HE Staining Experiment Hematoxylin-eosin (HE) staining is a classic method for observing tissue morphology. In this method, the alkaline dye hematoxylin stains the cell nucleus blue, and the acidic dye eosin stains the cytoplasm red, clearly distinguishing the microstructure and cell types of the tissue.

[0080] 1. Experimental method The animal model used in the experiment is the same as in Example Four. After the mice are sacrificed at the end of the experiment, the heart, liver, spleen, lung, and kidney tissues are collected for HE staining. The main steps are as follows: 1.1 Fixation: After washing with PBS, the tissue blocks are fixed in 4% paraformaldehyde at 4°C for 48 hours.

[0081] 1.2 Dehydration, embedding, and sectioning: After fixation, the tissues are dehydrated with gradient ethanol, cleared with xylene, embedded after wax immersion, and sectioned to a thickness of 3.5 μm.

[0082] 1.3 De-waxing and hydration: After de-waxing with xylene, the sections are hydrated through gradient ethanol to water.

[0083] 1.4 Staining: According to the standard procedure, first perform nuclear staining with hematoxylin and then cytoplasmic staining with eosin. Optimize the staining effect by differentiating with hydrochloric alcohol and returning to blue with running water.

[0084] 1.5 Dehydration, clearing, and mounting: After staining, the sections are dehydrated with gradient ethanol, cleared with xylene, and finally mounted with neutral gum.

[0085] 1.6 Observation of experimental results: Observe the mounted tissue sections under a light microscope, compare and analyze the tissue structure, cell morphology, and arrangement level of each organ in the two groups of mice, and evaluate the in vivo biological safety of P-MVs.

[0086] 2. Experimental results See Figure 10 (HE staining result figure), by Figure 10 It can be seen that compared with the control group, the heart, liver, spleen, lung, and kidney of the P-MVs treatment group mice showed no obvious pathological changes, the tissue structure was complete, and the cell morphology was normal. This result indicates that P-MVs exhibit good in vivo biological safety while exerting anti-tumor effects.

Claims

1. A Parabacteroides goldsteinii-derived membrane vesicle, characterized in that, The membrane vesicles are derived from Parabacteroides goldsteinii.

2. The membrane vesicle of claim 1, wherein, The average particle size of the membrane vesicles is 115 nm, and the Zeta potential is -12 mV.

3. A method of preparing Parabacteroides goldsteinii-derived membrane vesicles according to one of claims 1-2, characterized in that, After Parabacteroides goldsteinii is cultured to the late logarithmic growth phase, the culture supernatant is collected, gradient centrifugation and filtration are performed on the culture supernatant to remove bacteria and debris, the obtained filtrate is concentrated by ultrafiltration, and the concentrated solution is subjected to ultracentrifugation, the precipitate is collected and resuspended with a phosphate buffer to obtain the membrane vesicles.

4. Use of the Parabacteroides goldsteinii-derived membrane vesicles according to any one of claims 1-2 in the preparation of a medicament for preventing or treating breast cancer.

5. Use according to claim 4, characterized in that, The drug can play a role by activating the body's anti-tumor immune response, which is specifically manifested by being able to significantly increase the infiltration level of CD8 + T cells in tumor tissues, thereby inhibiting tumor growth.

6. Use according to claim 4, characterized in that, The medicament comprises a therapeutically effective amount of Parabacteroides goldsteinii-derived membrane vesicles and a pharmaceutically acceptable excipient, including one or more of a diluent, a stabilizer, a buffer, a preservative, or a carrier, and the concentration of the membrane vesicles in the medicament or pharmaceutical composition is not less than 2.69 x 10 11 ± 1.22 x 10 9 particles per milliliter.

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