Engineered bacterial membrane vesicle loaded with apatinib as well as preparation method and application of engineered bacterial membrane vesicle

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an apatinib-loaded engineered bacterial membrane vesicle and a preparation method and application thereof.The engineered bacterial membrane vesicle is prepared by mixing apatinib and a membrane vesicle derived from rodent bacillus in a PBS according to a specific mass ratio; incubating at 37 DEG C for 4 hours, removing free drugs by using a 100kDa ultrafiltration membrane, and concentrating; and washing with PBS to obtain a final product. Experimental results show that the engineered bacterial membrane vesicles can effectively inhibit malignant biological behaviors such as proliferation of breast cancer cells and significantly inhibit tumor growth in an animal model; mechanism research shows that the compound can effectively activate the anti-tumor immune response of the body; safety evaluation shows that the engineered bacterial membrane vesicle has no toxic or side effect; in conclusion, the engineered bacterial membrane vesicle provided by the invention has dual advantages of chemotherapy and immunotherapy, is good in safety, and has important application value in development of breast cancer treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biomedicine and microbiological technology, and particularly relates to an application of an engineered bacterial membrane vesicle loaded with apatinib in preparation of a product for or alleviating breast cancer. BACKGROUND

[0002] Breast cancer is the most common malignant tumor in women worldwide. Despite the continuous progress in treatment methods, there is still a lack of effective targeted treatment for some subtypes, such as triple-negative breast cancer (TNBC), and chemotherapy is still the main choice, but there are problems such as high toxicity and easy drug resistance; apatinib is a small molecule VEGFR-2 inhibitor that exerts an anti-tumor effect through anti-angiogenesis, but its poor water solubility and systemic toxicity (such as hypertension and proteinuria) limit its clinical application.

[0003] In recent years, microbial therapy, especially therapy based on probiotics and their active ingredients, has become a new strategy for tumor treatment; Faecalibaculum rodentium (F. rodentiums) is an important beneficial bacteria in the intestinal tract, and its anti-inflammatory and immunoregulatory functions have been confirmed; membrane vesicles (F-MVs) derived therefrom serve as natural nanometer delivery carriers, and have good biocompatibility and potential immunoregulatory capacity; however, there is no report on the use of F. rodentium membrane vesicles loaded with apatinib for breast cancer treatment, especially for playing a role by regulating lymph node immune cell subpopulations. SUMMARY

[0004] The present application provides an engineered bacterial membrane vesicle loaded with apatinib, a preparation method and application thereof, which can effectively inhibit the proliferation and other malignant biological behaviors of breast cancer cells, significantly inhibit tumor growth in animal models, effectively activate the body's anti-tumor immune response, and inhibit the progression of breast cancer.

[0005] The engineered bacterial membrane vesicle loaded with apatinib uses membrane vesicles derived from F. rodentium as a carrier, and internally loads the chemotherapeutic drug apatinib.

[0006] Preferably, the mass ratio of the membrane vesicles derived from F. rodentium to apatinib is 2:1.

[0007] The preparation method of the engineered bacterial membrane vesicles carrying apatinib comprises the following steps: extracting vesicles: after collecting the supernatant of rodent bacteria by gradient centrifugation, the bacterial supernatant is centrifuged by ultracentrifugation to obtain the precipitate, i.e., bacterial outer membrane vesicles, and resuspended with PBS; drug loading: the resuspended bacterial outer membrane vesicles are mixed with apatinib at a specific mass ratio in PBS, incubated at 37 DEG C for 4 hours, and then the free apatinib is removed using a 100KD ultrafiltration membrane and concentrated, and then washed several times with PBS to obtain the engineered bacterial membrane vesicles carrying apatinib.

[0008] The engineered bacterial membrane vesicles carrying apatinib are used for preparing a drug or a pharmaceutical composition for treating or relieving breast cancer.

[0009] Preferably, the engineered bacterial membrane vesicles can play a role by inhibiting tumor cell proliferation, migration and invasion, promoting tumor cell apoptosis and remodeling the tumor immune microenvironment.

[0010] The engineered bacterial membrane vesicles carrying apatinib and the preparation method thereof disclosed in the present application can achieve the following beneficial effects: in vivo and in vitro experiments have confirmed that the engineered bacterial membrane vesicles can effectively inhibit breast cancer cell proliferation, migration and invasion and induce apoptosis, and significantly inhibit tumor growth in animal models; mechanism research shows that its anti-tumor effect plays a role by remodeling the immune pattern of tumor draining lymph nodes, i.e., significantly increasing CD8+ T cells while reducing regulatory T cells, thereby fundamentally activating systemic anti-tumor immune response; safety evaluation shows that experimental animals treated with the drug delivery system do not have obvious toxic reactions, indicating that it has good biocompatibility at an effective dose; in summary, the drug delivery system has clear anti-tumor activity, immune regulation function and good safety, and is simple to prepare and widely available, especially providing a brand-new treatment strategy with immunotherapy characteristics for triple-negative breast cancer which lacks targeted drugs.

[0011] Other features and advantages of the present application will become apparent from the following detailed description of exemplary embodiments thereof, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0012] The accompanying drawings incorporated in and forming a part of the specification, illustrate embodiments of the present application and, together with the description, serve to explain the principles of the application.

[0013] Figure 1 is a preparation schematic diagram of FMVs / Apatinib provided by the embodiments of the present application; Figure 2 is a graph of in vitro drug release results of FMVs / Apatinib provided by the embodiments of the present application; Figure 3is a graph of the in vitro evaluation result of the effect of FMVs / Apatinib on the proliferation of breast cancer cells provided by the embodiment of the present application; Figure 4 is a graph of the detection result of the in vitro induction of breast cancer cell apoptosis by FMVs / Apatinib provided by the embodiment of the present application; Figure 5 is a graph of the in vitro evaluation result of the inhibition of breast cancer cell migration by FMVs / Apatinib provided by the embodiment of the present application; Figure 6 is a graph of the in vitro evaluation result of the inhibition of breast cancer cell invasion by FMVs / Apatinib provided by the embodiment of the present application; Figure 7 is a graph of the result of the FMVs / Apatinib HUVECs tube formation experiment provided by the embodiment of the present application; Figure 8 is a graph of the treatment effect of FMVs / Apatinib on mouse orthotopic breast cancer provided by the embodiment of the present application; Figure 9 is a graph of the flow cytometry analysis of breast cancer mouse lymph node CD8+ T cells provided by the embodiment of the present application; Figure 10 is a graph of the flow cytometry analysis of breast cancer mouse lymph node regulatory T cells provided by the embodiment of the present application; Figure 11 is a graph of the HE staining pathological analysis of the main organs of breast cancer mice provided by the embodiment of the present application. DETAILED DESCRIPTION

[0014] 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.

[0015] 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 that is not explicitly recited; and any lower limit can be combined with other lower limits to form a range that is not explicitly recited, and likewise any upper limit can be combined with any other upper limit to form a range that is not explicitly recited, and further, although not explicitly recited, every point or individual number within the range is included in the range, so that an explicit midpoint of the range and each point or individual number within the range are combined to form a subrange that is not explicitly recited.

[0016] In the description herein, it should be noted that, unless otherwise specified, "above", "below" include the number itself, and "several" means two or more.

[0017] The above summary of the application is not intended to describe every disclosed embodiment or implementation in the application, which is illustrated more particularly below by way of example. Throughout this application, guidance is provided by a series of examples, which can be used in various combinations, and in various instances, the recitations are merely representative groups, and should not be interpreted as exhaustive.

[0018] Example 1: Preparation of FMVs / Apatinib To obtain FMVs / Apatinib, first, rodent bacterium-derived membrane vesicles were obtained by ultracentrifugation, then apatinib and rodent bacterium-derived membrane vesicles were mixed in PBS at a specific mass ratio, incubated at 37°C for 4 hours, and then free drugs were removed using a 100KD ultrafiltration membrane and concentrated; after PBS washing, the final product was obtained.

[0019] Example 2: In vitro drug release experiment Drug in vitro release test is an experimental method for evaluating the rate and mode of drug release from a drug delivery system; through systematic study of drug in vitro release, researchers can understand and optimize the release behavior of the drug, and provide important basis for the development and evaluation of drug preparations.

[0020] 1. Experimental method In PBS containing 1% (v / v) Tween 80 at different pH (7.4 and 6.5), the PBS at two pH values simulates the normal physiological environment and the tumor micro-acidic environment; FMVs / Apatinib was dispersed in PBS, then shaken at 100 rpm in a shaking bed at 37°C, at different time points (0, 0.5, 1, 2, 6, 12, 24, 48, 72h), the filtrate was collected by 100KD ultrafiltration, the drug concentration was detected by ultraviolet spectrophotometer, and the following formula was used to calculate: Encapsulation efficiency=(actual drug loading amount / dose)x100% Drug loading=[actual drug loading mass / (actual drug loading mass+carrier mass)]x100% Actual drug loading amount according to the results of in vitro release 2. Experimental results Reference , Figure 2 , Figure 2 The in vitro drug release results of FMVs / Apatinib are shown in the figure, the drug in FMVs / Apatinib is more fully released in the micro-acidic environment of the tumor, and the drug is basically completely released (80-90%) within 72 hours.

[0021] Example Three: Inhibition of breast cancer cell proliferation by FMVs / Apatinib CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) is a membrane-permeable fluorescent dye that can be used for cell proliferation tracing. The dye itself has no fluorescence and can freely enter cells. Under the action of intracellular esterase, the acetic acid group is hydrolyzed to generate carboxyfluorescein succinimidyl ester with strong green fluorescence. At the same time, the product can covalently bind to the amino group of intracellular proteins to form stable fluorescent conjugates, thereby retaining the dye in the cell for a long time. During cell division, the fluorescent conjugates are evenly distributed to daughter cells, resulting in a gradual decrease in fluorescence intensity of daughter cells with increasing division generations. Based on this principle, changes in cell population fluorescence intensity can be detected by flow cytometry to quantitatively track cell division and proliferation behavior.

[0022] 1. Experimental method Centrifuge to collect cells, adjust cell concentration, mix cells with diluted CFSE suspension to a final concentration of 5 μM. Continue to incubate for 15 minutes, mix every 5 minutes, add cold complete medium to stop staining, incubate on ice for a few minutes, centrifuge and wash once with complete medium, resuspend the cells to the plating concentration, culture for 24 hours, collect the cells, detect cell proliferation by flow cytometry, and analyze with flow cytometry software.

[0023] 2. Experimental results Reference Figure 3 , Figure 3 The results of the CFSE cell proliferation experiment of breast cancer cells MDA-MB-231 cells, wherein flow cytometry was used to confirm that FMVs / Apatinib had an inhibitory effect on tumor cells MDA-MB-231, FMVs / Apatinib could inhibit the proliferation of breast cancer cells MDA-MB-231, and FMVs / Apatinib had the ability to resist breast cancer.

[0024] Example Four: Detection of FMVs / Apatinib-induced apoptosis of breast cancer cells in vitro Annexin V-PE / 7-AAD double staining method was used to detect cell apoptosis in this embodiment. The principle is based on: in early apoptosis, membrane phosphatidylserine (PS) is flipped from the inner side of the cell membrane, and Annexin V can specifically bind to the exposed PS in the presence of calcium ions; and 7-AAD, as a membrane-impermeable nucleic acid dye, can label late apoptotic and necrotic cells with lost membrane integrity; flow cytometry analysis can distinguish different cell states: Annexin V-PE single positive is early apoptotic cell; Annexin V-PE and 7-AAD double positive is late apoptotic or necrotic cell; double negative is living cell; this method can realize quantitative and staging detection of cell apoptosis process.

[0025] 1. Experimental method 1.1 Cell plating and intervention: Take the logarithmic growth period of breast cancer cell line MDA-MB-231 cells (American Type Culture Collection), digest and wash, then add 2×10 5 cells to 12-well plates in an incubator for 4 h, then add FMVs / Apatinib to 6 of the 12-well plates for 48 h, and continue normal culture for another 48 h in the other 6 wells; among them, the normal culture control group is recorded as Control group, and the bacterial intervention group is recorded as FMVs / Apatinib group. 1.2 Collect cells: Digest the cells with a digestion solution without EDTA, wash the collected cells with pre-cooled PBS twice, and resuspend with 100 uL Buffer. 1.3 Staining and detection: Add 2.5 uL Annexin V and 2.5 uL 7-AAD, mix gently, incubate at room temperature for 15 min, then add 300 uL PBS to resuspend the cells, and detect with flow cytometry. The results were analyzed using Flowjo.

[0026] 2. Experimental results Reference Figure 4 , Figure 4 The apoptosis experiment results of MDA-MB-231 cells are shown in the figure, wherein the number of apoptotic cells of MDA-MB-231 cells treated with FMVs / Apatinib is significantly greater than that of the Control group without intervention; this shows that the apoptosis of MDA-MB-231 cells treated with FMVs / Apatinib is increased, and FMVs / Apatinib can promote the apoptosis of breast cancer cells. It can be seen that FMVs / Apatinib has the ability to resist breast cancer.

[0027] Example Five: Detection of FMVs / Apatinib in vitro inhibition of breast cancer cell migration Transwell migration assay was used to detect the migration ability of the cells. In this assay, cells were seeded in the upper chamber (containing normal medium), and the lower chamber (containing complete medium) was separated from the upper chamber by a polycarbonate membrane. Since the membrane was permeable, the components in the lower chamber medium could affect the cells in the upper chamber. By counting the cells that migrated to the outside of the chamber, the migration ability of the cells could be evaluated.

[0028] 1. Experimental method 1.1 Cell culture: MDA-MB-231 breast cancer cells purchased from the American Type Culture Collection (ATCC) were divided into two groups. FMVs / Apatinib was added to one group for 48 hours, and the other group was cultured normally. This resulted in a normal culture control group (Control) and an intervention group (FMVs / Apatinib) of MDA-MB-231 cells. 1.2 Preparation of cell suspension: MDA-MB-231 cells in the logarithmic growth phase were digested and washed, then resuspended in serum-free medium. 3 x 10 5 cells were added to the upper chamber. 1.3 Cell inoculation: 750 μL of medium containing 10% FBS was added to the lower chamber of a 24-well plate, and 200 μL of cell suspension was added to the Transwell chamber. The Transwell chamber was placed in the 24-well plate using tweezers and incubated in an incubator for 48 hours. 1.4 Fixation, staining, and counting: The chamber was carefully removed with tweezers, and the culture medium was removed. The chamber was washed twice with PBS, and a new 24-well plate was prepared with 700 μL of 4% paraformaldehyde. The chamber was fixed for 30 minutes, then washed twice with PBS. Methanol was added and incubated at room temperature for 20 minutes, then washed twice with PBS. Crystal violet staining was performed for 15 minutes, followed by washing twice with PBS and microscopic counting.

[0029] 2. Experimental results Reference Figure 5 , Figure 5 The Transwell experiment results for MDA-MB-231 cells are shown in the figure. The number of MDA-MB-231 cells that migrated through the chamber after FMVs / Apatinib intervention was significantly less than the Control group without intervention. This indicates that the migration ability of MDA-MB-231 cells after FMVs / Apatinib intervention was significantly decreased. In summary, FMVs / Apatinib can inhibit the migration ability of breast cancer cells.

[0030] Example Six: In vitro detection of FMVs / Apatinib inhibition of breast cancer cell invasion This embodiment observes the invasion ability of cells by Transwell experiment. The Transwell experiment is to place a chamber into a well plate, the chamber contains dense small holes, cell suspension is added to the chamber, and the chamber is placed in a well plate with complete culture medium. Cells can pass through the holes in the chamber by deformation and run to the outside of the chamber where the nutrition is more abundant and adhere to the outside. By staining and counting the cells outside the chamber, the strength of the invasion ability of the cells can be determined.

[0031] 1. Experimental method 1.1 Cell culture: same as the cell culture part in Example Five; 1.2 Matrigel plating: dilute Matrigel matrix (ABW® Matrigengel basement membrane matrix, 0827065) with serum-free cell culture medium to 300 ug / mL, take 100 μL and add uniformly to the upper chamber of the bottom membrane of the Transwell chamber, incubate in a 37°C incubator for 30 minutes to make the matrix gel polymerize into a thin film, and after incubation, remove the excess liquid in the upper chamber; 1.3 Preparation of cell suspension: take MDA-MB-231 cells in logarithmic growth phase, digest and wash, resuspend the cells with serum-free medium, take 3×10 5 cells and add to the upper chamber; 1.4 Cell inoculation: generally add 750 uL of medium containing 10% FBS to the lower chamber of the 24-well plate, and 200 uL of cell suspension to the upper chamber of the Transwell chamber. Place the Transwell chamber in the 24-well plate with forceps and place it in the incubator for 48 h; 1.5 Fixation, staining, and counting: carefully remove the chamber with forceps, remove the culture medium, wash twice with PBS, take a new 24-well plate, add 700 uL of 4% paraformaldehyde, place the chamber in it and fix for 30 min, wash the fixed chamber twice with PBS, add methanol and place it at room temperature for 20 min, then wash twice with PBS, then stain with crystal violet for 15 min, wash twice with PBS, then wipe off the Matrigel in the upper chamber with a cotton swab, and finally examine and count under a microscope.

[0032] 2. Experimental results Reference Figure 6 , Figure 6 The Transwell experiment results of MDA-MB-231 cells are shown in the figure, wherein the number of MDA-MB-231 cells that pass through the chamber after FMVs / Apatinib intervention is significantly less than that of the control group without intervention; this indicates that the invasion ability of MDA-MB-231 cells after FMVs / Apatinib intervention is significantly decreased; in summary, FMVs / Apatinib can inhibit the invasion ability of breast cancer cells.

[0033] Example Seven: Inhibition of angiogenesis by FMVs / Apatinib This example uses a human umbilical vein endothelial cell (HUVEC) tube formation experiment to evaluate the effect of the sample on angiogenesis; this method is an in vitro angiogenesis model established based on the ability of endothelial cells to spontaneously differentiate, migrate and connect to form a three-dimensional capillary-like tube network structure on Matrigel; during the experiment, endothelial cells are cultured on Matrigel with reduced growth factors, and undergo differentiation, directional migration and connection, etc., and finally form a tubular polygonal network; by quantitatively analyzing the number, length and branch points of the tubular structure, the level of angiogenesis can be effectively evaluated.

[0034] 1. Experimental method Before the experiment, the yellow gun head and other consumables were pre-cooled at -20°C overnight; Matrigel was dissolved at 4°C, and 60 μL of Matrigel was inoculated in a 96-well plate, incubated at 37°C for 30 min-2 h, and 5x10 4 HUVECs were inoculated on Matrigel, and then co-incubated with FMVs / Apatinib, etc., with 3 replicate wells for each group; continue to culture in the incubator, observe the tube formation every 1-2 hours, and randomly select three fields under an inverted optical microscope to take pictures, a total of about 6-8 hours; and use ImageJ software to count the tube length, total node number, and total segment length, etc.

[0035] 2. Experimental results Reference Figure 7 , Figure 7 The results of the FMVs / Apatinib HUVECs tube formation experiment, wherein FMVs / Apatinib has an inhibitory effect on HUVECs tube formation, and FMVs / Apatinib has the ability to resist breast cancer.

[0036] Example Eight: Therapeutic effect of FMVs / Apatinib on mouse orthotopic breast cancer 1. Experimental animals SPF level 6-8 week old Balbc mice (female) were purchased from the Experimental Animal Center of Ningxia Medical University, and were fed in the barrier of the school's experimental animal center. The feeding environment is SPF level, and the barrier feeding environment is suitable; the mice drink water and feed are provided by the school's experimental animal center, and all animal experiments are strictly in accordance with the operation procedures of the school's experimental animal center, and have been approved by the Animal Ethics Committee of Ningxia Medical University (China Yinchuan, approval number: IACUC-NYLAC-2024-064).

[0037] Grouping: 5 mice in each group were randomly divided into 2 groups, breast cancer mouse control group, breast cancer mouse FMVs / Apatinib intervention group.

[0038] Modeling method: -5 days of subcutaneous injection of an appropriate amount of MDA-MB-231 cells, and when the tumor volume is greater than or equal to 100mm 3 Intervention.

[0039] Feeding: Both groups of mice were normally fed, and the FMVs / Apatinib group of mice was injected intratumorally with 15 μg of FMVs / Apatinib, once every three days, for a total of four consecutive interventions; among them, Faecalibaculum rodentium from Ningbo Mingzhou Biological Technology Co., Ltd., strain preservation number: JCM30274.

[0040] 2. Experimental method Using the above modeling and feeding methods, two groups of mice were fed for 3 weeks, the mice were weighed every two days and the mouse body weight was recorded, the mouse death was recorded every day, and at the end of the third week, all mice in the breast cancer mouse control group and the breast cancer mouse FMVs / Apatinib group were sacrificed by head and neck separation method, the mice were dissected and the tumor size of the mice was measured by vernier caliper to calculate the tumor volume of the mice.

[0041] 3. Experimental results See Figure 8 , which is the evaluation of the therapeutic effect of F-MVs / Apatinib on mouse orthotopic breast cancer. Figure 8 A is the body weight change curve of each group of mice during the experiment, and there is no significant difference between the F-MVs / Apatinib group and the control group, indicating that the preparation has good biological safety; Figure 8 B is a representative photo of the tumor of each group of mice; Figure 8 C and D are the tumor volume dynamic growth curve and tumor weight statistical results, and the results show that the tumor volume and weight of the F-MVs / Apatinib group of mice are significantly lower than those of the control group, indicating that F-MVs / Apatinib can effectively inhibit the growth of breast cancer tumors in mice.

[0042] Example Nine: Regulation of FMVs / Apatinib on the immune microenvironment of breast cancer mice This embodiment uses flow cytometry to quantitatively analyze the cell phenotype and function after sample processing; the specific method is as follows: first, prepare a single cell suspension, use specific fluorescently labeled antibodies to stain the cell surface or intracellular target, and then use a flow cytometer to quantitatively detect single cells; by analyzing the intensity of a specific fluorescent signal, different cell subpopulations can be identified, protein expression levels can be determined, and their proportion in a heterogeneous cell population can be accurately analyzed; this method has the advantages of high throughput and simultaneous detection of multiple parameters, and is suitable for precise evaluation of cell typing, functional status, and molecular expression levels.

[0043] 1. Experimental method Analysis of immune cells in the immune microenvironment by FCM: According to the design of different models, collect different groups of inguinal lymph nodes at specific times, isolate single nucleus cells from the tissue, and stain with fluorescent antibodies.

[0044] 1.1 Cell surface antigen staining Take the single cell suspension, mix 100 μL of the system per tube with 0.35 μL of the corresponding fluorescently labeled surface antibody, and incubate at 4°C in the dark for 30 minutes. After incubation, add 350 μL of PBS buffer containing 2% FBS and wash, centrifuge at 1500 rpm and 4°C for 5 minutes, then discard the supernatant to remove unbound antibodies.

[0045] 1.2. Intracellular antigen staining (membrane breaking treatment) Add 200 μL of membrane breaking working solution to the cell pellet after surface staining, mix well and resuspend, then incubate at 4°C in the dark for 30 minutes to achieve cell membrane permeability. Then add 350 μL of washing buffer, centrifuge at 1500 rpm and 4°C for 5 minutes, then discard the supernatant.

[0046] 1.3. Intracellular antibody labeling Resuspend the cells with 100 μL of washing buffer, add 1 μL of the corresponding fluorescently labeled intracellular antibody, and incubate at 4°C in the dark for 35 minutes. After incubation, wash and centrifuge with 350 μL of buffer, and discard the supernatant.

[0047] 1.4. Sample preparation and detection Resuspend the final cell pellet with 2% FBS in PBS buffer (250-400 μL), filter through a 200-mesh nylon screen, and then detect on a flow cytometer.

[0048] 1.5. Data analysis Use FlowJo software to analyze the changes in CD8 + T cells and other immune cells in the sample.

[0049] Experimental results See Figure 9IFN-γ + CD8 + The results of flow cytometry analysis of T cell infiltration levels showed that the proportion of IFN-γ + CD8 + T cells in the lymph nodes of the F-MVs / Apatinib treatment group mice was significantly increased compared with the control group.

[0050] See Figure 10 IFN-γ + Foxp3 + The results of flow cytometry analysis of the proportion of Treg cells (CD4 In combination Figure 9 With Figure 10 The results showed that F-MVs / Apatinib could simultaneously promote the activation of effector CD8 + T cells in the tumor-draining lymph nodes and reduce the proportion of immunosuppressive Treg cells, indicating that the preparation could exert an anti-tumor effect by bidirectional regulation of the tumor immune microenvironment.

[0051] Example Ten: Safety Evaluation of FMVs / Apatinib Hematoxylin-eosin staining, referred to as HE staining. Hematoxylin (H) is an alkaline dye that can stain cell nuclei and intracellular ribosomes blue-purple. Structures stained by alkaline dyes have basophilic properties. Eosin is an acid dye that can stain cytoplasm red or light red. Structures stained by acid dyes have acidophilic properties. In this embodiment, HE staining experiments were performed to observe the morphological characteristics such as tissue structure, tissue type, and cell level of the heart, liver, spleen, lung, and kidney of the three groups of mice.

[0052] The source of experimental animals and the modeling method were exactly the same as in Example Eight, and will not be repeated here.

[0053] 1. Experimental method Two groups of mice were raised for three weeks, and at the end of the third week, all mice in the breast cancer control group and the breast cancer FMVs / Apatinib group were sacrificed by head and neck separation method, and the heart, liver, spleen, lung, and kidney of the two groups of mice were dissected.

[0054] 1.1 Fixation: The heart, liver, spleen, lung, and kidney of the mice were washed with PBS, and then placed in 4% paraformaldehyde at 4°C for 48 hours to prevent autolysis or bacterial decomposition after cell death, thereby maintaining the original morphological structure of the cells.

[0055] 1.2 Dehydration, embedding and sectioning: Put the fixed tumor tissue into the embedding box, dehydrate it with low to high gradient ethanol, soak it in xylene for 2 hours at 56℃ paraffin. After the tissue is fixed, section it with a thickness of 3.5 μm.

[0056] 1.3 De-paraffinization and hydration: Bake the slide in an oven at 60° for 2 hours, then de-paraffinize and hydrate it. The procedure is as follows: xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 2 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and tap water for washing.

[0057] 1.4 Staining: Stain the tissue section with hematoxylin and eosin; specifically, dry the water on the slide, add hematoxylin staining solution, stain for 5 min, wash away the hematoxylin staining solution with running water, observe the staining degree under a microscope, differentiate with 1% hydrochloric acid alcohol for 1-3 s, wash with water, soak in tap water for 5-10 min for blue return, observe the color under a microscope, if the color is not deep enough, directly add hematoxylin staining solution for 1 min, then differentiate and wash with water immediately; if it is too deep, appropriately increase the differentiation time, then add 0.5% eosin staining solution, stain for 10-15 s, and observe under a microscope after washing with distilled water.

[0058] 1.5 Dehydration, mounting and microscopic examination: Dehydrate the slide by soaking it in the following reagents in sequence, 50% ethanol for 1 min, 75% ethanol for 1 min, 80% ethanol for 1 min, 85% ethanol for 1 min, 90% ethanol for 1 min, 95% ethanol for 1 min, xylene II for 2 min, and xylene I for 2 min. Take the tissue section out of the last jar, dry it, mount it with neutral resin, dry it in a fume hood, and then examine it under a microscope.

[0059] 2. Experimental results See Figure 11 , Figure 11 The results of the histological changes of the heart, liver, spleen, lung and kidney of the breast cancer mice; from Figure 11 As can be seen, the tissues of the FMVs / Apatinib group of breast cancer mice showed no obvious abnormalities compared with the control group, indicating that FMVs / Apatinib intervention in breast cancer mice does not cause damage to the heart, liver, spleen, lung and kidney, and FMVs / Apatinib has certain safety when used for treating or relieving breast cancer.

Claims

1. An engineered bacterial membrane vesicle loaded with apatinib, characterized in that, The engineered bacterial membrane vesicles take rod-shaped bacteria-derived membrane vesicles as carriers and internally encapsulate the chemotherapeutic drug apatinib.

2. The engineered bacterial membrane vesicle of claim 1, wherein, The mass ratio of the rod-shaped bacteria-derived membrane vesicles to apatinib is 2:

1.

3. The method of producing engineered bacterial membrane vesicles according to any one of claims 1-2, wherein, The method comprises the following steps: extracting vesicles: after collecting the supernatant of rod-shaped bacteria by gradient centrifugation, the bacterial supernatant is centrifuged by ultracentrifugation to obtain the precipitate, i.e. bacterial outer membrane vesicles, and resuspended with PBS; drug loading: the resuspended bacterial outer membrane vesicles are mixed with apatinib at a specific mass ratio in PBS, incubated at 37℃ for 4 hours, and then subjected to removal of free apatinib using a 100kD ultrafiltration membrane and concentration, and then washed several times with PBS to obtain apatinib-loaded engineered bacterial membrane vesicles.

4. Use of the apatinib-loaded engineered bacterial membrane vesicles according to any one of claims 1-2 in the preparation of a medicament or a pharmaceutical composition for treating or alleviating breast cancer.

5. Use according to claim 4, characterized in that, The engineered bacterial membrane vesicles can play a role by inhibiting tumor cell proliferation, migration and invasion, promoting tumor cell apoptosis, and remodeling the tumor immune microenvironment.

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