Bionic intestinal bacteria, and preparation method and application thereof

Abstract

The application discloses a kind of bionic intestinal bacteria and its preparation method and application, wherein the bionic intestinal bacteria include: bionic intestinal bacteria shell and intestinal bacterial metabolite loaded in the bionic intestinal bacteria shell;The bionic intestinal bacteria shell includes: outer membrane vesicle of intestinal bacterial source;The intestinal bacterial metabolite includes: one or more of short-chain fatty acid, oxidized trimethylamine, inosine, indole-3-methylaldehyde.The bionic intestinal bacteria of the application not only retains the immune regulation function of intestinal bacteria, can effectively regulate tumor immunosuppressive microenvironment into immune activation microenvironment, improve the effect of antitumor immunotherapy, but also can reduce the risk of intestinal flora direct administration.In addition, the preparation method of the bionic intestinal bacteria is simple, and has good biological safety at the same time, has potential application prospect in tumor treatment and industrial popularization.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a biomimetic gut bacterium, its preparation method, and its application. Background Technology

[0002] During the occurrence and development of tumors, tumors have evolved various mechanisms to evade tumor immune surveillance and suppress anti-tumor immune responses. The main mechanisms of tumor immune evasion involve the immune checkpoint pathway. Key immune checkpoints include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 / 2 and its ligands (PD-L1 / PD-L2 / PD-1). Tumor therapy targeting immune checkpoints is called immune checkpoint therapy (ICT). ICT aims to block inhibitory signals that activate T cells, promoting T cell recognition and killing of tumor cells. This treatment has improved the survival rate of patients with advanced malignant melanoma and advanced non-small cell lung cancer several times over. The clinical success of ICT has revolutionized the field of cancer immunotherapy and made it a pillar of cancer treatment alongside traditional therapies such as surgery, chemotherapy, and radiotherapy. However, it is worth noting that ICT is less effective against immunosuppressive "cold" tumors (such as breast cancer, pancreatic cancer, and glioma). Because ICT is primarily mediated by T cells, and "cold" tumors lack toxic T lymphocyte infiltration, the low number of toxic T cells is the main reason for the poor efficacy of ICT in "cold" tumors. Therefore, finding effective methods to enhance toxic T lymphocyte infiltration is key to improving the therapeutic effect of ICT in "cold" tumors.

[0003] In recent years, scientists have discovered in tumor immunology studies of mice and human patients that host gut bacteria have immunomodulatory effects on various solid tumors. Bifidobacteria-generated immune signals can stably regulate dendritic cell activation through the STING pathway, promoting antigen presentation and thus improving the effector function of cytotoxic T lymphocytes. Simultaneously, they can recruit cytotoxic T lymphocytes to infiltrate the tumor microenvironment. Combining Bifidobacteria with PD-L1 / PD-1 immune checkpoint therapy enhances the efficacy of PD-L1 / PD-1 therapy and effectively inhibits tumor proliferation. The enrichment of three bacterial groups—Xanthomonas pseudoepiploicum, Polysaccharidobacterium saccharidosis, and Streptomyces—within pancreatic cancer cells facilitates the recruitment and activation of cytotoxic T lymphocytes. In conclusion, the gut microbiota, through its own immunogenicity and the immune-activating effects of its metabolites, can successfully enhance the infiltration of cytotoxic T lymphocytes in "cold" tumors, representing an effective means of enhancing the efficacy of ICT (Invasive Clinical Trial) therapy. However, the tumor delivery of gut bacteria has the following drawbacks: (1) leakage risk: bacteria may leak into normal tissues, causing organ infection and sepsis; (2) poor operational flexibility; (3) the metabolites cannot be quantified and may contain other metabolites that are not conducive to tumor suppression; (4) high concentrations of metabolites are distributed throughout the body, inducing other diseases (such as TMAO, which is a high-risk factor for cardiovascular disease). These defects limit the application of gut bacteria in anti-tumor therapy. Therefore, it is necessary to find effective means to preserve the immunomodulatory function of gut bacteria while making up for the shortcomings of gut bacteria tumor delivery, which is of great significance for tumor ICT. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a biomimetic gut bacteria, its preparation method and application, in order to solve the problems of leakage risk and inability to quantify when using gut bacteria for tumor immunotherapy.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, a biomimetic gut bacterium is provided, the biomimetic gut bacterium comprising: a biomimetic gut bacterium shell and gut bacterium metabolites contained in the biomimetic gut bacterium shell;

[0007] The biomimetic gut bacteria shell includes: an outer membrane vesicle derived from gut bacteria;

[0008] The intestinal bacterial metabolites include one or more of the following: short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde.

[0009] In a preferred embodiment, the outer membrane vesicles derived from intestinal bacteria are outer membrane vesicles derived from Gram-negative intestinal bacteria.

[0010] In a preferred embodiment, the intestinal bacteria-derived outer membrane vesicles are derived from Escherichia coli.

[0011] In a preferred embodiment, the Escherichia coli is one or more of the strains BL21, Nissle1917, and DH-5α.

[0012] In a preferred embodiment, the mass ratio of the outer membrane vesicles derived from intestinal bacteria to the metabolites of intestinal bacteria is (1-50):(100-10000).

[0013] In a preferred embodiment, the biomimetic intestinal bacterial shell further includes a tumor-homing cell membrane; the tumor-homing cell membrane is one or more of a tumor cell membrane, a macrophage membrane, or a mesenchymal stem cell membrane.

[0014] In a preferred embodiment, the mass ratio of the outer membrane vesicles derived from intestinal bacteria, the tumor-homing cell membrane, and the metabolites of intestinal bacteria is (1-50):(0.1-5):(100-10000).

[0015] Secondly, a method for preparing biomimetic gut bacteria as described in the first aspect is provided, comprising the steps of:

[0016] The components of the biomimetic gut bacteria shell and gut bacteria metabolites are mixed and sonicated at 30-200W for 1-10 minutes to obtain a biomimetic gut bacteria immunomodulator system.

[0017] The components of the biomimetic gut bacteria shell include: outer membrane vesicles derived from gut bacteria;

[0018] The intestinal bacterial metabolites include one or more of the following: short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde.

[0019] In a preferred embodiment, the biomimetic intestinal bacterial shell further comprises: a tumor-homing cell membrane; the tumor-homing cell membrane is one or more of tumor cell membranes, macrophage membranes, or mesenchymal stem cell membranes.

[0020] Thirdly, the application of a biomimetic gut bacteria prepared by the method described in the first aspect or the method described in the second aspect in the preparation of tumor therapeutic drugs.

[0021] Beneficial Effects: This invention provides a biomimetic gut bacteria, its preparation method, and its application. The biomimetic gut bacteria are formed by the ultrasonic self-assembly of outer membrane vesicles and metabolites within gut bacteria, which possess tumor-enhancing immune-boosting properties. Experimental results show that the biomimetic gut bacteria constructed in this invention have uniform particle size and good stability, can regulate the tumor immunosuppressive microenvironment, and enhance intratumoral CD8 uptake. +This invention increases T-cell infiltration and improves the efficacy of immune checkpoint inhibitor therapy. When the biomimetic gut bacteria shell includes a tumor-homing cell membrane, the biomimetic gut bacteria of this invention can also specifically target tumor sites. This invention overcomes the risks of toxicity and induction of other diseases associated with direct delivery of gut bacteria and their metabolites into the bloodstream. Furthermore, the biomimetic gut bacteria provided by this invention have a simple synthesis process and good biosafety, showing potential applications in tumor treatment and industrial promotion. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the synthesis of ROMV / TMAO in Example 1.

[0023] Figure 2 These are the particle size statistics of OMV, ROMV, and ROMV / TMAO in Example 1, the characterization results of ROMV / TMAO in Example 2, the in vitro stability study results of ROMV / TMAO in Example 3, and the compositional analysis results of ROMV in Example 4; where a is the particle size statistics of OMV, ROMV, and ROMV / TMAO; b is the result of the change in hydrated particle size of ROMV / TMAO over time at 37℃; c is the result of the change in monodispersity of ROMV / TMAO over time at 37℃; d is the transmission electron microscopy characterization results of OMV, ROMV, and ROMV / TMAO, scale bar: 100nm; e is the protein bands of RV, OMV, and ROMV analyzed by SDS-PAGE; f is the fluorescence colocalization image of RV and OMV with ROMV.

[0024] Figure 3These are the in vivo targeting results of ROMV / TMAO in Example 5. G1 refers to the DiR group, G2 to the OMV-DiR group, G3 to the RV-DiR group, G4 to the ROMV-DiR group, and G5 to the ROMV / TMAO-DiR group. Specifically, a) shows the distribution of DiR, OMV-DiR, RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR in 4T1 breast cancer-bearing mice at different time points after administration; b) shows the distribution of DiR, OMV-DiR, RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR in 4T1 mice 24 hours after administration. Distribution fluorescence imaging within the tumor; c is the quantitative result of distribution fluorescence imaging of DiR, OMV-DiR, RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR within the 4T1 tumor 24 h after administration; d is the distribution fluorescence imaging of DiR, OMV-DiR, RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR within the tissue 24 h after administration; e is the quantitative result of distribution fluorescence imaging of DiR, OMV-DiR, RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR within the tissue 24 h after administration.

[0025] Figure 4 These are the results of the in vivo toxicity study of ROMV / TMAO in Example 6; where a is a schematic diagram of the administration of ROMV / TMAO in the in vivo toxicity study; b is the results of the study on the effect of different doses of ROMV / TMAO on the survival rate of mice; c is the results of the effect of 5 μg dose of ROMV / TMAO on the formation of atherosclerotic plaques in mice; and d is the effect of different doses of ROMV / TMAO on the liver and kidney function of mice.

[0026] Figure 5 This is the result of the in vivo tumor inhibition effect study of ROMV / TMAO in 4T1 breast cancer-bearing mice in Example 7. G1 refers to the PBS group, G2 refers to the TMAO group, G3 refers to the ROMV group, and G4 refers to the ROMV / TMAO group. Among them, a is the tumor volume curve of mice in different treatment groups; b is the statistical result of tumor weight of mice in different treatment groups; c is the H&E and TUNEL staining result of tumors in mice in different treatment groups.

[0027] Figure 6This refers to the results of the study on ROMV / TMAO remodeling of the tumor immune microenvironment in Example 8. G1 refers to the PBS group, G2 to the TMAO group, G3 to the ROMV group, and G4 to the ROMV / TMAO group. Specifically, a) is the flow cytometry analysis result of tumor macrophages in mice of different treatment groups; b) is the percentage of M1 macrophages in tumors of mice of different treatment groups; c) is the percentage of M1 / M2 macrophages in tumors of mice of different treatment groups; d) is the flow cytometry analysis result of tumor T cells in mice of different treatment groups; and e) is the percentage of CD45+ cells in tumors of mice of different treatment groups. + CD3 + CD8 + The percentage of T cells.

[0028] Figure 7 This refers to the results of ROMV / TMAO enhanced α-PD-L1 treatment of 4T1 tumors in Example 9. G1 refers to the PBS group, G2 refers to the α-PD-L1 group, G3 refers to the ROMV / TMAO group, and G4 refers to the ROMV / TMAO+α-PD-L1 group. Among them, a is a curve of tumor volume of a single mouse in different treatment groups; b is the statistical results of tumor weight of mice in different treatment groups; c is a representative photograph of tumor-bearing mice in different treatment groups at the treatment endpoint. Detailed Implementation

[0029] This invention provides a biomimetic intestinal bacterium, its preparation method, and its application. To make the purpose, technical solution, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0030] This invention provides a biomimetic gut bacterium, which includes: a biomimetic gut bacterium shell and gut bacterium metabolites encapsulated in the biomimetic gut bacterium shell;

[0031] The biomimetic gut bacteria shell includes: an outer membrane vesicle derived from gut bacteria;

[0032] The intestinal bacterial metabolites include one or more of the following: short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde.

[0033] Specifically, inspired by the role of gut bacteria in reshaping the immune microenvironment and enhancing anti-tumor immune responses within tumors, the inventors self-assembled outer membrane vesicles and metabolites (such as short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde) from gut bacteria to form biomimetic gut bacteria. These biomimetic gut bacteria retain the immunomodulatory functions of gut bacteria, effectively transforming the tumor immunosuppressive microenvironment into an immune-activating microenvironment, thus improving the efficacy of anti-tumor immunotherapy, while also reducing the risks associated with direct administration of gut microbiota.

[0034] Among them, the outer membrane vesicles derived from intestinal bacteria have an immunostimulatory effect, which can promote the polarization of macrophage M1 in the tumor microenvironment and promote intratumoral CD8. + T-cell infiltration.

[0035] In one embodiment, the intestinal bacterial metabolite is trimethylamine oxide (TMAO). TMAO can induce pyroptosis of tumor cells through endoplasmic reticulum stress and increase the infiltration of toxic T lymphocytes.

[0036] In one embodiment, the outer membrane vesicles derived from intestinal bacteria are outer membrane vesicles derived from Gram-negative intestinal bacteria.

[0037] In one embodiment, the intestinal bacteria-derived outer membrane vesicles are Escherichia coli-derived outer membrane vesicles.

[0038] In one embodiment, the Escherichia coli is one or more of BL21, Nissle1917, and DH-5α strains.

[0039] In one embodiment, the mass ratio of the outer membrane vesicles derived from intestinal bacteria to the metabolites of intestinal bacteria is (1-50):(100-10000); preferably, the mass ratio is (10-30):(2000-5000); more preferably, the mass ratio is (15-25):(3000-3500); and most preferably, the mass ratio is 18:3200. At these preferred mass ratios, the biomimetic intestinal bacteria exhibit a hydrated particle size of less than 100 nm and good monodispersity, which is beneficial for drug delivery.

[0040] In one embodiment, the biomimetic gut bacteria shell further includes a tumor-homing cell membrane; the tumor-homing cell membrane is one or more of a tumor cell membrane, a macrophage membrane, or a mesenchymal stem cell membrane.

[0041] In one embodiment, the mass ratio of the intestinal bacteria-derived outer membrane vesicles, the tumor-homing cell membrane, and the intestinal bacteria metabolites is (1-50):(0.1-5):(100-10000); preferably, the mass ratio is (10-30):(0.5-2):(2000-5000); more preferably, the mass ratio is (15-25):(0.8-1.2):(3000-3500); and most preferably, the mass ratio is 18:1:3200. At these preferred mass ratios, the biomimetic intestinal bacteria have a hydrated particle size of less than 100 nm and good monodispersity, which is beneficial for drug delivery.

[0042] In one embodiment, the biomimetic gut bacteria further includes a fluorescent marker.

[0043] In one embodiment, the fluorescent marker is selected from one or more of 1,1-bis(octadecyl)-3,3,3,3-tetramethylindocyanine perchlorate (DiI), 3,3'-bis(octadecyl)oxocarbazone perchlorate (DiO), and 1,1'-dioctyl-3,3,3',3'-tetramethylindocarbonylcyanine perchlorate (DiR).

[0044] In one embodiment, the hydrated particle size of the biomimetic gut bacteria ranges from 10 to 100 nm.

[0045] This invention provides a method for preparing the biomimetic gut bacteria as described above, comprising the following steps:

[0046] The components of the biomimetic gut bacteria shell and gut bacteria metabolites are mixed and sonicated at 30-200W for 1-10 minutes to obtain a biomimetic gut bacteria immunomodulator system.

[0047] The components of the biomimetic gut bacteria shell include: outer membrane vesicles derived from gut bacteria;

[0048] The intestinal bacterial metabolites include one or more of the following: short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde.

[0049] In one embodiment, the biomimetic gut bacterial shell further comprises: a tumor-homing cell membrane; the tumor-homing cell membrane is one or more of a tumor cell membrane, a macrophage membrane, or a mesenchymal stem cell membrane.

[0050] This invention provides an application of the biomimetic gut bacteria prepared by the above-described method in the preparation of tumor therapeutic drugs.

[0051] In one embodiment, the tumor treatment drug is used to treat common solid tumors, including one or more of lung cancer, gastric cancer, pancreatic cancer, colon cancer, uterine cancer, rectal cancer, pharyngeal cancer, and breast cancer; preferably, the solid tumor is one or more of breast cancer, lung cancer, and pancreatic cancer.

[0052] The present invention will be further described below through specific embodiments.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0054] Explanation of abbreviations or terms:

[0055] OMV: Bacterial extracellular vesicles.

[0056] RV: Macrophage membrane.

[0057] TMAO: Trimethylamine oxide.

[0058] ROMV: Bionic intestinal bacterial shell.

[0059] ROMV / TMAO: Biomimetic gut bacteria that can secrete TMAO.

[0060] In the embodiments, since the mass or concentration of OMV, RV and ROMV cannot be directly measured, they are all based on the protein mass or concentration, that is, the mass or concentration of protein is used as the mass or concentration of OMV, RV and ROMV.

[0061] Example 1: Preparation of biomimetic gut bacteria (ROMV / TMAO) capable of secreting TMAO

[0062] (1) Extraction of OMV

[0063] The BL21 strain was cultured in LB medium and incubated at 37°C on a rotary shaker (200 rpm) for 6 hours. The bacterial culture was then diluted 1:100 with fresh LB medium. When the OD600 value of the bacterial suspension reached approximately 1.0, the suspension was centrifuged at 8000 rpm for 30 minutes at 4°C to obtain the supernatant. The supernatant was filtered through a 0.22 μm sterile vacuum filter. The filtrate was concentrated and further centrifuged at 120,000 g for 2 hours at 4°C. Finally, the precipitate was resuspended in PBS (10 mM, pH 7.4). The protein concentration of the OMV solution was detected using a microBCA protein assay kit (Thermo Fisher Scientific). All samples were stored at -80°C for further experiments.

[0064] (2) RV extraction

[0065] The macrophage membrane-RAW264.7 cell membrane carrier (RV) was separated according to the instructions of the membrane protein extraction kit (Shanghai Beyotime Biotechnology Co., Ltd.). RAW264.7 cells were collected, centrifuged at 1200 rpm for 3 minutes at 4°C, and washed with PBS (10 mM, pH 7.4). The cells were resuspended in a mixture of reagent A from the membrane protein extraction kit and benzoyl fluoride (PMSF) solution (1 mM). The RAW264.7 cells were then placed on ice for 15 minutes. Cells were then lysed using a freeze-thaw method, followed by centrifugation at 6000 rpm for 10 minutes, and the supernatant was used for further experiments. The supernatant was centrifuged at 12000 rpm for 30 minutes to obtain a precipitate, which was resuspended in PBS (10 mM, pH 7.4). The protein concentration of the OMV solution was detected using the MicroBCA Protein Detection Kit (Thermo Fisher Scientific). All samples were stored at -80°C for further experiments.

[0066] (3) Preparation of biomimetic gut bacteria (ROMV / TMAO)

[0067] A biomimetic gut bacteria (ROMV / TMAO) capable of secreting the metabolite TMAO (Sigma-Aldrich) was prepared using an ultrasonic assembly method. Specifically, macrophage membranes (RV), bacterial extracellular vesicles (OMV), and trimethylamine oxide (TMAO) were prepared in different mass ratios for ROMV / TMAO. The ultrasonic power was 120W, and the ultrasonic time was 5 min, resulting in the biomimetic gut bacteria (ROMV / TMAO). A schematic diagram of the ROMV / TMAO synthesis is shown below. Figure 1As shown. The hydrated particle size and PDI of the prepared OMV, ROMV, and ROMV / TMAO at different mass ratios were determined using a Malvern particle size analyzer (model: nano series, manufacturer: Malvern). See details below. Figure 2 As shown in a and Table 1.

[0068] Table 1. Hydration particle size and PDI of OMV, ROMV, and ROMV / TMAO at different mass ratios.

[0069]

[0070] As shown in Table 1, the mass ratio of OMV, RV and TMAO has a significant impact on the hydrated particle size and PDI. When OMV:RV:TMAO = 18:1:3200, the hydrated particle size of ROMV / TMAO is less than 100 nm and has good monodispersity. This mass ratio is the best. Unless otherwise specified, this mass ratio will be used to prepare ROMV / TMAO in subsequent experiments.

[0071] Example 2: Characterization of biomimetic gut bacteria (ROMV / TMAO)

[0072] The OMV, ROMV, and ROMV / TMAO (OMV:RV:TMAO = 18:1:3200) solutions prepared in Example 1 were dropped onto a copper grid containing a Fanghua film and negatively stained with phosphotungstic acid. The morphology was then characterized using a transmission electron microscope (HT7700, Hitachi). The results are as follows: Figure 2 As shown in d. From Figure 2 As shown in Figure d, OMV, ROMV, and ROMV / TMAO are all uniformly spherical. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) showed that the encapsulation efficiency and drug loading of TMAO in the ROMV / TMAO prepared in Example 1 were 33.78 ± 2.97% and 98.26 ± 0.15%, respectively.

[0073] Example 3: Stability Study of Bionic Gut Bacteria (ROMV / TMAO)

[0074] To investigate the stability of the biomimetic gut bacteria (ROMV / TMAO), the biomimetic gut bacteria (ROMV / TMAO) prepared in Example 1 were dispersed in PBS and incubated at 37°C for 72 hours. Samples were taken at different time points, and changes in particle size and PDI were detected using a Malvern laser particle size analyzer. The detection results are as follows: Figure 2 As shown in b and c. From Figure 2 As shown in b and c, the particle size of ROMV / TMAO in PBS remained basically unchanged within 72 h under 37℃ conditions, and it also exhibited good monodispersity, indicating that ROMV / TMAO has good stability.

[0075] Example 4: Compositional Analysis of Bionic Intestinal Bacterial Capsid (ROMV)

[0076] To verify whether OMV and RV could fuse in Example 1, OMV was stained with 1,1-dioctadecyl-3,3,3,3-tetramethylindocyanine perchlorate (DiI, red, brand: Aladdin), and RV was stained with 3,3'-dioctadecyloxocarbazone perchlorate (DiO, green, brand: Solarbio). The final concentration of both DiI and DiO was 10 μM. Then, OMV-DiI and RV-DiO (mass ratio 18:1, ultrasonic power 120 W, ultrasonic time 5 min) were ultrasonically assembled to form a biomimetic intestinal bacterial shell (ROMV), and imaged under a confocal microscope. The results are as follows. Figure 2 As shown in f. From Figure 2 As can be seen from f, the fluorescence of OMV and RV is co-localized in ROMV, indicating that ROMV is composed of RV and OMV.

[0077] Furthermore, to demonstrate that the biomimetic gut bacterial capsid (ROMV) is formed by the fusion of bacterial extracellular vesicles (OMV) and macrophage membranes (RV), polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze membrane surface proteins. Specifically, 10 μL of bacterial extracellular vesicles (OMV), macrophage membranes (RV), and biomimetic gut bacterial capsid (ROMV) were added to three electrophoresis channels respectively. After electrophoresis, Coomassie brilliant blue solution was added for staining and incubation for 0.5–2 h. After washing with pure water 3–5 times, images were taken. The results are as follows: Figure 2 As shown in e. From Figure 2 As can be seen from e, ROMV has all the protein bands of RV and OMV.

[0078] Example 5: In vivo targeted study of biomimetic gut bacteria (ROMV / TMAO)

[0079] (1) Construction of a 4T1 breast cancer tumor model

[0080] Balb / c female mice (6–8 weeks old, 20–25 g) were provided by Guangdong Yaokang Biotechnology Co., Ltd. All mice were housed under 12 / 12h dark / light cycles and SPF (ambient temperature 25°C and humidity 55%) conditions. The animal research protocol was reviewed and approved by the Animal Care and Use Committee of Southern University of Science and Technology.

[0081] The number of 4T1 cells subcutaneously implanted in each mouse was 2 × 10⁶. 5 Cells were dispersed in 100 μL of PBS. The tumor volume reached 100 mm. 3 Within approximately 10 minutes, in vivo targeted studies of liposomes can be conducted.

[0082] (2) In vivo targeting studies of ROMV / TMAO

[0083] After establishing a 4T1 breast cancer tumor model, the model was divided into four groups: DiR (1,1'-dioctyl-3,3,3',3'-tetramethylindolecarbonylcyanine perchlorate, brand: Aladdin), OMV-DiR, RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR, with five mice in each group. OMV-DiR represents OMV labeled with DiR, RV-DiR represents macrophage membrane RV labeled with DiR, ROMV-DiR represents ROMV labeled with DiR, and ROMV / TMAO-DiR represents ROMV / TMAO labeled with DiR. Mice were injected intravenously with 200 μL DiR, OMV-DiR, RV-DiR, ROMV-DiR, or ROMV / TMAO-DiR (500 μg / kg DiR), respectively. In vivo imaging was performed on the mice at different time points after drug administration to observe the drug distribution at the tumor sites. Twenty-four hours after drug administration, the mice were euthanized, and their heart, liver, spleen, lungs, kidneys, and tumor tissues were removed for fluorescence signal analysis. The results are as follows: Figure 3 As shown. Figure 3 In the table, G1 refers to the DiR group, G2 refers to the OMV-DiR group, G3 refers to the RV-DiR group, G4 refers to the ROMV-DiR group, and G5 refers to the ROMV / TMAO-DiR group.

[0084] Depend on Figure 3 As shown in Figure a, the fluorescence intensity at the tumor site gradually increased with increasing administration time. After 24 hours of administration, the fluorescence intensity at the tumor site in the RV-DiR (G3), ROMV-DiR (G4), and ROMV / TMAO-DiR (G5) groups was higher than that in the DiR (G1) and OMV-DiR (G2) groups. Figure 3 As shown in b, the fluorescence imaging results of isolated mouse tumor tissue are consistent with those of in vivo imaging. Figure 3 As shown in Figure c, the accumulation levels of RV-DiR, ROMV-DiR, and ROMV / TMAO-DiR at the tumor site were significantly different from those in the control group, indicating that the macrophage membrane (RV) confers tumor targeting specificity to ROMV / TMAO. Figure 3 As shown in d and e, ROMV / TMAO is more distributed in the liver and spleen, indicating that it may be metabolized by the liver.

[0085] Example 6: In vivo toxicity study of biomimetic gut bacteria (ROMV / TMAO)

[0086] According to literature reports, 5 μg of ROMV administered via tail vein injection into mice exhibits significant toxicity. TMAO also carries a risk of inducing atherosclerosis. Therefore, this invention evaluated the in vivo toxicity of different doses of ROMV / TMAO.

[0087] Female Balb / c mice (6–8 weeks old, 20–25 g) were randomly divided into 5 groups of 5 mice each. Each group was injected via tail vein with different doses of ROMV / TMAO (0, 2, 3, 4, 5 μg OMV) prepared in Example 1, on days 0, 2, 4, 6, and 8, respectively, and the survival rate of the mice was monitored. On day 10, the mice were euthanized, and blood samples were collected for liver and kidney function analysis. Simultaneously, the aortic root was extracted, and sections of the aortic root were stained with H&E, Masson's red, and Oil Red to observe the atherosclerosis. The results are as follows: Figure 4 As shown.

[0088] Depend on Figure 4 As shown in sections b to d, ROMV / TMAO at a high dose of 5 μg OMV did not cause death in mice, and no atherosclerotic plaques were found at the aortic root. The liver and kidney functions of the mice were not significantly different from the control group and remained within the normal range. These results demonstrate the high biocompatibility of the biomimetic gut bacteria.

[0089] Example 7: In vivo antitumor effect study of biomimetic gut bacteria ROMV / TMAO

[0090] The 4T1 breast cancer model was constructed in the same way as in Example 5 (1), when the tumor volume reached 50-100 mm. 3 At approximately 14 days post-treatment, the model was divided into four groups: G1 (PBS), G2 (TMAO), G3 (ROMV), and G4 (ROMV / TMAO), with five mice in each group. Mice were administered the drug via tail vein (G1, G3, and G4, OMV: 5 μg / mouse, TMAO: 0.25 mg / g) or intraperitoneally (G2, TMAO: 0.25 mg / g), once every other day for a total of five administrations. Tumor volume was monitored. Fourteen days after the first administration, the mice were euthanized, and the tumors were removed and weighed. The results are as follows: Figure 5 As shown.

[0091] Depend on Figure 5 As shown in a and b, the tumor volume and weight of mice in the G2 (TMAO), G3 (ROMV), and G4 (ROMV / TMAO) groups were significantly lower than those in the G1 (PBS) group, and the G4 (ROMV / TMAO) group showed the best tumor-suppressing effect, which was significantly different from the other three groups. Furthermore, from Figure 5 As shown in Figure c, the G4 (ROMV / TMAO) group exhibited more significant tumor cell damage and a higher number of apoptotic cells. These results demonstrate that biomimetic gut bacteria (ROMV / TMAO) can effectively inhibit the growth of 4T1 tumors.

[0092] Example 8: The effect of biomimetic gut bacteria (ROMV / TMAO) on the tumor immune microenvironment. After weighing the tumor in Example 7, a portion of the tumor tissue was taken for analysis of the tumor immune microenvironment.

[0093] The study investigated the CD8+ in 4T1 tumors of mice treated with the biomimetic gut bacteria ROMV / TMAO using flow cytometry (model: BD FACSCanto SORP, manufacturer: BD). + The study investigated changes in T cells and macrophages, and observed the effects of ROMV / TMAO on the tumor immune microenvironment. Specifically, tumor tissue was cut into a paste-like state using ophthalmic scissors, collagenase (STEMCELL, 07912) was added, and the tissue was digested at 37°C for 30 minutes. The resulting suspension was then passed through a 70μm cell sieve to obtain a single-cell suspension. Then, the cells were incubated with the following antibodies: FITC-Anti-CD45 (BD Pharmingen, 553079), BB700-anti-CD3 (BD ​​Pharmingen, 566494), BV421-anti-CD4 (BD Pharmingen, 562891), BV510-anti-CD8 (BD Pharmingen, 563068), APC-Cy7-Fixable Viability Stain 780 (BD Pharmingen, 565388), PE-anti-CD11b (BDPharmingen, UF557397S), BV510-anti-GR-1 (BD Pharmingen, 563040), BV421-anti-F4 / 80 (BD Pharmingen, 565411), and PE-Cy7-anti-CD86 (BD Pharmingen, 563040). Pharmingen, 560582), AF647-anti-CD206 (BD Pharmingen, 568808). After staining, flow cytometry was used to analyze the proportion of M1 and M2 cells in macrophages, as well as CD8+. + The proportion of T cells, the results are as follows Figure 6 As shown.

[0094] Depend on Figure 6 As shown in a-c, the proportion of M1 macrophages in the tumor was significantly higher in the G4 (ROMV / TMAO) group than in other groups, while the proportion of M2 macrophages was significantly lower in the G1 (PBS) group. This indicates that ROMV / TMAO can promote the polarization of tumor macrophages from M2 to M1. Meanwhile, from Figure 6 As shown in d and e, CD8+ is present in the tumors of the G4 (ROMV / TMAO) group. +The proportion of T cells was also significantly higher than that in the control group. These results indicate that the biomimetic gut bacteria (ROMV / TMAO) have similar immune activation effects to gut bacteria and can regulate the tumor immune microenvironment.

[0095] Example 9: Effects of biomimetic gut bacteria (ROMV / TMAO) on α-PD-L1 therapy for 4T1 tumors

[0096] The 4T1 breast cancer model was constructed in the same way as in Example 5 (1), when the tumor volume reached 50-100 mm. 3 At approximately 14 days post-treatment, the model was divided into four groups: G1 (PBS), G2 (α-PD-L1), G3 (ROMV), and G4 (ROMV / TMAO+α-PD-L1), with six mice in each group. Mice were administered the drug via tail vein (G1, G3, and G4, OMV: 5 μg / mouse, TMAO: 0.25 mg / g) or intraperitoneally (G2, α-PD-L1: 100 μg / mouse), once every other day for a total of five administrations. Tumor volume was monitored. Fourteen days after the first administration, the mice were euthanized, and the tumors were removed and weighed. The results are as follows: Figure 7 As shown.

[0097] Depend on Figure 7 As shown in a and b, the combination of biomimetic gut bacteria (ROMV / TMAO) and the immune checkpoint inhibitor α-PD-L1 significantly delayed tumor growth and the tumor weight was also lower than that of other groups. Figure 7 Image c shows a photograph of tumor-bearing mice after treatment, visually reflecting the tumor-suppressing effect in each group. Combined with... Figure 6 The results indicate that the biomimetic gut bacteria ROMV / TMAO increases intratumoral CD8 levels by activating the tumor immune microenvironment. + The proportion of T cells, thereby enhancing the efficacy of α-PD-L1 therapy for 4T1 tumors.

[0098] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A biomimetic gut bacterium, characterized in that, The biomimetic gut bacteria include: a biomimetic gut bacteria shell and gut bacteria metabolites encapsulated in the biomimetic gut bacteria shell; The biomimetic gut bacteria shell includes: an outer membrane vesicle derived from gut bacteria and a cell membrane capable of tumor homing; The intestinal bacterial metabolites include: trimethylamine oxide; The mass ratio of the outer membrane vesicles derived from intestinal bacteria, the tumor-homing cell membrane, and the metabolites of intestinal bacteria is (1~50):(0.1~5):(100~10000).

2. The biomimetic gut bacteria according to claim 1, characterized in that, The outer membrane vesicles derived from intestinal bacteria are outer membrane vesicles derived from Gram-negative intestinal bacteria.

3. The biomimetic gut bacteria according to claim 1, characterized in that, The outer membrane vesicles derived from intestinal bacteria are outer membrane vesicles derived from Escherichia coli.

4. The biomimetic gut bacteria according to claim 3, characterized in that, The Escherichia coli strain is one or more of BL21, Nissle1917, and DH-5α.

5. The biomimetic gut bacteria according to claim 1, characterized in that, The tumor-homing cell membrane is one or more of the following: tumor cell membrane, macrophage membrane, or mesenchymal stem cell membrane.

6. A method for preparing biomimetic gut bacteria as described in claim 1, characterized in that, Including the following steps: The components of the biomimetic gut bacteria shell and gut bacteria metabolites are mixed and sonicated at 30-200W for 1-10 minutes to obtain a biomimetic gut bacteria immunomodulator system. The components of the biomimetic gut bacteria shell include: outer membrane vesicles derived from gut bacteria and a tumor-homing cell membrane; The intestinal bacterial metabolites include trimethylamine oxide.

7. The preparation method according to claim 6, characterized in that, The tumor-homing cell membrane is one or more of the following: tumor cell membrane, macrophage membrane, or mesenchymal stem cell membrane.

8. The use of a biomimetic gut bacterium as described in any one of claims 1 to 5, or a biomimetic gut bacterium prepared by the preparation method as described in any one of claims 6 to 7, in the preparation of tumor therapeutic drugs.

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