Bionic intestinal bacteria as well as preparation method and application thereof

Abstract

The invention discloses a bionic intestinal bacterium and a preparation method and application thereof, and the bionic intestinal bacterium comprises a bionic intestinal bacterium shell and an intestinal bacterium metabolite entrapped in the bionic intestinal bacterium shell; the bionic intestinal bacterial shell comprises: an intestinal bacterial sourced outer membrane vesicle; the intestinal bacterial metabolite comprises one or more of short-chain fatty acid, trimethylamine oxide, inosine and indole-3-formaldehyde. According to the bionic intestinal bacteria disclosed by the invention, the immune regulation function of the intestinal bacteria is reserved, a tumor immunosuppression microenvironment can be effectively regulated into an immune activation microenvironment, the anti-tumor immunotherapy effect is improved, and the risk of direct administration of intestinal flora can be reduced. Besides, the preparation method of the bionic intestinal bacteria is simple, and the bionic intestinal bacteria have good biological safety and potential application prospects in the aspects of tumor treatment and industrial popularization.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to bionic intestinal bacteria and a preparation method and application thereof. Background Art

[0002] During the occurrence and development of tumors, tumors evolve multiple mechanisms to escape tumor immune surveillance and suppress anti-tumor immune responses. The main mechanisms of tumor immune escape involve the participation of immune checkpoint pathways. The main immune checkpoints are cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 / 2 and their ligands (PD-L1 / PD-L2 / PD-1). The method of treating tumors by targeting immune checkpoints is called immune checkpoint therapy (ICT). ICT aims to block inhibitory signals of T cell activation and promote T cells to recognize and kill tumor cells. This treatment method has increased the survival rate of patients with advanced malignant melanoma and advanced non-small cell lung cancer by several times. The clinical success of ICT has completely changed the field of cancer immunotherapy and made ICT a mainstay of cancer treatment together with traditional treatments such as surgery, chemotherapy and radiotherapy. However, it is worth noting that ICT is less effective against "cold" tumors with immunosuppression (such as breast cancer, pancreatic cancer and brain glioma). Because ICT is mainly mediated by T cells, and "cold" tumors lack cytotoxic T lymphocyte infiltration, the low number of cytotoxic T cells is the main reason for the poor efficacy of ICT in "cold" tumors. Therefore, finding effective methods to enhance the infiltration of cytotoxic T lymphocytes is the key to improving the therapeutic effect of ICT in "cold" tumors.

[0003] In recent years, scientists have found in tumor immunology studies in mice and human patients that host intestinal bacteria have immunomodulatory effects on a variety of solid tumors. The immune signals produced by Bifidobacterium can stably regulate the activation of dendritic cells through the STING pathway, promote antigen presentation, thereby improving the effector function of cytotoxic T lymphocytes, and recruit cytotoxic T lymphocytes to infiltrate into the tumor microenvironment. Bifidobacterium combined with PD-L1 / PD-1 immune checkpoint therapy improves the efficacy of PD-L1 / PD-1 and effectively inhibits tumor proliferation. The enrichment of three bacterial communities, Pseudomonas, Saccharopolyspora, and Streptomyces, inside pancreatic cancer contributes to the recruitment and activation of cytotoxic T lymphocytes. In short, the intestinal flora can successfully enhance the infiltration of cytotoxic T lymphocytes in "cold" tumors by using its own immunogenicity and the immune activation effect of its metabolites, which is an effective means to enhance the therapeutic effect of ICT. However, the tumor delivery of intestinal bacteria has the following disadvantages: (1) leakage risk: bacteria can leak into normal tissues and cause organ infection and sepsis; (2) poor operational flexibility; (3) metabolites cannot be quantified and may contain other metabolites that are not conducive to tumor inhibition; (4) high concentrations of metabolites are distributed throughout the body and induce other diseases (such as TMAO, which is a high-risk factor for cardiovascular disease). These defects limit the application of intestinal bacteria in anti-tumor. Therefore, it is necessary to find effective means to retain the immune regulation function of intestinal bacteria and make up for the shortcomings of intestinal bacteria tumor delivery, which is of great significance for tumor ICT. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a bionic intestinal bacterium and a preparation method and application thereof, aiming to solve the problems of leakage risk and inability to quantify when using intestinal bacteria for tumor immunotherapy.

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

[0006] In a first aspect, a bionic intestinal bacterium is provided, wherein the bionic intestinal bacterium comprises: a bionic intestinal bacterium shell and an intestinal bacterium metabolite encapsulated in the bionic intestinal bacterium shell;

[0007] The bionic intestinal bacteria shell comprises: an outer membrane vesicle derived from intestinal bacteria;

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

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

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

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

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

[0013] According to a preferred technical solution, the bionic intestinal bacterial shell further comprises: a cell membrane capable of tumor homing; the cell membrane capable of tumor homing is one or more of a tumor cell membrane, a macrophage membrane or a mesenchymal stem cell membrane.

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

[0015] In a second aspect, a method for preparing the bionic intestinal bacteria according to the first aspect is provided, comprising the steps of:

[0016] The components of the bionic intestinal bacteria shell and the metabolites of the intestinal bacteria are mixed, and ultrasonicated at 30 to 200 W for 1 to 10 minutes to obtain a bionic intestinal bacteria immunomodulator system;

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

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

[0019] According to a preferred technical solution, the components of the bionic intestinal bacterial shell also include: a cell membrane capable of tumor homing; the cell membrane capable of tumor homing is one or more of a tumor cell membrane, a macrophage membrane or a mesenchymal stem cell membrane.

[0020] In a third aspect, there is provided a use of the bionic intestinal bacteria as described in the first aspect or the bionic intestinal bacteria prepared by the preparation method as described in the second aspect in the preparation of tumor therapeutic drugs.

[0021] Beneficial effects: The present invention provides a bionic intestinal bacteria and its preparation method and application, wherein the bionic intestinal bacteria are formed by ultrasonic self-assembly of the outer membrane vesicles and metabolites in the intestinal bacteria that have the effect of enhancing tumor immunity. The experimental results show that the bionic intestinal bacteria constructed by the present invention have uniform particle size and good stability, can regulate the tumor immunosuppressive microenvironment, and improve the CD8 +The number of T cell infiltrations improves the therapeutic effect of immune checkpoint inhibitors. When the bionic intestinal bacteria shell includes a cell membrane that can home to tumors, the bionic intestinal bacteria of the present invention can also specifically target the tumor site. The present invention overcomes the toxicity of directly delivering intestinal bacteria and intestinal bacterial metabolites in the blood circulation and the risk of inducing other diseases. In addition, the bionic intestinal bacteria provided by the present invention have simple synthesis steps and good biosafety, and have potential application prospects in tumor treatment and industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the synthesis of ROMV / TMAO in Example 1.

[0023] Figure 2 The figures 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 composition analysis results of ROMV in Example 4; wherein, a is the particle size statistics of OMV, ROMV and ROMV / TMAO; b is the result of the change of the hydrated particle size of ROMV / TMAO at 37°C over time; c is the result of the change of the monodispersity of ROMV / TMAO at 37°C over time; d is the transmission electron microscopy characterization results of OMV, ROMV and ROMV / TMAO, scale: 100 nm; e is the protein bands of RV, OMV and ROMV analyzed by SDS-PAGE; f is the fluorescence co-localization image of RV, OMV and ROMV.

[0024] Figure 3is the in vivo targeting study result of ROMV / TMAO in Example 5, 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; wherein a is the distribution diagram of DiR, OMV-DiR, RV-DiR, ROMV-DiR and ROMV / TMAO-DiR in 4T1 breast cancer tumor-bearing mice after administration at different times; b is the distribution diagram of DiR, OMV-DiR, RV-DiR, ROMV-DiR and ROMV / TMAO-DiR in 4T1 after administration for 24 hours. Distribution fluorescence imaging within the tumor; c is the quantitative results of the distribution fluorescence imaging of DiR, OMV-DiR, RV-DiR, ROMV-DiR and ROMV / TMAO-DiR in 4T1 tumors 24 hours after administration; d is the distribution fluorescence imaging of DiR, OMV-DiR, RV-DiR, ROMV-DiR and ROMV / TMAO-DiR in tissues 24 hours after administration; e is the quantitative results of the distribution fluorescence imaging of DiR, OMV-DiR, RV-DiR, ROMV-DiR and ROMV / TMAO-DiR in tissues 24 hours after administration.

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

[0026] Figure 5 The results of the study on the tumor inhibition effect 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; wherein, a is a curve of tumor volume of mice in different treatment groups; b is the statistical results of tumor weight of mice in different treatment groups; c is the H&E and TUNEL staining results of tumors of mice in different treatment groups.

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

[0028] Figure 7 It is the result of ROMV / TMAO enhancing α-PD-L1 in treating 4T1 tumor in Example 9, G1 refers to PBS group, G2 refers to α-PD-L1 group, G3 refers to ROMV / TMAO group, G4 refers to ROMV / TMAO+α-PD-L1 group; wherein, a is the tumor volume curve of a single mouse in different treatment groups; b is the statistical result of tumor weight of mice in different treatment groups; c is the representative photos of tumor-bearing mice in different treatment groups at the treatment endpoint. DETAILED DESCRIPTION

[0029] The present invention provides a bionic intestinal bacterium and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0030] The embodiment of the present invention provides a bionic intestinal bacterium, wherein the bionic intestinal bacterium comprises: a bionic intestinal bacterium shell and an intestinal bacterium metabolite encapsulated in the bionic intestinal bacterium shell;

[0031] The bionic intestinal bacteria shell comprises: an outer membrane vesicle derived from intestinal bacteria;

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

[0033] Specifically, inspired by the intestinal bacteria's ability to reshape the immune microenvironment and promote anti-tumor immunity in tumors, the inventors self-assembled the outer membrane vesicles and metabolites (such as short-chain fatty acids, trimethylamine oxide, inosine, indole-3-carboxaldehyde, etc.) in intestinal bacteria that have synergistic tumor immunity to form bionic intestinal bacteria. The bionic intestinal bacteria not only retain the immunomodulatory function of intestinal bacteria, but can also effectively adjust the tumor immunosuppressive microenvironment to an immune-activating microenvironment, improve the anti-tumor immunotherapy effect, and reduce the risk of direct administration of intestinal flora.

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

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

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

[0037] In one embodiment, the enteric 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 intestinal bacteria-derived outer membrane vesicles to the intestinal bacteria metabolites is (1-50): (100-10000); preferably, the mass ratio of the intestinal bacteria-derived outer membrane vesicles to the intestinal bacteria metabolites is (10-30): (2000-5000); further preferably, the mass ratio of the intestinal bacteria-derived outer membrane vesicles to the intestinal bacteria metabolites is (15-25): (3000-3500); most preferably, the mass ratio of the intestinal bacteria-derived outer membrane vesicles to the intestinal bacteria metabolites is 18: 3200. At the preferred mass ratio, the biomimetic intestinal bacteria has a hydrated particle size of less than 100 nm and good monodispersity, which is conducive to drug delivery.

[0040] In one embodiment, the bionic intestinal bacterial shell further comprises: a cell membrane capable of tumor homing; the cell membrane capable of tumor homing 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 of the intestinal bacteria-derived outer membrane vesicles, the tumor-homing cell membrane and the intestinal bacteria metabolites is (10-30): (0.5-2): (2000-5000); further preferably, the mass ratio of the intestinal bacteria-derived outer membrane vesicles, the tumor-homing cell membrane and the intestinal bacteria metabolites is (15-25): (0.8-1.2): (3000-3500); most preferably, the mass ratio of the intestinal bacteria-derived outer membrane vesicles, the tumor-homing cell membrane and the intestinal bacteria metabolites is 18: 1: 3200. At the preferred mass ratio, the biomimetic intestinal bacteria has a hydrated particle size of less than 100 nm and good monodispersity, which is conducive to drug delivery.

[0042] In one embodiment, the bionic intestinal bacteria further comprises: a fluorescent marker.

[0043] In one embodiment, the fluorescent marker is selected from one or more of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dioctadecyloxocarbocyanine perchlorate (DiO), and 1,1'-dioctyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiR).

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

[0045] The embodiment of the present invention provides a method for preparing the bionic intestinal bacteria as described above, comprising the steps of:

[0046] The components of the bionic intestinal bacteria shell and the metabolites of the intestinal bacteria are mixed, and ultrasonicated at 30 to 200 W for 1 to 10 minutes to obtain a bionic intestinal bacteria immunomodulator system;

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

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

[0049] In one embodiment, the components of the bionic intestinal bacteria shell also include: a cell membrane capable of tumor homing; the cell membrane capable of tumor homing is one or more of a tumor cell membrane, a macrophage membrane or a mesenchymal stem cell membrane.

[0050] An embodiment of the present invention provides a use of the bionic intestinal bacteria as described above or the bionic intestinal bacteria prepared by the preparation method as described above in preparing tumor therapeutic drugs.

[0051] In one embodiment, the tumor therapeutic 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, laryngeal 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 by means of specific examples.

[0053] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0054] Abbreviation or explanation of terms:

[0055] OMV: bacterial extramembrane vesicle.

[0056] RV: macrophage membrane.

[0057] TMAO: trimethylamine oxide.

[0058] ROMV: biomimetic intestinal bacterial envelope.

[0059] ROMV / TMAO: biomimetic intestinal bacteria that secrete TMAO.

[0060] In the examples, the mass or concentration of OMV, RV and ROMV cannot be directly measured and are therefore 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 intestinal bacteria (ROMV / TMAO) that can secrete TMAO

[0062] (1) Extraction of OMVs

[0063] The BL21 strain was cultured in LB medium and incubated in a rotary shaker (200 rpm) at 37°C for 6 hours. The bacterial culture medium was then diluted with fresh LB medium at a ratio of 1:100. When the OD600 value of the bacterial suspension reached about 1.0, the bacterial suspension was centrifuged at 8000 rpm for 30 minutes at 4°C to obtain a supernatant. The supernatant was filtered with a 0.22 μm sterile vacuum filter. The filtrate was concentrated, and the concentrated filtrate was 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 by the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). All samples were stored at -80°C for further experiments.

[0064] (2) Extraction of RV

[0065] The separation of macrophage membrane-RAW264.7 cell membrane carrier (RV) was carried out according to the instructions of the membrane protein extraction kit (Shanghai Biyuntian Biotechnology Co., Ltd.). RAW264.7 cells were collected, centrifuged at 1200rpm for 3 minutes at 4°C, and washed with PBS (10mM, pH 7.4). The cells were resuspended with a mixture of reagent A in the membrane protein extraction kit and phenylmethylsulfonyl fluoride (PMSF) solution (1mM). Then, the RAW264.7 cells were placed on ice for 15 minutes. The cells were then lysed by freeze-thaw method, then centrifuged at 6000rpm for 10 minutes, and the supernatant was taken for the next experiment. The supernatant was centrifuged at 12000rpm for 30 minutes to obtain a precipitate, and the precipitate was resuspended in PBS (10mM, pH 7.4). The protein concentration of the OMV solution was detected by micro BCA protein detection kit (Thermo Fisher Scientific). All samples were stored at -80°C for further experiments.

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

[0067] The biomimetic intestinal bacteria (ROMV / TMAO) that can secrete the metabolite TMAO (Sigma-Aldrich) were prepared by ultrasonic assembly. Specifically, macrophage membrane (RV), bacterial extracellular vesicle (OMV) and trimethylamine oxide (TMAO) were used to prepare ROMV / TMAO in different mass ratios, with an ultrasonic power of 120 W and an ultrasonic time of 5 min to obtain biomimetic intestinal bacteria (ROMV / TMAO). The schematic diagram of the synthesis of ROMV / TMAO is shown in the figure. Figure 1The hydrated particle size and PDI of the prepared OMV, ROMV and ROMV / TMAO with different mass ratios were measured by Malvern particle size analyzer (model: nano series, manufacturer: Malvern). 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] It can be seen from Table 1 that the mass ratio of OMV, RV and TMAO has a great influence 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 optimal. In subsequent experiments, this mass ratio is used to prepare ROMV / TMAO unless otherwise specified.

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

[0072] The OMV, ROMV and ROMV / TMAO (OMV:RV:TMAO=18:1:3200) solutions prepared in Example 1 were dripped onto a copper grid containing a fenvalerate membrane and negatively stained with phosphotungstic acid. The morphology was then characterized using a transmission electron microscope (model: HT7700, manufacturer: Hitachi). The results are shown in FIG. Figure 2 As shown in d. Figure 2 As shown in Figure d, OMV, ROMV and ROMV / TMAO are uniformly spherical. The encapsulation efficiency and drug loading of TMAO in ROMV / TMAO prepared in Example 1 were detected by ultra-high performance liquid chromatography-mass spectrometry to be 33.78±2.97% and 98.26±0.15%, respectively.

[0073] Example 3: Stability study of biomimetic intestinal bacteria (ROMV / TMAO)

[0074] In order to study the stability of bionic intestinal bacteria (ROMV / TMAO), the bionic intestinal bacteria (ROMV / TMAO) prepared in Example 1 were dispersed in PBS and incubated at 37°C for 72 h. Samples were taken at different time points and the changes in particle size and PDI were detected using a Malvern laser particle size analyzer. The test results are shown in Figure 2. Figure 2 As shown in b and c. Figure 2 As shown in b and c, the particle size of ROMV / TMAO in PBS at 37°C remained basically unchanged within 72 hours and had good monodispersity, indicating that ROMV / TMAO has good stability.

[0075] Example 4: Composition Analysis of Biomimetic Enteric Bacteria Shell (ROMV)

[0076] In order to verify whether OMV and RV can 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'-dioctadecyloxocarbonylcyanine perchlorate (DiO, green, brand: Solebao), and the final concentrations of DiI and DiO were both 10μM. Then OMV-DiI and RV-DiO (mass ratio of 18:1, ultrasonic power of 120W, ultrasonic time of 5min) were ultrasonically assembled to form a biomimetic intestinal bacterial shell (ROMV), and imaging was performed under a confocal microscope. The results are shown in Figure 2. Figure 2 As shown in f. Figure 2 As can be seen in figure f, the fluorescence of OMV and RV co-localizes in ROMV, indicating that ROMV is composed of RV and OMV.

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

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

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

[0080] Balb / c female mice (6-8 weeks, 20-25 g) were provided by Guangdong Yaokang Biotechnology Co., Ltd. All mice were housed under a 12 / 12 h dark / light cycle and SPF (ambient temperature 25 °C and humidity 55%) conditions. The animal study 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 implanted subcutaneously in each mouse was 2×10 5 The cells were dispersed in 100 μL PBS. The tumor volume reached 100 mm 3 With a volume of about 100 μl, in vivo targeting studies of liposomes can be carried out.

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

[0083] After constructing the 4T1 breast cancer tumor model, the model was divided into DiR (1,1'-dioctyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, brand: Aladdin), OMV-DiR, RV-DiR, ROMV-DiR and ROMV / TMAO-DiR groups, with 5 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. 200μLDiR, OMV-DiR, RV-DiR, ROMV-DiR or ROMV / TMAO-DiR (500μg / kgDiR) were injected into the tail vein, respectively. After different times of administration, the mice were imaged in vivo to observe the distribution of drugs in the tumor site of the mice. 24 hours after administration, the mice were euthanized, and the heart, liver, spleen, lung, kidney and tumor tissues were removed for fluorescence signal analysis. The results are as follows: Figure 3 shown. Figure 3 In the figure, 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, with the increase of administration time, the fluorescence intensity of the tumor site gradually increased. After 24 hours of administration, the fluorescence intensity of 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 Figure b, the fluorescence imaging results of mouse tumor tissue isolated from the body are consistent with those of in vivo imaging. Figure 3 As shown in Figure c, the accumulation of RV-DiR, ROMV-DiR and ROMV / TMAO-DiR in the tumor site was significantly different from that in the control group, and the macrophage membrane (RV) gave ROMV / TMAO tumor targeting. Figure 3 As shown in d and e, ROMV / TMAO is more distributed in the liver and spleen, indicating that it may be metabolized through the liver.

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

[0086] According to literature reports, 5 μg of OMV injected into the tail vein of mice has obvious toxicity. TMAO also has the risk of causing atherosclerosis. Therefore, the present invention evaluates the in vivo toxicity of different doses of ROMV / TMAO.

[0087] Balb / c female mice (6-8 weeks, 20-25g) were divided into 5 groups, 5 mice in each group, and different doses of ROMV / TMAO (0, 2, 3, 4, 5 μg OMV) prepared in Example 1 were injected into the tail vein on days 0, 2, 4, 6, and 8, respectively, and the survival rate of the mice was monitored. On the 10th day, the mice were euthanized, and blood was collected for liver and kidney function analysis. At the same time, the aortic root was removed, and the aortic root sections were stained with H&E, Masson and Oil Red to observe the atherosclerosis. The results are as follows Figure 4 shown.

[0088] Depend on Figure 4 As shown in b-d, ROMV / TMAO did not cause death in mice at a high dose of 5 μg of OMV, and there were no atherosclerotic plaques at the aortic root. The liver and kidney functions of mice were not significantly different from those of the control group and were within the normal range. The above results prove that biomimetic intestinal bacteria have high biological safety.

[0089] Example 7: Study on the anti-tumor effect of biomimetic intestinal bacteria ROMV / TMAO in vivo

[0090] The construction of the 4T1 breast cancer model was the same as in Example 5 (1). When the tumor volume reached 50-100 mm 3 When the mice were about 100 μg / day, the model was divided into 4 groups, namely G1 (PBS), G2 (TMAO), G3 (ROMV), and G4 (ROMV / TMAO), with 5 mice in each group. The mice were administered intravenously (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 5 times, and the tumor volume was monitored. The mice were euthanized 14 days after the first administration, and the tumors were removed and weighed. The results are as follows: Figure 5 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 had the best tumor inhibition effect, which was significantly different from the other three groups. Figure 5 As shown in Figure c, the tumor tissue cell damage in the G4 (ROMV / TMAO) group was more obvious, and there were more apoptotic cells. The above results prove that biomimetic intestinal bacteria (ROMV / TMAO) can effectively inhibit the growth of 4T1 tumors.

[0092] Example 8: Effect of biomimetic intestinal bacteria (ROMV / TMAO) on tumor immune microenvironment After the tumor in Example 7 was weighed, part of the tumor tissue was taken to analyze the tumor immune microenvironment.

[0093] The CD8 + The changes of T cells and macrophages were observed to observe the effects of ROMV / TMAO on the tumor immune microenvironment. Specifically, the tumor tissue was cut into minced meat with ophthalmic scissors, collagenase (STEMCELL, 07912) was added, and after digestion at 37°C for 30 minutes, a 70 μm cell sieve was passed to obtain a single cell suspension. The cells were then 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 (BD Pharmingen, UF557397S), BV510-anti-GR-1 (BD Pharmingen, 563040), BV421-anti-F4 / 80 (BD Pharmingen, 565411), PE-Cy7-anti-CD86 (BD Pharmingen, 563068). 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 shown.

[0094] Depend on Figure 6 From a to c, we can see that the proportion of M1 macrophages in the tumor of the G4 (ROMV / TMAO) group was significantly higher than that of the other groups, and the proportion of M2 macrophages was significantly lower than that of the G1 (PBS) group, indicating that ROMV / TMAO can promote the polarization of macrophages in tumors from M2 to M1. Figure 6 As shown in d and e, CD8 +The proportion of T cells was also significantly higher than that of the control group. The above results show that biomimetic intestinal bacteria (ROMV / TMAO) have immune activation effects similar to those of intestinal bacteria and can regulate the tumor immune microenvironment.

[0095] Example 9: Effect of biomimetic intestinal bacteria (ROMV / TMAO) on α-PD-L1 treatment of 4T1 tumors

[0096] The construction of the 4T1 breast cancer model was the same as in Example 5 (1). When the tumor volume reached 50-100 mm 3 When the mice reached 400 μg / kg, the model was divided into 4 groups, namely G1 (PBS), G2 (α-PD-L1), G3 (ROMV), and G4 (ROMV / TMAO+α-PD-L1), with 6 mice in each group. The mice were administered via tail vein (G1, G3, and G4, OMV: 5 μg / mouse, TMAO: 0.25 mg / g) or intraperitoneal administration (G2, α-PD-L1: 100 μg / mouse), once every other day, for a total of 5 times, and the tumor volume was monitored. 14 days after the first administration, the mice were euthanized, the tumors were removed, and the weight was measured. The results are as follows: Figure 7 shown.

[0097] Depend on Figure 7 As shown in a and b, the combination of bionic intestinal bacteria (ROMV / TMAO) and immune checkpoint inhibitor α-PD-L1 significantly delayed tumor growth, and the tumor weight was also lower than that of other groups. Figure 7 Figure c is a photo of tumor-bearing mice after treatment, which directly reflects the tumor inhibition effect of each group. Figure 6 The results showed that the biomimetic intestinal bacteria ROMV / TMAO activated the immune microenvironment in the tumor and increased the intratumoral CD8 + The proportion of T cells was increased, thereby enhancing the efficacy of α-PD-L1 in treating 4T1 tumors.

[0098] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A bionic intestinal bacterium, characterized in that: The bionic intestinal bacteria include: a bionic intestinal bacteria shell and intestinal bacteria metabolites contained in the bionic intestinal bacteria shell; The bionic intestinal bacteria shell comprises: an outer membrane vesicle derived from intestinal bacteria; The intestinal bacterial metabolites include: one or more of short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde.

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

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

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

5. The bionic intestinal bacteria according to claim 1, characterized in that: The mass ratio of the outer membrane vesicles derived from intestinal bacteria to the intestinal bacterial metabolites is (1-50):(100-10000).

6. The bionic intestinal bacteria according to claim 1, characterized in that: The bionic intestinal bacterial shell also includes: a cell membrane capable of tumor homing; the cell membrane capable of tumor homing is one or more of a tumor cell membrane, a macrophage membrane or a mesenchymal stem cell membrane.

7. The bionic intestinal bacteria according to claim 6, characterized in that: 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).

8. A method for preparing bionic intestinal bacteria as claimed in claim 1, characterized in that: Includes steps: The components of the bionic intestinal bacteria shell and the metabolites of the intestinal bacteria are mixed, and ultrasonicated at 30 to 200 W for 1 to 10 minutes to obtain a bionic intestinal bacteria immunomodulator system; The components of the bionic intestinal bacteria shell include: outer membrane vesicles derived from intestinal bacteria; The intestinal bacterial metabolites include: one or more of short-chain fatty acids, trimethylamine oxide, inosine, and indole-3-carboxaldehyde.

9. The preparation method according to claim 8, characterized in that: The components of the bionic intestinal bacteria shell also include: a cell membrane capable of tumor homing; the cell membrane capable of tumor homing is one or more of a tumor cell membrane, a macrophage membrane or a mesenchymal stem cell membrane.

10. Use of the bionic intestinal bacteria according to any one of claims 1 to 7 or the bionic intestinal bacteria prepared by the preparation method according to any one of claims 8 to 9 in preparing tumor therapeutic drugs.

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