Endogenous nanoprobe, preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to an endogenous nanoprobe, a preparation method and an application thereof. The nanoprobe is an outer membrane vesicle carrying a non-oxygen-dependent controllable and adjustable fluorescent label, has a natural bacterial outer membrane structure, and can visually identify, track or inhibit targets in an aerobic or anaerobic environment. The size of the nanoprobe is 40-100nm. The endogenous nanoprobe has strong specificity and high efficiency, and its fluorescence has the flexibility of on-demand switching and reversible switching of emission bands, which can be used for intelligent and dual-color imaging, thereby significantly improving the sensitivity and accuracy of detection, and is not harsh on the required detection equipment and sites, and has low cost, and has important research significance and application value. The application of the endogenous nanoprobe includes: the use of the nanoprobe in the preparation of drugs for preventing, slowing down or treating intestinal diseases; a biosensor system comprising at least the nanoprobe of the present invention; and a bioimaging system comprising at least the nanoprobe of the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an endogenous nanoprobe, a preparation method and application thereof. Specific applications include the application of the nanoprobe in biosensing, imaging, disease detection and pharmaceutical manufacturing. Background Art

[0002] The human intestinal microbiota is composed of approximately 10-100 trillion microbial cells. As a major effector component, it is estimated that more than 1,000 bacterial species maintain metabolic homeostasis through the gut-liver, gut-lung, gut-brain and other axes, regulate the host's immune and nervous systems, and play a key role in human health. Behind these intestinal bacteria, there are a large number of outer membrane vesicles (OMVs) with an average size of about 20-250nm. OMVs are released from the outer surface of bacteria and retain the inherent functions of the outer membrane. OMVs retain the intracellular substances associated with parent bacteria and play a vital role in the inter-species and intra-species biological behaviors of bacteria, such as intercellular communication, resistance to phage infection, and the output and toxicity of cellular metabolites. In addition to inter-kingdom communication, OMVs released by intestinal commensal microorganisms have been shown to be essential for the maturation of the immune system, while OMVs produced by pathogens can promote inflammation and infection in the host. A large number of studies have shown that OMVs derived from the intestinal flora can be transferred to other major organs and cause various functional disorders. It has also been reported that the accumulation levels of OMVs in serum and urine can reflect the pathogenesis and progression of various tumors.

[0003] Despite the important role of OMV, there are currently very limited suitable methods for tracking and analyzing intestinal flora-related OMVs to fundamentally understand their ultimate impact on host physiology and pathophysiology. Although Bittel, M. et al. used the expression of CRE-recombinase and reporter molecules in bacteria and host cells, respectively, to disclose a simple method for tracking OMVs in vivo (Visualizing transfer of microbial biomolecules by outer membranevesicles in microbe-host-communication in vivo, J Extracell Vesicles. 2021, 10, e12159.), it is understood that there is no disclosure or report on visualization strategies that can image and track OMV distribution and interactions between OMV and intestinal microorganisms and mammalian host cells. As a result, there has always been a demand for imageable probes that can be applied to living bodies, which is crucial for the visualization of complex biological processes.

[0004] Specifically, unlike small molecule probes, nanoprobes have been one of the research objects of researchers in this field due to their many advantages, such as inherent physicochemical tunability, easy functionalization, high sensitivity, specificity and targeting ability. So far, a variety of attractive nanoprobes have been prepared, mainly including inorganic carbon dots and quantum dots, organic polymers, micelles and vesicles, and inorganic-organic hybrid nanoparticles.

[0005] Considering the nanometer size and its effects, nanoprobes have the advantages of obtaining superior performance in vivo, such as deep penetration and adjustability of distribution in different tissues. In order to better visualize the dynamic processes of living biological systems, smart nanoprobes are designed to obtain real-time in situ information in complex physiological environments. In particular, stimuli-responsive probes that rely on the use of specific physiological markers as stimuli have been widely explored for sensing, bioimaging, and disease diagnosis. For example, reactive oxygen species (ROS)-sensitive nanoprobes have been developed to identify the occurrence and progression of acute liver failure and osteoarthritis; hypoxia, pH, and azoreductase-responsive nanoprobes have been produced to detect tumors in an efficient and specific manner. However, most of the disclosed nanoprobes lack fast on-demand signal switching and reversible variable signals and cannot be used for multimodal imaging.

[0006] Furthermore, traditional nanoprobes are exogenous substances and are not suitable for monitoring specific biological processes in living systems, such as the distribution of OMVs in the intestine and their interaction with the surrounding environment. Due to the presence of an anaerobic microenvironment in the intestine, the signal from the nanoprobe is still required to track OMVs independently of the report of oxygen molecules. Therefore, existing nanoprobes cannot be used directly to track and image OMVs in vivo, let alone detect specific biological processes in living systems. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art and the demand for endogenous probes and their imaging in the biomedical field, the present invention aims to provide an endogenous nanoprobe, a preparation method and applications thereof, and specific applications include the application of the nanoprobe in biosensing, imaging, disease detection and pharmaceutical manufacturing.

[0008] In the first aspect, the present invention provides an endogenous nanoprobe, a nanovesicle carrying a fluorescence-activating and absorption-shifting tag (FAST), wherein the nanovesicle is an outer membrane vesicle (OMV) produced by Escherichia coli (EcN) carrying pQE60-FAST-His, has a natural structure and an oxygen molecule-independent emission spectrum, and can visually identify, track or inhibit targets in an aerobic or anaerobic environment.

[0009] Furthermore, the nanovesicles are outer membrane vesicles produced by culturing Escherichia coli EcN carrying pQE60-FAST-His in a medium supplemented with ampicillin, inducing with IPTG, culturing, and adding kanamycin.

[0010] Furthermore, the target may be a bacterium in the intestinal flora, a pathogen or an intestinal epithelial cell.

[0011] In a second aspect, the present invention provides a method for preparing an endogenous nanoprobe, comprising:

[0012] Plasmid pQE60-FAST-His was constructed and transformed into Escherichia coli;

[0013] Screening to obtain ampicillin-resistant strains;

[0014] Induce, culture in a culture medium supplemented with kanamycin at a final concentration of 4.0-10.0 mg / l, and separate to obtain outer membrane vesicles FAST-OMV carrying the FAST tag, wherein the outer membrane vesicles are endogenous nanoprobes carrying the FAST tag.

[0015] Preferably, the final concentration of kanamycin is 6.0-9.0 mg / l, more preferably, 6.25 mg / l.

[0016] Furthermore, induction was performed under the inducer IPTG, and the final concentration of IPTG was 0.5M.

[0017] Furthermore, the outer membrane vesicle yield was 1.5×10 11 pcs / ml or more.

[0018] Furthermore, the nanovesicle has a natural outer membrane vesicle structure and an oxygen molecule-independent emission spectrum, which can visually identify, track or inhibit targets in an aerobic or anaerobic environment. Preferably, the interaction between bacterial outer membrane vesicles and intestinal flora or intestinal epithelial cells is tracked.

[0019] In a third aspect, the present invention also provides the use of the above-mentioned endogenous nanoprobes in the preparation of drugs for preventing, alleviating or treating intestinal diseases.

[0020] Further, the intestinal disease is inflammatory bowel disease and cancer caused by intestinal inflammation. More specifically, the intestinal disease is enteritis caused by bacterial infection. Preferably, the disease is enteritis induced by Salmonella.

[0021] In a fourth aspect, the present invention further provides a biosensor system, which at least includes the nanoprobe described in the present invention.

[0022] In a fifth aspect, the present invention also provides a biological imaging system, which at least includes the nanoprobe described in the present invention, and performs real-time tracking and imaging in vivo.

[0023] Beneficial Effects

[0024] The endogenous nanoprobe of the present invention has strong specificity and high efficiency, and has the flexibility of on-demand fluorescence switching and reversible switching of emission bands, and can be used for intelligent and dual-color imaging, thereby significantly improving the sensitivity and accuracy of detection. The required detection equipment and site are not highly demanding and the cost is low, and it has important research significance and application value.

[0025] The endogenous nanoprobe has a wide range of applications. The experiments of this application have confirmed the use of the nanoprobe in the preparation of drugs for preventing, alleviating or treating intestinal diseases; a biosensor system that at least includes the nanoprobe described in the present invention; and a bioimaging system that at least includes the nanoprobe of the present invention. Importantly, the nanoprobe of the present invention is an endogenous substance used in multimodal imaging and is suitable for monitoring specific biological processes in living systems, such as the distribution of OMVs in the intestine and their interaction with the surrounding environment. In the anaerobic microenvironment of the intestine, the oxygen molecule-independent signal from the nanoprobe can report and track OMVs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the design, preparation and characterization of FAST-OMV according to the present invention.

[0027] Figure 1 .a, Schematic diagram of the preparation of FAST-OMV;

[0028] Figure 1 .b, On-demand fluorescence switching and reversible switching of emission bands of FAST and FAST-OMV;

[0029] Figure 1 c, shows anaerobic tracking of FAST-OMVs in the intestine, which show selective fusion with intestinal flora and entry into intestinal epithelial cells;

[0030] Figure 1 .d, Size distribution of FAST-OMV measured by DLS (n=3);

[0031] Figure 1 .e, Representative TEM images of FAST-OMVs produced by EcN under the induction of IPTG and kanamycin, scale bar: 200 nm;

[0032] Figure 1.f, Western blot analysis of FAST-OMV using enzyme-labeled anti-His antibody (1:500), M is a pre-stained protein marker, and EcN-derived OMV carrying an empty vector was used as a negative control.

[0033] Figure 2 Characterize FAST-OMV for different methods, including:

[0034] Figure 2 .ab, Size distribution of FAST-OMVs obtained under different conditions measured by DLS, where purple represents OMVs derived from bacteria cultured in LB; green represents OMVs produced by bacteria cultured in LB medium using 0.5 M IPTG; and blue represents OMVs produced by bacteria cultured in LB medium containing 0.5 M IPTG and 6.25 mg / l kanamycin.

[0035] Figure 2 .c, The protein concentration of FAST-OMV obtained under different conditions was detected by BCA.

[0036] Figure 2 .d, The concentration of FAST-OMV obtained under different conditions was detected by NTA.

[0037] Figure 3 Characterization of the fluorescence activation of FAST-OMV in the present invention, wherein:

[0038] Figure 3 .a, Schematic diagram of fluorescence activation when FAST-OMV is combined with fluorescent agent;

[0039] Figure 3 .b, Fluorescence image of FAST-OMV suspension after adding corresponding fluorescent agent under dual-wavelength protein excitation light;

[0040] Figure 3 .cf, (c, e) confocal imaging and (d, f) flow cytometry analysis of FAST-OMV with 20 μM HMBR (left) or HBR-3,5-DOM (right). Scale bar: 10 μm;

[0041] Figure 3 .gj, Fluorescence spectra (g, i) and emission peak signal intensity data (h, j) of FAST-OMVs with 20 μM HMBR (left) or HBR-3,5-DOM (right) (n=3);

[0042] The significance was assessed using Student's t test and p values ​​were obtained, ***p<0.001.

[0043] Figure 4The in vitro tracking of the interaction of FAST-OMV with microbial cells in the present invention is shown, wherein:

[0044] Figure 4 .a, Enlarged LSCM image for visualization of the in vitro interaction between FAST-OMV and STm, red, FAST-OMV co-incubated with GFP-expressing STm at predetermined time intervals, where white arrows indicate HBR-3,5-DOM-activated FAST-OMV; blue arrows indicate STm-derived OMVs; white dashed lines indicate destroyed bacteria, scale bar: 2 μm;

[0045] Figure 4 .b, Flow cytometric analysis showing the interaction of FAST-OMVs with STm in vitro, where red FAST-OMVs were co-incubated with GFP-expressing STm at predetermined time points.

[0046] Figure 5 The fluorescence shut-off of FAST-OMV is shown, where:

[0047] Figure 5 .a and b, LSCM images (a) and flow cytometry histograms (b) after washing 1, 2, and 3 times, scale bar: 10 μm;

[0048] Figure 5 c and d, Quantitative analysis of the fluorescence shut-off process of pre-activated FAST-OMVs by washing out the fluorescent agent at the indicated time points, flow cytometric analysis (c) and fluorescence spectroscopy (d) (n = 3).

[0049] Figure 6 In vitro tracking of FAST-OMV interactions with different bacteria is shown, where:

[0050] Figure 6 a and b, Growth curves of BS and SA at 37°C with or without the addition of EcN-derived OMVs, and OD was recorded using a microplate reader. 600 (n=3);

[0051] Figure 6 c and d, Representative confocal images of BS and SA expressing GFP after being co-incubated with FAST-OMV for 3 h and then activated by HBR-3,5-DOM. Scale bar: 5 μm;

[0052] Figure 7 is the reversible switching of the emission band, where:

[0053] Figure 7.a and b, Representative LSCM images (a) and flow cytometry histograms (b) of FAST-OMVs pre-activated by HMBR in the absence or presence of fluorescence emission from green to red by replacing HBR-3,5-DOM, scale bar: 10 μm;

[0054] Figure 7 .c and d, Quantitative analysis of the λmax switch from 541 nm to 597 nm: flow cytometry (c) and fluorescence spectrometer measurement (d) (n = 3);

[0055] Figure 7 .e and f, Confocal imaging data (e) and flow cytometry analysis (f) data of FAST-OMV pre-activated by HBR-3,5-DOM under HMBR exposure without or with emission switching from 597 nm to 541 nm, scale bar: 10 μm;

[0056] Figure 7 .g and h, Quantitative analysis of the switching of red and green fluorescence signal emission as measured by flow cytometry (g) and fluorescence spectrometry (h) (n=3).

[0057] Figure 8 Representative IVIS images of the mouse intestine after oral administration of FAST-OMV to mice are shown, where the mice were administered FAST-OMV 2h before oral administration of green / red fluorescent agent, euthanized 2h later, and the intestine was collected for IVIS imaging through the corresponding channels. Mice were treated with FAST-OMV for 2h and then orally administered PBS for 2h, and IVIS imaging was performed through the GFP and mCherry channels as a control group (n=5).

[0058] Fig. 9 In vitro tracking of FAST-OMV interactions with microorganisms and mammalian cells is shown, where:

[0059] Fig. 9 .a and b, Typical confocal images (a) and flow cytometry histograms (b) of STm expressing GFP after co-incubation with FAST-OMVs and then activation by HBR-3,5-DOM at the indicated time points, scale bar: 5 μm. Gray dashed lines indicate magnified images, white arrows indicate FAST-OMVs activated by HBR-3,5-DOM, and blue arrows indicate STm-derived OMVs, scale bar: 2 μm;

[0060] Fig. 9 .c, using a microplate reader to record at 600nm (OD 600 ) Growth curves of STm with or without the addition of EcN-derived OMVs at 37°C (n=3);

[0061] Fig. 9 d, Confocal images of MODE-K cells after incubation with FAST-OMV at 37°C for 1 h and then activation with HMBR or HBR-3,5-DOM;

[0062] Fig. 9 .e, Confocal images of FAST-OMV-treated MODE-K cells at the indicated time points after washing with PBS, scale bar: 20 μm;

[0063] Fig. 9 .f and g, Confocal images of MODE-K cells treated with FAST-OMV preactivated with HBR-3 and 5-DOM and supplemented with HBR-3, 5-DOM (f) and HMBR (g), respectively. Scale bar: 20 μm.

[0064] Fig. 9 .h, Immunofluorescence images of Caco-2 cells treated with 0.1 mg / ml FAST-OMV and 5 μg / ml LPS at 37°C for 24 h, where red, green, and blue represent ZO-1, occludin, and cell nuclei, respectively. Scale bar: 25 μm.

[0065] Fig.10 A typical LSCM image of intestinal bacteria isolated from the colon of a mouse treated with FAST-OMV for 4 h is shown. The isolated bacteria were resuspended in PBS containing HBR-3,5-DOM for 5 min and then captured by LSCM. Scale bar: 5 μm. White dashed squares indicate intestinal bacteria illuminated by FAST-OMV, and black dashed squares indicate bacteria that FAST-OMV could not illuminate.

[0066] Fig.11 Tracking of OMVs in the intestine is shown, where:

[0067] Fig.11 .a, 4 h after oral administration of FAST-OMV without or with additional uptakeable fluorescent agents, the mouse intestine was sectioned and imaged using IVIS;

[0068] Fig.11 b, Confocal images of bacteria isolated from the small intestine and colon of mice 4 h after injection of FAST-OMV, and then stained with corresponding fluorescent agents. Scale bar: 5 μm;

[0069] Fig.11 c, Confocal images of cryosections stained with HMBR or HBR-3,5-DOM from FAST-OMV-injected mice, with nuclei stained with DAPI; scale bar: 20 μm.

[0070] Fig.12Shown is a typical LSCM image of intestinal bacteria sampled from the colon of mice treated with FAST-OMV for 4 h, where the isolated bacteria were resuspended in PBS with green HMBR for 5 min and then observed using LSCM. Scale bar: 5 μm. The white dotted squares represent bacteria illuminated by FAST-OMV, and the black dotted squares represent non-luminescent bacteria.

[0071] Fig.13 To demonstrate the in vivo function of OMVs, 5 × 10 8 After 2 days of infection with CFU STm, FAST-OMV and 1×10 8 Mice were intragastrically administered with CFU EcN or PBS daily. After 6 days, they were euthanized and samples were taken. Healthy mice served as the control group.

[0072] Fig.13 .a, STm bacteria were counted in the small intestine, cecum, and colon, respectively;

[0073] Fig.13 .b, The total amount of STm in the intestine after different treatments, data are mean ± SEM, n = 5, and the significance was evaluated using analysis of variance ANOVA test and Tukey's post hoc test to obtain the p value, ****p < 0.0001;

[0074] Fig.13 .ce, Immunofluorescence images of ileum, cecum, and colon tissues, where red, green, and blue represent ZO-1, occludin, and cell nuclei, the white dashed panes represent the normal expression and distribution of tight junction proteins, the large white arrows represent severe epithelial tissue destruction caused by loss of tight junctions and epithelial cell death, accompanied by obvious edema and inflammatory infiltration in the submucosal layer, the small white arrows represent villus shortening and structural damage, scale bar: 50 μm.

[0075] Fig.14 Confocal images of frozen sections sampled from mice administered with FAST-OMV or PBS are shown, wherein the small intestine and colon of mice treated with FAST-OMV were frozen and stained with DAPI and corresponding fluorescent agents; frozen sections sampled from mice administered with PBS were used as controls, scale bar: 50 μm.

[0076] Fig.15 The results of experiments on the treatment of STm-induced colitis with OMVs are shown, wherein:

[0077] Fig.15 .a, Treatment experimental design: mice were infected with 5×10 8 CFU STm, and PBS, 1×10 8CFU EcN or FAST-OMV treatment, and healthy mice served as controls;

[0078] Fig.15 .bd, ELISA method was used to measure the levels of IL-6, TNF-ɑ, and IFN-γ cytokines in serum;

[0079] Fig.15 .e, body weight fluctuation during treatment;

[0080] Fig.15 .f, Image of the colon tissue resected from the cecum to the rectum after processing;

[0081] Fig.15 .g, average length of colon after treatment;

[0082] Fig.15 .h, intestinal permeability was measured by recording the level of FITC-dextran in plasma;

[0083] Fig.15 .i, H&E staining images of ileum, cecum and colon, scale bar: 100 μm, data are mean ± SEM, n = 4 or 5. One-way ANOVA and Tukey's post hoc test were used to evaluate the significance, and p values ​​were given, *p < 0.05, **p < 0.01, ***p < 0.005. ns, not significant.

[0084] Fig.16 Immunofluorescence images of ZO-1 and occludin expression in ileal epithelial cells sampled from STm-infected mice are shown, scale bar: 100 μm.

[0085] Fig.17 Immunofluorescence images showing the expression of ZO-1 and occludin in cecal epithelial cells sampled from STm-infected mice, scale bars: 400 μm (left), 100 μm (right).

[0086] Fig.18 Immunofluorescence images of colonic epithelial cells sampled from STm-infected mice expressing ZO-1 and occludin are shown, scale bar: 100 μm.

[0087] Fig.19 Shown are typical H&E staining images of the ileum of colitis mice after different treatments, where black squares represent magnified images, scale bars: 400 μm (blue) and 50 μm (black).

[0088] Fig. 20Representative H&E-stained images of the cecum of colitis mice after treatment are shown, where the black panes represent magnified images, scale bars: 400 μm (blue) and 50 μm (black).

[0089] Fig.21 Shown are H&E staining images of the colon of colitis mice after treatment, where black squares represent the corresponding magnified images, black dotted squares represent the integrity of the epithelial cell layer, red arrows point to the healthy and normal muscle layer, black arrows represent the inflamed and damaged muscle layer, scale bars: 400 μm (blue) and 50 μm (black). DETAILED DESCRIPTION

[0090] The technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto. Without departing from the technical concept and technical solution of the present invention, any modification, adjustment or modification, or equivalent replacement method that can be implemented by a person of ordinary skill in the art to which the present invention belongs will fall within the scope of the protection claimed in the present invention.

[0091] Unless otherwise specified, the experimental methods used in the following examples are all conventional technical means or methods in the art. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0092] In the first aspect, the present invention provides an endogenous nanoprobe, a nanovesicle carrying a FAST tag, wherein the nanovesicle is an outer membrane vesicle produced by Escherichia coli EcN carrying pQE60-FAST-His, has a natural structure and an oxygen molecule-independent emission spectrum, and can visually identify, track or inhibit targets in an aerobic or anaerobic environment.

[0093] Furthermore, the nanovesicle is an outer membrane vesicle produced by culturing Escherichia coli EcN carrying pQE60-FAST-His in a medium supplemented with ampicillin, inducing with IPTG, culturing, and adding kanamycin.

[0094] Furthermore, the size of the nanovesicle is 40-100 nm.

[0095] In a second aspect, the present invention provides a method for preparing an endogenous nanoprobe, comprising:

[0096] Plasmid pQE-FAST-His was constructed and transformed into Escherichia coli;

[0097] Screening to obtain ampicillin-resistant strains;

[0098] Induce, culture in medium supplemented with kanamycin, and separate to obtain outer membrane vesicles (FAST-OMV) carrying the FAST tag.

[0099] Preferably, the final concentration of kanamycin is 6.0-9.0 mg / l, more preferably, 6.25 mg / l.

[0100] Furthermore, induction was performed under the inducer IPTG, and the final concentration of IPTG was 0.5M.

[0101] Furthermore, the outer membrane vesicle yield was 1.5×10 11 pcs / ml or more.

[0102] Furthermore, the nanovesicles have a natural outer membrane vesicle structure and an oxygen molecule-independent emission spectrum, which can visually identify, track or inhibit targets in an aerobic or anaerobic environment.

[0103] In a third aspect, the present invention also provides the use of the above-mentioned endogenous nanoprobes in the preparation of drugs for preventing, alleviating or treating intestinal diseases.

[0104] In a fourth aspect, the present invention also provides a kit for detecting intestinal diseases, which kit at least includes the endogenous nanoprobe.

[0105] In a fifth aspect, the present invention further provides a biosensor system, which at least includes the nanoprobe described in the present invention.

[0106] In a sixth aspect, the present invention further provides a biological imaging system, which at least includes the nanoprobe described in the present invention.

[0107] Materials and strains

[0108] Escherichia coli Nissle 1917 strain was purchased from China General Microbiological Culture Collection. FAST is a variant of the mutated photoactive yellow protein (PYP). The plasmid pQE60-FAST-His (ampicillin-resistant) capable of expressing FAST in EcN was constructed using the FAST gene template described by Li C et al. in "Dynamic multicolor protein labeling in living cells" (Li, C., Plamont, MA, Sladitschek, HL, Rodrigues, V., Aujard, I., Neveu, P., Le Saux, T., Jullien, L., Gautier, A., Dynamic multicolor protein labeling in living cells. Chem. Sci. 2017, 8, 5598-5605.). All bacterial strains were cultured in LB medium at 37°C with appropriate antibiotics. HMBR (green phosphor) and HBR-3,5-DOM (red phosphor) were gifted by Dr. Chenge Li and Professor Arnaudgautier.

[0109] Example 1. Extraction of FAST-OMV

[0110] EcN carrying pQE60-FAST-His was first cultured in LB medium supplemented with ampicillin. 600 When the value reaches 0.5-0.7, IPTG is added for induction. After 2h, 6.25mg / l of kanamycin is added to the bacterial culture to make the bacteria produce more OMVs. The bacterial culture supernatant is collected, centrifuged at 7000×g for 1h, the precipitate is removed, and then filtered with a 0.22μm filter to avoid unwanted bacterial cells. Finally, the filtered supernatant is centrifuged at 170000×g for 1h at 4°C to obtain the OMV precipitate, which is then resuspended in PBS (pH 7.2-7.4) for storage.

[0111] Example 2. Characterization of FAST-OMV

[0112] The morphology of OMVs was observed using transmission electron microscopy (HITACH, Japan). A drop of OMV solution was deposited on a carbon-coated copper grid. The grid was rinsed twice with double distilled water (DDH2O) for 5 min each time. The grid was dried and observed. The average size and concentration of OMVs were determined by DLS (Malvern Zetasizer nano ZS, UK) and NTA (Malvern nanosight NS300, UK). The OMV concentration was defined by the total protein concentration determined using a BCA assay kit (Thermo Scientific).

[0113] Example 3. Western blot analysis of FAST-His

[0114] OMVs were isolated from EcN carrying an empty vector and EcN carrying a FAST-His expression vector, purified and resuspended in PBS. 10 μl of purified FAST-OMVs were mixed with loading buffer, boiled and loaded on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The separated samples were transferred to polyvinylidene fluoride (PVDF) membranes by a Bio-rad semi-dry transfer turbine according to the pre-programmed program for 1.5 mm gels: 1.3A, 25V, 10min (Bio-rad, USA). The PVDF membranes were incubated in TBS / Tween containing 5% skim milk for 1h and then incubated with anti-His antibody (1:5000, AE028, Ab clone, China) conjugated to horseradish peroxidase (HRP). Then, the resulting membranes were incubated with 1 ml of enhanced chemiluminescence solution for 1min and captured using a chemiluminescence imager (Bio-rad, USA).

[0115] Example 4. In vitro priming

[0116] Green and red fluorescent agents (HMBR and HBR-3,5-DOM) were added to PBS containing FAST-OMV at room temperature. Luyoro-3415RG dual-wavelength fluorescent protein excitation light source and LSCM (Leica TCS SP8, Germany) were used for direct imaging, and the dynamic initiation process was analyzed by flow cytometry (Beckman CytoFlex, USA). Their intensity and λmax were measured by fluorescence spectrometer (Fluormax-4, HORIBA Scientific, Japan).

[0117] Example 5. In vitro closure

[0118] The FAST-carrying OMVs were incubated with green and red fluorescent agents at room temperature for several minutes to fully activate FAST fluorescence. In order to shut down FAST-OMVs by removing the bound fluorescent agent, the OMVs were resuspended and washed with PBS for a predetermined number of times. The dynamic shut-off process was analyzed using LSCM and flow cytometry, and the fluorescence intensity of green FAST-OMVs and red FAST-OMVs after removing the fluorescent agent in the PBS solution was recorded using a fluorescence spectrometer.

[0119] Example 6. In vitro reversibility (from green to red)

[0120] OMVs derived from EcN expressing FAST were activated by incubation with the green fluorescent agent HMBR for several minutes. Green FAST-OMVs were immobilized on agar plates and LSCM imaging was used as a starting point. To observe the reversibility of FAST-OMVs from green to red, 4 μl of red fluorescent agent was dropped into the pre-activated green FAST-OMVs and captured by GFP and mCherry channels until the green fluorescence disappeared. In addition, its reversibility was analyzed by measuring the green and red fluorescence intensities. The red fluorescent agent was added to the PBS solution of FAST-OMVs illuminated by green fluorescence and incubated at room temperature. The green, red fluorescence intensity and λmax switching of FAST-OMVs were recorded by flow cytometry and fluorescence spectrometer.

[0121] Example 7. In vitro reversibility (switching from red to green)

[0122] FAST-OMVs were activated by incubation with red fluorescer for a few minutes. Red OMVs were fixed on agar plates and imaged by LSCM as a starting point. To observe the reversibility of FAST-OMVs from red to green, 4 μl of green fluorescein was added to the red FAST-OMVs and captured by GFP and mCherry channels until the red fluorescence disappeared. Most likely, the reversibility from red to green was analyzed by measuring the green and red fluorescence intensities. Green fluorescer was added to the red fluorescer-activated FAST-OMV solution and co-incubated at room temperature. The green and red fluorescence intensities and λmax switching of FAST-OMVs were measured by flow cytometry and fluorescence spectrometer, respectively.

[0123] Example 8. Initiation of anaerobic gastrointestinal tract

[0124] All animal experiments were performed under the guidelines evaluated and approved by the Ethics Committee of the Animal Care and Use Professional Committee of Shanghai Jiao Tong University. Male ICR mice aged 6 to 8 weeks were selected for the experiments. The mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. and raised under SPF (specific pathogen free, SPF) conditions for 4 days. The mice were randomly divided into 3 groups (n = 5), and each ICR mouse was orally administered with 200 μl FAST-OMV before gavage with green / red fluorescent agent / PBS (100 μl) for 2 h. The mice were euthanized 2 h after administration. The entire intestine was extracted and imaged with IVIS through the GFP / mCherry channel. The fluorescence intensity of each intestine was recorded.

[0125] Example 9. Tracking the interaction between FAST-OMV and bacteria (in vitro)

[0126] To visualize the communication between OMVs and STm, FAST-OMVs were co-incubated with STm expressing GFP in LB / PBS solution (1:1) at 37°C. Samples were collected at different time points. STm were separated by centrifugation, and HBR-3,5-DOM was added to turn on the FAST-OMV fluorescence signal, which was then imaged by LSCM and analyzed by flow cytometry. The assay for the interaction of FAST-OMVs with SA, BS and other bacteria was similar.

[0127] Example 10. Tracking the interaction between FAST-OMV and bacteria (in vivo)

[0128] Three mice were fed water only for 12 h before the administration of FAST-OMV (200 μl) at the 0 h time point. To observe the interaction between FAST-OMV and intestinal bacteria in vivo, FAST-OMV was administered to the small intestine and colon of ICR mice, respectively. The mice were killed 4 h later, and intestinal bacteria were separated from the intestinal contents by gradient centrifugation, resuspended in sterile PBS, and observed by LSCM after incubation with the fluorescent agent for 5 min.

[0129] Example 11. FAST-OMV activation / deactivation in mammalian cells in vitro

[0130] The mouse intestinal cell line MODE-K was obtained from the American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin / penicillin at 37°C in a humidified atmosphere of 5% CO2. To further confirm the interaction between FAST-OMV and intestinal epithelial cells, MODE-K cells were seeded into confocal culture dishes one day in advance. 100 μl of FAST-OMV was added to the culture dish, and an equal amount of PBS was added to the control group. After 1 h of co-incubation, 10 μl of green / red fluorescent agent was added, FAST-OMV was fluorescently illuminated, and its interaction with cells was observed by LSCM. To turn off the fluorescence of FAST-OMV, the cells were rinsed with fresh PBS to remove the fluorescein, and then imaged by LSCM.

[0131] Example 12. Tracking the interaction between FAST-OMV and mammalian cells in vivo

[0132] Mice were treated according to the method described in the in vivo part of the interaction between FAST-OMV and bacteria disclosed by the inventor team of this application (Cao, Z., Wang, X., Pang, Y., Cheng, S., Liu, J., Biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment. Nat. Commun. 2019, 10 (1), 5783.). The intestines of different segments were frozen and sectioned for confirmation of the interaction between FAST-OMV and cells. Each section was stained with a 10-fold diluted concentration of fluorescent agent in a dark room for 15 minutes, and then rinsed with PBS 3 times to remove the residual fluorescent agent. After staining the nuclei with DAPI, LSCM imaging was performed.

[0133] Example 13. Construction of STm-induced colitis mouse model

[0134] The STm-induced colitis mouse model was constructed using the method disclosed by the inventor team of this application (Cao, Z., Wang, X., Pang, Y., Cheng, S., Liu, J., Biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment. Nat. Commun. 2019, 10(1), 5783.).

[0135] Female C57BL / 6 mice (6-8 weeks old) were purchased from SPF (Beijing) Biotechnology Co., Ltd. After 7 days of acclimatization, the mice were randomly divided into groups and infected with 5×10 8 24h before CFU STm, treat with 100μl (200mg / ml) of streptomycin solution twice. Mice were treated with OMV, EcN or PBS daily on the second day after infection and euthanized on the eighth day. Healthy mice were used as the control group. Tissues from the terminal ileum, cecum, and top of the colon were collected, fixed for dissection, sectioned, and stained with hematoxylin-eosin (H&E). The complete intestinal tissue was collected and homogenized in a glass homogenizer. Each homogenate was resuspended and serially diluted with PBS, and 50μl of each dilution was coated on an LB agar plate containing antibiotics, incubated overnight at 37°C, and the bacteria were counted.

[0136] Example 14. Cytokine Assay

[0137] Treated mice were euthanized on day 8 after infection. Blood from each mouse was taken from the eye socket and stored in a 1.5 ml Eppendorf tube without ethylenediaminetetraacetic acid (EDTA). Serum was incubated at 37°C for 0.5 h, centrifuged at 10,000 × g for 5 min, separated, and ELISA kits were used to detect IFN-γ, TNF-α, and IL-6.

[0138] Example 15. Immunofluorescence staining of tight junction proteins in vitro

[0139] Caco-2 cells were inoculated and cultured for 24 h before treatment with 10 μl of LPS at a concentration of 1 mg / ml. 100 μl of OMV, EcN and PBS were added and incubated for 24 h. The cells washed with PBS were fixed with 4% paraformaldehyde for 20 min and then blocked with 5% normal goat serum in PBS for 30 min to remove nonspecific binding. The cells were further incubated with primary antibodies (anti-ZO-1 and anti-occludin) at 37°C for 2 h, followed by secondary antibodies for 1 h. Images were visualized using LSCM after DAPI staining.

[0140] Example 16. Immunofluorescence staining of intestinal tissue

[0141] The samples of STm-induced colitis mouse model were fixed with 4% paraformaldehyde, processed according to the standard procedure of paraffin embedding, and sliced ​​at 4 μm. The sections were blocked with 5% normal goat serum in PBS for 30 min to remove nonspecific binding, and then incubated with primary antibodies (anti-ZO-1 and anti-occludin) at 4°C overnight. After that, PBS was rinsed 3 times and incubated with secondary antibodies for 2 h at room temperature. After sealing the tissue sections with anti-fading solution containing DAPI, images were visualized using LSCM.

[0142] Example 17. Intestinal Permeability Evaluation

[0143] Mice with STm-induced colitis treated with EcN-OMV, EcN, or PBS were gavaged with FITC-dextran at a dose of 400 mg / kg body weight on day 8 after infection. Mice were fasted for 12 h in advance. Blood samples were collected 4 h after FITC-dextran administration. Plasma samples were collected by centrifugation of blood specimens (2000 × g, 4 °C for 15 min). Then, the fluorescence intensity of FITC-dextran in plasma was recorded using a fluorescent plate fluorometer with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A standard curve was established using serially diluted FITC-dextran samples.

[0144] Statistics and reproducibility

[0145] All experiments were biologically replicated at least three times to demonstrate successful reproducibility. n is the number of mice used for in vivo experimental analysis, and it represents the number of biological replicates used for in vitro experiments, which are mentioned in the graphs or legends. Pairwise comparisons were performed using a two-tailed Student's t test, and multiple comparisons were performed using Tukey's post-hoc, one-way analysis of variance (ANOVA). p<0.05 was considered significant. Data analysis was performed using GraphPad software.

[0146] Results and Analysis

[0147] 1. Preparation of FAST-OMV

[0148] In the process of preparing FAST-OMV in Example 1, the vector carrying the FAST gene was converted into EcN and continuously expressed and fused with a 6× histidine tag (FAST-His). After overnight culture, the bacteria were removed by centrifugation and then filtered through a membrane with a pore size of 450 μm. FAST-OMV was collected and purified by ultracentrifugation.

[0149] To improve the yield of FAST-OMV, inducers and antibiotics were used to test the culture under different conditions.

[0150] The results showed that compared with the culture without addition or with addition of 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG), the FAST-OMV production induced by equal amounts of IPTG + 6.25mg / l kanamycin was the highest, measured by the bisphenol A (BCA) method to be 6.32mg / ml. Interestingly, the size of FAST-OMV varied with the induction conditions. As determined by dynamic light scattering (DLS), in the absence of an inducer, the particle size range of FAST-OMV was widely between 10 and 100nm. After the addition of IPTG, the particle size range was reduced to 20-100nm ( Figure 2 .ab). After adding IPTG and kanamycin, the size of FAST-OMV was more uniform, with a particle size of 40-100 nm and a yield of 1.5×10 11 / ml or more, the protein content reaches about 8.8mg / ml ( Figure 2 .cd). Compared with conventional techniques, the yield and protein content of outer membrane vesicles are significantly improved, reaching 1.4 times and 1.5 times or more of the conventional yield, respectively.

[0151] Transmission electron microscopy (TEM) characterization revealed that antibiotic-induced FAST-OMVs had a unique bilayer structure and spherical morphology ( Figure 1 .e). Western blot confirmed the existence of FAST, such as Figure 1 As indicated by the black arrow in .f, it was successfully expressed and encapsulated in OMV.

[0152] 2. FAST-OMV fluorescence on / off

[0153] As a small protein marker, FAST produces different fluorescence in a dynamic and reversible manner by combining with various fluorescent agents under anaerobic and aerobic conditions. After confirming the presence of FAST in OMV, the fluorescence properties of FAST-OMV were detected using 20 μM HMBR or HBR-3,5-DOM fluorescent agents in the examples of this application.

[0154] As expected, after adding HMBR or HBR-3,5-DOM to the FAST-OMV suspension, bright green (λmax=541nm) and red (λmax=597nm) fluorescence signals were immediately observed when directly exposed to the dual-wavelength fluorescent protein excitation light source ( Figure 3 .a and b).

[0155] The dynamic initiation process was analyzed by laser confocal microscopy and flow cytometry (Beckman CytoFlex, USA). After adding HMBR or HBR-3,5-DOM, green or red fluorescent spots were fully distributed in the field of view ( Figure 3 .c and e). Flow cytometry results showed that the fluorescence intensity in OMVs activated by HMBR or HBR-3,5-DOM consistently shifted to the right ( Figure 3 .d and f).

[0156] Microplate readers and fluorescence spectrometers can also measure the activated FAST-OMV signal. Figure 3 As shown in Figure .gj, the fluorescence signal intensity increased significantly after FAST-OMV was combined with the corresponding fluorescein. Quantitatively, the mean fluorescence intensity (MFI) of FAST-OMV illuminated by HMBR or HBR-3,5-DOM was more than 100 times higher than that of the blank control without added fluorescein ( Figure 3 .h and j). These results indicate that the signal of FAST-OMV can be rapidly switched on demand and has the ability to emit fluorescence in different wavelengths by adding fluorescein.

[0157] Considering the dynamic binding behavior of FAST-OMV, the inventors of the present application studied the fluorescence shutoff of FAST-OMV by controlling the concentration of fluorescent substances. The pre-activated FAST-OMV was resuspended in a blank PBS buffer solution, washed by ultracentrifugation, and the fluorescence was detected to determine the on-demand signal shutoff capability. Typically, confocal images of FAST-OMV pre-activated by HMBR or HBR-3,5-DOM showed that the fluorescence intensity gradually decreased with the increase of the number of washes ( Figure 5.a). The decrease in fluorescence of FAST-OMVs is attributed to the decrease in fluorescein concentration, which may trigger the dissociation of HMBR or HBR-3,5-DOM from FAST proteins. In contrast to the fluorescence turn-on, the flow cytometric histograms of the green and red fluorescence signals of pre-activated FAST-OMVs gradually shifted to the left, which further supports the decay of fluorescence intensity when the fluorescein concentration decreases ( Figure 5 .b). Quantification data based on flow cytometry analysis showed that the signal intensity of FAST-OMVs pre-activated by HMBR and HBR-3,5-DOM decreased by 67% and 75%, respectively, after resuspension in blank PBS ( Figure 5 .c). The fluorescence spectrometer further recorded the process of fluorescence shutdown, during which the signal intensity also showed a similar decay trend ( Figure 5 .d). Unlike HBR-3,5-DOM, the decrease in fluorescence intensity of HMBR pre-activated nanovesicles was delayed, which means a stronger binding ability with FAST. It is worth noting that after two washes with PBS, HBR-3,5-DOM pre-activated FAST-OMVs maintained negligible emission intensity. This indicates that the fluorescence off can be controlled by adjusting the concentration of the fluorescer.

[0158] 3. Reversible switching between different emission bands

[0159] Considering the selectivity of FAST binding to HMBR and HBR-3,5-DOM and its concentration-dependent dynamic binding mode, the inventor team of this application further studied the ability of FAST-OMV to switch emission bands. In order to test the switching of λmax from 541 to 597nm, 20μM HBR-3,5-DOM was added to the HMBR-luminescent FAST-OMV suspension, and the emission after mixing was measured using LSCM, flow cytometer and fluorescence spectrometer. Figure 7 As shown in .a, after replacing the pre-activated FAST-OMV with HBR-3,5-DOM, the green fluorescence disappeared and bright red fluorescence appeared under confocal imaging. At the same time, flow cytometry analysis showed that the green fluorescence channel shifted to the left and the red signal channel shifted to the right ( Figure 7 .b). The fluorescence intensity changes of the two channels were recorded, and the results confirmed that the green fluorescence emission was significantly reduced, while the red signal increased significantly ( Figure 7 .c). In addition, after adding HBR-3,5-DOM to the HMBR pre-activated FAST-OMV suspension, the fluorescence spectrum at 541 nm almost completely disappeared, while a new emission peak appeared at 597 nm ( Figure 7 .d). In pre-activated FAST-OMVs, when the HBR-3,5-DOM concentration was greater than 20 μM, the fluorescent agent could compete with the bound HMBR, resulting in a switch in the emission band.

[0160] The inventors of the present application also studied the switching of λmax from 597nm to 541nm by resuspending FAST-OMV pre-activated by HBR-3,5-DOM in 20μM HMBR. Figure 7 As shown in Figure .eh, all measurements confirmed that, under the same experimental conditions, the pre-activated red signal could be reversibly switched to green fluorescence after FAST-OMV was exposed to a higher concentration of HMBR. Taken together, these experimental data demonstrate that FAST-OMV has an amazing tunability in switching the fluorescence emission band through fluorophore exchange.

[0161] 4. Tracking the Interactions of FAST-OMV with Microorganisms and Mammalian Cells

[0162] To visualize the communication between OMVs and microbial cells, pathogenic STm was directly co-incubated with FAST-OMVs in vitro, as EcN has been reported to inhibit STm growth through the secretion of micromycin. STm was bioengineered to express green fluorescent protein (GFP) to facilitate the observation of their interaction.

[0163] After incubation for the indicated time points, STm was separated by centrifugation, and HBR-3,5-DOM was added to initiate the fluorescence signal of FAST-OMV. A typical LSCM image shows that after 1 h of incubation, limited FAST-OMVs bind to STm ( Fig. 9 .a and Figure 4 .a). As the incubation time was extended to 3h, more FAST-OMVs were bound to the surface of STm, and some FAST-OMVs even fused with bacteria. In particular, after 3h of incubation, it was found that most of the red fluorescent FAST-OMVs were preferentially attached to both ends of STm. As indicated by the change of the fluorescent signal from green to orange and even red, as the incubation time was further extended to 6h, most of the STm fused with FAST-OMV. The presence of a large number of dead and lysed bacteria proved that the OMVs derived from EcN can attach well to STm from both ends, fuse, and then transport the internal FAST and micromycin to the fused bacteria.

[0164] Flow cytometry analysis further confirmed the direct interaction between FAST-OMV and STm, and the results showed that after the addition of HBR-3,5-DOM, the intensity of the red fluorescence signal in STm increased with the increase of incubation time ( Fig. 9 .b and Figure 4 .b). Consistent with the results shown by LSCM, the continuous increase in bacterial fluorescence intensity indicated that OMVs mediated the communication between EcN and STm.

[0165] The inhibitory effect of OMVs derived from EcN on STm was observed by bacterial growth inhibition assay. Fig. 9 From the growth curve of .c, it can be seen that co-incubation with OMV can greatly inhibit the proliferation of STm compared with untreated bacteria.

[0166] Other bacteria including Staphylococcus aureus (SA) and Bacillus subtilis (BS) were co-incubated to test whether the interaction was specific. Figure 6 As shown, FAST-OMV had no significant effect on the growth of BS and SA, which indicates that the vesicle nanoprobe prepared in the present invention regulates the intestinal flora by inhibiting specific bacteria.

[0167] Mouse intestinal cell line MODE-K cells were co-incubated with FAST-OMV to study the interaction between probiotic-derived OMVs and intestinal cells in vitro. After adding HMBR or HBR-3,5-DOM, it was observed that FAST-OMV was able to effectively enter MODE-K cells, and after co-incubation at 37°C for 1 h, strong green or red fluorescence signals could be seen by LSCM ( Fig. 9 .d). The fluorescence was turned off by washing the cells with fresh medium without fluorinated substances to further ensure that FAST-OMVs had successfully entered the MODE-K cells ( Fig. 9 .e).

[0168] In addition to verifying the behavior of EcN-derived OMVs entering intestinal epithelial cells, the on-demand switching on / off of fluorescence by adding or removing the corresponding fluorescent agents also demonstrated the stronger and wider applicability of FAST-OMVs in intracellular imaging and tracking.

[0169] By changing the fluorescent agent, the intracellular signal is observed to switch from green to red or from red to green fluorescence ( Fig. 9 .f and g). In order to investigate the communication function of FAST-OMV with intestinal cells, considering the tight junction level which is crucial for maintaining intestinal homeostasis, which reflects the integrity of the epithelial barrier, the inventors of the present application performed immunofluorescence detection of tight junctions including zona occludens-1 (ZO-1) and anti-occludens.

[0170] Caco-2 cells, as the main epithelial cell line of the large intestine, were treated with 0.1 mg / ml FAST-OMV and 5 μg / ml LPS at 37°C for 24 h. The tight junction protein levels were captured by LSCM and Fig. 9 .h. Compared with co-incubation with LPS and PBS or EcN, the levels of ZO-1 and occludin in epithelial cells after LPS and FAST-OMV treatment were similar to those of untreated cells.

[0171] The experimental results show that in an external simulated environment, FAST-OMV has the ability to maintain the integrity of the physical barrier by entering and promoting tight junction expression. The intelligent two-color imaging achieved by FAST-OMV verifies that the nanovesicle probe of the present invention has the ability to work normally in living cells and reveals the positive interaction between probiotic-derived OMVs and intestinal cells.

[0172] 5. Tracking OMVs in the Intestine

[0173] Since FAST has an emission property independent of oxygen molecules, the inventors of the present application explored the anaerobic tracking of intestinal flora-derived OMVs in vivo. Mice were gavaged with FAST-OMV, and a fluorescent agent was applied 2 hours after ingestion. Mice treated with FAST-OMV were used as the experimental group, and mice treated with PBS were used as the control group. 4 hours after FAST-OMV administration, mice were euthanized, and samples were taken from the intestines of each mouse and observed using an in vivo imaging system (IVIS).

[0174] like Fig.11 .a and Figure 8 As shown in the figure, the intestine of mice treated with FAST-OMV+fluorescent agent showed bright fluorescence signals compared with the control group, especially in the small intestine and colon, which is consistent with the results of on-demand fluorescence activation after adding HMBR and HBR-3,5-DOM in vitro. Moreover, OMVs and intestinal flora can be observed in vivo.

[0175] Accordingly, 200 μl of FAST-OMV was injected into the small intestine and colon of mice, and 4 h after injection, the luminal contents were extracted and resuspended in PBS solution. The relevant bacteria were isolated by gradient centrifugation, activated with HMBR or HBR-3,5-DOM, and then fixed on agar plates for confocal imaging.

[0176] like Fig.11 As shown in Figure .b, many different bacteria were observed, which fluoresced green or red, depending on the type of fluorescent agent applied. More importantly, some bacteria did not fluoresce, indicating that the fusion of FAST-OMV with the intestinal flora was selective ( Fig.10 and Fig.11 ). These results demonstrate the potential of the probe prepared in the present invention to intelligently track gut microbiota-derived OMVs and visualize the interactions between these OMVs and different bacterial species in the intestine.

[0177] These experimental results prompted the inventors to test the ability of FAST-OMV to visualize the communication between OMVs and intestinal epithelial cells in vivo. The small intestine and colon tissues of mice were frozen and sectioned for immunohistochemical analysis. After staining with 4',6-diamidino-2-phenylindole (DAPI), HMBR or HBR-3,5-DOM were supplemented and the sections were imaged using LSCM.

[0178] like Fig.11 .c and Fig.14 As shown, FAST-OMVs resulted in enhanced intracellular fluorescence signals compared to PBS-treated control mice, suggesting that gut microbial OMVs can translocate into enterocytes located in the small intestine and colon.

[0179] Furthermore, the inventors of the present application also studied the effects of FAST-OMV on inhibiting specific pathogens and promoting the secretion of tight junction proteins in STm-infected mice. Starting from the second day after infection, FAST-OMV was gavaged daily. On the eighth day, the mice were euthanized and sampled. 1×10 8 CFU of EcN and PBS-treated mice were used as controls. Small intestine, cecum, and colon tissue samples and the corresponding luminal contents were homogenized and plated on Luria Bertani (LB) agar plates for bacterial counts. Fig.13 As shown in Figure .a, compared with the control group, the STm bacterial counts in different samples of mice treated with FAST-OMV were significantly reduced. Compared with the EcN and PBS treatment groups, the total number of STm in mice treated with FAST-OMV was reduced by 87% and 95%, respectively, which proves that FAST-OMV has a significant inhibitory effect on specific pathogens in the intestine ( Fig.13 .b).

[0180] In addition, the inventors of the present application also studied the communication of FAST-OMV with enterocytes in the intestine of STm-infected mice. Immunofluorescence detection of intestinal epithelial cells was performed to evaluate the role of FAST-OMV in maintaining the integrity of the intestinal physical barrier. Fig.13 .ce and Figure 16-18 As shown in the results, the levels of tight junction proteins were significantly increased compared with the PBS control group and EcN. It is certain that FAST-OMV can prevent the loss of tight junctions, epithelial cell shedding and death after intestinal infection, and can repair the integrity of the epithelial barrier.

[0181] 6. The therapeutic value of OMV in colitis

[0182] In view of the unprecedented advantages of FAST-OMV shown or demonstrated by the above experimental results, the inventors of the present application further tested its therapeutic value in colitis characterized by microbial structure disorder and impaired intestinal barrier.

[0183] Mice were infected with 5×10 8 CFU of STm, and then oral administration of FAST-OMV ( Fig.15 .a). Healthy mice treated with PBS and EcN and mice with STm-induced colitis were used as control groups, respectively. Mice were killed on day 8, and serum samples were collected via eye sockets for enzyme-linked immunosorbent assay (ELISA) to assess the level of inflammation. Fig.15 As shown in .bd, compared with mice treated with PBS and EcN, the levels of cytokines in the serum of mice treated with FAST-OMV were significantly reduced, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (IFN-γ). At the same time, the weight fluctuation of mice treated with FAST-OMV was smaller after treatment ( Fig.15 .e). EcN- and PBS-treated mice showed obvious histological inflammation, but FAST-OMV-treated mice showed no obvious histological inflammation ( Fig.15 .f,i and Figure 19-21 ).

[0184] The present application also analyzed the length of the colon, which was not significantly reduced in both FAST-OMV-treated mice and the healthy group ( Fig.15 .g). ​​Intestinal permeability test showed that the level of fluorescein isothiocyanate dextran (FITC-dextran, 4kDa) in serum samples of healthy mice and FAST-OMV-treated mice was significantly lower than that in the EcN group and PBS group, indicating that the intestinal barrier permeability was reduced after vesicle treatment ( Fig.15 .h). It is speculated that the reduction in intestinal permeability may be related to the ability of FAST-OMV to restore the integrity of the intestinal barrier, which is consistent with the enhanced expression of tight junction proteins such as ZO-1 and occludin in in vitro and in vivo immunofluorescence assays. In conclusion, FAST-OMV-enabled anaerobic tracking reveals the multifaceted functions of gut microbiota-derived vesicles, which can serve as targets for the treatment of intestinal diseases such as colitis.

[0185] Based on the above experiments and results, the present invention prepares FAST-OMV by stimulating the formation and release of bacterial vesicles through synthetic bioengineering and antibiotics. FAST-OMV has a natural structure and fluorescence emission that is independent of oxygen molecules, and can be used as an endogenous nanoprobe for anaerobic tracking of intestinal flora derivatives. Specifically, the intestinal probiotic EcN is genetically modified to express FAST, and a certain amount of ampicillin is used to induce during bacterial growth to improve the generation and separation of FAST-OMV. Utilizing the dynamic and reversible combination of FAST and HMBR or HBR-3,5-DOM, FAST-OMV shows that this type of vesicle has significant on-demand signal switching and reversible switching of emission wavelengths by supplementing, removing or exchanging the corresponding fluorescent agent. With the help of FAST intelligent two-color imaging technology, intestinal visualization can be achieved, and the fusion of intestinal microbial OMV with specific bacteria can be identified, inhibiting the growth of pathogens and entering intestinal epithelial cells, thereby maintaining the biological function of intestinal barrier integrity. The technical solution of the present invention has shown that the outer membrane vesicles can be used as a detection reagent, and can also be used as an effective ingredient of a drug for preventing, alleviating or treating intestinal diseases, both of which have great potential uses.

[0186] The above are only specific embodiments of the present invention, but the present invention is not limited thereto. All variations that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered to be included in the protection scope of the present invention.

Claims

1. An endogenous nanoprobe, characterized in that: The nanoprobe is a nanovesicle carrying an oxygen-independent controllable fluorescent label, wherein the nanovesicle is an outer membrane vesicle produced by Escherichia coli EcN carrying pQE60-FAST-His.

2. The endogenous nanoprobe according to claim 1, characterized in that: The size of the outer membrane vesicles is 40-100 nm.

3. A method for preparing an endogenous nanoprobe, characterized in that: The method comprises: Plasmid pQE60-FAST-His was constructed and transformed into Escherichia coli; Screening to obtain ampicillin-resistant strains; Induce, culture in a culture medium supplemented with 4.0-10.0 mg / l kanamycin, and separate to obtain outer membrane vesicles of uniform size, wherein the outer membrane vesicles are endogenous nanoprobes carrying FAST tags.

4. Use of the endogenous nanoprobe prepared by the method according to claim 3 in preparing drugs for preventing, alleviating or treating intestinal diseases, characterized in that: The intestinal disease is enteritis caused by bacterial infection.

5. A biosensor system, characterized in that: The biosensor system at least comprises the nanoprobe described in any one of claims 1 to 2 or the nanoprobe prepared by the method of claim 3.

6. A biological imaging system, characterized in that: The biological imaging system at least comprises the nanoprobe described in any one of claims 1 to 2 or the nanoprobe prepared by the method of claim 3.

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