CN116271107B

CN116271107B - Endogenous nano probe, preparation method and application thereof

Info

Publication number: CN116271107B
Application number: CN202211519745.XA
Authority: CN
Inventors: 刘尽尧; 曹浈萍; 刘蕊
Original assignee: Renji Hospital
Current assignee: Renji Hospital
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2025-05-13
Anticipated expiration: 2042-11-30
Also published as: CN116271107A

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

Endogenous nano probe, preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to an endogenous nano probe, a preparation method and application thereof, wherein the specific application comprises application of the nano probe in aspects of biological sensing, imaging, disease detection and pharmacy.

Background

The human intestinal microbiota consists of about 10-100 trillion microbial cells. As a major effector component, it is estimated that more than 1000 bacteria maintain metabolic homeostasis through the gut-liver, gut-lung, gut-brain, etc., regulate host immune and nervous systems, play a key role in human health. Behind these intestinal bacteria there are a large number of exogenous membrane vesicles (Outer Membrane Vesicles, OMV) of average size about 20-250nm, which are released from the outer surface of the bacteria and retain the functions inherent to the outer membrane. OMVs retain intracellular material associated with the parent bacteria and play a vital role in the interplanting and endophytic behaviour of bacteria, such as intercellular communication, resistance to phage infection, export of cellular metabolites and toxicity. In addition to microbial Wang Guojian communication (Inter-Kingdom Communication), OMVs released by gut commensal microorganisms have been shown to be critical for the maturation of the immune system, whereas OMVs produced by pathogens can promote inflammation and infection in the host. Numerous studies have shown that intestinal flora-derived OMVs can metastasize to other major organs and cause various dysfunctions. It has also been reported that the accumulation level of OMVs in serum and urine can reflect the pathogenesis and progression of a variety of tumors.

Despite the important role of OMVs, the current suitable methods by which intestinal flora-related OMVs can be tracked and analyzed to fundamentally understand their ultimate impact on host physiology and pathophysiology are very limited. Although Bittel, m.et al disclose a simple method (Visualizing transfer of microbial biomolecules by outer membrane vesicles in microbe-host-communication in vivo,J Extracell Vesicles.2021,10,e12159.), for tracking OMV in vivo in the context of expressing CRE-recombinase and reporter molecules in bacteria and host cells, respectively, it is understood that no visualization strategy has been disclosed or reported so far that is capable of imaging and tracking OMV distribution and interaction between OMVs and intestinal microorganisms and mammalian host cells in the gut. Thus, there is a continuing need for imageable probes that are applicable to living subjects, which is critical to the visualization of complex biological processes.

In particular, unlike small molecular probes, nanoprobes have been one of the subjects of research by researchers in the field due to their own advantages, such as inherent physicochemical adjustability, ease of functional modification, higher sensitivity, specificity, targeting ability, and the like. To date, 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.

In view of the nano-size and its effects, nanoprobes have been widely explored in vivo to obtain superior properties such as depth penetration and adjustability of distribution in different tissues, in order to better visualize dynamic processes of living biological systems, smart nanoprobes have been designed for acquiring real-time in situ information in complex physiological environments, in particular, stimulus-responsive probes relying on the use of specific physiological markers as stimulators have been widely explored for sensing, bioimaging and disease diagnosis. For example, reactive Oxygen Species (ROS) sensitive nanoprobes have been developed to recognize the occurrence and progression of acute liver failure and osteoarthritis, and hypoxia, pH and azo reductase responsive nanoprobes have been fabricated 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.

Furthermore, conventional nanoprobes are exogenous and are not suitable for monitoring specific biological processes in the living system, such as OMVs distribution in the intestinal tract and interactions with the surrounding environment. Because anaerobic microenvironments exist in the gut, there is still a need for reporting from nanoprobe signals independent of oxygen molecules to track OMVs, and thus existing nanoprobes cannot be used directly to track OMVs in vivo and image, and are not able to detect specific biological processes in the living system.

Disclosure of Invention

In view of the above-mentioned shortcomings in the prior art and the need for endogenous probes and imaging thereof in the biomedical field, the present invention aims to provide an endogenous nanoprobe, a preparation method and applications thereof, and specific applications include applications of the nanoprobe in biosensing, imaging, disease detection and pharmacy.

In a first aspect, the invention provides an endogenous nanoprobe, a nanoprobe carrying a Fluorescence-activated and Absorption-transfer tag (Fluorescence-ACTIVATING AND Absorption-SHIFTING TAG, FAST), wherein the nanoprobe is an Outer Membrane Vesicle (OMV) produced by pQE 60-FAST-His-carrying escherichia coli (EcN), having a native structure and an oxygen molecule independent emission spectrum, and the targets are visually recognized, tracked or inhibited in an aerobic or anaerobic environment.

Further, the nanovesicles are outer membrane vesicles produced by culturing E.coli EcN carrying pQE60-FAST-His in ampicillin-added medium, inducing with IPTG, culturing, and adding kanamycin.

Further, the target may be a bacterium, pathogen or intestinal epithelial cell in the intestinal flora.

In a second aspect, the present invention provides a method of preparing an endogenous nanoprobe, comprising:

constructing a plasmid pQE60-FAST-His, and converting the plasmid pQE60-FAST-His into escherichia coli;

screening to obtain ampicillin-resistant strains;

Inducing, culturing and separating in culture medium with final concentration of 4.0-10.0mg/l kanamycin to obtain FAST-labeled outer membrane vesicle FAST-OMV, wherein the outer membrane vesicle is endogenous nanometer probe carrying FAST label.

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

Further, induction was performed under the inducer IPTG, with a final concentration of 0.5M IPTG.

Further, the outer membrane vesicle yield is 1.5X10 ¹¹/ml or more.

Further, the nanovesicles have a natural outer membrane vesicle structure and an independent emission spectrum of oxygen molecules, and the targets are visually recognized, tracked or inhibited in an aerobic or anaerobic environment. Preferably, the interaction between the bacterial outer membrane vesicles and the intestinal flora or intestinal epithelial cells is followed.

In a third aspect, the present invention also provides an application of the endogenous nano probe in preparing a medicament for preventing, slowing down or treating intestinal diseases.

Further, the intestinal disease is an inflammatory bowel disease and a cancer caused by intestinal inflammation. More specifically, the intestinal disease is enteritis caused by bacterial infection. Preferably, the disease is salmonella-induced enteritis.

In a fourth aspect, the present invention also provides a biosensing system comprising at least a nanoprobe according to the present invention.

In a fifth aspect, the present invention also provides a biological imaging system, which at least comprises the nanoprobe of the present invention, and tracks and images in real time in an organism.

Advantageous effects

The endogenous nano probe has strong specificity and high efficiency, has the flexibility of on-demand fluorescent switch and reversible switching of emission wave bands, can be used for intelligent and bicolor imaging, thereby remarkably improving the sensitivity and accuracy of detection, has low severity of required detection equipment and sites, has low cost, and has important research significance and application value.

The application of the endogenous nano probe is wide, and experiments prove that the application of the nano probe in preparing medicines for preventing, slowing down or treating intestinal diseases, a biological sensing system at least comprising the nano probe and a biological imaging system at least comprising the nano probe are provided. Importantly, the nanoprobes of the present application are useful in multimodal imaging, which are endogenous substances, suitable for monitoring specific biological processes in the living system, such as OMV distribution in the intestinal tract and interactions with the surrounding environment. In the anaerobic microenvironment of the gut, the oxygen molecule independent signal from the nanoprobe can report and track OMVs.

Drawings

FIG. 1 is a schematic representation of the design, preparation and characterization of FAST-OMVs according to the present invention. Wherein:

FIG. 1.A, schematic representation of the preparation of FAST-OMVs;

FIG. 1.B, on-demand fluorescence switches and reversibly switchable emission bands of FAST and FAST-OMVs;

FIG. 1.C, showing anaerobic tracking of the intestinal FAST-OMVs, which shows selective fusion with intestinal flora and entry into intestinal epithelial cells;

Fig. 1.D, size distribution of FAST-OMVs measured by DLS (n=3);

FIG. 1.e, scale 200nm, shows EcN FAST production under IPTG and kanamycin induction, followed by isolation of representative TEM images of the FAST-OMVs produced;

FIG. 1.f Western blot analysis of FAST-OMVs with enzyme-labeled anti-His antibodies (1:500), M is a pre-stained protein tag, with EcN-derived OMVs carrying empty vector as negative control.

FIG. 2 is a representation of the detection of FAST-OMVs by a different method, wherein:

FIGS. 2.A-b, size distribution of FAST-OMVs obtained under different conditions, measured by DLS, wherein purple is the OMVs derived from bacteria cultivated in LB medium, green is the OMVs produced by bacteria cultivated in LB medium with 0.5M IPTG, and blue is the OMVs produced by bacteria cultivated in LB medium containing 0.5M IPTG and 6.25mg/l kanamycin.

FIG. 2.C, protein concentration of FAST-OMV obtained under different conditions was detected by BCA.

FIG. 2.D, concentration of FAST-OMV obtained under different conditions was detected by NTA.

FIG. 3 is a representation of fluorescence initiation of FAST-OMVs according to the invention wherein:

FIG. 3.A, schematic representation of fluorescence initiation of FAST-OMV binding to a fluorescent agent;

FIG. 3.b is a fluorescence image of FAST-OMV suspension with addition of corresponding fluorescent agent under dual wavelength protein excitation light;

FIG. 3.c-f, (c, e) confocal imaging and (d, f) flow cytometry analysis of FAST-OMVs with 20. Mu.M HMBR (left) or HBR-3,5-DOM (right). The scale bar is 10 mu m;

FIG. 3.g-j, fluorescence spectra (g, i) and data (h, j) of emission peak signal intensity using FAST-OMVs of 20 μM HMBR (left) or HBR-3,5-DOM (right) (n=3);

Significance was assessed using Student's t test, yielding p values p <0.001.

FIG. 4 shows in vitro tracking of FAST-OMV interactions with microbial cells in the present invention, wherein:

FIG. 4.A, enlarged LSCM image for visualization of in vitro interactions of FAST-OMV and STm, red, FAST-OMV co-incubated with GFP-expressing STm for a predetermined time interval, wherein white arrow indicates HBR-3,5-DOM activated FAST-OMV, blue arrow indicates STm-derived OMV, white dashed line indicates destroyed bacteria, scale: 2 μm;

FIG. 4.B, flow cytometry analysis shows interactions of FAST-OMVs with STm in vitro, wherein red FAST-OMVs were co-incubated with GFP-expressing STm at predetermined time points.

FIG. 5 shows fluorescence shut down of FAST-OMVs, wherein:

FIGS. 5.a and b, LSCM image (a) and flow cytometer histogram (b) after 1, 2, 3 flushes, scale bar 10 μm;

fig. 5.C and d, flow cytometry analysis (c) and fluorescence spectrum (d) by quantitative analysis of fluorescence shut-off procedure of pre-activated FAST-OMV by flushing the fluorescent agent at the indicated time points (n=3).

Figure 6 shows in vitro tracking of FAST-OMV interactions with different bacteria, wherein:

Figures 6.a and b, growth curves of BS and SA at 37 ℃, with or without addition of EcN derived OMVs, OD ₆₀₀ (n=3) was recorded using a microplate reader;

FIGS. 6.C and d, typical confocal images of GFP expression after incubation of BS and SA with FAST-OMV for 3h and subsequent activation by HBR-3, 5-DOM. Scale bar 5 μm;

fig. 7 is a reversible switching of the transmit band, wherein:

FIGS. 7.a and b, by substituting HBR-3,5-DOM, HMBR pre-activated FAST-OMV, on a scale of 10 μm with representative LSCM image (a) and flow cytometer histogram (b) with no or fluorescent emission from green to red;

fig. 7.C and d, quantitative analysis of switching λmax from 541nm to 597nm, flow cytometry (c) and fluorescence spectrometer measurement (d) (n=3);

FIGS. 7.e and f, confocal imaging data (e) and flow cytometry analysis (f) data, scale: 10 μm, of HBR-3,5-DOM pre-activated FAST-OMV without or with 597nm to 541nm emission switch under HMBR exposure;

fig. 7.g and h, quantitative analysis of red and green fluorescence signal emission switching (n=3) measured by flow cytometry (g) and fluorescence spectrometer (h).

Fig. 8 shows representative IVIS images of the mouse gut after lavage with FAST-OMV, wherein mice were euthanized 2h before gastric lavage with green/red fluorescent agent, 2h after administration of FAST-OMV, and the gut was collected for IVIS imaging through the corresponding channel. Mice were treated with FAST-OMV for 2h, followed by oral PBS for 2h, IVIS imaging via GFP and mCherry channels as control group (n=5).

FIG. 9 shows in vitro tracking of FAST-OMVs interactions with microorganisms and mammalian cells, wherein:

FIG. 9.a and b, representative confocal images of STm expressing GFP (a) and flow cytometer histograms (b) at indicated time points after co-incubation with FAST-OMVs and subsequent activation by HBR-3,5-DOM, scale bar 5 μm. The grey dotted line represents the enlarged picture, the white arrow is the HBR-3,5-DOM activated FAST-OMV, the blue arrow is the STm derived OMV, the scale bar is 2 μm;

fig. 9.c, a microplate reader was used to record the STm growth curve (n=3) at 37 ℃ with or without EcN derived OMVs added at 600nm (OD ₆₀₀);

FIG. 9.d confocal images of MODE-K cells after incubation with FAST-OMV for 1h at 37℃and subsequent activation with HMBR or HBR-3, 5-DOM;

FIG. 9.e confocal images of FAST-OMV treated MODE-K cells at indicated time points after rinsing with PBS, scale bar 20 μm;

FIGS. 9.f and g, in a scale bar of 20 μm, confocal images of MODE-K cells treated with HBR-3,5-DOM pre-activated FAST-OMV and supplemented with HBR-3,5-DOM (f) and HMBR (g), respectively;

FIG. 9.h immunofluorescence images of Caco-2 cells after 24h treatment at 37℃with 0.1mg/ml FAST-OMV and 5. Mu.g/ml LPS, where red, green and blue are ZO-1, occludin and nuclei, respectively. The scale bar is 25 μm.

Fig. 10 shows a typical LSCM image of intestinal bacteria isolated from the colon of a FAST-OMV treated 4h mouse. The isolated bacteria were resuspended in PBS containing HBR-3,5-DOM for 5min and then captured by LSCM. Scale bar 5 μm. White dotted squares represent intestinal bacteria illuminated by the FAST-OMV and black dotted squares represent bacteria not illuminated by the FAST-OMV.

Fig. 11 shows tracking OMVs in the intestinal tract, wherein:

FIG. 11.A, section of mouse intestinal tract 4h after FAST-OMV lavage with no or additional absorbable fluorescent agent, and imaging with IVIS;

FIG. 11b, bacterial confocal images isolated from mice small intestine and colon 4h after FAST-OMV injection, followed by staining with the corresponding fluorescent agent. Scale bar 5 μm;

FIG. 11c, confocal images of frozen sections stained with HMBR or HBR-3,5-DOM, taken from FAST-OMV injected mice, with nuclei stained with DAPI, scale bar 20 μm.

FIG. 12 shows a typical LSCM image of intestinal bacteria sampled from the colon of a FAST-OMV treated mice for 4 hours, wherein isolated bacteria were resuspended in PBS with green HMBR for 5min and then observed with LSCM, scale bar 5 μm, white dotted squares represent bacteria illuminated by FAST-OMV, and black dotted squares represent non-luminescent bacteria.

FIG. 13 shows the in vivo function of OMVs, after 2d infection with 5X10 ⁸ CFU STm, mice were daily gastrected with FAST-OMV, 1X 10 ⁸ CFU EcN or PBS respectively, and after 6d, euthanized and sampled, healthy mice were used as control groups, wherein:

FIG. 13.A, STm bacteria were counted for small intestine, cecum and colon, respectively;

fig. 13 b, total amount of STm in the intestine after different treatments, data are mean ± SEM, n=5, using analysis of variance ANOVA test and Tukey's post hoc test to evaluate significance, resulting in p-value, ×p <0.0001;

FIGS. 13.C-e, immunofluorescence images of ileum, cecum and colon tissues, wherein red, green and blue represent ZO-1, occludin and nucleus, white dashed panels represent normal expression and distribution of tight junction proteins, white large arrows represent severe epithelial tissue destruction due to loss of tight junction and death of epithelial cells, accompanied by significant edema and inflammatory infiltrates of submucosa, white small arrows represent villus shortening and structural damage, scale: 50 μm.

FIG. 14 shows confocal images of frozen sections taken from mice administered with FAST-OMV or PBS, in which the small intestine and colon of mice treated with FAST-OMV were frozen and stained with DAPI and the corresponding fluorescent agent, and frozen sections taken from mice administered with PBS as controls, in a scale of 50. Mu.m.

Fig. 15 shows experimental results of OMVs in terms of treatment of STm-induced colitis, wherein:

FIG. 15.A, treatment experiments were designed with mice infected with 5X 10 ⁸ CFU STm and treated with PBS, 1X 10 ⁸ CFU EcN or FAST-OMV, healthy mice as controls;

FIG. 15.B-d, ELISA assay for IL-6, TNF-alpha, IFN-gamma cytokine levels in serum;

fig. 15 e, body weight fluctuation during treatment;

Fig. 15.F, images of colon tissue from cecum to rectum resected after treatment;

fig. 15.G, average length of colon after treatment;

FIG. 15.H, intestinal permeability was determined by recording FITC-dextran levels in plasma;

fig. 15 i, H & E staining images of ileum, cecum and colon, scale: 100 μm, data are mean ± SEM, n=4 or 5. Significance was assessed using one-way ANOVA and Tukey's post hoc test to give p values p <0.05, p <0.01, p <0.005.ns, no significance.

FIG. 16 shows immunofluorescence images of ZO-1 and occludin expression in ileal epithelial cells sampled from STm infected mice, scale bar 100 μm.

FIG. 17 shows immunofluorescence images of ZO-1 and occludin expression in cecal epithelial cells sampled from STm infected mice, scale bar 400 μm (left), 100 μm (right).

FIG. 18 shows immunofluorescence images of ZO-1 and occludins expressed by colonic epithelial cells sampled from STm infected mice, scale bar 100 μm.

Figure 19 shows typical H & E stained images of ileum after different treatments of colon mice, wherein the black squares represent magnified pictures, scale bars 400 μm (blue) and 50 μm (black).

FIG. 20 shows representative H & E stained images of ceca from a colon inflammatory mice after treatment, with black panes representing magnified pictures, scale bars 400 μm (blue) and 50 μm (black).

Fig. 21 shows an H & E stained image of colon after treatment of colitis mice, wherein the black boxes represent the corresponding enlarged image, the black dotted boxes represent the integrity of the epithelial cell layer, the red arrows point to healthy and normal muscle layers, the black arrows represent inflammatory and disrupted muscle layers, the scales 400 μm (blue) and 50 μm (black).

Detailed Description

The technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto. Any modification, adjustment or modification, or equivalent replacement method that can be implemented by those skilled in the art to which the present invention pertains will fall within the scope of the claimed invention without departing from the technical idea and technical solution of the present invention.

The experimental methods used in the following examples are conventional means or methods in the art unless specifically indicated. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In a first aspect, the present invention provides an endogenous nanoprobe, a FAST-tagged nanovesicle, wherein the nanovesicle is an outer membrane vesicle produced by pQE 60-FAST-His-bearing escherichia coli EcN, having a native structure and an oxygen molecule independent emission spectrum, and wherein targets are visually recognized, tracked, or inhibited in an aerobic or anaerobic environment.

Further, the nanovesicles were outer membrane vesicles produced by culturing pQE60-FAST-His E.coli EcN in ampicillin-added medium, induction with IPTG, culture, and kanamycin addition.

Further, the size of the nano vesicles is 40-100nm.

constructing a plasmid pQE-FAST-His, and converting the plasmid pQE-FAST-His into escherichia coli;

screening to obtain ampicillin-resistant strains;

Inducing, culturing in a culture medium added with kanamycin, and separating to obtain the outer membrane vesicle FAST-OMV carrying the FAST label.

Further, the outer membrane vesicle yield is 1.5X10 ¹¹/ml or more.

Further, the nanovesicles have a natural outer membrane vesicle structure and an independent emission spectrum of oxygen molecules, and the targets are visually recognized, tracked or inhibited in an aerobic or anaerobic environment.

In a fourth aspect, the invention also provides a kit for detecting an intestinal disease, the kit comprising at least the endogenous nanoprobe.

In a fifth aspect, the present invention also provides a biosensing system comprising at least a nanoprobe according to the present invention.

In a sixth aspect, the present invention also provides a biological imaging system, which at least includes the nanoprobe of the present invention.

Materials and strains

The Escherichia coli Nissle 1917 strain was purchased from China center for general microbiological culture collection center. FAST is a variant of mutated Photoactive Yellow Protein (PYP). A plasmid pQE60-FAST-His (ampicillin-resistant) capable of expressing FAST in EcN was constructed using a FAST gene template described in LiC et al, dynamic multicolor protein marker of living cells, (Li,C.,Plamont,M.A.,Sladitschek,H.L.,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 cultivated in LB medium at 37℃and appropriate amounts of antibiotics were added. HMBR (green fluorescer) and HBR-3,5-DOM (red fluorescer) were taught by Chenge Li doctor and Arnaud gautier.

EXAMPLE 1 FAST-OMV extraction

EcN carrying pQE60-FAST-His was first cultured in LB medium supplemented with ampicillin until OD ₆₀₀ reached 0.5-0.7, and then induced by addition of IPTG. After 2h, 6.25mg/l kanamycin was added to the bacterial cultures to allow the bacteria to produce more OMVs. Bacterial culture supernatants were collected, centrifuged at 7000 Xg for 1h, the pellet removed, and then filtered with a 0.22 μm filter to avoid undesired bacterial cells. Finally, the filtered supernatant was centrifuged at 170000Xg for 1h at 4℃to obtain OMV pellet, which was then stored in PBS (pH 7.2-7.4) in a resuspended state.

EXAMPLE 2 characterization of FAST-OMV

The morphology of the OMVs was visualized using a transmission electron microscope (HITACH, japan). A drop of OMV solution was deposited on the copper grid of the carbon coating. Washing with double distilled water (DDH ₂ O) 2 times for 5min each. And (5) observing after airing. The average size and concentration of OMVs were determined from DLS (Malvern Zetasizer nano ZS, UK) and NTA (Malvern nanosight NS, UK). OMV concentration is defined by total protein concentration measured using BCA assay kit (Thermo Scientific).

EXAMPLE 3 Western blot analysis of FAST-His

OMVs were isolated from EcN carrying empty vector and EcN carrying FAST-His expression vector, respectively, and resuspended in PBS after purification. Mu.l of purified FAST-OMV was mixed with loading buffer, boiled and loaded on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The isolated samples were transferred to polyvinylidene fluoride (PVDF) membranes by a Bio-rad semi-dry transfer turbine according to the preprogrammed procedure for a 1.5mm gel of 1.3A,25V,10min (Bio-rad, USA). PVDF membranes were incubated for 1h in TBS/Tween with 5% skim milk, followed by incubation with horseradish peroxidase (HRP) -conjugated anti-His antibodies (1:5000, AE028, ab clone, china). The synthesized membrane was then incubated with 1ml of enhanced chemiluminescent solution for 1min, and then captured with a chemiluminescent imager (Bio-rad, USA).

Example 4 in vitro Start-Up

Green and red fluorescers (HMBR and HBR-3, 5-DOM) were added to PBS containing FAST-OMV at room temperature. The dynamic initiation process was directly imaged using Luyoro-3415RG dual wavelength fluorescent protein excitation light source and LSCM (Lycra TCS SP8, germany) and analyzed by flow cytometry (Beckman CytoFlex, USA). Their intensities and λmax were measured using a fluorescence spectrometer (Fluormax-4,HORIBA Scientific,Japan).

Example 5 in vitro shut off

The FAST-carrying OMVs were incubated with green and red fluorescent agents, respectively, for several minutes at room temperature to fully activate FAST fluorescence. To shut down the FAST-OMVs by removing bound fluorescent agent, the OMVs were resuspended and washed a predetermined number of times with PBS. The dynamic shutdown procedure was analyzed using LSCM and flow cytometry, and fluorescence intensities of green FAST-OMV and red FAST-OMV after removal of the fluorescent agent in PBS solution were recorded using a fluorescence spectrometer.

Example 6 in vitro reversibility (from green to red)

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

Example 7 in vitro reversibility (switching green from red)

FAST-OMVs were activated by incubation with red fluorescent agent for several minutes. Red OMVs were immobilized on agar plates starting with LSCM imaging. To observe the reversibility of FAST-OMVs from red to green, 4 μl of green fluorescein was added to the red FAST-OMVs, captured by GFP and mCherry channels, until the red fluorescence disappeared. It is likely that the reversibility from red to green is analyzed by measuring green and red fluorescence intensities. Green fluorescent agent was added to the red fluorescent agent activated FAST-OMV solution and incubated at room temperature. And (3) respectively measuring the green and red fluorescence intensities and the lambda max switching of the FAST-OMV by using a flow cytometer and a fluorescence spectrometer.

Example 8 initiation in anaerobic gastrointestinal tract

All animal experiments were conducted under guidelines of the university of Shanghai transportation animal protection and evaluation and approval using the ethical committee of the professional committee. Experiments were performed using 6-8 week old male ICR mice purchased from SPF (Beijing) biotechnology Co., ltd, and kept under SPF (specific pathogenfree, SPF) conditions for 4 days. The mice were randomly divided into 3 groups (n=5) and 200 μl FAST-OMV was orally administered per ICR mouse before 2h gavage with green/red fluorescent agent/PBS (100 μl). Mice were euthanized 2h after dosing. The whole intestine was extracted via GFP/mCherry channel and imaged with IVIS. The fluorescence intensity of each intestine was recorded.

Example 9 tracking interactions between FAST-OMVs and bacteria (in vitro)

To visualize communication between OMVs and STm, FAST-OMVs were incubated with GFP-expressing STm in LB/PBS solution (1:1) at 37 ℃. Samples were taken at different time points. STm was centrifuged, HBR-3,5-DOM was added to turn on FAST-OMV fluorescent signal, then imaged with LSCM and analyzed using flow cytometry. The method for determining the interaction of FAST-OMV with SA, BS and other bacteria is similar.

Example 10 tracking interactions between FAST-OMVs and bacteria (in vivo)

Only 3 mice were fed water within 12 hours prior to administration of FAST-OMV (200 μl) at the 0h time point. To observe the interactions of FAST-OMV with intestinal bacteria in vivo, the small intestine and colon of ICR mice were given FAST-OMV, respectively, after 4h mice were sacrificed, intestinal bacteria were isolated from the intestinal content using gradient centrifugation, resuspended in sterile PBS, and after 5min incubation with fluorescent agent, observed using LSCM.

EXAMPLE 11 FAST-OMV Start/stop in mammalian cells in vitro

The mouse intestinal cell line MODE-K was obtained from American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin at 37℃in a humidified atmosphere of 5% CO ₂. To further confirm the interaction between FAST-OMVs and intestinal epithelial cells, MODE-K cells were seeded one day in advance into confocal dishes. 100 μl of FAST-OMV was added to the petri dish, and the control group was added with an equal amount of PBS. After 1h of co-incubation, 10 μl of green/red fluorescent agent was added, the FAST-OMV was fluorescent irradiated and its interaction with the cells was observed by LSCM. To shut down the fluorescence of FAST-OMVs, cells were washed with fresh PBS to remove fluorescein, then imaged with LSCM.

Example 12 in vivo tracking of interactions between FAST-OMVs and mammalian cells

Mice were treated according to the method described in vivo part (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.) of the interaction between FAST-OMVs and bacteria disclosed by the team of inventors. Different sections of the intestinal tract were frozen for confirmation of interactions between FAST-OMV-cells. Each section was stained with 10-fold dilution of the fluorescent agent in a dark room for 15min, then washed 3 times with PBS to remove residual fluorescent agent. After staining nuclei with DAPI, LSCM imaging was performed.

EXAMPLE 13 construction of STm-induced colitis mouse model

A mouse model of STm-induced colitis was constructed using the method (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.), disclosed by the team of inventors.

Female C57BL/6 mice (6-8 weeks) were purchased from SPF (Beijing) Biotech Co. After 7d of acclimation, the mice were randomized and treated 2 times with 100 μl (200 mg/ml) of streptomycin solution 24h prior to 5×10 ⁸ CFU STm. Mice were given OMV, ecN or PBS treatment daily on day 2 post infection and euthanized on day 8. Healthy mice were used as control group. The ileal terminal, cecal and colonic top tissues were collected, fixed for dissection, sectioned, and stained with hematoxylin-eosin (H & E). The whole intestinal tissue was collected and homogenized in a glass homogenizer. Each homogenate was resuspended and serially diluted with PBS, 50. Mu.l of each dilution was plated on LB agar plates containing antibiotics, incubated overnight at 37℃and bacteria were counted.

EXAMPLE 14 cytokine determination

Mice receiving treatment were euthanized on day 8 post-infection. Blood from each mouse was removed from the orbital, and stored in 1.5ml Eppendorf tubes without ethylenediamine tetraacetic acid (EDTA). After incubation of serum at 37℃for 0.5h, 10000 Xg was centrifuged for 5min, separated and assayed for IFN-. Gamma.TNF-. Alpha.and IL-6 using ELISA kits.

Example 15 immunofluorescent staining of tight junctions in vitro

Caco-2 cells were seeded and cultured for 24h before treatment with 10. Mu.l of LPS at a concentration of 1 mg/ml. Mu.l of OMV, ecN and PBS were added simultaneously and incubated for 24h, respectively. Cells washed with PBS were fixed with 4% paraformaldehyde for 20min and then blocked with 5% normal goat serum in PBS for 30min to remove non-specific binding. Cells were further incubated with primary antibodies (anti-ZO-1) and anti-obliterators (anti-occludin) for 2h at 37 ℃, followed by 1h incubation with secondary antibodies. After DAPI staining, LSCM was used for image visualization.

EXAMPLE 16 immunofluorescent staining of intestinal tissue

The STm-induced colitis mouse model slice samples were fixed with 4% paraformaldehyde, treated according to paraffin embedding standard procedure, and sectioned for 4 μm. Sections were blocked with 5% normal goat serum in PBS for 30min to remove non-specific binding, then incubated with primary antibodies (anti-ZO-1) and anti-obliterating (anti-occludin)) overnight at 4 ℃. After that, PBS was washed 3 times and incubated with the secondary antibody for 2h at room temperature. After sealing the tissue sections with the anti-fade solution containing DAPI, image visualization was performed using LSCM.

EXAMPLE 17 evaluation of intestinal permeability

STm-induced colitis mice treated with EcN-OMV, ecN or PBS mice were perfused with FITC-tagged dextran at a dose standard of 400mg/kg body weight on day 8 post-infection. Mice were fasted for 12h in advance. Blood samples were collected 4h after administration of FITC-labeled dextran. Blood sample collection was performed by centrifugation (2000 Xg, 4 ℃ C. For 15 min). Then, the fluorescence intensity of FITC-labeled dextran in the plasma was recorded with a fluorescence plate fluorometer having an excitation wavelength of 485nm and an emission wavelength of 530 nm. A standard curve was established using serially diluted FITC-labeled dextran samples.

Statistics and reproducibility

All experiments were performed at least 3 biological replicates to demonstrate successful reproducibility. n is the number of mice used for in vivo experimental analysis and represents the number of biological replicates used for in vitro experiments, all of which are mentioned in the diagrams or legends. Two-by-two comparisons were tested using a two-tailed Student's t test, multiple comparisons were tested using Tukey's posterior, one-way analysis of variance (ANOVA). p <0.05 was considered significant. Data analysis was performed using GraphPad software.

Results and analysis

1. Preparation of FAST-OMV

In the preparation of FAST-OMVs in example 1, the vector carrying the FAST gene was transformed into EcN, and fusion of the 6 Xhistidine tag (FAST-His) was expressed consecutively. After overnight incubation, the bacteria were removed by centrifugation and then filtered through a membrane having a pore size of 450 μm. FAST-OMV was collected and ultracentrifuged for purification.

In order to increase the yield of FAST-OMV, experiments were performed with inducer and antibiotic under different culture conditions.

The results showed that the highest FAST-OMV production was induced using equal amounts of IPTG+6.25mg/l kanamycin, as determined by the bisphenol diacid (BCA) method, compared to cultures without or with 0.5mM isopropyl-beta-D-thiogalactopyranoside (IPTG), which was determined to be 6.32mg/ml. Interestingly, the size of FAST-OMVs varies with the induction conditions. As measured by Dynamic Light Scattering (DLS), the particle size range of FAST-OMV was widely between 10-100 nm without inducer, and the particle size range was reduced to 20-100nm after addition of IPTG (FIG. 2. A-b). After addition of IPTG and kanamycin, FAST-OMV was more uniform in size, 40-100 nm in particle size, 1.5X10 ¹¹/ml or more in yield, and protein content reached about 8.8mg/ml (FIG. 2. C-d). The yield and protein content of outer membrane vesicles are significantly improved compared to conventional techniques, and can be 1.4 times and 1.5 times or more, respectively, than conventional yields.

Transmission Electron Microscopy (TEM) characterization showed that antibiotic-induced FAST-OMVs have a unique bilayer structure and spherical morphology (FIG. 1.e). Western blot verifies the presence of FAST, as indicated by the black arrow in fig. 1.f, indicating that it was successfully expressed and encapsulated in OMVs.

FAST-OMV fluorescence on/off

FAST as a small protein marker, generates different fluorescence in a dynamic and reversible manner by binding to various fluorescent agents in anaerobic and aerobic environments. After confirming the presence of FAST in the OMVs, the fluorescent properties of FAST-OMVs were tested in the examples of the application using 20. Mu.M HMBR or HBR-3,5-DOM fluorescent agent.

As expected from the previously reported protocol synthesis, immediately after addition of HMBR or HBR-3,5-DOM, respectively, to the FAST-OMV suspension, bright green (λmax=541 nm) and red (λmax=597 nm) fluorescent signals were observed upon direct exposure to the dual-wavelength fluorescent protein excitation light source (fig. 3.A and b).

Dynamic initiation was analyzed by confocal laser microscopy and flow cytometry (Beckman CytoFlex, U.S.) and after addition of HMBR or HBR-3,5-DOM, the green or red spots were well distributed in the field of view (fig. 3.c and e). Flow cytometry detection showed consistent rightward shift in fluorescence intensity in HMBR or HBR-3,5-DOM activated OMVs (fig. 3.D and f).

Microplate readers and fluorescence spectrometers can also measure activated FAST-OMV signals. As shown in FIG. 3.g-j, fluorescence signal intensity increased significantly after FAST-OMVs were bound to the corresponding luciferins. Quantitatively, the Mean Fluorescence Intensity (MFI) of the HMBR or HBR-3,5-DOM lit FAST-OMVs was more than 100-fold higher than that of the non-fluorescein added blank (FIGS. 3.h and j). These results indicate that the FAST-OMV signal can be turned on rapidly as required and that the addition of fluorescein has the ability to emit fluorescence in different bands.

In view of the dynamic binding behavior of FAST-OMVs, the present inventors studied the fluorescent shutdown of FAST-OMVs by controlling the concentration of fluorescent substances. The pre-activated FAST-OMVs were resuspended in blank PBS buffer solution, washed by ultracentrifugation, and fluorescence detected to determine the on-demand signal-off capability. Typically, the HMBR or HBR-3,5-DOM pre-activated FAST-OMV confocal images showed a gradual decrease in fluorescence intensity with increasing number of washes (fig. 5.a). The decrease in fluorescence of FAST-OMVs is due to a decrease in fluorescein concentration, which may trigger dissociation of HMBR or HBR-3,5-DOM in FAST protein. In contrast to fluorescence on, the flow cytometer histograms of the green and red fluorescence signals of the pre-activated FAST-OMVs gradually move to the left, which further supports the decay of fluorescence intensity as the concentration of the fluorescent agent decreases (fig. 5. B). Quantitative data based on flow cytometry analysis showed a 67% and 75% decrease in signal intensity of FAST-OMVs pre-activated with HMBR and HBR-3,5-DOM, respectively, after re-suspension in blank PBS (fig. 5. C). The fluorescence spectrometer further recorded the fluorescent off process, during which the signal intensity also had a similar tendency to decay (fig. 5. D). Unlike HBR-3,5-DOM, the decrease in fluorescence intensity of HMBR pre-activated nanovesicles is delayed, which means a stronger binding capacity to FAST. Notably, the HBR-3,5-DOM pre-activated FAST-OMVs maintained negligible emittance intensity after two washes with PBS. This indicates that the fluorescence can be controlled to be off by adjusting the concentration of the fluorescent agent.

3. Reversible switching between different transmit bands

Considering the selectivity of FAST binding to HMBR and HBR-3,5-DOM and its dynamic binding mode with concentration dependence, the inventors team further studied the ability of FAST-OMV to switch the emission band. To test the switching of λmax from 541 to 597nm, 20 μM HBR-3,5-DOM was added to the HMBR luminescent FAST-OMV suspension and the mixed emissions were measured using LSCM, flow cytometer and fluorescence spectrometer. As shown in FIG. 7.a, after the pre-activated FAST-OMV was replaced with HBR-3,5-DOM, the green fluorescence disappeared and a bright red fluorescence appeared under confocal imaging. Meanwhile, flow cytometry analysis showed that the green fluorescent channel shifted left and the red signal channel shifted right (fig. 7. B). The change in fluorescence intensity of the two channels was recorded and the result confirmed that the green fluorescence emission was significantly reduced, while the red signal was greatly increased (fig. 7. C). Furthermore, after addition of HBR-3,5-DOM to the HMBR pre-activated FAST-OMV suspension, the fluorescence spectrum almost completely disappeared at 541nm, while a new emission peak appeared at 597nm (FIG. 7. D). In pre-activated FAST-OMVs, at HBR-3,5-DOM concentrations greater than 20 μm, the fluorescent agent may compete with HMBR in the bound state, resulting in switching of the emission band.

The inventors also studied the detection of switching of λmax from 597nm to 541nm by re-suspending HBR-3,5-DOM pre-activated FAST-OMV in 20. Mu.M HMBR. Likewise, as shown in fig. 7.e-h, all measurements demonstrated that under the same experimental conditions, the pre-activated red signal could be reversibly switched to green fluorescence after FAST-OMV exposure to higher concentrations of HMBR. Taken together, these experimental data demonstrate the surprising adjustability of FAST-OMVs in fluorescence emission band switching through fluorescent agent exchange.

4. Tracking interactions of FAST-OMVs with microorganisms and mammalian cells

In order to visualize communication between OMVs and microbial cells, pathogenic STm has been co-incubated directly with FAST-OMVs in vitro, as EcN has been reported to be able to inhibit STm growth by secretion of micromycin. STm was bioengineered to express Green Fluorescent Protein (GFP) to facilitate observation of interactions between them.

After incubation to the indicated time point, STm was isolated by centrifugation and HBR-3,5-DOM was added to initiate fluorescence signal of FAST-OMV. Typical LSCM images show limited FAST-OMV binding to STm after 1h incubation (fig. 9.a and fig. 4. A). As the incubation time was extended to 3h, more FAST-OMVs were bound to the STm surface, and part of FAST-OMVs were even fused with bacteria. In particular, after 3h incubation, most of the red fluorescent FAST-OMVs were found to preferentially attach to both ends of STm. As the fluorescence signal is shown by the green to orange and even red change, most STm fuses with FAST-OMV as the incubation time is further extended to 6 h. The presence of a large number of dead and lytic bacteria demonstrated that OMVs derived from EcN can be well attached to STm from both ends, fused, and then the internal FAST and micromycin transported into the fusion bacteria.

Flow cytometry analysis further confirmed the direct interaction between FAST-OMV and STm, which showed that the intensity of the red fluorescent signal in STm increased with increasing incubation time after addition of HBR-3,5-DOM (fig. 9.b and fig. 4. B). Consistent with the results shown in LSCM, a sustained increase in bacterial fluorescence intensity suggests that OMVs mediate communication between EcN and STm.

Inhibition of STm by EcN-derived OMVs was observed by bacterial growth inhibition assays. As can be seen from the growth curve of figure 9.c, incubation with OMVs greatly inhibited STm proliferation compared to untreated bacteria.

Other bacteria include staphylococcus aureus (Staphylococcus aureus, SA) and bacillus subtilis (Bacillus subtilis, BS) were co-incubated to test whether the interaction was specific. As shown in fig. 6, the FAST-OMV did not significantly affect the growth of BS and SA, indicating that the vesicle nanoprobe prepared according to the present invention regulates intestinal flora by inhibiting specific bacteria.

The mouse intestinal cell line MODE-K cells were incubated with FAST-OMVs to study the interaction of probiotic OMVs with intestinal cells in vitro. After addition of HMBR or HBR-3,5-DOM, FAST-OMVs were observed to be able to efficiently enter MODE-K cells, and after co-incubation for 1h at 37 ℃, LSCM could see strong green or red fluorescent signals (fig. 9.d). Fluorescence was turned off by washing the cells with fresh medium without fluoride, further ensuring that FAST-OMVs successfully entered MODE-K cells (fig. 9.e).

In addition to validating EcN-derived OMVs' behavior into intestinal epithelial cells, on-demand activation/deactivation of fluorescence by addition or removal of corresponding fluorescent agents also demonstrates greater and wider applicability of FAST-OMVs in intracellular imaging and tracking.

The switching of the intracellular signal from green to red or red to green fluorescence was observed by changing the fluorescent agent (fig. 9.f and g). To investigate the communication function of FAST-OMVs with intestinal cells, the inventors of the present application performed immunofluorescence assays involving tight junctions of occlusion band-1 (ZO-1) and anti-occlusion elements, taking into account the level of tight junctions critical to maintain intestinal homeostasis, which reflects the integrity of the epithelial barrier.

Caco-2 cells, as the major large intestine epithelial cell line, were treated with 0.1mg/ml FAST-OMV and 5. Mu.g/ml LPS at 37℃for 24h, with LSCM capturing the tight junction protein levels and shown in FIG. 9.h. ZO-1 and obliteratin (occludin) levels of epithelial cells after LPS and FAST-OMV treatment were similar to untreated cells compared to LPS and PBS or EcN co-incubations.

The experimental results demonstrate that FAST-OMVs have the ability to maintain physical barrier integrity by entering and promoting tight junction expression in an external simulated environment. Intelligent bicolor imaging by FAST-OMV demonstrated that the nanovesicle probes of the invention have the ability to function properly in living cells and revealed positive interactions between probiotic-derived OMVs and intestinal cells.

5. Tracking OMVs in the gut

Since FAST has an emissivity independent of oxygen molecules, the inventors explored the in vivo anaerobic tracking of intestinal flora derived OMVs. Mice were lavaged using FAST-OMV and after ingestion for 2 hours, fluorescent agent was applied. FAST-OMV treated mice served as the experimental group and PBS treated mice served as the control group. 4h after FAST-OMV administration, mice were euthanized, sampled from the intestinal tract of each mouse, and observed with a live imaging system (in vivo IMAGING SYSTEM, IVIS).

As shown in fig. 11.A and fig. 8, the gut of the FAST-OMV + fluorescer treated mice showed bright fluorescent signals compared to the control group, especially in the small intestine and colon, which is very consistent with the on-demand fluorescence initiation results after in vitro addition of HMBR and HBR-3, 5-DOM. Furthermore, OMVs and intestinal flora in vivo can be observed.

Accordingly, 200 μl of FAST-OMV was injected into the small intestine and colon of the mice, respectively, and the contents of the extraction chamber were resuspended in PBS solution 4h after injection. Relevant bacteria were isolated by gradient centrifugation, then activated with HMBR or HBR-3,5-DOM, and then immobilized on agar plates for confocal imaging.

As shown in fig. 11.B, a number of different bacteria were observed, which fluoresce green or red depending on the type of fluorescent agent applied. More importantly, some bacteria did not exhibit fluorescence, indicating that fusion of FAST-OMVs with intestinal flora was selective (fig. 10 and 11). These results demonstrate the potential of the probes prepared according to the invention to intelligently track intestinal flora derived OMVs and visualize the interactions between these OMVs and different bacterial species in the gut.

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

As shown in FIGS. 11.C and 14, FAST-OMVs resulted in an increase in intracellular fluorescent signals compared to PBS-treated control mice, indicating that intestinal microbial OMVs can be transferred into intestinal epithelial cells located in the small intestine and colon.

Furthermore, the inventors of the present application have studied the effect of FAST-OMV in inhibiting specific pathogenic bacteria and promoting the secretion of claudin in STm-infected mice. From day 2 post infection, the stomach FAST-OMV was lavaged daily, samples were euthanized on day 8, and mice treated with 1x10 ⁸ CFU EcN and PBS served as controls. Small intestine, cecum and colon tissue samples and the corresponding luminal contents were homogenized and bacterial counts were performed on Luria Bertani (LB) agar plates. As shown in fig. 13.A, the STm bacterial count in the different samples was significantly reduced in the mice that applied FAST-OMV compared to the control group. The total STm of the FAST-OMV treated mice was reduced by 87% and 95% compared to EcN and PBS treated mice, respectively, demonstrating significant inhibition of specific pathogens in the gut by FAST-OMVs (fig. 13. B).

In addition, the inventors of the present application studied the communication of FAST-OMVs with intestinal cells in the intestinal tract of STm-infected mice. Immunofluorescence assays were performed on intestinal epithelial cells to assess the role of FAST-OMVs in maintaining the integrity of the physical barrier of the intestinal tract. As shown in fig. 13.C-e and fig. 16-18, the results demonstrate a significant increase in the level of the tight junction protein compared to PBS control and EcN. It is believed that FAST-OMVs prevent loss of tight junctions, epithelial cell shedding and death after intestinal infection, and repair the integrity of the epithelial barrier.

Therapeutic value of omvs in colitis

In view of the unprecedented advantages of FAST-OMVs shown or demonstrated by the above experimental results, the inventors further tested their therapeutic value in colitis characterized by a disturbed microbial structure and an impaired intestinal barrier.

Mice were infected with STm at 5 x 10 ⁸ CFU on day 1 (defined as day 0) after streptomycin treatment, and then orally FAST-OMV daily starting on day 2 post-infection (fig. 15. A). Healthy mice treated with PBS and EcN and STm-induced colitis mice served as control groups, respectively. Mice were sacrificed on day 8, serum specimens were collected via the orbital sockets, and enzyme-linked immunosorbent assay (ELISA) was performed to assess the level of inflammation. As shown in FIGS. 15.B-d, the levels of cytokines in serum of FAST-OMV treated mice were significantly reduced compared to PBS and EcN treated mice, including tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma). Meanwhile, the mice administered FAST-OMV had less fluctuation in body weight after treatment (fig. 15. E). EcN and PBS treated mice showed significant histological inflammation, but FAST-OMV treated mice did not (fig. 15.F, i and fig. 19-21).

The application also analyzed the length of the colon, with no significant decrease in FAST-OMV treated mice and healthy groups (fig. 15. G). Intestinal permeability experiments showed that the levels of fluorescein isothiocyanate dextran (FITC-dextran, 4 kDa) were significantly lower in serum samples of healthy mice and FAST-OMV treated mice than in EcN and PBS groups, indicating a decrease in intestinal barrier permeability after vesicle treatment (fig. 15. H). It is speculated that the decrease in intestinal permeability may be related to the ability of FAST-OMVs to restore intestinal barrier integrity, consistent with enhanced expression of tight junction proteins such as ZO-1 and occludin in immunofluorescence assays in vitro and in vivo. In summary, FAST-OMV-supported anaerobic tracking reveals a versatile function of gut flora-derived vesicles that can be targeted for the treatment of gut diseases such as colitis.

In view of the above experiments and results, the present invention prepares FAST-OMVs by stimulating bacterial vesicle formation and release through synthetic bioengineering and antibiotics. FAST-OMVs have a natural structure and fluorescent emission independent of oxygen molecules and can be used as endogenous nanoprobes for anaerobic tracking of intestinal flora derivatives. Specifically, intestinal probiotics EcN are genetically modified to express FAST and induced with an amount of ampicillin during bacterial growth to improve FAST-OMV production and isolation. By utilizing the dynamic and reversible combination of FAST and HMBR or HBR-3,5-DOM, FAST-OMV shows that the vesicle has obvious capability of switching on-demand signal switch and reversible switching emission wavelength through supplementing, removing or exchanging corresponding fluorescent agent. By means of the FAST intelligent bicolor imaging technology, intestinal tract visualization can be achieved, intestinal tract microorganism OMVs can be identified to be fused with specific bacteria, growth of bacteria is inhibited, and the bacteria enter intestinal epithelial cells, so that the biological function of intestinal tract barrier integrity is maintained. The technical scheme of the invention shows that the outer membrane vesicle can be used as a detection reagent and also can be used as an effective component of a medicine for preventing, relieving or treating intestinal diseases, and has huge potential application.

The foregoing is merely exemplary of the invention. But are not intended to limit the invention in this regard. All modifications which may be directly derived or suggested to one skilled in the art from the present disclosure are deemed to be included within the 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 vesicle 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 according to the method of claim 3 in the preparation of 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 comprises at least the nanoprobe according to any one of claims 1 to 2 or the nanoprobe prepared by the method according to claim 3.

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