Engineered bacterial outer vesicle loaded with lipophilic drug as well as preparation method and application of engineered bacterial outer vesicle

Abstract

The invention discloses an engineered bacterial outer vesicle loaded with a lipophilic drug as well as a preparation method and application of the engineered bacterial outer vesicle. The preparation method comprises the following steps: firstly, extracting bacterial exovesicles OMV by adopting a differential centrifugation method, then loading lipophilic drugs on the bacterial exovesicles OMV, and then sequentially coating the bacterial exovesicles OMV loaded with the lipophilic drugs with polysaccharides and weakly acidic small molecules through layer-by-layer self-assembly under a neutral condition to obtain the engineering bacterial exovesicles loaded with the lipophilic drugs. The stability of the engineered bacterial outer vesicle in a gastric acid environment and the responsiveness to intestinal pH are beneficial to accurate release and adhesion of drugs, so that the oral bioavailability and the treatment effect are improved, and the engineered bacterial outer vesicle can be used for treating acute and chronic ulcerative colitis and intestinal tissue fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to an engineered bacterial exovesicle loaded with a lipophilic drug, its preparation method and application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing intestinal disease that primarily affects the distal colon and rectum. Its progression begins with disruption of the intestinal barrier; damage to the intestinal barrier, altered gut microbiota, and increased intestinal inflammation all contribute to the disease's progression. As a lifelong condition, UC is currently incurable. Current treatments primarily focus on suppressing inflammation while neglecting the synergistic effects between gut microbiota, intestinal inflammation, and gut bacteria. Furthermore, there is still no effective treatment for intestinal fibrosis caused by chronic inflammation. Therefore, given the complex medical challenge of UC, there is an urgent need to develop novel anti-inflammatory treatment strategies to improve efficacy and reduce side effects, thereby providing patients with more lasting disease remission and a better quality of life.

[0003] Oral administration has always been the most commonly used route of drug delivery due to its convenience, speed, and good patient compliance. The development of oral colon-targeted drug delivery systems (OCTDDS) has greatly compensated for the shortcomings of traditional oral formulations in targeting the colon. This technology utilizes the unique digestive environment of the colon (such as pH, intestinal flora, and metabolic enzymes) to ensure that the drug is not disintegrated and released in the upper gastrointestinal tract, but gradually disintegrates and dissolves upon reaching the ileocecal junction, ultimately exerting a local or systemic therapeutic effect. However, oral administration still has many drawbacks. For example, protein and peptide drugs are easily degraded by digestive enzymes in the gastrointestinal tract after oral administration, losing their efficacy. In intestinal diseases, oral medications are often released and absorbed before reaching the disease site, leading to reduced efficacy and increased side effects. Therefore, overcoming the difficulty of targeting the colon with oral medication has been a focus of attention.

[0004] Upadacitinib is a lipophilic, selective Janus kinase 1 (JAK1) inhibitor approved for the treatment of autoimmune diseases such as rheumatoid arthritis. It has shown good efficacy in inhibiting inflammatory cytokine signaling pathways and reducing immune responses, and has been extensively studied for the treatment of inflammatory bowel disease in recent years. However, the systemic adverse reactions and instability of utadacitinib in the intestinal environment limit its widespread application. Therefore, developing a delivery system with targeted and controlled-release capabilities is expected to improve the local therapeutic effect of utadacitinib in the colon and reduce systemic toxicity, which is of great significance for the anti-ulcerative colitis effect of utadacitinib.

[0005] Bacterial outer membrane vesicles (OMVs) are negatively charged nanoscale vesicles secreted by Gram-negative bacteria. Rich in lipopolysaccharides, membrane proteins, phospholipids, and specific microbial-associated molecular patterns (MAMPs), OMVs possess natural immunomodulatory, tissue-targeting, and mucosal penetration capabilities. OMVs inherit bioactive components from their parent bacteria, exhibiting significant advantages in maintaining intestinal homeostasis, regulating inflammatory responses, and repairing the epithelial barrier. As naturally derived biological carriers, OMVs possess excellent biocompatibility and intestinal adaptability, making them an ideal platform for constructing targeted drug delivery systems for inflammatory bowel disease. However, direct oral administration of unmodified conventional bacterial outer membrane vesicles often fails to withstand the harsh acidic conditions of gastric acid, leading to reduced bioactivity, premature drug release, or drug penetration. Therefore, constructing an OMV system capable of effectively crossing the gastric acid barrier and achieving stable delivery to specific sites in the intestine remains a critical challenge.

[0006] Currently, no domestic or international literature or patents have reported any studies on the use of engineered bacterial exovesicles loaded with utpatinib, prepared using chitosan and trans-cinnamic acid modification, for the oral treatment of ulcerative colitis. Furthermore, no reports have been found on the synergistic effects of bacterial exovesicles and utpatinib in regulating the intestinal barrier, modulating the gut microbiota, and reducing inflammation in the treatment of ulcerative colitis. In conclusion, developing an effective colon-targeted drug delivery formulation for utpatinib is of great significance for the multifunctional treatment of ulcerative colitis. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing engineered bacterial exovesicles loaded with lipophilic drugs.

[0008] Another object of the present invention is to provide engineered bacterial exovesicles loaded with lipophilic drugs prepared by the method.

[0009] Another object of the present invention is to provide the application of the engineered bacterial exovesicles loaded with the lipophilic drug.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A method for preparing engineered bacterial exovesicles loaded with lipophilic drugs includes the following steps: sequentially encapsulating polysaccharides and weakly acidic small molecules onto (primary) bacterial exovesicles loaded with lipophilic drugs through layer-by-layer self-assembly under neutral conditions; specifically including the following steps:

[0012] (1) Extraction of bacterial extravesicle OMV: Escherichia coli was inoculated into a culture medium for culture, and then the extravesicles of Escherichia coli were extracted by differential centrifugation to obtain bacterial extravesicle OMV;

[0013] (2) Drug loading: The lipophilic drug and the bacterial exovesicles OMV obtained in step (1) were mixed and incubated, then centrifuged and washed to obtain drug-loaded bacterial exovesicles.

[0014] (3) Layer-by-layer self-assembly modification and synthesis of engineered bacterial exovesicles: The drug-loaded bacterial exovesicles obtained in step (2) are mixed with polysaccharide solution and incubated. Then, the precipitate is centrifuged to remove the unbound part, washed, and the precipitate is resuspended and incubated with a weakly acidic small molecule solution. After centrifugation and washing (removing the unbound part), engineered bacterial exoves loaded with lipophilic drugs are obtained.

[0015] The bacterial extravesicular OMV in step (1) is preferably obtained by extraction using the following method:

[0016] (i) Escherichia coli is inoculated into a culture medium and cultured to obtain an Escherichia coli culture;

[0017] (ii) Centrifuge the Escherichia coli culture at low speed, filter the supernatant, and concentrate it to obtain a concentrated solution;

[0018] (iii) The concentrate was centrifuged for the first time (ultracentrifugation), the precipitate was collected, washed, and resuspended, and then centrifuged for the second time (ultracentrifugation) to collect the precipitate and obtain bacterial exovesicles OMV.

[0019] The Escherichia coli mentioned in step (i) is preferably Escherichia coli Nissle 1917.

[0020] The Escherichia coli culture mentioned in step (i) is preferably obtained by the following method:

[0021] Escherichia coli is inoculated into a culture medium and cultured at 37±1℃ and 150-200 rpm for 10-20 hours (preferably 180 rpm for 10 hours) to obtain a primary culture medium. The primary culture medium is then cultured at 37±1℃ and 150-200 rpm for another 4-10 hours (preferably 180 rpm for 4 hours) to obtain a secondary culture medium, which is the Escherichia coli culture.

[0022] The culture medium mentioned in step (i) is preferably LB culture medium.

[0023] The ultraviolet absorbance OD value of the Escherichia coli culture described in step (i) is 0.8 to 1.2; preferably, the ultraviolet absorbance OD value is 1.

[0024] The conditions for low-speed centrifugation in step (ii) are: centrifugation at 3550-5000g and 4°C for 25-35 minutes; preferably: centrifugation at 3550g and 4°C for 30 minutes.

[0025] The filtration described in step (ii) involves sequentially using 0.45 μm and 0.22 μm filter membranes (sterile filters).

[0026] The concentration described in step (ii) is performed using an ultrafiltration tube with a molecular weight cutoff of 100 kD.

[0027] In step (iii), the conditions for the first and second ultracentrifugation are: centrifugation at 150,000g and 4°C for 70 to 90 minutes; preferably: centrifugation at 150,000g and 4°C for 70 minutes.

[0028] The washing described in step (iii) is performed using PBS buffer; preferably, the washing is performed more than twice using PBS buffer.

[0029] The resuspension described in step (iii) is performed using PBS buffer.

[0030] The lipophilic drug mentioned in step (2) is preferably utpatinib.

[0031] The mass ratio of the lipophilic drug to bacterial exovesicle OMV in step (2) is (0.25-5):1; preferably 1:1.

[0032] The incubation conditions described in step (2) are: 60-100 rpm, 37±1℃ for 2 hours, or 4℃ overnight; preferably: 80 rpm, 37℃ for 2 hours.

[0033] The centrifugation conditions described in steps (2) and (3) are: 150,000g, centrifugation at 4°C for 70-90 minutes; preferably: 150,000g, centrifugation at 4°C for 70 minutes.

[0034] The washing described in steps (2) and (3) is performed using PBS buffer; preferably, the washing is performed more than twice using PBS buffer.

[0035] The concentration of the drug-loaded bacterial vesicles in step (3) is 1–3 mg / ml; preferably 2 mg / ml.

[0036] The polysaccharide mentioned in step (3) is a cationic polysaccharide; preferably chitosan; more preferably chitosan with a viscosity-average molecular weight (Mv) < 4000 (Da).

[0037] The polysaccharide solution mentioned in step (3) is a chitosan solution with pH = 6.5 ± 0.1 and a concentration of 4 to 6 mg / ml; preferably, it is a chitosan solution with pH = 6.5 and a concentration of 5 mg / ml.

[0038] The polysaccharide mentioned in step (3) is coated by an over-addition method; the mass ratio of polysaccharide to drug-loaded bacterial vesicles is preferably (1-10):(1-10); further preferably (1-5):(1-5); and even further preferably 5:1.

[0039] The incubation conditions described in step (3) are: incubation at 4°C for 25-35 minutes; preferably: incubation at 4°C for 30 minutes.

[0040] The resuspension mentioned in step (3) is to resuspend the suspension using PBS buffer solution.

[0041] The weakly acidic small molecule mentioned in step (3) is preferably trans-cinnamic acid.

[0042] The weakly acidic small molecule solution mentioned in step (3) is a trans-cinnamic acid solution with pH = 6.5 ± 0.1 and a concentration of 4 to 6 mg / ml; preferably, it is a trans-cinnamic acid solution with pH = 6.5 ± 0.1 and a concentration of 5 mg / ml.

[0043] The weakly acidic small molecules mentioned in step (3) are coated by an over-addition method; the preferred mass ratio of the weakly acidic small molecules to the polysaccharide is (1-2):(1-2); more preferably (1.5-2):1, that is, it can be added at 1.5-2 times the upper limit of the mass ratio of the weakly acidic small molecules to the polysaccharide. After the coating is completed, the unbound weakly acidic small molecules are removed by washing to ensure the stability of the coating structure.

[0044] An engineered bacterial exovesicle loaded with a lipophilic drug is prepared by any of the methods described above.

[0045] The average particle size of the engineered bacterial extravesicles is about 184.33±53.39 nm, and obvious bacterial extravesicle structures were observed under a transmission electron microscope.

[0046] The application of the engineered bacterial exovesicles (nanomaterials) loaded with lipophilic drugs in the preparation of drugs for treating ulcerative colitis and / or intestinal fibrosis.

[0047] The ulcerative colitis mentioned includes acute ulcerative colitis (acute colitis) and chronic ulcerative colitis (chronic colitis).

[0048] The intestinal fibrosis mentioned refers to intestinal tissue fibrosis caused by ulcerative colitis.

[0049] The drug also includes pharmaceutically acceptable excipients.

[0050] The excipients may be at least one of carriers, excipients, diluents, etc.

[0051] The dosage form of the drug is tablets, pills, capsules (soft capsules), or oral liquid, etc.

[0052] The preferred route of administration for the drug is oral.

[0053] The treatment of ulcerative colitis or intestinal fibrosis is achieved by regulating the intestinal flora, restoring the intestinal barrier, and reducing intestinal inflammation.

[0054] A drug for treating ulcerative colitis and / or intestinal fibrosis (oral delivery platform), the active ingredient comprising engineered bacterial exovesicles loaded with the aforementioned lipophilic drug.

[0055] The ulcerative colitis mentioned includes acute ulcerative colitis (acute colitis) and chronic ulcerative colitis (chronic colitis).

[0056] The intestinal fibrosis mentioned refers to intestinal tissue fibrosis caused by ulcerative colitis.

[0057] The present invention has the following advantages and effects compared with the prior art:

[0058] 1. This invention utilizes a layer-by-layer self-assembly modification technique based on chitosan and trans-cinnamic acid. By leveraging electrostatic interactions, hydrogen bonds, and chemical cross-linking, multi-layer encapsulation modification is achieved, enhancing the stability of the encapsulated drug in the gastrointestinal tract and enabling precise responsive release and intestinal retention in the colon, thereby improving drug bioavailability and targeting. Therefore, this invention selects chitosan and trans-cinnamic acid to modify bacterial exovesicles to obtain a pH-responsive oral colon-targeting formulation.

[0059] 2. This invention, by designing a multilayer coating structure with "pH responsiveness and surface protection," significantly enhances the stability of OMVs in the gastrointestinal environment and improves their targeted release capability. In this invention, a multilayer self-assembled system is constructed, using a natural cationic polysaccharide as the inner protective layer and a weakly acidic small molecule as the outer barrier, to coat OMVs. The cationic polysaccharide possesses good biocompatibility and intestinal adhesion, and can be adsorbed onto the surface of OMVs through electrostatic interactions to form a primary protective layer. The outer component has low solubility under acidic conditions, effectively blocking gastric acid and allowing release after entering the intestine. This design not only improves the stability of vesicles in the acidic gastric environment but also endows them with pH responsiveness in the intestine, facilitating precise release and adhesion, thereby improving oral bioavailability and therapeutic efficacy.

[0060] 3. This invention provides a bacterial exovesicle nanomaterial. First, bacterial exovesicles are extracted from cultured *E. coli* 1917 using differential centrifugation. Then, the JAK1 inhibitor utpatinib is loaded directly through incubation. Chitosan (positively charged) and trans-cinnamic acid (negatively charged) are sequentially coated onto the bacterial exoves using a layer-by-layer encapsulation method. The resulting utpatinib-loaded, layer-by-layer-encapsulated bacterial exovesicle nanomaterial is purified by ultracentrifugation. The engineered exovesicles have an average particle size of approximately 184.33 ± 53.39 nm, and a distinct bacterial exovesicle structure is observed under a transmission electron microscope.

[0061] 4. In vivo experiments of this invention have demonstrated that engineered bacterial exovesicles not only have colon-targeting properties in mice, but also regulate the gut microbiota, restore the intestinal barrier, and reduce intestinal inflammation. They can synergistically work with loaded drugs to improve efficacy and reduce side effects. These engineered bacterial exovesicles provide a new, safe, and effective biotherapy approach for treating acute and chronic ulcerative colitis and fibrosis.

[0062] 5. This invention provides an orally administered, colon-targeting engineered bacterial exovesicle delivery system. Using bacterial exovesicles with ulcerative colitis-relieving functions as carriers, the bacterial exovesicles are loaded with utpatinib and further functionalized through layer-by-layer encapsulation. This engineered nanomaterial exhibits pH responsiveness and colon-targeting properties. Simultaneously, it can treat acute and chronic ulcerative colitis and intestinal fibrosis by regulating the intestinal barrier, intestinal flora, and reducing intestinal inflammation. The preparation process is simple, convenient, and easily scalable for industrial production. The colon-targeting engineered bacterial exovesicles constructed using the method disclosed in this invention will have promising applications as biomedical materials in drug delivery, bioimaging, disease prevention and diagnosis, and other fields. Attached Figure Description

[0063] Figure 1 This is a flowchart of the synthesis of layer-by-layer encapsulation of bacterial exovesicles (OMV-LbL@Upa) loaded with utpatinib in Example 1.

[0064] Figure 2 The images shown are transmission electron microscope (TEM) images of the engineered bacterial extravesicles prepared in Example 1; where A is a TEM image of bacterial extravesicles (OMV); and B is a TEM image of engineered bacterial extravesicles (OMV-LbL@Upa).

[0065] Figure 3 This is a graph showing the change in Zeta potential during the preparation of engineered bacterial exovesicles in Example 1.

[0066] Figure 4 The image shows the zeta potential and particle size distribution of the engineered bacterial vesicles prepared in Example 1.

[0067] Figure 5 This is a diagram showing the Coomassie brilliant blue staining results of the engineered bacterial vesicles prepared in Example 1.

[0068] Figure 6 This is a graph showing the release curve of utpatinib from engineered bacterial vesicles in Example 2.

[0069] Figure 7 The graph shows the in vitro stability test results of engineered bacterial exovesicles (OMV-LbL@Upa) and unmodified bacterial exovesicles (OMV@Upa) in Example 2. In the graph, A is the OMV retention rate of OMV-LbL@Upa in simulated gastric fluid for 4 hours; B is the OMV retention rate of OMV@Upa in simulated gastric fluid for 4 hours; C is the particle size of OMV-LbL@Upa in simulated gastric fluid for 4 hours; and D is the particle size of OMV@Upa in simulated gastric fluid for 4 hours.

[0070] Figure 8 The images are in vivo images of mice 2, 4, 8, 16, and 20 hours after oral administration of DiR-labeled engineered bacterial exovesicles in Example 3 (where OMV@DiR represents unmodified fluorescently labeled bacterial exovesicles; OMV-LbL@DiR represents fluorescently labeled bacterial exovesicles that have undergone layer-by-layer encapsulation modification).

[0071] Figure 9 The images show the in vitro fluorescence distribution and quantitative results of the main organs at different time points in Example 3; where A represents the in vitro fluorescence distribution of the main organs at different time points; and B represents the average in vitro fluorescence intensity of the main organs at different time points.

[0072] Figure 10 This image shows the fluorescence distribution of unlabeled DiO and DiO-labeled engineered bacterial extravesicles (free DiO, OMV-LbL@DiO) in mice after oral administration in Example 3 at 8 hours.

[0073] Figure 11 This is a cell uptake diagram of RAW264.7 macrophages after co-incubation with engineered bacterial vesicles labeled with PKH26 for 24 hours, as shown in Example 4.

[0074] Figure 12 The image shows the survival of RAW264.7 macrophages after co-incubation for 24 hours with different concentrations of utpatinib (pure Upa), OMV-LbL, and OMV-LbL@Upa in Example 4.

[0075] Figure 13Example 4 shows a comparison of the main inflammatory factors assessed by ELISA and RT-qPCR in RAW264.7 macrophages stimulated by LPS in Example 1 and engineered bacterial outer vesicles after 24 h. In this example, A represents the content of IL-6, TNF-α and IL-10; B represents the expression levels of IL-6, TNF-α and IL-10.

[0076] Figure 14 The following is a graph showing the detection results of six biochemical indicators in mouse blood under the action of utpatinib, OMV-LbL, and OMV-LbL@Upa in Example 5: A is the hematoxylin-eosin (H&E) staining image of the major organs of mice: heart, liver, spleen, lung and kidney; B is the detection results of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), uric acid (UA), blood urea nitrogen (UREA) and creatinine (CREA).

[0077] Figure 15 Figure 6 shows the results of mouse body weight, colon length, and DAI score after treatment with 5-aminosalicylic acid (5-ASA), utpatinib, OMV-LbL, and OMV-LbL@Upa in mice with acute colitis. In the figure, A shows the changes in DAI score of mice in each group; B shows the changes in body weight of mice in each group; C shows the changes in colon length of mice in each group; and D shows the colon of mice in each group.

[0078] Figure 16 Figure 7 shows the results of ELISA of inflammatory factors, oxidative stress level, macrophage polarization, and tissue H&E staining assessment of colon tissue and serum from mice in Example 6. In this figure, A represents H&E staining results; B represents H&E pathological scores; C, D, and E represent the expression levels of inflammatory factors IL-6, IL-10, and TNF-α in serum, respectively; F, G, and H represent the expression levels of inflammatory factors IL-6, IL-10, and TNF-α in colon supernatant, respectively; I represents macrophage polarization results; J represents GSH analysis results; K represents MDA analysis results; and L represents NO analysis results.

[0079] Figure 17 The figure shows the fluorescence changes and intestinal permeability assessment results of the key intestinal barrier proteins ZO-1 and Occuldin in mice from Example 6 in Example 8; where A is the immunofluorescence result of intestinal barrier marker proteins; and B is the result of intestinal permeability assessment by FITC-glucan.

[0080] Figure 18The diagram shows the impact of different treatment groups on fecal microbiota in mice from Example 6 in Example 9; where A is the Chao1 index; B is the Simpson index; C is the β diversity analysis (PCoA) results; D is the analysis results at the phylum level; E is the analysis results at the genus level; F is the relative abundance difference between beneficial and pathogenic bacteria associated with intestinal inflammation; and G is the ratio of Firmicutes to Bacteroidetes.

[0081] Figure 19 The diagram shows the action pathway and validation results of OMV-LbL@Upa screened by RNA sequencing of mouse colon tissue in Example 6 in Example 10; where A is the result of differential gene analysis; B is the Venn diagram; C is the result of GO functional enrichment analysis; D is the result of KEGG pathway enrichment; E is the result of GSEA analysis; and F is the expression level of JAK-STAT pathway core protein in colon tissue detected by Western blot.

[0082] Figure 20 The figure shows the changes in relative colon weight, intestinal inflammation, and expression of inflammatory factors in mice with chronic colitis after treatment with the control group, model group, fasudil, utpatinib, OMV-LbL, and OMV-LbL@Upa in Example 11. In the figure, A is the result of H&E staining; B is the relative weight of mouse colon; C is the case score; D is the expression level of pro-inflammatory factor IL-6; and E is the expression level of anti-inflammatory factor IL-10.

[0083] Figure 21 This is a histofluorescence analysis of the fibrosis area and fibrosis-related proteins in the colon of mice from Example 11, as shown in Example 12. A represents the Masson staining result; B represents the quantitative analysis result after Masson staining; and C represents the immunofluorescence staining result.

[0084] Figure 22 The graph shows the JAK-STAT pathway validation results of utpatinib, OMV-LbL, and OMV-LbL@Upa in Example 13. Detailed Implementation

[0085] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. It should be understood that the terminology used in the present invention is merely for describing particular embodiments and is not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0086] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe the methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail. Unless otherwise stated, all reagents and raw materials used in this invention are commercially available.

[0087] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0088] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0089] like Figure 1 As shown, this invention loads utpatinib onto the surface of bacterial exovesicles through direct incubation, enabling utpatinib to penetrate the intestinal barrier and target colonic lesions. Simultaneously, the vesicles possess the ability to restore the intestinal barrier, alter gut microbiota, and regulate intestinal immunity. Furthermore, the vesicles are functionalized through layer-by-layer modification, giving them resistance to gastric acid digestion and the ability to be released into the colon, ensuring that more bioactive vesicles reach the colon. This results in the preparation of a multifunctional nanomaterial based on bacterial exovesicles that can be used to treat acute and chronic ulcerative colitis and fibrosis.

[0090] The RAW264.7 macrophages used in the following examples were purchased from Wuhan Pronosei Biotechnology Co., Ltd., the experimental mice were purchased from Zhuhai Best Biotechnology Co., Ltd., and utpatinib (CAS No.: 1310726-60-3) can be obtained through conventional commercial channels.

[0091] Example 1: Preparation and characterization of OMV-LbL@Upa

[0092] 1. Preparation of nanomaterials:

[0093] (1) Extraction of bacterial extravesicular vesicle OMV:

[0094] Escherichia coli (E. coli Nissle 1917) strain (purchased from Beijing Beina Biotechnology Co., Ltd.) was added to LB medium and cultured at 37°C and 180 rpm for 10 hours to obtain primary culture medium. The primary culture medium was then cultured for another 4 hours under the same conditions to obtain secondary culture medium, completing the bacterial culture. The E. coli Nissle 1917 culture medium with an absorbance of 1.0 was collected, centrifuged at 3550g and 4°C for 30 minutes, and the supernatant was filtered through 0.45μm and 0.22μm filters, respectively. The supernatant was then concentrated using a 100kD ultrafiltration tube. After concentration to a suitable volume, the medium was ultracentrifuged at 150000g and 4°C for 70 minutes. The medium was washed twice with PBS, and the ultracentrifugation was repeated. The medium was then resuspended in PBS for BCA protein quantification, and the extracted OMV was stored at -80°C.

[0095] (2) Drug loading of utpatinib: Upatinib and OMV at different mass ratios (mass ratio range of 0.25 to 5:1) were directly incubated at 80 rpm and 37°C for 2 hours, and then subjected to ultracentrifugation again (150,000 g, 4°C, 70 minutes). After washing twice with PBS, drug-loaded bacterial vesicles OMV@Upa were obtained, and the optimal ratio was determined by ultraviolet spectroscopy (the optimal mass ratio in this example is 1:1).

[0096] (3) Synthesis of OMV-LbL@Upa: 2 mg / ml OMV@Upa and 5 mg / ml chitosan (Mv < 4000) solution at pH 6.5 were mixed uniformly at a mass ratio of 1:5 (OMV@Upa: chitosan) to form 3 ml solution. The mixture was incubated at 4°C on a vertical shaker for 30 min, then centrifuged at 150000 g at 4°C for 70 min, and washed twice with PBS. The precipitate after ultracentrifugation was resuspended in 1 ml PBS, and then resuspended again in 2 ml of 5 mg / ml trans-cinnamic acid solution at pH 6.5. After incubation at 4°C with vertical shaking for 30 min, OMV-LbL@Upa was extracted by ultracentrifugation (150000 g at 4°C for 70 min). The nanomaterial was resuspended in PBS and stored at -80°C.

[0097] 2. Characterization of nanomaterials

[0098] The OMV-LbL@Upa prepared in step (3) of this embodiment was characterized, with the bacterial exovesicles OMV and OMV@Upa prepared in steps (1) and (2) as controls.

[0099] Characterization results:

[0100] TEM images of the layer-by-layer encapsulated bacterial exovesicles (OMV-LbL@Upa) loaded with utpatinib prepared in this embodiment are shown below. Figure 2 As shown: From Figure 2 A and Figure 2 As shown in Figure B, the pure bacterial exovesicles OMV are smooth, while the surface of OMV-LbL@Upa is rough, indicating that chitosan and trans-cinnamic acid were successfully modified on the surface of OMV@Upa, and OMV-LbL@Upa was successfully synthesized.

[0101] The zeta potential change spectrum during the preparation of engineered bacterial exovesicles in this embodiment is as follows: Figure 3 As shown: From Figure 3 As can be seen, the OMV loaded with utpatinib is negatively charged (-27.79±1.28mV). After being encapsulated with chitosan, the potential approaches zero, with a potential of (-2.94±0.085mV). After being encapsulated with trans-cinnamic acid, the potential decreases to (-10.47±1.93mV), indicating that the layer-by-layer encapsulation modification was successful and OMV-LbL@Upa was successfully synthesized.

[0102] The zeta potential and particle size of the engineered bacterial exovesicles and the raw material OMV prepared in this embodiment are as follows: Figure 4 As shown, the pure OMV potential is -12.09±0.83mV and the particle size is 120.7±21.97nm. After modification and drug loading, the potential is -10.47±1.93mV and the particle size is 184.33±53.35nm. This indicates that the particle size of the bacterial exovesicles increases and the voltage decreases after modification, and the engineered bacterial exovesicles are successfully synthesized.

[0103] The engineered bacterial extravesicles OMV-LbL@Upa, pure extravesicle OMV, and bacterial lysate prepared in this embodiment were subjected to Western blot analysis and stained with Coomassie brilliant blue. The bacterial lysate was prepared as follows: Following step (1) of Example 1, *Escherichia coli* strain *E. coli* Nissle 1917 was cultured, the bacterial solution was collected, and then centrifuged at 3550g and 4℃ for 30 minutes. The precipitate was collected, 5× loading buffer was added, and the sample was boiled in a 100℃ metal bath for 10 minutes. It was then centrifuged at 12000g and room temperature for 10 minutes to obtain the lysate. The protein map is shown below. Figure 5 As shown, there were no significant changes in protein expression in engineered exovesicles, pure exovesicles, and bacterial lysates. This indicates that the bacterial exovesicles inherited the protein expression of the parent bacteria. At the same time, the engineered vesicles did not change the vesicle protein expression, indicating that the engineered vesicles retained the protein function of the vesicles.

[0104] Example 2: Drug release profile and in vitro stability of OMV-LbL@Upa

[0105] (1) Drug release assay: Dialysis bags containing the same concentration of pure utpatinib, OMV@Upa, and OMV-LbL@Upa (molecular weight cutoff 100kD) were sequentially immersed in different in vitro simulated digestive solutions (simulated gastric juice SGF 2h, simulated small intestinal juice SIF 6h, simulated colonic juice SCF 16h) (all simulated digestive solutions were purchased from Guangzhou Danoan Biotechnology Co., Ltd.). A quantitative amount of extradialyzed fluid was collected every two hours. The concentration of utpatinib was calculated by high performance liquid chromatography (mobile phase: A phase: water, B phase: acetonitrile; detection wavelength: 260nm; flow rate: 1ml / min, gradient elution with A phase: B phase = 3:7 for the first 10 minutes, and A phase: B phase = 7:3 from the 10th to the 60th minute). Fresh PBS was added to replenish the original volume. The experiment was repeated three times.

[0106] (2) In vitro stability: Bacterial exovesicles of the same concentration, OMV@Upa and OMV-LbL@Upa, were placed in simulated gastric fluid SGF or PBS, respectively. The bacterial exovesicles were re-extracted every hour by ultracentrifugation (150,000 g, 4 °C), and the changes in particle size and concentration were measured by a particle size analyzer and BCA, respectively. The experiment was set up in triplicate.

[0107] (3) Results:

[0108] The release curve of utpatinib evaluated in this example is as follows: Figure 6 As shown, under the protection of LbL (chitosan and trans-cinnamic acid) and OMV, the release of utpatinib in gastric juice was significantly reduced, and the release rate was also significantly decreased. Upatinib reached its peak release at 8 hours, while OMV-LbL@Upa was still releasing at 25 hours. These results indicate that the protection of OMV and LbL effectively resists gastric acid consumption, while giving utpatinib pH-responsive release and increasing the amount of utpatinib released in the colon.

[0109] The in vitro stability of bacterial extravesicles evaluated in this example is as follows: Figure 7 As shown: Single-bacterial exovesicles loaded with OMV@Upa exhibited significant changes in particle size and retention rate within 4 hours in simulated gastric fluid. Figure 7 B Figure 7 D), while the retention rate and particle size of OMV-LbL@Upa after LbL modification did not change significantly. Figure 7 A, Figure 7 C) The above results indicate that LbL modification can effectively resist gastric acid digestion and improve the preservation rate of bacterial exovesicles.

[0110] Example 3: Distribution of OMV-LbL@Upa in vivo, in vitro biodistribution, and tissue fluorescence in colitis mice

[0111] (1) Establishment of a mouse model of colitis: An acute ulcerative colitis model was established in C57BL / 6 mice (6 weeks old, female, purchased from Zhuhai Best Biotechnology Co., Ltd.) by free access to 3.5% sodium dextran sulfate (DSS) in drinking water for 7 days. On the 4th day after model establishment, the mice were randomly divided into 2 groups of 3 mice each according to their body weight.

[0112] (2) 100 μM of the fluorescent dye DiR was diluted to 10 μM with 2 mg / ml OMV or OMV-LbL (refer to the method in Example 1, first extract bacterial exovesicles OMV, then replace OMV@Upa in step (3) with OMV, obtained by layer-by-layer modification with chitosan and trans-cinnamic acid), and OMV (OMV@DiR) and OMV-LbL (OMV-LbL@DiR) were labeled and administered orally to mice with colitis. In vivo fluorescence imaging was performed at 2, 4, 8, and 20 h. After imaging, in vitro fluorescence imaging was performed on the major organs of the mice. In addition, pure DiO and DiO-labeled OMV-LbL were administered orally, and the mice were sacrificed at 8 hours to perform frozen fluorescence sections of the colon.

[0113] (3) Results:

[0114] The in vivo imaging of mice evaluated in this example is as follows: Figure 8 As shown, the in vitro imaging and quantitative results are as follows: Figure 9 As shown in the figure, the results indicate that LbL protection can prolong the retention time of OMV in the intestine. Unmodified OMV was almost completely metabolized after 8 hours, while LbL-modified OMV showed significant retention even after 20 hours. In vitro imaging and quantification further confirmed the long retention capacity of LbL-modified OMV in the colon, and no significant fluorescence accumulation was observed in major organs within 20 hours. These results demonstrate the good targeting ability of OMV to the colon.

[0115] 8-hour colon tissue fluorescence Figure 10 As shown, unlabeled DiO did not accumulate significantly in the colon, while OMV-LbL was not only significantly absorbed by the colon, but could also cross the mucosal layer to approach the submucosa (white line area). This indicates that engineered OMV did not change the mucosal permeability of OMV, which helps to increase the amount of utpatinib reaching the lesion.

[0116] Example 4: Detection of cellular uptake capacity, anti-inflammatory activity and cell compatibility of OMV-LbL@Upa nanomaterials

[0117] (1) Cell uptake: 2 mg / ml OMV / LbL@Upa was labeled with the fluorescent dye PKH26 (100 μM). Then, pure PKH26 (100 μM) or PKH26-labeled OMV / LbL@Upa was incubated with RAW264.7 macrophages stimulated with 1 mg / ml lipopolysaccharide (LPS) for 24 hours. The fluorescence position of PKH26 was then observed using confocal microscopy. The experiment was repeated three times.

[0118] (2) Cell compatibility: Cytotoxicity of RAW264.7 macrophages after co-incubation with different concentrations of drugs for 24 h was assessed using CCK8 assay. The drugs used were utpatinib at final concentrations of 20–60 nM, OMV-LbL@Upa (i.e., concentration of 20–60 nM based on utpatinib), and OMV-LbL (prepared using the same method as in Example 3). The dosage of OMV-LbL was consistent with the dosage of OMV-LbL contained in OMV-LbL@Upa. The experiment was performed in triplicate.

[0119] (3) Anti-inflammatory capacity: The expression of inflammatory factors in RAW264.7 macrophages stimulated with 1 mg / ml LPS for 24 hours after co-incubation with the drug was assessed by ELISA and RT-qPCR. The inflammatory factors assessed were IL-6, IL-10, and TNF-α. The drug groups were: model group (LPS) (with an equal volume of PBS), utpatinib monotherapy group, OMV-LbL (preparation method as in Example 3) group, and OMV-LbL@Upa group. The drug concentration was set at 40 nM for utpatinib, OMV-LbL@Upa with the same utpatinib concentration, and OMV-LbL (preparation method as in Example 3) (the drug concentration of OMV-LbL was the same as the dose of OMV-LbL contained in OMV-LbL@Upa). The experiment was repeated three times.

[0120] (4) Results:

[0121] The cellular uptake results assessed in this example are as follows: Figure 11 As shown, pure PKH26 was not taken up by macrophages after 24 hours of incubation, while PKH26-labeled OMV-LbL@Upa was taken up by macrophages and mainly accumulated around the cell nucleus. This indicates that engineered extravesicles did not change the characteristics of macrophage uptake and helped to increase the amount of utpatinib entering macrophages.

[0122] Cell compatibility results as follows Figure 12As shown, 50-60 nM OMV-LbL@Upa has certain toxicity to cells, while the corresponding OMV-LbL and utpatinib have no obvious toxicity to cells. This indirectly indicates that vesicle loading of utpatinib promotes the amount of utpatinib entering cells. Therefore, we chose a concentration of 40 nM utpatinib as the dosage.

[0123] The results of the assessment of cellular inflammatory factors are as follows: Figure 13 As shown in the results, whether using ELISA or RT-qPCR, OMV-LbL, utpatinib, and OMV-LbL@Upa all increased the content and expression of the anti-inflammatory factor IL-10 and decreased the content and expression of the pro-inflammatory factors TNF-α and IL-6. The anti-inflammatory effect of the utpatinib group was better than that of OMV-LbL, but OMV-LbL@Upa provided the best anti-inflammatory effect.

[0124] Example 5: In vivo biocompatibility of OMV-LbL@Upa nanomaterials

[0125] (1) Six-week-old female C57BL / 6 mice were acclimatized to their enclosures for one week. The mice were randomly assigned to four groups: a model group (animal model construction method as in Example 3), an utpatinib monotherapy group (Upa), an OMV-LbL (preparation method as in Example 3) group, and an OMV-LbL@Upa group, with five mice in each group. Subsequently, from day 0 to day 15, mice were orally administered an equal volume of PBS, 5.63 mg / kg Upa, OMV-LbL@Upa (where Upa concentration was 5.63 mg / kg), or OMV-LbL (preparation method as in Example 3) (the OMV-LBL concentration was consistent with the OMV-LbL concentration in OMV-LbL@Upa). Administration was once daily via gavage. On day 14, blood samples were collected from the eyes for rapid assessment of blood biochemical parameters. After euthanasia, major organs such as the heart, liver, lungs, kidneys, and spleen were collected for histopathological analysis.

[0126] (2) Results:

[0127] In vivo biocompatibility results can be as follows Figure 14 As shown. After drug administration, there were no statistically significant changes in AST, ALP, ALT, UA, UREA, CREA, and the model group. Figure 14 B). H&E staining of major organs ( Figure 14 A) No obvious damage was observed, indicating that all drugs at this concentration have good biocompatibility in vivo.

[0128] Example 6: Evaluation of the therapeutic effect of OMV-LbL@Upa nanomaterials on acute colitis

[0129] (1) An acute ulcerative colitis model was established in C57BL / 6 mice (6 weeks old, female) using 2.5% sodium dextran sulfate (DSS) in free drinking water for 7 days. On day 4 of model establishment, mice were randomly divided into 6 groups (n=5 per group) according to body weight and administered PBS, 5-ASA (5-aminosalicylic acid) (150 mg / kg), utpatinib (5.63 mg / kg), OMV-LbL, and OMV-LbL@Upa (utpatinib concentration 5.63 mg / kg), respectively. The dosage of OMV-LBL (prepared in the same way as in Example 3) was the same as that of OMV-LbL contained in OMV-LbL@Upa. The mice were administered orally once daily by gavage for 7 consecutive days. Normal C57BL / 6 mice served as the control group. Daily changes in body weight, fecal characteristics, and fecal hematoma were recorded to calculate the disease activity index (DAI). Animals were euthanized on day 12 of modeling, and their colons were harvested to measure their length. The tissue was photographed and preserved for subsequent analysis.

[0130] (2) Results:

[0131] The treatment effect evaluated in this case is as follows: Figure 15 As shown, the model group exhibited a significant increase in DAI score ( Figure 15 A) Weight loss ( Figure 15 B) and colonic shortening ( Figure 15 C Figure 15 D). OMV-LbL, utpatinib, and OMV-LbL@Upa can all inhibit weight loss, colon shortening, and DAI scores to varying degrees. Among them, OMV-LbL@Upa has the best therapeutic effect, similar to the treatment effect of 5-ASA in the positive control group, while OMV-LbL has the weakest effect.

[0132] Example 7: Evaluation of the anti-inflammatory effect of OMV-LbL@Upa nanomaterials on acute colitis

[0133] (1) After treatment, mice in each group of Example 6 underwent treatment. Distal colon tissue was collected, and H&E staining was performed to observe histological changes and conduct pathological scoring. Serum and colon tissue were collected, and the levels of inflammatory factors (TNF-α, IL-1β, IL-6) were detected and evaluated using ELISA. Macrophage polarization status was further analyzed, and the polarization distribution and relative expression levels were observed by immunohistochemical labeling of M1 type (IRF5) and M2 type (IRF4) markers. At the same time, oxidative stress changes in colon tissue were analyzed using GSH (glutathione) and MDA kits, and systemic oxidative stress changes were assessed by serum NO.

[0134] (2) Results:

[0135] The efficacy of inflammatory treatment evaluated in this case is as follows: Figure 16As shown. In the control group, intact colonic tissue was observed, while in the model group, significant inflammatory cell proliferation, mucosal erosion, and a decrease in goblet cells were observed. After treatment, varying degrees of recovery were observed. Figure 16 A), H&E pathology scores showed that OMV-LbL@Upa had the best treatment effect, followed by utpatinib, and lastly OMV-LbL ( Figure 16 B). Assessment of inflammatory factors in serum and colonic supernatant showed that OMV-LbL@Upa effectively reduced the production of systemic pro-inflammatory factors and promoted the production of anti-inflammatory factors, with a better regulatory effect than utpatinib. OMV-LbL also locally promoted the secretion of the anti-inflammatory factor IL-10 in the colon, partially contributing to the anti-inflammatory effect. Figure 16 C~ Figure 16 H).

[0136] Macrophage polarization results as follows Figure 16 As shown in Figure I, OMV-LbL@Upa and 5-ASA have similar therapeutic effects, significantly inhibiting the differentiation of M1 cells (IRF5) and promoting the production of M2 cells (IRF4).

[0137] Studies have shown that common free radicals attack their own tissues after injury, activating and acting as mediators of inflammation. MDA, a product of lipid peroxidation, is associated with damage promoted by common free radicals. GSH, on the other hand, is one of the most important antioxidants in the body, capable of inactivating oxygen free radicals and helping to maintain immune regulation. NO is closely related to the degree of inflammation and oxidative stress; therefore, assessing changes in MDA, GSH, and NO can indirectly reflect the degree of inflammation suppression. Figure 16 J~ Figure 16 As shown in Figure L, MDA and NO levels were significantly elevated and GSH levels were significantly decreased in the modeling group. After treatment with OMV-LbL@Upa, MDA, NO, and GSH levels reached similar levels to 5-ASA. Upatinib showed better antioxidant stress response than OMV-LbL.

[0138] Example 8: Evaluation of the effect of OMV-LbL@Upa nanomaterials on intestinal barrier restoration in acute colitis

[0139] (1) In Example 6, the colon tissues of mice after treatment were used to detect the expression levels of epithelial tight junction proteins (ZO-1, Occludin), and the integrity of the intestinal barrier was assessed by immunofluorescence staining. Serum was collected and fluorescence intensity was measured using a FITC-Dextran (4kDa) gavage leakage assay to assess intestinal permeability. The recovery of mucosal structure and the integrity of glandular pits were observed simultaneously.

[0140] (2) Results:

[0141] Immunofluorescence results of intestinal barrier marker proteins as follows Figure 17As shown in Figure A, after treatment with different drugs, ZO-1 and Occludin showed varying degrees of recovery. Among them, the expression levels of ZO-1 and Occludin in OMV-LbL@Upa and 5-ASA were similar to those in the control group, with the strongest expression. OMV-LbL was the second strongest, followed by utpatinib. These results indicate that OMV-LbL@Upa and 5-ASA have similar intestinal recovery abilities, but OMV-LbL has better intestinal recovery ability than utpatinib.

[0142] The results of FITC-glucan assessment of intestinal permeability are as follows: Figure 17 As shown in Figure B, similar to immunofluorescence, the group with the lowest FITC-glucan expression in serum was OMV-LbL@Upa. The OMV-LbL content was similar to that of OMV-LbL@Upa, while the utpatinib group had the highest content. This indicates that OMV-LbL and OMV-LbL@upa have similar intestinal barrier restoration capabilities, and OMV-LbL treats colitis by restoring the intestinal barrier.

[0143] Example 9: Evaluation of the effect of OMV-LbL@Upa nanomaterials on the regulation of intestinal flora in acute colitis

[0144] (1) Fecal samples were collected from mice treated in each group in Example 6. Total DNA was extracted, and the bacterial community structure was analyzed using 16S rRNA high-throughput sequencing. Changes in α-diversity (e.g., Shannon index), β-diversity (PCA analysis), and key phyla and genus levels were compared among the groups. The relative abundance differences of beneficial bacteria (e.g., Lactobacillus, Bifidobacterium) and pathogenic bacteria (e.g., Clostridium perfringens) associated with intestinal inflammation were analyzed to evaluate the ability of utpatinib, OMV-LbL, and OMV-LbL@Upa to improve dysbiosis.

[0145] (2) Results:

[0146] Gut microbiota analysis results as follows Figure 18 As shown. Compared with the model group, OMV-LbL@Upa significantly improved the α-diversity index of the gut microbiota, with both the Chao1 and Simpson indices being significantly higher than those in the model group ( Figure 18 A, Figure 18 B) indicates that it helps restore the abundance and evenness of the bacterial community. β-diversity analysis (PCoA) further showed that the OMV-LbL@Upa group samples were more similar to the normal group in terms of bacterial community composition. Figure 18 C) Secondly, OMV-LbL, indicating that it has a strong restorative ability on the overall microecological structure, also showing that OMV-LbL can alleviate acute colitis by regulating the flora.

[0147] At the taxonomic level, OMV-LbL@Upa treatment significantly improved the dysbiosis of the gut microbiota. Heatmaps at the phylum and genus levels are shown below. Figure 18 D and Figure 18 As shown in E. Phylum-level analysis showed that it significantly reduced the ratio of pathogenic Firmicutes / Bacteroidetes (E). Figure 18 G), which was positively correlated with inflammation relief. At the genus level, the OMV-LbL@Upa group significantly increased the relative abundance of probiotics such as Akkermansia and Lactobacillus, and decreased the abundance of potentially pathogenic bacteria such as Sutterella and Bacteroides. Figure 18 F), and the improvement effect was better than that of the utpatinib monotherapy group and the OMV-LbL group.

[0148] In summary, the results show that the OMV-LbL@Upa nanosystem provided by this invention not only significantly alleviates intestinal flora imbalance in an acute colitis model, but is also superior to utpatinib alone or the OMV-LbL group, demonstrating good potential for intestinal microecological reconstruction and showcasing its comprehensive advantages in synergistically regulating the intestinal microenvironment and treating intestinal inflammation.

[0149] Example 10: Exploring the therapeutic pathway of OMV-LbL@Upa nanomaterials in acute colitis

[0150] (1) Transcriptome RNA sequencing (RNA-seq) analysis was performed on the colon tissues of mice treated in each group in Example 6 to screen differentially expressed genes and their enrichment pathways. Western blot analysis was used to verify changes in protein expression levels of key signaling pathways.

[0151] (2) Results:

[0152] Tissue sequencing results as follows Figure 19 As shown. Differential gene analysis revealed that, compared to the model group (PBS), the OMV-LbL@Upa treatment group upregulated 527 genes and downregulated 644 genes. Figure 19 A), some of which regulate inflammatory pathways, immune responses, and cell activation. Venn diagrams further show that OMV-LbL@Upa reduces 85 genes ( Figure 19 B).

[0153] GO functional enrichment analysis showed that OMV-LbL@Upa treatment significantly affected biological processes such as "immune system processes," "lymphocyte activation," "T cell activation," and "cellular immune response." Figure 19C) KEGG pathway enrichment further confirmed that differentially expressed genes were significantly enriched in key signaling pathways closely related to inflammation, such as the JAK-STAT signaling pathway, cytokine-receptor interaction, and Th17 cell differentiation. Figure 19 D). Based on the important role of utpatinib in the JAK-STAT pathway and its role in intestinal fibrosis, we selected the JAK-STAT pathway for further research. GSEA analysis showed that the JAK-STAT pathway was significantly negatively enriched in the OMV-LbL@Upa group ( Figure 19 E) suggests that the system can effectively inhibit the activation of this pathway.

[0154] To further validate the above analytical results, Western blot was used to detect the expression levels of core proteins in the JAK-STAT pathway in colon tissue. The results showed that, compared with the model group, OMV-LbL@Upa treatment significantly inhibited the phosphorylation levels of JAK1, STAT1, and STAT3. Figure 19 F), while the total protein level did not change significantly, indicating that the treatment strategy mainly exerts its anti-inflammatory effect by inhibiting the activation state of JAK-STAT signaling. Compared with utpatinib treatment alone, the OMV-LbL@Upa treatment group showed more significant inhibition of p-JAK1, p-STAT1, and p-STAT3, indicating that the nanodelivery system not only enhances drug release but also synergizes with the regulatory function of OMV itself.

[0155] In summary, the OMV-LbL@Upa system can achieve synergistic anti-inflammatory effects in an acute colitis model by downregulating the expression of inflammation-related genes, inhibiting the activity of the JAK-STAT signaling pathway, directly restoring the intestinal barrier, regulating the intestinal flora, and loading utpatinib with OMV, thus verifying the significant therapeutic advantages of this invention at the molecular mechanism level.

[0156] Example 11: Evaluation of the anti-inflammatory therapeutic effect of OMV-LbL@Upa nanomaterials on chronic colitis

[0157] (1) A chronic colitis model was established using 6-week-old female C57BL / 6 mice induced by periodic DSS free drinking (1.5% DSS for 7 days + clean water for 7 days as one cycle, for a total of 4 cycles). During the last two weeks, the mice were orally administered the following nanomaterials daily: PBS, fasudil (2 mg / kg), utpatinib (5.63 mg / kg), OMV-LbL, and OMV-LbL@Upa (utpatinib concentration 5.63 mg / kg), respectively. The dosage of OMV-LBL (prepared in the same way as in Example 3) was the same as that of OMV-LbL contained in OMV-LbL@Upa. Colon length and histological changes were observed, and the anti-inflammatory effects of each group were evaluated in conjunction with changes in the expression of inflammatory factors.

[0158] (2) Results:

[0159] The outcome of anti-inflammatory treatment for chronic colitis Figure 20 As shown. Compared with the PBS group, the OMV-LbL@Upa group showed a significantly reduced decrease in the relative weight of the colon ( ). Figure 20 B) indicates a reduction in the degree of inflammation. Histological analysis showed that OMV-LbL@Upa treatment could alleviate mucosal structural damage, glandular loss, and inflammatory cell infiltration. Figure 20 A, Figure 20 C), the improvement effect was superior to that of utpatinib monotherapy or the OMV-LbL group. Inflammatory factor detection results showed that OMV-LbL@Upa significantly reduced the level of the pro-inflammatory factor IL-6 (C). Figure 20 D), while simultaneously increasing the expression of the anti-inflammatory factor IL-10 (D). Figure 20 E) suggests that it has excellent anti-inflammatory effects in chronic colitis.

[0160] Example 12: Evaluation of the anti-fibrotic ability of OMV-LbL@Upa nanomaterials against chronic colitis

[0161] (1) Colonic tissues from mice with chronic colitis treated in Example 11 were collected. Masson's trichrome staining was used to observe collagen deposition, and the proportion of fibrotic area was calculated. Immunofluorescence was used to detect the expression levels of fibrosis-related genes and proteins. The study analyzed whether OMV-LbL@Upa inhibited excessive fibrosis, reduced mucosal matrix deposition, and improved chronic intestinal remodeling.

[0162] (2) Results:

[0163] Results of antifibrotic treatment in mice with chronic colitis: Figure 21 As shown in the figure. Masson staining results showed that the PBS group mice had significant collagen deposition in the colonic mucosa, presenting extensive blue fibrotic areas, while the OMV-LbL@Upa group had significantly reduced fibrosis deposition. Figure 21A). Quantitative analysis shows that this system can significantly reduce the proportion of fibrous area ( Figure 21 B). Immunofluorescence staining was used to further detect the expression of fibrosis marker proteins α-SMA and Vimentin. The results showed that their expression levels were significantly reduced in the OMV-LbL@Upa treatment group. Figure 21 C) shows that the system can effectively inhibit intestinal fibrosis caused by chronic inflammation, and has the ability to protect tissue structure and improve intestinal remodeling.

[0164] Example 13: Validation of the therapeutic pathway of OMV-LbL@Upa nanomaterials in chronic colitis

[0165] (1) In the chronic colitis mice treated with the drug in Example 11, the expression of JKA-STAT pathway proteins such as p-JAK1, p-STAT1, and p-STAT3 in the chronic colitis model was detected by Western blot.

[0166] (2) Results:

[0167] Results of mechanism of action studies, such as Figure 22 As shown in the figure, compared with the PBS group, OMV-LbL@Upa significantly inhibited the expression levels of p-JAK1, p-STAT1, and p-STAT3, while the total protein level showed no significant change, suggesting that its mechanism of action is mainly the downregulation of pathway activation. Compared with the utpatinib group, OMV-LbL@Upa showed stronger performance in downregulating phosphorylated proteins, indicating that its delivery system not only enhances drug bioavailability but also achieves more effective inhibition of signaling pathways through synergy with the regulatory function of bacterial exovesicles.

[0168] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing engineered bacterial exovesicles loaded with a lipophilic drug, characterized in that, The process includes the following steps: encapsulating polysaccharides and weakly acidic small molecules onto bacterial exovesicles carrying lipophilic drugs through layer-by-layer self-assembly under neutral conditions.

2. The method according to claim 1, characterized in that, Specifically, the steps include the following: (1) Extraction of bacterial extravesicle OMV: Escherichia coli was inoculated into a culture medium for culture, and then the extravesicles of Escherichia coli were extracted by differential centrifugation to obtain bacterial extravesicle OMV; (2) Drug loading: The lipophilic drug and the bacterial exovesicles OMV obtained in step (1) were mixed and incubated, then centrifuged and washed to obtain drug-loaded bacterial exovesicles. (3) Layer-by-layer self-assembly modification and synthesis of engineered bacterial vesicles: The drug-loaded bacterial vesicles obtained in step (2) are mixed with polysaccharide solution and incubated. Then, the precipitate is centrifuged, washed, and the precipitate is resuspended and incubated with a weakly acidic small molecule solution. After centrifugation and washing, engineered bacterial vesicles loaded with lipophilic drugs are obtained.

3. The method according to claim 2, characterized in that: The lipophilic drug mentioned in step (2) is utpatinib; The polysaccharide mentioned in step (3) is a cationic polysaccharide; further, it is chitosan; and even further, it is chitosan with a viscosity-average molecular weight of <4000 Da; The weakly acidic small molecule mentioned in step (3) is trans-cinnamic acid.

4. The method according to claim 2, characterized in that: The bacterial extravesicular vesicles (OMV) in step (1) were extracted using the following method: (i) Escherichia coli is inoculated into a culture medium and cultured to obtain an Escherichia coli culture; (ii) Centrifuge the E. coli culture at low speed, filter the supernatant, and concentrate it to obtain the concentrated solution; (iii) The concentrate was subjected to a first ultracentrifugation, the precipitate was collected, washed, and resuspended, and then subjected to a second ultracentrifugation to collect the precipitate, thus obtaining bacterial exovesicles OMV. The conditions for low-speed centrifugation described in step (ii) are: 3550-5000g, centrifugation at 4℃ for 25-35 minutes; The filtration described in step (ii) involves sequentially using 0.45 μm and 0.22 μm filter membranes; The concentration described in step (ii) is performed using an ultrafiltration tube with a molecular weight cutoff of 100 kD; In step (iii), the conditions for the first and second ultracentrifugation are: 150,000g, centrifugation at 4℃ for 70–90 minutes.

5. The method according to claim 2, characterized in that: The mass ratio of the lipophilic drug to bacterial exovesicle OMV in step (2) is 0.25–5:1; The mass ratio of the polysaccharide to the drug-loaded bacterial exovesicles in step (3) is 1-10:1-10; further, it is 1-5:1-5; and even further, it is 5:

1. The mass ratio of the weakly acidic small molecule to the polysaccharide in step (3) is 1-2:1-2; further, it is 1.5-2:

1.

6. The method according to claim 2, characterized in that: The incubation conditions described in step (2) are: 60-100 rpm, 37±1℃ for 2 hours, or 4℃ overnight; The centrifugation conditions described in steps (2) and (3) are: 150,000g, centrifuged at 4°C for 70-90 minutes; The washing described in steps (2) and (3) is performed using PBS buffer. The concentration of the drug-loaded bacterial vesicles mentioned in step (3) is 1–3 mg / ml; The incubation conditions described in step (3) are all: incubation at 4°C for 25–35 min; The resuspension mentioned in step (3) is to resuspend the suspension using PBS buffer solution.

7. An engineered bacterial exovesicle loaded with a lipophilic drug, characterized in that: It is prepared by the method described in any one of claims 1 to 6.

8. The use of the engineered bacterial exovesicles loaded with the lipophilic drug as described in claim 7 in the preparation of medicaments for treating ulcerative colitis and / or intestinal fibrosis.

9. The application according to claim 8, characterized in that: The ulcerative colitis mentioned includes acute ulcerative colitis and chronic ulcerative colitis; The intestinal fibrosis mentioned refers to intestinal tissue fibrosis caused by ulcerative colitis; The medication is administered orally.

10. A medicament for treating ulcerative colitis and / or intestinal fibrosis, characterized in that: The active ingredient comprises engineered bacterial exovesicles loaded with a lipophilic drug as described in claim 7.

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