A dl-tryptophan-loaded bacterial outer membrane vesicle delivery system, and a preparation method and application thereof

Abstract

The application relates to a DL-tryptophan-loaded bacterial outer membrane vesicle delivery system and a preparation method and application thereof, the delivery system comprising a delivery carrier and an active ingredient loaded in the delivery carrier; the delivery carrier is a bacterial outer membrane vesicle separated from culture supernatant of Escherichia coli Nissle 1917, and the active ingredient is DL-tryptophan; the drug loading amount of the DL-tryptophan is 5-12 mu g / mg of bacterial outer membrane vesicle protein. The application firstly establishes a delivery system by taking the bacterial outer membrane vesicle from Escherichia coli Nissle 1917 as a delivery carrier, the carrier component is clear, the targeting property is strong, and the safety is high; the internal DL-tryptophan presents a slow-release release characteristic under the conditions of pH 3.0 and pH 7.0, and the utilization efficiency and treatment effect of the active ingredient are improved.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and nanodelivery technology, specifically to a bacterial outer membrane vesicle delivery system loaded with DL-tryptophan, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including ulcerative colitis and Crohn's disease, is a chronic, relapsing inflammatory bowel disease. Patients often experience symptoms such as abdominal pain, diarrhea, rectal bleeding, and weight loss. The disease is chronic and prone to relapse, severely impacting quality of life. Current treatments include aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics, but these generally suffer from significant individual variability in efficacy, marked adverse reactions with long-term use, and high relapse rates after discontinuation. For example, current IBD treatments are often single-target (e.g., targeting only inflammation or only immunity), failing to simultaneously repair the intestinal barrier, inhibit inflammasome activation, and regulate the immune microenvironment. Furthermore, free active ingredients (such as tryptophan and polyphenols) have low oral bioavailability and are easily degraded in the intestine, making it difficult to reach the lesion site.

[0003] In recent years, postbiotics (referring to inactivated probiotics and their metabolites) and their derived systems have become an important research direction for the intervention of inflammatory bowel disease due to their high safety, good stability, and ability to regulate intestinal immunity and intestinal barrier function. In particular, extracellular vesicles derived from probiotics (such as bacterial outer membrane vesicles, OMVs) can serve as delivery carriers for active molecules due to their natural nanoscale size and good biocompatibility. However, existing crude postbiotic extraction systems often have unclear compositions, are difficult to control in terms of quality, and unoptimized vesicles have limited enrichment efficiency at inflammatory sites and unclear target sites, making it difficult to meet the needs of precision treatment and limiting their therapeutic effects in inflammatory bowel disease. Summary of the Invention

[0004] Based on the above, this application aims to solve the problems of existing postbiotic carriers having complex components, low delivery efficiency of active ingredients, and inability to simultaneously regulate the inflammasome-barrier-immune axis. It provides a targeted delivery system based on probiotic-derived bacterial exosomes (i.e., exosomes). By loading specific active ingredients (DL-tryptophan), it utilizes the natural intestinal homing characteristics of bacterial exosomes to achieve the goal of efficient, safe, and multi-target treatment of inflammatory bowel disease.

[0005] To achieve the above objectives, this application provides a bacterial outer membrane vesicle delivery system loaded with DL-tryptophan, comprising a delivery carrier and an active ingredient loaded inside the delivery carrier; The delivery vector is bacterial outer membrane vesicles isolated from the culture supernatant of Escherichia coli Nissle 1917, and the active ingredient is DL-tryptophan; The drug loading of DL-tryptophan is 5~12 μg / mg bacterial outer membrane vesicle protein.

[0006] Preferably, the bacterial outer membrane vesicles have an average particle size of 100~150 nm and a Zeta potential of -30~-20 mV.

[0007] More preferably, the encapsulation rate of the DL-tryptophan is 70-90%.

[0008] Preferably, the release of DL-tryptophan is maintained at 24.25% ± 2.57% after 50 hours at pH 3.0. The cumulative release of DL-tryptophan was 65.47% ± 2.01% after 50 hours at pH 7.0.

[0009] To achieve the above objectives, this application also provides a method for preparing a bacterial outer membrane vesicle delivery system loaded with DL-tryptophan, comprising the following steps: S1. After culturing Escherichia coli Nissle 1917 in LB liquid medium until stable, collect the supernatant containing no bacterial cells; S2. Take the supernatant and centrifuge it at ultracentrifugation, resuspend the precipitate obtained by ultracentrifugation, wash it and obtain the bacterial outer membrane vesicles of Escherichia coli Nissle 1917; S3. Prepare a PBS solution of DL-tryptophan, wherein the concentration of DL-tryptophan is 3~8 mg / mL; S4. The bacterial outer membrane vesicles and the PBS solution of DL-tryptophan are mixed at a ratio of bacterial outer membrane vesicle protein mass: drug mass = 1:7~1:3 (e.g. 1:7, 1:6, 1:5, 1:4, 1:3). The mixture is sonicated in an ice bath to obtain bacterial outer membrane vesicles loaded with DL-tryptophan. S5. Remove the free DL-tryptophan from the system in step S4 to obtain the DL-tryptophan-loaded bacterial outer membrane vesicle delivery system.

[0010] Preferably, in step S1, the method for removing the bacterial cells from the supernatant is as follows: The culture system was centrifuged at 4℃ and 8000×g for 20 min. The supernatant after centrifugation was then filtered through a 0.45 μm filter membrane to obtain the supernatant free of bacteria.

[0011] More preferably, step S2 is: S21. The supernatant without bacteria is centrifuged at 4°C and 150,000 × g for 2 h to obtain a precipitate; S22. Resuspend the precipitate in PBS solution; S23. The resuspension system obtained in step S22 is subjected to ultracentrifugation to precipitate the bacterial outer membrane vesicles.

[0012] Preferably, the ultrasonic conditions in step S4 are: 20 kHz, 20% power, 6 cycles, with each cycle consisting of 30 s on and 30 s off.

[0013] More preferably, step S5 is: The system obtained in step S4 was centrifuged at 4°C and 150,000×g for 2 h, and the precipitate was resuspended in PBS solution. The resuspended system was filtered through a 0.22 μm filter membrane for sterilization to obtain the bacterial outer membrane vesicle delivery system loaded with DL-tryptophan.

[0014] This application also provides the use of the bacterial outer membrane vesicle delivery system carrying DL-tryptophan described in any of the preceding claims in the preparation of a medicament for treating inflammatory bowel disease.

[0015] The technical solution claimed in this application achieves the following beneficial effects: 1. For the first time, a delivery system was established using bacterial outer membrane vesicles derived from Escherichia coli Nissle 1917 as a delivery carrier. Compared with existing crude metagenic extraction systems with complex components, the carrier composition is more defined. The DL-tryptophan inside exhibits sustained-release characteristics under pH 3.0 and pH 7.0 conditions, which improves the utilization efficiency of the active ingredient. Moreover, the delivery system can be prepared using mature bacterial outer membrane vesicle extraction and drug loading methods, which is simple and easy to scale up.

[0016] 2. The obtained delivery system utilizes the natural intestinal homing ability of bacterial outer membrane vesicles, exhibiting a tendency to accumulate at lesion sites in the inflammatory bowel disease model, increasing local drug concentration, improving disease activity, reducing inflammatory damage to colonic tissue, regulating the expression of inflammation-related factors, and promoting the expression of intestinal barrier-related proteins. It has the advantages of both delivery carrier and bioactivity regulation, and has the potential to be further developed into a therapeutic drug for inflammatory bowel disease.

[0017] 3. It achieves synergistic treatment through multiple mechanisms, simultaneously inhibiting inflammasome activation, blocking the NF-κB pathway, and repairing the intestinal barrier, thus achieving multi-target therapy; and after repeated administration for 30 days, no significant pathological changes were observed in major organs such as the heart, liver, spleen, lungs, and kidneys, demonstrating high safety. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The physicochemical characterization results of bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles are shown. Figure 1 In the figure, A represents the particle size distribution of bacterial outer membrane vesicles. Figure 1 In the image, B represents a transmission electron microscope image of a bacterial outer membrane vesicle. Figure 1 In the figure, C represents the zeta potential detection result of bacterial outer membrane vesicles. Figure 1 DE in the figure represents the Western blot identification results of bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles. Figure 1 In the figure, F represents the storage stability test result of bacterial outer membrane vesicles. Figure 1 In the figure, G represents the drug loading and encapsulation efficiency of bacterial outer membrane vesicles loaded with DL-tryptophan. Figure 1 H in the figure represents the cumulative drug release curve of bacterial outer membrane vesicles loaded with DL-tryptophan.

[0020] Figure 2 The results of targeted delivery characterization of bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles are presented. Figure 2 In the image, A represents a confocal microscopy observation of PKH67-labeled bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles. Figure 2 In the figure, B represents the fluorescence intensity of PKH67-labeled bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles. Figure 2 In the figure, C represents the in vivo distribution of bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles in a DSS (dextran sulfate sodium salt)-induced IBD mouse model. Figure 2 In the figure, D represents the quantitative fluorescence detection results of bacterial outer membrane vesicles and DL-tryptophan-loaded bacterial outer membrane vesicles in a mouse model.

[0021] Figure 3 A schematic diagram illustrating the therapeutic effect of DL-tryptophan-loaded bacterial outer membrane vesicles on a DSS-induced inflammatory bowel disease model is shown. Figure 3 In the figure, A represents the colon length results of different experimental groups. Figure 3 In the figure, B represents the disease activity index score results of different experimental groups. Figure 3 In the figure, C represents the histological score of different experimental groups. Figure 3In the diagram, D represents the H&E staining results of colon tissue from different experimental groups. Figure 3 In the figure, E represents the expression results of the pro-inflammatory factor TNF-α in different experimental groups. Figure 3 F in the figure represents the expression results of the pro-inflammatory factor IL-6 in different experimental groups. Figure 3 In the figure, G represents the expression results of the pro-inflammatory factor IL-1β in different experimental groups. Figure 3 H in the figure represents the expression results of the pro-inflammatory factor IL-1O in different experimental groups.

[0022] Figure 4 The safety evaluation results of the bacterial outer membrane vesicle delivery system loaded with DL-tryptophan are shown.

[0023] Figure 5 Results related to the mechanism of action of the bacterial outer membrane vesicle delivery system loaded with DL-tryptophan are presented. Figure 5 In the figure, A represents the Western blot (protein immunoblotting) results of each experimental group. Figure 5 In the figure, B represents the expression results of Cleaved Caspase-1 protein in each experimental group. Figure 5 In the figure, C represents the NLRP3 protein expression results of each experimental group. Figure 5 In the figure, D represents the IL-1β protein expression results of each experimental group. Figure 5 In the figure, E represents the p-NF-κB protein expression results of each experimental group. Figure 5 F in the figure represents the ZO-1 protein expression results of each experimental group. Figure 5 In the figure, G represents the expression results of Occludin protein in each experimental group; Figure 5 In the figure, H represents the NLRP3 mRNA expression result detected by qPCR in each experimental group. Figure 5 In the figure, I represents the Cleaved Caspase-1 mRNA expression results detected by qPCR in each experimental group. Figure 5 In the figure, J represents the IL-1β mRNA expression results detected by qPCR in each experimental group. Figure 5 In this context, K represents the TNF-α mRNA expression results detected by qPCR in each experimental group. Figure 5 In this context, L represents the ZO-1 mRNA expression results detected by qPCR in each experimental group. Figure 5 M in the figure represents the Occludin mRNA expression results detected by qPCR in each experimental group. Detailed Implementation

[0024] To make the objectives, technical solutions, and beneficial effects of the embodiments in this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0025] <Example 1> This embodiment provides the preparation of bacterial outer membrane vesicles, and the specific preparation steps are as follows: S1. Strain culture: Escherichia coli Nissle 1917 was inoculated into LB liquid medium and cultured at 37℃ and 200 rpm until the stationary phase (OD600≈1.8). S2. Cell removal: Centrifuge at 4℃ and 8000×g for 20 min, collect the supernatant, and filter the supernatant through a 0.45 μm filter membrane; S3. Ultracentrifugation: Take the filtrate filtered through the filter membrane and centrifuge at 4℃ and 150,000×g for 2 h to precipitate bacterial outer membrane vesicles; collect the bacterial outer membrane vesicles, resuspend them in PBS, and wash them again by ultracentrifugation to obtain natural bacterial outer membrane vesicles (OMV).

[0026] The bacterial outer membrane vesicles obtained in this embodiment were characterized as follows: 1. Dimensional tracking analysis The particle size distribution and concentration of extracted bacterial outer membrane vesicles were determined using a NanoSight NS300, Malvern Panalytical. The bacterial outer membrane vesicle samples were diluted with PBS to an appropriate concentration (1×10⁻⁶). 8 Particles per mL were injected into the sample cell and detected using a 488 nm laser source at 25°C. Each sample was recorded three times (60 seconds each time). The Brownian motion trajectory of the particles was analyzed using NTA 3.0 software to calculate the average particle size and particle size distribution.

[0027] 2. Morphological observation 10 μL of bacterial outer membrane vesicle suspension was dropped onto a copper mesh (covered with a carbon film) and allowed to stand at room temperature for 2 min. Excess liquid was then blotted away with filter paper. Next, 10 μL of 2% phosphotungstic acid (pH 7.0) negative staining solution was added, and staining was performed for 1 min. The solution was then blotted dry with filter paper. After thorough drying, the vesicles were observed and imaged using a transmission electron microscope (HT7700, Hitachi) at an accelerating voltage of 80 kV. Typical vesicle structures are characterized by a cup-shaped morphology.

[0028] 3. Zeta potential measurement Bacterial outer membrane vesicles were resuspended in deionized water (pH 7.4) and the concentration was adjusted to approximately 0.1 mg protein / mL. Measurements were performed using a Zeta potential analyzer (Zetasizer Nano ZS, Malvern Panalytical) at 25°C. Results are expressed as mean ± standard deviation of Zeta potential.

[0029] 4. Western Blot identification of bacterial outer membrane vesicle markers Extracted bacterial outer membrane vesicle samples were lysed on ice for 30 min using RIPA lysis buffer (containing protease inhibitors), and protein concentration was determined by the BCA method. An equal volume of protein (20 μg) was separated by SDS-PAGE electrophoresis and then wet-transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4°C with the following primary antibodies: anti-OmpA (E. coli outer membrane protein A, Thermo Fisher, PA5-144390, 1:1000), anti-LPS (lipopolysaccharide, GeneTex, GTX41231, 1:500), anti-TSG101 (AffinityBiosciences, DF8427, 1:1000), and anti-CD63 (Novus Biologicals, NBP2-80654, 1:1000). The following day, the sample was washed three times with TBST, incubated with HRP-labeled secondary antibody (e.g., goat anti-rabbit / goat anti-mouse IgG, 1:5000) at room temperature for 1 h, and then developed using ECL chemiluminescence. Mammalian cell exosomes were used as a positive control to verify that the extract was bacterial outer membrane vesicles rather than mammalian exosomes.

[0030] 5. Short-term stability test (polydispersity index) Freshly prepared bacterial outer membrane vesicle suspensions were aliquoted into sterile EP tubes and stored at 4°C. Samples were taken on days 0, 1, 2, 3, 4, and 5, and the polydispersity index (PDI) was determined using a dynamic light scattering particle size analyzer (Zetasizer Nano ZS). A PDI < 0.15 was used as the criterion for good stability.

[0031] Figure 1 The characterization results of the bacterial outer membrane vesicles (Free drug group) obtained in this embodiment are shown. Figure 1 The results showed that the average particle size of bacterial outer membrane vesicles was approximately 120 nm, with a uniform distribution and a unimodal pattern (see reference). Figure 1 (A) Transmission electron microscopy revealed that the bacterial outer membrane vesicles exhibited a typical goblet-shaped vesicle structure, with intact morphology and no aggregation (see reference). Figure 1(B in the text); Zeta potential detection showed that the surface of bacterial outer membrane vesicles carried a negative charge of approximately -25 mV, exhibiting good colloidal stability (reference). Figure 1 (C in the middle).

[0032] Western blot analysis showed strong positive expression of OmpA and LPS in bacterial outer membrane vesicles, while the mammalian exosome markers TSG101 and CD63 were negative, indicating that the extract obtained from the culture supernatant was a high-purity bacterial outer membrane vesicle (reference). Figure 1 The polydispersity index (PDI) of bacterial outer membrane vesicles showed no significant change within 5 days under storage conditions at 4°C, remaining less than 0.15 (PDI < 0.15), indicating good short-term storage stability (see reference). Figure 1 (F in the text). <Example 2> This embodiment provides the preparation of a drug-loaded bacterial outer membrane vesicle delivery system, which is carried out according to the following steps: S1. Dissolve DL-tryptophan in PBS to prepare a 5 mg / mL solution; S2. Mix bacterial outer membrane vesicles with DL-tryptophan solution at a mass ratio of 1:5 (bacterial outer membrane vesicle protein mass: drug mass), and sonicate under ice bath conditions to load DL-tryptophan into bacterial outer membrane vesicles. The sonication parameters are 20 kHz, 20% power, 6 cycles, with each cycle being 30 s on and 30 s off. S3. After loading, centrifuge at 4℃ and 150,000×g for 2 h to remove free drug. The precipitate is the DL-tryptophan-loaded bacterial outer membrane vesicle (DL-OMV). The precipitate is resuspended in PBS, filtered through a 0.22 μm filter membrane for sterilization, and stored at -80℃.

[0033] The outer membrane vesicles of the drug-loaded bacteria obtained in this embodiment were characterized as follows: 1. HPLC was used to determine drug loading and encapsulation efficiency. Drug-loaded bacterial outer membrane vesicles were ultracentrifuged (150,000 × g, 2 h) to remove free DL-tryptophan. The precipitate was resuspended in PBS and lysed. DL-tryptophan content was determined using a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity). Chromatographic conditions: C18 reversed-phase column (4.6 × 250 mm, 5 μm), mobile phase: methanol:water:glacial acetic acid (20:80:0.1, v / v / v), flow rate: 1.0 mL / min, detection wavelength: 280 nm, column temperature: 30 °C. Drug loading was defined as the number of micrograms of drug (μg / mg protein) contained in each milligram of bacterial outer membrane vesicle protein. Encapsulation efficiency was calculated as: (amount of drug loaded into bacterial outer membrane vesicles / total amount of drug added) × 100%.

[0034] 2. In vitro release curve determination The in vitro release behavior of drug-loaded bacterial outer membrane vesicles was determined by dialysis. One mL of the drug-loaded bacterial outer membrane vesicle suspension (containing approximately 80 μg of DL-tryptophan) was placed in a dialysis bag (molecular weight cutoff 100 kDa) and immersed in 50 mL of PBS release medium at pH 3.0 (simulating gastric acid environment) and pH 7.0 (simulating intestinal / inflammatory environment), respectively, and incubated at 37°C and 100 rpm in a constant temperature shaker. At preset time points (0, 1, 2, 4, 6, 8, 12, 18, 25, and 50 h), 1 mL of the dialysis fluid was collected, and an equal volume of fresh medium was immediately added. The concentration of DL-tryptophan in the released fluid was determined by high-performance liquid chromatography (HPLC), and the cumulative release percentage at each time point was calculated. Results are expressed as mean ± standard deviation.

[0035] 3. Observe bacterial outer membrane vesicle uptake using an inverted fluorescence microscope. RAW264.7 macrophages were seeded in confocal culture dishes (density 1×10⁻⁶). 5 (Various vesicles / plate), cultured overnight at 37°C and 5% CO2 in DMEM medium containing 10% fetal bovine serum. Label the outer membrane vesicles of the drug-loaded bacteria with PKH67 green fluorescent dye according to the manufacturer's instructions: 100 μg of drug-loaded bacterial outer membrane vesicles were dissolved in 1 mL of dilution buffer C, and 2 μL of PKH67 dye was added. After mixing, the mixture was incubated at room temperature for 5 min, and the reaction was terminated by adding an equal volume of 1% BSA. Free dye was removed by ultracentrifugation (150,000×g, 1 h). PKH67-labeled drug-loaded bacterial outer membrane vesicles (final concentration 50 μg bacterial outer membrane vesicle protein / mL) were added to a macrophage culture system and incubated for 4 h. After incubation, the cells were washed three times with PBS to remove untaken bacterial outer membrane vesicles and fixed with 4% paraformaldehyde for 15 min. The cell nuclei were then stained with DAPI (1 μg / mL, room temperature, 5 min), washed with PBS, and observed under a laser scanning confocal microscope. The excitation wavelength of PKH67 was 488 nm, and the emission wavelength was 502 nm; the excitation wavelength of DAPI was 405 nm, and the emission wavelength was 461 nm. After image acquisition, fluorescence quantification analysis was performed using ImageJ software.

[0036] 4. In vivo imaging and quantitative fluorescence of isolated colon cells Drug-loaded bacterial outer membrane vesicles were labeled with DiR fluorescent dye (according to the kit instructions: 1 mg DiR was mixed with 10 mg of drug-loaded bacterial outer membrane vesicle protein, incubated at room temperature for 30 min, and free dye was removed by ultracentrifugation). A DSS-induced IBD mouse model was established. On day 7 of modeling, the labeled drug-loaded bacterial outer membrane vesicles were administered via tail vein injection (dose based on bacterial outer membrane vesicle protein 5 mg / kg). 24 h after administration, mice were subjected to fluorescence imaging using an IVIS Spectrum (PerkinElmer) small animal in vivo imaging system with an excitation wavelength of 745 nm, an emission wavelength of 800 nm, and an exposure time of 5 s. After imaging, mice were euthanized, colon tissue was dissected, and the colon was laid flat on an imaging plate for in vitro fluorescence imaging under the same conditions. Fluorescence intensity was analyzed using Living Image software, expressed as average radiometric efficiency (photons / s / cm²). 2 The results are expressed as mean ± standard deviation, and a two-tailed t-test was used for comparisons between the two groups.

[0037] Figure 1 The characterization results of the drug-loaded bacterial outer membrane vesicles (Loaded OMV group) obtained in this embodiment are shown. From Figure 1 It can be seen that the drug delivery process did not change the protein composition of bacterial outer membrane vesicles (see reference). Figure 1 (DE in the text). HPLC analysis showed that the drug loading capacity of DL-tryptophan in bacterial outer membrane vesicles was approximately 8.0 μg / mg of drug-loaded bacterial outer membrane vesicle protein, with an encapsulation efficiency of approximately 80%, indicating that bacterial outer membrane vesicles have a good loading capacity for the active ingredient (reference). Figure 1 (G in the text). The in vitro release behavior of the drug in the delivery system was detected by dialysis under different pH conditions. The results showed that the release was stable at pH 3.0, remaining essentially unchanged at 24.25% ± 2.57% over 50 h; at pH 7.0, the cumulative release increased from 40% to 60% in the first 10 h, then tended to plateau, reaching approximately 65% ​​cumulative release at 50 h. This indicates that the delivery system has sustained-release properties in an inflamed intestinal environment (reference...). Figure 1 (H in the text).

[0038] Figure 2 The targeting performance of DL-tryptophan-loaded bacterial outer membrane vesicles is shown. Confocal microscopy revealed that after co-incubation of PKH67-labeled green fluorescent bacterial outer membrane vesicles with macrophages, the fluorescence signal was mainly distributed within the cytoplasm, indicating that the bacterial outer membrane vesicles were effectively taken up and internalized by macrophages (reference). Figure 2 (AB in the text). Further in vivo imaging was used to detect the distribution of drug-loaded bacterial outer membrane vesicles in a DSS-induced IBD mouse model. The results showed that the drug-loaded bacterial outer membrane vesicle group exhibited significant fluorescence enrichment in the colon (reference). Figure 2 (CD in the text). The above results indicate that bacterial outer membrane vesicles have good targeting ability to intestinal inflammation sites, and can effectively deliver active ingredients to the lesion area. <Example 3> This embodiment uses a DSS-induced IBD mouse model to evaluate the therapeutic effect of the drug-loaded bacterial outer membrane vesicles obtained in Example 2. The process is as follows: Experimental animals selected: C57BL / 6 mice, 8-10 weeks old, male; Model establishment: Add 3% DSS to the drinking water of experimental mice and feed them for 7 consecutive days; Grouping: Mice were divided into a normal control group (no drug, Normal group), a model group, an empty drug group (bacterial outer membrane vesicles only, Free Drug group), a free drug group (DL-tryptophan only, Empty OMV group), and a drug-loaded bacterial outer membrane vesicle group (containing DL-tryptophan, Loaded OMV group), with 10 mice in each group; The details of each experimental group and the medication used are shown in the table below:

[0039] Administration: Starting from day 1 of modeling, administer the medication via tail vein injection every 2 days according to the dosage shown in the table above, for a total of 4 administrations; Evaluation: The weight, fecal characteristics, and blood in the stool of mice in each experimental group were recorded daily, and the DAI (Disease Activity Index) score was calculated. On day 8, the mice were sacrificed, the colon length was measured, and colon tissue was taken for subsequent H&E, ELISA, WB, and qPCR tests. The heart, liver, spleen, lung, and kidney of mice in each experimental group were also taken for H&E staining to evaluate safety.

[0040] The specific evaluation methods are as follows: 1. Colon length measurement Mice were euthanized on day 8, and the abdominal cavity was immediately opened. The colon was completely dissected from the ileocecal junction to the anus. The colon was laid flat on moistened filter paper, kept in a naturally extended state, and the length from the ileocecal junction to the anus was measured using a ruler (accurate to 0.1 cm). Each sample was measured by two independent experimenters, and the average value was taken. Data are expressed as mean ± standard deviation of each group, and statistical comparisons were performed using one-way ANOVA.

[0041] 2. Disease Activity Index Score From day 1 of modeling until sacrifice on day 8, daily changes in body weight, fecal characteristics, and fecal blood in mice were recorded for each group. The disease activity index scoring criteria were as follows: weight loss (0: no loss; 1: 1-5%; 2: 5-10%; 3: >10%), fecal characteristics (0: normal formed stool; 2: soft stool; 4: watery stool), and fecal blood (0: none; 2: occult blood positive; 4: gross bloody stool). The daily total score was the sum of the three scores divided by 3. Repeated measures ANOVA was used for comparisons between groups.

[0042] 3. H&E staining and histological scoring of colon tissue Approximately 1 cm of distal colon tissue was collected, fixed with 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare 4 μm thick serial sections. The sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope. Histological scoring was performed independently by two pathologists unaware of the group assignments. A 0-5 scoring system was used: 0 points: normal colon tissue, no inflammatory cell infiltration, intact crypt structure; 1 point: mild inflammatory cell infiltration, mild crypt destruction, confined to the mucosal layer; 2 points: moderate inflammatory cell infiltration, crypt destruction <25%, lesion extending to the submucosa; 3 points: severe inflammatory cell infiltration, crypt destruction 25-50%, transmural inflammation; 4 points: severe inflammatory cell infiltration with crypt destruction >50%, ulceration visible; 5 points: extensive transmural inflammation, complete crypt destruction, accompanied by large-area ulceration or tissue necrosis. Each group is expressed as mean ± standard deviation.

[0043] 4. ELISA detection of inflammatory factors in colon tissue Approximately 50 mg of fresh colon tissue was taken and added to RIPA lysis buffer containing protease inhibitors (tissue:lysis buffer = 1:10, w / v). The tissue was homogenized thoroughly on ice using a tissue homogenizer. The homogenate was centrifuged at 12000×g for 15 min at 4°C, and the supernatant was collected. The total protein concentration was determined using the BCA method, and the concentration was adjusted to the same level (1 mg / mL). Detection was performed according to the instructions of the commercially available ELISA kit (Biosharp). Standards and samples were added to a 96-well plate pre-coated with antibodies and incubated at 37°C for 2 h. After washing, the detection antibody was added and incubated for 1 h. After washing again, HRP-labeled secondary antibody was added and incubated for 30 min. TMB chromogenic solution was added, and the reaction was carried out in the dark for 15 min. After adding stop solution, the absorbance was immediately measured at 450 nm using a microplate reader. The concentrations of each inflammatory factor were calculated based on the standard curve and expressed as picograms (pg / mg protein) of inflammatory factors per milligram of total protein. Each group is expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and Tukey post-hoc test was used for pairwise comparisons.

[0044] 5. H&E staining of major organs and safety assessment After the DSS-induced IBD mouse model treatment experiment concluded (victims sacrificed on day 8), five mice were randomly selected from both the normal control group and the drug-loaded bacterial outer membrane vesicle group. Heart, liver, spleen, lung, and kidney were rapidly removed after dissection. Each organ was rinsed with pre-cooled physiological saline to remove residual blood, and tissue blocks approximately 0.5 cm × 0.5 cm × 0.3 cm in size were cut and immediately fixed in 4% paraformaldehyde fixative for 48 h. The fixed tissues underwent graded dehydration with ethanol (70%, 80%, 90%, 95%, and 100% for 1 h each), clearing with xylene (twice, 15 min each time), paraffin embedding, and other treatments. Continuous sections with a thickness of 4 μm were prepared using a microtome, spread, mounted on glass slides, and baked at 60℃ for 2 h. After dewaxing to water, the sections were stained with hematoxylin for 5 min, then blued with tap water; followed by eosin staining for 1 min, dehydration, clearing, and mounting. The morphological changes of each organ in each group were observed under an optical microscope.

[0045] Figure 3 The results of the therapeutic effect of a bacterial outer membrane vesicle delivery system loaded with DL-tryptophan on a DSS-induced inflammatory bowel disease model are presented. From Figure 3 The colon length measurement results showed that the colon length in the model group was significantly shortened (5.2 cm), while the colon length in the drug-loaded bacterial outer membrane vesicle group was significantly restored (7.6 cm), approaching the level of the normal control group (reference). Figure 3 (A) The disease activity index score showed a significant decrease in the score of the drug-loaded bacterial outer membrane vesicle group, indicating a significant improvement in disease symptoms (reference). Figure 3 (B)

[0046] H&E staining and histological scoring of colon tissue showed that the model group exhibited extensive inflammatory cell infiltration, severe crypt structure destruction, and a significantly elevated histological score; the drug-loaded bacterial outer membrane vesicle group showed significantly reduced inflammatory infiltration, partial recovery of crypt structure, and a decreased histological score, demonstrating significantly better efficacy than the free drug group (reference). Figure 3 (CD in the text). ELISA detection of inflammatory factor expression in colonic tissue showed that, compared with the model group, the pro-inflammatory factors TNF-α, IL-6, and IL-1β were decreased in the drug-loaded bacterial outer membrane vesicle group, while the anti-inflammatory factor IL-10 was increased (reference). Figure 3 (EH in). The above results indicate that the bacterial outer membrane vesicle delivery system loaded with DL-tryptophan can effectively alleviate DSS-induced colitis and improve the intestinal inflammatory state.

[0047] Figure 4 The H&E staining analysis of major organs in mice from the normal control group and the drug-loaded bacterial outer membrane vesicle group is shown. Results are as follows: Figure 4As shown, no obvious pathological changes, including tissue necrosis, inflammatory cell infiltration, fibrosis, or structural abnormalities, were observed in the heart, liver, spleen, lung, and kidney tissues of both groups of mice. These results indicate that the drug-loaded bacterial outer membrane vesicles have good biocompatibility in vivo. <Example 4> This embodiment investigates the mechanism of DL-tryptophan-loaded bacterial outer membrane vesicles in treating IBD. Western blotting and qPCR were used to detect the expression levels of colon-related proteins and mRNAs in each experimental group in Example 3. The results are as follows: Figure 5 As shown in the figure. Since Western blotting and qPCR are both standard methods in this field, their specific implementation processes will not be described in detail here.

[0048] Depend on Figure 5 The results showed that, according to Western blotting, compared with the model group, the expression of NLRP3 protein was significantly downregulated in the drug-loaded bacterial outer membrane vesicle group, and the levels of Cleaved Caspase-1 (activated Caspase-1) B, p-NF-κB, NLRP3C, and IL-1βD were significantly reduced, indicating that NLRP3 inflammasome activation and the NF-κB pathway were effectively inhibited. Simultaneously, the expression of tight junction proteins ZO-1F and OccludinG was significantly upregulated, suggesting that intestinal barrier function was repaired (reference). Figure 5 (AG in the middle).

[0049] qPCR results showed that the mRNA expression levels of NLRP3, Cleaved Caspase-1, IL-1β, and TNF-α were significantly downregulated in the drug-loaded bacterial outer membrane vesicle group, while the mRNA expression levels of ZO-1 and Occludin were significantly upregulated, consistent with the trend of protein level changes (reference). Figure 5 (HM in the text). The above results indicate that the bacterial outer membrane vesicle delivery system loaded with DL-tryptophan exerts a therapeutic effect on IBD by inhibiting NLRP3 inflammasome activation and the NF-κB pathway, downregulating the expression of inflammatory factors, and repairing the intestinal epithelial barrier function. A targeted delivery system for bacterial outer membrane vesicles after drug loading was developed. Particle size distribution and transmission electron microscopy analysis confirmed that the outer membrane vesicles extracted from the culture supernatant of *E. coli* Nissle 1917 exhibited a typical cup-shaped structure with an average particle size of approximately 120 nm and a Zeta potential of approximately -25 mV, demonstrating good colloidal stability. Western blotting confirmed the identification markers of bacterial outer membrane vesicles (strongly positive for OmpA and LPS), while the mammalian exosome marker CD63 was negative, indicating that the extract consisted of high-purity bacterial outer membrane vesicles.

[0050] HPLC analysis showed that the drug loading of DL-tryptophan in bacterial outer membrane vesicles was approximately 8.0 μg / mg of bacterial outer membrane vesicle protein, with an encapsulation efficiency of approximately 80%. In vitro release experiments demonstrated that the delivery system exhibited sustained-release characteristics at pH 7.0. The protocol described in this application confirms that drug-loaded bacterial outer membrane vesicles effectively alleviated the DSS-induced colitis phenotype in mice through a multi-target mechanism, significantly restored colon length (7.6 cm, compared to only 5.2 cm in the model group), reduced the disease activity index score (only 1.8, compared to 3.8 in the model group), and alleviated inflammatory infiltration and crypt damage in colonic tissue.

[0051] ELISA analysis showed that drug-loaded bacterial outer membrane vesicles significantly inhibited the expression of key pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) while upregulating the level of the anti-inflammatory factor IL-10. Mechanistically, Western blot and qPCR results revealed that the drug-loaded bacterial outer membrane vesicles regulate the inflammatory response by inhibiting NLRP3 inflammasome activation (downregulation of NLRP3, Cleaved Caspase-1, and mature IL-1β expression) and the NF-κB pathway (downregulation of the p-P65 / P65 ratio), while simultaneously upregulating the expression of tight junction proteins ZO-1 and Occludin, thus repairing intestinal barrier function. Safety assessments showed that the drug-loaded bacterial outer membrane vesicles had no significant tissue toxicity to major organs such as the heart, liver, spleen, lungs, and kidneys. In other words, the drug-loaded bacterial outer membrane vesicle delivery system provided in this application offers a safe and efficient novel therapeutic strategy for inflammatory bowel disease through a multi-target mechanism involving inhibition of the NLRP3 inflammasome-NF-κB pathway, downregulation of inflammatory factors, and repair of the intestinal barrier. The embodiments and application examples described above are merely illustrative descriptions of this application and are not intended to limit the scope of this application. Any modifications and improvements made to the technical solutions of this application by those skilled in the art without departing from the spirit of this application should fall within the protection scope defined by this application.

Claims

1. A bacterial outer membrane vesicle delivery system loaded with DL-tryptophan, characterized in that, Includes a delivery carrier and an active ingredient loaded inside the delivery carrier; The delivery vector is bacterial outer membrane vesicles isolated from the culture supernatant of Escherichia coli Nissle 1917, and the active ingredient is DL-tryptophan; The drug loading of DL-tryptophan is 5~12 μg / mg bacterial outer membrane vesicle protein.

2. The delivery system according to claim 1, characterized in that, The bacterial outer membrane vesicles have an average particle size of 100~150 nm and a zeta potential of -30~-20 mV.

3. The delivery system according to claim 1, characterized in that, The encapsulation rate of the DL-tryptophan is 70-90%.

4. The delivery system according to any one of claims 1 to 3, characterized in that, At pH 3.0 for 50 hours, the cumulative release of DL-tryptophan remained at 24.25% ± 2.57%. The cumulative release of DL-tryptophan was 65.47% ± 2.01% after 50 hours at pH 7.

0.

5. A method for preparing a bacterial outer membrane vesicle delivery system loaded with DL-tryptophan, characterized in that, Includes the following steps: S1. After culturing Escherichia coli Nissle 1917 in LB liquid medium until stable, collect the supernatant containing no bacterial cells; S2. Take the supernatant and centrifuge it at ultracentrifugation, resuspend the precipitate obtained by ultracentrifugation, wash it and obtain the bacterial outer membrane vesicles of Escherichia coli Nissle 1917; S3. Prepare a PBS solution of DL-tryptophan, wherein the concentration of DL-tryptophan is 3~8 mg / mL; S4. The bacterial outer membrane vesicles and the PBS solution of DL-tryptophan are mixed at a ratio of bacterial outer membrane vesicle protein mass: drug mass = 1:7~1:

3. The mixture is sonicated in an ice bath to obtain bacterial outer membrane vesicles loaded with DL-tryptophan. S5. Remove the free DL-tryptophan from the system in step S4 to obtain the DL-tryptophan-loaded bacterial outer membrane vesicle delivery system.

6. The preparation method according to claim 5, characterized in that, In step S1, the method for removing the bacterial cells from the supernatant is as follows: The culture system was centrifuged at 4℃ and 8000×g for 20 min. The supernatant after centrifugation was then filtered through a 0.45 μm filter membrane to obtain the supernatant free of bacteria.

7. The preparation method according to claim 5, characterized in that, Step S2 is as follows: S21. The supernatant without bacteria is centrifuged at 4°C and 150,000 × g for 2 h to obtain a precipitate; S22. Resuspend the precipitate in PBS solution; S23. The resuspension system obtained in step S22 is subjected to ultracentrifugation to precipitate the bacterial outer membrane vesicles.

8. The preparation method according to claim 5, characterized in that, The ultrasound conditions in step S4 are: 20 kHz, 20% power, 6 cycles, with each cycle consisting of 30 s on and 30 s off.

9. The preparation method according to claim 5, characterized in that, Step S5 is as follows: The system obtained in step S4 was centrifuged at 4°C and 150,000×g for 2 h, and the precipitate was resuspended in PBS solution. The resuspended system was filtered through a 0.22 μm filter membrane for sterilization to obtain the bacterial outer membrane vesicle delivery system loaded with DL-tryptophan.

10. The use of the bacterial outer membrane vesicle delivery system carrying DL-tryptophan according to any one of claims 1 to 4 in the preparation of a medicament for treating inflammatory bowel disease.

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