STA-5326-loaded hybrid exosome oral preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to an STA-5326-loaded heterozygous exosome oral preparation which comprises an STA-5326-loaded heterozygous exosome and an acid-resistant shell covering the surface of the heterozygous exosome, and the heterozygous exosome is at least fused with a Treg cell exosome and an MSCs cell exosome. Experiments show that the STA-5326-loaded hybrid exosome oral preparation has a good treatment effect on ulcerative colitis, can regulate the immune environment in a UC colon part, has good stability, and can be enriched in an inflammation area to repair an intestinal damaged barrier.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an oral preparation of a heterozygous exosome loaded with STA-5326, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing inflammatory bowel diseases, mainly including ulcerative colitis (UC), Crohn's disease (CD), and indeterminate colitis (IC). The pathogenesis of IBD is not fully understood, but it is generally believed to be caused by a combination of factors, including genetic susceptibility, environmental factors, immune system dysregulation, and gut microbiota dysbiosis. Patients clinically present with symptoms such as diarrhea, abdominal pain, bloody and mucous stools, weight loss, and fever, severely impacting their quality of life. The prolonged and recurring course of the disease places a heavy economic and psychological burden on patients and their families.

[0003] Currently, the diagnosis of IBD mainly relies on a combination of clinical symptoms, endoscopic examination, imaging assessment, and pathological biopsy. In terms of treatment, drug therapy is the core strategy, including: (1) aminosalicylic acid preparations (such as mesalazine), which are suitable for induction and maintenance therapy in patients with mild to moderate UC; (2) glucocorticoids (such as prednisone), which are used for symptom control in the acute phase, but long-term use can easily lead to serious side effects; (4) immunosuppressants (such as azathioprine), which are often used for maintenance therapy in patients who are hormone dependent or resistant; and (5) biological agents (such as the anti-TNF-α monoclonal antibody infliximab), which provide targeted therapy options for patients with moderate to severe IBD.

[0004] However, traditional drug treatments have limitations, including limited efficacy, systemic side effects, and non-response or secondary failure in some patients. For patients with drug-resistant bowel conditions or serious complications (such as massive bleeding, perforation, or cancer), surgical resection of the affected bowel segment becomes the final option, but postoperative challenges may include recurrence or decreased quality of life. Therefore, developing safer and more effective targeted therapy strategies is crucial.

[0005] Recent studies have revealed that interleukin-23 (IL-23) plays a crucial role in the pathogenesis of IBD. IL-23 is a heterodimeric cytokine that shares the p40 subunit with IL-12 but possesses a unique p19 subunit. IL-23 drives a chronic inflammatory response in the intestinal mucosa by promoting Th17 cell differentiation and the secretion of pro-inflammatory factors such as IL-17 and IL-22. Genetic studies have shown that IL23R gene polymorphism is significantly associated with IBD susceptibility, and animal models have confirmed that inhibiting IL-23 signaling can alleviate colitis symptoms. Based on this, monoclonal antibodies targeting the IL-12 / IL-23 p40 subunit (such as ustekinumab) and monoclonal antibodies specifically targeting the IL-23 p19 subunit (such as restazolidone and milizumab) have demonstrated efficacy in clinical trials for Crohn's disease and psoriasis, providing new directions for the treatment of UC.

[0006] Despite this, existing IL-23 inhibitors still face limitations in administration methods and side effects due to systemic exposure. For example, current IL-23 inhibitors (such as anti-p19 or anti-p40 monoclonal antibodies) are mainly administered intravenously (IV) or subcutaneously (SC). The drugs need to be distributed to the intestinal lesion site via the bloodstream; however, due to differences in vascular permeability and drug penetration barriers in the intestinal inflamed area, local drug concentrations are insufficient, affecting efficacy. The development of oral or intestinal local delivery systems remains challenging. Therefore, it is necessary to develop new immunomodulators and targeted therapies, as well as efficient drug delivery methods, to provide more effective treatment options for ulcerative colitis. Summary of the Invention

[0007] To address the problems mentioned above in the background art, the first objective of this invention is to provide an oral formulation of a heterozygous exosome loaded with STA-5326, comprising a heterozygous exosome loaded with STA-5326 and an acid-resistant shell coating the surface of the heterozygous exosome, wherein the heterozygous exosome is fused with at least Treg cell exosomes and MSCs cell exosomes.

[0008] This application demonstrates through experiments that the oral preparation of heterozygous exosomes loaded with STA-5326 has a good therapeutic effect on ulcerative colitis, can regulate the immune environment in the colon of UC, and has good stability, can accumulate in the inflammatory area, and repair the damaged intestinal barrier.

[0009] Preferably, the average particle size of the hybrid exosomes is 200 nm.

[0010] Preferably, the mass ratio of Treg cell exosomes to mesenchymal stem cell exosomes is 2:1.

[0011] Preferably, the mass ratio of the hybrid exosome loaded with STA-5326 to the acid-resistant shell is 1:(1-2), and the acid-resistant shell comprises chitosan, sodium alginate, and calcium chloride.

[0012] Preferably, the average particle size of the oral formulation of the hybrid exosome loaded with STA-5326 is 500 nm.

[0013] The second objective of this invention is to provide a method for an oral formulation of a heterozygous exosome loaded with STA-5326, comprising the following steps:

[0014] S1. Preparation of hybrid exosomes: Treg cell exosomes and mesenchymal stem cell exosomes are extruded together through a filter membrane;

[0015] S2. Loading STA-5326: STA-5326 is loaded into hybrid exosomes using pulsed ultrasound.

[0016] S3, Acid-resistant layer blending and coating: The hybrid exosome loaded with STA-5326 is mixed with the acid-resistant layer solution and placed in a magnetic stirrer, so that the acid-resistant layer solution coats the surface of the hybrid exosome through intermolecular forces. After the acid-resistant layer is dried, it forms an acid-resistant shell.

[0017] Preferably, the method for obtaining Treg cell exosomes is as follows: Treg cells are obtained by inducing immature CD4+ T cells, and their culture supernatant is collected and purified by gradient centrifugation.

[0018] The specific steps of gradient centrifugation purification are as follows: first, centrifuge at 300g for 10min to remove intact cells, then centrifuge at 2000g for 15min to remove cell debris, and then centrifuge at 3000g for 20min for further purification; after collecting the supernatant, use 100,000g ultracentrifugation for 70min to precipitate exosomes, and finally collect the precipitate and resuspend it with PBS buffer to obtain purified Treg cell exosomes.

[0019] Preferably, the method for obtaining the MSCs cell exosomes is as follows: collecting the MSCs cell supernatant and purifying it by gradient centrifugation;

[0020] The specific steps of gradient centrifugation purification are as follows: first, centrifuge at 300g for 10min to remove intact cells, then centrifuge at 2000g for 15min to remove cell debris, and then centrifuge at 3000g for 20min for further purification; after collecting the supernatant, use 100,000g ultracentrifugation for 70min to precipitate exosomes, and finally collect the precipitate and resuspend it with PBS buffer to obtain purified MSCs cell exosomes.

[0021] The third objective of this invention is to provide the application of an oral preparation of a heterozygous exosome loaded with STA-5326 in the preparation of a medicament for treating ulcerative colitis.

[0022] Preferably, the oral formulation of the STA-5326-loaded heterozygous exosome promotes intestinal barrier repair by reducing the expression of ZO-1 in the colonic tissue of mice with ulcerative colitis and increasing the expression of Occludin.

[0023] In summary, the beneficial effects of the present invention are as follows:

[0024] 1. This application achieves efficient fusion of Treg-exo and MSCs-exo through physical extrusion technology, constructing an engineered hybrid exosome that can not only regulate the immune environment in the colon of UC, but also repair the damaged intestinal barrier.

[0025] 2. The method for constructing heterozygous exosomes in this application is simple, efficient, and easy to operate, and the resulting structures are stable and uniform. Furthermore, the oral formulation of this application can accumulate in the colon of UC for a long time, effectively avoiding premature drug dissociation in the biological environment.

[0026] 3. In this application, immature CD4+ T cells are isolated from the spleen of healthy mice, activated and induced to become regulatory T cells, and regulatory T cell vesicles are obtained after identification, isolation and purification of the regulatory T cells.

[0027] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0028] Figure 1 This is a laser confocal fluorescence image of TMexo, formed by the extrusion fusion of Treg exosomes (Treg-exo) and MSC exosomes (MSCs-exo) in this invention.

[0029] Figure 2 The image shows the ultraviolet absorption spectrum (Absorbance; Wavelength) of STA-5326, where part A is the ultraviolet absorption spectrum of STA-5326 and part B is the standard curve of STA-5326.

[0030] Figure 3The determination of STA-5326 loading rate and drug loading amount (mass ratio) in TMexo-S is as follows: Part A is the determination of STA-5326 loading rate in TMexo-S; Part B is the determination of STA-5326 drug loading amount in TMexo-S.

[0031] Figure 4 This study analyzes the changes in particle size and potential during drug loading and coating of chitosan and sodium alginate. Part A is the particle size change analysis, and Part B is the potential change analysis.

[0032] Figure 5 The potential changes of TMexo-S@LBL in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively;

[0033] Figure 6 The particle size and potential of TMexo-S@ (extracted by centrifugation after incubation in SIF for 6 h) and TMexo-S were determined.

[0034] Figure 7 The images are TEM images of TMexo-S@ and TMexo-S, where part A is the TEM image of TMexo-S@ and part B is the TEM image of TMexo-S.

[0035] Figure 8 Western blotting of TMexo-S@ and TMexo-S;

[0036] Figure 9 Particle size determination of TMexo-S@LBL within 7 days;

[0037] Figure 10 In vivo imaging of small animals at different time points after drug administration;

[0038] Figure 11 Imaging of ex vivo organs at different time points after drug administration;

[0039] Figure 12 This is a schematic diagram of an experiment to treat acute ulcerative colitis in mice. The groups are, respectively, the blank control PBS group, the DSS model group, the Treg-exo@LBL, MSCs-exo@LBL, STA-5326, TMexo@LBL, Treg-exo-S@LBL, MSCs-exo-S@LBL and TMexo-S@LBL treatment groups.

[0040] Figure 13The analysis included changes in body weight and DAI over 9 days in different drug administration groups. Part A shows daily body weight records, Part B shows daily DAI statistics, and Part C shows spleen index statistics for each group on day 9.

[0041] Figure 14 The analysis of colon length in different drug administration groups is presented in Part A, which shows colon images of different drug administration groups, and Part B, which shows a statistical graph of colon length.

[0042] Figure 15 H&E sections of colon tissue from different drug administration groups;

[0043] Figure 16 Analysis of Treg cell counts in colon tissue of different drug administration groups;

[0044] Figure 17 Analysis of the number of Th1 cells in colon tissue of different drug administration groups;

[0045] Figure 18 Analysis of the number of Th17 cells in colon tissue of different drug administration groups;

[0046] Figure 19 Analysis of IL-12 content in colon tissue of different drug administration groups;

[0047] Figure 20 Analysis of MPO content in colon tissue of different drug administration groups;

[0048] Figure 21 Analysis of ZO-1 expression in colon tissue of different drug administration groups;

[0049] Figure 22 Analysis of Occludin expression in colon tissues of different drug administration groups. Detailed Implementation

[0050] To make the content of this invention easier to understand, the invention will be further described below with reference to specific embodiments and accompanying drawings.

[0051] Regulatory T cells (Tregs) play a crucial role in the development and progression of autoimmune diseases. Tregs are specialized immune cells whose primary functions are maintaining immune homeostasis and preventing immune responses against their own tissues. These cells achieve immunosuppression through various mechanisms, including direct interaction with effector T cells (such as Th1 and Th17 cells) and the secretion of anti-inflammatory cytokines. Dysfunction or a reduction in the number of Tregs is considered a significant factor in the pathological process of autoimmune diseases. Tregs play a vital role in immune regulation through their vesicles. These vesicles contain various bioactive molecules, such as proteins, lipids, and RNA, which effectively transmit information to target cells, thereby influencing the function and behavior of recipient cells.

[0052] Mesenchymal stem cells (MSCs) are pluripotent stem cells with the ability to self-renew and differentiate into multiple lineages. MSCs possess unique biological characteristics. First, they can differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, playing a crucial role in tissue repair and regeneration. Second, MSCs have immunomodulatory functions, capable of inhibiting excessive activation of immune cells and alleviating inflammatory responses. Compared to MSCs, the vesicles secreted by MSCs have advantages such as low immunogenicity, ease of preservation, and transport, making them highly sought after in disease treatment and regenerative medicine.

[0053] This application utilizes physical extrusion technology to achieve efficient fusion of Treg-exo and MSCs-exo, constructing a stable and homogeneous engineered hybrid exosome that simultaneously possesses immunomodulatory and tissue repair properties.

[0054] Traditional STA-5326 formulations have multiple limitations: low bioavailability (<15%) after oral administration, leading to systemic exposure and off-target effects; and technical bottlenecks such as insufficient drug accumulation at colonic lesion sites and poor chemical stability, making it difficult to achieve effective colonic targeted drug delivery and maintain therapeutic concentrations, ultimately resulting in clinical efficacy falling short of expectations.

[0055] This application utilizes pulsed ultrasound to load STA-5326 onto hybrid exosomes, thereby enhancing the oral bioavailability of STA-5326, achieving targeted enrichment at the colonic lesion site, prolonging the drug's residence time at the inflammatory site, and synergizing with the anti-inflammatory and barrier repair functions of the hybrid exosomes themselves, thus improving its therapeutic effect on ulcerative colitis.

[0056] Example 1: Preparation of Treg-exo, MSCs-exo and hybrid exosomes (TMexo)

[0057] 1. Preparation of Treg-exo

[0058] 1.1 Mouse Treg cell culture

[0059] Using immature CD4 + T-cell isolation kits were used to isolate immature CD4 cells from the spleens of healthy C57BL / 6 mice. + T cells were obtained from a healthy C57BL / 6 mouse. 5 × 10⁵ cells were collected. 6 An initial CD4 + T cells were seeded in X-VIVO medium containing 1% penicillin-streptomycin, 10% vesicle-free serum, IL-2 (20 ng / mL) and TGF-β (5 ng / mL), and anti-CD3 and anti-CD28 antibodies to stimulate T cell activation and expansion were added. The cells were cultured for 7 days.

[0060] 1.2 Extraction of Treg exosomes

[0061] Collect the culture supernatant containing Treg cells, centrifuge at 300g for 10 min to remove intact cells, and centrifuge at 2000g for 10 min to remove cell debris. After collecting the supernatant, centrifuge at 10000g for 30 min at 4°C to remove impurities. Collect the supernatant again, and ultracentrifuge at 100,000g for 70 min at 4°C. Repeat twice. Collect the Treg-exo precipitate, resuspend it in PBS, and finally determine the exosome protein concentration using the BCA method.

[0062] 2. Preparation of MSCs-exo

[0063] Collect the culture supernatant of MSCs, centrifuge at 300g for 10 min to remove intact cells, and centrifuge at 2000g for 10 min to remove cell debris. After collecting the supernatant, centrifuge at 10000g for 30 min at 4°C to remove impurities. Collect the supernatant again, and ultracentrifuge at 100,000g for 70 min at 4°C. Repeat twice. Collect MSCs-exo and resuspend in PBS. Finally, determine the exosome protein concentration using the BCA method.

[0064] 3. Preparation of TMexo

[0065] Using an extrusion apparatus, hybrid exosomes TMexo were prepared by 21 extrusion processes using a 200 nm pore size filter membrane at a Treg-exo to MSCs-exo mass ratio of 2:1. Example 2: Preparation and characterization of STA-5326-loaded hybrid exosomes (TMexo-S).

[0066] 1. Preparation of TMexo-S

[0067] 1.1 Using the TMexo prepared in Example 1, prepare three TMexo solutions with the same protein concentration (1 mg / mL). Take 100 μL of each solution and add it to three different EP tubes, which are labeled as tubes 1-3 respectively. Add solutions containing 50 μg, 100 μg and 200 μg STA-5326 to each tube respectively.

[0068] 1.2 The mixed solution was treated with pulsed ultrasonication using an ultrasonic cleaner. The specific operation was as follows: the EP tube was inserted into the float, making it suspend in the ultrasonic cleaner. The ultrasonic power was adjusted to 40kHz and the temperature was set to 4℃. Pulsed ultrasonication was performed, with 3 minutes on and 3 minutes off, and this was repeated 5 times before the tube was removed.

[0069] 1.3 After the ultrasound is completed, the EP tube is placed in a water bath constant temperature pot, the temperature is adjusted to 37℃, and incubated for 1 hour to restore membrane stability.

[0070] 1.4 After the water bath constant temperature incubation is completed, the mixed solution of drug and heterozygous exosomes is transferred to a 100kD ultrafiltration tube and centrifuged at 4,500g for 15min to remove excess drug molecules.

[0071] 2. Determination of drug loading in TMexo-S

[0072] The drug loading of STA-5326 was determined using a UV spectrophotometer.

[0073] 2.1 Prepare standard solutions by dissolving STA-5326 in PBS solution, with concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL and 50 μg / mL.

[0074] 2.2 Determination of drug loading

[0075] The filtrate of TMexo-S after 1.4°C ultrafiltration was placed in a cuvette, and its UV absorption peak was measured. The results showed that the drug loading was 10%, 20%, 25%, and 18% when the Exo:STA-5326 mass ratio was 1:3, 1:2, 1:1, and 2:1, respectively. The drug loading increased with increasing drug concentration, reaching its maximum at a mass ratio of 1:1. Therefore, a mass ratio of 1:1 for Exo:STA-5326 was used for subsequent experiments.

[0076] Example 3: Preparation of oral drug-loaded hybrid exosomes TMexo-S@LBL

[0077] 1. Solution preparation for acid-resistant shell

[0078] Chitosan was dissolved in 1% acetic acid solution; sodium alginate and calcium chloride were dissolved in PBS buffer.

[0079] 2. Preparation of TMexo-S@LBL

[0080] A 1:1 mass ratio mixture of TMexo-S and chitosan solution was stirred at 500 rpm for 30 min using a magnetic stirrer. 1 ml of PBS buffer was added, followed by centrifugation at 11000 rpm for 15 min. After washing three times, the mixture was resuspended in PBS buffer to obtain chitosan-encapsulated hybrid exosomes (TMexo-S@L). TMexo-S@L was then mixed with sodium alginate solution at a 1:2 mass ratio and stirred for 30 min. 1 ml of PBS buffer was added, and the mixture was centrifuged at 11000 rpm for 15 min. After washing three times, the mixture was resuspended in PBS buffer. An equal mass of calcium chloride solution was added, and the mixture was cross-linked at 37°C for 10 min. The mixture was then centrifuged at 11000 rpm for 15 min and washed three times to obtain oral-loaded drug-loaded hybrid exosomes (TMexo-S@LBL) layered with chitosan, sodium alginate, and calcium chloride.

[0081] The time-dependent effect of TMexo-S@LBL in simulated gastric juice (SGF) and simulated intestinal juice (SIF) was investigated. Samples were taken at time points of 0, 2, 4, 6, 8, 10, and 12 (h). The obtained samples were subjected to particle size and potential analysis. The results are as follows: Figure 5 As shown, TMexo-S@LBL can exist stably in simulated gastric juice.

[0082] Example 4: Characterization of TMexo-S@LBL after in vitro release

[0083] 1.1 Transmission electron microscopy observation

[0084] Preparation of TMexo-S@: TMexo-S was incubated in SIF for 6 h, centrifuged and purified, and the precipitate was collected to obtain TMexo-S@.

[0085] Take 10 μL of TMexo-S and TMexo-S@ precipitate resuspended in PBS, add them to a copper grid, let stand for 5 min, and remove the residual liquid through the edge of filter paper; then perform negative staining with uranium acetate for 30 s, remove excess staining solution again, and air dry for 10 min.

[0086] After negative staining and drying, the vesicle morphology of the samples was observed under a transmission electron microscope with an accelerating voltage of 120kV.

[0087] The results are as follows Figure 6 As shown, after incubation with SIF, the shells of chitosan, sodium alginate, and calcium chloride can be dissolved without destroying the morphology of TMexo-S.

[0088] 1.2 Measurement of Particle Size and Potential

[0089] The potential and particle size of vesicles were determined using a dynamic light scattering particle size analyzer: 1 mL of TMexo-S and TMexo-S@ solutions with a concentration greater than 200 μg / mL were taken, blown evenly, and placed into the detection slot of the dynamic light scattering particle size analyzer for detection.

[0090] The results are as follows Figure 7 As shown, SIF does not affect the particle size and surface potential of TMexo-S.

[0091] 1.3 Western blot detection

[0092] Protein concentrations in TMexo-S and TMexo-S samples were determined using the BCA method. Samples were separated by SDS-PAGE using an 8-12% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was washed with TBST solution (10 mM Tris-HCl pH 7.6, 150 mM NaCl, and 0.05% Tween-20), blocked with 5% skim milk for 30 min, and incubated overnight at 4°C with an appropriate primary antibody. After washing twice with TBST solution, horseradish peroxidase-conjugated secondary antibody was added and incubated for 2 h. Bands were detected using a high-sensitivity ECL chemiluminescence assay kit, and images were obtained using a gel imaging system.

[0093] The results are as follows Figure 8 As shown, SIF does not affect the surface proteins of TMexo-S.

[0094] 1.4TMexo-S@LBL Stability Test

[0095] The particle size of TMexo-S@LBL was measured on days 1, 2, 3, 4, 5, 6, and 7, respectively. The results... Figure 9 As shown, the particle size of TMexo-S@LBL remained around 500nm within 7 days, demonstrating good stability and making it suitable for long-term storage.

[0096] Example 5: Targeting effect of TMexo-S@LBL

[0097] 1. Establishment of a mouse model of ulcerative colitis

[0098] 1.1 Prepare 6 female C57BL / 6 mice aged 8-9 weeks.

[0099] 1.2 Ulcerative Colitis Model: Mice were housed in a pathogen-free animal facility, and an ulcerative colitis mouse model was established by adding 3% dextran sulfate sodium salt (DSS) to their drinking water. All ulcerative colitis mice had free access to water, which was changed every 2 days. After 7 days, the water was replaced with normal water without 3% DSS.

[0100] 2. Targeting effect determination

[0101] DiR staining was performed on TMexo-S to obtain DiR-stained TMexo-S; the DiR-labeled TMexo-S was coated with an acid-resistant shell, the steps of which were the same as those in Example 3, and then DiR-stained TMexo-S@LBL was obtained.

[0102] Mice were randomly divided into two groups of three each: the TMexo-S group and the TMexo-S@LBL group. Mice in the TMexo-S group were administered DiR-stained TMexo-S via intragastric perfusion at a dose of 20 mg / kg, while mice in the TMexo-S@LBL group were administered DiR-stained TMexo-S@LBL via intragastric perfusion at a dose of 20 mg / kg.

[0103] In vivo fluorescence photography of mice was performed at 0H, 2H, 4H, 6H, 12H, 24H and 48H after gavage.

[0104] The results are as follows Figure 10 As shown, at 48 hours, most of the drug in the TMexo-S group had been metabolized, while some drug remained in the colon in the TMexo-S@LBL group. Figure 11 As shown, TMexo-S@LBL reached its maximum concentration in the mouse colon at 6 hours, indicating that TMexo-S@LBL exhibits a stronger accumulation capacity at sites of inflammation.

[0105] Example 6: TMexo-S@LBL for the treatment of ulcerative colitis

[0106] 1. Treatment of ulcerative colitis model

[0107] 1.1 Ulcerative Colitis Model: Mice were housed in a pathogen-free animal facility, and an ulcerative colitis mouse model was established by adding 3% DSS to their drinking water. All ulcerative colitis mice had free access to water, which was changed every 2 days. After 7 days, the water was replaced with normal water without 3% DSS.

[0108] 1.2 Mouse grouping: Mice were randomly divided into 6 groups of 6 mice each. The groups were: blank control PBS group, DSS model group, TMexo@LBL, Treg-exo-S@LBL, MSCs-exo-S@LBL and TMexo-S@LBL treatment group.

[0109] The control group mice were treated by regularly administering 20 mg / kg of PBS orally.

[0110] The DSS model mice were treated as follows: after establishing the ulcerative colitis mouse model, they were regularly given 20 mg / kg of PBS orally.

[0111] The treatment method for TMexo@LBL group mice was as follows: after establishing the ulcerative colitis mouse model, TMexo@LBL was administered orally at regular intervals at a dose of 20 mg / kg.

[0112] The treatment method for mice in the Treg-exo-S@LBL group was as follows: after establishing a mouse model of ulcerative colitis, mice were regularly administered 20 mg / kg of Treg-exo-S@LBL orally.

[0113] The treatment method for mice in the MSCs-exo-S@LBL group was as follows: after establishing a mouse model of ulcerative colitis, MSCs-exo-S@LBL were administered orally at regular intervals at a dose of 20 mg / kg.

[0114] The treatment of mice in the TMexo-S@LBL treatment group was as follows: after establishing a mouse model of ulcerative colitis, TMexo-S@LBL was administered orally at regular intervals at a dose of 20 mg / kg.

[0115] The preparation methods for each group of oral drugs were the same as in Example 3. Treatment was administered orally to mice on days 2, 4, and 6 after modeling.

[0116] 2. Recording of mouse body weight, disease activity index score, and spleen index.

[0117] During treatment, the mice's weight and disease activity index were measured and recorded daily. The disease activity index (DAI) was determined by recording weight, stool consistency, and fecal occult blood daily. A comprehensive score was calculated by combining the percentage of weight loss (0 for no change in weight, 1-5 for 1 point, 5-10 for 2 points, 10-15 for 3 points, and greater than 15 for 4 points), stool consistency (0 for normal, 2 for loose stool, and 4 for diarrhea), and fecal bleeding (0 for normal, 2 for positive occult blood, and 4 for overt bleeding). The total score of these three results was divided by 3 to obtain the statistical analysis result of the DAI value. The results are as follows: Figure 13As shown in Part A, after treatment with TMexo-S@LBL, body weight recovered relative to the DSS group. Figure 13 As shown in Part B, the disease severity index decreased significantly in the later stages after TMexo-S@LBL treatment.

[0118] Spleen index determination: All mice were euthanized on day 9, and spleens were harvested and their weights were recorded. Results are as follows: Figure 13 As shown in section C, after treatment with TMexo-S@LBL, the spleen index was closer to that of the PBS group.

[0119] 3. Histological analysis of the colon

[0120] Colon length determination: All mice were euthanized on day 9, and the cecum and colon were extracted, photographed, and colon length was measured.

[0121] Histological analysis: Colonic tissue was collected at the end of the experiment and fixed with 4% paraformaldehyde, then embedded in paraffin. After dewaxing and hydration, 4 mm thick sections were stained with hematoxylin and eosin (H&E), respectively, and then observed under a microscope at 400x magnification. Results are as follows: Figure 15 As shown, H&E results indicate that TMexo-S@LBL treatment helps maintain the integrity of the colonic epithelium.

[0122] Example 7: Mechanism Study of TMexo-S@LBL in Improving UC

[0123] 1. Immunological analysis

[0124] 1.1 After the ulcerative colitis model treatment experiment in Example 6 was completed, the mice were euthanized, the colon was quickly separated, the mesentery was removed, and the colon was placed in a pre-cooled sterile PBS culture dish; the colon was longitudinally cut along the mesentery, and vigorously shaken in PBS to thoroughly remove the intestinal contents; the cleaned colonic mucosa was cut into pieces of approximately 1 mm. 3Small pieces of minced mucosal tissue were transferred to a preheated sterile culture flask containing calcium- and magnesium-free HBSS (5% FBS and 5mM EDTA) solution. The mixture was then incubated at 37°C and 250 rpm for 20 min, the liquid was discarded, and this process was repeated twice. After the second incubation, the mixture was filtered, and excess liquid was blotted away with filter paper (to ensure the removal of residual EDTA). The colon was transferred to a 1.5 mL EP tube and minced. The mucosal tissue fragments were transferred to a sterile culture flask containing colonic lamina propria digestion solution and digested for 30 min (37°C, 250 rpm). After digestion, the mixture was filtered through a 70 μm cell filter, and the supernatant was collected into a 1.5 mL centrifuge tube and centrifuged at 1500 rpm for 10 min at 4°C. The supernatant was discarded, and the separated cell pellet was resuspended in pre-chilled calcium- and magnesium-free HBSS / FBS buffer. The sample was placed on ice, and appropriate volumes of blocking antibody and fluorescent antibody were added according to the cell volume before analysis.

[0125] Colon tissue was taken for flow cytometry analysis, and the results are as follows: Figure 16 As shown, after treatment on days 2, 4, and 6, the increase in Treg cell content in colonic tissue varied depending on the drug treatment. The Treg-exo-S@LBL, MSCs-exo-S@LBL, and TMexo@LBL groups all showed significantly higher Treg cell counts compared to the DSS model group, indicating that Treg-exo-S@LBL, MSCs-exo-S@LBL, and TMexo@LBL all have therapeutic effects on the colonic tissue of DSS mice. Further comparison revealed that the TMexo-S@LBL treatment group had a higher Treg cell count than the Treg-exo-S@LBL and MSCs-exo-S@LBL treatment groups, almost matching the PBS group. The TMexo-S@LBL treatment group demonstrated a significant advantage in increasing Treg cell levels. This result not only confirms the individual therapeutic effects of Treg-exo-S@LBL and MSCs-exo-S@LBL, but more importantly, reveals the synergistic effect of the combined application of the three components.

[0126] 1.2 Measurement of Th1 and Th17 cell content: Compared with the control group, UC increased the mRNA expression levels of Th1 cell-related transcription factors and the cytokine IFN-γ in colonic tissue, indicating an increased number of Th1 cells in UC colonic tissue. Furthermore, compared with the control group, the proportion of CD4+IL-17+ T cells in UC was also significantly increased, indicating an increased number of Th17 cells in UC colonic tissue. Th1 and Th17 cells have a synergistic pathogenic effect in the pathogenesis of UC, influencing each other and jointly exacerbating intestinal inflammatory damage.

[0127] After the experimental control treatment in Example 6, the content of Th1 cells and Th17 cells in the colon tissue was measured.

[0128] The results are as follows Figure 17 As shown, compared with the DSS model group, the proportion of CD4+IFN-γ+ T cells in the colon tissue of mice in the Treg-exo-S@LBL group, MSCs-exo-S@LBL group and TMexo-S@LBL treatment group was significantly reduced, with the TMexo-S@LBL treatment group showing the lowest proportion of CD4+IFN-γ+ T cells in the colon tissue of mice.

[0129] like Figure 18 As shown, compared with the DSS model group, the proportion of CD4+IL-17+ T cells in the colon tissue of mice in the Treg-exo-S@LBL group, MSCs-exo-S@LBL group and TMexo-S@LBL treatment group was significantly reduced, with the TMexo-S@LBL treatment group showing the lowest proportion of CD4+IL-17+ T cells in the colon tissue of mice.

[0130] The above experiments all demonstrate that TMexo-S@LBL can synergistically regulate the immune environment in the colon tissue of UC model mice.

[0131] 2. Evaluation of anti-inflammatory effects

[0132] On day 9 after treatment in the ulcerative colitis model of Example 6, all mice were euthanized, and colonic tissue was extracted. A suitable amount of colonic tissue was taken, washed in pre-chilled PBS to remove blood, weighed, and transferred to a glass homogenizer. 5-10 mL of pre-chilled PBS was added for thorough homogenization (this process should be performed on ice). The resulting homogenate was further processed by ultrasonic disruption or repeated freeze-thaw cycles. The prepared homogenate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected for analysis. All samples were adjusted to the same protein concentration, and the levels of IL-12 and MPO in the colon were detected using an ELISA kit.

[0133] like Figure 19 As shown, compared with the DSS model group, the expression level of IL-12 in the colon tissue of mice in the Treg-exo-S@LBL group, MSCs-exo-S@LBL group and TMexo-S@LBL treatment group was significantly reduced.

[0134] like Figure 20As shown, compared with the DSS model group, the expression levels of IL-12 in the colon tissue of mice in the Treg-exo-S@LBL group, MSCs-exo-S@LBL group and TMexo-S@LBL treatment group were all reduced. Among them, the reduction of MPO content in the colon tissue of mice in the TMexo-S@LBL treatment group was the most significant, and it showed the same trend as the IL-12 content in the colon tissue. This indicates that the oral formulation of TMexo-S@LBL can effectively inhibit the inflammatory response and reduce intestinal damage.

[0135] 3. Evaluation of intestinal barrier repair efficacy

[0136] At the end of the experiment, colon tissue was collected, sections were prepared, fixed with 4% paraformaldehyde, and then embedded in paraffin. After dewaxing and hydration, the sections were incubated with ZO-1 and Occludin antibodies, respectively, and blocked with serum at room temperature for 30 min. The obtained samples were then incubated overnight at 4°C with the corresponding primary antibodies. Subsequently, HRP-conjugated secondary antibodies were added, and the sections were incubated at room temperature for 15 min. Random field images of the sections were obtained using a microscope.

[0137] The results are as follows Figure 21 As shown, the expression level of ZO-1 in the colonic tissue of mice in the DSS group was decreased, while the expression level of ZO-1 in the colonic tissue of mice increased after treatment with TMexo-S@LBL. These results indicate that TMexo-S@LBL can maintain the integrity of intestinal epithelial cells, keep the first line of defense of intestinal immunity intact, and alleviate colitis.

[0138] Occludin is a membrane-integrated protein that plays a role in the formation and regulation of the permeability barrier surrounding tight junction cells. A deficiency of Occludin in colon tissue cells induces adhesion in the colon tissue.

[0139] like Figure 22 As shown, compared with the DSS group mice, the expression level of Occludin in the colon tissue of the TMexo-S@LBL group mice was increased, which promoted the repair of the intestinal barrier.

[0140] The embodiments described above are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention shall fall within the scope of protection of the present invention.

Claims

1. An oral formulation of a heterozygous exosome loaded with STA-5326, characterized in that, It includes a hybrid exosome loaded with STA-5326 and an acid-resistant shell covering the surface of the hybrid exosome, wherein the hybrid exosome is fused with at least Treg cell exosomes and MSC cell exosomes.

2. The oral formulation of a heterozygous exosome loaded with STA-5326 according to claim 1, characterized in that, The average particle size of the hybrid exosomes is 200 nm.

3. The oral formulation of a heterozygous exosome loaded with STA-5326 according to claim 1, characterized in that, The mass ratio of Treg cell exosomes to mesenchymal stem cell exosomes is 2:

1.

4. The oral formulation of a heterozygous exosome loaded with STA-5326 according to claim 1, characterized in that, The mass ratio of the hybrid exosomes loaded with STA-5326 to the acid-resistant shell is 1:(1-2), and the acid-resistant shell comprises chitosan, sodium alginate and calcium chloride.

5. The oral formulation of a heterozygous exosome loaded with STA-5326 according to claim 4, characterized in that, The average particle size of the oral formulation of the hybrid exosome loaded with STA-5326 is 500 nm.

6. A method for preparing an oral formulation of a hybrid exosome loaded with STA-5326 according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of hybrid exosomes: Treg cell exosomes and mesenchymal stem cell exosomes are extruded together through a filter membrane; S2. Loading STA-5326: STA-5326 is loaded into hybrid exosomes using pulsed ultrasound. S3, Acid-resistant layer blending and coating: The hybrid exosome loaded with STA-5326 is mixed with the acid-resistant layer solution and placed in a magnetic stirrer, so that the acid-resistant layer solution coats the surface of the hybrid exosome through intermolecular forces. After the acid-resistant layer is dried, it forms an acid-resistant shell.

7. The method according to claim 6, characterized in that, The method for obtaining Treg cell exosomes is as follows: Treg cells are obtained by inducing immature CD4+ T cells, and their culture supernatant is collected and purified by gradient centrifugation. The specific steps of gradient centrifugation purification are as follows: first, centrifuge at 300g for 10min to remove intact cells, then centrifuge at 2000g for 15min to remove cell debris, and then centrifuge at 3000g for 20min for further purification; after collecting the supernatant, use 100,000g ultracentrifugation for 70min to precipitate exosomes, and finally collect the precipitate and resuspend it with PBS buffer to obtain purified Treg cell exosomes.

8. The method according to claim 6, characterized in that, The method for obtaining MSCs cell exosomes is as follows: collect MSCs cell supernatant and perform gradient centrifugation purification; The specific steps of gradient centrifugation purification are as follows: first, centrifuge at 300g for 10min to remove intact cells, then centrifuge at 2000g for 15min to remove cell debris, and then centrifuge at 3000g for 20min for further purification; after collecting the supernatant, use 100,000g ultracentrifugation for 70min to precipitate exosomes, and finally collect the precipitate and resuspend it with PBS buffer to obtain purified MSCs cell exosomes.

9. The use of an oral formulation of a heterozygous exosome loaded with STA-5326 in the preparation of a medicament for treating ulcerative colitis, characterized in that, The oral formulation of the hybrid exosome as described in any one of claims 1-5 is used.

10. The application according to claim 9, characterized in that, The oral formulation of the STA-5326-loaded heterozygous exosomes promotes intestinal barrier repair by reducing the expression of ZO-1 and increasing the expression of Occludin in the colonic tissue of mice with ulcerative colitis.

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