Extracellular vesicle complex encapsulating thymosin beta 4, preparation method and application thereof

Abstract

This invention relates to an extracellular vesicle complex encapsulating thymosin β4, its preparation method, and its application. Mesenchymal stem cell small extracellular vesicles (MSC-sEVs) are extracted, mixed with thymosin β4 (Tβ4), and incubated. After ultrasound-assisted loading, MSC-sEVs-Tβ4 encapsulated in MSC-sEVs is obtained, which can be used in formulations or drugs targeting autoimmune dry eye. The beneficial effects of this invention are: by encapsulating Tβ4 in MSC-sEVs, the stability problem of Tβ4 in aqueous solution is effectively solved, allowing it to maintain its medicinal properties for a long time; furthermore, Tβ4 can synergistically interact with MSC-sEVs to produce a better therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an extracellular vesicle complex containing thymosin β4, its preparation method, and its application. Background Technology

[0002] Dry eye is a chronic ocular surface disease caused by multiple factors, with a high incidence rate in the population. It often causes symptoms such as dry eyes, foreign body sensation, and visual disturbances, with inflammation as the main pathogenic mechanism. The pathogenesis of dry eye is complex. Studies have shown that tear film instability, high tear osmotic pressure, and ocular surface inflammation are the main causes. Among them, Sjögren's syndrome-related dry eye, as a more severe and difficult-to-treat type, is often accompanied by the infiltration of a large number of lymphocytes and the formation of autoantibodies. In severe cases, it can even lead to secondary corneal ulcers or perforation, impairing vision. The pathological characteristics of SS-related dry eye are mainly manifested by the infiltration of a large number of activated immune cells (including T cells, macrophages, etc.) and increased expression of inflammatory factors in the lacrimal glands and ocular surface, leading to glandular structure destruction and ocular surface tissue damage, ultimately causing dry eye symptoms. The pathogenic factors of SS-related dry eye are complex and diverse, requiring more effective treatment methods.

[0003] According to the 2018 guidelines and consensus of the International Society for Extracellular Vesicles, extracellular vesicles with a diameter <200 nm in the supernatant of mesenchymal stem cells obtained by differential ultracentrifugation are referred to as small extracellular vesicles (sEVs) to replace exosomes, because current isolation methods are unable to completely remove non-exosome vesicles. MSC-sEVs refer to small extracellular vesicles with a lipid bilayer structure secreted by mesenchymal stem cells, with a diameter of approximately 30-150 nm. They carry abundant cell-specific bioactive molecules and can deliver their contents (proteins, lipids, RNAs, and DNAs) to recipient cells through macropinocytosis, phagocytosis, receptor-mediated endocytosis, or direct fusion with the plasma membrane, thereby regulating the function of the recipient cells. MSC-sEVs have biological functions similar to their parent cells, can migrate to sites of tissue inflammation and injury, regulate various immune cells, including T lymphocytes and macrophages, and thus inhibit excessive immune inflammatory responses.

[0004] Thymosin β4 (Tβ4) is a highly conserved, water-soluble small-molecule actin chelate peptide composed of 43 amino acids. First isolated from calf thymus in the 1980s, it is widely distributed in eukaryotic cells throughout the body. Exogenous Tβ4 can inhibit local corneal inflammation, reduce corneal and conjunctival epithelial cell apoptosis, promote cell migration and regeneration, and possesses certain immunomodulatory functions, reducing the infiltration of macrophages and CD4+ T cells. Tβ4 has significant potential for use in autoimmune inflammatory diseases. However, as a small-molecule peptide, it suffers from a short half-life and is easily hydrolyzed and denatured. Furthermore, the inherent biological barriers in the eye further limit its bioavailability.

[0005] Currently, there is no ideal treatment for dry eye. Clinically available medications for dry eye mainly include artificial tears, corticosteroids, 0.05% cyclosporine A, and autologous serum. Artificial tears primarily provide lubrication and relieve symptoms, but cannot cure dry eye. Corticosteroids and immunosuppressants such as cyclosporine A can significantly improve dry eye symptoms, but long-term use of these drugs has significant side effects on the eyes. Autologous serum contains various bioactive components that promote corneal epithelial repair, but its demanding preparation and storage conditions limit its widespread application. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an extracellular vesicle complex containing thymosin β4, its preparation method, and its application.

[0007] The technical solution adopted in this invention is: the application of an extracellular vesicle complex loaded with thymosin β4 in the preparation of a drug for treating autoimmune diseases, wherein the drug includes a nanocomposite obtained by loading thymosin β4 into extracellular vesicles.

[0008] Preferably, the extracellular vesicles are small extracellular vesicles (sEVs) derived from umbilical cord mesenchymal stem cells, and an extracellular vesicle complex MSC-sEVs-Tβ4 loaded with thymosin β4 is prepared.

[0009] Preferably, a drug for treating autoimmune dry eye is prepared.

[0010] Preferably, MSC-sEVs-Tβ4 alleviates macrophage inflammation by inhibiting the activation of the JNK / ERK / p38 MAPK pathway.

[0011] Preferably, thymosin β4 is loaded into extracellular vesicles using an ultrasound method.

[0012] Preferably, saponins are added to the forward ultrasonic reaction system, and cholesterol solution is added to the backward ultrasonic reaction system.

[0013] Preferably, the specific preparation method is as follows:

[0014] Step 1: Mix extracellular vesicles with thymosin β4 at a protein ratio of 2-10:1, add PBS to form a reaction system, add 0.01-0.03% (w / v) saponin to the reaction system, and incubate on ice for pre-osmosis;

[0015] Step 2: Sonicate the reaction system for 1-10 minutes;

[0016] Step 3: Dilute with an equal volume of PBS buffer, then add cholesterol solution to a final concentration of 0.1-0.2 mg / mL, and incubate at 37°C for 60-90 min;

[0017] Step 4: Remove free thymosin β4 to obtain a suspension containing MSC-sEVs-Tβ4.

[0018] Preferably, in step two, one cycle consists of 1-20 seconds of ultrasound, 1-20 seconds of pause, and 1-5 minutes of cooling on ice, and a total of 4-6 cycles are performed.

[0019] Preferably, the cholesterol solution is a complex solution of cholesterol and methyl-β-cyclodextrin.

[0020] The advantages and positive effects of this invention are as follows: By loading mesenchymal stem cell extracellular vesicles (sEVs) into the complex, the complex exhibits better stability and sustained-release effect in vivo, effectively solving the stability problem of Tβ4 in aqueous solution and maintaining its medicinal properties for a long time. Furthermore, Tβ4 and MSC-sEVs can synergistically enhance the therapeutic effect, resulting in better therapeutic efficacy at the same dosage, demonstrating strong clinical application value. Using MSC-sEVs as a carrier, the contents (proteins, lipids, RNAs, and DNAs) can be delivered to recipient cells through macropinocytosis, phagocytosis, receptor-mediated endocytosis, or direct plasma membrane fusion, thereby regulating the function of recipient cells. This preparation method is simple, time-saving, and has a high drug loading rate, making it suitable for large-scale production.

[0021] MSC-sEVs-Tβ4 can be used to treat autoimmune diseases, especially autoimmune dry eye. Using sEVs as a carrier, the complex is given better stability and sustained-release effect in vivo, which enhances the multiple therapeutic effects of Tβ4 in the treatment of autoimmune dry eye, such as anti-inflammatory, immunomodulatory, anti-fibrotic, and tissue repair promotion. Attached Figure Description

[0022] Figure 1A shows the characterization of MSC-sEVs; B shows the morphological observation of MSCs; C shows a representative transmission electron microscope image of MSC-sEVs; D shows the expression of surface antigens in MSCs cultured to the third generation by reverse transcription PCR.

[0023] Figure 2 This refers to the particle diameter distribution of MSC-sEVs;

[0024] Figure 3 This is a Western Blot analysis of the expression levels of CD81, CD63 and TSG101 proteins in MSCs, MSC-sEVs and MSC-sEVs-Tβ4.

[0025] Figure 4 The flow cytometry was used to detect the encapsulation efficiency of Tβ4 in small extracellular vesicles.

[0026] Figure 5 These are transmission electron microscope images of MSC-sEVs-Tβ4;

[0027] Figure 6 This refers to the particle diameter distribution of MSC-sEVs-Tβ4.

[0028] Figure 7 It is the linear regression equation of the Tβ4 standard curve;

[0029] Figure 8 The fluorescence expression of PKH-26-MSC-sEVs-Tβ4-FITC in mouse lacrimal glands;

[0030] Figure 9 The fluorescence expression of PKH-26-MSC-sEVs-Tβ4-FITC in mouse conjunctiva;

[0031] Figure 10 The fluorescence expression of PKH-26-MSC-sEVs-Tβ4-FITC in the cervical draining lymph nodes of mice;

[0032] Figure 11 The tear secretion of mice in the PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEV treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) was measured before treatment and at 2 and 4 weeks after treatment (*p<0.05).

[0033] Figure 12These are images of corneal fluorescein staining in mice in the PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) before treatment, 2 weeks after treatment, and 4 weeks after treatment.

[0034] Figure 13 This study compares the corneal fluorescein staining scores of mice in the PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) before treatment, at 2 weeks after treatment, and at 4 weeks after treatment (**p<0.01, ***p<0.001, ****p<0.0001).

[0035] Figure 14 These are H&E stained histopathological sections of the lacrimal glands of mice in the PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4), bar=100μm.

[0036] Figure 15 This is a comparison of the lymphocyte infiltration area in the lacrimal gland H&E stained histopathological sections of mice in the PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4).

[0037] Figure 16 These are ocular surface and sodium fluorescein staining images of mice in the blank control group, PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4).

[0038] Figure 17 These are H&E stained histopathological sections of the lacrimal glands, eyeballs, liver, heart, spleen, lungs, and kidneys of mice in the blank control group, PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4).

[0039] Figure 18The changes in body weight during treatment were observed in mice in the blank control group, PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4).

[0040] Figure 19 These are immunofluorescence images of PKH-26, FITC-labeled MSC-sEVs-Tβ4, and RAW cells after 24 hours of co-culture.

[0041] Figure 20 The results of the CCK-8 assay on RAW cell proliferation were obtained from MSC-sEVs-Tβ4 treatment.

[0042] Figure 21 The mRNA expression levels of inflammation-related genes IL-6 and IL-1β in RAW cell blank control group (BLANK), LPS experimental group (LPS), PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) under LPS inflammatory stimulation;

[0043] Figure 22 These are immunofluorescence images of PKH-26, FITC-labeled MSC-sEVs-Tβ4, and HCEC cells after 24 hours of co-culture.

[0044] Figure 23 The mRNA expression levels of inflammation-related genes IL-6 and TNF-α in HCEC cells under hyperosmolar stimulation were measured in the blank control group (BLANK), hyperosmolar stimulation experimental group (HS), PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4).

[0045] Figure 24 The mRNA expression levels of IFN-γ, T-BET, RORC, and IL-17 after treatment with PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) were measured.

[0046] Figure 25The expression level of IL-17, a marker protein of Th17 cells, after treatment with PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) for T cell differentiation.

[0047] Figure 26 The mRNA expression levels of M1 and M2 macrophage markers iNOS, Arg1, and IL-10 in the lacrimal glands of mice in the PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) were measured.

[0048] Figure 27 The polarization ratio of M1 cells under M1 polarization-induced conditions was determined by flow cytometry in the following groups: RAW cell blank control group (BLANK), LPS experimental group (LPS), PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4).

[0049] Figure 28 The mRNA expression levels of M2 macrophage-specific transcription factors Arg1 and CD206 in RAW cell blank control group (BLANK), LPS experimental group (LPS), PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4) under LPS inflammatory stimulation;

[0050] Figure 29 The protein expression levels of MAPK pathway-related proteins p-JNK, p-ERK, and p-p38 in RAW cells under LPS inflammatory stimulation were measured in the blank control group (BLANK), LPS experimental group (LPS), PBS control group (PBS), Tβ4 treatment group (Tβ4), MSC-sEVs treatment group (MSC-sEVs), and MSC-sEVs-Tβ4 treatment group (MSC-sEVs-Tβ4). Detailed Implementation

[0051] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0052] This invention relates to an extracellular vesicle complex loaded with thymosin β4, its preparation method, and its application. The extracellular vesicle complex loaded with thymosin β4, obtained by loading thymosin β4 into extracellular vesicles, can be used to treat autoimmune dry eye. The extracellular vesicles can be small extracellular vesicles from mesenchymal stem cells. These small extracellular vesicles are extracted from mesenchymal stem cells, mixed with Tβ4, incubated, and then ultrasonically loaded to obtain MSC-sEVs-Tβ4 loaded with MSC-sEVs. Based on the advantages of MSC-sEVs, such as their double-membrane structure protecting the contents from degradation, strong transmembrane transport capacity, and low immunogenicity, using MSC-sEVs as a carrier of Tβ4 can effectively improve the stability and penetration of Tβ4 in the eye, increasing its bioavailability, thus becoming a more effective treatment for autoimmune dry eye.

[0053] MSC-sEVs-Tβ4 combines the advantages of both Tβ4 and sEVs. Using sEVs as a carrier effectively solves the stability problem of Tβ4 in aqueous solution, maintaining its medicinal properties for a long time. In the treatment of autoimmune dry eye, Tβ4 endows the nanocomposite with multiple therapeutic effects, including anti-inflammatory, immunomodulatory, anti-fibrotic, and tissue repair-promoting effects. The sEVs carrier provides the nanocomposite with better stability and sustained-release effect in vivo. Simultaneously, Tβ4 can synergistically interact with mesenchymal stem cell small extracellular vesicles to produce better therapeutic effects. Compared with Tβ4 or sEVs alone, the MSC-sEVs-Tβ4 nanocomposite exhibits better therapeutic effects at the same dosage, demonstrating strong clinical application value.

[0054] Mesenchymal stem cells (MSCs) can load Tβ4 into their extracellular vesicles. MSCs secrete small extracellular vesicles (sEVs) containing abundant cell-specific bioactive molecules. These vesicles can deliver their contents (proteins, lipids, RNAs, and DNAs) to recipient cells via macropinocytosis, phagocytosis, receptor-mediated endocytosis, or direct membrane fusion, thereby regulating the function of the recipient cells. MSCs-sEVs possess biological functions similar to their parent cells, migrating to sites of tissue inflammation and damage, regulating various immune cells, and thus inhibiting excessive immune inflammatory responses. Based on the advantages of MSCs-sEVs, such as their double-membrane structure protecting their contents from degradation, strong transmembrane transport capacity, and low immunogenicity, using MSCs-sEVs as a Tβ4 carrier can effectively improve the stability and penetration of Tβ4 in the eye, increasing its bioavailability and thus becoming a more effective treatment for autoimmune diseases.

[0055] During preparation, Tβ4 was added to small extracellular vesicles of mesenchymal stem cells and mixed and incubated. In order to further improve the encapsulation rate of Tβ4 in small extracellular vesicles, the mixed solution was sonicated to promote the entry of Tβ4 into the small extracellular vesicles. After sonication, the mixture was incubated at 37°C to restore the stability of the small extracellular vesicle membrane, thus obtaining MSC-sEVs-Tβ4 that can be used to prepare drugs.

[0056] In some embodiments of the present invention, the specific preparation method of MSC-sEVs-Tβ4 is as follows: First, small extracellular vesicles of mesenchymal stem cells are obtained. The small extracellular vesicles and Tβ4 are mixed at a protein ratio of 10-2:1, with the preferred protein concentration ratio of extracellular vesicles to Tβ4 being 2:1. After mixing, the mixture is added to PBS, and saponins are added to the mixture to a final concentration of 0.01–0.03% (w / v). The mixture is then placed on ice for 5 min for pre-permeation. On ice, the small extracellular vesicles are loaded with drug using ultrasound technology. For example, the sample is sonicated for 1 min using an ultrasound instrument at 20-50% power, with a 5-second pause every 5 seconds of sonication. This constitutes one cycle. After one cycle, the sample is cooled on ice for 1 min. A total of 6 cycles are performed before ending the sonication. After sonication, the sample is diluted with an equal volume of PBS buffer, and cholesterol working solution is added to a final concentration of 0.1–0.2 mg / mL. The mixture is then placed at room temperature for 10 min (to allow cholesterol to bind with saponins, helping to restore membrane cholesterol and permeability). The mixture is then placed at 37°C. Incubate for 60–90 min to restore membrane stability; centrifuge using a 100 kDa ultrafiltration tube at 14000 g for 10 min to remove free Tβ4 from the liquid and obtain a suspension of MSC-sEVs-Tβ4.

[0057] The cholesterol working solution was a composite solution (Chol-MβCD) prepared by mixing cholesterol and methyl-β-cyclodextrin at a molar ratio of 1:6. After dissolving and evaporating the cholesterol to form a film, it was mixed with the MβCD solution (stirred in a 60°C water bath) until clear, and then cooled and diluted to a final volume in PBS. This solution was added to the sample to bring the final cholesterol concentration to 0.1-0.2 mg / mL.

[0058] Tβ4 carries a negative charge at pH 7.4 and exhibits strong hydrophilicity, resulting in electrorepulsion with negatively charged extracellular vesicles, making spontaneous transmembrane loading difficult. By combining saponins with ultrasound, channels are established on the surface of extracellular vesicles, allowing Tβ4 to be successfully loaded. Further reversible cholesterol-dependent porosimetry then re-encapsulates the loaded extracellular vesicles, preserving their biological characteristics. This method achieves efficient Tβ4 loading into extracellular vesicles, reducing false-positive (extra-adsorption) loading, and ensures the Tβ4-extracellular vesicle complex maintains its complete delivery function and drug efficacy.

[0059] The prepared MSC-sEVs-Tβ4 can be used to prepare drugs for treating autoimmune dry eye. It can be mixed with other pharmaceutically acceptable excipients to prepare liquid drug formulations, such as eye drops. Autoimmune dry eye includes dry eye symptoms caused by autoimmune factors, such as dry eye associated with Sjögren's syndrome, experimental autoimmune dry eye, and dry eye caused by autoimmune dacryoadenitis.

[0060] The prepared MSC-sEVs-Tβ4 formulation was used in a mouse model of dry eye. Clinical dry eye symptoms and lacrimal gland histopathology were assessed in the autoimmune dry eye model mice. The results showed that the model mice gradually exhibited decreased tear secretion, punctate or diffuse sodium fluorescein staining of the cornea, corneal epithelial roughness, and corneal epithelial defects. H&E staining of lacrimal gland tissue sections revealed large areas of lymphocytic infiltration, destruction of glandular ducts and acini, acinar cell degeneration, and gradual atrophy of the acinar surface area. MSC-sEVs-Tβ4 was effective in treating dry eye. Subconjunctival injection of s-Tβ4 significantly improved dry eye symptoms, as evidenced by decreased corneal fluorescein staining scores, reduced corneal epithelial damage, significantly increased tear secretion, and a substantial reduction in lymphocyte infiltration in the lacrimal gland tissue. This suggests that using MSC-sEVs as a carrier to encapsulate Tβ4 significantly enhances the therapeutic effect of Tβ4 or MSC-sEVs alone. MSC-sEVs-Tβ4 can alleviate clinical symptoms of dry eye in model mice, improve pathological changes in lacrimal gland tissue, and has the potential to delay disease progression, indicating promising clinical applications. Furthermore, using the same treatment regimen in healthy male Balbc mice, ocular surface indicators were observed. Slit-lamp examination 28 days after local subconjunctival injection revealed no eye irritation, no corneal epithelial damage, and normal corneal structure, epithelial thickness, and stromal fiber arrangement. No inflammatory cell infiltration or significant neovascularization was observed in the corneal tissue. Histological analysis of major organs revealed no obvious histopathological abnormalities or lesions in the heart, liver, kidneys, spleen, and lungs. During the safety test, the body weight of mice in all groups remained stable without significant change, and no observable behavioral abnormalities were observed, suggesting that MSC-sEVs-Tβ4 has biocompatibility as a treatment for dry eye.

[0061] The immunomodulatory effects of MSC-sEVs-Tβ4 on cells closely related to various autoimmune dry eye pathogenesis mechanisms, namely macrophages, corneal epithelial cells, and T cells, were investigated. It was found that in vitro, MSC-sEVs-Tβ4 can significantly inhibit the inflammatory response of macrophages and corneal epithelial cells under inflammatory stimulation, and significantly inhibit the differentiation of T cells into pro-inflammatory cells Th1 and Th17. This indicates that the constructed MSC-sEVs-Tβ4 has good cell biocompatibility, can be effectively taken up by HCECs and RAW cells, and has stronger immunomodulatory properties than using Tβ4 and / or MSC-sEVs alone.

[0062] This study investigated the cellular and molecular mechanisms of MSC-sEVs-Tβ4 in treating autoimmune dry eye. Firstly, after 4 weeks of subconjunctival injection, the expression of the M1 macrophage-specific transcription factor iNOS was significantly decreased, while the expression of the M2 macrophage-specific transcription factor Arg1 and the marker cytokine IL-10 was significantly increased. In addition to MSC-sEVs-Tβ4, LPS and IFN-γ were added to unpolarized RAW cells (M0) to induce M1 polarization. Flow cytometry results showed that MSC-sEVs-Tβ4 treatment inhibited the conversion of RAW cells to M1 cells, indicating that MSC-sEVs-Tβ4 can alleviate inflammation by inhibiting the polarization of macrophages into inflammatory M1 cells. RAW cells (M0) were stimulated with MSC-sEVs-Tβ4 followed by LPS for inflammatory treatment. RT-qPCR showed that under inflammatory stimulation, the expression levels of Arg1 and CD206, specific transcription factors of anti-inflammatory M2 macrophages, were significantly decreased in the LPS group, but significantly increased in the MSC-sEVs-Tβ4 group. Further investigation into the molecular mechanism by which MSC-sEVs-Tβ4 inhibits macrophage inflammation was conducted using Western blotting to analyze its effect on the MAPK pathway. Compared to M0 cells, RAW cells treated with LPS showed increased expression of MAPK pathway-related proteins p-JNK, p-ERK, and p-p38, confirming that LPS stimulation significantly activated the MAPK inflammatory signaling pathway. Treatment with MSC-sEVs-Tβ4 significantly reduced the protein expression of p-ERK / p-JNK / p-p38 in RAW cells, indicating that MSC-sEVs-Tβ4 alleviates LPS-induced macrophage inflammation by inhibiting the activation of the JNK / ERK / p38 MAPK pathway. These results suggest that MSC-sEVs-Tβ4 inhibits macrophage polarization into inflammatory M1 cells and promotes their transformation into M2 cells by blocking MAPK signaling pathway activation, thereby suppressing inflammation.

[0063] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0064] Example 1: Preparation of MSC-sEVs

[0065] 1.1 Isolation, culture and identification of mesenchymal stem cells

[0066] After obtaining informed consent from the family and having them sign the informed consent form, the umbilical cord of a healthy full-term newborn was harvested. In a clean bench, it was placed in a 10cm diameter cell culture dish, cut into 3cm pieces, washed twice with PBS, and the blood clots were squeezed out. The tissue was then soaked in PBS containing 1% penicillin and streptomycin for 10 minutes. The soaked umbilical cord tissue was then cut to 1-2mm lengths with scissors. 3 The timer was set for 30 minutes. The chopped tissue was placed in a 500mL autoclaved glass bottle, a sterile rotor was added, along with 150mL PBS and 0.05g type II collagenase. The mixture was heated in a water bath using a magnetic stirrer at 37°C for 1 hour. After digestion, the mixture was filtered through a 200-mesh filter, and the filtrate was collected and stored at 4°C. Undigested tissue fragments were returned to the original bottle and digested again with 0.125% trypsin at 37°C for 20 minutes (100mL). Fetal bovine serum (FBS) was added to stop the digestion, and the filtrate was collected. The two filtrates were diluted with appropriate amounts of PBS and aliquoted into 50mL centrifuge tubes. The tubes were centrifuged at 1800rpm for 10 minutes at 4°C, and the supernatant was discarded. This process was repeated several times until the lower tissue precipitate was combined into a single 50mL centrifuge tube. The precipitate was resuspended in 20mL of complete stem cell culture medium, gently pipetted, and then seeded into a 175cm³ centrifuge tube. 2 The cells were placed in cell culture flasks, shaken well, and incubated at 37°C in a cell culture incubator with 5% CO2. After 2-3 days, the cell culture flasks were removed, the old culture medium was aspirated in a clean bench, and 25 mL of fresh complete culture medium was added. The flasks were then placed back in the original cell culture incubator, and the medium was changed every 2-3 days. The cells were observed under a microscope to assess their growth and morphological characteristics. When the cell confluence reached 80-90%, the cells were digested with 0.25% trypsin, passaged, and cells from passages 3-7 that showed good growth and were identified were used for experiments.

[0067] The prepared cells were identified using MSCs-specific antigen molecules, and total RNA was extracted from MSCs: Third-generation MSCs in good growth condition were collected. After aspirating the culture medium, the flask walls were washed with an appropriate amount of PBS. 3 mL of PBS and 3 mL of 0.25% trypsin were added to each cell culture flask, and the flasks were incubated at 37°C for 1 min for digestion. When most cells became rounded and some cells detached from the culture wall under a microscope, 4 mL of complete stem cell culture medium was added to stop the digestion. The cell suspension in the flasks was collected into 50 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min. The cell pellet at the bottom of the tube was collected, and total RNA from the MSCs was extracted using an RNA extraction kit. cDNA was synthesized by reverse transcription using the extracted total RNA from the MSCs. The synthesized cDNA was amplified by polymerase chain reaction and identified by agarose gel electrophoresis.

[0068] MSCs were passaged to the third generation and observed under an inverted microscope. The results are as follows: Figure 1 As shown in Figure A, the cells are elongated spindle-shaped and arranged in a whorled pattern. The results of the identification of MSC surface antigen molecule mRNA levels are as follows. Figure 1 As shown in Figure C, reverse transcription PCR results revealed that third-generation MSCs highly expressed the mesenchymal stem cell marker surface antigens CD29, CD105, CD44, and CD73, while expressing low levels or no hematopoietic stem cell markers CD45, HLA-DR, and CD34. These results confirm that the extracted cultured cells were mesenchymal stem cells.

[0069] 1.2 Extraction of small extracellular vesicles derived from mesenchymal stem cells

[0070] Well-developed MSCs (generations 3-7) were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) pre-exposed to remove small extracellular vesicles. After 48 hours, the supernatant was collected. The supernatant was then centrifuged sequentially at 300g for 10 minutes, 2000g for 10 minutes, and 10000g for 10 minutes at 4°C to remove cells, cell debris, and large extracellular vesicles. An ultracentrifuge was then performed at 110000g for 70 minutes at 4°C. The pellet was washed with cold PBS and resuspended, then centrifuged again at 110000g for 70 minutes. The pellet was resuspended in cold PBS and filtered through a 0.22μm filter. Protein concentrations in the small extracellular vesicle samples were determined using the BCA method, recorded, aliquoted, and stored at -80°C for later use.

[0071] Identification of MSC-sEVs morphology under transmission electron microscopy: Take 10 μL of each of the above sample suspensions, dilute 10-fold with PBS, and take 30 μL of the diluted liquid. Fix the liquid with 4% paraformaldehyde. Place 5 μL of the liquid on each copper grid, preparing 3 copper grids for each sample. Allow them to air dry for 20 minutes, then add phosphotungstic acid for negative staining for 2 minutes. Gently absorb excess staining solution from the edges of the copper grids with filter paper. Place the samples under an incandescent lamp and bake for 10 minutes. Observe the samples under a transmission electron microscope and photograph them. Results are as follows: Figure 1 As shown in Figure B, the ultrastructure of the sample was observed using a transmission electron microscope, revealing that MSC-sEVs appear as round or elliptical bilayered membrane vesicles.

[0072] The particle size distribution of MSC-sEVs was detected using a NanoSight nanoparticle tracking analyzer. 2 μL of each of the above sample suspensions was diluted 500 times with purified water and thoroughly mixed. 1 mL of the diluted liquid was aspirated into a 1 mL syringe, and the sample was loaded into the NanoSight nanoparticle tracking analyzer using a syringe pump for data collection. Three consecutive collections were performed, and the results were combined and statistically analyzed as a single result. The results are as follows... Figure 2 As shown, the results indicate that the particle size of MSC-sEVs conforms to the normal range (30-150 nm). Further identification of MSC-sEV surface proteins using Western blotting yielded the following results. Figure 3 As shown, the results indicate that MSC-sEVs highly express small extracellular vesicle-specific proteins CD81, CD63, and TSG101.

[0073] Example 2: Construction and analysis of MSC-sEVs-Tβ4

[0074] 2.1 Construction of MSC-sEVs-Tβ4

[0075] Take the small extracellular vesicle suspension prepared in Example 1, dilute it with PBS to a total protein concentration of 0.1 μg / μL, and pipette 300 μL into a 1.5 mL EP tube. Then, add Tβ4 to the EP tube at a protein ratio of 1:3, and add saponins to a final concentration of 0.01–0.03% (w / v). Incubate on ice for 5 min for pre-permeation, and gently pipette to mix. Under light-protected conditions, sonicate the sample on ice at 20% power for 3 min, pausing for 1 min every 30 s. After sonication, dilute with an equal volume of PBS buffer, and add cholesterol working solution to a final concentration of 0.2 mg / mL. Incubate at room temperature for 10 min. Then, incubate at 37°C for 60 min to restore membrane stability. Centrifuge using a 100 kDa ultrafiltration tube at 14000 g for 10 min to remove free Tβ4 from the liquid, obtaining a suspension of MSC-sEVs-Tβ4.

[0076] In this process, cholesterol is dissolved and evaporated to form a film, then mixed with MβCD solution. The molar ratio of cholesterol to methyl-β-cyclodextrin is 1:6. The mixture is stirred in a water bath at 60°C until it becomes clear, thus obtaining a cholesterol working solution.

[0077] 2.2 Validation of MSC-sEVs-Tβ4

[0078] To verify that a structurally stable MSC-sEVs-Tβ4 complex can be constructed using the above method, MSC-sEVs-Tβ4 was constructed using Tβ4 linked to FITC fluorescent protein according to the above method, and the prepared MSC-sEVs-Tβ4 was detected and verified.

[0079] MSC-sEVs suspension loaded with FITC-labeled Tβ4 were analyzed by flow cytometry to detect the proportion of FITC fluorescence expression, with ordinary MSC-sEVs used as a negative control. Flow cytometry was used to assess whether Tβ4 could be effectively loaded into MSC-sEVs, and the results are as follows: Figure 4 As shown, the proportion of MSC-sEVs loaded with FITC-labeled Tβ4 reached over 80%, demonstrating that ultrasound can effectively encapsulate Tβ4 into small extracellular vesicles.

[0080] The morphology of MSC-sEVs-Tβ4 was identified by transmission electron microscopy. 10 μL of each MSC-sEVs sample suspension loaded with FITC-labeled Tβ4 was diluted 10-fold with PBS, and 30 μL of the diluted liquid was fixed with 4% paraformaldehyde. 5 μL of the liquid was placed on each copper mesh, and three copper meshes were prepared for each sample. After allowing them to air dry for 20 minutes, phosphotungstic acid was added for negative staining for 2 minutes. Excess staining was gently blotted from the edges of the copper mesh with filter paper, and the samples were placed under an incandescent lamp for 10 minutes. The samples were observed and photographed under a transmission electron microscope. The results are as follows: Figure 5 As shown, MSC-sEVs-Tβ4 appears as round or oval bimembranous vesicles, and the small extracellular vesicles carrying Tβ4 retain their small extracellular vesicle shape.

[0081] The particle size distribution of MSC-sEVs-Tβ4 was detected using a NanoSight nanoparticle tracking analyzer. 2 μL of each of the above sample suspensions was diluted 500 times with purified water and thoroughly mixed. 1 mL of the diluted liquid was aspirated into a 1 mL syringe, and the sample was loaded into the NanoSight nanoparticle tracking analyzer using a syringe pump for data collection. Three consecutive collections were performed, and the results were combined and statistically analyzed as a single result. The results are as follows... Figure 6 As shown, the particle size of MSC-sEVs-Tβ4 is within the normal range (30-150 nm) for MSC-sEVs. Transmission electron microscopy and particle size analysis results indicate that the drug loading process did not affect the shape of the small extracellular vesicles.

[0082] The surface protein of MSC-sEVs-Tβ4 was then detected, and the results are as follows: Figure 3 As shown, MSC-sEVs-Tβ4 highly expresses the small extracellular vesicle-specific proteins CD81, CD63, and TSG101; and the results are consistent with those of the MSC-sEVs group. This demonstrates that loading Tβ4 does not affect the biological function of small extracellular vesicles.

[0083] 2.3 Identification of drug loading rate of MSC-sEVs-Tβ4

[0084] A series of FITC-labeled Tβ4 solutions with concentrations ranging from 0 to 25 μg / mL were prepared. A standard curve was plotted with fluorescence value on the x-axis and Tβ4 solution concentration on the y-axis, and the regression equation was calculated. The MSC-sEVs-Tβ4 suspension obtained as described above was diluted 5-fold with PBS, and the fluorescence value was detected using a microplate reader. The fluorescence value was then substituted into the regression equation to calculate the concentration of Tβ4 in MSC-sEVs-Tβ4. Simultaneously, the protein concentration was quantitatively determined using the BCA method. The formula for calculating the drug loading rate is as follows:

[0085] Drug loading rate (%) = (Drug loading amount) / (Drug loading amount + Total protein content of MSC-sEVs) × 100%

[0086] After examination and calculation, the linear regression equation of the Tβ4 standard curve is y = 5E-07x + 0.0002, R0 2 =0.9987, such as Figure 7 As shown in the figure, calculations show that the drug loading rate of Tβ4 in MSC-sEVs-Tβ4 is 11.8%.

[0087] The experimental results show that the prepared MSC-sEVs-Tβ4 nanocomposite material has a particle size mainly distributed between 100-200 nm and is relatively concentrated, which is consistent with the normal particle size range of MSC-sEVs. Transmission electron microscopy observation of the MSC-sEVs-Tβ4 morphology reveals that it consists of round or elliptical bilayered membrane vesicles, and the Tβ4-loaded extracellular vesicles retain their small extracellular vesicle shape. Flow cytometry results show that ultrasound can effectively encapsulate Tβ4 into the small extracellular vesicles, successfully constructing the MSC-sEVs-Tβ4 nanocomposite; the Tβ4-loaded mesenchymal extracellular vesicles MSC-sEVs-Tβ4 still possess the characteristics of MSC-sEVs. Furthermore, the concentration-fluorescence standard curve of Tβ4 shows a high drug loading rate of Tβ4 in MSC-sEVs-Tβ4.

[0088] Example 3: In vivo tracing of MSC-sEVs-Tβ4

[0089] NOD / ShiLtj mice are an animal model of spontaneous Sjögren's syndrome (SS). Pathological manifestations such as inflammatory cell infiltration and decreased secretory function of the lacrimal glands begin to appear at 8-12 weeks of age, and characteristic autoantibodies can be detected in the serum. Fifteen-week-old male NOD mice were used as the dry eye model mice.

[0090] PKH-26-labeled sEVs were used, and FITC-labeled Tβ4 was encapsulated in PKH-26-labeled sEVs using the method described in Example 2. At the peak disease incidence period (15 weeks of age) in NOD mice, MSC-sEVs-Tβ4 was injected subconjunctively into the mice. In vivo distribution tracking: NOD model mice were injected subconjunctively with a single therapeutic dose of PKH-26-MSC-sEVs-Tβ4-FITC at the peak disease incidence period (15 weeks of age). Mice were sacrificed at 24h, 48h, 72h, and 96h post-injection. Lacrimal gland, eyeball, and lymph node tissues were collected, embedded in OCT, and frozen sections were prepared. After DAPI staining, the red fluorescence expression representing sEVs and the green fluorescence expression of Tβ4 were observed under a confocal microscope.

[0091] The results are as follows Figures 8-10 As shown, after subconjunctival injection of PKH-26 and FITC-labeled MSC-sEVs-Tβ4 in NOD mice, PKH-26-MSC-sEVs-Tβ4-FITC was observed to be distributed in the lacrimal gland, conjunctiva, and lymph nodes at 24 h, and the distribution decreased over time at 96 h.

[0092] Example 4: Treatment of NOD autoimmune dry eye mouse model with MSC-sEVs-Tβ4

[0093] Fifteen-week-old male NOD mice were used as dry eye model mice. After one week of acclimatization, the mice were randomly divided into four groups: a PBS control group, a Tβ4 treatment group, an MSC-sEVs treatment group, and an MSC-sEVs-Tβ4 treatment group. The corresponding drugs were injected subconjunctivally into each group twice a week for a total of four weeks. Clinical indicators of dry eye were examined before treatment and at two and four weeks after treatment to evaluate the therapeutic effect of MSC-sEVs-Tβ4.

[0094] 4.1 Tear secretion test

[0095] The volume of tears in mice was measured using the traditional Schirmer I test. During measurement, the lower eyelid of the mouse was gently pulled open with toothless forceps, and a phenol red cotton thread was placed in the middle and outer third of the palpebral conjunctival surface of the lower conjunctival sac. The forceps were then released, leaving the thread in the conjunctival sac, and the measurement was timed for 30 seconds, recording the length of the reddened area. Each measurement was repeated three times. The average of the three measurements was taken as the final result.

[0096] Phenolic red cotton thread was used to measure basal tear secretion, reflecting the secretory function of the lacrimal gland. Phenolic red cotton thread is less irritating to the cornea than tear detection filter paper strips, reducing the production of irritating tears. It also has a fixed absorbency, is easy to use, and has high repeatability. Tear secretion in mice was quantitatively assessed using phenol red cotton thread before treatment, at 2 weeks of treatment, and at 4 weeks of treatment. Results are as follows... Figure 11 As shown, there were no statistically significant differences among the four groups before treatment. After 2 and 4 weeks of subconjunctival injection treatment, the MSC-sEVs-Tβ4 treatment group showed a significant increase in tear secretion compared to the other three groups, and the difference was statistically significant.

[0097] 4.2 Corneal fluorescein staining:

[0098] Mydriasis was achieved using compound tropicamide eye drops. 1 μL of 4% sodium fluorescein was instilled into the ocular surface of mice per eye, and the instillation was timed for three minutes. After the time was completed, the ocular surfaces were observed under cobalt blue light using a slit-lamp microscope, and photographs were taken and saved. Corneal epithelial punctation was graded according to the scoring criteria. The scoring criteria for sodium fluorescein staining of the cornea are shown in Table 1.

[0099] Table 1 Corneal fluorescein staining scoring criteria

[0100]

[0101] Mice in each group underwent corneal fluorescein staining before treatment, at 2 weeks of treatment, and at 4 weeks of treatment to observe changes in corneal punctation. Results are as follows: Figure 12 As shown, before treatment, all four groups of mice exhibited punctate or diffuse sodium fluorescein staining, with rough corneal epithelium and corneal epithelial defects, and there was no statistically significant difference in corneal fluorescein staining scores among the groups. After 2 and 4 weeks of subconjunctival injection treatment, the MSC-sEVs-Tβ4 treatment group showed significantly reduced corneal epithelial punctation compared to the other three groups, with a smoother and more intact corneal epithelium. Fluorescein staining scores were then calculated for each group, and the results are as follows: Figure 13 As shown, the fluorescein staining score of the MSC-sEVs-Tβ4 treatment group was significantly reduced, and the difference was statistically significant.

[0102] 4.3 Histopathological observation of the lacrimal gland:

[0103] After four weeks of continuous treatment, the mice were euthanized, and the lacrimal glands were removed using ophthalmic surgical instruments. The tissue was thoroughly immersed in 4% formaldehyde solution and fixed at room temperature for 72 hours before paraffin sections were prepared. After dewaxing and hydration, the sections were stained with H&E, dehydrated, cleared, mounted, and dried. The sections were then observed under an optical microscope and photographed.

[0104] like Figure 14 As shown, extensive lymphocyte infiltration and destruction of structures such as glandular ducts and acini were observed in the lacrimal gland tissue of mice in the PBS group; acinar cells showed degeneration, and the acinar area shrank. In contrast, the lymphocyte infiltration area in the lacrimal gland of the MSC-sEVs-Tβ4 group was significantly reduced, and the degree of destruction of some lacrimal gland ducts and acini was lessened; acinar cells showed mild degeneration and remained relatively intact. The lymphocyte infiltration areas of lacrimal gland tissue in each group were analyzed and compared, and the statistical results are shown below. Figure 15 As shown, the infiltration area in the MSC-sEVs-Tβ4 treatment group was significantly lower than that in other groups.

[0105] Example 5: Ocular and systemic safety assessment of MSC-sEVs-Tβ4

[0106] Healthy male Balbc mice aged 15 weeks were used as normal controls. After one week of acclimatization, the mice were randomly divided into a blank control group, a PBS control group, a Tβ4 treatment group, an MSC-sEVs treatment group, and an MSC-sEVs-Tβ4 treatment group. All mice were treated with the same regimen as before, and ocular surface parameters were observed to assess the safety of MSC-sEVs-Tβ4 treatment.

[0107] Slit-lamp observation was performed on the four treatment groups 28 days after local subconjunctival injection. The results were as follows: Figure 16 As shown, no abnormal clinical signs such as tear turbidity, corneal defects, turbidity, corneal neovascularization, conjunctival hyperemia, or inflammation were observed in mice in different groups. Fluorescein staining showed no obvious corneal epithelial defects in any group.

[0108] After four weeks of continuous treatment, the mice were euthanized, and the lacrimal glands, eyeballs, liver, heart, spleen, lungs, and kidneys were removed using ophthalmic surgical instruments. The tissues were thoroughly immersed in 4% formaldehyde solution and fixed at room temperature for 72 hours before paraffin sections were prepared. After dewaxing and hydration, the sections were stained with H&E, dehydrated, cleared, mounted, and dried. They were then observed under an optical microscope and photographed for record-keeping.

[0109] The results are as follows Figure 17 As shown, HE staining of tissues and organs revealed that after subconjunctival injection, the corneal morphology, epithelial thickness, and stromal fiber arrangement remained normal in all groups. The lacrimal gland tissue showed no obvious inflammatory cell infiltration, and the structures of glandular ducts and acini were basically normal. Histological analysis of the main organs showed no obvious histopathological abnormalities or lesions in the heart, liver, kidney, spleen, and lungs.

[0110] During the 28-day period of local subconjunctival injection in the four treatment groups, the results were as follows: Figure 18 As shown, the body weight of mice in each group remained stable without significant change during the safety test, and no observable behavioral abnormalities were observed.

[0111] Example 6: Cellular biosafety and immunomodulatory properties of MSC-sEVs-Tβ4.

[0112] To clarify whether the constructed MSC-sEVs-Tβ4 possesses the dual immunomodulatory properties of Tβ4 and MSC-sEVs, the immunomodulatory effects of MSC-sEVs-Tβ4 on cells closely related to the pathogenesis of various autoimmune dry eye diseases were investigated in vitro.

[0113] 6.1 Immunomodulatory effects of MSC-sEVs-Tβ4 on macrophages

[0114] Macrophages play a crucial role in the pathogenesis of various autoimmune diseases, including autoimmune dry eye. This study investigated the effects of the RAW macrophage line on the cytotoxicity, in vitro uptake, and inflammatory response of MSC-sEVs-Tβ4. First, MSC-sEVs were stained with PKH-26 dye and then coated with FITC-labeled Tβ4. After incubation for 24 hours in RAW cell culture medium, cell slides were collected for immunofluorescence analysis. The results are as follows: Figure 19As shown, PKH-26-MSC-sEVs with red fluorescence overlapped with FITC-Tβ4 with green fluorescence, and both were taken up by RAW cells, indicating that Tβ4 can be encapsulated by MSC-sEVs in vitro and that MSC-sEVs-Tβ4 can be effectively taken up by RAW cells. CCK-8 results showed that MSC-sEVs-Tβ4 had no significant cytotoxic effect. Figure 20 Macrophages play a crucial role in inducing tissue inflammation; activated macrophages can promote the secretion of inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-1β (IL-1β). RAW cells were divided into LPS, PBS, Tβ4, MSC-sEVs, MSC-sEVs-Tβ4, and a blank control (BLANK). After pretreatment with the drugs from each group for 24 hours, 1 μg / ml LPS was added to the culture medium of each group for inflammatory stimulation. Cells were collected after 24 hours for RT-qPCR analysis. Figure 21 As shown, compared with the blank control group, the expression levels of inflammation-related genes IL-6 and IL-1β in RAW cells of LPS group were significantly increased under inflammatory stimulation, while the expression levels in MSC-sEVs-Tβ4 group were significantly decreased (p<0.05).

[0115] 6.2 Immunomodulatory effects of MSC-sEVs-Tβ4 on corneal epithelial cells

[0116] Corneal epithelial cells play a crucial role in the development and progression of dry eye. Patients with autoimmune dry eye typically exhibit reduced tear secretion, leading to tear hyperosmolarity, ocular surface irritation, inflammation, and corneal barrier disruption, further resulting in cell damage, apoptosis, and goblet cell loss. Corneal epithelial cells are also a core component of the ocular surface's innate immune system, directly responding to exposure to adverse environments. Tβ4 possesses anti-inflammatory and wound-healing properties, and MSC-sEVs can significantly promote corneal epithelial cell proliferation, in vitro migration, and in vivo corneal epithelial wound healing. This study further investigated the regulatory effect of MSC-sEVs-Tβ4 on corneal epithelial cells using the human corneal epithelial cell line HCEC.

[0117] To determine whether MSC-sEVs-Tβ4 could be effectively taken up by human corneal epithelial cells (HCECs), MSC-sEVs were stained with PKH-26 dye and then coated with FITC-labeled Tβ4. The PKH-26-MSC-sEVs-Tβ4-FITC mixture was added to the culture medium for HCECs and incubated for 24 hours. Cell slides were then subjected to immunofluorescence. The results are as follows: Figure 22As shown, PKH-26-MSC-sEVs with red fluorescence overlapped with FITC-Tβ4 with green fluorescence and were both taken up by HCEC cells, indicating that MSC-sEVs-Tβ4 can be effectively taken up by HCEC cells. Next, sodium chloride was added to serum-free medium to achieve an osmotic pressure of 500 mOsM to simulate the hypertonic stress state of tear film in autoimmune dry eye patients. HCEC cells were divided into HS group, PBS group, Tβ4 group, MSC-sEVs group, MSC-sEVs-Tβ4 group, and untreated blank control group (BLANK group). When the HCEC cell confluence reached approximately 70% and the cell condition was good, the medium for each group was replaced with serum-free medium for starvation treatment. Drug was added to the experimental groups for pretreatment. After 16 hours, the medium for the experimental groups was replaced with 500 mOsM serum-free medium for hypertonic stress treatment. Cells were collected 4 hours later for RT-qPCR analysis. Figure 23 As shown, compared with the untreated Blank group, the expression levels of inflammation-related genes IL-6 and TNF-α in HCEC cells were significantly increased under hyperosmolar stress, while the expression levels were significantly decreased in the group treated with MSC-sEVs-Tβ4. This indicates that hyperosmolar stress induces HCEC cells to secrete inflammatory cytokines, while MSC-sEVs-Tβ4 treatment can significantly reduce the inflammatory response induced by hyperosmolarity in HCEC cells, and the difference is statistically significant (p<0.05).

[0118] 6.3 Immunomodulatory effects of MSC-sEVs-Tβ4 on rabbit autoreactive Th1 and Th17 cells in vitro

[0119] Studies show that CD4 + T cells are central to the immune-mediated inflammatory damage in patients with autoimmune diseases. During the immune response, CD4 cells, stimulated by different antigen signals, [are involved]. + T cells can be activated, proliferate, and differentiate into different subsets, mainly including Th1, Th2, Th17, and Treg cells. Th1 and Th17 cells, as pro-inflammatory cell populations, play a crucial role in the pathogenesis of SS-related dry eye, inducing inflammation by infiltrating glands and promoting disease progression. The proportion of Th1 and Th17 cells is significantly increased in the peripheral blood of SS patients and is closely related to the disease activity parameter ESSDAI. Previous studies have also shown that Th1 and Th17 cells play an important role in the disease progression of a rabbit autoimmune dry eye model, and regulating Th1 and Th17 cell responses can effectively alleviate the development of autoimmune dry eye.

[0120] Blood was collected from the middle auricular artery of rabbits using a venipuncture needle. 20 ml of heparin-anticoagulated blood was drawn from each rabbit and diluted 5 times with PBS. 40 ml of diluted blood was slowly added to 10 ml of lymphocyte separation medium and centrifuged at 2000 rpm for 20 min. After centrifugation, the tube separated into 3 layers. At the interface between the upper and middle layers, there was a narrow white, cloudy band, mainly composed of mononuclear cells, including lymphocytes and monocytes. This layer of cells was aspirated, washed with PBS, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded to obtain rabbit peripheral blood mononuclear cells (PBMCs). The cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum.

[0121] 6.3.2 Establishment of a co-culture system of lacrimal gland epithelial cells and autologous peripheral blood lymphocytes

[0122] After anesthetizing the rabbit, the left inferior lacrimal gland was harvested, and the rabbit lacrimal gland epithelial cells were isolated, purified, and adjusted to a cell density of 1×10⁻⁶. 5 MSCs were seeded at a density of 5 μg / ml in 96-well plates. After 2 days of culture, the cells were irradiated with 25 Gy γ-rays and then co-cultured with PBMCs of the same density for 3 days. Then, MSC-sEVs, MSC-sEVs-Tβ4, and Tβ4 were added to each well at a final concentration of 5 μg / ml (previous studies have shown that 5 μg / ml MSC-sEVs can inhibit the proliferation of peripheral blood T lymphocytes). An equal volume of PBS was added as a PBS control group. The cells were cultured for another 48 hours, and then PBMCs were collected.

[0123] Total RNA was extracted from cells co-cultured with lacrimal gland epithelial cells and autologous peripheral blood lymphocytes using Trizol, and cDNA was prepared by reverse transcription. The expression levels of Th17 cell differentiation-related genes IL-17 and RORC, and Th1 cell differentiation-related genes IFN-γ and T-BET mRNA were detected by Q-PCR. Primer sequences are as follows:

[0124] GAPDH: Upstream primer: 5'-GGGTGGTGGACCTCATGGT-3', (SEQ ID No. 1);

[0125] Downstream primer: 5'-CGGTGGTTTGAGGGCTCTTA-3', (SEQ ID No. 2);

[0126] IL-17: Upstream primer: 5'-GGAATGAGGACCACCACATGA-3', (SEQ ID No. 3);

[0127] Downstream primer: 5'-CTGCGTAGGACCAGGATCTCTT-3', (SEQ ID No. 4);

[0128] RORC: Upstream primer: 5'-GGCCTACCACGCCGA-3', (SEQ ID No. 5);

[0129] Downstream primer: 5'-TCCATGCCACCGTATTTGC-3', (SEQ ID No. 6);

[0130] IFN-γ: Upstream primer: 5'-TTGGCTTTGCAGCTCTTCCT-3', (SEQ ID No. 7);

[0131] Downstream primer: 5'-TGACTGTGCCGTGGCAGTA-3', (SEQ ID No. 8);

[0132] T-BET: Upstream primer: 5'-ACCTGTTGTGGTCCAAGTTCAA-3', (SEQ ID No. 9);

[0133] Downstream primer: 5'-GCCGTCCTTGCTTAGTGATGA-3' (SEQ ID No. 10).

[0134] The relative quantitative results were analyzed using the 2 - ΔΔCt method.

[0135] RT-qPCR test results are as follows Figure 24 As shown, compared with the PBS group, the MSC-sEVs-Tβ4 group showed significantly decreased expression of Th1 cell transcription factor T-BET and the marker cytokine IFN-γ in lymphocytes, as well as significantly decreased expression of Th17-specific transcription factor RORC and the marker cytokine IL-17, with statistically significant differences. Furthermore, the MSC-sEVs-Tβ4 group exhibited a stronger inhibitory effect on the expression of the Th17 cell marker protein IL-17 than the PBS and Tβ4 groups, with statistically significant differences. Figure 25 This suggests that MSC-sEVs-Tβ4 has a stronger immunomodulatory effect compared to Tβ4 and MSC-sEVs.

[0136] In summary, the constructed MSC-sEVs-Tβ4 exhibits excellent cellular biocompatibility, can be effectively taken up by HCECs and RAW cells, and possesses stronger immunomodulatory properties compared to Tβ4 and MSC-sEVs.

[0137] Example 7: Investigation into the cellular and molecular mechanisms of MSC-sEVs-Tβ4 in the treatment of autoimmune dry eye

[0138] Macrophages play a crucial role in the pathogenesis of autoimmune dry eye. They can differentiate into two distinct functional phenotypes in response to tissue microenvironment factors, such as damaged cells, activated lymphocytes, or microbial products: classically activated macrophages (M1) and alternately activated macrophages (M2). Classically activated macrophages (M1) exert pro-inflammatory activity, induced alone or in combination with microbial stimulation and / or inflammatory cytokines by interferon-γ (IFN-γ). Alternatively activated macrophages (M2) participate in anti-inflammatory responses by inducing cytokines such as IL-4 and IL-13. Macrophage polarization is essential for tissue regeneration and homeostasis. Numerous studies have shown that small extracellular vesicles derived from MSCs promote M1 to M2 polarization, increasing anti-inflammatory cytokines and chemokines, a process that alleviates inflammation.

[0139] The effects of MSC-sEVs-Tβ4 on macrophage phenotype were investigated in vivo. RT-qPCR was performed on lacrimal gland tissues from mice in the PBS, Tβ4, MSC-sEVs, and MSC-sEVs-Tβ4 groups after 4 weeks of subconjunctival injection treatment. Results are as follows: Figure 26 As shown, compared with the PBS group, the expression of iNOS, a specific transcription factor of M1 macrophages, was significantly decreased in the MSC-sEVs-Tβ4 group, while the expression of Arg1, a specific transcription factor of M2 macrophages, and IL-10, a marker cytokine, was significantly increased, and the differences were statistically significant.

[0140] Unpolarized RAW cells (M0) were divided into experimental groups: M1 group, PBS group, Tβ4 group, MSC-sEVs group, and MSC-sEVs-Tβ4 group; and a control group (M0 group) without treatment. In the experimental groups, the culture medium was pretreated with the drug for 24 h, followed by the addition of 250 ng / ml LPS and 100 ng / ml IFN-γ to induce polarization towards M1 cells. Flow cytometry analysis was performed 24 h later, and the results are as follows: Figure 27 As shown, under M1-induced stimulation, RAW cells significantly polarized from M0 to M1, while MSC-sEVs-Tβ4 treatment inhibited the conversion of RAW cells to M1 cells. The difference was statistically significant (**p<0.01, ****p<0.0001), indicating that MSC-sEVs-Tβ4 reduces inflammation by inhibiting the polarization of macrophages to inflammatory M1 cells.

[0141] Untreated RAW cells (M0) were divided into experimental groups (LPS, PBS, Tβ4, MSC-sEVs, and MSC-sEVs-Tβ4) and a blank control group (M0). In the experimental groups, the culture medium was pretreated with the drugs for 24 hours, followed by inflammatory stimulation with 1 μg / ml LPS. Cells were collected 24 hours later for RT-qPCR analysis. Figure 28 As shown, compared with the control group M0, the expression levels of Arg1 and CD206, specific transcription factors of anti-inflammatory M2 macrophages, were significantly reduced in the LPS group under inflammatory stimulation, but their expression levels were significantly increased in the MSC-sEVs-Tβ4 group (p<0.05).

[0142] Tβ4 can exert an anti-inflammatory effect by blocking the activation of the MAPK inflammatory signaling pathway, and MSC-sEVs may inhibit the inflammatory response through the NF-κB / MAPK signaling pathway. At the same time, the MAPK pathway is a crucial signaling pathway for macrophages to participate in the inflammatory process.

[0143] To further investigate the molecular mechanism by which MSC-sEVs-Tβ4 inhibits macrophage inflammation, Western blotting was used to analyze the effect of MSC-sEVs-Tβ4 on the MAPK pathway. Results are as follows: Figure 29 As shown, compared with unstimulated M0 cells, RAW cells treated with LPS showed increased protein expression of MAPK pathway-related proteins p-JNK, p-ERK, and p-p38, confirming that LPS stimulation significantly activated the MAPK inflammatory signaling pathway. Conversely, treatment with MSC-sEVs-Tβ4 significantly reduced the protein expression of p-ERK / p-JNK / p-p38 in RAW cells, indicating that MSC-sEVs-Tβ4 alleviates LPS-induced macrophage inflammation by inhibiting the activation of the JNK / ERK / p38 MAPK pathway.

[0144] The above results indicate that MSC-sEVs-Tβ4 inhibits the polarization of macrophages into inflammatory M1 cells and promotes their transformation into M2 cells by blocking the activation of the MAPK signaling pathway, thereby playing a role in suppressing inflammation.

[0145] The above experimental results demonstrate that MSC-sEVs encapsulating Tβ4 can effectively deliver Tβ4 to the conjunctiva and lacrimal gland. Compared with the PBS control group, the Tβ4 group, and the MSC-sEVs-only treatment group, the MSC-sEVs-Tβ4 treatment group showed a significantly lower corneal fluorescein staining score, a smaller fluorescein sodium staining area, and a significantly improved corneal epithelial smoothness. Simultaneously, tear secretion was also significantly increased, with statistically significant differences. Histologically, the MSC-sEVs-Tβ4 treatment group also showed a significant reduction in inflammatory cell infiltration in the lacrimal gland tissue. Safety results indicate that MSC-sEVs-Tβ4 treatment has reliable biocompatibility and cellular safety. In terms of molecular mechanisms, MSC-sEVs-Tβ4 can be effectively taken up by HCECs and RAW cells, and compared with Tβ4 and MSC-sEVs, it has stronger anti-inflammatory and immunomodulatory properties in macrophages, corneal epithelial cells, and T cells. In vivo treatment, MSC-sEVs-Tβ4 affects the macrophage phenotype in the lacrimal glands of autoimmune dry eye mice by inhibiting the M1 phenotype and promoting the M2 phenotype. MSC-sEVs-Tβ4 can effectively reduce the polarization level of M1 macrophages in vitro and in vivo, and the effect is better than that of the Tβ4 treatment group and the MSC-sEVs treatment group alone. Under inflammatory stimulation, MSC-sEVs-Tβ4 can significantly increase the inhibitory effect of LPS on the expression of M2 macrophage-specific transcription factors Arg1 and CD206. Compared with uninflammated M0 cells, RAW cells treated with LPS showed increased expression of MAPK pathway-related proteins p-JNK, p-ERK, and p-p38. After treatment with MSC-sEVs-Tβ4, the expression of p-ERK / p-JNK / p-p38 in RAW cells was significantly reduced. This suggests that MSC-sEVs-Tβ4 may inhibit the polarization of macrophages into inflammatory M1 cells and promote their transformation into M2 cells by blocking the activation of the MAPK signaling pathway, thereby playing a role in inhibiting inflammation.

[0146] In summary, the results demonstrate that MSC-sEVs-Tβ4 treatment can significantly alleviate the clinical manifestations and histopathological changes of autoimmune dry eye. Compared with the MSC-sEVs treatment group and the Tβ4 treatment group alone, it has more outstanding therapeutic effects at all levels, and no local or systemic toxicity was observed, indicating great potential for clinical application.

[0147] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. The application of an extracellular vesicle complex encapsulating thymosin β4 in the preparation of a drug for treating autoimmune dry eye, characterized in that: The drug includes a nanocomposite obtained by loading thymosin β4 into extracellular vesicles; the extracellular vesicles are small extracellular vesicles (sEVs) derived from umbilical cord mesenchymal stem cells. Thymosin β4 is loaded into extracellular vesicles using an ultrasonic method. Saponins are added to the ultrasonic-forward reaction system, and cholesterol solution is added to the ultrasonic-backward reaction system to prepare the extracellular vesicle complex MSC-sEVs-Tβ4 loaded with thymosin β4. The preparation method of the MSC-sEVs-Tβ4 is as follows: Step 1: Mix extracellular vesicles with thymosin β4 at a protein ratio of 2-10:1, add PBS to form a reaction system, add 0.01-0.03% saponin by mass / volume to the reaction system, and incubate on ice for pre-osmosis; Step 2: Sonicate the reaction system for 1-10 minutes; one cycle consists of sonication for 1-20 seconds, pause for 1-20 seconds, and cooling on ice for 1-5 minutes, and a total of 4-6 cycles are performed; Step 3: Dilute with an equal volume of PBS buffer, then add cholesterol solution with a final concentration of 0.1-0.2 mg / mL, and incubate at 37°C for 60-90 min; the cholesterol solution is a complex solution of cholesterol and methyl-β-cyclodextrin. Step 4: Remove free thymosin β4 to obtain a suspension containing MSC-sEVs-Tβ4.

2. The application according to claim 1, characterized in that: MSC-sEVs-Tβ4 reduces macrophage inflammation by inhibiting the activation of the JNK / ERK / p38 MAPK pathway.

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