Bradyrhizobium sp. Exosome and application thereof in preparation of product for treating dry eye
By using exosomes from *Rhizobium tumefaciens* to prepare eye drops, sprays, or gels, the side effects of chemical drugs in the treatment of dry eye have been resolved, achieving safe and effective anti-inflammatory effects, reducing production costs, and improving dry eye symptoms.
Patent Information
- Application Number
- CN202610707503.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-21
- Publication Date
- 2026-08-25
AI Technical Summary
Current treatments for dry eye rely on chemical drugs, which have side effects and drug toxicity issues, and have failed to effectively address symptoms such as tear film instability and ocular surface inflammation. There is a lack of safe and effective natural treatment options.
Omniform exosomes (OMV) from slow-growing rhizobia are extracted by ultrafiltration centrifugation and combined with pharmaceutically acceptable adjuvants to prepare eye drops, sprays, or gels for the treatment of dry eye syndrome, promoting the transformation of anti-inflammatory M2 macrophages, reducing pro-inflammatory factors, and increasing the level of anti-inflammatory factors.
Slow-growing rhizobium exosomes significantly improve dry eye symptoms, reduce ocular tissue inflammation, lower the risk of immune rejection, reduce production costs, and improve treatment efficacy.
Smart Images
Figure CN122624533A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a type of exosome of *Staphylococcus aureus* and its application in the preparation of products for treating dry eye syndrome. Background Technology
[0002] Dry eye disease (DED) is a chronic ocular surface disease caused by abnormalities in tear quality, quantity, and dynamics, leading to decreased tear film stability or an imbalance in the ocular surface microenvironment. Due to the expanding aging population and lifestyle changes influenced by the widespread use of information technology devices, the number of patients is increasing annually. The main causes include tear film instability, tear hypertonicity, ocular surface inflammation and damage, and abnormal nerve sensation. Mild cases primarily manifest as eye discomfort such as eye fatigue, foreign body sensation, and dryness, while severe cases can lead to decreased vision or even visual disturbances.
[0003] Delayed keratoconjunctivitis (DED) is considered an incurable disease requiring long-term medication to rebuild and maintain ocular surface homeostasis. Treatment primarily focuses on replacement therapy and symptomatic relief. Medications mainly concentrate on anti-inflammatory and antioxidant effects, but the limitations and side effects of chemical drugs are difficult to overcome in clinical application. Currently, the clinical treatment of DED often involves the combined use of multiple medications, which can easily lead to drug-induced keratoconjunctivitis and even opportunistic infections such as Staphylococcus epidermidis, resulting in corneal ulcers. Therefore, in-depth research into the pathogenesis of DED and the search for safe and effective natural drug treatments are urgently needed.
[0004] The interaction of the ocular surface microbiome produces antimicrobial peptides that resist the invasion of harmful bacteria into ocular surface epithelial cells. This is crucial for maintaining the normal immune status of the ocular surface and preventing dry eye and allergies caused by excessive immune imbalance. Bacterial exosomes, also known as outer membrane vesicles (OMVs), are extracellular carriers secreted by Gram-negative bacteria and are always present around the bacteria. Outer membrane vesicles are composed of various components, including but not limited to phospholipids, lipopolysaccharides, proteins, RNA, or DNA. As small, biologically active molecules produced by bacteria, bacterial outer membrane vesicles are considered key mediators of interactions between bacteria and between bacteria and the host due to their structural stability and long effective circulation time. They not only participate in various physiological and pathological processes of bacteria but also play an indispensable role in the life activities of the host. This invention investigates the therapeutic effects of microbially extracted exosomes on dry eye syndrome, aiming to find new treatment options for dry eye. Summary of the Invention
[0005] The purpose of this invention is to provide *Rhizobium stoloniferum* exosomes for the treatment of dry eye syndrome. In vitro cell experiments and mouse experiments using a dry eye model were conducted on exosomes extracted from this microbial strain, confirming that *Rhizobium stoloniferum* exosomes have a therapeutic effect on dry eye syndrome. Specifically, this invention provides the following technical solution:
[0006] This invention provides the use of *Staphylococcus aureus* exosomes in the preparation of products for the prevention and / or treatment of dry eye syndrome.
[0007] Preferably, the slow-growing rhizobium is Bradyrhizobium elkanii.
[0008] Furthermore, the exosomes have a diameter of 20-300 nm and are positive for expression of outer membrane proteins OmpA, OmpC, and OmpF.
[0009] Furthermore, the exosomes are extracted from the culture medium of *Rhizobium stoloniferum* by ultrafiltration centrifugation. Preferably, the culture medium is obtained by inoculating *Rhizobium stoloniferum* with TY liquid medium.
[0010] Furthermore, the exosomes can promote the transformation and increase of anti-inflammatory M2 macrophages, reduce the expression levels of pro-inflammatory factors such as TNF-α, IL-6, IL-1β and IL-23 in M1 macrophages, and increase the expression levels of anti-inflammatory factors such as IL-10, TGF-β1, IL-13 and G-CSF in M2 macrophages.
[0011] In some embodiments, the present invention provides an ophthalmic pharmaceutical composition comprising the aforementioned *Rhizobium tumefaciens* exosomes. In specific embodiments, the *Rhizobium tumefaciens* exosomes provided by the present invention can be administered in combination with other active ingredients proven effective for dry eye syndrome.
[0012] In some embodiments, the present invention provides an ophthalmic drug comprising an effective amount of the aforementioned *Rhizobium stomatum* exosomes, and pharmaceutically acceptable adjuvants or carriers. In some specific embodiments, the ophthalmic drug uses the *Rhizobium stomatum* exosomes provided by the present invention as the active ingredient, and is administered after being formulated with pharmaceutically acceptable adjuvants or carriers. Adjuvants include osmotic pressure regulators (such as sodium chloride, glucose, mannitol, etc.), pH adjusters and buffers (such as phosphates, borates, acetates, citrates, etc.), viscosity enhancers (such as hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, sodium carboxymethyl cellulose, sodium hyaluronate, trehalose, glycerin, propylene glycol, carbomer, hydroxypropyl guar gum, etc.), surfactants (such as polysorbate 80, Tyloxapol, Poloxamer 188, etc.), stabilizers (such as disodium edetate (EDTA), glycine, sorbitol, ascorbic acid derivatives, etc.); excipients include mineral oil, white petrolatum, etc.
[0013] Furthermore, the pharmaceutical composition or dosage form includes eye drops, sprays, or gels.
[0014] In one specific embodiment, the present invention provides an eye drop containing an effective amount of the aforementioned *Staphylococcus aureus* exosomes.
[0015] In one specific embodiment, the present invention provides an eye spray containing an effective amount of the aforementioned Slow-growing Rhizobium exosomes.
[0016] In one specific embodiment, the present invention provides an eye gel containing an effective amount of the aforementioned Slow-growing Rhizobium exosomes.
[0017] The technical effects achieved by this invention are as follows:
[0018] Bradyrhizobium is a core microorganism inherent to the ocular surface of healthy individuals, discovered through analysis of 16S rRNA high-throughput sequencing data. It is not a human pathogen but an exosome derived from the core microorganisms of the eye. Compared to exosomes from other sources, it is more easily "recognized" by the local mucosal immune system. As a natural "resident," it can reduce immunogenicity and decrease the probability of immune rejection.
[0019] The OMV of the slow-growing rhizobium in this invention is derived from the secretion of the slow-growing rhizobium itself. It has a short fermentation cycle, low culture medium cost, and high exosome yield. Compared with mammalian cells or stem cells, it is easier to scale up and standardize, thereby reducing the production cost of ophthalmic products such as eye drops or ophthalmic gels.
[0020] In this invention, the OMV of *Rhizobium stoloniferum* can promote the transformation of macrophages to the M2 phenotype in vitro, significantly reducing the levels of M1-type pro-inflammatory factors TNF-α, IL-6, IL-1β, and IL-23, and significantly increasing the levels of M2-type anti-inflammatory factors IL-10, TGF-β1, IL-13, and G-CSF. When the OMV of *Rhizobium stoloniferum* was formulated into eye drops and applied to the ocular surface, its efficacy was compared with that of saline eye drops. The results showed that the OMV of *Rhizobium stoloniferum* improved the dry eye phenotype in a mouse model of dry eye. Furthermore, pathological sections and inflammatory factor detection also revealed that the OMV of *Rhizobium stoloniferum* reduced inflammatory damage in the ocular tissues of the dry eye model mice, indicating that the OMV of *Rhizobium stoloniferum* provided by this invention can significantly improve the symptoms and signs of dry eye. Attached Figure Description
[0021] Figure 1 The images shown are electron micrographs of OMV extracted from the slow-growing rhizobium strain of this invention. In the image, A shows the morphology of OMV under an electron microscope, and B shows the expression of bacterial outer membrane proteins A, C, and F (OmpA, OmpC, and OmpF).
[0022] Figure 2 Image showing the macrophage proliferation activity of slow-growing rhizobium OMV (detected by CCK-8 assay).
[0023] Figure 3 This is a graph showing the secretion levels of pro-inflammatory factors in M1 macrophages and anti-inflammatory factors in M2 macrophages.
[0024] Figure 4 A graph showing the proportion of M1 and M2 phenotypes in macrophages from each group as detected by dual-label flow cytometry (M1: CD86+; M2: CD206+).
[0025] Figure 5 Fluorescence staining of corneal epithelium at different time points in each group of dry-eye mice.
[0026] Figure 6 HE staining images of meibomian gland pathological sections from each group of mice. Detailed Implementation
[0027] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the testing methods, purchased goods, unless otherwise specified, shall be used under conventional conditions or conditions recommended by the manufacturer. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in the practice and testing of this disclosure.
[0028] The Bradyrhizobium sp. strain provided in this invention is a standard strain purchased from ATCC, strain number: ATCC49852 Bradyrhizobium elkanii.
[0029] Example 1
[0030] I. The extraction and preparation method of exosomes from slow-growing rhizobia includes the following steps:
[0031] ① Culture of OMV (Originally Demodex rhizobium exosomes):
[0032] Slow-growing rhizobia were grown in TY liquid medium (8 g / L peptone, 10 g / L glucose, 3 g / L yeast extract, 0.1 g / L CaCl2·6H2O, 3 mL / L Rh trace elements, pH 7.0) for 12 to 24 hours. The culture was centrifuged to separate the whole bacteria from the supernatant containing exosomes (OMV). The supernatant was filtered through a 0.22 or 0.45 μm filter and then centrifuged at high speed (ultracentrifugation) to obtain OMV particles. Specific preparation method: OMV was prepared using ultrafiltration centrifugation. 500 mL of bacterial culture was centrifuged at 5000 × g for 10 min at 4 °C, the supernatant was collected, and then filtered through a 0.45 μm filter membrane. The filtrate was concentrated using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100,000. The concentrate was ultracentrifuged at 4°C (150,000 × g) for 4 h, and the precipitate was resuspended in 50 mmol / L HEPES (pH 6.8 buffer and stored at -20°C).
[0033] OMV protein concentration was determined using the BCA protein concentration assay kit; OMV endotoxin content was determined using the Limulus amebocyte lysate (LAL) reagent, and the procedures were strictly followed according to the instructions.
[0034] ②SDS-PAGE Analysis
[0035] Take 4 μL of OMV solution, add 16 μL of loading buffer, mix well, and boil in a water bath at 100 °C for 3 min. Perform SDS-PAGE protein electrophoresis using a 5% stacking gel and a 15% separating gel at a constant speed of 60 V and 120 V, respectively. Evaluate the presence of bacterial outer membrane proteins in the extracted OMV based on the electrophoresis results.
[0036] ③Western blot was used to detect the level of bacterial outer membrane protein F in the extracted OMV samples.
[0037] Total protein was extracted from cells using a protein extraction kit, and protein concentration was determined using a BCA kit. The procedure was strictly followed according to the kit instructions. Images were captured using a chemiluminescence imager, and the results were analyzed after data acquisition.
[0038] ④ Electron microscopy observation of the isolated slow-growing rhizobium OMV.
[0039] After removing the OMV sample from the -80℃ freezer, place it in an ice box to thaw, then vortex and centrifuge. Adjust the sample concentration or viscosity according to its properties. Use a pipette to aspirate approximately 15 μL of the sample onto a copper grid and let it stand for 1 min. Stain with 2% uranium acetate staining solution at room temperature for 1 min, then observe and photograph.
[0040] OMVs exhibit a circular, vesicle-like structure with varying diameters, ranging from 20 to 300 nm. Further Western blot analysis revealed positive expression of bacterial outer membrane proteins A, C, and F (OmpA, OmpC, and OmpF). Figure 1 ).
[0041] II. Results
[0042] 1. Effects of OMV (Obstemia ulmoides) on macrophage activity, immune function, and phenotype
[0043] Experimental groups: Control group (M0); M1 macrophage group; M0+OMV treatment group; M1+OMV treatment group;
[0044] THP-1 monocytes were cultured and induced to differentiate into macrophages (M0 / Mφ) using PMA (100 ng / ml). Further induction with IFN-γ (20 ng / ml) and LPS (100 ng / ml) yielded M1 macrophages.
[0045] Assessing cell viability:
[0046] (1) Detection of cell proliferation activity by CCK-8 assay
[0047] After trypsin digestion, cells were collected, counted, and divided into groups of 9 × 10⁶ cells per well. 3 Cells were seeded in 96-well cell culture plates, with 3-5 replicates per group. Cells were incubated overnight in a CO2 incubator to allow cell adhesion. After treatment according to the groups, cells were cultured in a CO2 incubator for 24, 48, and 72 h respectively. 10 μL of CCK-8 solution was added to each well and mixed thoroughly. Blank wells (containing no cells, culture medium, or CCK-8 solution) were also included. Incubation was continued in a CO2 incubator for 40 min. Absorbance values were measured at 450 nm using a microplate reader. The results showed that OMV of *O. truncatula* did not significantly affect cell proliferation activity compared to M1 cells at the same time points. Figure 2 ).
[0048] (2) ELISA was used to detect the secretion levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β, IL-23) in M1 macrophages and anti-inflammatory factors (IL-10, TGF-β1, IL-13, G-CSF) in M2 macrophages;
[0049] Transfer the cell supernatant to a sterile centrifuge tube and centrifuge at 1000×g for 10 min at 4°C. Aliquot the supernatant into small EP tubes and store below -20°C. After removing the kit from the refrigerator, allow it to equilibrate at room temperature (25-28ºC) for 30 minutes. Prepare an appropriate amount of washing buffer and add 1 mL of standard diluent to the standard. Incubate at room temperature for 10 minutes, gently mix, and pipette several times to completely dissolve the standard. Take a sterile 1.5 mL centrifuge tube and add standard diluent to each tube. Dilute sequentially to obtain the corresponding standard concentration. Add 100 µL of standard or test sample to the reaction wells and incubate at 37°C for 60 minutes. Add 100 µL of biotinylated antibody per well and incubate at 37°C for 60 minutes. Add 100 µL of biotinylated antibody per well and incubate at 37°C for 60 minutes. Add μL of enzyme conjugate working solution and incubate at 37°C for 30 minutes. Add 90 μL of substrate solution to the reaction wells and incubate at 37°C in the dark for 15 minutes. Add 50 µL of stop solution per well, mix well, and immediately measure the OD value at 450 nm using a microplate reader.
[0050] The results showed that OMV from *Rhizobium stoloniferum* could promote the transformation of macrophages to the M2 phenotype in vitro, significantly reduce the levels of M1 pro-inflammatory factors TNF-α, IL-6, IL-1β, and IL-23, and significantly increase the levels of M2 anti-inflammatory factors IL-10, TGF-β1, IL-13, and G-CSF. Figure 3 ).
[0051] (3) The proportion of M1 and M2 phenotypes in macrophages in each group was detected by dual-label flow cytometry (M1: CD86+CD80+; M2: CD163+CD206+).
[0052] After cell processing, aspirate the cell culture medium into a suitable centrifuge tube, add an appropriate amount of trypsin cell digestion solution to digest the cells, add cell culture medium, gently pipette the cells, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them, and take 1×10⁶ cells. 6 Centrifuge the resuspended cells at 1000 rpm for 5 min, discard the supernatant, gently resuspend the cells in 100 µL of 1×Annexin V binding buffer, add 2 µL LCD86 and 1.5 µL LCD206 and mix well. Incubate at room temperature for 30 min, add 400 µL of 0.5% BSA solution, add 2 µL of Annexin V-FITC and mix gently, add 1.5 µL of Annexin V-PE staining solution and mix gently. Simultaneously set up a negative control without dye and a single-stain tube with only one dye. Incubate in the dark on ice for 15 min. After incubation, resuspend the cells directly in 400 µL of 1×Annexin V binding buffer and immediately perform instrumental analysis.
[0053] The results showed that the proportions of M1 and M2 phenotypes in macrophages in each group were detected by dual-label flow cytometry (M1:CD86). + M2: CD206 + The proportion of the M1 phenotype in the M1 macrophage group was significantly higher than that in the M2 phenotype; the proportions of the M1 and M2 phenotypes were similar in the M0+OMV and M1+OMV groups. Figure 4 ).
[0054] 2. Observation on the therapeutic effect of OMV (Obstemia ulmoides) on dry-eye mice.
[0055] A dry eye model was established in C57 BL / 6 mice by subcutaneous injection of scopolamine. Mice in each experimental group were injected subcutaneously into the back of the neck with scopolamine at a concentration of 2.5 mg / mL, at a dose of 0.3 ml / time, four times a day (9:00, 12:00, 15:00, 18:00) for one week. All animals were housed in a dry environment for two weeks, successfully establishing a mouse dry eye model.
[0056] Experimental groups: normal group, dry eye model group, saline treatment group, and OMV treatment group. The saline group received saline eye drops four times a day, 50 μL each time, for two consecutive weeks; the OMV intervention group received OMV eye drops four times a day, 50 μL each time, for two consecutive weeks. Ocular tissues from rats in each group were collected 14 days after the start of the experiment for histopathological analysis.
[0057] Results showed that significant fluorescent staining of the corneal epithelium was visible in the dry eye model group, and the epithelium was incomplete, proving the successful modeling. Corneal fluorescent staining and photography were performed at 3, 5, and 7 days post-treatment. In the OMV treatment group, corneal epithelium showed significant healing at 7 days post-treatment, with fluorescent staining disappearing and the epithelium remaining intact and smooth. In the saline group, at 7 days post-treatment, most of the corneal epithelium still showed areas of fluorescent staining due to epithelial detachment. Figure 5 ).
[0058] After 14 days of treatment, HE staining of meibomian gland tissue sections from mice in each group revealed that in the normal group, the meibomian gland acinar cells were morphologically intact with centrally located nuclei and no obvious inflammatory cells. In the dry eye model group, numerous inflammatory cells infiltrated around the meibomian gland acinar cells, and the acinar cells tended towards a vacuolar appearance. The histopathological changes in the saline treatment group were similar to those in the dry eye model group. In the OMV treatment group, the meibomian gland acinar cells were more morphologically intact, and the inflammatory cell infiltration was significantly reduced compared to the dry eye model group. Figure 6 ).
Claims
1. Application of Slow-growing rhizobium exosomes in the preparation of products for the prevention and / or treatment of dry eye syndrome.
2. The application as described in claim 1, characterized in that, The slow-growing rhizobium mentioned is Bradyrhizobium elkanii.
3. The application as described in claim 1 or 2, characterized in that, The exosomes have a diameter of 20-300 nm and are positive for expression of outer membrane proteins OmpA, OmpC, and OmpF.
4. The application as described in claim 3, characterized in that, The exosomes were extracted from the culture medium of *Rhizobium stoloniferum* by ultrafiltration centrifugation; the culture medium was obtained by inoculating *Rhizobium stoloniferum* with TY liquid medium.
5. An ophthalmic pharmaceutical composition, characterized in that, It contains the exosomes of slow-growing rhizobium as described in any one of claims 1-4.
6. An ophthalmic drug, characterized in that, It contains an effective amount of the slow-growing rhizobium exosomes as described in any one of claims 1-4, and a pharmaceutically acceptable adjuvant or carrier.
7. The pharmaceutical composition of claim 5 or the medicament of claim 6, characterized in that, The pharmaceutical composition or dosage form includes eye drops, sprays, or gels.
8. An eye drop, characterized in that, It contains an effective amount of the slow-growing rhizobium exosomes as described in any one of claims 1-4.
9. An eye spray, characterized in that, It contains an effective amount of the slow-growing rhizobium exosomes as described in any one of claims 1-4.
10. An eye gel, characterized in that, It contains an effective amount of the slow-growing rhizobium exosomes as described in any one of claims 1-4.