Lactiplantibacillus plantarum ssp. plantarum, membrane vesicles thereof, and uses thereof for ameliorating eye diseases

Abstract

To provide pharmaceutical compositions for use to ameliorate eye diseases.SOLUTION: Provided herein are a strain of Lactiplantibacillus plantarum ssp. plantarum, membrane vesicles thereof, and uses thereof for ameliorating eye diseases. The Lactiplantibacillus plantarum ssp. plantarum EP21 and bacterial membrane vesicles are derived from the Lactiplantibacillus plantarum ssp. plantarum EP21. Both the Lactiplantibacillus plantarum ssp. plantarum strain EP21 and bacterial membrane vesicles derived therefrom can suppress inflammation by inhibiting inflammation factor thereby can ameliorate eye diseases.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to Lactiplantibacillus plantarum subsp. plantarum strains, bacterial membrane vesicles (MVs) derived from Lactiplantibacillus plantarum subsp. plantarum strains, in particular compositions comprising Lactiplantibacillus plantarum subsp. plantarum strain EP21, compositions comprising bacterial MVs derived from Lactiplantibacillus plantarum subsp. plantarum strain EP21, and their use in the improvement of eye diseases.

Background Art

[0002] Myopia is increasing worldwide, and myopia is spreading in East Asia. Recent publications estimate that by 2050, nearly half (49.8% (95% CI 43.4 - 55.7)) of the world's population will be myopic (≤ -0.5 D) and 9.8% (95% CI 5.7 - 19.4) will be highly myopic (≤ -5 D). High myopia is one of the important risk factors for blindness because it increases the risk of retinal detachment, macular choroidopathy, early-onset cataract, and glaucoma. The incidence of neovascularization in patients with high myopia is 9 times higher than that in normal people. In Taiwan, the prevalence of myopia in 6-year-old children is 9.4%, and it reaches 75% in 15-year-old youths. In 18-year-old youths, the prevalence rises to 80% - 90%, and 10% - 20% of this age group are highly myopic. Therefore, prevention of myopia to prevent blindness has become an important global issue.

[0003] Axial myopia is the most common type among myopia. Due to the excessive elongation of the vitreous chamber, the axial length of the eye increases more than the increase in corneal refractive power. In addition, remodeling of the scleral tissue is associated with the elongation of the axial length and myopia. This is because the synthesis of connective tissue decreases and the degradation of type I collagen is promoted. As myopia progresses, the composition and ductility of the sclera change. However, the exact pathogenesis of myopia remains unclear. For example, atropine, dopamine, pirenzepine, 7-methylxanthine can suppress the progression of myopia, but the underlying molecular mechanism is still unknown. The onset of myopia is related to both environmental factors and genetic factors. Recent studies have shown that environmental factors have an important impact on the progression of myopia. Many hypotheses predict that genetic factors affect the molecular mechanism of the onset of myopia, and environmental factors (such as the time spent outdoors) do not have a direct impact on myopia. Current research has shown that retinal signaling plays an important role in the growth and axial regulation of the eye. Signals that accelerate the remodeling of scleral tissue are emitted from the retina and affect photoreceptors and retinal pigment epithelium (RPE). The characteristics of myopia include a longer axial length (longer eyeball), a deeper vitreous chamber, a thinner lens, and a flatter cornea.

[0004] In addition to environmental and genetic factors, inflammation has also been found to be associated with the onset of myopia. The first paper mentioning myopia and inflammation was published in 2011. This article examined the correlation between myopia and intraocular inflammation. Some reports have suggested an association between myopia and allergic conjunctivitis. In the prior art, atropine is widely used clinically to slow the progression of myopia by reducing the expression levels of c-Fos, NFκB, IL-6, and TNF-α involved in chronic inflammation. This paper was cited by the American Academy of Optometry, indicating that atropine may suppress the progression of myopia by suppressing inflammation. In an animal model of allergic conjunctivitis, allergic inflammation (especially allergic conjunctivitis) promotes the progression of myopia. Also, when PM2.5 is directly applied to the ocular surface of Syrian hamsters, the progression of myopia is accelerated. Therefore, exposure to PM2.5 not only increases eye inflammation but also induces myopia. Several studies have shown that retinal dopamine is recognized as a signal that halts normal eye growth and also plays a role in the onset of myopia. Acupuncture treatment reduces inflammation by activating dopa decarboxylase and increasing dopamine levels. Dopamine can suppress the expression of TNF-α and IL6, further suppressing inflammation. That is, acupuncture treatment suppresses the progression of myopia by suppressing inflammation caused by the activation of the dopamine-D1R signaling pathway. Currently, there are several anti-inflammatory drugs that suppress the progression of myopia, such as resveratrol, dicumarol, and herbal formulations containing two types of herbs. Other researchers have also reported that when bovine lactoferrin is given to mice, the onset of lens-induced myopia in mice is prevented via the IL-6-MMP-2 axis. Furthermore, in a clinical trial, as a result of using crocetina, a potent anti-inflammatory compound, in the treatment of pediatric myopia, it has been shown that crocetina has the effect of suppressing the progression of pediatric myopia. In 2021, inflammation is one of the myopia risk factors of the IMI (International Myopia Institute). Although prior art has provided clinical and experimental data showing the association between myopia and inflammation, the cause of the inflammatory response remains unclear. There are no diseases or environmental factors that cause such a high incidence of myopia (in some regions, the incidence of myopia in people under 18 years old is over 90%).

[0005] It has been reported that various mediators are involved in the onset of myopia. In myopia, transforming growth factor beta (TGF-β) and matrix metalloproteinase-2 (MMP2) increase, while type I collagen decreases. The transforming growth factor beta (TGF-β) signaling pathway involves multiple different reactions related to inflammatory responses, tissue fibrosis, and remodeling. Therefore, TGF-β is closely related to the onset of myopia. There are three different isoforms of TGF-β in mammals. In ocular tissues, the expression level of TGF-β2 is the highest. The expressions of TGF-β1, TGF-2, and TGF-3 are all positively correlated with the optical axis.

[0006] In addition, matrix metalloproteinases (MMPs) are responsible for the degradation of ECM (extracellular matrix) proteins such as collagen, gelatin, fibronectin, and aggrecan, and also act as mediators of angiogenesis during inflammation. Therefore, cooperation among the MMP family is thought to be involved in the etiological changes of myopia. In the sclera of chickens and the guinea pig model, it can be seen from the results of form deprivation myopia (FDM) that the expression of MMP2 increases. In human samples, myopic eyes with high expression of MMP2 have also been confirmed. Furthermore, studies have shown that TGF-β affects the expression of MMP2 through the activation of NFκB.

[0007] The complement system is also involved in the onset and mechanism of myopia. In patients with pathological myopia (-8D to -25D), the expression levels of C3 (p = 0.004) and CH50 (p < 0.001) increase dramatically. In the sclera of guinea pigs with myopia caused by defocusing with negative lenses, the expression levels of C1q, C3, and C5b-9 increase significantly. CD55 inhibits the formation of convertases and promotes their degradation, thereby inhibiting the activities of C3 and C5 convertases. CD55 prevents the activation of the complement system, and an increase in the expression level of CD55 can suppress the progression of myopia.

[0008] Microorganisms in different organs / tissues are different and constantly changing. According to current research, the composition and balance of microorganisms play important roles in our health and diseases. In an autoimmune uveitis model, Horai et al. (2015) discovered that gut microbiota induce autoreactive T helper 17 (Th17) cells and trigger retinal-related uveitis. The use of antibiotics can alleviate uveitis and reduce the amount of autoreactive Th17 cells. The replenishment of broad-spectrum antibiotics can not only upregulate regulatory T cells but also suppress autoreactive effector T cells. Also, human leukocyte antigen B27 is known to affect the immune system by increasing intestinal permeability and is associated with autoimmune uveitis and abnormal gut microbiota. Furthermore, the disruption of gut microbiota is one of the factors involved in the progression of type 2 diabetes (DM), increasing the concentrations of lipids, fatty acids, and glucose in plasma, changing immune cells, and inducing the secretion of inflammatory molecules (TNFα, IL-1β, IL-6, IFN-γ, inflammatory adipokines, chemokines). These factors also affect diabetic retinopathy (DR). Chronic low-level inflammation associated with metabolic disorders and oxidative stress is also related to changes in the composition of gut microbiota. Gut microbiota is associated with age-related macular degeneration (AMD). Compared with healthy people, patients with end-stage AMD also show abnormal gut microbiota.

[0009] The mammalian intestine harbors a large number of symbiotic bacteria (indigenous microbiota) that interact with the host via secreted proteins, metabolites, and bacterial membrane vesicles (BMVs). BMVs are produced at various stages of bacterial growth. Therefore, BMVs are thought to be a long-distance communication and signaling system that is involved in the interaction between the host and bacteria or between bacteria. Membrane vesicles released from the outer membrane of Gram-positive bacteria are called outer membrane vesicles (OMVs), and Gram-positive bacteria release membrane vesicles (MVs). Studies have suggested that BMVs are associated with microbial pathogenicity and diseases. In the presence of antibiotics, BMVs enhance the survival rate of bacteria and transmit antibiotic resistance genes or antibiotic-degrading enzymes to other bacteria, causing diseases. BMVs transmit microbe-associated molecular patterns (MAMPs) to host cells, which may induce inflammatory or anti-inflammatory responses. Generally, BMVs derived from pathogenic bacteria have an inflammatory-inducing effect on host cells, while probiotic BMVs have an immunomodulatory effect. Furthermore, BMVs can deliver DNA or RNA into cells and induce a cell protection mechanism through the regulation of pattern recognition receptors or host cell gene expression. OMVs are detected in cerebrospinal fluid, indicating that OMVs can cross the blood-brain barrier. Metagenomic analysis of BMVs can be used to determine the interaction between bacteria and the host. Metagenomic analysis of serum extracellular vesicles has revealed differences in the gut microbiota between psoriasis patients and asthma patients.

Summary of the Invention

[0010] Therefore, the main object of the present invention is to provide Lactiplantibacillus plantarum subsp. plantarum strain EP21, which is used for the improvement of eye diseases and has been deposited with the Food Industry Research and Development Institute (FIRDI) under the accession number BCRC911210.

[0011] Preferably, the Lactiplantibacillus plantarum subsp. plantarum strain EP21 contains the sequence segment shown in SEQ ID NO: 3.

[0012] Another object of the present invention is to provide Lactiplantibacillus plantarum subsp. plantarum strain EP21 for use in the preparation of a pharmaceutical composition for improving eye diseases, and to provide a pharmaceutical composition comprising Lactiplantibacillus plantarum subsp. plantarum strain EP21.

[0013] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

[0014] Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, a solvent, buffer, emulsifier, suspending agent, decomposer, disintegrant, dispersant, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption retardant, liposome, and analogs and alternatives thereof. Those skilled in the art can select appropriate excipients and adjust the ratios according to specialized or general techniques.

[0015] Preferably, the carriers suitable for the present invention include, but are not limited to, water, physiological saline, phosphate buffered saline (PBS), aqueous solutions containing alcohol, and combinations thereof.

[0016] Preferably, the food additive is a substance added to food materials for preparing products that can be eaten by animals (including humans). The use and ratio of food additives are determined by those skilled in the art according to specialized or general techniques. The food additives include natural and artificial sweeteners, colorants, pickling and souring agents, flavors, emulsifiers, fat substitutes, hardening agents, swelling agents, lubricants, moisturizing agents, preservatives, stabilizers, and thickening agents.

[0017] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, solutions, suspensions, capsules, tablets, lozenges, troches, powders, lyophilized powders, lotions, emulsions, suppositories, slurries, and ointments.

[0018] More preferably, an isolated strain of Lactiplantibacillus plantarum subsp. plantarum or its subculture is prepared with a pharmaceutically acceptable vehicle by a technique known to those skilled in the art to form a dosage form suitable for oral administration.

[0019] Preferably, the effective dose of the pharmaceutical composition is to administer 1×10 6 cfu / kg to 1×10 10 cfu / kg to a subject.

[0020] More preferably, the effective dose of the pharmaceutical composition is to administer 5×10 8 cfu / kg to 5×10 9 cfu / kg to a subject.

[0021] Preferably, the eye diseases include, but are not limited to, myopia, dry eye syndrome (DES), eye strain, keratitis, macular degeneration, retinitis pigmentosa (RP), proliferative diabetic retinopathy (PDR), ischemic retinopathy, choroidal neovascularization, glaucoma, systemic lupus erythematosus (SLE), and Sjögren's syndrome (SS).

[0022] More preferably, the ischemic retinopathy includes diabetic retinopathy (DR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO), and ischemic optic neuropathy (ION).

[0023] Another object of the present invention is to provide a composition containing bacterial membrane vesicles for improving eye diseases. The bacterial membrane vesicles (MV) are derived from the Lactiplantibacillus plantarum subsp. plantarum strain EP21.

[0024] Preferably, the vesicle diameter of the MV is from 100 nm to 300 nm.

[0025] Preferably, the vesicle diameter of the MV is from 120 to 255 nm.

[0026] Another object of the present invention is to provide a pharmaceutical composition containing the above-mentioned microvesicles (MVs) for improving eye diseases.

[0027] After administering Lactiplantibacillus plantarum subsp. plantarum strain EP21 of the present invention and the MVs of Lactiplantibacillus plantarum subsp. plantarum strain EP21 to a subject, eye diseases can be improved by suppressing excessive increase in axial length and refractive changes. The expression of activated p-NFκB, NFκB, NLRP3, IL1β, TNFα, and IL6 is also inhibited, and the expression of the anti-inflammatory target IL10 increases.

[0028] Preferably, the pharmaceutical composition containing the above-mentioned microvesicles (MVs) is prepared in a dosage form suitable for application to a subject as an injection or eye drops. The types of injections include intravenous injection (IV), intraperitoneal injection (IP), and tail vein injection.

[0029] Preferably, the effective dose of the pharmaceutical composition containing the above-mentioned MVs is to administer 1×10 5 particles / kg to 1×10 11 particles / kg to a subject per day.

[0030] More preferably, the optimal effective dose of the pharmaceutical composition containing the above-mentioned MVs is to administer 2.7×10 8 particles / kg to 5.3×10 9 particles / kg to a subject per day.

[0031] Hereinafter, a method for preparing a composition containing Lactiplantibacillus plantarum subsp. plantarum strain EP21 and membrane vesicles (MV) derived from Lactiplantibacillus plantarum subsp. plantarum strain EP21, a test of a pharmaceutical composition prepared using Lactiplantibacillus plantarum subsp. plantarum strain EP21 or its membrane vesicles (MV) for improving eye diseases, and a test and analysis of the inhibitory effect of a pharmaceutical composition prepared using Lactiplantibacillus plantarum subsp. plantarum strain EP21 or its membrane vesicles (MV) on inflammatory factors will be described in detail. Therefore, it has been proven that the composition containing Lactiplantibacillus plantarum subsp. plantarum strain EP21 or its membrane vesicles (MV) can indeed improve eye diseases. Furthermore, the said membrane vesicles (MV) and its pharmaceutical composition can pass through the blood-retinal barrier (BRB) via the bloodstream, enhance the immunomodulatory properties of cells, reduce retinal inflammation caused by TGF-β2, and thereby further delay the progression of myopia caused by TGF-β2.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0083] The following embodiments are for further explaining the present invention and do not limit the content of the present invention. Those skilled in the art can make certain improvements and modifications without departing from the scope of the present invention. Unless otherwise specified, the following terms used in this specification and each claim have the definitions shown below.

[0084] The singular forms "a" used in the description and claims are intended to cover not only one item but also one or more items, such as at least one, at least two, or at least three. Further, the terms "comprising" and "having" used in the description and claims of the present invention are unrestricted and do not exclude additional unnecessary elements or components described in the description and claims. Also, note that unless explicitly indicated otherwise in this specification, the term "or" usually includes "and / or". The terms "about" or "substantially" used in the specification and claims of this application are used to correct possible slight errors, but such slight changes do not change the nature of the present invention.

[0085] Probiotics or probiotic bacteria are microorganisms whose cells, mixed strains, extracts, or metabolites have beneficial effects on the health of the host. These are usually produced from the human body or supplements and are beneficial to humans.

[0086] As used herein, "pre-test measurement" refers to measuring the axial length and refractive power of each experimental animal's eyes. The pre-test measurement includes the following procedures. Inject 50 - 80 mg / kg of a liquid anesthetic (Zoletil (registered trademark)-50) intraperitoneally into the experimental animal for chemical fixation. Measure the refraction and axial length using a handheld strip retinal detector and an A-scan ultrasonic biometer, respectively. According to the measurement results, the difference in axial length and refraction between both eyes is expected to be 0.02 mm and 2 (D) or less, respectively. Mice found to have congenital eye defects during the pre-test measurement are not counted in the statistics in the next experiment because it may cause errors in the experimental results.

[0087] The flowcharts and procedures of each embodiment comply with laboratory safety guidelines. The term "effective dose" referred to in this specification refers to the dosage range obtained according to the experimental data and results of each embodiment of the present invention. Those skilled in the art can select appropriate receptors according to specialized or general techniques, adjust the dosage ratio according to the type of receptor applied and the dosage form, and convert the dosage.

[0088] Preparation Embodiment 1: Source and Preparation of Test Strains

[0089] The strain according to the present invention is collected and isolated from the feces of hamsters fed with Chinese herbal medicine. This strain is inoculated onto De Man, Rogosa, Sharpe (MRS) agar medium (BD) by the quadrant streaking method and cultured for 16 - 18 hours. Then, the bacterial solution is diluted 100 - fold and cultured in 40 ml of MRS at 37°C for 16 - 18 hours. Next, the obtained bacterial solution is diluted 100 - fold and cultured in 1000 ml of MRS at 37°C for 24 hours. One probiotic colony on the plate is selected, and a part of the sequence of its 16S rRNA gene is amplified with a pair of bacterial - specific primers such as SEQ ID NO: 1 (forward primer: 8F primer) and SEQ ID NO: 2 (reverse primer: 1492R primer) to obtain a nucleic acid segment such as the sequence shown in SEQ ID NO: 3. The total RNA extraction method is well - known to those skilled in the art and will not be described in further detail. Comparing the sequence of the nucleic acid segment (SEQ ID NO: 3) with the six 16S rRNA gene sequences of Lactiplantibacillus plantarum subsp. plantarum (accession numbers KX057658.1, ON506095.1, MK524162.1, MT613641.1, MT597711.1, and MT597692.1) in the GenBank of the National Center for Biotechnology Information (NCBI), the sequence shows 99% similarity. Therefore, the strain of the present invention is Lactiplantibacillus plantarum subsp. plantarum (L.plantarum), hereinafter abbreviated as strain EP21.

[0090] The following are the physiological characteristics of strain EP21. Growth temperature: 35°C to 40°C; growth pH: 4.0 - 7.0; oxygen requirement level: facultative anaerobic. The strain EP21 of the present invention shows a positive result in the Gram staining test.

[0091] This strain EP21 was deposited on November 21, 2023, with the accession number BCRC 911210 at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (331 Shih-Pin Rd., Hsinchu 300, Taiwan).

[0092] Preparation Embodiment 2: Extraction of Membrane Vesicles (MV)

[0093] The bacterial solution of strain EP21 (hereinafter abbreviated as EP21) is centrifuged at 8000 rpm for 1 hour, and the supernatant is collected. The supernatant is filtered through a 0.22 μm filter cup and concentrated to 10 times the volume with a centrifugal filter. It is placed in an ultracentrifuge and centrifuged at 28000 rpm at 4°C for 6 hours to collect the pellet. Next, the pellet is dissolved in a 50% OptiPrep TM solution, and 45%, 40%, 35%, 30%, 25%, 20% OptiPrep TM solutions are added to the ultracentrifuge tube in sequence, and density gradient centrifugation (28000 rpm, 4°C, overnight) is performed for density gradient separation. The isolated EP21 membrane vesicles (MV) are stored at -80°C. After preparation, the morphology of MV is detected using a transmission electron microscope (JEM 1400 FLASH). EP21 MV is negatively stained with 1% PTA (phosphotungstic acid) and applied to a 400 mesh copper grid (model: 01754-F, TED PELLA) for 1 minute, then the excess solution is removed with filter paper and placed in a dryer before observation. The transmission electron microscope uses 120 KV to accelerate the electron beam passing through the MV. In this way, the different fractions (ODG fractions) after density gradient separation and the morphology of EP21 MV are observed. The results show that the vesicle diameter of EP21 MV of the present invention is about 177.95 ± 32.6 nm.

[0094] Nanoparticle Tracking Analysis (NTA) (version NTA3.4 build 3.4.003) is used to detect the size and number of vesicles (particles) of EP21 MV. The device is set to SOP standard measurement for detection, and the dilution rate is multiplied by the result obtained for application in the following experiment to estimate the number of vesicles of EP21 MV. The NTA result shows that the vesicle diameter of EP21 MV of the present invention is about 156.36 ± 25.7 nm.

[0095] The prepared EP21 MV is rapidly frozen in liquid nitrogen and its morphology is fixed on a 0.2 μm PC film (model: GTTP02500, Isopore TM )). After platinum coating on the surface, a cryo-scanning electron microscope (JEOL JSM-7800F) is used to observe the vesicle size and morphology of EP21 MV. The purified EP21 MV is hydrophobically labeled with 5 μm fluorescent dye DIL (CAS number: 41085-99-8, Alfa Aesar, Switzerland) (for 30 minutes, 37 °C), and unbound dye is removed by ultracentrifugation (28,000 rpm, 2 hours at 4 °C), and then frozen at -80 °C for subsequent phagocytosis of MV and tracking in in vivo experiments of MV. The results of the cryo-scanning electron microscope show that the vesicle diameter of EP21 MV is about 153.04 ± 17.6 nm, and the vesicle diameter of isolated EP21 MV is about 195.28 ± 59.5 nm.

[0096] Embodiment 1: Vitamin EP21 Administration Test for Myopia Inhibition

[0097] Three-week-old Brown Norway rats are used as test animals. After pre-test measurement of the animals, 250 ng / ml of TGF-β2 (transforming growth factor beta 2) is injected into the upper eyelid of the right eye once a week for 3 weeks to induce inflammation. In the negative control group, balanced salt solution (BSS, the eye wash buffer used for the preparation of the injection drug) is injected and administered intragastrically with 1X PBS for 3 weeks. In the positive control group, TGF-β2 is injected and 1X PBS is administered intragastrically for 3 weeks. The treatment group is injected with TGF-β2 and logarithmic-phase strain EP21 (2.5 - 3×10 9Administer it via the trachea at a dose of 9 CFU / 0.1 ml / day) for 3 weeks. Measure the axial length and refraction of the eye before and after treatment, and confirm the changes between each group. After collecting the entire eyeball as a sample, perform Western blotting, immunohistochemical staining, immunohistochemistry (IHC) analysis, and analysis of the dopamine content. Measure the growth curve for 24 hours using INFINITE M NANO (TECAN), select the strain EP21 in the logarithmic phase, and administer it to the animals. Collect the colonies of the EP21 strain, culture them in 6 ml of MRS (model: 288130, BD) at 37 °C for 16 - 18 hours. Every day, dilute the EP21 bacterial solution 100-fold, culture it in 40 ml of MRS at 37 °C for 6 hours, and culture it until the logarithmic phase. Next, centrifuge the EP21 bacterial solution at 3500 rpm for 15 minutes, and wash the EP21 pellet with 1X PBS. After centrifugation, dissolve it in 1X PBS to form EP21, and give 0.1 ml (2.5 - 3×10 9 CFU / ml) of EP21 to each rat.

[0098] As shown in FIGS. 1A and 1B, TGF-β2 significantly increases the axial length of the rat's eye and changes the refraction. Administration of EP21 suppresses the excessive increase in axial length and the excessive change in refraction. In this embodiment, one-way analysis of variance (ANOVA) is used as a statistical method. * means a P value < 0.05. ** means a p value < 0.01. *** means a p value < 0.001. **** means a p value < 0.0001.

[0099] After inducing myopia in the eyes with TGF-β2, the effects of EP21 administration on TGF-β, MMP2 (matrix metalloproteinase-2), and COL1A1 (alpha-1 type I collagen) are analyzed by immunofluorescence (IF). Fixed tissues are embedded in paraffin, deparaffinized, rehydrated, and boiled with an epitope exposure reagent after sectioning. Nonspecific binding sites on the tissues are blocked with 1% BSA at room temperature. After reacting the primary antibody overnight at 4°C, the secondary antibody is reacted for 1 hour at room temperature. Nuclear staining is performed using DAPI (4',6-diamidino-2-phenylindole) (1:1000) and reacted for 5 minutes in the dark. After encapsulating with an anti-bleaching reagent, fluorescence excitation is performed with a fluorescence microscope and images are taken. Quantitative analysis of the experimental results is performed with Image J, and one-way analysis of variance of the numerical values is carried out with GraphPad Prism (statistical software). A P value < 0.05 (represented by *) is usually considered statistically significant. The results are shown in Figures 2A - 4B. TGF-β2 increases the expression of TGF-β, MMP2, and COL1A1, whereas administration of EP21 inhibits TGF-β (Figures 2A - 2B), MMP2 (Figures 3A - 3B), and COL1A1 (Figures 4A - 4B).

[0100] The expression of the TGF-β2-induced transcription factor NFκB in the eyes is analyzed by the above immunofluorescence method. Quantitative analysis of the experimental results is performed with Image J, and the obtained numerical values are analyzed by one-way analysis of variance with GraphPad Prism (statistical software). A P value < 0.05 is usually considered statistically significant. The results are shown in Figures 5A - 6B. TGF-β2 promotes the expression of NFκB and TNF-α, whereas administration of EP21 of the present invention inhibits NFκB (Figures 5A - 5B) and TNF-α (Figures 6A - 6B).

[0101] Next, the expression level of TGF-β2-induced transcription factor p-NFκB in the eyeball is analyzed by Western blotting. Add radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor, and phosphatase inhibitor) to the eyeball or retinal tissue and cut it finely. Extract the proteins in the tissue by sonication, centrifuge at 12,000 rpm at 4°C for 15 minutes, and then collect the supernatant. Quantify the protein in the sample using Bradford reagent (Bio-Rad, #5000006). Use 2 mg / ml BSA as the standard solution, and the linear range of the calibration curve is 10 μg (micrograms) to 1 μg. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is used for protein separation. After electrophoresis, transfer the protein sample from the gel to a 0.45 μm polyvinylidene fluoride (PVDF) membrane for 50 minutes. Treat the transferred membrane with 5% skim milk at room temperature for 1 hour to fill and block the non-specific binding sites on the membrane. Incubate overnight at 4°C with the primary antibody, then add the secondary antibody and react at room temperature for 1 hour. Next, add enhanced chemiluminescence (ECL) reagent, record the results using a luminometer, and quantify the results using Image J. Calibrate the expression by the internal standard method using the quantification results of the target protein. Use GAPDH as the loading control antibody and perform quantitative analysis of the experimental results by Image J. Statistically analyze the obtained numerical values (p-NFκB / NFκB / GAPDH), and perform one-way analysis of variance of the numerical values using GraphPad Prism. A P value < 0.05 is generally considered statistically significant. The results are shown in FIGS. 7A-7B. TGF-β2 activates the expression of NFκB, while administration of EP21 of the present invention inhibits the activation of NFκB.

[0102] Embodiment 2: Cell viability test

[0103] Seed 3×10 retinal pigment epithelial cells RPE1 in a 96-well plate 3Inoculate at a density of [number] cells per well and culture for approximately 16 - 18 hours. After cell adhesion, add different numbers of EP21 membrane vesicles (MVs) containing [number], [number], [number], [number], and [number], and treat for 24 hours. React with a cell viability reagent (20 μl of PMS + 2 ml of MTS) for 2 hours and measure the absorbance at 490 nm. The survival rate is (treatment / control) × 100%, and it is used to evaluate the cytotoxicity of EP21 MV on human retinal pigment epithelial cells RPE1. 8 Inoculate at a density of [number] cells per well and culture for approximately 16 - 18 hours. After cell adhesion, add different numbers of EP21 membrane vesicles (MVs) containing [number], [number], [number], [number], and [number], and treat for 24 hours. React with a cell viability reagent (20 μl of PMS + 2 ml of MTS) for 2 hours and measure the absorbance at 490 nm. The survival rate is (treatment / control) × 100%, and it is used to evaluate the cytotoxicity of EP21 MV on human retinal pigment epithelial cells RPE1. 8 Inoculate at a density of [number] cells per well and culture for approximately 16 - 18 hours. After cell adhesion, add different numbers of EP21 membrane vesicles (MVs) containing [number], [number], [number], [number], and [number], and treat for 24 hours. React with a cell viability reagent (20 μl of PMS + 2 ml of MTS) for 2 hours and measure the absorbance at 490 nm. The survival rate is (treatment / control) × 100%, and it is used to evaluate the cytotoxicity of EP21 MV on human retinal pigment epithelial cells RPE1. 8 Inoculate at a density of [number] cells per well and culture for approximately 16 - 18 hours. After cell adhesion, add different numbers of EP21 membrane vesicles (MVs) containing [number], [number], [number], [number], and [number], and treat for 24 hours. React with a cell viability reagent (20 μl of PMS + 2 ml of MTS) for 2 hours and measure the absorbance at 490 nm. The survival rate is (treatment / control) × 100%, and it is used to evaluate the cytotoxicity of EP21 MV on human retinal pigment epithelial cells RPE1. 8 Inoculate at a density of [number] cells per well and culture for approximately 16 - 18 hours. After cell adhesion, add different numbers of EP21 membrane vesicles (MVs) containing [number], [number], [number], [number], and [number], and treat for 24 hours. React with a cell viability reagent (20 μl of PMS + 2 ml of MTS) for 2 hours and measure the absorbance at 490 nm. The survival rate is (treatment / control) × 100%, and it is used to evaluate the cytotoxicity of EP21 MV on human retinal pigment epithelial cells RPE1.

[0104] Use EP21 MVs with different numbers of vesicles obtained in Preparation Embodiment 2 to treat RPE1 cells for 24 hours and observe cell proliferation. Analyze the results using a cell viability reagent and GraphPad Prism. The results are shown in Figure 8. After treatment with EP21 MVs with different numbers of vesicles, there is no significant change in the cell viability of RPE1 cells (ns means no significant difference compared to the control group). Therefore, the EP21 MV of the present invention does not show cytotoxicity.

[0105] Embodiment 3: Evaluation of the effect of EP21 MV on the activation of transcription factor NFκB

[0106] Inoculate three types of human retinal pigment epithelial cells (RPE1, ARPE 19, hRPEtiC) in 6-well plates at a density of [number] cells per well and culture for approximately 16 - 18 hours. Next, pretreat with EP21 MV ([number] vesicles / particles) and further treat with IL-1β (interleukin-1 beta) (1.25 ng / ml) for 10 minutes. 5 Inoculate three types of human retinal pigment epithelial cells (RPE1, ARPE 19, hRPEtiC) in 6-well plates at a density of [number] cells per well and culture for approximately 16 - 18 hours. Next, pretreat with EP21 MV ([number] vesicles / particles) and further treat with IL-1β (interleukin-1 beta) (1.25 ng / ml) for 10 minutes. 6 Inoculate three types of human retinal pigment epithelial cells (RPE1, ARPE 19, hRPEtiC) in 6-well plates at a density of [number] cells per well and culture for approximately 16 - 18 hours. Next, pretreat with EP21 MV ([number] vesicles / particles) and further treat with IL-1β (interleukin-1 beta) (1.25 ng / ml) for 10 minutes.

[0107] It should be noted that the specific numbers in the text are replaced with [number] in the translation as the original numbers are not clearly presented in the question. You may need to substitute the actual numbers according to the original text.The expression level of the transcription factor p-NFκB is analyzed by Western blotting. GAPDH is used as a loading control antibody, and the experimental results are quantified with Image J. The obtained numerical values (p-NFκB / NFκB / GAPDH) are statistically analyzed, and one-way ANOVA of the numerical values is performed with GraphPad Prism. A P value < 0.05 is generally regarded as statistically significant. The results are shown in Figures 9A - 9F. All groups treated with EP21 MV indicate that the activation of the IL-1β-induced transcription factor NFκB is inhibited.

[0108] Embodiment 4: Influence of nuclease on EP21 MV

[0109] EP21 MV treated with nuclease (DNase I and RNase A) is added to human retinal pigment epithelial cells RPE1 at a final concentration of 100 ng and pretreated for 2 hours. Next, the inflammatory substance IL-1β (concentration 1.25 ng / ml) is added to confirm whether EP21 MV can still exert its anti-inflammatory ability on human retinal pigment epithelial cells after nuclease treatment. The expression of the transcription factor p-NFκB is analyzed by Western blotting. GAPDH is used as a loading control antibody, and the experimental results are quantified with Image J. The obtained numerical values (p-NFκB / NFκB / GAPDH) are statistically analyzed, and one-way ANOVA of the numerical values is performed with GraphPad Prism. A P value < 0.05 is generally regarded as statistically significant. The results shown in Figures 10A - 10B indicate that the activation of the IL-1β-induced transcription factor NFκB is inhibited in both the group administered with EP21 MV and the group administered with nuclease.

[0110] Embodiment 5: Cytokine inhibition by EP21 MV

[0111] To test whether the EP21 MV of the present invention can suppress the secretion of inflammatory cytokines over a long period, the inflammation of retinal pigment epithelial cells RPE-1 is induced for 24 hours using 2.5 ng / ml of TNFα or 1.25 ng / ml of IL1β. 4.4×10 6Adding the EP21 MV of individual particles suppresses the IL6 secretion induced by TNFα or IL1β. As shown in Figure 11A, the EP21 MV of the present invention can suppress the IL6 secretion induced by TNFα. As shown in Figure 11B, the current EP21 MV can suppress the IL6 secretion induced by IL1β.

[0112] To test whether the RNA of the EP21 MV of the present invention can suppress the secretion of inflammatory cytokines for a long time, the EP21 RNA strain and the EP21 MV RNA strain (1.5 μg each) were introduced (transfected) into retinal pigment epithelial cells RPE-1, and inflammation was induced for 24 hours using 2.5 ng / ml of TNFα. Next, enzyme-linked immunosorbent assay (ELISA) was used to detect whether the cells after transfection could suppress the IL6 secretion induced by TNFα. ELISA includes the following steps. Dilute the antibody (IL6) with 1X PBS on an ELISA plate (at 4°C overnight), add blocking buffer and react for 1 hour. Add the sample and bind for 2 hours, add horseradish peroxidase (HRP) and react for 30 minutes. Next, add 3,3'5,5'-tetramethylbenzidine (TMB) substrate to develop color. Between two adjacent steps, wash the plate 3 times with 1X PBS. According to the standard color development at different dilution concentrations, add 1N HCL to stop the reaction and read the absorbance at 450 nm. As shown in Figure 12, the EP21 RNA and EP21 MV RNA of the present invention can also suppress the IL6 secretion induced by TNFα.

[0113] Embodiment 6: Effect of an eye drop containing EP21 MV on TGF-β2-induced myopic rats

[0114] Three-week-old Brown Norway rats are used as test animals. After pre-test measurement of the animals, 250 ng / ml of TGF-β2 is injected once a week into the upper eyelid of the right eye to induce inflammation for 3 weeks. In the negative control group, balanced salt solution (BSS, used for preparing eye wash buffer and injection drug) is injected, and 1X PBS eye drops are administered for 3 weeks. In the positive control group, TGF-β2 is injected, and 1X PBS eye drops are administered for 3 weeks. In the first treatment group, TGF-β2 is injected, and eye drops containing EP21 MV are administered for 3 weeks (eye drops with a high concentration of a total of 3.3×10 9 particles are instilled twice a week for 3 weeks). In the second treatment group, TGF-β2 is injected, and eye drops containing EP21 MV are administered for 3 weeks (eye drops with a low concentration of a total of 9.9×10 8 particles are instilled seven times a week for 3 weeks). The axial length and refraction of the eyes are measured before and after treatment to confirm the changes between groups. As shown in FIGS. 13A and 13B, the results show that TGF-β2 significantly increases the axial length of the rat eyes and changes the refraction. In the treatment groups, instilling eye drops containing different numbers of EP21 MV particles suppresses the excessive increase in axial length and excessive change in refraction.

[0115] Embodiment 7: Effect of EP21 MV on TGF-β2-induced myopic rats

[0116] 1. Intravenous administration of EP21 MV via the tail vein

[0117] Three-week-old Brown Norway rats are used as test animals. After pre-test measurement of the animals, 250 ng / ml of TGF-β2 is injected once a week into the upper eyelid of the right eye to induce inflammation for 3 weeks. In the negative control group, balanced salt solution (BSS, used for preparing eye wash buffer and injection drug) is injected, and 1X PBS is injected into the tail vein for 3 weeks. In the positive control group, TGF-β2 is injected, and 1X PBS is injected into the tail vein for 3 weeks. In the first treatment group, TGF-β2 is injected, and EP21 MV is administered via tail vein injection for 3 weeks (at a high concentration twice a week, with a total dose of 3.3×10 9(Inject individual particles). Inject TGF-β2 into the second treatment group, and administer EP21 MV via tail vein injection for 3 weeks (twice a week at a low concentration, with a total dose of 3.3×10 8 (Inject individual particles). Measure the axial length and refraction of the eyes before and after treatment, and confirm the changes between groups. As shown in FIGS. 14A and 14B, the results show that TGF-β2 significantly increases the axial length of the rats and changes the refraction. In the treatment groups administered different numbers of particles of EP21 MV by tail vein injection, both excessive increase in axial length and change in refraction are suppressed.

[0118] 2. Intraperitoneal administration of EP21 MV

[0119] Use 3-week-old Brown Norway rats as test animals. After pre-test measurement of the animals, inject 250 ng / ml of TGF-β2 into the upper eyelid of the right eye once a week for 3 weeks to induce inflammation. In the negative control group, inject balanced salt solution (BSS, used for preparing eye wash buffer and injection drug), and intraperitoneally inject 1X PBS for 3 weeks. In the positive control group, inject TGF-β2 and intraperitoneally inject 1X PBS for 3 weeks. Inject TGF-β2 into the first treatment group and intraperitoneally inject EP21 MV for 3 weeks (twice a week at a high concentration, with a total dose of 3.3×10 9 (Inject individual particles). Inject TGF-β2 into the second treatment group and intraperitoneally inject EP21 MV for 3 weeks (twice a week at a low concentration, with a total dose of 3.3×10 8 (Inject individual particles). Measure the axial length and refraction of the eyes before and after treatment, and confirm the changes between groups. Collect the entire eyeball as a sample and perform Western blotting and immunohistochemistry (IHC) analysis. As shown in FIGS. 15A and 15B, the results show that TGF-β2 significantly increases the axial length of the rats and changes the refraction. In the treatment groups, by intraperitoneally injecting different numbers of particles of EP21 MV, excessive increase in axial length and excessive change in refraction are suppressed.

[0120] Samples of intraperitoneal administration of P21 MV in Embodiment 7 (rat eyeballs) were added with RIPA lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor and phosphatase inhibitor), minced, and subjected to Western blotting. As shown in Fig. 15C, TGF-β2 activates the expression of inflammatory targets including p-NFκB, NFκB, NLRP3, IL1β, and TNFα, and decreases the expression of the anti-inflammatory target IL10. Therefore, intraperitoneal injection of EP21 MV of the present invention can inhibit the activation of the expression of inflammatory targets including p-NFκB, NFκB, NLRP3, IL1β, and TNFα, and increase the expression of the anti-inflammatory target IL10.

[0121] Samples of intraperitoneal administration of P21 MV in Embodiment 7 (rat eyeballs) were analyzed by immunofluorescence and quantified using Image J. Next, the obtained numerical values were interpreted by one-way ANOVA of GraphPad Prism. A P value < 0.05 is usually considered statistically significant. As a result shown in Figs. 16A-16K, TGF-β2 activates the expression of inflammatory targets including p-NFκB, NFκB (Figs. 16B-16C), NLRP3 (Figs. 16H-16I), IL1β (Figs. 16J-16K), and TNFα (Figs. 16F-16G), and decreases the expression of the anti-inflammatory target IL10 (Figs. 16D-16E). Therefore, intraperitoneal injection of EP21 MV of the present invention can inhibit the activation of the expression of inflammatory targets including p-NFκB, NFκB, NLRP3, IL1β, and TNFα, and increase the expression of the anti-inflammatory target IL10.

[0122] Embodiment 8: Phagocytosis assay

[0123] Retinal pigment epithelial cells RPE1 (1×10 4 ) were seeded in a chamber slide (PEZGS0816, MilliporeSigma (trademark)) and cultured for about 16-18 hours. After cell adhesion, EP21 MV labeled with DIL obtained in Preparation Embodiment 2 (4×10 8) Treat RPE1 cells with it for 24 hours. Next, use a modular DMi8 inverted microscope (Leica / CCD, Andor ZYLA 4.2 Plus) for fluorescence excitation to observe the EP21 MV taken up by the cells. For MV tracking in vivo, use Sprague-Dawley (SD) rats as test animals. Inject the EP21 MV labeled with DIL (1×10 10 ) into the tail of the rat. After 24 hours, sacrifice the SD rat, collect the eyeballs, and fix the tissues with formalin. Next, dehydrate with 30% sucrose solution for 2 hours, embed with optimal cutting temperature compound (OCT, FSC 22 Clear, 3801480, Leica), and freeze and store at -80 °C. Use a tissue chopper with both the machine and the carrier at -20 °C to prepare tissue sections, and the thickness of each section is 12 μm. Stain the nuclei with DAPI (1:1000) and react for 5 minutes in the dark. After enclosing with a bleaching inhibitor, perform fluorescence excitation with a fluorescence microscope and take images.

[0124] As shown in Figure 17, the EP21 MV of the present invention is taken up by retinal pigment epithelial cells RPE1.

[0125] Embodiment 9: Test for the EP21 MV to Pass through the Blood-Retinal Barrier (BRB)

[0126] Inject the EP21 MV labeled with DIL (1×10 10 ) into the tail of the SD rat. After 24 hours, sacrifice the SD rat, collect the eyeballs, and fix the tissues with formalin. Next, dehydrate with 30% sucrose solution for 2 hours, embed with optimal cutting temperature compound (OCT, FSC 22 Clear, 3801480, Leica), and freeze and store at -80 °C. Use a tissue chopper with both the machine and the carrier at -20 °C to prepare tissue sections, and the thickness of each section is 12 μm. Stain the nuclei with DAPI (1:1000) and react for 5 minutes in the dark. After enclosing with a bleaching inhibitor, perform fluorescence excitation with a fluorescence microscope and take images. As shown in Figures 18A and 18B, the results show that the EP21 MV of the present invention can pass through the BRB through the bloodstream.

[0127] Example 10: Test of Other Probiotics for Suppressing Inflammation

[0128] Three-week-old Brown Norway rats are used as test animals. After pre-test measurement of the animals, TGF-β2 at 250 ng / ml is injected into the upper eyelid of the right eye once a week for 3 weeks (days 1, 8, and 15 respectively) to induce inflammation. In the negative control group, a balanced salt solution (BSS, used for the preparation of the eye wash buffer and injection drug) is injected together with 1X PBS via gavage for 3 weeks. In the positive control group, TGF-β2 and 1X PBS are injected for 3 weeks. The treatment group is injected with TGF-β2, and the logarithmic-phase probiotics (2.5 - 3×10 9 CFU / 0.1 ml / day) are administered via gavage for 3 weeks. In addition to EP21 of Preparation Embodiment 1, the other three groups of probiotics are Lactobacillus casei Shirota, Lactobacillus paracasei NTU 101, and Lactiplantibacillus plantarum subsp. plantarum PS128. The axial length and refraction of the eye are measured before and after treatment to confirm the changes between groups. After collecting the whole eyeball as a sample, Western blotting and immunohistochemistry (IHC) analysis are performed.

[0129] As a result shown in Fig. 19A, TGF-β2 significantly increases the axial length of the rat eye and changes the refraction. In the treatment group administered with EP21, the excessive increase in axial length and the excessive change in refraction are suppressed. Furthermore, during the progression of myopia, the genes of two tissue remodeling proteins, TGF-β and MMP2, are upregulated. MMP2 is an extracellular matrix enzyme that degrades type I collagen (collagen 1) on the sclera. As myopia progresses, the eyeball gradually becomes longer, and an image is formed in front of the retina when light enters the eye. This causes myopia. Fig. 19B shows the results of immunohistochemistry (IHC) analysis used to detect the amounts of TGF-β, MMP2, and collagen 1 in the retina. The intensity of the color after color development represents the amount of the target protein on the retina. The control shown in Fig. 19B means the group treated with TGF-β2. The results indicate that in the TGF-β2-induced group with myopia, the expressions of TGF-β2 and MMP2 increase, and the expression of collagen 1 decreases. When EP21 is administered, the expressions of TGF-β2 and MMP2 decrease, and the expression of collagen 1 increases. From the above changes, it can be seen that TGF-β2-induced myopia is improved by the administration of EP21. The results of Western blotting are shown in Figs. 19C and 19D. The administration of EP21 according to the present invention significantly inhibits the activation of the transcription factor NFκB. As can be seen from the results of the above tests, the strain EP21 of the present invention has an anti-inflammatory effect compared with other lactic acid bacteria (Lactobacillus) and can delay the progression of myopia.

Claims

1. Lactiplantibacillus plantarum subsp. plantarum strain EP21, deposited at the Biosource Collection and Research Center (BCRC) under accession number 911210, for use in improving eye diseases.

2. 2. The Lactiplantibacillus plantarum subsp. plantarum strain EP21 of claim 1, wherein the strain comprises a segment of the sequence set forth in SEQ ID NO:

3.

3. Use of the Lactiplantibacillus plantarum subsp. plantarum strain EP21 according to claim 1 or 2 in the preparation of a pharmaceutical composition for improving eye diseases.

4. The use according to claim 3, wherein the pharmaceutical composition further comprises pharma- ceutically acceptable excipients, carriers, adjuvants and food additives.

5. The use according to claim 3, wherein the dosage form of the pharmaceutical composition comprises a solution, a suspension, a capsule, a tablet, a lozenge, a troche, a powder, a lyophilized powder, a lotion, an emulsion, a suppository, a slurry, an ointment.

6. The use according to claim 3 , wherein the pharmaceutical composition is applied to a subject by oral administration.

7. The effective dose of the pharmaceutical composition is 1×10 ６ cfu / kg to 1 x 10 １０ 7. The use according to claim 6, wherein the cfu / kg is administered to a subject.

8. The effective dose of the pharmaceutical composition is 5×10 ８ cfu / kg to 5 x 10 ９ 8. The use according to claim 7, wherein the cfu / kg is administered to a subject.

9. The use according to claim 3, wherein the eye disease comprises myopia, dry eye syndrome (DES), eye fatigue, keratitis, macular degeneration, retinitis pigmentosa (RP), proliferative diabetic retinopathy (PDR), ischemic retinopathy, choroidal neovascularization, glaucoma, systemic lupus erythematosus (SLE), and Sjogren's syndrome (SS).

10. The use according to claim 9, wherein the ischemic retinopathy includes diabetic retinopathy (DR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO) and ischemic optic neuropathy (ION).

11. A composition comprising bacterial membrane vesicles for use in improving eye diseases, comprising bacterial membrane vesicles (MVs) derived from Lactiplantibacillus plantarum subsp. plantarum strain EP21 according to claim 1.

12. The composition of claim 11, wherein the vesicle diameter of the MVs is between 100 nm and 300 nm.

13. The composition of claim 12, wherein the vesicle diameter of the MVs is between 120 nm and 255 nm.

14. A composition comprising bacterial membrane vesicles for use in improving eye diseases, comprising bacterial membrane vesicles (MVs) derived from Lactiplantibacillus plantarum subsp. plantarum strain EP21 as described in claim 2.

15. The composition of claim 14, wherein the vesicle diameter of the MVs is between 100 nm and 300 nm.

16. The composition of claim 15, wherein the vesicle diameter of the MVs is between 120 nm and 255 nm.

17. Use of a composition comprising a bacterial membrane vesicle according to any one of claims 11 to 16 in the preparation of a pharmaceutical composition for improving an eye disease.

18. The use according to claim 17, wherein the pharmaceutical composition further comprises pharma- ceutically acceptable excipients, carriers, adjuvants and food additives.

19. The use according to claim 17, wherein the dosage form of the pharmaceutical composition comprises a solution, a suspension, a capsule, a tablet, a lozenge, a troche, a powder, a lyophilized powder, a lotion, an emulsion, a suppository, a slurry, an ointment.

20. The use according to claim 17, wherein the pharmaceutical composition is applied to a subject by injection or eye drops.

21. 21. The use of claim 20, wherein the injection comprises intravenous injection (IV), intraperitoneal injection (IP), and tail vein injection.

22. The effective dose of the pharmaceutical composition is 1×10 ５ Particles / kg to 1 x 10 １１ 21. The use according to claim 20, wherein the particles / kg are administered to a subject.

23. The effective dose of the pharmaceutical composition is 2.7×10 ８ particles / kg to 5.3 x 10 ９ 23. The use according to claim 22, wherein the particles / kg are administered to a subject.

24. The use according to claim 17, wherein the eye disease comprises myopia, dry eye syndrome (DES), eye strain, keratitis, macular degeneration, retinitis pigmentosa (RP), proliferative diabetic retinopathy (PDR), ischemic retinopathy, choroidal neovascularization, glaucoma, systemic lupus erythematosus (SLE), and Sjogren's syndrome (SS).

25. 25. The use according to claim 24, wherein the ischemic retinopathies include diabetic retinopathy (DR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO) and ischemic optic neuropathy (ION).

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