WO2023033500A1 - Composition for preventing or treating ocular surface inflammatory disorders, containing extracellular vesicles - Google Patents

Abstract

The present invention relates to a composition containing extracellular vesicles derived from mesenchymal stem cells, wherein the extracellular vesicles are exosomes isolated from bone marrow-derived mesenchymal stem cells using an aqueous two-phase system separation method and have excellent effects in ameliorating dry eye symptoms, corneal damage recovery, and inhibiting ocular surface inflammation, and thus can be preferably applied in uses for preventing and treating ocular surface inflammatory disorders or corneal damage.

Description

Composition for preventing or treating inflammatory diseases of the ocular surface containing extracellular vesicles

The present invention relates to a composition for preventing or treating inflammatory diseases of the ocular surface or corneal damage containing extracellular vesicles isolated from mesenchymal stem cells using a biphasic aqueous solution separation method.

Inflammatory diseases of the ocular surface are defined as instability of the tear film due to various causes, and are accompanied by inflammation of the ocular surface. The causes of this are aging, corneal inflammation, drug use, ophthalmic surgery history, contact lens wearing history and rheumatoid arthritis, Sjogren's syndrome (a disease in which inflammation or dryness occurs in the mucous membranes throughout the body, such as the mouth and eyes), lupus, scleroderma, There are systemic factors such as comorbid diseases such as diabetes and vitamin A deficiency, thyroid disease, and decrease in female hormones.

Commonly known symptoms of ocular surface inflammatory disease are expressed in various symptoms such as eye irritation, foreign body sensation like sand rolling, burning sensation in the eyes, eye discomfort that feels dark, itching, glare, and sudden excessive tears. make life uncomfortable. In particular, when inflammatory diseases of the ocular surface become severe, inflammation of the ocular surface (cornea and conjunctiva) and instability of the tear layer damage the ocular surface, resulting in permanent visual impairment due to pain, irregular corneal surface, blurred and fluctuating vision, and corneal ulcers. may occur

Existing treatment methods include artificial tear instillation, punctum occlusion, and non-specific anti-inflammatory drugs such as steroids and cyclosporine. Long-term use of steroid eye drops can cause complications such as glaucoma and cataracts, and cyclosporine eye drops have a therapeutic effect on steroids. The prevalence of patients with ocular surface inflammatory diseases is over 30% and is continuously increasing, but there is no effective and safe treatment that can surpass steroids yet.

US2019-0015452 A1 separates extracellular vesicles from cell-derived microparticle mesenchymal stem cells through centrifugation, and discloses that the separated extracellular vesicles are effective for ocular surface inflammatory diseases, and in WO2017/160884 A1 It is disclosed that eye diseases organically damaged by alkaline agents can be improved by ultracentrifuging extracellular vesicles derived from stem cells and using the separated extracellular vesicles. These patents only mention the possibility of treating inflammatory diseases of the ocular surface of extracellular vesicles, but do not specifically prove them.

On the other hand, the CN109431985A patent is a mixture of 2.5% extracellular vesicles derived from mesenchymal stem cells, 0.2% vitamin E, 0.2% carbomer, and 2% NaCl powder to prepare an eye drop, and this eye drop can be used for dry eye treatment. It is mentioned that it can.

According to the contents of this patent, the separation of extracellular vesicles from stem cells is carried out by first centrifugation at 300g for 10min, taking the upper layer, centrifugation for 2000g, 10min, taking the upper layer again, centrifugation for 10000g, 30min, and finally taking the upper layer. After 100000g, 70min ultracentrifugation is performed. This (ultra)centrifugation method is the most representative separation method, but it is difficult to separate extracellular vesicles with high purity and efficiency, and the gravitational acceleration actually received by cells reaches up to 100,000 g, which is highly likely to damage cells, so it is practically ocular. Confidence in the therapeutic effect of desiccation is low.

The inventors of the present invention studied to develop a therapeutic agent related to ocular surface inflammatory diseases using mesenchymal stem cell culture medium-derived extracellular vesicles, and as a result, in a method for obtaining stem cell-derived extracellular vesicles isolated from their culture, It was confirmed that by using the aqueous solution-based composition as a method, damage to extracellular vesicles can be minimized and separated with high purity, so that it can be effectively applied to the treatment and recovery of inflammatory diseases of the ocular surface or corneal damage.

An object of the present invention is to provide a composition for preventing or treating inflammatory diseases of the ocular surface comprising the extracellular vesicles as an active ingredient.

Another object of the present invention is to provide a composition for preventing or treating corneal damage comprising the extracellular vesicles as an active ingredient.

In order to achieve the above purpose,

The present invention provides a composition for preventing or treating inflammatory diseases of the ocular surface, comprising, as an active ingredient, extracellular vesicles having an average particle diameter of 50 nm to 1000 nm, derived from mesenchymal stem cells or separated from a culture medium thereof.

In addition, the present invention provides a composition for preventing or treating corneal damage comprising, as an active ingredient, extracellular vesicles derived from mesenchymal stem cells or separated from their culture medium and having an average particle diameter of 50 nm to 1000 nm.

In one embodiment, the extracellular vesicles are produced by culturing stem cells to produce extracellular vesicles; culturing the extracellular endoplasmic reticulum; and isolating extracellular vesicles using a two-phase separation method in an aqueous solution.

In one embodiment, the extracellular vesicles are exosomes, wherein the exosomes may have CD9, CD63, and CD81 membrane protein markers, and the ratio of CD63/CD9 among the membrane protein markers satisfies 2.5 or more. can

In one embodiment, the number of exosomes separated per 1 L of the extracellular vesicles is 1x10 ⁹ to 1X10 ¹² , and the amount of protein may be 0.1 mg to 20 mg.

In one embodiment, the mesenchymal stem cells may be human or animal tissue-derived induced pluripotent stem cells (iPSC), autologous and allogeneic mesenchymal stem cells, or cell lines derived from mesenchymal stem cells.

In one embodiment, the human or animal tissue may be isolated from any one or more tissues selected from bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood, and amniotic fluid.

In one embodiment, the ocular surface inflammatory disease may be dry eye.

In one embodiment, the ocular surface inflammatory disease is an autoimmune disease such as anorexia, Sjogren's syndrome, keratoconjunctivitis sicca, Stevens-Johnson syndrome, eye-like blister, ophthalmic surgery, allergic conjunctivitis, VDT ( (Visual Display Terminal) Workers' tear reduction, dry room due to air conditioning, toxicity due to long-term drug use, long-term use of contact lenses, systemic drugs and systemic diseases that reduce tear secretion, eye burns and chronic eye transplants It may be caused or accompanied by any one or more selected from the group consisting of host reaction.

In one embodiment, the composition may contain 0.0001 to 95% by weight of extracellular vesicles based on the total weight of the composition.

In one embodiment, the composition may be administered by an intraocular, intravitreal or intradermal route.

In one embodiment, the composition is any one selected from the group consisting of eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, drops, solutions, contact lens cleaners and contact lens lubricants. It may be a form.

The composition according to the present invention includes stem cell-derived exosomes and vesicles, which do not contain cellular waste products, and inflow and proliferation to other organs, problems in body distribution, and tumorigenicity that occur in conventional stem cell-based therapeutics. And it has the advantage that there is no problem of immunotoxicity, and the effect of improving dry eye symptoms, recovering corneal damage, and inhibiting ocular surface inflammation is excellent, so it can be preferably applied for prevention and treatment of ocular surface inflammatory diseases or corneal damage. do.

1 is a graph showing the particle size distribution of bone marrow-derived exosomes separated by an aqueous solution biphasic (ATPS) separation method of Example 1 of the present invention.

Figure 2 is a result of measuring the number and purity of bone marrow-derived exosomes extracted after separating extracellular vesicles from the culture medium by the aqueous phase system (ATPS) method of the present invention or the existing ultracentrifugation (Gradient) method.

3 is a result of measuring the ratio of markers expressed by bone marrow-derived extracellular vesicles (exosomes) isolated by the ATPS method of the present invention using a total reflection microscope.

Figure 4 is an image showing the degree of corneal damage through lissamine green staining on day 0 and day 7 when a control group was injected under the conjunctiva of a mouse.

5 is an image showing the degree of corneal damage through lissamine green staining on day 0 and day 7 of subconjunctival injection of bone marrow-derived exosomes.

6 is a graph comparing the NEI scoring of a control group injected under the conjunctiva of mice and a group administered with bone marrow-derived exosome.

7 is a graph comparing tear secretion in a control group injected subconjunctivally of mice and a bone marrow-derived exosome-administered group through a phenol red thread.

8 is an image showing the lacrimal gland staining results of a control group injected under the conjunctiva of a mouse and a bone marrow-derived exosome-administered group.

Figure 9 shows the expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP9 in the cornea of a control group and a bone marrow-derived exosome-administered group injected under the conjunctiva of mice as fold values. it's a graph

10 shows the expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP9 in the lacrimal glands of a control group and a bone marrow-derived exosome-administered group injected under the conjunctiva of mice as fold values. it's a graph

11 is an image showing the degree of corneal damage through lissamine green staining on day 0 and day 14 of the control group treated with eye drop administration of mice.

12 is an image showing the degree of corneal damage through lissamine green staining on day 0 and day 14 after treatment with bone marrow-derived exosomes by eye drop administration to mice for 14 days.

13 is a graph comparing the NEI scoring of a control group treated with mouse eye drop administration and a bone marrow-derived exosome-administered group.

14 is a graph comparing the tear secretion of a control group treated with mouse eye drop administration and a bone marrow-derived exosome-administered group through a phenol red thread.

15 is an image showing the lacrimal gland staining results of a control group treated with mouse eye drop administration and a bone marrow-derived exosome-administered group.

16 shows fold expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP-9 in the corneas of control groups and bone marrow-derived exosome-administered groups treated by eye drop administration of mice. It is a graph of values.

17 shows the fold expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP-9 in the lacrimal glands of the control group and bone marrow-derived exosome-administered groups treated by eye drop administration of mice. It is a graph of values.

18 is an image showing the degree of corneal damage through lissamine green staining on day 0 and day 14 of the control group treated with eye drop administration of mice.

19 is an image showing the degree of corneal damage through lissamine green staining on day 0 and day 14 after treatment with umbilical cord blood-derived exosomes by eye drop administration to mice for 14 days.

20 is a graph comparing the NEI scoring of a control group treated with mouse eye drop administration and a group administered with umbilical cord blood-derived exosome.

21 is a graph comparing the tear secretion of a control group treated with mouse eye drop administration and a group administered with umbilical cord blood-derived exosome through a phenol red thread.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

Throughout this specification, '%' used to indicate the concentration of a particular substance is solid/solid (w/w) %, solid/liquid (w/v) %, and Liquid/liquid is (v/v) %.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

In one aspect, the present invention relates to a composition for preventing or treating inflammatory diseases of the ocular surface, comprising as an active ingredient extracellular vesicles derived from mesenchymal stem cells or separated from a culture thereof and having an average particle diameter of 50 nm to 1000 nm.

In another aspect, the present invention relates to a composition for preventing or treating corneal damage comprising, as an active ingredient, extracellular vesicles derived from mesenchymal stem cells or separated from a culture medium thereof and having an average particle diameter of 50 nm to 1000 nm.

As used herein, the term 'culture medium' refers to a culture medium obtained by culturing stem cells in a culture medium, or a dried, filtered and/or concentrated product of the culture medium. The culture may or may not contain stem cells.

In the present specification, the term 'extracellular vesicles (EVs)' is a membrane structure composed of a lipid-bilayer secreted by cells or present in cells extracellularly, and is present in almost all eukaryotic body fluids. exist. The extracellular vesicles are classified into exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, and nanovesicles based on their origin, secretion mechanism, and size. It is used interchangeably with terms such as nanovesicles and outer membrane vesicles, and is a concept encompassing them. Unless specifically stated herein, the extracellular vesicles may be exosomes. The extracellular vesicles have a diameter of 50 nm to 1000 nm, and are released from the cell when the multivesicular bodies fuse with the cell membrane or are released directly from the cell membrane. It is well known that the extracellular vesicle serves to transport intracellular biomolecules such as protein, bioactive lipid and RNA in order to perform functional roles of mediating coagulation, cell-cell communication and cellular immunity. CD9, CD63, CD81, etc. are known as marker proteins of the extracellular endoplasmic reticulum, and other cell surface receptors such as EGFR, molecules related to signal transduction, cell adhesion related proteins, mesenchymal stem cell (MSC) related antigens, Proteins such as heat shock protein and Alix related to vesicle formation are known.

In the present invention, 'exosome' refers to biological nanoparticles present in stem cells or secreted into their culture.

As used herein, the term 'comprising as an active ingredient' includes an amount sufficient to achieve the improvement, prevention or treatment activity of ocular surface inflammatory diseases or corneal damage of stem cells or their culture medium or extracellular vesicles isolated therefrom. means that

As used herein, the term 'ocular surface inflammatory disease' refers to an abnormal condition or symptom occurring in the eye, particularly, the ocular surface.

In the present specification, 'corneal damage' is not limited thereto, but is, for example, damage to the cornea due to pathogens, inflammation, physical stimulation (eg, contact lenses, ultraviolet rays), chemical stimulation (eg, drugs), nerve damage, or fatigue accumulation. This means that it is applied, and may be accompanied by symptoms such as pain, congestion, corneal opacity, glare, and foreign body sensation.

As used herein, the term 'prevention' refers to any action that inhibits or delays the progression of inflammatory diseases on the ocular surface or corneal damage by administration of the composition of the present invention.

As used herein, the term 'treatment' includes (a) inhibition of the development of ocular surface inflammatory diseases or corneal damage; (b) reduction of ocular surface inflammatory disease or corneal damage; and (c) elimination of ocular surface inflammatory disease or corneal damage.

In one embodiment, the extracellular vesicles of the present invention can be produced by culturing mesenchymal stem cells in a medium.

In one embodiment, the extracellular vesicles are produced by culturing mesenchymal stem cells to produce extracellular vesicles; culturing the extracellular endoplasmic reticulum; and isolating extracellular vesicles using a two-phase separation method in an aqueous solution.

As used herein, the term "medium" refers to the growth and proliferation of cells, such as sugars, amino acids, various nutrients, serum, growth factors, minerals, etc. A mixture for growth and multiplication. In particular, the medium of the present invention is a medium for culturing mesenchymal stem cells. The above "mesenchymal stem cells" are cells isolated from stem cells derived from mammals, including humans, and have the ability to proliferate indefinitely and various cell types (eg, fat cells, chondrocytes, muscle cells, cells that can differentiate into bone cells, etc.). The above "cultivation" is meant to include the growth and proliferation of mesenchymal stem cells.

The "basic medium" of the present invention is a mixture containing essential sugars, amino acids, water, etc. required for cell survival, excluding serum, nutrients, and various mesenchymal stem cell growth factors. The basal medium of the present invention may be artificially synthesized and used, or a commercially produced medium may be used. Commercially prepared media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Mesenchymal Stem Cell Growth Medium (MSCGM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F- 12, α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium, but are not limited thereto.

Serum is a supernatant obtained by centrifugation from animal or human blood. The serum contains trace elements including various factors, such as various inorganic salts, polypeptide growth factors, and polypeptide hormones, which are essential for growth but have not been clearly identified, in addition to essential nutrients necessary for cell growth.

The "serum" of the present invention is fetal bovine serum, but commercial human serum, commercial human fetal serum, and autologous serum can be used.

The concentration of serum used in the medium for culturing the cells of the present invention is within 30% of the total medium composition, preferably within 25%, and more preferably within 20%.

The culture medium of the present invention may further include a nutrient mixture. The nutritional mixture is a mixture containing various amino acids, vitamins, inorganic salts, etc. commonly used in cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, etc., or a commercially produced nutritional mixture. Commercially prepared nutrient mixtures include, for example, F-12, M199, MCDB110, MCDB202, MCDB302, etc., preferably F-12 Nutrient Mixture, M199, MCDB culture medium, etc. can The nutrient mixture may be diluted in a basal medium at a ratio of 1:1 to 10:1, preferably diluted at a ratio of 1:1 to 5:1. In addition, Transferrin, Selenium, Glutamine, etc. can be mixed and used.

As a specific embodiment, the cell culture medium of the present invention may contain mesenchymal stem cell growth factors that affect the growth of mesenchymal stem cells in addition to or instead of serum. Growth factors of mesenchymal stem cells include, for example, insulin, hydrocortisone, EGF (Epidermal Growth Factor), LIF (Leukemia Inhibitory Factor), GM-CSF (Granulocyte-macrophage colony stimulating factor), EPO (Erythropoietin), FGF (Fibroblast Growth Factor), IGF (Insulin-like growth factor), PDGF (Platelet-derived growth factor), SCF (Stem cell factor), TGF (Transforming growth factor), and the like. A general culture medium for cells and a separate culture medium for separation of extracellular vesicles in the last step are distinguished. The culture medium for separating extracellular vesicles contains serum from which extracellular vesicles are removed, or preferably does not contain serum. However, it may contain nutrient mixtures and growth factors.

As a specific aspect, the production of extracellular vesicles from the mesenchymal stem cell-derived or the culture thereof of the present invention can be made by a method comprising the following steps.

First, mesenchymal stem cells are cultured in a cell culture medium. As an example, mesenchymal stem cells are cultured in a cell culture dish/flask containing the general culture medium of the present invention at a concentration of 1 to 10,000 cells/cm ² , preferably 50 to 6000 cells/cm ² . do. It is generally carried out under a temperature of 30°C to 38°C, 2% to 7% CO ₂ environment. Generally, passage number 8, preferably passage number 6, is not exceeded, but the date of passage and maintenance of passage of stem cells and stem cell lines is not particularly limited as long as it is suitable for each cell. The cell culture is such that the passage interval does not exceed 150 hours in any passage.

Next, the normal culture medium is replaced with the culture medium for isolating extracellular vesicles. The replacement time is not limited, but preferably does not exceed passage number 6, and the culture solution for isolating extracellular vesicles is collected within 1 to 4 days after the replacement of the culture solution.

As the culture medium for the isolation of extracellular vesicles, fetal serum from which vesicles have been removed is used, or a serum-free medium from which fetal serum is removed step by step is used. Mixtures of influences, growth factors may be included.

The serum-free medium is a medium that does not contain animal serum as an additive, and is not particularly limited. Compositions containing other additives other than animal serum in a known basal medium may be accepted and used. The composition of the basal medium can be appropriately selected according to the type of cells to be cultured.

In the embodiment of the present invention, at the time of the last passage, it was replaced with an extracellular vesicle isolation culture medium, bone marrow mesenchymal stem cell culture, passage number 6 or higher was not used, but it is possible to culture with other combinations such as specific stem cells or stem cell lines, Therefore, in the present invention, there is no limitation on a specific passage number. Also, the final incubation does not exceed 100 hours. The collected culture medium for isolation of extracellular vesicles is centrifuged and the supernatant is frozen for isolation of extracellular vesicles. Centrifugation is a step for removing dead cells and cell debris. In the present invention, 500 × g, 10 minutes, 2,000 × g, 10 minutes, or 10,000 × g, 10 minutes were used, but the combination of centrifugation and time Appropriate application can be used.

The separation process may be an aqueous two-phase separation method, an ultracentrifugation method, an antibody affinity separation method, a polymer precipitation method, a filtration method, or a tangential flow filtration method. method may be used. The aqueous two-phase separation method is a separation method using a phenomenon in which different particles are separated differently by two different phases, and is easy to remove impurities and macromolecules outside the extracellular vesicles.

Specifically, the aqueous two-phase phase separation composition according to the present invention comprises a first aqueous liquid phase forming a dispersed phase; And a second aqueous solution phase separated from the dispersed phase and forming a continuous phase, wherein the tension (γ) at the interface between the first aqueous phase and the second aqueous liquid phase is phase-separated is to use a composition satisfying Equation 1 below. .

[Equation 1]

2×10 ^-7 J/m ² ≤ γ ≤ 50×10 ^-5 J/m ²

The first aqueous phase containing extracellular vesicles has a bulk form and exists in a state of flux in the second aqueous phase due to gravitational force or buoyancy. Extracellular vesicles are trapped at the interface where the first aqueous phase and the second aqueous phase come into contact, and only proteins with small particle sizes escape the interface and migrate to the second aqueous phase, thereby residing in the first aqueous phase. The outer vesicles can be recovered with high purity. This separation method can control the critical particle size by adjusting the interfacial tension, so that the separation ability can separate impurities and extracellular vesicles with a size difference of about 10 nm. For example, the first aqueous phase contains dextran, and the second aqueous phase contains polyethylene glycol.

According to one embodiment of the present invention, the step of separating extracellular vesicles using the aqueous phase separation method is to separate 1 to 5 of the first aqueous phase containing dextran relative to 100 parts by volume of the supernatant of the culture medium for separation of extracellular vesicles. recovering extracellular vesicles from the phase-separated first aqueous phase by mixing and dissolving 3 to 10 parts by volume of the second aqueous phase containing polyethylene glycol; And 40 to 60 parts by volume of the second aqueous phase containing polyethylene glycol with respect to 100 parts by volume of the supernatant for the isolation of the extracellular vesicles in the first aqueous phase remaining after recovering the extracellular vesicles, and then mixing and dissolving , recovering extracellular vesicles from the phase-separated first aqueous phase by centrifugation; and the above process may be repeated twice or more.

The separation process using the aqueous solution phase system is performed to sufficiently recover the extracellular vesicles, preferably within 2 hours, preferably within 5 minutes to 1 hour, more preferably within 5 minutes to 30 minutes, most preferably within 15 minutes do. This time has the advantage of significantly reducing the time compared to the conventional ultracentrifugation process that takes at least 2 hours or more.

In the case of the existing method using ultracentrifugation, the size of extracellular vesicles is as small as hundreds of nm and the difference in density from protein is not large, so there is a disadvantage in that the separation efficiency of extracellular vesicles is low with high purity and efficiency from plasma. When using , there is a problem that the gravitational acceleration actually received by the cells reaches 100,000 × g, and there is a high possibility of damaging the endoplasmic reticulum. This is because the separation process using the aqueous two-phase system can solve the problems of these existing separation methods.

The separated extracellular vesicles can be analyzed for exosome size, number, distribution of membrane protein markers, etc. through a nanoparticle tracking analysis device and total internal fluorescence (TIRF) analysis.

At this time, the quality of extracellular vesicles can be confirmed by the number of extracellular vesicles separated per 1 L of culture medium, the amount of protein in extracellular vesicles, and the number of extracellular vesicles per unit protein.

According to one embodiment of the present invention, the number of exosomes separated per 1 L of the culture medium is 1×10 ⁹ to 1×10 ¹² , and the amount of protein is 0.1 mg to 20 mg. can be Depending on the stem cell type, culture passage number, and type of culture medium, the number and protein amount of separated extracellular vesicles may vary, and the finally separated extracellular vesicles have the same or similar final size distribution, purity, and the same or similar physiological activity. If it has, the present invention is not limited to a specific separation method.

Preferably, the extracellular vesicles may have an average particle diameter of 50 nm to 1000 nm, and may have a membrane protein marker (ie, an extracellular vesicle-specific marker, more specifically, an exosome-specific marker).

At this time, the extracellular vesicles present in the composition are 90% by weight or more, preferably 95% by weight or more, of the total amount of extracellular vesicles having a particle size distribution of 10 nm or more and 300 nm or less. If the particle size is less than 10 nm or more than 10% by weight of extracellular vesicles having a diameter of 300 nm or more, it is not preferable because there is a problem of mixing with high molecular proteins or apoptotic bodies (dead bodies).

The suitable/proper cell culture method used in this example and the separation by the two-phase aqueous solution minimize the impurities of the polymer protein and the impurities of the apoptotic body. In addition, in order to minimize and remove impurities from apoptotic bodies, in an embodiment of the present invention, filtering is performed to a desired size (250 μm or 450 μm), and nanoparticles having a desired size range are produced.

In one embodiment, the extracellular vesicles are exosomes, and in this case, the exosomes may have CD9, CD63, or CD81 membrane protein markers.

The term "membrane protein marker" of the present invention refers to a protein abundantly present in the membrane of extracellular endoplasmic reticulum. The extracellular vesicles, in particular, the membrane protein markers of the exosomes may be CD9, CD63, CD81, etc., and the isolated exosomes have 25% or less of the CD9 membrane protein markers compared to the number of CD9 or CD63-expressing exosomes, preferably 20% or less, most preferably 15% or less. In addition, the isolated exosomes express 10% or more, preferably 30% or more, more preferably 50% or more, and most preferably 70% or more of the CD63 membrane protein marker relative to the number of CD9 or CD63-expressing exosomes. .

In one embodiment, the ratio of CD63/CD9 among the membrane protein markers may satisfy 2.5 or more.

According to one embodiment of the present invention, CD9 is in the range of 2-25%, CD63 is in the range of 13-70%, and CD81 is in the range of 10-60% relative to the total sum of CD9, CD63 and CD81. In particular, there is a difference in efficacy depending on the ratio of CD63 and CD9, and when the ratio of CD63/CD9 is 2.5 or more, preferably 3.5 or more, more preferably 5.0 or more, the effect of inflammatory diseases of the ocular surface can be further enhanced. . A CD63/CD9 ratio of 5.0 to 6.0 may be most preferred.

In addition, when the number of extracellular vesicles present in the composition is within a specific number range, the effect of inflammatory diseases of the ocular surface can be further enhanced. Quantification of protein is quantitatively analyzed by methods such as Bradford (Bradford) or Bicinchoninic acid (BCA). Referring to Example 2 of the present invention, the extracellular vesicles have a purity of 5×10 ⁷ or more and 5×10 ⁸ or less per 1 μg of Bradford quantitative standard protein. It became.

In one embodiment, the mesenchymal stem cells may be human or animal tissue-derived induced pluripotent stem cells (iPSC), autologous and allogeneic mesenchymal stem cells, or cell lines derived from mesenchymal stem cells.

In one embodiment, the human or animal tissue may be isolated from a tissue selected from any one or more of bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood, and amniotic fluid.

In the present specification, the term 'mesenchymal stem cells' refers to stem cells having multipotent ability to differentiate into cells of fat, cartilage, bone, muscle, skin, nerve, and the like. The mesenchymal stem cells may be differentiated from induced pluripotent stem cells or isolated from bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood, synovium, amniotic fluid, and the like.

In one embodiment, preferably, the mesenchymal stem cells of the present invention may be bone marrow or cord blood-derived mesenchymal stem cells.

'Induced pluripotent stem cell (iPSC)' is a cell that returns to its initial undifferentiated state by inducing dedifferentiation in already differentiated cells such as somatic cells and has pluripotency. it means. The dedifferentiation may be induced by introducing and expressing a specific gene (eg, Sox2, c-Myc, Klf4, Oct-4, etc.) or by injecting a dedifferentiation inducing protein made in a cell into which the specific gene has been introduced. .

The pluripotency means the ability to differentiate into tissues or organs derived from the three germ layers constituting the living body, that is, endoderm, mesoderm, and ectoderm.

The induced pluripotent stem cells of the present invention include induced pluripotent stem cells derived from all mammals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, but preferably human-derived pluripotent stem cells. is a cell

In one embodiment, the ocular surface inflammatory disease of the present invention may be a disease occurring in the eye, preferably dry eye.

As used herein, the term 'dry eye syndrome (dry eye syndrome or dry eye syndrome)' is one of the inflammatory diseases of the ocular surface, and the tear layer is damaged due to instability of the tear film, hyperosmotic pressure of tears, damage and inflammation of the ocular surface, and sensory nerve abnormalities. It means that homeostasis is lost, accompanied by symptoms such as dryness, damage or inflammation of the ocular surface.

In one embodiment, the ocular surface inflammatory disease is an autoimmune disease such as anorexia, Sjogren's syndrome, keratoconjunctivitis sicca, Stevens-Johnson syndrome, eye-like blister, ophthalmic surgery, allergic conjunctivitis, VDT (Visual Display Terminal)) reduction of tears in workers, dry rooms due to air conditioning, toxicity due to long-term drug use, long-term use of contact lenses, systemic drugs and systemic diseases that reduce tear secretion, eye burns, and chronic eye graft-versus-host reactions It may be an ocular surface inflammatory disease-like condition caused or accompanied by any one or more selected from the group. The ocular surface inflammatory disease-like state may include a tear reduction symptom without any systemic symptom, and may mean a dry eye symptom. Preferably, it may be dry eye caused by or accompanied by Sjogren's syndrome.

The mesenchymal stem cells or extracellular vesicles isolated from the mesenchymal stem cells or their culture solution presented in the present invention are used for the prevention or treatment of inflammatory diseases of the ocular surface or corneal damage, wherein the inflammatory diseases of the ocular surface are used to inhibit damage and disease or corneal damage associated with the disease. or used for relief and recovery.

Specifically, extracellular endoplasmic reticulum may exert a disease control effect by improving ocular surface inflammatory diseases or corneal damage and regulating immune responses. According to one embodiment of the present invention, an effect of improving corneal epithelial defect and tear amount was shown after administration of extracellular vesicles in a dry eye animal model in which inflammatory ocular surface inflammatory disease was induced, and extracellular vesicles were observed in lacrimal tissue of a dry eye animal model. It was confirmed that administration can reduce inflammatory foci. In addition, as a result of confirming the expression of inflammatory cytokines (IL-6, IL-1β, TNFα, IFN-γ, IL-17A, MMP9) in lacrimal and corneal tissues, it was observed that extracellular endoplasmic reticulum inhibited inflammatory gene expression. (See Examples 4 and 5).

The extracellular vesicles of the present invention have a function as a nano-drug delivery system, in particular, retention within the vitreous of the eye, distribution within the retina, and functions as a drug nano-delivery system, and the efficacy of the drug is expected to be maintained for a long period of time , It is possible to reduce the single dose concentration and frequency of administration of the loaded drug, reducing complications that may occur during administration, and reducing the patient's visit to the hospital and medical expenses.

As used herein, the term "administration" means providing a substance to a subject by any suitable method. The administration route of the composition of the present invention can be administered orally or parenterally through all general routes as long as it can reach the target tissue. In addition, the composition of the present invention may be administered using any device capable of delivering active ingredients to target cells or organs.

The term "subject" as used herein is not particularly limited, but includes, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or It includes guinea pigs, preferably mammals and more preferably humans.

The composition containing extracellular vesicles according to the present invention contains 0.0001 to 95% by weight of extracellular vesicles within the total weight of the composition (total 100% by weight) and is formulated into a pharmaceutical preparation and administered or combined with other drugs. It can be administered simultaneously, or can be administered at an appropriate time before or after administration of other drugs.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous systemic administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transdermal administration, ocular administration, or ocular topical administration can be administered. there is. Ocular topical administration can be administered e.g. directly into the eye by eye drop, intraocular, periocular, retrobulbar, subretinal, central retinal, fovea external, subconjunctival, This includes intravitreous, intracameral, or suprachoroidal administration. Compositions of the present invention may also be administered through an intraocular implant device. Preferably, the composition of the present invention may be administered by an intraocular, intravitreal or intradermal route.

The preventive or therapeutic composition of the present invention can be made into an appropriate preparation or dosage form by a conventional method. The dosage form of the preparation may be a solid preparation such as a powder or granule, but from the viewpoint of obtaining an excellent prophylactic treatment effect, eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, drops, solutions, and contact It is preferably any one formulation selected from the group consisting of lens cleaners and contact lens lubricants. In particular, when preparing eye injections and eye drops, solutions are preferred. As the liquid preparation method, for example, a method of mixing previously prepared stem cell-derived microparticles or a stem cell culture solution (eg, culture supernatant) with a solvent, or a method of further mixing a suspending agent or emulsifying agent can be suitably exemplified. there is.

As described above, in the formulation of the pharmaceutical composition of the present invention, appropriate pharmaceutically acceptable carriers, for example, excipients, binders, solvents, dissolution aids, suspending agents, emulsifiers, tonicity agents, buffers, and stabilizers according to the needs of the formulation , pain relievers, preservatives, antioxidants, coloring agents, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, diluents and the like may be blended with arbitrary components. In addition, when the composition of the present invention contains cells, components acceptable for cell preparations may be incorporated.

The dosage of the preventive or therapeutic composition of the present invention may vary depending on the type of disease, the degree of symptoms thereof, the dosage form, the weight of the subject to be administered, etc. The range of pg/kg to 100 mg/kg can be exemplified suitably, and the range of 100 pg/kg to 10 mg/kg can be exemplified more suitably.

In addition, administration of the composition for prevention or treatment of the present invention may be divided into one to several times a day in the case of eye drops. In addition, single administration and multiple administration are possible, and in the case of multiple administration, for example, it can be administered twice or more continuously with a frequency of one or more times every 7 days, and in particular, two or more administrations with a frequency of one time or more every 3 days. Preferably, it is preferably administered 3 times or more at a frequency of 1 time or more per 2 days, and it is more preferable to continuously administer 2 or more times at a frequency of 1 time or more per day.

If the formulation of the composition for prevention or treatment of the present invention is intraocular administration and eye drops, it can be prepared using a technique commonly used for intraocular administration and eye drops, using pharmaceutically acceptable additives as necessary. .

tonicity agents such as sodium chloride and glycerin; pH adjusters such as hydrochloric acid and sodium hydroxide; buffering agents such as sodium phosphate and sodium acetate; surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, and polyoxyethylene hydrogenated castor oil; Stabilizers, such as sodium citrate and sodium edetate; Preservatives such as benzalkonium chloride and parabens can be prepared by selecting and using them as needed. When administered into the vitreous body of the eye, the pH of the sterile preparation is usually 7.3, and the pH of the eye drops should be within the acceptable range for ophthalmic preparations, but the range of pH 4 to 8 is generally preferred.

Compositions of the present invention may be dissolved in any of a variety of buffers. Suitable buffers include, for example, phosphate buffered saline (PBS), normal saline, Tris buffer, and sodium phosphate (eg 150 mM sodium phosphate). The insoluble polynucleotide may be dissolved in a weak acid or base and then diluted to a desired volume using a buffer. The pH of the buffer solution can be appropriately adjusted. In addition, appropriate osmotic properties may be provided using pharmaceutically acceptable additives. Such additives are within the purview of those skilled in the art. When the composition is used in vivo, sterilized, pyrogen-free water may be used. Such preparations may contain an effective amount of the polynucleotide in combination with a suitable amount of an aqueous solution to prepare a composition suitable for administration to humans. In particular, as a buffer solution for the vitreous body of the eye and the eye drop, a commercially available balanced salt solution (BSS) for the eye, which is manufactured similarly thereto, may be used. The pH is an isotonic solution of 6.8-7.4, the osmotic pressure is about 300 mOsm/kg, and the composition is a solution containing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide, and the like.

The animal to which the prophylactic or therapeutic composition of the present invention is administered is not particularly limited, but is preferably human, monkey, mouse, rat, hamster, marmot, cow, horse, rabbit, sheep, goat, cat, dog, etc. Humans are more preferable. In addition, when the composition for prevention or treatment of the present invention contains cells and/or their culture, the formulation of the composition for prevention or treatment of the present invention consistent with the type of animal to be administered is more stable against the disease. It is preferable from the viewpoint of obtaining excellent prophylactic and/or therapeutic effects.

On the other hand, the composition for preventing or treating inflammatory diseases of the ocular surface comprising the extracellular vesicles of the present invention can also be applied as a functional food composition or animal feed containing this and a functional food-acceptable carrier.

As used herein, the term 'functional food composition' includes food products, foodstuffs, dietary supplements, nutritional supplements, or supplemental compositions for food products or foodstuffs.

The animal feed comprising the pet food composition is not only a treat (eg dog biscuits) or other food supplement, but advantageously a food supplying a necessary dietary requirement, such as a supplement such as gravies, drinks, yogurt , powder, suspension, chews, treats (eg biscuits) or any other delivery form.

Dietary supplements of the present invention may be delivered in any suitable format. In a preferred embodiment, it may be a liquid, gel, gelcap, capsule, powder, solid tablet (coated or uncoated), tea, and the like.

In one aspect, the present invention relates to a method for preventing or treating inflammatory diseases of the ocular surface comprising administering the composition for preventing or treating inflammatory diseases of the ocular surface of the present invention to a non-human subject.

In another aspect, the present invention relates to a method for preventing or treating corneal damage comprising administering the pharmaceutical composition for preventing or treating corneal damage of the present invention to a non-human subject.

The treatment method of the present invention comprises administering to a subject in a therapeutically effective amount a composition for preventing or treating inflammatory diseases of the ocular surface or corneal damage, respectively. A specific therapeutically effective amount for a specific individual depends on the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the age, weight, general health condition, sex and diet of the individual, the time of administration, It is preferable to apply differently according to various factors including the route of administration and secretion rate of the composition, treatment period, drugs used together with or concurrently used with the specific composition, and similar factors well known in the medical field. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above.

The subject (subject) is applicable to any mammal, and the mammal is not only humans and primates, but also monkeys, mice, rats, hamsters, marmots, cows, pigs, horses, rabbits, sheep, goats, cats, and dogs. such as livestock or pets.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1: Isolation of extracellular vesicles from bone marrow-derived stem cells

1-1. stem cell culture

After diluting 11 ml of bone marrow donated from a normal healthy person or from the patient himself with DMEM medium at a ratio of 1:2, it was placed on a previously prepared 10 ml Histopaque (Sigma, density 1.077 g/ml) layer. and centrifuged at 400 × g for 30 minutes at room temperature. The mononuclear cell layer was separated, DMEM medium was added, centrifuged at 400×g for 5 minutes at room temperature, and the mononuclear cells obtained were washed twice with DMEM medium, maintained at 37° C. and 5% C0 ₂ in MSCGM™ medium, cultured. After 48 hours of culture, cells that did not adhere to the bottom of the flask were removed by exchanging with a fresh medium, and cells attached to the bottom of the flask were cultured while changing the medium once every 3 to 4 days. When the cultured cells have grown to about 80%, they are subcultured into a new flask, and some of the cultured cells are continued to be subcultured, and some of them are cryopreservative containing MSCGM™, 10% fetal bovine serum (FBS), and 5% DMSO. Or it was turbid in a Stem Cell Banker and stored in -176℃ liquid nitrogen.

Specifically, mesenchymal stem cells (Catholic Seoul St. Mary's Hospital Cell Therapy Project Group) were purchased (sold), stored in a nitrogen tank at the head office, and used within one year after purchase. Cells of passage number 2 were purchased and cultured in DMEM culture medium containing 20% FBS (fetal bovine serum) and penicillin/streptomycin/amphotegicin B. After culturing using a general culture medium up to passage number 4, replacing the culture medium with a separate culture medium before extracting exosomes and vesicles, and culturing for 48 hours, the cell culture supernatant was collected.

The collected cell culture supernatant was collected after 10 minutes at 500 × g, centrifuged again at 2,000 × g for 10 minutes, and the supernatant was aliquoted and stored frozen. Specifically, the cryopreserved cell culture supernatant is a culture medium from which cells and cell debris are removed, and contains 1×10 to 1×10 ³ cell-secreted exosomes per ml of culture medium, and the protein concentration per ml of culture medium is 100 μg. to 5000 μg were used.

1-2. Separation of extracellular vesicles using an aqueous biphasic phase separation composition

Polyethylene glycol and dextran were dissolved in phosphate buffered saline (PBS) at concentrations of 10.5 wt% and 45 wt%, respectively, to prepare an aqueous biphasic system.

2 L of the culture medium obtained in Example 1-1 (cell culture supernatant containing exosomes from which cells and cell debris were removed) was mixed with 20 ml of dextran aqueous solution and 50 ml of polyethylene aqueous solution, dissolved for 1 hour, and For separation, centrifugation was performed at 3,000 g for 10 minutes. The final phase-separated aqueous solution system had an interfacial tension of 5.36×10 ⁻⁶ J/m ² between the dextran aqueous solution and the polyethylene glycol aqueous solution. After extraction (separation and recovery) of extracellular vesicles from the dextran aqueous solution layer of the phase-separated sample with a pipette, the dextran aqueous solution layer was mixed again with 1 L of polyethylene aqueous solution. For phase separation of the mixed solution, centrifugation was performed at 3000 g for 10 minutes. In the phase-separated aqueous two-phase system, the interfacial tension between the aqueous dextran solution and the aqueous polyethylene glycol solution was set to 5.36×10 ⁻⁶ J/m ² . Extracellular vesicles were extracted from the phase-separated dextran layer with a pipette. In order to increase the purity of the number of extracellular vesicles per protein, that is, to completely remove residual proteins present in the culture medium, extracellular vesicles were isolated and obtained (recovered) by repeating this process two or more times.

Subsequently, the characteristics were analyzed by measuring the number and protein concentration of the recovered extracellular vesicles.

Example 2: Confirmation of isolated extracellular endoplasmic reticulum characteristics

In order to analyze the characteristics of the bone marrow-derived extracellular vesicles isolated in Example 1, the results of measuring the particle size (diameter) are shown in FIG. 1 . 1 is a graph showing the particle size distribution of extracellular vesicles isolated in Example 1. At this time, the extracellular vesicles were exosomes, and the particle diameter distribution of the isolated exosomes was 1000 nm or less, but it was confirmed that they were densely distributed over 96% by weight in the range of 50 nm to 300 nm.

In order to analyze the characteristics of isolated exosomes (the number of exosomes extracted from 1 L media, the content of exosomes in protein per 1 L, and the number of exosomes per μg of protein), density gradient μLtracentrifugation (sec. As a result of applying separation using a centrifugal separation) and an aqueous phase system (ATPS), the number and purity of exosomes were analyzed and shown in FIG. 2 . At this time, the number of exosomes was measured using nano tracking analysis, and the purity was measured by the number of exosomes per unit protein.

In addition, as a result of measuring the distribution and content of membrane protein markers in the isolated exosomes, CD9, CD63, and CD81 were mainly present, but 14.3%, 75.79%, and 2.5%, respectively, and among the membrane protein markers, CD63 and CD9 The ratio of was 5.3 (FIG. 3).

Example 3: Preparation of mouse dry eye model and administration of extracellular vesicles

3-1. Establishment of mouse dry eye model

The NOD/LtJ dry eye mouse model was used, and 6-week-old female and male mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA), raised up to 18 weeks, and then used for experiments. All mice were reared on sterile feed and ad libitum in a pathogen-free facility in the animal breeding room of The Catholic University of Korea (Seoul, Korea). In order to confirm the time point at which the dry eye model was formed, the cornea was stained with lissamine green, a reagent for staining the corneal conjunctiva and tear amount, from 13 weeks to confirm the degree of corneal damage. The time point when the tear amount decreased and the corneal staining increased was the time when dry eye was induced in the mouse, and 18-week-old mice were used in the experiment.

3-2. Administration of Extracellular Vesicles to the Mouse Dry Eye Model

At the time when the dry eye model was confirmed, the bone marrow-derived extracellular vesicles of Example 1 were treated with two methods: subconjunctival injection (injection solution administration) or eye drop administration (eye drop administration).

The control group (10 μL/per eye) administered with extracellular vesicles by subconjunctival injection, and the extracellular vesicles treatment group (10 μL/per eye) were administered once and sacrificed on the 7th day. That is, mice were treated by injection under the conjunctiva of control (control group) or extracellular vesicles (exosome) at 10 μL in each eye, 20 μL per mouse once a day, and sacrificed on the 7th day.

Control (5μL/per eye) was administered to the control group administered with extracellular vesicles by eye drop, and extracellular vesicles (5μL/per eye) were administered to the extracellular vesicles treatment group and sacrificed on the 14th day. That is, control (control group) or extracellular vesicles (exosome) were treated with 5 μL in each eye and 10 μL in each eye for 14 days by eye drop administration to the eyes of mice.

At this time, the extracellular endoplasmic reticulum sample to be administered is prepared by extracting exosomes from the medium in which the bone marrow-derived stem cells obtained by the method of Example 1 were cultured in an aqueous solution system, and the sample administered as a control cultured stem cells It was prepared by proceeding with the separation in the same way using an aqueous two-phase system in an untreated medium. The medium in which stem cells are not cultured has no extracellular vesicles, but contains other impurities, so the final extracted control sample contains a very small amount of impurities. By comparing the efficacy of the control sample and the extracellular vesicle sample, it can be confirmed whether the factor inducing the efficacy is the extracellular vesicle, not an impurity.

Tear secretion was measured using a phenol red thread before administration and before sacrifice.

In Examples 4 to 5 and the drawings related thereto, Control is the control group, and exosome is the administration group to which the bone marrow-derived extracellular vesicles of Example 1 were administered.

Example 4. Effect on dry eye according to administration of injection solution of extracellular vesicles

4-1. Test Methods

Experiments were performed as follows to determine the effect of the administration of the injection solution on the extracellular vesicles.

The mice of Example 3 were administered with exosomes or control by subconjunctival injection once, and then sacrificed 7 days later. Changes in clinical indicators according to the administration of extracellular vesicles (exosomes) were confirmed in animal models of ocular surface inflammatory diseases. Before subconjunctival injection of control and extracellular vesicles (exosome), the amount of tear secretion was confirmed using phenol red thread and the degree of corneal epithelial defect was confirmed by lissamine green staining. After sacrifice, the lacrimal gland and cornea of the mouse were surgically excised, and inflammatory factor analysis using hematoxylin & eosin staining (H & E staining) and inflammatory factor gene expression analysis were performed through Real-Time PCR.

4-2. Comparative analysis of corneal images

4 is an image showing the cornea on day 0 and day 7 of the control group, and FIG. 5 is an image showing the cornea on day 0 and day 7 after exosome injection into the eyeball once.

Referring to FIGS. 4 and 5, when the exosome according to the present invention is administered once to the conjunctiva, compared to the injection control, which is a control group, defects in the corneal epithelium are reduced, thereby improving the inflammatory disease of the ocular surface. .

4-3. NEI Scoring Comparative Analysis

6 is a graph comparing the NEI scoring of a control group and an exosome-administered group.

There was no significant difference between the scores of the control group and the extracellular vesicles administration group on day 0 (black bar), but looking at the scores on day 7 (hatched bar), the score of the extracellular vesicles administration group decreased significantly compared to the control group. showed up

4-4. Tear secretion analysis

7 is a graph comparing tear secretion between a control group and an exosome-administered group. Tear secretion was checked before administration and before sacrifice using a phenol red thread.

There was no significant difference between the scores of the control group and the extracellular vesicles administration group on Day 0 (black bar), but looking at the values on day 7 (hatched bar), it was observed that the extracellular vesicles administration group significantly increased compared to the control group. . Through this, it was confirmed that the inflammatory disease of the ocular surface of the mouse was improved by administering the extracellular vesicles.

4-5. H&E Stain (lacrimal glands) analysis

The surgically excised mouse lacrimal glands were fixed in 10% formalin and embedded in paraffin. The paraffin tissue was finely cut with a microtome (RM2255, Leica Biosystems, Nussloch, Germany) to prepare 4 μm corneal sections, and then hematoxylin & eosin staining (H&E staining) was performed to determine the number of inflammatory foci and size was analyzed.

8 is an image showing lacrimal gland staining of a control group and an exosome-administered group. As shown in FIG. 8, in the case of the control group, it was confirmed that the number and size of the inflammatory foci were large, and the lacrimal foci score of the lacrimal gland was large, whereas in the group administered with extracellular vesicles, the inflammatory foci index of the lacrimal gland The number was significantly reduced.

4-6. Gene expression analysis through real-time PCR

The gene expression of inflammatory factors in the surgically excised mouse cornea was observed as a fold value using Real-Time PCR analysis, and is shown in FIG. 9 . In the group administered with extracellular vesicles, the expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP9 was significantly decreased in the cornea compared to the control group.

10 is a graph showing the expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP9 in the lacrimal gland. 10 and 9, in the case of the administration group administered with extracellular vesicles, the gene expression of inflammatory factors was significantly reduced compared to the control group. From these results, it was confirmed that administration of the injection of the extracellular vesicles of the present invention can suppress gene expression of inflammatory factors in relation to the improvement of inflammatory diseases of the ocular surface.

Example 5. Effect on dry eye according to administration of eye drops from extracellular vesicles

In order to examine the effect of the administration of eye drops on extracellular vesicles, it was performed as follows.

5-1. Test Methods

The mice of Example 3 were intraocularly administered with extracellular vesicles or control for 2 weeks, and then sacrificed on the 14th day. Changes in clinical indicators according to the administration of extracellular vesicles were confirmed in animal models of ocular surface inflammatory diseases. Before administration of control and extracellular vesicles (exosome) by eye drop, the amount of tear secretion was confirmed using phenol red thread and the degree of corneal epithelial defect was confirmed by lissamine green staining. After sacrifice, the lacrimal gland and cornea of the mouse were surgically excised, and inflammatory factor analysis using hematoxylin & eosin staining (H & E staining) and inflammatory factor gene expression analysis were performed through Real-Time PCR.

5-2. Comparative analysis of corneal images

FIG. 11 is images showing the corneas on day 0 and day 14 of the control group, and FIG. 12 is images showing the corneas on day 0 and day 14 after treating the eye with exosomes for 14 days.

11 and 12, when the extracellular vesicles according to the present invention were administered as eye drops for 14 days, compared to the control Eyedrop control, corneal epithelial defects were reduced, resulting in improvement of ocular surface inflammatory diseases.

5-3. NEI Scoring Comparative Analysis

13 is a graph comparing NEI scoring of a control group and an exosome-administered group.

There was no significant difference between the scores of the control group and the extracellular vesicles administration group on day 0 (black bar), but on day 14 (hatched bar), it was observed that the extracellular vesicles administration group significantly decreased compared to the control group. .

5-4. Tear secretion analysis

14 is a graph comparing tear secretion between a control group and an exosome-administered group. Tear secretion was checked before administration and before sacrifice using a phenol red thread.

There was no significant difference between the scores of the control group and the extracellular vesicles-administered group on Day 0 (black bar), but the scores on Day 14 (hatched bar) showed an increase in the extracellular vesicles-administered group compared to the control group. Through this, it was confirmed that the inflammatory disease of the ocular surface of the mouse was improved by administering the extracellular vesicles.

5-5. H&E Stain (lacrimal glands) analysis

The surgically excised mouse lacrimal glands were fixed in 10% formalin and embedded in paraffin. The paraffin tissue was finely cut with a microtome (RM2255, Leica Biosystems, Nussloch, Germany) to prepare 4 μm corneal sections, and then hematoxylin & eosin staining was performed to analyze the number and size of inflammatory foci. .

15 is an image showing lacrimal gland staining of a control group and an exosome-administered group. As shown in FIG. 15, in the case of the control group, it was confirmed that the number and size of inflammatory foci were large, resulting in a large lacrimal foci score, whereas in the group administered with cell vesicles, the lacrimal foci score was large. The number was significantly reduced.

5-6. Gene expression analysis through real-time PCR

The gene expression of inflammatory factors in the surgically resected mouse cornea was observed in fold values and shown in FIG. 16 . In the group administered with extracellular vesicles, the expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP9 was significantly decreased in the cornea compared to the control group.

17 is a graph showing the expression of IL-6, IL-1β, TNF-a, IFN-g, IL-17A, and MMP9 in the lacrimal gland. FIG. 17 In addition, as shown in FIG. 16, in the case of the administration group administered with extracellular vesicles, the gene expression of inflammatory factors was significantly reduced compared to the control group. From these results, it was confirmed that the administration of the eye drop of the extracellular vesicles of the present invention can suppress the gene expression of inflammatory factors in relation to the improvement of ocular surface inflammatory diseases.

Example 6. Isolation of Extracellular Vesicles from Cord Blood-Derived Stem Cells

6-1. Cord blood-derived stem cell culture

Cell culture was performed using cord blood-derived mesenchymal stem cells distributed by Medipost Co., Ltd. (Korea). Using the cells of passage number 2, they were cultured in a DMEM culture medium containing 20% FBS (fetal bovine serum) and penicillin/streptomycin/amphotericin B. After culturing using a general culture medium up to passage number 4, replacing the culture medium with a separate culture medium before extracting exosomes and vesicles, and culturing for 48 hours, the cell culture supernatant was collected.

The collected cell culture supernatant was collected after 10 minutes at 500 × g, centrifuged again at 2,000 × g for 10 minutes, and the supernatant was aliquoted and stored frozen. Specifically, the cryopreserved cell culture supernatant is a culture medium from which cells and cell debris are removed, and contains 1×10 to 1×10 ³ cell-secreted exosomes per ml of culture medium, and the protein concentration per ml of culture medium is 100 μg. to 5000 μg were used.

6-2. Separation of extracellular vesicles using an aqueous biphasic phase separation composition

Using the culture medium obtained in Example 6-1 (cell culture supernatant containing exosomes from which cells and cell debris were removed), the same aqueous phase separation method as in Example 1-2 was performed to obtain phase-separated dextran Cord blood-derived extracellular vesicles were extracted from the layer with a pipette. In order to increase the purity of the number of extracellular vesicles per protein, i.e., to completely remove residual proteins present in the culture medium, extracellular vesicles derived from cord blood were isolated and obtained (recovered) by repeating this process two or more times.

Example 7. Effects of cord blood-derived extracellular vesicles on dry eyes following administration of eye drops

7-1. Test Methods

In order to examine the effect of cord blood-derived extracellular vesicles on dry eye according to the administration of eye drops, changes in clinical indicators according to the administration of extracellular vesicles were confirmed in the animal model of inflammatory diseases of the ocular surface in the same manner as in Examples 3 and 5 above. . Before administering control and cord blood-derived extracellular vesicles (exosomes) by eye drop, the amount of tear secretion was confirmed using phenol red thread and the degree of corneal epithelial defect was confirmed by lissamine green staining.

7-2. Comparative analysis of corneal images

18 is an image showing the cornea on day 0 and day 14 of a control group, and FIG. 19 is an image showing the cornea on day 0 and day 14 after dropwise administration of cord blood-derived exosomes to the eye for 14 days.

18 and 19, when the umbilical cord blood-derived extracellular vesicles according to the present invention were administered as eye drops for 14 days, compared to the control Eyedrop, the corneal epithelial defect was reduced, and the inflammatory disease of the ocular surface was improved. .

7-3. NEI Scoring Comparative Analysis

20 is a graph comparing the NEI scoring of a control group and a group administered with cord blood-derived extracellular vesicles (exosome).

There was no significant difference between the scores of the control group and cord blood-derived extracellular vesicles administration group on Day 0 (black bar), but on Day 14 (checkered bar), the corneal staining score of the cord blood-derived extracellular vesicles administration group was higher than that of the control group. A decrease could be observed.

7-4. Tear secretion analysis

21 is a graph comparing tear secretion between a control group and a group administered with cord blood-derived extracellular vesicles (exosome). Tear secretion was checked before administration and before sacrifice using a phenol red thread.

There was no significant difference between the scores of the control group and cord blood-derived extracellular vesicles administration group on Day 0 (black bar), but the scores on Day 14 (checkered bar) showed a significant increase in the cord blood-derived extracellular vesicles administration group compared to the control group showed

Through this, it was confirmed that the inflammatory disease of the ocular surface of the mouse was improved by administering the cord blood-derived extracellular vesicles.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (17)

1. A composition for the prevention or treatment of inflammatory diseases on the ocular surface comprising, as an active ingredient, extracellular vesicles derived from mesenchymal stem cells or separated from a culture thereof and having an average particle diameter of 50 nm to 1000 nm.
2. According to claim 1,

   The extracellular vesicles are produced by culturing mesenchymal stem cells to produce extracellular vesicles; culturing the extracellular endoplasmic reticulum; And separating the extracellular vesicles using a two-phase separation method in an aqueous solution.
3. According to claim 1,

   The extracellular vesicles are exosomes, and the exosomes have CD9, CD63, CD81 membrane protein markers, the composition for preventing or treating ocular surface inflammatory diseases.
4. According to claim 3,

   Among the membrane protein markers, the ratio of CD63/CD9 satisfies 2.5 or more, a composition for preventing or treating ocular surface inflammatory diseases.
5. According to claim 3,

   The extracellular vesicles have a number of exosomes separated per 1 L of the culture medium of 1 × 10 ⁹ to 1 × 10 ¹² and a protein amount of 0.1 mg to 20 mg, a composition for preventing or treating ocular surface inflammatory diseases.
6. According to claim 1,

   The mesenchymal stem cells are human or animal tissue-derived induced pluripotent stem cells (iPSC), autologous and allogeneic mesenchymal stem cells, or cell lines derived from mesenchymal stem cells, preventing or treating ocular surface inflammatory diseases composition for.
7. According to claim 6,

   The human or animal tissue is bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood and amniotic fluid, which is isolated from any one or more tissues selected from, the composition for preventing or treating inflammatory diseases of the ocular surface.
8. According to claim 1,

   The inflammatory disease of the ocular surface is dry eye, the composition for preventing or treating inflammatory diseases of the ocular surface.
9. According to claim 8,

   The ocular surface inflammatory diseases include anorexia, autoimmune diseases such as Sjogren's syndrome, keratoconjunctivitis sicca, Steven-Johnson syndrome, ocular blister, ophthalmic surgery, allergic conjunctivitis, VDT (Visual Display Terminal) workers Tear reduction, dry room due to air conditioning, toxicity due to long-term drug use, long-term use of contact lenses, systemic drugs that reduce tear secretion, ocular burns, and chronic ocular graft-versus-host reactions A composition for preventing or treating ocular surface inflammatory diseases caused by or accompanied by.
10. According to claim 1,

    The composition contains extracellular vesicles in an amount of 0.0001 to 95% by weight within 100% by weight of the total composition, a composition for preventing or treating ocular surface inflammatory diseases.
11. According to claim 1,

    The composition is administered by intraocular, intravitreal or intradermal route, composition for preventing or treating ocular surface inflammatory diseases.
12. According to claim 1,

    The composition is any one formulation selected from the group consisting of eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, drops, solutions, contact lens cleaners, and contact lens lubricants. A composition for preventing or treating inflammatory diseases.
13. A pharmaceutical composition for preventing or treating corneal damage comprising, as an active ingredient, extracellular vesicles derived from mesenchymal stem cells or separated from their culture medium and having an average particle diameter of 50 nm to 1000 nm.
14. According to claim 13,

    The extracellular vesicles are exosomes, and the exosomes have CD9, CD63, CD81 membrane protein markers, a pharmaceutical composition for preventing or treating corneal damage.
15. According to claim 14,

    Among the membrane protein markers, the ratio of CD63/CD9 satisfies 2.5 or more, a pharmaceutical composition for preventing or treating corneal damage.
16. A method for preventing or treating inflammatory diseases of the ocular surface comprising administering the composition for preventing or treating inflammatory diseases of the ocular surface of claim 1 to a non-human subject.
17. A method for preventing or treating corneal damage comprising administering the pharmaceutical composition for preventing or treating corneal damage of claim 13 to a non-human subject.

Images