COMPOSITION FOR ANTI-INFLAMMATION, WOUND HEALING OR WOUND HEALING PROMOTION, COMPRISING ROSE STEM CELL-DERIVED EXOSOMES AS ACTIVE INGREDIENT

Abstract

A composition containing rose stem cell-derived exosomes as an active ingredient is provided for anti-inflammation, wound healing or wound healing acceleration. The composition has excellent effects on anti-inflammation, wound healing and/or wound healing acceleration.

Claims

1. A method for preventing, suppressing, alleviating, ameliorating or treating inflammation of skin of a subject in need thereof, the method comprising treating an inflammatory area or a wounded area in the skin of the subject with an anti-inflammatory composition, wherein the anti-inflammatory composition comprises exosomes derived from rose stem cells as an active ingredient.

2. The method of claim 1, wherein the inflammatory area or the wounded area is treated with the anti-inflammatory composition by injection, microneedling, iontophoresis, application, or a combination thereof.

3. The method of claim 1, wherein the anti-inflammatory composition is an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

4. The method of claim 1, wherein the exosomes are isolated from a conditioned medium of rose plant cells, a conditioned medium of rose callus, or a biological solution of rose equivalent thereto.

5. The method of claim 1, wherein the rose stem cells are obtained by inducing a callus from rose embryos or leaves and then culturing cells of the callus.

6. The method of claim 1, wherein the subject is at least one selected from the group consisting of humans, dogs, cats, rodents, horses, cattle, monkeys and pigs.

7. A method for preventing, suppressing, alleviating, ameliorating or treating inflammation of skin of a subject in need thereof, the method comprising applying an anti-inflammatory composition to an inflammatory area or a wounded area in the skin of the subject, wherein the anti-inflammatory composition comprises exosomes derived from rose stem cells as an active ingredient.

8. The method of claim 7, wherein the anti-inflammatory composition is applied to the inflammatory area or the wounded area by microneedling, iontophoresis, application, or a combination thereof.

9. The method of claim 7, wherein the anti-inflammatory composition is a spray formulation, a liquid formulation, or a patch formulation.

10. The method of claim 7, wherein the exosomes are isolated from a conditioned medium of rose plant cells, a conditioned medium of rose callus, or a biological solution of rose equivalent thereto.

11. The method of claim 7, wherein the rose stem cells are obtained by inducing a callus from rose embryos or leaves and then culturing cells of the callus.

12. The method of claim 7, wherein the subject is at least one selected from the group consisting of humans, dogs, cats, rodents, horses, cattle, monkeys and pigs.

13. A method for preventing, suppressing, alleviating, ameliorating or treating inflammation of a subject in need thereof, the method comprising administering an effective amount of an anti-inflammatory composition to the subject, wherein the anti-inflammatory composition comprises exosomes derived from rose stem cells as an active ingredient.

14. The method of claim 13, wherein the anti-inflammatory composition is administered to the subject by injection, microneedling, iontophoresis, or a combination thereof.

15. The method of claim 13, wherein the anti-inflammatory composition is an injectable formulation, an infusion formulation, a spray formulation, or a liquid formulation.

16. The method of claim 13, wherein the exosomes are isolated from a conditioned medium of rose plant cells, a conditioned medium of rose callus, or a biological solution of rose equivalent thereto.

17. The method of claim 13, wherein the rose stem cells are obtained by inducing a callus from rose embryos or leaves and then culturing cells of the callus.

18. The method of claim 13, wherein the subject is at least one selected from the group consisting of humans, dogs, cats, rodents, horses, cattle, monkeys and pigs.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] FIG. 1A shows particle images obtained by transmitted electron microscopy (TEM) of rose stem cell-derived exosomes obtained according to one embodiment of the present invention.

[0023] FIG. 1B shows enlarged particle images of FIG. 1A.

[0024] FIG. 2 is a graph showing the results of NTA analysis of rose stem cell-derived exosomes.

[0025] FIG. 3 depicts fluorescence microscopic images of cells showing that fluorescence-stained exosomes are delivered into human dermal fibroblasts (green: exosomes delivered into cells; blue: cell nucleus; and red: cell membrane).

[0026] FIGS. 4A to 4C are graphs showing that the migration of human dermal fibroblasts remarkably increased after scratch-wounds of human dermal fibroblasts were treated with rose stem cell-derived exosomes.

[0027] FIG. 5 is a graph showing that IL-6 induced in RAW 264.7 cells by LPS remarkably decreased when RAW 264.7 cells were treated with rose stem cell-derived exosomes.

[0028] FIG. 6 is a graph showing that rose stem cell-derived exosomes have an excellent effect of reducing IL-6 (inflammatory cytokine), i.e., an excellent anti-inflammatory effect, as compared with conditioned media of rose stem cells (conditioned media of rose callus).

DETAILED DESCRIPTION OF INVENTION

[0029] The present invention provides a composition for anti-inflammation, wound healing or wound healing acceleration containing rose stem cell-derived exosomes as an active ingredient.

[0030] As used herein, the term “rose (Rosa spp.)” refers to plants belonging to the genus Rosa, in the family Rosaceae, the order Rosales and the class Dicotyledoneae, and includes all of wild species and cultivated garden species.

[0031] As used herein, the term “exosomes” refers to nano-sized vesicles secreted or released from plant cells into extracellular spaces and having a membrane structure, and is also referred to as exosome-like vesicles or exosome-like particles.

[0032] As used herein, the term “anti-inflammation” means prevention, suppression, alleviation, amelioration or treatment of inflammation. As an example, not limiting the present invention, examples of the inflammatory diseases include dermatitis, atopic dermatitis, eczema, inflammation caused by bacterial, viral or fungal infections, burns, inflammation caused by burns, wounds, inflammation caused by wounds, and the like.

[0033] As used herein, the term “wound” means a condition in which a part or all of the body is injured, and is intended to encompass pathological conditions in which a tissue constituting an inside or an external surface of the body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ, or blood vessel tissue, is damaged or destroyed. As an example, not limiting the present invention, examples of the wound include abrasion, laceration, stab wound, incised wound, avulsion, bedsore, tissue destruction caused by irradiation, penetrated wound, gunshot wound, burn, frostbite, surgical wound, sutures after plastic surgery, wound caused by chemical substance and so on, and may include any damage to any part of an individual.

[0034] As used herein, the term “iontophoresis” refers to a method of flowing a microcurrent through a skin to which an active ingredient has been applied, generating a potential difference thereby and changing the electrical environment of the skin, and thus allowing an ionized active ingredient to penetrate the skin by electrical repulsion. Examples of iontophoresis that is used in one embodiment of the present invention include: a method of introducing a microcurrent into a skin by allowing the microcurrent to flow from an external power source into an electrode patch on the skin, the microcurrent generated by the external power source; a method of introducing a microcurrent into a skin, the microcurrent generated by a battery provided in an electrode patch on the skin; and a method of introducing a microcurrent into a skin through a patch on the skin provided with a reverse electrodialysis device, the microcurrent generated by the concentration difference between high concentration electrolyte solution and low concentration electrolyte solution in the reverse electrodialysis device. However, the present invention is not limited thereto, and various types of iontophoresis may, of course, be used.

[0035] As used herein, the term “rose stem cell-derived exosomes” is meant to include all exosomes isolated from, for example, a conditioned medium of rose plant cells, a conditioned medium of rose callus, or a biological solution of rose equivalent thereto.

[0036] The composition containing rose stem cell-derived exosomes as an active ingredient according to one embodiment of the present invention may exhibit at least one effect selected from the group consisting of anti-inflammation, wound healing, and wound healing acceleration.

[0037] In the composition according to one embodiment of the present invention, the rose stem cells may be obtained by inducing a callus derived from rose embryos or leaves and then culturing cells of the callus.

[0038] For example, the composition containing rose stem cell-derived exosomes as an active ingredient according to the present invention may be a pharmaceutical composition for anti-inflammation, wound healing or wound healing acceleration. In addition, the composition containing rose stem cell-derived exosomes as an active ingredient according to the present invention may be used as a functional cosmetic composition, a skin external preparation or a quasi-drug for anti-inflammation, wound healing or wound healing acceleration.

[0039] As an example, not limiting the present invention, the composition according to one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof. For example, the composition may be an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

[0040] In one embodiment of the present invention, when the composition is used as a pharmaceutical composition, it may include pharmaceutically acceptable carriers, excipients or diluents. The carriers, excipients and dilutes include, but are not limited to, lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the effective amount of the pharmaceutical composition according to one embodiment of the present invention means the amount required for administration in order to achieve the effects of anti-inflammation, wound healing and/or wound healing acceleration.

[0041] The content of the pharmaceutical composition according to one embodiment in a formulation may be suitably selected depending on the kind, amount, form and the like of additional components as described above. For example, the pharmaceutical composition of the present invention may be contained in an amount of about 0.1 to 99 wt %, preferably about 10 to 90 wt %, based on the total weight of an injectable formulation. Furthermore, the suitable dose of the pharmaceutical composition according to one embodiment of the present invention may be adjusted depending on the severity of disease, the type of formulation, formulating method, patient's age, sex, body weight, health condition, diet, excretion rate, the period of administration, and the regime of administration. For example, when the pharmaceutical composition according to one embodiment of the present invention is administered to an adult, it may be administered once to several times at a dose of 0.001 mg/kg to 100 mg/kg per day.

[0042] In addition, the present invention provides a method for treating inflammation, healing wound or accelerating wound healing, the method comprising administering a therapeutically effective amount of the composition to a mammal, or applying the composition to a skin, an inflammatory area, or a wounded area. The mammal may be at least one mammal selected from the group consisting of humans, dogs, cats, rodents, horses, cattle, monkeys and pigs.

[0043] The composition of the present invention is less likely to contain impurities such as growth regulation factors than conventional rose filtrates, rose extracts, conditioned media of rose callus, or filtrates or extracts thereof, and has excellent effects on anti-inflammation, wound healing and/or wound healing acceleration.

[0044] It should be understood that the scope of the present invention is not limited to the aforementioned effects.

EXAMPLES

[0045] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only to illustrate the present invention and are not intended to limit or restrict the scope of the present invention. Those that can be easily inferred by those skilled in the art from the detailed description and examples of the present invention are interpreted as falling within the scope of the present invention. References referred to in the present invention are incorporated herein by reference.

[0046] Throughout the present specification, it is to be understood that, when any part is referred to as “comprising” any component, it does not exclude other components, but may further include other components, unless otherwise specified.

Example 1: Preparation of Rose Stem Cells

[0047] According to preparation and culture methods of plant stem cells known in the art, calluses were induced from rose embryos and/or leaves, and cells of the induced callus were cultured. In addition, a callus having a good growth state was selected and cultured in large amounts, thereby preparing conditioned media of rose stem cells (conditioned media of rose callus).

Example 2: Preparation of Rose Stem Cell-Derived Exosomes

[0048] The conditioned media of rose stem cells (conditioned media of rose callus) prepared as described in Example 1 were purchased from BIO-FD&C Co., Ltd. (located in Incheon, Korea and supplying conditioned media of Damask Rose callus). The conditioned media of rose stem cells were filtered through a 0.22 μm filter to remove impurities such as cell debris, waste products and large particles. Rose stem cell-derived exosomes were isolated from the filtered conditioned media by tangential flow filtration (TFF) method.

[0049] The size of the isolated rose stem cell-derived exosomes was analyzed by transmitted electron microscopy (TEM). As shown in FIGS. 1A and 1B, it was confirmed that the isolated rose stem cell-derived exosomes were nano-sized vesicles. The size and concentration of the rose stem cell-derived exosomes were analyzed by nanoparticle tracking analysis (NTA) using NS300 (purchased from Malvern Panalytical) (FIG. 2).

Example 3: Evaluation of Delivery Ability of Rose Stem Cell-Derived Exosomes into Dermal Fibroblasts

[0050] In order to examine whether the rose stem cell-derived exosomes would be delivered into human dermal fibroblasts (purchased from CEFO Co., Ltd.), the following analysis was performed. To fluorescence-stain the membrane of the rose stem cell-derived exosomes prepared in Example 2, the exosomes were allowed to react with PKH67 fluorescence dye (purchased from Sigma-Aldrich). After the reaction, the reaction solution was fractionated with an MW3000 column (purchased from ThermoFisher Scientific) to remove free PHK67 that was not stained in the exosome membrane. A negative control was prepared by allowing PKH67 fluorescence dye to react with a buffered solution and fractionating the reaction product with the MW3000 column. The exosomes stained with PKH67 were incubated with pre-cultured human dermal fibroblasts, and then whether the exosomes would be delivered into the cells over time was observed using a fluorescence microscope. Hoechst fluorescence dye (purchased from Sigma-Aldrich) was used to stain the cell nucleus, and CellMask Orange Plasma Membrane Stain fluorescence dye was used to stain the cell membrane. As a result of examining whether the exosomes would be delivered into the cells, it was confirmed that the fluorescence-stained exosomes were delivered into the cells and green fluorescence accumulated in the cells over time (FIG. 3).

Example 4: Evaluation of Skin Regeneration Effect Using Dermal Fibroblasts

[0051] To evaluate whether the rose stem cell-derived exosomes prepared as described in Example 2 promotes wound healing in human dermal fibroblasts, scratch-wound assay was performed. Human dermal fibroblasts dispersed in a DMEM containing fetal bovine serum were seeded into a culture plate for wound induction (ImageLock Plate; purchased from EssenBio) at a density of 10,000 cells/well and cultured for 24 hours under 5% CO.sub.2 and 37° C. After the cells reached a confluency of 90% or more, scratches were made using a WoundMaker (purchased from EssenBio). To evaluate the skin regeneration effect using human dermal fibroblasts, experimental groups was classified as follows:

[0052] (1) Negative control (NC): an experimental group treated with a serum-free medium alone;

[0053] (2) Positive control (PC): an experimental group treated with a medium containing 10% fetal bovine serum;

[0054] (3) Conditioned media of rose stem cells (CM): an experimental group treated with the conditioned media of rose stem cells (conditioned media of rose callus), which were prepared in Example 2 and diluted with a serum-free medium (treatment concentration of CM based on the number of particles: 1.0×10.sup.9 particles/mL); and

[0055] (4) Rose stem cell-derived exosomes (EXO): an experimental group treated with the rose stem cell-derived exosomes, which were prepared in Example 2 and diluted with a serum-free medium (treatment concentration of EXO based on the number of particles: 1.0×10.sup.9 particles/mL).

[0056] After that, each of the experimental groups was subjected to Scratch-Wound assay, and the human dermal fibroblasts were cultured at 37° C. under 5% CO.sub.2 for 24 hours. At each of 6, 12 and 24 hours after the treatment of each experimental group, the wound healing efficacy in each experimental group was measured using Incucyte (purchased from Sartorius), respectively.

[0057] As a result of measuring the wound healing efficacy, it was confirmed that the treatment with the rose stem cell-derived exosomes of the present invention remarkably increased the migration of the human dermal fibroblast compared to the positive control at 6 hours after the treatment, and also remarkably increased the migration of the human dermal fibroblasts compared to the negative control and the conditioned media of rose stem cells at all the time points of measurement (at 6 hours, 12 hours and 24 hours after the treatment) (FIGS. 4A to 4C).

[0058] As can be seen from the above results, the rose stem cell-derived exosomes of the present invention have an excellent effect for promoting the migration of human dermal fibroblasts, that is, an excellent wound healing effect, as compared with the conditioned media of rose stem cells (conditioned media of rose callus).

[0059] Therefore, the rose stem cell-derived exosomes of the present invention are useful as an active ingredient of a composition for wound healing or wound healing acceleration.

Example 5. Evaluation 1 of Anti-Inflammatory Effect of Rose Stem Cell-Derived Exosomes

[0060] To evaluate whether the rose stem cell-derived exosomes prepared as described in Example 2 exhibit an anti-inflammatory effect, RAW 264.7 cells were suspended in 10% FBS-containing DMEM, and then seeded into each well of a multi-well plate to reach a confluency of 80 to 90%. On the next day, the RAW 264.7 cells were treated for 24 hours with the rose stem cell-derived exosomes diluted in a LPS-containing fresh medium (DMEM containing 1% FBS and 200 nM LPS). Experimental groups for evaluation of the anti-inflammatory effect were classified as follows:

[0061] (1) Negative control: an experimental group in which RAW 264.7 cells were treated with LPS alone;

[0062] (2) Positive control (PC): an experimental group in which RAW 264.7 cells were treated with LPS plus 200 μM dexamethasone; and

[0063] (3) Rose stem cell-derived exosomes (EXO): an experimental group in which RAW 264.7 cells were treated with the rose stem cell-derived exosomes, which were prepared in Example 2 and diluted with the LPS medium (treatment concentration of EXO based on the number of particles: 1.0×10.sup.9 particles/mL).

[0064] After the completion of culturing the RAW 264.7 cells of each experimental group, the culture supernatant was collected, and the inflammatory response was checked by measuring the inflammatory cytokines present in the culture supernatant. The amount of inflammatory cytokines in the culture supernatant was measured using an IL-6 ELISA kit. The amount of IL-6 (inflammatory cytokine) produced in the group treated with LPS alone, and the amount of IL-6 (inflammatory cytokine) produced in the respective group treated with LPS plus each of dexamethasone and the rose stem cell-derived exosomes were measured using the ELISA kit (purchased from R&D system) according to the manufacturer's manual.

[0065] As shown in FIG. 5, it was confirmed that, when RAW 264.7 cells, which are mouse macrophages, were treated with LPS plus the rose stem cell-derived exosomes of the present invention, the production of IL-6 was remarkably inhibited compared to that in the negative control group in which the RAW 264.7 cells were treated with LPS alone, and the production of IL-6 decreased to a level similar to that in the positive control group in which RAW 264.7 cells were treated with LPS plus dexamethasone.

[0066] As can be seen from the above results, the rose stem cell-derived exosomes of the present invention have an excellent anti-inflammatory effect. Thus, the rose stem cell-derived exosomes of the present invention are useful for preventing, suppressing, alleviating, ameliorating or treating inflammatory diseases.

Example 6. Evaluation 2 of Anti-Inflammatory Effect of Rose Stem Cell-Derived Exosomes

[0067] To evaluate whether the rose stem cell-derived exosomes prepared as described in Example 2 exhibit an anti-inflammatory effect, RAW 264.7 cells were suspended in 10% FBS-containing DMEM, and then seeded into each well of a multiwell plate to reach a confluency of 80 to 90%. On the next day, the RAW 264.7 cells were treated for 24 hours with the rose stem cell-derived exosomes diluted in a LPS-containing fresh medium (DMEM containing 1% FBS and 200 nM LPS). Experimental groups for evaluation of the anti-inflammatory effect were classified as follows:

[0068] (1) Negative control (NC): an experimental group in which RAW 264.7 cells were treated with LPS alone;

[0069] (2) Positive control (PC): an experimental group in which RAW 264.7 cells were treated with LPS plus 200 μM dexamethasone;

[0070] (3) Conditioned media of rose stem cells (CM): an experimental group in which RAW 264.7 cells were treated with the conditioned media of rose stem cells (conditioned media of rose callus), which were prepared in Example 2 and diluted with the LPS medium (treatment concentration of CM based on the protein content: 5 μg/mL, and 50 μg/mL); and

[0071] (4) Rose stem cell-derived exosomes (EXO): an experimental group in which RAW 264.7 cells were treated with the rose stem cell-derived exosomes, which were prepared in Example 2 and diluted with the LPS medium (treatment concentration of EXO based on the protein content: 5 μg/mL, and 50 μg/mL).

[0072] After the completion of culturing the RAW 264.7 cells of each experimental group, the culture supernatant was collected, and the inflammatory response was checked by measuring the inflammatory cytokines present in the culture supernatant. The amount of inflammatory cytokines in the culture supernatant was measured using an IL-6 ELISA kit (purchased from R&D system). The amount of IL-6 (inflammatory cytokine) produced in the group treated with LPS alone, and the amount of IL-6 (inflammatory cytokine) produced in the respective group treated with LPS plus each of dexamethasone, the conditioned media of rose stem cells (conditioned media of rose callus) and the rose stem cell-derived exosomes were measured using the ELISA kit according to the manufacturer's manual. The remaining cells were subjected to cell viability measurement using MTT assay (purchased from Sigma-Aldrich), and the measured values of IL-6 were normalized using the cell viability.

[0073] As shown in FIG. 6, it was confirmed that, when RAW 264.7 cells, which are mouse macrophages, were treated with LPS plus the rose stem cell-derived exosomes of the present invention, the production of IL-6 was remarkably inhibited compared to that in the negative control group in which RAW 264.7 cells were treated with LPS alone, and the production of IL-6 also decreased to a level superior to that in the positive control group in which RAW 264.7 cells were treated with LPS plus dexamethasone.

[0074] In addition, it was confirmed that, when RAW 264.7 cells were treated with LPS plus the rose stem cell-derived exosomes of the present invention, the production of IL-6 decreased to a level superior to that in the CM group in which RAW 264.7 cells were treated with LPS plus the same concentration of the conditioned media of rose stem cells (conditioned media of rose callus). In particular, it was confirmed that, when RAW 264.7 cells were treated with LPS plus the rose stem cell-derived exosomes of the present invention at a concentration of 5 μg/mL, the production of IL-6 was remarkably inhibited, as compared with the CM group in which RAW 264.7 cells were treated with LPS plus the same concentration of the conditioned media of rose stem cells (conditioned media of rose callus).

[0075] As can be seen from the above results, the rose stem cell-derived exosomes of the present invention have an excellent anti-inflammatory effect, as compared with the conditioned media of rose stem cells (conditioned media of rose callus). Thus, the rose stem cell-derived exosomes of the present invention are useful for preventing, suppressing, alleviating, ameliorating or treating inflammatory diseases.

[0076] Although the present invention has been described with reference to the embodiments, the scope of the present invention is not limited to these embodiments. Any person skilled in the art will appreciate that various modifications and changes are possible without departing from the spirit and scope of the present invention and these modifications and changes also fall within the scope of the present invention.