WO2021137398A1 - Conditioned medium comprising high concentration of extracellular vesicles bound to lactoferrin - Google Patents

Abstract

The present invention relates to a conditioned medium comprising a high concentration of extracellular vesicles bound to lactoferrin obtained by culturing cells in a culture medium comprising lactoferrin. In addition, the present invention relates to a cosmetic composition and a pharmaceutical composition comprising the conditioned medium. In addition, the present invention relates to a cosmetic composition and a pharmaceutical composition comprising the conditioned medium.

Description

Conditioned culture medium containing a high concentration of extracellular vesicles bound to lactoferrin

The present invention relates to a conditioned medium containing a high concentration of extracellular vesicles binding to lactoferrin obtained by culturing cells in a culture medium containing lactoferrin. The present invention also relates to a cosmetic composition and a pharmaceutical composition comprising the conditioned culture solution.

The skin consists of three layers: the epidermis, the dermis, and the subcutis, and the layer deeply related to skin aging and regeneration is the dermis. Dermal fibroblasts present in the dermal layer maintain the structure of the dermal layer or produce collagen, Fibronectin, and elastin, which are major factors of the extracellular matrix that give elasticity. It synthesizes or produces proteins such as TIMP (Tissue Inhibitor of Metalloproteinase) that help it. However, damage to fibroblasts reduces the extracellular matrix or generates a matrix metalloproteinase (MMP) that breaks down cross links. The strength and elasticity of the skin is maintained due to the balance of the synthesis and decomposition of the extracellular matrix in the dermal layer, and aging due to intrinsic aging or external stimuli occurs when the dermal layer is not maintained beyond the synthesis.

Extracellular vesicles have recently been in the spotlight. Extracellular vesicles are vesicles having a phospholipid bilayer structure with a size of 30 nm to 1 μm that originates from the cell and is discharged out of the cell. Extracellular vesicles are composed of exosomes and microvesicles, and the two pathways are different. Exosomes are generated in various sizes in multivesicular endosomes and then released out of the cell, and microvesicles are released as the plasma membrane of the cell forms an endoplasmic reticulum directly outside the cell. In recent years, the efficacy of the extracellular vesicles on skin regeneration or hair growth has been consistently presented (J Extracell Vesicles. 2019 Jan 20;8(1):1565885, Sci Rep. 2017 Nov 14;7(1):15560).

Since the conventional cell culture medium has a low ratio of extracellular vesicles, there has been a problem in terms of mass production or cost of extracellular vesicles. The present inventors confirmed that, through previous studies, when cells were cultured by adding a lactoferrin/calcium composition to the culture medium, the intracellular calcium concentration increased and the amount of extracellular vesicles excreted thereby increased significantly. However, the efficacy or effect of the conditioned culture medium obtained by the above method has not been specifically confirmed to date.

In addition to the previous finding that lactoferrin increases the production signal of extracellular vesicles, we hypothesized that lactoferrin could bind to a lactoferrin receptor called GAPDH present on the surface of extracellular vesicles and be released together with the extracellular vesicles. [Biochem Cell Biol. 2012 Jun;90(3):329-38. doi: 10.1139/o11-058. Epub 2012 Jan 31.]. In addition, further studies were conducted on whether positively charged lactoferrin could induce a charge change in extracellular vesicles.

The present inventors predicted that lactoferrin would enhance the skin regeneration effect of extracellular vesicles by improving the binding capacity of extracellular vesicles with skin cells by increasing the number of extracellular vesicles in the culture medium as well as decreasing the negative charge of the extracellular vesicles.

Accordingly, an object of the present invention is to provide a conditioned culture medium that increases the content of extracellular vesicles and enhances the skin regeneration efficacy of extracellular vesicles.

The present inventors studied to solve the above problems, and as a result, lactoferrin binds to the extracellular vesicles and reduces the strong membrane negative charge of the extracellular vesicles, thereby improving the binding ability of the extracellular vesicles with the skin cells. It was found to enhance the skin regeneration effect.

Furthermore, the present inventors have found that a conditioned culture medium containing a high concentration of extracellular vesicles binding to lactoferrin obtained by culturing cells in a culture medium containing lactoferrin can enhance skin regeneration and skin barrier, and completed the present invention. did it

The conditioned culture medium containing a high concentration of extracellular vesicles bound to lactoferrin of the present invention has a positive effect of increasing skin regeneration efficacy due to the significantly higher number of extracellular vesicles in the culture medium, and the positive charge of lactoferrin reduces the negative charge of extracellular vesicles This has the advantage of maximizing the skin regeneration effect by increasing the binding ability of the extracellular vesicles with the skin cells, thereby increasing the skin regeneration effect synergistically.

In addition, the conditioned culture medium containing a high concentration of extracellular vesicles bound to lactoferrin of the present invention increases the factors responsible for the skin barrier and moisturizing role, thereby showing the effect of restoring the damaged skin barrier.

Therefore, the conditioned culture medium of the present invention can be used as a raw material for cosmetics and pharmaceuticals for skin regeneration strengthening and skin barrier strengthening.

1 is a result of measuring the number of extracellular vesicles generated by a serum substitute alone and a serum substitute/lactoferrin/calcium combination with a nanoparticle tracking analyzer.

Figure 2 is the result of measuring the size of the extracellular vesicles generated by the combination of serum replacement agent alone and serum replacement agent/lactoferrin/calcium with a nanoparticle tracking analyzer.

3 is a result of observing the shape of extracellular vesicles generated by using a serum substitute alone and a serum substitute/lactoferrin/calcium combination through a transmission electron microscope.

4 is a result of observing lactoferrin bound to extracellular vesicles produced by using a serum substitute alone and a serum substitute/lactoferrin/calcium combination through a transmission electron microscope.

5 is a graph quantifying the amount of lactoferrin bound to the extracellular vesicles produced by the combination of serum substitute alone and serum substitute/lactoferrin/calcium by ELISA.

FIG. 6 is a result of measuring the membrane charge of extracellular vesicles generated by a serum substitute alone and a serum substitute/lactoferrin/calcium combination using a zeta potential & particle size analyzer.

7 is a result of comparing the binding ability of extracellular vesicles with fibroblasts by using a serum substitute alone and a serum substitute/lactoferrin/calcium combination with FACS.

8 is a result of confirming the shape and proliferation rate by MTT assay when fibroblasts were treated with a culture medium cultured with a serum replacement agent alone and a serum replacement agent/lactoferrin/calcium combination.

9 is an RT-PCR result showing the change in the expression of extracellular matrix in fibroblasts by a culture medium cultured with a serum substitute alone and a serum substitute/lactoferrin/calcium combination.

10 is a result of confirming the change in proliferation of fibroblasts by MTT assay after removing extracellular vesicles from the culture medium cultured with serum replacement alone and serum replacement/lactoferrin/calcium combination.

11 is a result of an MTT assay confirming the proliferation rate of fibroblasts by comparing the number of extracellular vesicles generated by the combination of serum substitute alone and serum substitute/lactoferrin/calcium.

12 is a fluorescence microscope observation of the skin tissue permeability of extracellular vesicles generated by using a serum substitute alone and a serum substitute/lactoferrin/calcium combination, and H&E results of observing the tissue shape.

13 is an ELISA result confirming changes in the extracellular matrix and metalloprotease (MMP) secreted by the skin tissue by a culture medium cultured with a serum substitute alone and a serum substitute/lactoferrin/calcium combination.

14 is a result of observing changes in filaggrin secreted by keratinocytes by a fluorescence microscope in a culture medium cultured with a combination of an additive-free medium and a serum substitute/lactoferrin/calcium.

15 is a result of observation by IHC of the degree of filaggrin recovery in skin epidermal tissue by a culture medium cultured with a combination of an additive-free medium and a serum substitute/lactoferrin/calcium.

The present invention relates to a conditioned medium containing a high concentration of extracellular vesicles binding to lactoferrin obtained by culturing cells in a culture medium containing lactoferrin.

According to one embodiment of the present invention, the binding of extracellular vesicles to lactoferrin can enhance the binding of extracellular vesicles to cells. As will be confirmed in Examples to be described later, extracellular vesicles in a culture medium obtained by culturing cells in a culture medium to which lactoferrin is added have a lactoferrin receptor, and thus have a possibility of binding lactoferrin to extracellular vesicles. This binding reduces the negative charge of the extracellular vesicle and induces a phenomenon that maximizes the binding between the extracellular vesicle and the cell.

As used herein, the term "culture medium (Culture medium)" refers to the medium before culturing the cells, and "culture medium or conditioned medium" refers to the medium obtained after culturing the cells.

As used herein, the term "extracellular vesicles" includes all of the nano-sized (30 ~ 2,000 nm) vesicles released to the outside of the cell composed of a phospholipid bilayer, which is the same component as the structure of the cell membrane. Thus, it encompasses "Exosome" and "Microvesicle" that are released from the cell. Furthermore, the term "extracellular vesicle" is meant to encompass Ectosome, Microparticle, Tolerosomes, Prostatosomes, Cardiosomes and Vexosomes. is also used as

In one embodiment, the number of extracellular vesicles in the conditioned culture medium of the present invention ^{may be in the range of 1.0 × 10 8} /ml to 1.0 × 10 ¹¹ /ml, specifically 1.0 × 10 ⁹ /ml to 1.0 × 10 ¹¹ /ml may be in the range.

In one embodiment, the conditioned culture medium of the present invention may contain lactoferrin at a concentration of 0.1 μg/ml to 1 mg/ml in the culture medium before culturing, and 0.01 μg/ml to 1 mg/ml of lactoferrin in the conditioned culture medium after culturing may be present at a concentration of

In one embodiment, the lactoferrin in the conditioned culture medium of the present invention may be selected from the group consisting of Holo-lactoferrin, Apo-lactoferrin and Pis-lactoferrin. Specifically, the lactoferrin may be synthesized or obtained by extraction, and includes both human and non-human animals.

In one embodiment, calcium may be additionally added to the culture medium prior to culture in the conditioned culture medium of the present invention. In one embodiment, the calcium may be added to the culture medium before culturing at a concentration of 0.2 μM to 10 mM, and calcium in the conditioned culture medium after culture may be present at a concentration of 0.02 μM to 10 mM. In one embodiment, the calcium ^{may be added in the form of calcium ions (Ca 2+} ), and the source of the calcium ions includes all salts capable of supplying calcium ions.

In one embodiment, in the conditioned culture medium of the present invention, one or more substances selected from the group consisting of basal media, serum replacement and serum may be additionally added to the culture medium before culture. . The "serum substitute" refers to a single substance or a composition substance that replaces serum for preparing a serum-free medium.

In one embodiment, the cells used in the preparation of the conditioned culture medium of the present invention may be all types of cells derived from humans or animals. In one embodiment, the cell is a cell having the ability to regenerate the skin, specifically, the cell having the ability to regenerate the skin may be a stem cell, but is not necessarily limited to a stem cell. In Examples to be described later, "human adipose-derived mesenchymal stem cells" were used as the cells, but the present invention is not limited thereto. As one example not limiting the present invention, mesenchymal stem cells, for example, adipose (Adipose), umbilical cord (Umbilical cord), umbilical cord blood (Umbilical cord blood), bone marrow (Bone marrow), placenta-derived, embryonic stem cells , iPS-derived stem cells, or skin stem cells. In one embodiment, fibroblasts, which are cells that have confirmed binding to extracellular vesicles, include both negatively charged human and non-human animal-derived cells.

The present invention also relates to a cosmetic composition comprising the conditioned culture medium and a cosmetically acceptable additive or carrier. In one embodiment, the cosmetic composition may have a use for strengthening skin regeneration or strengthening skin barrier.

When the conditioned culture solution of the present invention is prepared as a cosmetic composition, the cosmetic composition may include components commonly used in the cosmetic composition as well as the conditioned culture solution of the present invention, for example, antioxidants, stabilizers, solubilizers, vitamins, customary adjuvants such as pigments and fragrances, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, surfactant-containing cleansing oil, emulsion It may be formulated as a foundation, a wax foundation, and a spray, but is not limited thereto. More specifically, lotion, nourishing lotion, nourishing cream, moisture cream, massage cream, eye cream, essence, paste, mask pack, patch, gel, cream, lotion, powder, soap, cleanser, oil, foundation, makeup base, It can be prepared in the form of a wax or spray.

The present invention also relates to a pharmaceutical composition comprising the conditioned culture medium and a pharmaceutically acceptable additive or carrier. In one embodiment, the pharmaceutical composition may have a use for strengthening skin regeneration or strengthening the skin barrier.

The pharmaceutical composition of the present invention may be formulated using a pharmaceutically acceptable additive, carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may additionally contain a dispersant or stabilizer. Specifically, the pharmaceutical composition may be formulated for external use for the skin, and more specifically, may be prepared in the form of ointments, creams, gels, sprays, skin patches, dressings, masks, non-adhesive gauze, and the like. In addition, the pharmaceutical composition may be formulated as an injection, and more specifically, may be prepared in the form of subcutaneous injection, intradermal injection, and the like. In addition, the pharmaceutical composition may be prepared for administration to a mammal, more preferably for administration to a human.

Pharmaceutically acceptable carriers may be solid or liquid, and include excipients, antioxidants, buffers, bacteriostats, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release controlling agents, wetting agents, It may be at least one selected from a stabilizer, a suspending agent, and a lubricant. In addition, the pharmaceutically acceptable carrier may be selected from saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

In one embodiment, the pharmaceutical composition may be formulated for external use for skin. In another embodiment, the pharmaceutical composition may be formulated for subcutaneous injection or intradermal injection.

The method of separating extracellular vesicles used herein, that is, the Size Exclusion Columns method, is a method of separating other extracellular vesicles, for example, ultracentrifuge, density gradient, ultracentrifugation. It may be filtration (Ultrafiltration), immunoaffinity purification (Immunoaffinity purification), but is not limited to the method specified above (Mol Ther Nucleic Acids. 2018 Mar 2;10:131-141).

As used herein, the term "skin regeneration" refers to a skin regeneration effect of the dermal layer of the skin, and an increase in the proliferation rate of dermal fibroblasts in the dermal layer and an increase in extracellular matrix synthesis comprehensively. However, based on the results from the human tissue model, it may be the effect of the epidermal layer rather than the dermal layer alone.

As used herein, the term "Nutrient Full media-Conditioned medium (NF-CM)" refers to a culture medium obtained after culturing in a culture medium containing a serum substitute, and "NFL-CM (Nutrient Full media with Lactoferrin-Conditioned medium)" " means a culture medium obtained after culturing in a culture medium containing a serum substitute and a lactoferrin/calcium composition. In the same vein, extracellular vesicles isolated from NF-CM are expressed as “NF-EVs (NF-Extracellular vesicles)”, and extracellular vesicles isolated from NFL-CM are expressed as “NFL-EVs (NFL-Extracellular vesicles)”. express it as

As confirmed in the Examples to be described later, the present inventors found that the lactoferrin added to the culture medium binds to the increased extracellular vesicles in the culture medium, and this binding reduces the negative charge of the extracellular vesicles and increases the binding ability with the cells. Confirmed.

In addition, the present inventors confirmed that the conditioned culture medium containing a high concentration of extracellular vesicles bound to lactoferrin enhances the regeneration efficacy of skin fibroblasts by increasing the proliferation rate of skin fibroblasts and increasing the expression of extracellular matrix.

In addition, the present inventors have demonstrated that the skin regeneration enhancing effect of the conditioned medium is due to the quantitative and qualitative change of extracellular vesicles in the culture medium.

In addition, the present inventors confirmed that the extracellular vesicles in the conditioned culture medium showed increased skin tissue permeability and increased the amount of extracellular matrix secreted by the skin tissue using a skin tissue model.

In addition, the present inventors confirmed the effect of maintaining the skin barrier and increasing skin barrier recovery of the conditioned culture medium.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Confirmation of extracellular vesicles derived from adipose-derived stem cell culture medium

In order to proceed with the experiment, a culture medium was first obtained, and the method is as follows.

Human adipose-derived mesenchymal stem cells were cultured for 72 hours in a 175 cm 2 cell culture dish (T175 flask). After 72 hours, it was washed with a phosphate buffer solution, and an additive-free medium (Alpha-Minimum Essential Medium, alpha-MEM) was added and incubated for 24 hours. _{Thereafter, calcium (CaCl 2} , sigma, USA) and lactoferrin (Lactoferrin, aspira scientific, USA) were mixed in a medium containing serum replacement and the cells were treated. In addition, the group containing only serum replacement was also conducted. After 48 hours, the culture solution was collected and filtered through a 0.22-μm filter to obtain a final culture solution. The culture medium obtained after cell culture with lactoferrin/calcium composition/serum substitute was expressed as Nutrient Full media with Lactoferrin-Conditioned medium (NFL-CM), and the culture medium obtained after cell culture with only serum substitute was NF-CM (Nutrient Full media-Conditioned medium). medium) was expressed.

In order to separate the extracellular vesicles in the culture medium, a size-based column (Size Exclusion Columns, IZONscience, New Zealand) was used. The size-based column separates particles with a size of 35 - 400 nm or more, and removes small proteins not associated with exosomes in the culture medium. Extracellular vesicles isolated from NF-CM were expressed as NF-EVs, and extracellular vesicles isolated from NFL-CM were expressed as NFL-EVs. The number and size of extracellular vesicles in NF-EVs and NFL-EVs were measured with a nanoparticle tracking analysis (Nanoparticle tracking analysis, NTA, Nanosight NS300, Malvern, UK). A graph of is shown in FIG. 2 .

As shown in FIG. 1 , when the lactoferrin/calcium composition was added to the culture medium, it was confirmed that the extracellular vesicles in the culture medium increased. The number of extracellular vesicles per cell was expressed as 100% of NF-CM, and it was confirmed that NFL-CM contained about 4 times more extracellular vesicles compared to NF-CM (Unparried t-test, *** ; p < 0.0001). On the other hand, it was confirmed that there was no significant change in the size of the extracellular vesicles (FIG. 2).

Negative staining was performed to observe the shape of the isolated extracellular vesicles. The separated extracellular vesicles are washed with a washing solution in the exosome staining kit (Exosome-TEM-easy kit, 101bio, USA). Thereafter, negative staining was performed with uranyl acetate in the kit for 10 minutes, dried at room temperature, and observed at 120 kV with a transmission electron microscope (Transmission Electron Microscope, TEM, Talos L120c, FEI, Czech).

As shown in FIG. 3, both NF-EVs and NFL-EVs could be observed to be surrounded by the shape of a lipid bilayer, and it was observed that they had a cup shape, which is the shape of a conventional extracellular vesicle.

In conclusion, it can be confirmed that the lactoferrin/calcium composition increases the number of extracellular vesicles in the culture medium, but does not affect the size or shape of the extracellular vesicles.

Example 2: Confirmation of increased binding ability with skin cells by binding of lactoferrin to extracellular vesicles produced by the lactoferrin/calcium composition

The experiment was carried out thinking that the lactoferrin added to the culture medium would bind to the increased extracellular vesicles in the culture medium and increase the binding ability with skin cells.

As in Example 1, extracellular vesicles isolated from the culture medium were reacted with lactoferrin-gold antibody and observed with a transmission electron microscope.

Specifically, extracellular vesicles were fixed (4% paraformaldehyde), blocked (0.4% bovine serum albumin), lactoferrin antibody reaction, and finally negative staining was performed with uranyl acetate. After drying at room temperature, it was observed with a transmission electron microscope at 120 kV, and is shown in FIG. 4 . Extracellular vesicles isolated from the culture medium obtained after cell culture with lactoferrin/calcium composition/serum substitute were expressed as NFL-EVs, and extracellular vesicles isolated from the culture medium obtained after cell culture with serum replacement were expressed as NF-EVs. .

In the case of NF-EVs, it was observed that the lactoferrin-gold particles did not bind to the extracellular vesicles, whereas in the NFL-EVs, it was observed that the lactoferrin-gold particles were attached to the extracellular vesicles.

The amount of lactoferrin bound to the extracellular vesicles was confirmed using an enzyme-linked immunosorbent assay (ELISA), and the method is as follows.

As in Example 1, lactoferrin and various proteins not bound to extracellular vesicles in the culture medium were removed with a size-based column, and the number of separated extracellular vesicles was measured by the method of nanoparticle tracking analysis (NTA). . Based on the measured number, ^{the amount of lactoferrin bound to 2×10 9} NF-EVs and NFL-EVs was measured using a Lactoferrin ELISA kit (NOVUS, USA).

Specifically, the quantitative substance of the lactoferrin ELISA kit and the extracellular vesicles were put into a 96-well plate coated thereto, and then reacted at 37° C. for 2 hours. After that, the first antibody (Detection antibody) was attached and the second antibody (HRP antibody) was attached, and washing was performed at least 3 times with a phosphate buffer solution between each process. Finally, a substrate and a stop buffer were put, and absorbance was measured at 450 nm with an ELISA reader (Molecular devices, USA), and the quantitative value is shown in FIG. 5 .

The amount of lactoferrin bound to 2×10 ⁹ NFL-EVs was 135 ng/ml, while the amount of lactoferrin bound to 2×10 ⁹ NF-EVs was 4.5 ng/ml. It was found that lactoferrin bound to NFL-EVs was 30 times that of lactoferrin bound to NF-EVs. expected to induce functional enhancement (unpaired T-test, ***;p < 0.0001).

In order to measure the surface charge of extracellular vesicles separated by a size-based column, Zeta potential & particle size analysis was used, and the results are shown in FIG. 6 .

As shown in FIG. 6 , the negative charge of NFL-EVs cultured in a culture medium containing lactoferrin was significantly weaker than that of NF-EVs. NF-EVs confirmed a charge of -60 ~ 64 mV, but in the case of NFL-EVs, a charge of -20 ~ 30 mV was confirmed, so that when lactoferrin was incubated with lactoferrin, extracellular vesicles were combined with positive lactoferrin to reduce the negative charge. It was found to be consistent with one result (unpaired T-test, **;p < 0.001).

The decrease in the negative charge of the extracellular vesicles was expected to show an increase in the binding of NFL-EVs to the cell membrane, and the experiment was carried out. The difference in binding ability between NFL-EVs and NF-EVs with human dermal fibroblasts was confirmed by fluorescence activated cell sorting (FACS, ACEA, USA).

Specifically, after staining the extracellular vesicles with a lipophilic dye (Lipophilic dye, DiO, Invitrogen, USA), they were separated by a size-based column as in Example 1. The stained extracellular vesicles ^{were treated with 5×10 6} fibroblasts per group for 3 hours, washed with phosphate buffer solution, and cells were washed with 1×trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA, welgene, Korea) solution. It was isolated from the culture dish. After fixing the cells with 4% paraformaldehyde for 10 minutes, the cells were washed and diluted with a phosphate buffer solution, and the green fibroblasts binding to the extracellular vesicles were confirmed by fluorescence-activated cell isolation.

As shown in Figure 7, it was confirmed that the binding force of NFL-EVs with fibroblasts was increased than that of NF-EVs. Cells bound to NF-EVs accounted for 46.72% of the total cells, whereas fibroblasts bound to NFL-EVs accounted for 82.02% of the total cells. Therefore, it can be seen that NFL-EVs bind to about 1.8 times more fibroblasts.

In conclusion, it was confirmed that the lactoferrin added to the culture medium binds to the increased extracellular vesicles in the culture medium, and this binding reduces the negative charge of the extracellular vesicles and increases the binding ability with the cells.

Example 3: Confirmation of increase in skin cell regeneration ability by lactoferrin/calcium composition culture medium

Fibroblasts present in the dermal layer of the skin are a major source of proteins of the extracellular matrix of the dermal layer, such as collagen, piperonectin, and elastin. The extracellular matrix plays a major role in maintaining skin structure and elasticity, and thus the proliferation of fibroblasts and the increase of extracellular matrix proteins play an important role in preventing skin aging (Mol Cell Biochem. 2019 Oct 10).

In order to confirm the effect of the culture medium prepared in Example 1 on the proliferation of fibroblasts, the MTT assay method was performed. The serum replacement culture medium containing the lactoferrin/calcium composition was expressed as Nutrient Full media with Lactoferrin-Conditioned medium (NFL-CM), and the culture medium containing only the serum replacement medium was expressed as NF-CM (Nutrient Full media-Conditioned medium).

More specifically, 250 μl of a fibroblast suspension ^{of 2 × 10 4} cells/ml was placed in a 48-well plate with 48 holes and cultured for 24 hours. After removing the medium (Fibroblast Growth Media, FGM, Lonza, USA), 250 μl of NF-CM and NFL-CM were treated, and an additive-free medium (SF) was used as a negative control. After inoculation, each medium was removed after incubation at 37° C. for 48 hours and washed three times with a phosphate buffer solution. The MTT solution (0.5 mg/ml) was reacted for 2 hours and mixed, and then the absorbance was measured at 570 nm by stirring in an isopropyl alcohol solution for 30 minutes. Result values are graphed with relative values when SF is set to 100%. In addition, the cell state was observed under a microscope (inverted microscope, Olympus corporation, Japan) and shown in FIG. 8 together.

It was confirmed that the culture medium obtained after lactoferrin was added to the culture medium induces more fibroblast proliferation. As shown in FIG. 8 , when treated with NF-CM, a proliferation rate of 137% was shown, whereas when treated with NFL-CM, a cell proliferation rate of 174% could be confirmed. It was confirmed that the difference between the results was significant (Unparried T-test, ***;p < 0.0001).

Changes in collagen, elastin, and fibronectin, which are major factors of matrix secreted by fibroblasts by NFL-CM, were observed through reverse transcriptase polymerase chain reaction (RT-PCR).

In a 6-well plate with 6 holes, ^{2 ml of a suspension of 3 × 10 4} cells/ml fibroblasts was placed and cultured for 24 hours. After 24 hours, NF-CM, NFL-CM, and ascorbic acid (L-Ascorbic acid, Sigma, USA) as a positive control were diluted to a final concentration of 100 uM and treated with 1 ml each. After 48 hours, the fibroblasts were washed with a phosphate buffer solution, and the cells were separated from the dish with 1×trypsin-EDTA solution. Thereafter, intracellular RNA was isolated using an RNA extraction kit (TAKARA MiniBEST universal RNA extraction kit, TAKARA, Japan). It was then synthesized with this cDNA (Complementary DNA), cDNA Synthesis Kit ^{(PrimeScript 1 st strand cDNA synthesis kit} , TAKARA, Japan) in the same amount of alen. Then, the synthesized cDNA was quantitatively analyzed for pro-collagen type 1, elastin, and fibronectin by reverse transcriptase chain reaction.

As a result, as shown in FIG. 9 , it was confirmed that the increase in matrix was significant in fibroblasts treated with NFL-CM compared to fibroblasts treated with NF-CM. In NF-CM, the gene levels of procollagen type 1, elastin, and fibronectin were increased by 136%, 187%, and 149%, respectively, compared to the negative control group in NF-CM, and the gene levels in NFL-CM were 174%, 244%, and 187%, respectively. this increased. It was confirmed that the result value of the three genes of NFL-CM increased with a significant difference from the result value increased in NF-CM (Unpaired T-test, ****; p < 0.0001).

Example 4: Confirmation that the increase in skin cell proliferation rate by the NFL-CM culture is due to quantitative and qualitative changes in extracellular vesicles in the culture medium

In Example 3, an experiment was prepared to prove that the increase in the proliferation rate of fibroblasts by the culture medium produced through the lactoferrin/calcium composition is due to the extracellular vesicles present in the culture medium.

In order to confirm whether the effect of increasing the fibroblast proliferation rate of NFL-CM is due to the increased extracellular vesicles in the culture medium, an experiment was performed after removing the extracellular vesicles in the culture medium and comparing the results with before removal. The method of removing extracellular vesicles in the culture medium is as follows.

A filtration spin column (Vivaspin20, Sartorius, UK) of 300,000 MWCO (Molecular Weight Cut-Off), which is generally used for concentrating the culture medium, was used. After putting the culture solution in the column, the culture solution was filtered through centrifugation. filter The filtered culture solution was used as a culture solution from which extracellular vesicles were removed, and the culture solution from which extracellular vesicles were removed from NFL-CM in FIG. 10 was called “EVs-depleted NFL-CM”, and the culture solution from which extracellular vesicles had been removed from NF-CM was used. Each was expressed as "EVs-depleted NF-CM".

First, after removing extracellular vesicles as described above, fibroblasts were treated with the same amount of NF-CM, EVs-depleted NF-CM, NFL-CM, and EVs-depleted NFL-CM for two days. sey proceeded. The method is the same as in FIG. 8 of Example 3.

As shown in Figure 10, NF-CM and EVs-depleted NF-CM did not show a significant difference in cell proliferation rate, whereas in the case of EVs-depleted NFL-CM from which extracellular vesicles were removed from NFL-CM, the proliferation rate of fibroblasts was decreased significantly (Unpaired T-test, NS; p > 0.05, ***; p < 0.0001). As a result, the reason why NFL-CM showed a large difference in fibroblast proliferation compared to NF-CM was demonstrated that NFL-EVs of NFL-CM acted as a major factor.

An experiment was prepared to confirm that the increase in the proliferation rate of fibroblasts by NFL-EVs is caused by a change in the number of extracellular vesicles as well as a change in the surface charge of the extracellular vesicles. The same number (2×10 ⁸ ) of NF-EVs and NFL-EVs were treated in skin fibroblasts. In addition, ^{after treatment with 4×10 8} NFL-EVs, differences in fibroblast proliferation due to changes in the number and surface charge were analyzed through MTT assay. As in Example 1, after separating extracellular vesicles from NF-CM and NFL-CM using a size-based column, the number of each extracellular vesicle was measured using a nanoparticle tracking analyzer. Thereafter, as in Example 3, the difference in proliferation of fibroblasts was compared, and the results are shown in FIG. 11 .

As shown in FIG. 11 , ^{when treated with 2×10 8} NF-EVs, an increase of 104.5% was observed, whereas when treated with the same number of NFL-EVs, a cell increase of 109% was confirmed (Unpaired T-test) , *: p < 0.01). In addition, 4×10 ⁸ NFL-EVs could confirm a 112% cell increase. This is 2×10 ⁸ There was a significant difference with NFL-EVs (Unpaired T-test, **: p < 0.001).

Based on the above results, it is indirectly indicated that the excellent proliferation rate of fibroblasts shown by NFL-EVs is not only due to the increased number of NFL-EVs, but also a synergistic effect with lactoferrin binding together as shown in Example 2.

Example 5: Confirmation of increased regenerative capacity due to increased permeability of extracellular vesicles in lactoferrin/calcium composition culture medium in skin tissue model

While fibroblasts synthesize extracellular matrix such as collagen, elastin, and fibronectin in the dermal layer, when cells are damaged, they secrete metalloprotase that breaks down collagen, a major factor in the extracellular matrix, to form the extracellular matrix. metalloproteinase is regulated by thymp, an inhibitor produced by fibroblasts. In this regard, skin structure and elasticity are maintained through the balance of various elements created by fibroblasts, and when the balance is broken, skin aging is induced (Exp Cell Res. 2001 Dec 10;271(2):249-62) .

It is well known that extracellular vesicles are highly permeable to the skin (BBRC, 2017, Nov 18; 493(2): 1102-1108), and furthermore, as confirmed in Example 2, extracellular vesicles in the conditioned culture medium of the present invention are Since the number of cells is large and the ability to bind to skin cells is high due to a decrease in negative charge, the conditioned culture medium was prepared for an experiment in anticipation of high skin penetration ability and regenerative effect.

After staining the extracellular vesicles with a lipophilic dye, they were separated by a size-based column as in Example 1. After transferring a three-dimensional skin model (KeraSkin™-FT, Biosolution, Korea) to a 6-well plate containing assay-media, 100 μl of each of the stained extracellular vesicles was treated. After 6 hours, the tissue was fixed (4% formaldehyde), washed, dehydration, clearing, infiltration, and embedding to make a paraffin block. The paraffin block was sectioned with a microtome (Automated microtome, Leica, Germany).

The samples were later run in two ways. For the fluorescence experiment, the tissue was subjected to nuclear staining and tissue protection using a mounting solution (Mounting medium, Vector laboratories, USA) containing DAPI (DAPI, 4',6-diamidino-2-phenylindole), followed by a fluorescence microscope (DM4000B). , fluorescence microscopy, Leica, Germany) was used for tissue observation and imaging. Alternatively, after staining with hematoxylin & eosin (H&E, Hematoxylin and eosin), observation was made at 400 times magnification through a tissue observation microscope (BX41, Olympus, Japan), and images were taken at the same magnification.

As shown in Figure 12, NFL-EVs showed excellent tissue permeability. Although it showed a lot of NFL-EVs in the epidermis, it was confirmed that the NFL-EVs penetrated inside and accumulated in the dermis.

In order to confirm whether NFL-EVs, which have the characteristic of accumulating in the dermis, induce regeneration, an experiment in which NFL-CM including NFL-EVs is treated in a human tissue model in the same way as above was conducted, and the assay media was analyzed 48 hours later. obtained. Procollagen 1 type, a substrate present in the assay media, and metalloproteinase that degrades the substrate and changes in thymp regulating it were investigated with a procollagen type 1 ELISA kit (PIP ELISA kit, TAKARA, Japan) and thymp-1. It was measured using an ELISA kit (TIMP-1 ELISA kit, R&D system, USA) and a metalloproteinase-1 ELISA kit (Human total MMP-1 kit duoset, R&D system, USA) and proceeded according to the method of the kit. After the SF group was 100%, the relative value is shown in FIG. 13 .

As shown in FIG. 13 , in the case of the NFL-CM-treated group, compared to the group not treated with anything, an increase of 142% procollagen type 1, 391% thymp-1 and a 77% decrease in metalloproteinase-1 were confirmed. did. On the other hand, the positive control ascorbic acid (1 mM) showed an increase of 87% procollagen type 1 and 137% thymp-1, which was lower than that of NFL-CM (Unpaired T-test, ***: p < 0.0001), and ascorbic acid was also known to be effective in reducing metalloproteinase-1, but it only showed a 90% reduction in metalloproteinase-1, so it showed a relatively weak effect compared to NFL-CM.

In conclusion, when cells are incubated with lactoferrin/calcium composition, more extracellular vesicles are produced and lactoferrin reduces the negative charge of extracellular vesicles, so that more extracellular vesicles reach the skin tissue and increase the amount of extracellular matrix secreted by skin cells. It was confirmed that the amount increased. These results are consistent with the results of Example 3, and as a result, it shows that NFL-CM enhances the skin regeneration effect.

Example 6: Confirmation of filaggrin changes by lactoferrin/calcium composition culture medium in skin cells and human tissue models

Filaggrin affects the physical support of the skin barrier by linking keratin with each other in the outermost layer of the skin that protects the human body from the outside. In addition, the final product of filaggrin is a natural moisturizing factor and has the function of maintaining skin moisture, so filaggrin acts as a major factor in skin barrier and moisturizing.

In order to confirm the effect of the culture medium prepared in Example 1 on filaggrin in keratinocytes, filaggrin fluorescence staining (immunofluorescence) was performed. The culture medium obtained after cell culture with lactoferrin/calcium composition/serum substitute was expressed as Nutrient Full media with Lactoferrin-Conditioned medium (NFL-CM).

Specifically, in a 24-well plate with 24 holes, put a cover slip of 12Φ into the hole, and then ^{put 500 μl of a 2 × 10 4} cells/ml keratinocyte (HaCaT) suspension. Incubated for 48 hours. After removing the culture medium (Dulbecco's Modified Eagle Medium, DMEM, Welgene, Korea), 250 μl of the culture medium and 250 μl of NFL-CM were treated, and an additive-free medium was used as a negative control. After inoculation, each medium was removed and stained with filaggrin after incubation for 5 days at 37°C. Fixative (2% formaldehyde, sigma, USA), permeabilization (0.2% Triton-X 100, MP biomedical, Germany), Blocking (5% fetal bovine serum), antibody reaction (Filaggrin antibody, Thermofisher, USA), second antibody reaction ( Fluorescein horse anti-mouse IgG antibodies, vector laboratories, USA), and finally, nuclear staining and cell protection were performed using a mounting solution (Mounting medium, Vector laboratories, USA). The results were confirmed with a fluorescence microscope (DM4000B, fluorescence microscopy, Leica, Germany).

As shown in FIG. 14 , it was confirmed that more filaggrin was stained in the group treated with NFL-CM than in the negative control group, and it was confirmed that NFL-CM is effective in generating filaggrin by keratinocytes.

In order to confirm the effect of the culture medium prepared in Example 1 on filaggrin in skin tissue, filaggrin immunostaining (Immunohistochemistry) was performed. Specifically, a 3D human skin epidermal model (KeraSkin™, Biosolution, Korea) was treated with 10% sodium dodecyl sulfate (SDS, Sodium Dodecyl Sulfate) to break the cell barrier, and then treated with NFL-CM. After 4 days, the tissues were fixed in a fixative solution (4% formaldehyde, sigma, USA) for 4 hours and washed with running water for 4 hours to remove the fixative solution. Next, as in Example 5, dehydration, clearing, infiltration, embedding, thinning, drying, deparaffinization, hydration, permeation (0.2% Triton-X) 100), and then washed 3 times with a phosphate buffer solution. In order to eliminate non-specific staining of antibodies due to endogenous hydrogen peroxide activity in tissues, after reacting in 0.3% water peroxide for 5 minutes, the antigen was exposed with 0.01M sodium citrate. Thereafter, blocking, Filaggrin antibody reaction (Filaggrin antibody, Thermofisher, USA), and biotinylated secondary antibody [biotinylated universal (Anti-Mouse IgG/Rabbit IgG) antibody] were reacted. Each process included a washing process. After 30 minutes of reaction with ABC reagent in the kit, it was reacted with DAB substrate (VECTOR laboratory, SK4100, USA) and the tissue was protected with a mounting solution. The tissue was observed under a microscope (BX41, Olympus, Japan) at 400 times magnification and taken at the same magnification. The results are shown in FIG. 15 .

As shown in FIG. 15 , filaggrin was stained brown. When sodium dodecyl sulfate was treated alone, it was confirmed that the skin epidermal tissue was damaged, and the amount of filaggrin was also small. On the other hand, when incubated with NFL-CM after treatment with sodium dodecyl sulfate, the damaged epidermal tissue was recovered as much as the existing skin tissue, and it was observed that the amount of filaggrin was also increased.

In conclusion, it was confirmed that NFL-CM has the effect of restoring the damaged skin barrier by increasing filaggrin, which is a major factor in skin barrier and moisturizing.

Claims (17)

1. A conditioned medium containing a high concentration of extracellular vesicles binding to lactoferrin obtained by culturing cells in a culture medium containing lactoferrin.
2. The conditioned culture medium according to claim 1, wherein the number of extracellular vesicles is in ^{the range of 1.0 × 10 8} /ml to 1.0 × 10 ^{11 /ml.}
3. The conditioned culture medium according to claim 1, wherein the culture medium contains lactoferrin at a concentration of 0.1 μg/ml to 1 mg/ml before culturing.
4. The conditioned culture medium according to claim 1, wherein the lactoferrin is present in a concentration of 0.01 μg/ml to 1 mg/ml in the conditioned medium after culturing.
5. The conditioned culture medium according to claim 1, wherein the lactoferrin is selected from the group consisting of Holo-lactoferrin, Apo-lactoferrin and Pis-lactoferrin.
6. The conditioned culture medium of claim 1, wherein calcium is additionally added to the culture medium before culturing.
7. 7. The conditioned culture medium according to claim 6, wherein calcium is added at a concentration of 0.2 μM to 10 mM.
8. The conditioned culture medium of claim 1, wherein calcium is present in a concentration of 0.02 µM to 10 mM in the conditioned culture medium after culturing.
9. The conditioned culture medium of claim 1, wherein one or more substances selected from the group consisting of basal media, serum replacement and serum are additionally added to the culture medium prior to culture.
10. The conditioned culture medium according to claim 1, wherein the cells are cells having skin regeneration ability.
11. The conditioned culture medium according to claim 10, wherein the cells having skin regeneration ability are stem cells.
12. A cosmetic composition comprising the conditioned culture medium of any one of claims 1 to 11 and a cosmetically acceptable additive or carrier.
13. The cosmetic composition according to claim 12, which is for strengthening skin regeneration or strengthening skin barrier.
14. 12. A pharmaceutical composition comprising the conditioned culture medium of any one of claims 1 to 11 and a pharmaceutically acceptable additive or carrier.
15. 15. The pharmaceutical composition according to claim 14, for strengthening skin regeneration or strengthening the skin barrier.
16. 15. The pharmaceutical composition according to claim 14, formulated for external application to the skin.
17. 15. The pharmaceutical composition of claim 14, formulated for intradermal injection or subcutaneous injection.

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