Exosome composition formed as double-layered membrane with enhanced penetration and stability, and preparation method therefor

Abstract

The present invention relates to an exosome composition formed as a double-layered membrane with enhanced penetration and stability and a preparation method therefor. Addressing the limitations of conventional exosomes in terms of insufficient penetration and stability, the present invention can provide excellent effects such as improved penetration (significantly improved delivery efficiency to a target tissue by forming a double-layered structure through the addition of an outer membrane composed of the same lipid bilayer component as the outer membrane of the exosome), strengthened structural stability (strengthened resistance to external environmental changes and prolonged in vivo half-life by increasing the physical and chemical stability of the exosome), improved drug delivery efficiency (providing stability capable of more safely protecting bioactive substances such as drugs, proteins, and nucleic acids within the exosome, and enabling precise delivery to the target tissue), maintaining biocompatibility (maintaining biocompatibility by utilizing the lipid bilayer component, without inducing immune responses or toxicity in vivo), and increased commercial applicability (providing substantial and innovative applicability in exosome-based cosmetics, skin treatment agents, hair loss treatment agents, precision medical technologies, drug delivery systems, and the like).

Description

Exosome composition formed with a double membrane having improved penetration power and stability, and method for manufacturing the same

[0001] The present invention relates to an exosome composition formed with a double membrane that improves penetration power and stability, and a method for manufacturing the same.

[0002] Recently, exosomes have garnered attention in the life sciences and medical fields for their role in intercellular signaling and the transport of biomaterials, and their potential applications are expanding in various areas such as drug delivery systems, skin treatment, and hair regeneration. In particular, exosomes are nanoparticles naturally secreted by cells, and due to their excellent biocompatibility and drug delivery efficiency, many research and commercialization efforts are underway.

[0003] However, there are several limitations to the conventional use of exosomes. First, due to a lack of penetration, the delivery efficiency of exosomes to target tissues is limited, making it difficult to utilize exosomes effectively. For example, when attempting to deliver exosomes to target sites such as skin or hair follicles, exosomes often fail to penetrate sufficiently into the tissue and are lost at the surface. Second, due to a lack of structural stability, exosomes rapidly degrade or denature within the body, sometimes failing to fully exert the desired effects. Due to these limitations, the commercial application of exosome-based technologies is restricted, and there is a need for the development of technologies to maximize existing drug delivery and therapeutic effects.

[0004] Conventional exosome compositions consist of a single lipid bilayer, making them vulnerable to external environmental changes (e.g., temperature, pH, enzymes) or physical shock. Consequently, they have short half-lives in the body and are prone to degradation and denaturation. Additionally, since the penetration power of exosomes relies primarily on the endocytosis mechanism of a single membrane, delivery efficiency into target tissues has been limited. In particular, for fields requiring precise in vivo application, such as skin treatment, hair loss treatment, and drug delivery, technology that simultaneously ensures both the penetration power and stability of exosomes is essential.

[0005] To solve these problems, a technology utilizing a double-layered structure to simultaneously improve the penetration power and stability of exosomes is being proposed. The present invention provides a novel exosome composition and a method for manufacturing it, which maximize the physical and chemical stability of exosomes and significantly improve their penetration power into target tissues by additionally wrapping the outer membrane of the exosome with the same lipid bilayer component to form a double-layered structure. Exosomes formed with a double layer can not only overcome the limitations of conventional exosomes but also maximize the physiological efficacy of exosomes in drug delivery and skin and hair follicle treatment.

[0006] <Prior Art Literature>

[0007] <Patent Document 1> Korean Registered Patent Publication No. 10-2660197 (Registered on April 19, 2024)

[0008] The present invention aims to solve the aforementioned problems and provides an exosome composition formed with a double membrane that improves penetration power and stability, and a method for manufacturing the same.

[0009]

[0010] The problems solved by the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0011] In order to solve the aforementioned problems,

[0012] The present invention provides an exosome composition having improved penetration power and stability, wherein the exosome composition has an added outer membrane, and the outer membrane is formed as a double membrane characterized by being composed of the same lipid bilayer component as the outer membrane of the exosome composition and forming a micelle structure.

[0013] In addition, the above outer membrane is formed as a double membrane characterized by forming a single membrane (a single layer membrane), thereby providing an exosome composition with improved penetration power and stability.

[0014] In addition, the above lipid bilayer component is characterized by being formed as a double membrane comprising one or more components selected from the group consisting of phospholipids, cholesterol, sphingolipids, and lipid rafts, thereby providing an exosome composition with improved penetration power and stability.

[0015] A method for preparing an exosome composition is provided, comprising the steps of: extracting exosomes; and adding an outer membrane to the extracted exosomes; and a method for preparing an exosome composition formed of a double membrane with improved penetration power and stability.

[0016] In addition, the step of extracting the exosomes is characterized by using Tangential Flow Filtration (TFF), thereby providing a method for preparing an exosome composition formed of a double membrane with improved penetration power and stability.

[0017] In addition, the above tangential flow filtration method provides a method for manufacturing an exosome composition formed with a double membrane having improved penetration power and stability, characterized by using a filtration filter having 100,000 Da to 500,000 Da.

[0018] In addition, the step of adding an outer membrane to the extracted exosome is characterized by mixing lipid bilayer components identical to the exosome in a 2:1 ratio, thereby providing a method for preparing an exosome composition formed as a double membrane with improved penetration power and stability.

[0019] The present invention solves the problem of insufficient penetration and stability of conventional exosomes, thereby providing excellent effects such as improved penetration (significantly improving delivery efficiency to target tissues by forming a double membrane structure with an outer membrane composed of the same lipid bilayer component), enhanced structural stability (increasing the physical and chemical stability of exosomes to strengthen resistance to changes in the external environment and increase half-life in the body), improved drug delivery efficiency (providing stability that can more safely protect bioactive substances such as drugs, proteins, and nucleic acids inside exosomes, and enabling precise delivery to target tissues), maintenance of biocompatibility (maintaining biocompatibility by utilizing lipid bilayer components and not inducing immune reactions or toxicity in the body), and increased commercial applicability (providing practical and innovative application possibilities in exosome-based cosmetics, skin treatments, hair loss treatments, precision medical technology, and drug delivery systems).

[0020]

[0021] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0022] The attached drawings are intended to explain the contents of the present invention in more detail to those skilled in the art, and the technical concept of the present invention is not limited thereto.

[0023] FIG. 1 is a drawing showing an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability.

[0024] FIG. 2 is a diagram showing a method for manufacturing an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability.

[0025] FIG. 3 is a diagram showing the improved penetration power of an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability.

[0026] <Explanation of Symbols>

[0027] 100: Method for preparing an exosome composition formed with a double membrane, with improved penetration power and stability

[0028] S110: Step for extracting exosomes

[0029] S120: Step of adding an outer membrane to extracted exosomes

[0030] Hereinafter, an exosome composition formed with a double membrane according to the present invention, with improved penetration power and stability, and a method for manufacturing the same will be described in detail, but the scope of the exosome composition formed with a double membrane, with improved penetration power and stability, and the method for manufacturing the same is not limited by the following description.

[0031] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0032] The terms used in this specification are for describing embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other components in addition to the components mentioned. Throughout the specification, the same reference numerals refer to the same components, and "and / or" includes each of the mentioned components and all combinations of one or more. Although terms such as "first," "second," etc., are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, the first component mentioned below may be the second component within the technical scope of the invention.

[0033] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0034] Spatially relative terms such as "below," "beneath," "lower," "above," and "upper" may be used to facilitate the description of the relationship between one component and other components as illustrated in the drawings. Spatially relative terms should be understood as encompassing different orientations of components during use or operation, in addition to the orientations depicted in the drawings. For example, if a component depicted in a drawing is inverted, a component described as "below" or "beneath" of another component may be placed "above" of that component. Therefore, the exemplary term "below" may encompass both the lower and upper directions. Components may also be oriented in other directions, and accordingly, spatially relative terms may be interpreted according to the orientation.

[0035]

[0036] FIG. 1 is a drawing showing an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability.

[0037] Referring to FIG. 1, an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability, is an exosome composition with an added outer membrane, wherein the outer membrane is composed of lipid bilayer components identical to the outer membrane of the exosome composition and can be formed in a micelle structure.

[0038] Here, micelle structures are specific nanostructures composed of amphiphilic molecules, which are structures spontaneously formed in water or other solvent environments by materials that simultaneously possess hydrophobic and hydrophilic parts; micelle structures mainly exist in a spherical shape, but can be deformed into various forms (cylindrical, layered structures, etc.) depending on the environment.

[0039] The exosomes included in the above exosome composition may be derived from stem cells or dermal papilla cells.

[0040] The outer membrane may be formed as a single membrane (a single layer). Additionally, if necessary, the outer membrane may be formed as a double membrane or a multilayer membrane.

[0041] In addition, the outer membrane may include a growth factor to promote the proliferation of hair papilla cells and hair growth, and the growth factor may include one or more factors selected from the group consisting of BMP, TGF-β, KGF, EGF and IL-6, but is not limited thereto.

[0042] In addition, the outer membrane may include a specific ligand to selectively bind to cells surrounding the hair follicle, and the ligand may include one or more ligands selected from the group consisting of VEGF ligand, FGF ligand, HGF ligand, RGD peptide, and ECM-binding ligand, but is not limited thereto.

[0043] In addition, the outer membrane may include a surfactant that improves skin permeability to increase absorption in the scalp, and the surfactant may include one or more surfactants selected from the group consisting of lecithin, saponin, cholesterol, SDS, PEG, DPPC, PLGA, DMSO, and lauryl glucoside, but is not limited thereto.

[0044] The above lipid bilayer component may include one or more components selected from the group consisting of phospholipids, cholesterol, sphingolipids, and lipid rafts, but is not limited thereto.

[0045] Here, the phospholipid is a core component of the lipid bilayer, has amphiphilic properties, and can provide physical stability and selective permeability. Additionally, the phospholipid may include one or more components selected from the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI), but is not limited thereto.

[0046] Cholesterol can regulate the fluidity and stability of the lipid bilayer.

[0047] Sphingolipids are specialized lipids found in cell membranes and exosome outer membranes that can provide structural stability and signal transduction. Additionally, sphingolipids may include one or more components selected from the group consisting of sphingomyelin and glycosphingolipids, but are not limited thereto.

[0048] In addition, lipid microstructure can refer to nanodomains within the lipid bilayer where specific lipids (cholesterol, sphingolipids, etc.) and proteins are densely packed.

[0049] The lipid bilayer components described above can perform various roles, such as structural stability of biological membranes, fluidity control, signal transduction, and interaction with pathogens. These lipid bilayer components are essential in biological systems such as exosomes, cell membranes, and liposomes, and can provide an important foundation for drug delivery and biotechnological applications.

[0050]

[0051] FIG. 2 is a drawing showing a method (100) for manufacturing an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability.

[0052] Referring to FIG. 2, a method (100) for manufacturing an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability, may include the step of extracting exosomes (S110) and the step of adding an outer membrane to the extracted exosomes (S120).

[0053] The exosomes in the step (S110) of extracting the exosomes are a collective term for extracellular vesicles having a size of about 50 nm to 200 nm separated from a cell culture medium, and the exosomes were separated and extracted from a culture medium in which human dermal papilla cells were cultured for 3 days.

[0054] To isolate and extract the above exosomes, centrifugation, ultracentrifugation, density gradient centrifugation, chromatography, filtration, ultrafiltration, polymer-based precipitation, and immunoaffinity separation methods may be used; however, it is appropriate to use tangential flow filtration (TFF), which allows for easy quality control of the exosomes and mass production. Accordingly, in the present invention, the exosomes were extracted using the above tangential flow filtration method.

[0055] Here, the tangential flow filtration method is a method in which a liquid sample is flowed parallel to the surface of a filter membrane to filter out particles larger than a certain size through the filter membrane while allowing small particles to pass through. This is an efficient and useful filtration method widely used for the separation and concentration of exosomes, proteins, viruses, and nanoparticles.

[0056] In addition, the tangential flow filtration method used in the present invention is a filtration method suitable for filtering out small impurities present in the solution and separating large exosomes, in which the solution flows perpendicular to the filtration membrane. Furthermore, when exosomes are separated from a cell culture medium, the cell culture medium is concentrated to 1 / 100 of its volume, resulting in a state containing exosomes at a high concentration.

[0057] In addition, the exosomes obtained by the above tangential flow filtration method are 1 x 10 per 1 ml volume 7 Up to 1X10 9 It has several particles.

[0058] In addition, in the above tangential flow filtration method, a TFF filter having 100,000 Da to 500,000 Da can be used.

[0059] The exosomes separated and extracted in the step of extracting exosomes (S110) can be stored in a freezer at a temperature of -70℃.

[0060] The step (S120) of adding an outer membrane to the extracted exosome can be performed by mixing lipid bilayer components identical to the exosome in a 2:1 ratio to add and form an outer membrane to the extracted exosome.

[0061]

[0062] The present invention will be explained in more detail below through specific manufacturing examples and embodiments. However, these manufacturing examples and embodiments are intended to illustrate the invention and the scope of the invention is not limited to these manufacturing examples and embodiments.

[0063]

[0064] [Example] Preparation of an exosome composition (A) formed with a double membrane, with improved penetration power and stability

[0065] (1) Exosomes are isolated and extracted from a culture medium of human dermal papilla cells cultured for 3 days using tangential flow filtration. The extracted exosomes are 1 x 10⁶ per 1 ml volume. 7 ~ 1X10 9 It has several particles.

[0066] (2) The size and number of particles of the separated exosomes are determined using a nanoparticle analyzer, and the dry weight of some exosomes is measured to determine the correlation between the number and weight of particles, 1X10 7It was confirmed that the number of exosome particles had an average weight of 1 μg, allowing the exosomes to be expressed by particle count or weight, and they were stored frozen at -70℃ until used in the examples.

[0067] (3) A lipid bilayer component identical to the exosome that was frozen is mixed in a 2:1 ratio at a temperature of 4°C for 24 hours to add and form an outer membrane to the exosome.

[0068]

[0069] [Comparative Example 1] Preparation of an exosome composition (B) formed as a double membrane with improved penetration power and stability

[0070] Compared to the preparation of the exosome composition (A) formed with a double membrane of the above example and having improved penetration power and stability, an exosome composition was prepared in the same manner as the example, except that lipid bilayer components identical to exosomes were mixed in a 2:1 ratio at a temperature of 10°C for 24 hours.

[0071]

[0072] [Comparative Example 2] Preparation of an exosome composition (C) formed as a double membrane with improved penetration power and stability

[0073]

[0074] Compared to the preparation of the exosome composition (A) formed with a double membrane of the above example and having improved penetration power and stability, an exosome composition was prepared in the same manner as the example, except that lipid bilayer components identical to exosomes were mixed in a 2:1 ratio at a temperature of 20°C for 24 hours.

[0075]

[0076] [Comparative Example 3] Preparation of an exosome composition (D) formed as a double membrane with improved penetration power and stability

[0077]

[0078] Compared to the preparation of the exosome composition (A) formed with a double membrane of the above example and having improved penetration power and stability, an exosome composition was prepared in the same manner as the example, except that lipid bilayer components identical to exosomes were mixed in a 2:1 ratio at a temperature of 4°C for 12 hours.

[0079]

[0080] [Comparative Example 4] Preparation of an exosome composition (E) formed as a double membrane with improved penetration power and stability

[0081]

[0082] Compared to the preparation of the exosome composition (A) formed with a double membrane of the above example and having improved penetration power and stability, an exosome composition was prepared in the same manner as the example, except that lipid bilayer components identical to exosomes were mixed in a 2:1 ratio at a temperature of 4°C for 18 hours.

[0083]

[0084] [Comparative Example 5] Preparation of an exosome composition (F) formed as a double membrane with improved penetration power and stability

[0085]

[0086] Compared to the preparation of the exosome composition (A) formed with a double membrane of the above example and having improved penetration power and stability, an exosome composition was prepared in the same manner as the example, except that lipid bilayer components identical to exosomes were mixed in a 2:1 ratio at a temperature of 10°C for 12 hours.

[0087]

[0088] [Comparative Example 6] Preparation of an exosome composition (G) formed as a double membrane with improved penetration power and stability

[0089]

[0090] Compared to the preparation of the exosome composition (A) formed with a double membrane of the above example and having improved penetration power and stability, an exosome composition was prepared in the same manner as the example, except that lipid bilayer components identical to exosomes were mixed in a 2:1 ratio at a temperature of 10°C for 18 hours.

[0091]

[0092] [Experimental Example] Preparation of an exosome composition formed with a double membrane

[0093]

[0094] Composition Example (A) Comparative Example 1 (B) Comparative Example 2 (C) Comparative Example 3 (D) Comparative Example 4 (E) Comparative Example 5 (F) Comparative Example 6 (G) Exosome 2222222 Lipid bilayer 1111111

[0095] [Table 1] above is a table showing the mixing ratio of lipid bilayer components identical to the exosomes.

[0096] Condition Example (A) Comparative Example 1 (B) Comparative Example 2 (C) Comparative Example 3 (D) Comparative Example 4 (E) Comparative Example 5 (F) Comparative Example 6 (G) Temperature (°C) 4 10 20 4 4 10 10 Hour 2 4 2 4 2 4 12 18 12 18

[0097] [Table 2] above is a table showing the time and temperature conditions under which the lipid bilayer component identical to the exosome is mixed.

[0098] Classification Example (A) Comparative Example 1 (B) Comparative Example 2 (C) Comparative Example 3 (D) Comparative Example 4 (E) Comparative Example 5 (F) Comparative Example 6 (G) Manufacturing Status Success Failure Failure Failure Failure Failure Failure

[0099] [Table 3] above is a table indicating whether the exosome composition formed with a double membrane was manufactured as a success or failure.

[0100] As can be seen in [Table 3] above, it was found that in order to prepare an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability, the lipid bilayer component identical to the exosome must be mixed in a 2:1 ratio at a temperature of 4°C for 24 hours so that an outer membrane (double membrane) can be formed on the exosome.

[0101]

[0102] FIG. 3 is a diagram showing the improved penetration power of an exosome composition formed with a double membrane according to one embodiment of the present invention, with improved penetration power and stability.

[0103] Referring to Fig. 3, the bottom two images highlighted by red boxes show that the penetration power of the exosome composition has been improved. Additionally, it can be seen that the amount of the exosome composition penetrating the artificial skin has improved by 6 times compared to conventional exosomes.

[0104] In addition, it can be confirmed that the stability of the above exosome has increased by more than 5 times compared to existing exosomes.

[0105]

[0106] Accordingly, the exosome composition formed with a double membrane according to one embodiment of the present invention, which has improved penetration power and stability, and the method for manufacturing the same, can solve the problem of insufficient penetration power and stability of conventional exosomes and provide excellent effects such as improved penetration power (significantly improved delivery efficiency to target tissues by forming a double membrane structure by adding an outer membrane composed of the same lipid bilayer component to the outer membrane of the exosome), enhanced structural stability (increased physical and chemical stability of the exosome, thereby strengthening resistance to changes in the external environment and increasing the half-life in the body), improved drug delivery efficiency (providing stability that can more safely protect bioactive substances such as drugs, proteins, and nucleic acids inside the exosome, and enabling precise delivery to target tissues), maintenance of biocompatibility (maintaining biocompatibility by utilizing lipid bilayer components and not inducing immune reactions or toxicity in the body), and increased commercial applicability (providing practical and innovative application possibilities in exosome-based cosmetics, skin treatments, hair loss treatments, precision medical technology, and drug delivery systems).

[0107]

[0108] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0109] The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and the concept of equivalents thereof should be interpreted as being included within the scope of the present invention.

Claims

1. An exosome composition having an added outer membrane, An exosome composition having improved penetration power and stability, characterized by being formed as a double membrane in which the outer membrane is composed of the same lipid bilayer component as the outer membrane of the exosome composition and forms a micelle structure.

2. In Paragraph 1, The above outer membrane is, An exosome composition formed as a double membrane characterized by forming a single membrane (one layer membrane), with improved penetration power and stability.

3. In Paragraph 1, The above lipid bilayer component is, An exosome composition formed as a double membrane with improved penetration power and stability, characterized by comprising one or more components selected from the group consisting of phospholipids, cholesterol, sphingolipids, and lipid rafts.

4. A method for preparing an exosome composition, Step of extracting exosomes; and A method for preparing an exosome composition formed of a double membrane with improved penetration power and stability, characterized by including the step of adding an outer membrane to the extracted exosome.

5. In Paragraph 4, The step of extracting the exosomes above is, A method for preparing an exosome composition with improved penetration power and stability, formed with a double membrane characterized by using tangential flow filtration (TFF).

6. In Paragraph 5, In the above tangential flow filtration method, A method for preparing an exosome composition formed with a double membrane having improved penetration power and stability, characterized by using a filtration filter having 100,000 Da to 500,000 Da.

7. In Paragraph 4, The step of adding an outer membrane to the extracted exosomes is, A method for preparing an exosome composition formed as a double membrane with improved penetration power and stability, characterized by mixing lipid bilayer components identical to the exosome in a 2:1 ratio.

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