Method for producing extracellular vesicles for wound healing using electroporation

Abstract

The present invention relates to a method for producing extracellular vesicles into which a therapeutic gene is introduced by electroporation, a wound-healing pharmaceutical composition produced by the method, and a wound-healing pharmaceutical preparation or quasi-drug preparation comprising same as an active ingredient. The extracellular vesicles derived from mesenchymal stem cells (MSCs), fibroblasts, and colostrum, produced by the present method, are all non-cytotoxic, have excellent nucleic acid delivery efficiency, and thus allow for stable expression. In particular, by introducing a vascular endothelial growth factor (VEGF) gene, which is known to have a wound-healing effect, into extracellular vesicles derived from mesenchymal stem cells (MSCs), fibroblasts, and colostrum through electroporation, VEGF gene-introduced extracellular vesicles produced by the production method of the present invention can be used as a gene therapy agent for wound healing.

Description

Method for producing extracellular vesicles for wound healing using electroporation

[0001] The present invention relates to a method for producing extracellular vesicles into which a therapeutic gene has been introduced using electroporation, a pharmaceutical composition for wound healing produced by the method, and a pharmaceutical preparation and quasi-drug preparation for wound healing containing the composition as an active ingredient.

[0002] All cells must exchange information with their surroundings and other cells in order to survive. To exchange information, cells secrete various substances outside the cells. In addition to soluble factors such as cytokines, hormones, and neurotransmitters, extracellular vesicles, also known as exosomes, have recently been attracting attention as a new type of intercellular information exchanger.

[0003] Extracellular vesicles (EVs) are microscopic particles ranging from several nm to several μm in size secreted from cells or cells into which genetic material has been introduced, and are surrounded by a lipid bilayer. Recently, various studies have been conducted on the important functions of EVs.

[0004] Secretion of extracellular vesicles is an evolutionarily conserved phenomenon across all living organisms, from bacteria to archaea to eukaryotes.

[0005] In particular, extracellular vesicles, which contain DNA, RNA, functional proteins, and antigens, are used as a new method of intracellular communication. Extracellular vesicles are known to contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. Because they are fundamentally cell-derived, extracellular vesicles are biocompatible, unlike other nanoparticles. Furthermore, they can be loaded with or labeled with drugs or biologically active ingredients inside or on their surface. Therefore, ongoing attempts are being made to use them as drug delivery vehicles or raw materials for cosmetics and pharmaceuticals.

[0006] However, development is difficult due to uneconomical productivity (high cost, low yield) and lack of efficient therapeutic agent loading technology, and it is difficult to diversify the administration route of extracellular vesicles, and especially, it is difficult to ensure the stability and safety of the substance when administered orally.

[0007] Viruses, lipid nanoparticles, and virus-like particles (VLPs) have been developed as delivery vehicles for gene therapy. However, existing virus-based delivery systems suffer from side effects, such as reduced efficacy due to immune responses to various viruses. Lipid nanoparticles, as synthetic materials, are prone to immune responses with repeated administration. Furthermore, naked gene delivery systems have very low delivery efficiency, limiting their use as therapeutics.

[0008]

[0009] The present invention relates to a method for producing extracellular vesicles into which therapeutic genes are introduced using electroporation, a pharmaceutical composition for wound healing produced by the method, and a pharmaceutical preparation and quasi-drug preparation for wound healing containing the composition as an active ingredient, and is intended to improve the above-mentioned problems.

[0010] Electroporation is a method that uses an external electric field to apply an electric shock to cells to temporarily create holes in the cell membrane in order to efficiently introduce external genetic materials such as DNA, RNA, and proteins into cells. It is a widely used experimental method because it can be applied to animal, plant, and microbial cells and allows for high-efficiency gene introduction.

[0011] When an electric field is applied to a cell, lipid molecules in the cell membrane shift and change position, creating nanometer-sized conductive paths composed of hydrophilic pores. When cells are suspended in a DNA solution and a high-voltage DC pulse is applied, pores are created in the cell membrane, and DNA molecules are introduced into the cell through electrophoresis.

[0012] To improve these problems, the inventors conducted extensive research and found that:

[0013] The present invention was designed to effectively deliver various types of genes (gene expression vectors, RNA, small RNA) by loading them into extracellular vesicles (EV) produced by cells in the body using electroporation, and to verify the therapeutic efficacy in various disease models.

[0014] In addition, extracellular vesicles isolated from milk and colostrum can be introduced into genes similar to extracellular vesicles isolated from cells and have therapeutic efficacy, so they were developed as oral gene delivery vehicles.

[0015]

[0016] Accordingly, the present invention

[0017] i) Step of preparing extracellular vesicles (EV);

[0018] ⅱ) A step of preparing the vascular endothelial growth factor (VEGF) gene, which is a therapeutic gene to be introduced into extracellular vesicles; and

[0019] ⅲ) A step of introducing a therapeutic gene, VEGF gene, into extracellular vesicles using electroporation;

[0020] The purpose of the present invention is to provide a method for producing an extracellular vesicle for wound healing, including:

[0021] In addition, the present invention aims to provide a pharmaceutical composition for wound healing, which comprises extracellular vesicles manufactured by the above manufacturing method as an active ingredient.

[0022] In addition, the present invention aims to provide a pharmaceutical preparation for wound healing, which comprises the pharmaceutical composition as an active ingredient.

[0023] In addition, the present invention aims to provide a wound healing quasi-drug preparation comprising the pharmaceutical composition as an active ingredient.

[0024] In order to achieve the above purpose, the present invention

[0025] i) Step of preparing extracellular vesicles (EV);

[0026] ⅱ) A step of preparing the vascular endothelial growth factor (VEGF) gene, which is a therapeutic gene to be introduced into extracellular vesicles; and

[0027] ⅲ) A step of introducing a therapeutic gene, VEGF gene, into extracellular vesicles using electroporation;

[0028] A method for producing an extracellular vesicle for wound healing, including:

[0029] In one embodiment of the present invention, the extracellular vesicles may be derived from nucleated cells or milk, and may be characterized in that they are not derived from bacteria.

[0030] In another embodiment of the present invention, the nucleated cells may be selected from the group consisting of mesenchymal stem cells (MSCs) and fibroblasts.

[0031] In another embodiment of the present invention, the mesenchymal stem cells may be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0032] In another embodiment of the present invention, the milk may be colostrum.

[0033] In another embodiment of the present invention, the electroporation method may be performed under conditions in which the voltage ranges from 1 volt to 3000 volts and the number of pulses ranges from 1 to 20.

[0034] In another embodiment of the present invention, the wound healing may be due to angiogenesis, in which the growth of vascular endothelial cells is promoted by VEGF.

[0035]

[0036] In addition, the present invention provides a pharmaceutical composition for wound healing, which comprises extracellular vesicles manufactured by the above manufacturing method as an active ingredient.

[0037] In one embodiment of the present invention, the pharmaceutical composition may be characterized in that it is in the form of an oral formulation.

[0038] In another embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0039] In another embodiment of the present invention, the pharmaceutical composition may further comprise an auxiliary component selected from the group consisting of cytokines, growth factors, and genes.

[0040] In another embodiment of the present invention, the wound healing may be due to angiogenesis, in which the growth of vascular endothelial cells is promoted by VEGF.

[0041]

[0042] In addition, the present invention provides a pharmaceutical preparation for wound healing, comprising the pharmaceutical composition as an active ingredient.

[0043] In one embodiment of the present invention, the formulation may be in the form of an injection, an infusion, a spray, a liquid or a patch.

[0044]

[0045] In addition, the present invention provides a wound healing quasi-drug preparation comprising the pharmaceutical composition as an active ingredient.

[0046]

[0047] In addition, the present invention provides a method for preventing or treating a wound, comprising a step of administering to a subject an extracellular vesicle manufactured by the above manufacturing method.

[0048] In addition, the present invention provides a use of extracellular vesicles manufactured by the above manufacturing method for preventing or treating wounds.

[0049] Mesenchymal stem cells (MSCs), fibroblasts, and colostrum-derived extracellular vesicles manufactured by this method are all non-cytotoxic and have excellent nucleic acid introduction efficiency, resulting in stable expression.

[0050] In particular, by introducing the vascular endothelial growth factor (VEGF) gene, which is known to have a wound healing effect, into mesenchymal stem cells (MSCs), fibroblasts, and colostrum-derived extracellular vesicles by electroporation, the VEGF gene-introduced extracellular vesicles manufactured by the manufacturing method of the present invention can be used as a gene therapy agent for wound healing.

[0051]

[0052] It should be understood that the effects of the present invention are not limited to the effects mentioned above, but include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0053] Figure 1 is a simplified schematic diagram of the entire process of the manufacturing method of the present invention.

[0054] Figure 2 shows the results of confirming the cytotoxicity of extracellular vesicles containing VEGF introduced by the manufacturing method of the present invention according to concentration.

[0055] Figure 3 is a cell image result confirming whether the gene is stably expressed in target cells after introducing the GFP gene into extracellular vesicles using the manufacturing method of the present invention.

[0056] Figure 4 is a cell image result confirming whether mRNA is stably expressed in target cells after introducing GFP mRNA into extracellular vesicles using the manufacturing method of the present invention.

[0057] Figure 5 shows the results of confirming whether VEGF is stably expressed in target cells after introducing a therapeutic gene (VEGF) into extracellular vesicles using the manufacturing method of the present invention.

[0058] Figure 6 shows the results of confirming the wound healing effect in target cells using a tube formation assay after introducing a therapeutic gene (VEGF) into extracellular vesicles using the manufacturing method of the present invention.

[0059] Hereinafter, the present invention will be described in detail.

[0060] The present invention

[0061] i) Step of preparing extracellular vesicles (EV);

[0062] ⅱ) A step of preparing the vascular endothelial growth factor (VEGF) gene, which is a therapeutic gene to be introduced into extracellular vesicles; and

[0063] ⅲ) A step of introducing a therapeutic gene, VEGF gene, into extracellular vesicles using electroporation;

[0064] A method for producing an extracellular vesicle for wound healing, including:

[0065] The above term "extracellular vesicle (EV)" refers to a substance surrounded by a lipid bilayer and a microscopic particle ranging from several nm to several μm in size secreted from a cell or a cell into which genetic material has been introduced. Recently, various studies are being conducted on the important functions of extracellular vesicles.

[0066] Secretion of extracellular vesicles is an evolutionarily conserved phenomenon across all living organisms, from bacteria to archaea to eukaryotes.

[0067] In particular, extracellular vesicles, which contain DNA, RNA, functional proteins, and antigens, are used as a new method of intracellular communication. Extracellular vesicles are known to contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. Because they are fundamentally cell-derived, extracellular vesicles are biocompatible, unlike other nanoparticles. Furthermore, they can be loaded with or labeled with drugs or biologically active ingredients inside or on their surface. Therefore, ongoing attempts are being made to use them as drug delivery vehicles or raw materials for cosmetics and pharmaceuticals.

[0068] The term "VEGF" refers to vascular endothelial growth factor (VEGF), a potent growth and angiogenic cytokine that stimulates the growth, differentiation, and survival of vascular endothelial cells, promoting angiogenesis and vascular permeability, and primarily functions to form new blood vessels. VEGF is known to play a crucial role in promoting angiogenesis and providing adequate blood flow to tissues and organs.

[0069] Meanwhile, the formation of new blood vessels during the wound healing process is regulated by various growth factors and cytokines secreted from cells surrounding the wound, including Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor beta (TGF-β), Platelet Derived Growth Factor (PDGF), and Epidermal Growth Factor (EGF).

[0070] In particular, VEGF is the most potent endothelial cell mitogen and is involved in new angiogenesis. It functions by binding to receptor kinases on endothelial cells to change vascular permeability and induce intracellular signaling that progressively forms blood vessels. VEGF is involved in angiogenesis in various physiological and pathological situations, such as embryogenesis, tumor growth, and wound healing. In particular, during the wound healing process, the promotion of angiogenesis by increased VEGF production can induce the creation and maturation of granulation tissue, increase elastic fibers, and cause rapid wound healing. Previous studies have shown that various drugs known to be effective in wound healing promote angiogenesis by increasing the expression of VEGF.

[0071] The above term "electroporation" refers to a method of applying an electric shock to cells using an external electric field to temporarily create holes in the cell membrane in order to efficiently introduce external genetic materials such as DNA, RNA, and proteins into cells. It is a widely used experimental method because it can be applied to animal, plant, and microbial cells and allows for high-efficiency gene introduction.

[0072] When an electric field is applied to a cell, lipid molecules in the cell membrane shift and change position, creating nanometer-sized conductive paths composed of hydrophilic pores. When cells are suspended in a DNA solution and a high-voltage DC pulse is applied, pores are created in the cell membrane, and DNA molecules are introduced into the cell through electrophoresis.

[0073] In one embodiment of the present invention, the extracellular vesicles may be derived from nucleated cells or milk, and may be characterized in that they are not derived from bacteria.

[0074] The term "eukaryotic cell" refers to a cell with a nucleus, surrounded by a nuclear membrane, and containing its genetic material. Eukaryotic cells are found in a wide variety of tissues and organisms, including many organisms such as animals, plants, and fungi. This contrasts with prokaryotic cells, which have a relatively simple cell structure.

[0075] The term "Milk" above is an opaque liquid containing proteins, fats, lactose, and vitamins and minerals produced by the mammary glands of mature mammals to provide nutrition to their young.

[0076] In another embodiment of the present invention, the nucleated cells may be selected from the group consisting of mesenchymal stem cells (MSCs) and fibroblasts.

[0077] The above term "mesenchymal stem cell (MSC)" refers to stem cells that exist in the stroma of cartilage, bone tissue, adipose tissue, and bone marrow, differentiated from the mesoderm formed by the division of a fertilized egg. In this case, the "mesenchymal stem cell" may be a mesenchymal stem cell derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, placenta, and other tissues, but is not limited thereto.

[0078] The above term "Fibroblast" is one of the cells that plays an important role in connective tissue and is found in various tissues such as skin, cartilage, bone, muscle, and blood vessel walls.

[0079] Fibroblasts are involved in the production of collagen and elastin fibers, extracellular matrix production, tissue regeneration and damage repair, and immune responses. These functions make them crucial for maintaining tissue health and stability. Furthermore, fibroblasts and their extracellular matrix are known to have therapeutic and regenerative applications in various research and application fields.

[0080] In another embodiment of the present invention, the mesenchymal stem cells may be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0081] The above term "umbilical cord" refers to the baby's umbilical cord.

[0082] The term "umbilical cord blood" refers to blood collected from the umbilical vein, which connects the placenta and the fetus. Umbilical cord blood is a natural byproduct of childbirth and is much easier to collect than bone marrow and other mesenchymal tissues, which require multiple surgeries. Furthermore, compared to bone marrow transplants, the cord blood storage industry has been developed, and the infrastructure has already been established, making it easier to find donors. Furthermore, cord blood-derived cells do not express the human leukocyte antigen (HLA-DR) class II, the most important cause of rejection in tissue or organ transplants. Therefore, they can minimize or eliminate immune responses, such as rejection, that have been problematic in conventional transplant surgeries.

[0083] The term "bone marrow" refers to a flexible tissue located in the inner space of the bone, and is also a hematopoietic organ that produces most of the blood in adults. Bone marrow is divided into red bone marrow and white bone marrow according to the ratio of its constituent cells. Red bone marrow is composed mostly of hematopoietic cells, and white bone marrow is composed mostly of adipose tissue.

[0084] The term "fat" is one of the three major nutrients, along with carbohydrates and proteins. Fat is mainly composed of carbon and hydrogen atoms, so it is hydrophobic, soluble in organic solvents, and insoluble in water. Fat is one of the types of lipids, along with phospholipids and cholesterol.

[0085] The term "muscle" refers to both tendons and flesh, and is the organ responsible for animal movement. Functionally, it is divided into voluntary skeletal muscle and involuntary visceral muscle, and structurally, it is divided into striated and smooth muscle. Muscles are divided into three types: skeletal muscle, cardiac muscle, and smooth muscle, based on their shape, cell signaling pathways, the way in which contractile force changes, contraction patterns (rhythmic or differential), and the role of the nervous system in muscle function.

[0086] The term "nerve" refers to the organ that allows organisms to sense and respond to their surrounding environment and stimuli. It constitutes the nervous system, particularly the peripheral nervous system, and consists of multiple axons of nerve cells bundled together. Because a single nerve primarily consists of axons originating from adjacent nerve cells, it often transmits only specific information.

[0087] The term "skin" refers to the largest tissue in the integumentary system, composed of numerous epithelial tissues that protect the muscles and organs within the body. When exposed to the external environment, the skin plays a crucial role in protecting the body from pathogens. Other key functions of the skin include insulation, temperature regulation, sensory functions, and the synthesis of vitamin D and the protection of vitamin B folates.

[0088] The term "amnion" refers to the membrane that covers the embryo, covering its outer surface within the uterus. The amniotic sac is filled with a fluid called amniotic fluid, which expands the amniotic sac to form a sac called the amniotic sac, providing a protective environment for the embryo as it grows.

[0089] The term "placenta" refers to a reproductive organ in mammals that transfers maternal nutrients to the fetus and fetal waste products to the mother during development. The placenta is formed through the differentiation and division of embryonic trophectoderm implanted in the maternal endometrium, the subsequent differentiation of endometrial basal cells and vascular cells (endocytes), and the growth of spiral arteries.

[0090] In another embodiment of the present invention, the milk may be colostrum.

[0091] The above term "Colostrum" refers to milk secreted from the end of pregnancy to a few days after birth in mammals. Unlike regular milk, it is thicker and contains more nutrients (protein, fat, ash, lactose, etc.) and antibodies (IgG, IgA, IgM) necessary for survival and growth, and is characterized by a yellowish color.

[0092] Also, human colostrum has a slightly yellowish tint compared to breast milk secreted later, and is produced in smaller quantities, but it contains a lot of lactoferrin, which has an antibacterial effect that kills bacteria such as Bacillus, E. coli, and Salmonella, and secretory immunoglobulin A, an immunoglobulin that prevents bacterial invasion from outside the body, helping with immunity and sterilization in the newborn's throat and digestive organs.

[0093] In another embodiment of the present invention, the electroporation method may be performed under conditions in which the voltage ranges from 1 volt to 3000 volts and the number of pulses ranges from 1 to 20.

[0094] In another embodiment of the present invention, the wound healing may be due to angiogenesis, in which the growth of vascular endothelial cells is promoted by VEGF.

[0095] The term "angiogenesis" above refers to the phenomenon in which endothelial cells of existing blood vessels degrade the extracellular matrix, migrate, divide, and differentiate to form new capillaries, and is a normal and important process in growth and development, wound healing, and granulation tissue formation.

[0096] Angiogenesis is primarily driven by various angiogenic proteins, such as integrins and prostaglandins, along with growth factors such as vascular endothelial growth factor (VEGF), which stimulate endothelial cells to form new blood vessels.

[0097] Angiogenesis is an essential physiological process of wound healing, and by assessing the effects of angiogenesis, wound healing can be verified. In other words, angiogenesis is essential for the wound healing process, which is essential for the regeneration of injured skin tissue. In the initial stages of wound healing, an inflammatory response occurs due to cell necrosis and blood vessel destruction. This inflammatory response is followed by a series of events, including devascularization of blood components, platelet activation, and blood coagulation, along with the formation of biological mediators such as kallikrein, thrombin, and plasmin.

[0098]

[0099] In addition, the present invention provides a pharmaceutical composition for wound healing, which comprises extracellular vesicles manufactured by the above manufacturing method as an active ingredient.

[0100] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by a person skilled in the art.

[0101] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0102] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitantly administered drugs, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0103] In one embodiment of the present invention, the pharmaceutical composition may be characterized in that it is in the form of an oral formulation.

[0104] In another embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0105] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in formulations, and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further comprise other conventional additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated using the methods disclosed in Remington's literature.

[0106] In another embodiment of the present invention, the pharmaceutical composition may further comprise an auxiliary component selected from the group consisting of cytokines, growth factors, and genes.

[0107] The term "cytokine" refers to a relatively small immune protein found in the bloodstream. It is a broad, loosely packed protein secreted by immune cells and plays a crucial role in cell signaling. After being secreted, cytokines can affect other cells or the cells themselves.

[0108] The above term "growth factor" refers to a protein that affects the survival, growth, differentiation, and maintenance of cell function, and various growth factors interact with each other in physiological and developmental processes to help normal development and maintenance of tissues.

[0109] The above term "gene" is the basic unit of heredity, and genes contain the information necessary to form and maintain the cells of an organism and to form organic relationships between them, and are passed down to offspring through reproduction.

[0110] In modern genetics, a gene is defined as "a segment of the genome sequence that constitutes a unit of heredity." Within the genome sequence, a gene constitutes a segment of DNA sequence and is comprised of regulatory regions, transcription regions, and other functional regions.

[0111] In another embodiment of the present invention, the wound healing may be due to angiogenesis, in which the growth of vascular endothelial cells is promoted by VEGF.

[0112] Angiogenesis is primarily driven by various angiogenic proteins, such as integrins and prostaglandins, along with growth factors such as vascular endothelial growth factor (VEGF), which stimulate endothelial cells to form new blood vessels.

[0113] Angiogenesis is an essential physiological process of wound healing, and by assessing the effects of angiogenesis, wound healing can be verified. In other words, angiogenesis is essential for the wound healing process, which is essential for the regeneration of injured skin tissue. In the initial stages of wound healing, an inflammatory response occurs due to cell necrosis and blood vessel destruction. This inflammatory response is followed by a series of events, including devascularization of blood components, platelet activation, and blood coagulation, along with the formation of biological mediators such as kallikrein, thrombin, and plasmin.

[0114]

[0115] In addition, the present invention provides a pharmaceutical preparation for wound healing, comprising the pharmaceutical composition as an active ingredient.

[0116] The term "preparations" above refers to products made in a specific form according to a prescription, such as tablets, capsules, or suppositories containing active ingredients, and are final products that are actually administered. Preparations come in various dosage forms.

[0117] In one embodiment of the present invention, the formulation may be in the form of an injection, an infusion, a spray, a liquid or a patch.

[0118] The above term "injection" usually refers to a solution, suspension, emulsion, or solid sterile preparation that is directly administered to body tissues or organs such as subcutaneously, intramuscularly, or into blood vessels, or that is dissolved or suspended in a solvent when used.

[0119] The above term "injectable" refers to a medicine that is inserted into the body through the urethra, anus, vagina, etc. and is dissolved by body temperature or secretions before its effect is revealed.

[0120] The above term "aerosol" refers to a medicine that is sprayed like a mist using a device.

[0121] The above term "liquid formulation" refers to a pharmaceutical form that provides the drug in liquid form, and refers to a formulation that contains the active ingredient in liquid form.

[0122] The term "patch" refers to a preparation that adheres to the skin and provides sustained therapeutic effects. Patches have a lower risk of adverse effects, such as gastrointestinal or liver damage, due to oral medications. They can be administered to patients who have difficulty taking oral medications. Furthermore, they are absorbed directly into the bloodstream through the skin, bypassing liver metabolism. Therefore, compared to oral medications, they can be administered in lower doses while maintaining the same efficacy without adverse effects.

[0123]

[0124] In addition, the present invention provides a wound healing quasi-drug preparation comprising the pharmaceutical composition as an active ingredient.

[0125] The above term “quasi-drug” refers to any of the following products designated by the Commissioner of the Ministry of Food and Drug Safety: i) fibers, rubber products or similar products used for the purpose of treating, alleviating, managing or preventing diseases of humans or animals; ii) products that have a weak effect on the human body or do not directly affect the human body and are not instruments or machines or similar products; and iii) preparations used for sterilization, insecticide and similar purposes to prevent infectious diseases.

[0126]

[0127] To facilitate understanding of the present invention, the following examples will be described in more detail. However, these examples are intended only to exemplify the content of the present invention and are not intended to limit the scope of the present invention. These examples are provided to more fully explain the present invention to those with average knowledge in the technical field to which the invention pertains.

[0128]

[0129] [Example 1] Preparation of extracellular vesicles containing genes using electroporation

[0130] 1. Isolation of extracellular vesicles

[0131] (1) Method for isolating extracellular vesicles in cell culture medium

[0132] Mesenchymal stem cells (MSC) and fibroblasts (1X10) 7 Using the Tangential Flow Filtration (TFF) method in serum-free conditioned media cultured for more than 10 10 Separated at a concentration of particle / ml or more.

[0133] (2) Method for isolating extracellular vesicles in colostrum

[0134] After slowly thawing frozen colostrum at low temperature, the fat is removed using centrifugation, and after acid treatment and milk protein removal, the tangential flow filtration (TFF) method is used to remove 10 10 Separated at a concentration of particle / ml or more.

[0135]

[0136] 2. Preparation of the GFP gene to be introduced into extracellular vesicles

[0137] The GFP (Green Fluorescent Protein) vector is a GFP gene expression vector measuring approximately 8 Kb. Plasmid DNA was isolated by introducing it into Escherichia coli and culturing the cells in large quantities. A plasmid DNA isolation kit was used for this purpose, and absorbance values ​​were measured to quantify the amount of isolated DNA. GFP mRNA was purchased commercially and used in the experiments.

[0138]

[0139] 3. Gene introduction into extracellular vesicles using electroporation

[0140] For the separated extracellular vesicles, the voltage range is adjusted from 1 volt to 3000 volts, and the pulse number is set from 1 to 20 times, and the GFP or VEGF gene is introduced into the extracellular vesicles using an electroporation device such as NEPA at 100 volts and 3 pulses.

[0141]

[0142] [Example 2] Confirmation of the effectiveness of extracellular vesicles containing genes using electroporation.

[0143] 1. Concentration-dependent cytotoxicity of extracellular vesicles containing the VEGF gene

[0144] To determine the cytotoxicity according to the concentration of extracellular vesicles into which the VEGF gene was introduced using electroporation, the extracellular vesicles were 10 6 particles / ml to 10 9 MRC-5 cells (human fibroblasts) were treated at a concentration of 100 particles / ml. Cytotoxicity tests were conducted using extracellular vesicles isolated from mesenchymal stem cells (MSCs), fibroblasts, and colostrum.

[0145] The viability of cells was measured using the CCK-8 assay after 24 hours, as shown in Fig. 2. 9 It was confirmed that there was no cytotoxicity up to a particle / ml concentration, and that cell growth increased as the concentration increased.

[0146]

[0147] 2. Confirmation of gene expression after introducing the GFP gene into extracellular vesicles

[0148] After introducing a gene expression vector containing a cloned GFP gene into extracellular vesicles isolated from mesenchymal stem cells (MSCs), fibroblasts, and colostrum using electroporation, 1 × 107 The test was conducted by treating MRC-5 cells (human fibroblasts) and HEK293 cells (Human Embryonic Kidney 293, human embryonic kidney cells) at a particle / ml concentration.

[0149]

[0150] As shown in Fig. 3, the degree of intracellular GFP expression was confirmed using a fluorescence microscope 48 hours later, and GFP was confirmed to be expressed in MRC-5 cells and HEK293 cells compared to the untreated control group.

[0151] These results indicate that DNA can be introduced into extracellular vesicles via a GFP expression vector using electroporation, that DNA can be delivered into cells via extracellular vesicles, and that gene expression can be stably achieved.

[0152]

[0153] 3. Confirmation of gene expression after introduction of GFP mRNA into extracellular vesicles

[0154] GFP mRNA was introduced into extracellular vesicles isolated from mesenchymal stem cells (MSCs), fibroblasts, and colostrum using electroporation, and 1 × 10 7 The test was conducted by treating MRC-5 cells (human fibroblasts) at a particle / ml concentration.

[0155] As shown in Fig. 4, the degree of GFP expression in MRC-5 cells was confirmed using a fluorescence microscope 24 hours later, and it was confirmed that GFP was expressed in MRC-5 cells compared to the untreated control group.

[0156] These results indicate that GFP mRNA can be introduced into extracellular vesicles using electroporation, that RNA can be delivered into cells via extracellular vesicles, and that gene expression can be stably achieved.

[0157]

[0158] 4. Analysis of the amount of VEGF protein in the medium after treatment with extracellular vesicles containing the therapeutic gene (VEGF)

[0159] After introducing a gene expression vector containing the therapeutic gene VEGF into the extracellular vesicles isolated from mesenchymal stem cells (MSCs), fibroblasts, and colostrum, respectively, using electroporation, 1 × 10 8 The test was conducted by treating MRC-5 cells (human fibroblasts) at a particle / ml concentration.

[0160] After 48 hours, the amount of VEGF protein expression in the cell culture medium was quantitatively measured using the Enzyme-Linked Immunosorbent Assay (ELISA) method.

[0161] As a result, as shown in Fig. 5, it was confirmed that the amount of VEGF expression in the culture medium increased when the extracellular vesicles into which the therapeutic gene (VEGF) was introduced were treated compared to the untreated control group.

[0162] These results indicate that introduction of a therapeutic gene (VEGF) using electroporation promotes VEGF expression within cells, thereby promoting wound healing.

[0163]

[0164] 5. Confirmation of wound healing effect through HUVEC tube formation assay after treatment with extracellular vesicles containing therapeutic gene (VEGF)

[0165] After introducing a gene expression vector containing the therapeutic gene VEGF into the extracellular vesicles isolated from mesenchymal stem cells (MSCs), fibroblasts, and colostrum, respectively, using electroporation, 1 × 10 8The test was conducted by treating HUVEC (Human Umbilical Vein Endothelial Cells) at a particle / ml concentration.

[0166] After 48 hours, a tube formation test was conducted using HUVECs, and the results of the experiment were confirmed by analyzing the degree to which HUVECs grew densely like a net through a microscope.

[0167] As a result, as shown in Fig. 6, it was confirmed that tube formation occurred more in HUVECs treated with extracellular vesicles introduced with therapeutic genes (VEGF) compared to the untreated control group.

[0168] These results indicate that introduction of a therapeutic gene (VEGF) using electroporation promotes wound healing by promoting vascular tube formation in vascular endothelial cells (HUVECs).

[0169]

[0170] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. ⅰ) Step of preparing extracellular vesicles (EV); ⅱ) A step of preparing the vascular endothelial growth factor (VEGF) gene, which is a therapeutic gene to be introduced into extracellular vesicles; and ⅲ) A step of introducing a therapeutic gene, VEGF gene, into extracellular vesicles using electroporation; A method for producing an extracellular vesicle for wound healing, comprising:

2. In claim 1, A manufacturing method characterized in that the extracellular vesicles are derived from nucleated cells or milk and not from bacteria.

3. In claim 2, A manufacturing method characterized in that the above nucleated cells are selected from the group consisting of mesenchymal stem cells (MSCs) and fibroblasts.

4. In claim 3, A manufacturing method, characterized in that the above mesenchymal stem cells are derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane or placenta.

5. In claim 2, A manufacturing method characterized in that the above milk is colostrum 6. In claim 1, A manufacturing method characterized in that the above electroporation method is performed under conditions in which the voltage is 1 volt to 3000 volts and the number of pulses is 1 to 20.

7. In claim 1, A manufacturing method characterized in that the above wound healing is due to angiogenesis, in which the growth of vascular endothelial cells is promoted by VEGF.

8. A pharmaceutical composition for wound healing, comprising as an active ingredient an extracellular vesicle manufactured by the manufacturing method of any one of claims 1 to 7.

9. In claim 8, A pharmaceutical composition, characterized in that the above pharmaceutical composition is in the form of an oral formulation.

10. In claim 8, A pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

11. In claim 8, A pharmaceutical composition, characterized in that the pharmaceutical composition further comprises an auxiliary ingredient selected from the group consisting of cytokines, growth factors, and genes.

12. In claim 8, A pharmaceutical composition characterized in that the above wound healing is due to angiogenesis, in which the growth of vascular endothelial cells is promoted by VEGF.

13. A pharmaceutical preparation for wound healing, comprising the pharmaceutical composition of claim 8 as an active ingredient.

14. In claim 13, A pharmaceutical preparation characterized in that the above preparation is in the form of an injection, infusion, spray, liquid or patch.

15. A wound healing quasi-drug preparation comprising the pharmaceutical composition of claim 8 as an active ingredient.

16. A method for preventing or treating a wound, comprising a step of administering to a subject an extracellular vesicle manufactured by the manufacturing method of any one of claims 1 to 7.

17. Use of an extracellular vesicle manufactured by the manufacturing method of any one of claims 1 to 7 for preventing or treating wounds.

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