Method for inducing extracellular vesicles in stem cells using focused ultrasound

Abstract

In conventional methods for isolating extracellular vesicles (EVs), extracellular vesicle secretion is induced for 24 hours or more using a medium containing EV-depleted FBS. In this process, it is difficult to isolate pure extracellular vesicles due to the presence of various waste products, proteins, cytokines, animal-derived serum components, and the like secreted by growing cells. In addition, since the secretion of the extracellular vesicles takes a long time, mother cells can be exposed to various environments, thereby increasing the possibility of secreting non-uniform extracellular vesicles. The present invention stimulates stem cells using ultrasonic energy, and can induce extracellular vesicle secretion through fluid shear stress in the stem cells. In particular, by inducing extracellular vesicles by stimulating the stem cells for a short period of no longer than 10 minutes, it is possible to obtain relatively homogeneous extracellular vesicles, and the same technique can be applied to various types of stem cells to extract extracellular vesicles having different functions depending on the objective, thus providing limitless opportunities for application.

Description

A method for inducing extracellular vesicles in stem cells using focused ultrasound

[0001] The present invention relates to a method for inducing secretion of extracellular vesicles in stem cells using focused ultrasound.

[0002]

[0003] Stem cell therapy is widely studied for tissue regeneration, and its therapeutic mechanism is known to be mediated by various paracrine factors secreted by stem cells. Extracellular vesicles (EVEs) are attracting attention as mediators for the delivery of these secreted factors. With the recent advancement of separation and analysis techniques and a deeper understanding of EVEs, stem cell EVE-based therapies are being studied in various fields.

[0004] Extracellular vesicles (EVEs) are divided into exosomes and microvesicles based on their secretion mechanism. Exosomes are known to be produced by the cell's endosomal system, while microvesicles are formed by budding of the cell membrane. EVEs are known to share cellular characteristics while being secreted from cells. Exploiting these properties, attempts are being made to isolate EVEs from mesenchymal stem cells, known for their immune-modulating and regenerative effects, and apply them to therapeutic applications.

[0005] Stem cell therapy is a promising field that continues to attract attention within biotechnology, but it still faces many challenges. There are concerns that transplanted stem cells can form abnormal tissues, and cases of stem cells transplanted into tiny blood vessels, such as pulmonary arteries, causing blockages have been reported. Furthermore, autologous stem cells present challenges in obtaining sufficient cells to achieve therapeutic efficacy, and the use of allogeneic stem cells carries the risk of immune rejection. However, utilizing stem cell-derived extracellular vesicles (ESVs) allows for the therapeutic benefits of stem cells while avoiding the potential risks associated with cell transplantation, as the cells themselves are not directly harvested. Furthermore, because EVs are a byproduct of cell culture, they can create new added value within the existing stem cell industry.

[0006] Currently, laboratories around the world are isolating extracellular vesicles from various stem cells and applying them to treat various diseases.

[0007]

[0008] One object of the present invention is to provide a method for inducing secretion of extracellular vesicles in cells separated from a subject by treating the cells separated from the subject with focused ultrasound.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune-related diseases, comprising extracellular vesicles obtained by the above method.

[0010] Another object of the present invention is to provide a wound treatment composition comprising extracellular vesicles obtained by the above method.

[0011] Another object of the present invention is to provide a regenerative composition comprising extracellular vesicles obtained by the above method.

[0012] Another object of the present invention is to provide a focused ultrasound device used to induce secretion of extracellular vesicles, comprising a function generator; an amplifier; a coupler; and a transducer.

[0013]

[0014] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0015]

[0016] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0017]

[0018] 1. Method for inducing secretion of extracellular vesicles

[0019] In one embodiment of the present invention, a method is provided for inducing secretion of extracellular vesicles in cells separated from a subject by treating the cells separated from the subject with focused ultrasound.

[0020]

[0021] The “focused ultrasound or FUS” of the present invention can be divided into high-intensity focused ultrasound (HIFU) and low-intensity focused ultrasound (LIFU) depending on the intensity. High-intensity focused ultrasound is used for direct treatment such as necrosis of living tissues such as cancer cells, tumors, and lesions, whereas low-intensity focused ultrasound is known to be able to obtain medical effects without necrosis of body tissues. In the present invention, it is preferable to use “low-intensity ultrasound”, but the present invention is not limited thereto.

[0022] Although the standard for the type of ultrasound of the present invention has not yet been clearly defined, generally, “low-intensity ultrasound” means ultrasound that has an acoustic intensity of less than 3 W / ㎠ of the time-averaged highest peak acoustic intensity (Ispta) in accordance with the US FDA standards and European safety standards, and is in a range that does not cause damage to the body, and ultrasound that has a time-averaged highest peak acoustic intensity of 3 W / ㎠ or more can be classified as “high-intensity ultrasound”, and in the present invention, it is preferable to use “low-intensity ultrasound”, but is not limited thereto.

[0023] In addition, in the present invention, the “focused ultrasound (FUS)” is largely classified into high-intensity focused ultrasound (HIFU) and low-intensity focused ultrasound (LIFUS) depending on the intensity. HIFU generates a temperature high enough to denature proteins and coagulate tissues, and is frequently used to remove fibroids, cancers, or skull tumors. Unlike HIFU, LIFUS can temporarily and reversibly open the BBB when used with microbubbles. There are also research results showing that LIFUS promotes differentiation of pluripotent stem cells, resulting in neurogenesis. In particular, one study reported that only LIFUS conditions sufficient to induce and regulate an increase in BBB permeability can induce neurogenesis.

[0024]

[0025] In the present invention, the "extracellular vesicles" or "extracellular vesicles" or "extracellular vesicles (EVs)" are nano-sized vesicles secreted by all cells into the external environment for the exchange of information between cells. EVs contain various biologically active substances, such as proteins, lipids, nucleic acids, and metabolites. Recently, the substances constituting EVs have been identified through high-throughput analysis. Furthermore, EVs reflect the state of the cells they originate from and have been found in various body fluids. EVs interact with target cells to perform physiological functions in normal situations, as well as pathological functions that induce diseases. Recently, research into the diagnosis and treatment of diseases using EVs has also been actively conducted.

[0026] Extracellular vesicles are called by various names such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles based on their origin, secretion mechanism, and size, but the extracellular vesicles in the present invention are not limited to these. Mammalian extracellular vesicles are classified into two types: extracellular vesicles, which are created when multivesicular endosomes (MVEs) mature, and intraluminal vesicles are formed when the endosomal membrane inwardly moves, and then the multivesicular endosomes fuse with the cell surface and the intraluminal vesicles are secreted out of the cell, and microvesicles, which are secreted out of the cell when the plasma membrane protrudes outward and separate. However, it is difficult to distinguish between them once they are secreted out of the cell.

[0027] In addition, extracellular vesicles (EVEs) contain various biologically active substances such as proteins, lipids, nucleic acids, and metabolites, and reflect the state of the cells from which they are derived. In addition, EVEs exist in various body fluids such as cell culture media, blood, urine, saliva, tears, semen, breast milk, and ascites, and perform various physiological and pathological functions. For example, EVEs derived from cancer cells affect cancer growth, metastasis, and angiogenesis, while EVEs derived from bacteria are involved in antibiotic resistance, elimination of competing bacteria, delivery of virulence factors, and host cell regulation.

[0028] Extracellular vesicles (EVs) have recently attracted attention because of their potential for disease diagnosis and treatment. Recent efforts are focused on utilizing noninvasive liquid biopsies for disease diagnosis. One approach involves using EVs, which can be isolated from various bodily fluids, to discover new diagnostic markers and use them to diagnose diseases. Furthermore, because EVs perform physiological functions and can be delivered to specific cells or tissues, they can be utilized as therapeutic agents, drug delivery systems, and vaccines.

[0029] Furthermore, the treatment using extracellular vesicles (ECVs) has gained attention due to the discovery that EVCs are a crucial component of mesenchymal stem cell (MSC)-mediated cardioprotection. Subsequent studies using EVCs to regenerate heart and liver tissue have demonstrated numerous advantages over living cells for tissue regeneration. Because EVCs are not living cells, they can be stored and transported for long periods of time. Furthermore, because they do not divide, mammalian EVCs pose a lower risk of tumorigenesis, while bacterial EVCs pose a lower risk of infection. Furthermore, EVCs offer several advantages over other nanocarriers. Because they are derived from cells, they are less immunogenic. Furthermore, because they share the same membrane topology as cells, they can be genetically engineered to express various membrane proteins, enabling the delivery of drugs and vaccines to specific cells or tissues. Furthermore, because extracellular vesicles possess a membrane and lumen, they can be loaded with various drugs, allowing them to be easily delivered to target cells through the cell membrane. However, because dying cells producing extracellular vesicles can secrete apoptotic bodies or intracellular components, managing the survival rate of extracellular vesicle-producing cells is crucial.

[0030] However, because the amount of naturally secreted extracellular vesicles is limited, treatments using extracellular vesicle mimics are being attempted. To manufacture extracellular vesicle mimics, a method can be used in which cells are treated with drugs to load them, extrusion is performed, and only the drug-loaded extracellular vesicle mimics are separated through density gradient ultracentrifugation. Extracellular vesicle mimics are attracting attention as an alternative to extracellular vesicle-based treatments because they have a much higher yield and similar drug delivery efficiency to naturally secreted extracellular vesicles. Based on this, there is a study that used cells and vesicle mimics created from pancreatic beta cells to create insulin-producing cells from bone marrow-derived cells in mice.

[0031]

[0032] In addition, extracellular vesicles can be separated from various biological materials, such as blood, obtained from a subject according to a method known in the art. The extracellular vesicles can be separated by any separation method known in the art. For example, there are many methods for separating extracellular vesicles, but the precipitation method using polyethylene glycol (PEG), differential ultra-centrifugation (UC), ultrafiltration, and tangential flow filtration (TFF) are the most commonly used because they can obtain a large amount of extracellular vesicles. However, residual proteins and substances other than extracellular vesicles are separated together, resulting in poor purity. In order to obtain high-purity extracellular vesicles, separation methods such as size exclusion chromatography (SEC), density gradient ultracentrifugation (Gradient UC or density gradient ultracentrifugation), and affinity isolation are applied. However, despite the high purity, the amount of extracellular vesicles obtained is small, making it difficult to enter non-clinical and clinical applications. When selecting a separation method, factors to consider include sufficient yield of extracellular vesicles, therapeutic effect, and purity. The most important of these are sufficient quantity and clear therapeutic effect. Purity should be managed so that it does not lead to other side effects and stability issues.

[0033]

[0034] Qualitative and quantitative analyses are key to managing the quality and properties of EVE therapeutics. To measure the total amount of EVEs, the number of EVE particles or the total protein content are measured. The most commonly used method for measuring particle counts is nanoparticle tracking analysis (NTA). NTA has the advantage of measuring both the size distribution and the number of EVEs. However, it has the disadvantage of having a large error value because aggregates of substances other than EVEs can be detected as particles during measurement, and EVEs can also be clumped together. Even when measuring total protein content, there is often no strong correlation with the number of particles.

[0035]

[0036] In addition, tetraspanins such as CD63, CD81, and CD9 are known as surface proteins of extracellular vesicles, and these are representative markers for identifying extracellular vesicles. To measure the amount of these markers, it is possible to measure it using a specific antibody binding method using the western blotting method, but it is close to semi-quantitative and requires a considerable amount of extracellular vesicles, more than 5 μg. Most enzyme-linked immunosorbent assay (ELISA) kits on the market can detect significant values ​​from nanogram-scale proteins. In addition, flow cytometry, which has been used in the existing cell therapy field, can only analyze sizes greater than 500 nm, so there are some limitations in analyzing extracellular vesicles. To overcome this, methods are being developed to capture molecules expressed on the surface of extracellular vesicles by binding antibodies to beads of about 5 um or 9 um and analyze them using a flow cytometer.

[0037]

[0038] In addition, cells or stem cells can be isolated from various biological materials, such as blood, obtained from a subject according to methods known in the art. The cells or stem cells can be isolated using any conventionally known separation method. Examples include density gradient fractionation, immunoselection, and differential adhesion separation. In particular, all methods previously used, including the method of Republic of Korea Patent Application No. 10-2009-0023821, can be used for isolating and culturing stem cells from umbilical cord blood.

[0039] In addition, the culture of the cells or stem cells separated above can be performed in a cell culture medium known in the art, and examples thereof include, but are not limited to, DMEM medium, McCoys 5A medium, Eagles' basal medium, CMRL medium, Glasgow's minimal essential medium, Ham's F-12 medium, Iscove's modified Dulbecco's medium, Liebovitz' L-15 medium, RPMI 1640 medium, etc. In addition, in the present invention, one or more auxiliary components may be added to the cell culture medium as needed, and may include fetal bovine serum, horse or human serum, as well as antibiotics and antifungal agents to prevent microbial contamination.

[0040] Isolated or cultured cells or stem cells can be stored by methods known in the art until use. Generally, cells or stem cells can be frozen and stored after cryoprotection. The cryoprotection can be performed using cryoprotectants known in the art, such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, or choline chloride.

[0041]

[0042] In another embodiment of the present invention, a method for inducing secretion of extracellular vesicles is provided, wherein the cell is a stem cell.

[0043] In the present invention, the term “stem cell” refers to undifferentiated cells before they are differentiated into each cell constituting a tissue, and differentiation into specific cells proceeds through specific differentiation stimuli (environments). Unlike differentiated cells that have stopped dividing, stem cells have the characteristic of proliferation (expansion) by being able to produce cells identical to themselves through cell division (self-renewal). In addition, when a differentiation stimulus is applied, they differentiate into specific cells, but can also differentiate into other cells under different environments or different differentiation stimuli, and are characterized by having flexibility in differentiation (plasticity). These stem cells can be divided into embryonic stem cells and adult stem cells depending on their origin. In the present invention, it is preferable to use adult stem cells rather than embryonic stem cells, which have many biological, ethical, and legal issues and thus have many limitations in clinical application.

[0044] In the present invention, the "adult stem cell" is a stem cell extracted from grown body tissue, and is a primitive cell just before differentiating into cells of a specific organ. Adult stem cells have difficulty in proliferation and a strong tendency to differentiate easily, but can differentiate into tissue-specific progenitor cells inherent in the human body. Adult stem cells can differentiate into cells with various characteristics and have the ability to generate replacement cells for widely located tissues and organs, such as the heart, pancreas, nervous tissue, muscle, and cartilage.

[0045] In the present invention, the "neural stem cells" refer to cells that can perform cell division 20-30 times or more and maintain differentiation potency to produce neurons and glial cells. Preferably, the cells can perform cell division 40 times or more, more preferably 50 times or more, and most preferably unlimitedly. The neural stem cells are defined as multipotent, that is, they can differentiate into multiple neural cell types (e.g., neurons / glial cells). Neural stem cells can be obtained by primary culture of tissues of the central nervous system (CNS) and peripheral nervous system (PNS), and these cells differentiate into glial lineage and neural lineage under specific differentiation conditions.

[0046]

[0047] In another embodiment of the present invention, a method for inducing secretion of extracellular vesicles is provided, wherein the cell is a mesenchymal stem cell.

[0048] In the present invention, the above-mentioned “mesenchymal stem cell” or “mesenchymal stem cell” is a stromal cell with multipotency (a cell that surrounds and supports cells or tissues (parenchyma) that perform their functions in organs such as the thymus or bone marrow) and can differentiate into various cells including osteoblasts (bone cells), chondrocytes, muscle cells, and adipocytes (adipocytes that form bone marrow adipose tissue). The above-mentioned mesenchymal stem cell has an excellent ability to self-renew while maintaining multipotency. In particular, the mesenchymal stem cell appears to differentiate even into cells such as neurons.

[0049] Numerous studies have shown that human mesenchymal stem cells avoid transplant antigen recognition (the recognition of transplanted antigens as autologous or foreign, resulting in an allogenic response or rejection), interfere with the function of dendritic cells and T cells, and create a local immunosuppressive microenvironment by secreting cytokines. In addition, mesenchymal stem cells have immunomodulatory abilities such as suppressing the activity and proliferation of T lymphocytes and B lymphocytes, inhibiting the activity of natural killer cells (NK cells), and regulating the function of dendritic cells and macrophages, making allotransplantation and xenotransplantation possible.

[0050] In addition, mesenchymal stem cells have the ability to differentiate into various connective tissues such as cartilage, bone tissue, ligaments, and bone marrow stroma. In addition to these structural support functions, they also have immunomodulatory and inhibitory functions, inhibiting inflammatory responses and making them suitable stem cells for tissue regeneration. Articular cartilage damage occurs when degeneration and degenerative changes around the articular cartilage progress, invading the subchondral bone and causing joint inflammation. This occurs because the supply of progenitor cells from the blood or bone marrow and the supply of articular chondrocytes have difficulty migrating to the lesion surrounded by the matrix around the joint, preventing them from secreting appropriate new joint matrix. This disease can be treated and prevented using mesenchymal stem cells. In addition, adipose-derived mesenchymal stem cells derived from adipose tissue are further subdivided into adipose-derived stem / stromal cells (ASCs) and adipose-derived adult stem cells (ADAS), and can be used to produce biomaterials to treat soft tissue defects caused by trauma, tumor removal surgery, burns, etc. When mesenchymal stem cells are injected into the ventricles, these cells migrate to the frontal cortex and cerebellum and produce neurofilament. This is evidence that cells of mesodermal origin can differentiate into neural tissue, and due to this advantage, active research is being conducted to apply them to degenerative neurological diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, and spinal cord injuries. Stem cell therapy can be a very effective treatment for neurological diseases such as liver diseases, where active cell division makes it difficult to treat tissue damage and regeneration is rare.

[0051]

[0052] In another embodiment of the present invention, the focused ultrasound treatment provides a method for inducing secretion of extracellular vesicles, wherein the duty cycle among the focused ultrasound parameters is 10 to 90%.

[0053] In the present invention, the "duty cycle" is a concept widely used in machine control processes. It is a numerical value representing the percentage of time a signal is on within one signal period. A period refers to the time it takes for a signal to complete one full on-and-off cycle. Duty cycle can be expressed using the following formula:

[0054] D=T / P x 100%

[0055]

[0056] Here, D is the duty cycle, T is the time the signal is on, and P is the period of the signal.

[0057] The above duty cycle is preferably 10 to 90%, more preferably 10 to 85%, or 10 to 75%, or 10 to 50%, or 10 to 25%, but is not limited thereto.

[0058]

[0059] In another embodiment of the present invention, the focused ultrasound treatment provides a method for inducing secretion of extracellular vesicles, wherein the focused ultrasound parameters have a pulse repetition frequency (PRF) of 10 to 200 Hz.

[0060] In the present invention, the “pulse repetition frequency (PRF)” refers to the number of transmission pulses transmitted per second. Therefore, the pulse repetition frequency is defined by the following equation.

[0061] PRF=1 / t1

[0062] The pulse repetition frequency is preferably 10 to 200 Hz, more preferably 50 to 150 Hz or 75 to 125 Hz, but is not limited thereto.

[0063]

[0064] In another embodiment of the present invention, the focused ultrasound treatment provides a method for inducing secretion of extracellular vesicles, wherein the central frequency of the transducer among the focused ultrasound parameters is 100 to 1000 KHz.

[0065] The center frequency of the above converter is preferably, but not limited to, 100 to 1000 KHz, 250 to 750 KHz, or more preferably 400 to 600 KHz.

[0066] In another embodiment of the present invention, the focused ultrasound treatment provides a method for inducing secretion of extracellular vesicles, wherein the amplitude among the focused ultrasound parameters is 1 to 10 Mpa.

[0067] The above amplitude may be preferably 1 to 10 Mpa, or 1 to 7 Mpa, or more preferably 1 to 5 Mpa, or more preferably 2 to 4 Mpa, but is not limited thereto.

[0068]

[0069] In another embodiment of the present invention, a method for inducing secretion of extracellular vesicles is provided, wherein the focused ultrasound treatment is performed with a total exposure time (sonication time) of 10 to 300 seconds among the focused ultrasound parameters.

[0070] The total exposure time may preferably be 10 to 300 seconds, or 30 to 200 seconds, or more preferably 60 to 120 seconds, but is not limited thereto.

[0071]

[0072] In another embodiment of the present invention, the focused ultrasound treatment provides a method for inducing secretion of extracellular vesicles, wherein the pulse repetition period (PRP) among the focused ultrasound parameters is 1 to 100 ms.

[0073] In the present invention, the “pulse repetition period (PRP)” refers to the time it takes from when one pulse is fired until the next pulse is fired.

[0074] The pulse repetition period is preferably 1 to 100 ms, more preferably 2 to 60 ms or 3 to 40 ms, and more preferably 5 to 20 ms, but is not limited thereto.

[0075]

[0076] 2. Focused ultrasound device for inducing secretion of extracellular vesicles

[0077] In one embodiment of the present invention, a focused ultrasound device used to induce secretion of extracellular vesicles according to the above method is provided, comprising: a function generator; an amplifier; a coupler; and a transducer.

[0078]

[0079] In the present invention, the “waveform generator” is a type of electronic test equipment or software, and is a device for generating a waveform, which is an electronic signal. The most common waveforms include sine waves, square waves, triangular waves, sawtooth waves, etc. It is usually used to create periodic signals. High-performance equipment can be connected to a computer to create single-shot waveforms, but is not limited thereto.

[0080] In the present invention, the "amplifier (AMP)" or "amplifier" is an electronic device that increases the power of a signal. It can strengthen the intensity of a physical or electronic signal to obtain a desired output, but is not limited thereto.

[0081] In the present invention, a "transducer" or "converter" is a device that converts energy from one form to another. Generally, a transducer converts a signal of one energy form into a signal of another form. Transducers are often used at the boundaries of automation, measurement, and control systems where an electrical signal is converted into another physical quantity, but are not limited thereto.

[0082]

[0083] In another embodiment of the present invention, there is provided a focused ultrasound device that further includes an oscilloscope.

[0084] In the present invention, the "oscilloscope" is equipment that visually shows periodic changes such as waves, and in particular, is a device that visually displays changes in voltage as a signal, but is not limited thereto.

[0085]

[0086] 3. A pharmaceutical composition for preventing or treating an immune-related disease, comprising extracellular vesicles

[0087] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating an immune-related disease, comprising extracellular vesicles obtained by the above method.

[0088] In another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating an immune-related disease, wherein the immune-related disease is at least one selected from the group consisting of autoimmune diseases, graft-versus-host diseases, organ transplant rejection reactions, asthma, atopy, and acute or chronic inflammatory diseases.

[0089] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating an immune-related disease is provided, wherein the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

[0090]

[0091] 4. Composition for wound treatment or regeneration containing extracellular vesicles

[0092] In one embodiment of the present invention, a wound treatment composition comprising extracellular vesicles obtained by the above method is provided.

[0093] In another embodiment of the present invention, a composition for treating a wound is provided, wherein the wound is selected from the group consisting of a wound, a bedsore, a burn, an abrasion, a puncture ulcer, a stab wound, a chronic skin wound caused by active oxygen, a bruise, a cut, a wound of the mucous membrane of the throat or mouth, a laceration, a diabetic ulcer, a leg ulcer, a hypertensive ischemic ulcer, a venous ulcer, and a foot ulcer.

[0094] In one embodiment of the present invention, a regenerative composition comprising extracellular vesicles obtained by the above method is provided.

[0095] In one embodiment of the present invention, the regeneration is selected from the group consisting of a mandibular disorder, a gastric ulcer by endoscopic submucosal dissection, human autologous fat grafting, treatment of knee osteoarthritis, treatment of chronic non-healing ulcers, hair regeneration therapy for androgenetic alopecia, treatment of Achilles tendon rupture, high-risk proliferative diabetic retinopathy, high-risk proliferative diabetic retinopathy, knee OA, patellar dislocation, lumbar facet joint syndrome, retinitis pigmentosa, diabetic macular edema, traumatic bone cyst, high-risk proliferative diabetic retinopathy, scars, dry eye, Sjogren's syndrome, osteoarthritis, meniscus lesions, freckles, muscle damage, elbow ulnar neuropathy, chronic ulcers, alopecia, epicondylitis, bone resorption, rotator cuff tear, thin endometrium, striae gravidarum, plantar fasciitis, osteoarthritis, muscle rupture, tennis elbow, skin grafting, aging, and knee arthritis, and regeneration is required. A composition for regeneration is provided.

[0096]

[0097] In the present invention, "drug delivery methods" include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. Oral or parenteral administration is preferred.

[0098] The "dosage and administration" of the composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, pharmaceutical form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a sugar-coated tablet, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0099] The "subject" of the present invention refers to an individual whose disease onset is uncertain, but who has a high probability of developing the disease. In particular, the disease refers to, but is not limited to, nerve damage or a neurological disorder.

[0100]

[0101] In addition, the composition provided in the present invention can be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0102] The above “prevention” of the present invention may include, without limitation, any act that can block, suppress or delay symptoms caused by a disease by using the composition of the present invention.

[0103] The above “treatment” and “improvement” of the present invention may include, without limitation, any act that can improve or benefit symptoms caused by a disease by using the composition of the present invention.

[0104] In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being for human use.

[0105] The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections. In the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0106] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0107] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0108] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0109] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0110] A food composition comprising the composition of the present invention as an active ingredient can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts with almost no toxicity or side effects, it can be used with confidence even when taken for long periods of time for preventive purposes.

[0111] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0112] Here, when the food composition is manufactured in the form of a beverage, there are no special restrictions other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as additional ingredients like in a typical beverage. That is, as a natural carbohydrate, it may include a monosaccharide such as glucose, a disaccharide such as fructose, a polysaccharide such as sucrose, a dextrin, a cyclodextrin, a typical sugar such as dextrin, a cyclodextrin, a sugar alcohol such as xylitol, sorbitol, and erythritol. As the flavoring agent, there may be mentioned a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).

[0113] In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0114] These components can be used independently or in combination. The proportions of these additives are not particularly critical, but are typically selected within the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0115] In the present invention, the "subject" refers to a subject suspected of having a disease, and the subject suspected of having a disease refers to a mammal including a human, a rat, a livestock, etc. that has developed or can develop the disease, but a subject that can be treated with the effective substance provided in the present invention is included without limitation.

[0116] The neurological disease treated by the method of the present invention may be a neurodegenerative disease or a neuroinflammatory disease, and specific examples thereof include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and May be selected from the group consisting of, but not limited to, tauopathies.

[0117]

[0118] Previous methods for isolating extracellular vesicles (EVs) used a medium containing EV-depleted FBS to induce EV secretion for more than 24 hours. This process contained various waste products, proteins, cytokines, and animal-derived serum components secreted by growing cells, making it difficult to isolate pure EVs. Furthermore, the prolonged secretion time exposed parent cells to various environments, increasing the likelihood of heterogeneous EV secretion.

[0119] The present invention utilizes ultrasonic energy to stimulate stem cells and induce the secretion of extracellular vesicles (EVEs) through fluid shear stress in the stem cells. Specifically, by inducing EVEs with stimulation for a short period of time, less than 10 minutes, relatively homogeneous EVEs can be obtained. Furthermore, the same technology can be applied to various types of stem cells, offering limitless potential for extracting EVEs with different functions depending on the intended purpose.

[0120]

[0121] Figure 1 illustrates the results of extracellular vesicle extraction according to changes in focused ultrasound parameters. The results show that the highest concentration of extracellular vesicles can be extracted under condition 5.

[0122] Figure 2 shows the results of cytotoxicity tests according to changes in focused ultrasound parameters. The results show that conditions 5 and 6 exhibit the lowest cytotoxicity.

[0123] Figure 3 is a schematic diagram showing a method for separating extracellular vesicles through extracellular vesicle precipitation in Example 3.

[0124] FIG. 4 illustrates a focused ultrasound device used to induce secretion of extracellular vesicles according to the present invention, comprising a function generator; an amplifier; a coupler; and a transducer. As can be seen in FIG. 4, an oscilloscope may additionally be included.

[0125]

[0126] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0127]

[0128] Example 1: Stem cell stimulation using focused ultrasound

[0129] First, the experiment was prepared in a water-filled tank so that the focused ultrasound energy output from the transducer (H-107 model from Sonic Concept) could be well transmitted to the cell culture plate. Then, the cells spread on the bottom of the plate were stimulated with focused ultrasound while changing the duty cycle and pulse repetition frequency. The focused ultrasound parameters are shown in Table 1 below. Next, in order to characterize the extracellular vesicles, first, under appropriate cell culture conditions, 90% confluency was maintained, and the medium was exchanged for serum-free medium for 12 hours before focused ultrasound treatment to ensure that the extracellular vesicles could be stably and efficiently induced out of the cells.

[0130] Parameter Value Acoustic pressure amplitude (Mpa) 3 Central frequency of transducer (kHz) 5 15 Pulse repetition frequency (Hz) 10, 25, 50, 75, 100 Total exposure time (Sonication time, Sec) 90 Duty cycle (Duty cycle, %) 10, 25, 50, 75 Pulse repetition period (ms) 10

[0131]

[0132] Example 2: Exosome precipitation

[0133] 1. The medium containing extracellular vesicles treated with focused ultrasound according to Example 1 was centrifuged at 3000 × g for 15 minutes to remove cells and cell debris. 2. The supernatant was transferred to a sterile container, an appropriate amount of ExoQuick-TC™ was added, and the tube was inverted or gently tapped to mix well. 3. The tube was stored upright in the refrigerator overnight (at least 12 hours) at 4°C without inverting or mixing. 4. The ExoQuick-TC™ / biological fluid mixture was centrifuged at 1500 × g for 30 minutes. Centrifugation was performed at room temperature or 4°C to obtain similar results, and after centrifugation, the extracellular vesicles appeared as a beige or white pellet at the bottom of the container. 5. Aspirate the supernatant, taking special care not to disturb the precipitated EVEs in the pellet, and spin down the remaining ExoQuick-TC™ solution by centrifugation at 1500 × g for 5 minutes. 6. Resuspend the EVE pellet in 100-500 μL of sterile 1X PBS or a specific buffer depending on the downstream application.

[0134]

[0135] Example 3: Morphological analysis of extracellular vesicles

[0136] In Example 2, the expression pattern of CD63 / CD81, known as a tetraspanin family member, in the precipitated and isolated extracellular vesicles was confirmed. Specifically, Western blotting was performed using anti-CD63 antibody (Santa Cruz Biotechnology, Inc., #sc-5275, 1:1000 dilution) and anti-CD81 antibody (Santa Cruz Biotechnology, Inc., #sc-7637, 1:1000 dilution). Subsequently, morphological analysis was performed using phase-contrast electron micrographs. Specifically, the morphology was confirmed by phase-contrast electron micrographs before and after FUS irradiation, and cell viability was confirmed using Cell Counting Kit-8 (CCK-8, Dojindo, #CK04). After investigating FUS, CCK-8 solution was treated on the cells, and the 450 nm absorbance was measured at 0 and 2 hours to evaluate the difference, which is shown in Fig. 2.

[0137]

[0138] As a result, the concentration of extracellular vesicles according to the change in various focused ultrasound parameters is shown in Figure 1, and the cytotoxicity of stem cells is shown in Figure 2. Specifically, the cells were divided into 7 groups and the focused ultrasound parameters were varied, which are shown in Table 2 below.

[0139] ParameterResultsNumberDuty Cycle (%)BurstPulse Repetition Frequency (PRF) (Hz)Cytotoxicity (%)Concentration (mg / ml)110107.1550.017225258.7260.087350503.8970.082475753.0830.0985101002.4430.1456751001.3960.105

[0140]

[0141] conclusion

[0142] As a result, the extraction result of extracellular vesicles according to the focused ultrasound parameters was 0.145 mg / ml in condition 5, which was the highest concentration of extracellular vesicles compared to other conditions, and 0.105 mg / ml was also possible in condition 6, which was a relatively high concentration of extracellular vesicles compared to other conditions.

[0143] Additionally, the cytotoxicity test results according to the focused ultrasound parameters showed that the cytotoxicity was the lowest under conditions 5 and 6.

[0144] This found the optimal combination that enables high-concentration extraction of extracellular vesicles with the lowest cytotoxicity, especially depending on the duty cycle and pulse repetition frequency (PRF) among various focused ultrasound parameters. That is, under conditions 5 and 6, the mechanical energy of the focused ultrasound stimulates stem cells, inducing shear stress in the stem cells, which induces the secretion of extracellular vesicles well, but the shear stress is not so strong as to damage the stem cells, so damage to the stem cells can be minimized.

[0145]

[0146] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for inducing secretion of extracellular vesicles in cells separated from a subject by treating the cells separated from the subject with focused ultrasound.

2. In paragraph 1, A method for inducing secretion of extracellular vesicles, wherein the above cells are stem cells.

3. In paragraph 2, A method for inducing secretion of extracellular vesicles, wherein the above cells are mesenchymal stem cells.

4. In paragraph 3, The above focused ultrasound treatment is a method for inducing secretion of extracellular vesicles, wherein the duty cycle among the focused ultrasound parameters is 10 to 75%.

5. In paragraph 4, The above focused ultrasound treatment is a method for inducing secretion of extracellular vesicles, wherein the pulse repetition frequency (PRF) among the focused ultrasound parameters is 10 to 200 Hz.

6. In paragraph 5, The above focused ultrasound treatment is a method for inducing secretion of extracellular vesicles, wherein the central frequency of the transducer among the focused ultrasound parameters is 100 to 1000 KHz.

7. In paragraph 6, The above focused ultrasound treatment is a method for inducing secretion of extracellular vesicles, wherein the amplitude among the focused ultrasound parameters is 1 to 10 Mpa.

8. In paragraph 7, The above focused ultrasound treatment is a method for inducing secretion of extracellular vesicles, wherein the total exposure time (sonication time) among the focused ultrasound parameters is 10 to 300 seconds.

9. In paragraph 8, The above focused ultrasound treatment is a method for inducing secretion of extracellular vesicles, wherein the pulse repetition period among the focused ultrasound parameters is 1 to 100 ms.

10. A device used to induce secretion of extracellular vesicles according to any one of claims 1 to 9, Function generator; Amplifier; Coupler; and Including a transducer, A focused ultrasound device used to induce secretion of extracellular vesicles.

11. In paragraph 10, A focused ultrasound device, wherein the focused ultrasound device further comprises an oscilloscope.

12. In paragraph 11, The above focused ultrasound device is a focused ultrasound device having a duty cycle of 10 to 75% among the focused ultrasound parameters.

13. In paragraph 12, The above focused ultrasound device is a focused ultrasound device having a pulse repetition frequency (PRF) of 10 to 200 Hz among the focused ultrasound parameters.

14. In paragraph 13, The above focused ultrasound device is a focused ultrasound device, wherein the central frequency of the transducer among the focused ultrasound parameters is 100 to 1000 KHz.

15. In paragraph 14, The above focused ultrasound device is a focused ultrasound device, wherein the amplitude among the focused ultrasound parameters is 1 to 10 Mpa.

16. In paragraph 15, The above-mentioned focused ultrasound device is a focused ultrasound device, wherein the total exposure time (sonication time) among the focused ultrasound parameters is 10 to 300 seconds.

17. In paragraph 16, The above focused ultrasound device is a focused ultrasound device having a pulse repetition period of 1 to 100 ms among the focused ultrasound parameters.

Images