A method for promoting the generation of microvesicles containing mitochondria and its medical applications.

Abstract

This invention provides a method for promoting the generation of mitochondria-containing microvesicles and its medical applications. Specifically, it provides a method for generating an extracellular vesicle composition by using a mitochondrial synthesis promoting factor and an extracellular vesicle synthesis promoting factor in combination. By co-culturing cells with the mitochondrial synthesis promoting factor and the extracellular vesicle synthesis promoting factor added, the resulting extracellular vesicle composition is rich in mitochondria-containing microvesicles. By adding the mitochondrial synthesis promoting factor and the extracellular vesicle synthesis promoting factor, cells can be made to generate mitochondria-containing microvesicles, and since the collected mitochondria are mitochondria enclosed in microvesicles, the preservation and intracellular transfer of mitochondria becomes easier. Furthermore, the invention provides applications for the manufacture of isolated extracellular vesicle compositions, pharmaceutical compositions containing them, and drugs for treating diabetes and / or kidney damage.

Description

[Technical Field]

[0001] This invention relates to the combined use of a mitochondrial synthesis promoting factor and an extracellular vesicle synthesis promoting factor. It also relates to a method for generating an extracellular vesicle composition rich in microvesicles containing mitochondria in cells, an isolated extracellular vesicle composition, a pharmaceutical product containing the same, and its uses. [Background technology]

[0002] Stem cells are undifferentiated parent cells that have the ability to differentiate into cells with different functions and can generate even more identical stem cells through replication. Because stem cells have the ability to replicate and differentiate into other types of cells, humans recognized their potential use in treating diseases and began to experiment with using stem cells as drugs. Stem cell therapy is defined as treating various diseases using stem cells, and its earliest origins can be traced back to bone marrow transplantation in 1968. However, bone marrow transplantation technology involves transplanting "hematopoietic stem cells," which differs from today's mesenchymal stem cell transplantation therapy, although it can still be considered the beginning of a type of allogeneic stem cell therapy. After many animal experiments yielded promising results, attempts to treat human diseases with stem cells have gradually been made since 2000. For example, in 2003, "bone marrow stem cells" were transplanted into the left ventricle to repair myocardial tissue. In applications to human spinal cord injury, it has been possible to reliably bring about a clear recovery of motor function, and in stroke treatment, it can reduce acute inflammation and long-term brain degeneration and promote long-term functional recovery. In brain injury research, it has been confirmed that it alleviates symptoms and improves activity levels and long-term memory. Later, adult stem cells derived from adipose tissue were also found to be effective in treating neurological disorders. Although time and evidence have proven the effectiveness of stem cell therapy, the therapeutic principle of mesenchymal stem cells has not been definitively established because the original differentiation substitution theory always contradicts experimental research results.

[0003] Recent research has demonstrated the principle behind the therapeutic effects of stem cells. Among the substances secreted by stem cells, mitochondria are transferred to damaged cells through mechanisms such as tunneling, gap junctions, microvesicles, and cell fusion. The acquisition of these exogenous mitochondria by the damaged cells is the main reason why stem cells generate their therapeutic effects. Many papers, such as Paliwal et al. (2018), have also verified that the regenerative capacity of mesenchymal stem cells originates from mitochondrial transport. In the same year, Wang et al. (2018) also proposed that mitochondrial transport by stem cells is a novel therapeutic technology for tissue damage.

[0004] Current research has confirmed that stem cells with superior mitochondrial quality possess better therapeutic capabilities. Furthermore, it has been directly confirmed that, in subsequent treatments, it is not necessary to use the entire stem cell, and isolated mitochondria can be directly injected into the lungs to treat pulmonary inflammation and damage and restore fibrotic alveoli. Alternatively, direct injection of free mitochondria into the brain can treat cerebral infarction, reduce the area of ​​cerebral infarction, treat multiple system atrophy (MSA), reduce brain damage, and improve functional capacity.

[0005] Therefore, by delivering healthy mitochondria into damaged cells, it is possible to promote cell regeneration, accelerate cell growth and activation, and improve aging or various degenerative diseases caused by mitochondrial damage. However, the biggest technical obstacle to such an effective treatment method is that there is still no way to mass-produce mitochondria, and isolated mitochondria are quickly damaged and die once they leave the intracellular environment, making them difficult to store. Furthermore, the manufacture, mass production, and long-term storage of purified mitochondrial preparations remain difficult. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Given that existing technologies cannot mass-produce mitochondria, and even when they are produced, they are difficult to store, the innovation of this invention lies in the simple, large-scale, and continuous mass production of mitochondrial preparations. Furthermore, the produced mitochondria are easier to store for longer periods, and the structure of the vesicles and the components within them not only protect the mitochondria from damage but also promote their entry into damaged cells, thereby improving therapeutic capacity and simultaneously solving all the technical obstacles in the practical application of purified mitochondrial preparations. [Means for solving the problem]

[0007] To achieve the aforementioned objectives, the present invention provides an application for the combined use of a mitochondrial synthesis promoter and an extracellular vesicle synthesis promoter, which are used to promote the generation of mitochondrial microvesicles by cells containing mitochondria.

[0008] The present invention may be used for non-therapeutic purposes. By using a combination of a mitochondrial synthesis promoter and an extracellular vesicle synthesis promoter, the present invention can generate a large number of microvesicles containing mitochondria. The microvesicle structure promotes the entry of mitochondria into damaged cells and facilitates repair. Furthermore, the collected mitochondria are already enclosed in microvesicles and have protection from small ribonucleic acid molecules inside, making them easier to store.

[0009] According to the present invention, the cell having mitochondria can generate extracellular vesicles and microvesicles having mitochondria.

[0010] Preferably, the ratio of the amounts of the mitochondrial synthesis promoting factor and the extracellular vesicle synthesis promoting factor added is 1:1 to 1:1000, for example, 1:1 to 1:8, 1:1 to 1:6, 1:1 to 1:4, or 1:100, 1:300, 1:600, or 1:900. More preferably, the ratio of the amounts of the mitochondrial synthesis promoting factor and the extracellular vesicle synthesis promoting factor added is 1:1 to 1:5.

[0011] Preferably, the mitochondrial synthesis promoting factor can be selected from the group consisting of heme oxygenase (HO-1), CoPPIX (Co-protoporphyrin IX), hemin, hemin derivatives such as chlorohemin, hemoprotein, fragmented red blood cells, mitochondrial structural fragments, cell tissue fragments, and culture in an oxygen concentration environment of 5-20%.

[0012] Preferably, the extracellular vesicle generation promoting factor can be selected from the group consisting of ethanol, EP4 receptor antagonists (EP4 antagonists), such as GW627368X, endosomal sorting complex required for transport (ESCRT), ARRDC1 (arrestin domain-containing protein 1), associated protein's tumor suppressor gene 101 (TSG101), and cytochalasin B. The extracellular vesicle generation promoting factor can promote the generation of microvesicles and exosomes by cells.

[0013] Preferably, the cells having mitochondria are mesenchymal stem cells, hematopoietic stem cells, bone marrow stem cells, hepatocytes, or other somatic cells. More preferably, the cells having mitochondria are mesenchymal stem cells. Preferably, the mesenchymal stem cells are neonatal mesenchymal stem cells or embryonic stem cells.

[0014] To achieve the aforementioned objectives, the present invention further provides: The process of preparing cells containing mitochondria (1), (2) A step in which a mitochondrial synthesis promoting factor and an extracellular vesicle synthesis promoting factor are added to the culture medium of the cells having mitochondria and co-culture is performed to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, The extracellular vesicle composition, which includes and is rich in microvesicles having mitochondria, provides a method for producing an extracellular vesicle composition rich in microvesicles having mitochondria, wherein the total number of extracellular vesicles contains 0.2% or more microvesicles having mitochondria.

[0015] Preferably, step (2) is The process (2-1) involves adding a mitochondrial synthesis promoting factor and an extracellular vesicle synthesis promoting factor to the culture medium of the cells having mitochondria and co-culturing them, (2-2) A step of separating the extracellular vesicle composition from the co-cultured culture medium to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, An extracellular vesicle composition containing the above-mentioned microvesicles having mitochondria has 0.2% or more of mitochondria-containing microvesicles, based on the total number of extracellular vesicles. That is, the extracellular vesicle composition is obtained by removing cells and culture medium containing mitochondria. That is, 0.2% or more of the extracellular vesicles are microvesicles having mitochondria.

[0016] Preferably, step (2) involves first adding a mitochondrial synthesis promoting factor to the cells having mitochondria, then adding an extracellular vesicle synthesis promoting factor to the culture medium and performing co-culturing to obtain an extracellular vesicle composition rich in mitochondrial microvesicles, and the extracellular vesicle composition rich in mitochondrial microvesicles contains 0.2% or more mitochondrial microvesicles based on the total number of extracellular vesicles. In another example, step (2) involves first adding an extracellular vesicle synthesis promoting factor to the cells having mitochondria, then adding a mitochondrial synthesis promoting factor to the culture medium and performing co-culturing to obtain an extracellular vesicle composition rich in mitochondrial microvesicles, and the extracellular vesicle composition rich in mitochondrial microvesicles contains 0.2% or more mitochondrial microvesicles based on the total number of extracellular vesicles. That is, 0.2% or more of the extracellular vesicles are mitochondrial microvesicles.

[0017] Preferably, the amount of mitochondrial synthesis promoting factor added is such that the concentration of the mitochondrial synthesis promoting factor in the culture medium is 0.1 to 100,000 ng / mL. Preferably, the amount of mitochondrial synthesis promoting factor added is such that the concentration of the mitochondrial synthesis promoting factor in the culture medium is 1 to 10,000 ng / mL, for example, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, or 1,000 ng / mL.

[0018] Preferably, the mitochondrial synthesis promoting factor can be selected from the group consisting of heme oxygenase-1, CoPPIX, hemin, hemin derivatives, such as chlorohemin, hemoprotein, fragmented red blood cells, mitochondrial structural fragments, cell tissue fragments, and culture in an oxygen concentration environment of 5-20%. Here, the oxygen concentration can be 7-18% or 10-15%, or 12%, 13%, or 14%.

[0019] Preferably, the addition amount of the heme oxygenase-1 is such that the concentration of the heme oxygenase-1 in the medium is 0.1 to 300 ng / mL. Preferably, the addition amount of the heme oxygenase-1 is such that the concentration of the heme oxygenase-1 in the medium is 1 to 100 ng / mL, and may be, for example, 10 ng / mL, 30 ng / mL, 50 ng / mL or 70 ng / mL.

[0020] Preferably, the addition amount of the CoPPIX is such that the concentration of the CoPPIX in the medium is 0.2 to 100 μmol / kg. Preferably, the addition amount of the CoPPIX is such that the concentration of the CoPPIX in the medium is 1 to 70 μmol / kg, and may be, for example, 5 μmol / kg, 10 μmol / kg, 30 μmol / kg or 50 μmol / kg.

[0021] Preferably, the addition amount of the hemin or the hemin derivative is such that the concentration of the hemin or the hemin derivative in the medium is 0.2 to 100 μmol / kg. Preferably, the addition amount of the hemin or the hemin derivative is such that the concentration of the hemin or the hemin derivative in the medium is 1 to 80 μmol / kg, and may be, for example, 10 μmol / kg, 20 μmol / kg, 50 μmol / kg or 70 μmol / kg. <ocke="" style="box-sizing: border-box; outline: none; cursor: pointer; padding: 0px; margin: 0px; border: 0px; font-size: 100%; vertical-align: baseline; background: transparent;">

[0022] Preferably, the addition amount of the extracellular vesicle production promoting factor is such that the concentration of the extracellular vesicle production promoting factor in the medium is 10 -4 , -2 , -7 , -5 ,

[0023] , , , , ,

[0022] millimoles per liter (mM) to 800 mM. Preferably, the addition amount of the extracellular vesicle production promoting factor is such that the concentration of the extracellular vesicle production promoting factor in the medium is 10 -5 to 500 mM, and may be, for example, 10 -4 mM, 10 -2 mM, 1 mM, 10 mM, 50 mM, 100 mM or 300 mM.

[0023] Preferably, the extracellular vesicle-promoting factor can be selected from the group consisting of ethanol, EP4 receptor antagonists such as GW627368X, endosomal sorting transport complexes, ARRDC1, tumor suppressor genes of related proteins, and cytochalasin B. The extracellular vesicle-promoting factor can promote the generation of microvesicles and exosomes by cells.

[0024] Preferably, the amount of ethanol added is such that the ethanol concentration in the culture medium is 10 to 100 mM. More preferably, the amount of ethanol added is such that the ethanol concentration in the culture medium is 20 to 70 mM, for example, 30 mM, 40 mM, 50 mM, or 60 mM.

[0025] Preferably, the amount of EP4 receptor antagonist added is such that the concentration of the EP4 receptor antagonist in the culture medium is 0.2 to 500 μg / mL. Preferably, the amount of EP4 receptor antagonist added is such that the concentration of the EP4 receptor antagonist in the culture medium is 1 to 200 μg / mL, for example, 10 μg / mL, 50 μg / mL, 70 μg / mL, 100 μg / mL, or 150 μg / mL.

[0026] Preferably, the amount of endosome sorting and transport complex added is such that the concentration of the endosome sorting and transport complex in the culture medium is between 0.2 nanomoles per liter (nM) and 500 nM. Preferably, the amount of endosome sorting and transport complex added is such that the concentration of the endosome sorting and transport complex in the culture medium is between 1 and 300 nM, for example, 5 nM, 10 nM, 50 nM, or 100 nM.

[0027] Preferably, the amount of ARRDC1 added is such that the ARRDC1 concentration in the culture medium is between 0.3 micromoles per liter (μM) and 600 μM. Preferably, the amount of ARRDC1 added is such that the ARRDC1 concentration in the culture medium is between 1 and 300 μM, for example, 10 μM, 50 μM, or 150 μM.

[0028] Preferably, the added amount of the tumor suppressor gene of the related protein is such that the concentration of the tumor suppressor gene of the related protein in the culture medium is 10 -10 mol per liter (M) to 10 -6 M. Preferably, the added amount of the tumor suppressor gene of the related protein is such that the concentration of the tumor suppressor gene of the related protein in the culture medium is 10 -9 M to 10 -7 M, for example, it may be 10 -8 M.

[0029] Preferably, the added amount of the cytochalasin B is such that the concentration of the cytochalasin B in the culture medium is 10 -7 ~10 -4 M. Preferably, the added amount of the cytochalasin B is such that the concentration of the cytochalasin B in the culture medium is 10 -6 ~10 -5 M.

[0030] According to the present invention, in the method for producing an extracellular vesicle composition rich in microvesicles having mitochondria, the culture conditions of the cells having mitochondria can be adjusted by those having ordinary knowledge in the technical field as needed.

[0031] Preferably, the co-culture is carried out by culturing at 5% carbon dioxide, 35°C to 39°C for 60 to 80 hours. Preferably, it is cultured for 70 to 75 hours.

[0032] To achieve the above object, the present invention further provides a separated extracellular vesicle composition having microvesicles having mitochondria of 0.2% or more based on the total number of extracellular vesicles.

[0033] The isolated extracellular vesicle composition further contains microvesicles and exosomes. Microvesicles are extracellular vesicles capable of enveloping mitochondria. Microvesicles containing mitochondria can enhance their therapeutic effect when combined with other microvesicles or exosomes.

[0034] Preferably, the isolated extracellular vesicle composition has microvesicles having 0.5%, 1%, 2%, or 3% or more mitochondria, based on the total number of extracellular vesicles. More preferably, the isolated extracellular vesicle composition has microvesicles having 4% or more mitochondria, based on the total number of extracellular vesicles, and may have, for example, 4.2%, 4.4%, 4.6%, or 4.8% or more mitochondria.

[0035] The present invention further provides an extracellular vesicle composition containing the isolated mitochondria-containing microvesicles, and a pharmaceutical composition containing a pharmaceutically acceptable carrier.

[0036] The present invention further provides an application for producing a drug for treating or alleviating diabetes, wherein the drug comprises an effective dose of an isolated extracellular vesicle composition and a pharmaceutically acceptable carrier, and the isolated extracellular vesicle composition has microvesicles having 0.2% or more mitochondria based on the total number of extracellular vesicles.

[0037] Preferably, the diabetes is type 2 diabetes.

[0038] The effective dose described in this invention refers to an amount effective in achieving the desired treatment or alleviation of diabetes in terms of dosage and time, and according to this invention, administering a specific range of amounts of the isolated extracellular vesicle composition can lower fasting blood glucose levels and / or insulin concentrations in diabetic rats and improve insulin resistance.

[0039] Preferably, the target of administration of the drug is a warm-blooded animal or a human. Preferably, the warm-blooded animal is a mammal or a bird, and the mammal may be a rat or a mouse.

[0040] The present invention further provides an application for a separated mitochondrial-containing extracellular vesicle composition for the manufacture of a drug for treating or alleviating kidney injury, wherein the drug contains an effective dose of a separated extracellular vesicle composition and a pharmaceutically acceptable carrier, and the separated extracellular vesicle composition has microvesicles having 0.2% or more mitochondria based on the total number of extracellular vesicles.

[0041] The effective dose described in the present invention refers to an amount effective in achieving the desired treatment or alleviation of chronic kidney disease in terms of dosage and time, and according to the present invention, it is possible to reduce blood creatinine and urea nitrogen levels in mice with chronic kidney disease by administering a specific range of amounts of isolated mitochondrial-containing extracellular vesicle composition.

[0042] Preferably, the kidney injury may be a chronic kidney disease or an acute kidney injury.

[0043] Preferably, the target of administration of the drug is a warm-blooded animal or a human. Preferably, the warm-blooded animal is a mammal or a bird, and the mammal may be a rat or a mouse.

[0044] The “pharmaceutical composition or drug” described in the present invention may exist in a variety of forms, including, but not limited to, liquid, semi-solid, and solid drug forms, such as solutions, emulsions, suspensions, and other similar or dosage forms applicable to the present invention.

[0045] In one embodiment, the pharmaceutical composition of the present invention is in the form of an injectable dosage form, and the injectable dosage form is administered by intravenous injection. [Effects of the Invention]

[0046] The advantages of the present invention are that it enables the easy, large-scale, and continuous production of formulations containing mitochondrial preparations, and that it allows for the direct separation of mitochondrial-containing microvesicles from the culture medium, eliminating the need to further disrupt cells to obtain mitochondria, and thus eliminating the costly and time-consuming process of high-speed mitochondrial centrifugation, thereby achieving cost and time savings. Furthermore, the collected mitochondria are already mitochondrial vesicles enclosed in microvesicles and are also protected by the small molecule ribonucleic acid of exosomes, making them easier to cryopreserve.

[0047] The isolated extracellular vesicle composition of the present invention can reliably lower fasting blood glucose levels and / or insulin concentrations in diabetic rats, improve insulin resistance, and effectively treat or alleviate diabetes. Furthermore, it can lower creatinine and urea nitrogen levels in the blood of mice with chronic kidney disease, and effectively treat or alleviate renal damage. [Brief explanation of the drawing]

[0048] [Figure 1] This indicates the number of mitochondria in extracellular vesicles generated by treating human mesenchymal stem cells with mitochondrial synthesis promoters, extracellular vesicle synthesis promoters, or a combination thereof. * represents a confidence interval >95%. [Figure 2] The percentage of microvesicles containing mitochondria generated by treating human mesenchymal stem cells with mitochondrial synthesis-promoting factors, extracellular vesicle-promoting factors, or a combination thereof, is shown for the total extracellular vesicles generated in each treatment group. * indicates a confidence interval >95%. [Figure 3] Extracellular vesicles of mitochondrial synthesis-promoting factors, extracellular vesicle groups of extracellular vesicles of extracellular vesicle synthesis-promoting factors, and co-treated extracellular vesicles showed an effect of lowering fasting blood glucose levels in type 2 diabetic rats, with * indicating a confidence interval >95%. [Figure 4]Extracellular vesicles of mitochondrial synthesis-promoting factors, extracellular vesicle groups of extracellular vesicles of extracellular vesicle synthesis-promoting factors, and co-treated extracellular vesicles showed the effect of lowering fasting insulin levels in type 2 diabetic rats, with * indicating a confidence interval >95% and ** indicating a confidence interval >90%. [Figure 5] Extracellular vesicles of mitochondrial synthesis-promoting factors, extracellular vesicle groups of extracellular vesicles of extracellular vesicle synthesis-promoting factors, and co-treated extracellular vesicles showed the effect of reducing blood creatinine in mice with chronic kidney disease, with * indicating a confidence interval >95%. [Figure 6] Extracellular vesicles of mitochondrial synthesis-promoting factors, extracellular vesicle groups of extracellular vesicles of extracellular vesicle synthesis-promoting factors, and co-treated extracellular vesicles showed the effect of reducing blood urea nitrogen in mice with chronic kidney disease, with * indicating a confidence interval >95%. [Modes for carrying out the invention]

[0049] The present invention will be further described by the following embodiments, which are not intended to limit the scope of the above-mentioned disclosures. Those skilled in the art can make some improvements and modifications without departing from the scope of the present invention.

[0050] Example 1: Mesenchymal stem cells are cultured to generate extracellular vesicles (EVs).

[0051] Human mesenchymal stem cells isolated from the umbilical cord of a healthy donor were cultured in 150 mm culture dishes in high-sugar (glucose concentration 4500 mg / L) Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were cultured for 24-48 hours at 37°C with 5% carbon dioxide until the cells reached 40-60% confluence. After washing the cells twice with the aforementioned DMEM medium, the cells were treated in four groups by adding 10% FBS to the aforementioned DMEM medium. The control group consisted of cells cultured in culture medium only, with no additional treatment. In the mitochondrial synthesis promoter treatment group, the mitochondrial synthesis promoter was added, and the concentration of the mitochondrial synthesis promoter in the medium was set to 40 nanograms / milliliter (ng / mL). In this example, heme oxygenase-1 (Enzo LifeSciences, Ann Arbor, MI, USA) was selected as the mitochondrial synthesis promoter. In the extracellular vesicle generation promoting factor treatment group, the concentration of the extracellular vesicle generation promoting factor in the culture medium was set to 50 mM after adding the extracellular vesicle generation promoting factor. In this example, ethanol was selected as the extracellular vesicle generation promoting factor. Alternatively, there was a joint treatment group of mitochondrial generation promoting factor and extracellular vesicle generation promoting factor (hereinafter abbreviated as the joint treatment group), in which the mitochondrial generation promoting factor was added first, followed by the extracellular vesicle generation promoting factor. Specifically, mitochondrial generation promoting factor-heme oxygenase-1 was added 24 hours after culturing human mesenchymal stem cells, setting the concentration of heme oxygenase-1 in the culture medium to 40 ng / mL. At 36 hours of culture, extracellular vesicle generation promoting factor-ethanol was added, setting the concentration of ethanol in the culture medium to 50 mM. After 72 hours of culture, the supernatant was collected, and extracellular vesicles in the conditioned medium (CM) generated in each treatment group were collected. Specifically, the conditional media generated in each treatment group were centrifuged at 3000g for 10 minutes to remove dead cells or larger cell fragments. Subsequently, the supernatant of the conditional media from each group was pre-washed using an Extracellular vesicle Isolation Pre-Clearing Column.The supernatant after preliminary washing was transferred to an extracellular vesicle isolation column (Capturem™) and centrifuged at 1000g for 2-4 minutes at room temperature. The column was then washed once with Extracellular Vesicle Isolation Wash Buffer, and the extracellular vesicles from each group were eluted using the Extracellular Vesicle Isolation Elution Buffer provided in the kit.

[0052] Test Example 1: Nanoparticle Tracking Analysis

[0053] Extracellular vesicles obtained by processing each group in Example 1 were diluted with phosphate-buffered saline (PBS) to prepare dilutions containing 20 to 100 particles in the field of view. The number of microvesicles containing mitochondria was measured by nanoparticle tracking analysis (NTA). The final count of microvesicles containing mitochondria was performed by fluorescent nanoparticle tracking analysis (fNTA). Extracellular vesicles separated in Example 1 were labeled with TMRE fluorescent stain (tetramethylrhodamine ethyl ester) to detect mitochondria in the extracellular vesicles. The excitation light and synchrotron radiation wavelengths of the TMRE fluorescent stain were 550 nanometers (nm) and 575 nm, respectively. The size of extracellular vesicles and microvesicles containing mitochondria was measured using a nanoparticle size analyzer (NanoSight LM10-HS system, Malvern, UK), and the data was analyzed using nanoparticle tracking software (version 2.3).

[0054] Figure 1 shows the results of mitochondrial count measurement for each treatment group. Microvesicles containing mitochondria obtained from the mitochondrial synthesis-promoting factor treatment group, the extracellular vesicle synthesis-promoting factor treatment group, and the combined treatment group were confirmed to reliably promote the generation of microvesicles with more mitochondria compared to the control group. Therefore, combined treatment of human mesenchymal stem cells with mitochondrial synthesis-promoting factor, extracellular vesicle synthesis-promoting factor, or both mitochondrial synthesis-promoting factor and extracellular vesicle synthesis-promoting factor can all promote the generation of microvesicles with more mitochondria by human mesenchymal stem cells. Of these, the mitochondrial synthesis-promoting factor treatment group and the combined treatment group were able to further promote the generation of microvesicles with more mitochondria by human mesenchymal stem cells, at 16 times and 132 times, respectively, compared to the control group.

[0055] Furthermore, by dividing the results in Figure 2 by the number of extracellular vesicles obtained by each treatment group, the ratio of microvesicles containing mitochondria to the extracellular vesicles generated in each treatment group was determined, and the results are shown in Figure 2. The extracellular vesicles obtained by the mitochondrial synthesis-promoting factor treatment group, the extracellular vesicle-promoting factor treatment group, and the combined treatment group were confirmed to have a significantly higher proportion of mitochondria compared to the control group. Therefore, combined treatment of human mesenchymal stem cells with mitochondrial synthesis-promoting factor, extracellular vesicle-promoting factor, or both mitochondrial synthesis-promoting factor and extracellular vesicle-promoting factor can all promote the presence of a higher proportion of mitochondria in the extracellular vesicles generated by human mesenchymal stem cells. Among these, the mitochondrial synthesis-promoting factor treatment group and the combined treatment group enabled the presence of a higher proportion of mitochondria in the extracellular vesicles generated by human mesenchymal stem cells, at 1.89% and 4.9%, respectively, which were 14.44 times and 37.40 times higher than the control group, respectively.

[0056] Therefore, by using a combination of a mitochondrial synthesis promoting factor and an extracellular vesicle synthesis promoting factor, it is possible to reliably promote the generation of mitochondrial microvesicles in cells that possess mitochondria, and the method of the present invention can reliably generate a large amount of mitochondrial microvesicles.

[0057] Example 2: Animal experiment using a rat model of type 2 diabetes.

[0058] Seven male albino rats weighing 150-200 grams (g) were housed in cages and kept under temperature control (22°C-25°C) and a 12-hour light-dark cycle (light exposure from 08:00 to 20:00). The experimental animals had free access to food and water. On day 0 of the experiment, the experimental animals were randomly divided into two groups: a normal control group (n=7) fed a standard experimental diet for two weeks, and a diabetic group fed a high-fat diet (HFD; 20% protein, 60% fat, and 20% carbohydrates) for two weeks. On day 14, type 2 diabetes was induced in the diabetic group by intraperitoneal injection of a low dose of streptozotocin (STZ) at a dose of 45 mg / kg (mg / kg). Both low-dose streptozotocin and a high-fat diet are necessary elements for inducing insulin resistance in type 2 diabetes. Therefore, all rats were able to freely consume the food and water corresponding to their respective groups until the end of the experiment. On day 21 of the experiment, fasting blood glucose (FBG) and insulin levels were measured in the control group and the diabetic group (Control group) that had been fasted overnight. The type 2 diabetic model rats were further divided into: a diabetes control group, a group of extracellular vesicles of mitochondrial synthesis promoters, a group of extracellular vesicles of extracellular vesicles of extracellular vesicle synthesis promoters, and a group of jointly treated extracellular vesicles (n=7 in each group). Each group of rats received tail vein injections of PBS buffered solution (diabetes control group) twice a week for two weeks, or tail vein injections of the extracellular vesicles of each treatment group obtained in Example 1 twice a week, with a dose of 3 × 10⁶ per kg of body weight each time. 8Individual extracellular vesicles were administered and the treatment was carried out over a period of two weeks. During the experiment, all rats were fed a normal diet, and blood samples were collected from the rats three weeks after administration.

[0059] Figures 3 and 4 show the improvement in fasting blood glucose and insulin resistance in diabetic rats after each treatment, respectively. Figure 3 shows that fasting blood glucose levels in all treatment groups—the mitochondrial synthesis-promoting extracellular vesicle group, the extracellular vesicle-promoting extracellular vesicle group, and the co-treatment extracellular vesicle group—were lower than those in the diabetes control group. Among these, the mitochondrial synthesis-promoting extracellular vesicle group and the co-treatment extracellular vesicle group showed the best results. The mitochondrial synthesis-promoting extracellular vesicle group showed a 61.78% reduction in fasting blood glucose compared to the diabetes control group, while the co-treatment extracellular vesicle group showed a further 65.35% reduction in fasting blood glucose compared to the diabetes control group, and was similar to that of the normal control group.

[0060] Figure 4 shows that the fasting insulin concentrations in diabetic rats in all treatment groups—the mitochondrial synthesis-promoting extracellular vesicle group, the extracellular vesicle-promoting extracellular vesicle group, and the co-treated extracellular vesicle group—were all lower than those in the diabetes control group. Among these, the mitochondrial synthesis-promoting extracellular vesicle group and the co-treated extracellular vesicle group showed the best effects. The mitochondrial synthesis-promoting extracellular vesicle group showed a 53.70% reduction in fasting insulin concentration compared to the diabetes control group, while the co-treated extracellular vesicle group showed a further 56% reduction in fasting insulin concentration compared to the diabetes control group, and was similar to that of the normal control group of rats.

[0061] Therefore, the microvesicles and extracellular vesicle compositions containing mitochondria produced by the method of the present invention can reliably treat type 2 diabetes. They can lower fasting blood glucose levels and improve the high insulin concentrations caused by insulin resistance in diabetic rats.

[0062] Example 3: Animal experiments using a mouse model of chronic kidney disease.

[0063] All animal experiments were conducted in accordance with the guidelines for the management and use of experimental animals. Six-week-old BALB / c mice were housed in a constant temperature (25°C) environment with a 12-hour light-dark cycle. The mice were divided into the following five groups (n=5 in each group): (1) normal control group, (2) chronic kidney disease control group (treated with PBS), (3) mitochondrial synthesis-promoting extracellular vesicle group, (4) extracellular vesicle-promoting extracellular vesicle group, and (5) co-treated extracellular vesicle group. Chronic kidney disease was induced in the mice by feeding them a diet containing 0.25% adenine. After one week, each group of mice received tail vein injections of PBS buffered solution (chronic kidney disease control group) twice a week. Alternatively, extracellular vesicles from each treatment group obtained in Example 1 were injected into the tail vein twice a week, with 3 × 10⁶ units per kg of body weight each time. 8 Each mouse was administered an extracellular vesicle for a total of two weeks. During the experiment, all mice were fed a normal diet, and blood samples were taken from the mice three weeks after administration.

[0064] After collecting mouse blood, it was centrifuged at 1500 rpm for 30 minutes. The upper layer serum was then taken and its blood creatinine and blood urea nitrogen (BUN) levels were measured in combination with enzyme-linked immunosorbent assay (ELISA). The absorbance at 450 nm was then measured using a microplate analyzer (Thermo Fisher Scientific).

[0065] Figures 5 and 6 show the improvement in blood creatinine and urea nitrogen levels in mice with chronic kidney disease after each treatment, respectively. From Figure 5, it can be seen that blood creatinine levels in all treatment groups—the group with extracellular vesicles of mitochondrial synthesis promoters, the group with extracellular vesicles of mitochondrial synthesis promoters, and the group with co-treated extracellular vesicles—were lower than in the control group of mice with chronic kidney disease. Among these, the groups with extracellular vesicles of mitochondrial synthesis promoters and the group with co-treated extracellular vesicles showed particularly good effects. The group with extracellular vesicles of mitochondrial synthesis promoters reduced creatinine levels by approximately 54.06% compared to the control group with chronic kidney disease, and the group with co-treated extracellular vesicles reduced creatinine levels by 67.96% compared to the control group with chronic kidney disease, which was similar to the normal control group.

[0066] Figure 6 shows that blood urea nitrogen levels in chronic kidney disease mice were lower in all treatment groups: the mitochondrial synthesis-promoting extracellular vesicle group, the extracellular vesicle-promoting extracellular vesicle group, and the co-treated extracellular vesicle group, compared to the chronic kidney disease control group. Among these, the mitochondrial synthesis-promoting extracellular vesicle group and the co-treated extracellular vesicle group showed particularly good effects. The mitochondrial synthesis-promoting extracellular vesicle group reduced urea nitrogen levels by approximately 66.80% compared to the chronic kidney disease control group, and the co-treated extracellular vesicle group reduced urea nitrogen levels by 79.42% compared to the chronic kidney disease control group, similar to the normal control group.

[0067] Therefore, microvesicles having mitochondria produced by the method of the present invention can reliably treat and improve diabetes, and can reduce creatinine and urea nitrogen levels in the blood of mice with renal injury and chronic kidney disease.

[0068] As described above, the method of the present invention can reliably produce large quantities of microvesicles containing mitochondria. Furthermore, since the isolated extracellular vesicle composition produced by the method of the present invention contains a large amount of microvesicles containing mitochondria, it can reliably treat or alleviate diabetes, such as type 2 diabetes, and kidney injury, such as chronic kidney disease.

[0069] The foregoing is merely to illustrate preferred embodiments of the present invention and does not limit the scope of the invention; the scope of rights claimed by the present invention should be primarily those described in the claims.

Claims

1. An application that uses a combination of mitochondrial synthesis promoters and extracellular vesicle synthesis promoters to promote the generation of mitochondrial microvesicles in cells containing mitochondria.

2. The mitochondrial synthesis promoting factor described in claim 1 is selected from the group consisting of heme oxygenase-1, CoPPIX, hemin, hemin derivatives, mitochondrial structural fragments, cell tissue fragments, and culture in a 5-20% oxygen concentration environment, and is used in combination with the extracellular vesicle synthesis promoting factor.

3. The use of the mitochondrial synthesis promoter and the extracellular vesicle synthesis promoter according to claim 1, wherein the extracellular vesicle synthesis promoter is selected from the group consisting of ethanol, an EP4 receptor antagonist, an endosomal protein sorting and transport complex, ARRDC1, a tumor suppressor gene of a related protein, and cell relaxant B.

4. The use of the mitochondrial synthesis promoting factor and the extracellular vesicle synthesis promoting factor according to claim 1 in combination, wherein the cells having mitochondria are mesenchymal stem cells, hematopoietic stem cells, bone marrow stem cells, or liver cells.

5. Step (1) to provide cells having mitochondria, Step (2) involves co-culturing cells having mitochondria by adding a mitochondrial synthesis promoting factor and an extracellular vesicle synthesis promoting factor to the culture medium to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, Includes, A method for producing an extracellular vesicle composition rich in mitochondria, wherein the extracellular vesicle composition rich in mitochondria contains 0.2% or more mitochondria-containing microvesicles based on the total number of extracellular vesicles.

6. A method for producing an extracellular vesicle composition rich in mitochondrial microvesicles according to claim 5, wherein the amount of the mitochondrial synthesis promoting factor added is such that the concentration of the mitochondrial synthesis promoting factor in the culture medium is 0.1 to 100,000 ng / mL.

7. The amount of the extracellular vesicle formation promoting factor added is such that the concentration of the extracellular vesicle formation promoting factor in the culture medium is 10 －７ A method for producing an extracellular vesicle composition rich in mitochondria-containing microvesicles according to claim 5 or 6, wherein the concentration is ~800 mmol / liter (mM).

8. A pre-isolated extracellular vesicle composition having microvesicles containing 0.2% or more mitochondria, based on the total number of extracellular vesicles.

9. The isolated extracellular vesicle composition according to claim 8, wherein the isolated extracellular vesicle composition is produced by the method described in any one of claims 5 to 7 and then isolated.

10. A pharmaceutical composition comprising the isolated extracellular vesicle composition according to claim 8 or 9, and a pharmaceutically acceptable carrier.

11. An application for manufacturing a drug for treating or alleviating diabetes, wherein the drug contains an effective dose of an isolated extracellular vesicle composition and a pharmaceutically acceptable carrier, and the isolated extracellular vesicle composition has microvesicles having 0.2% or more mitochondria based on the total number of extracellular vesicles.

12. Uses of the isolated extracellular vesicle composition according to claim 11, wherein the diabetes is type 2 diabetes.

13. Uses of the isolated extracellular vesicle composition according to claim 11 or 12, wherein the target of administration of the drug is a warm-blooded animal or a human.

14. An application for manufacturing a drug for treating or alleviating kidney injury, wherein the drug contains an effective dose of an isolated extracellular vesicle composition and a pharmaceutically acceptable carrier, and the isolated extracellular vesicle composition has microvesicles having 0.2% or more mitochondria based on the total number of extracellular vesicles.

15. Uses of the isolated extracellular vesicle composition according to claim 14, wherein the kidney injury is a chronic kidney disease.

16. Uses of the isolated extracellular vesicle composition according to claim 14 or 15, wherein the target of administration of the drug is a warm-blooded animal or a human.

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