Patent Application
20240215568
The present invention relates to a composition for freeze-dry protecting extracellular vesicles, capable of preserving the functions and characteristics of extracellular vesicles, and an extracellular vesicle freeze-drying method using the same.
Treatments using stem cells, particularly mesenchymal stem cells (MSCs), and positive clinical results have been reported for various diseases. However, a stem cell therapeutic agent has a risk of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders, and efficacy verification through clinical trials is still required. It is known that a paracrine effect of stem cells induces the promotion of the regeneration and vascular regeneration of surrounding skin cells, and in particular, extracellular vesicles (EV) are known as a major efficacious factor in the paracrine effect.
The extracellular vesicles are classified into exosomes and microvesicles depending on a size, and the exosomes have a diameter of 30 to 150 nm, and the microvesicles have a size of 100 to 1,000 nm. The extracellular vesicles are parts of the cell membrane that have been separated into the blood, and are known to mediate intercellular communication by containing both protein and nuclear components. Using extracellular vesicles instead of stem cells not only increases safety by minimizing the side effects by using stem cells, but also is advantageous in terms of biodistribution and production process.
However, despite the usefulness of extracellular vesicles, there is still a lack of research on technologies and stable formulations for a method capable of isolating and then stably maintaining and storing extracellular vesicles, which are difficult to obtain. Therefore, there is a need for a new preservation method to stably maintain and store extracellular vesicles that can be used as therapeutic agents for various diseases.
Therefore, the present inventors have studied technology for stably maintaining and storing extracellular vesicles, identified a new freeze-dry protective agent capable of effectively improving problems that may occur during a freeze-drying process and preserving unique properties and functions of extracellular vesicles, and then completed the present invention.
Accordingly, an object of the present invention is to provide a composition for freeze-dry protecting extracellular vesicles including trehalose and sucrose, and a method for freeze-drying extracellular vesicles using the same.
In order to achieve the object, the present invention provides a composition for freeze-dry protecting extracellular vesicles including trehalose and sucrose.
The present invention also provides a freeze-dry protective agent for extracellular vesicles, including trehalose and sucrose.
In addition, the present invention provides a composition for freeze-drying extracellular vesicles including a mixture of trehalose and sucrose as a freeze-dry protective agent: and extracellular vesicles.
In addition, the present invention provides a freeze-drying method of extracellular vesicles including 1) mixing a mixture of trehalose and sucrose, as a freeze-dry protective agent, with extracellular vesicles; and 2) freeze-drying the mixture in step 1).
According to the present invention, trehalose and sucrose are added to an extracellular vesicle freeze-drying process to solve the problems of crystal growth and redissolution capacity degradation, which are problems in an existing freeze-dry protective agent, and also maintain the unique marker, functional factor expressions and therapeutic effects of the extracellular vesicles, and thus may be variously utilized for producing various therapeutic agents using extracellular vesicles.
The present invention provides a composition for freeze-dry protecting extracellular vesicles including trehalose and sucrose, and a method for freeze-drying extracellular vesicles using the same.
According to the present invention, the composition for freeze-dry protecting extracellular vesicles may solve the problems of crystal growth and redissolution capacity degradation, which are problems in an existing freeze-dry protective agent, and also maintain or increase the unique marker, functional factor expressions and therapeutic effects of the extracellular vesicles.
Hereinafter, the present invention will be described in detail.
The present invention provides a composition for freeze-dry protecting extracellular vesicles including trehalose and sucrose and an extracellular vesicle freeze-dry protective agent including trehalose and sucrose, which is characterized by including trehalose and sucrose as active ingredients as an extracellular vesicle freeze-dry protective agent. As such, when trehalose and sucrose are mixed and treated to extracellular vesicles, it is possible to not only solve the problems of forming unidentified nanoparticles where single ingredients such as trehalose, mannitol, and sucrose form salts and crystals by a PBS component during the freeze-drying process, but also to increase the dissolution rate when redissolving the freeze-dried extracellular vesicles.
Accordingly, the present invention provides a composition for freeze-drying extracellular vesicles including a mixture of trehalose and sucrose as a freeze-dry protective agent: and extracellular vesicles.
The trehalose and the sucrose may be 1 to 10% (w/v), preferably 2 to 9% (w/v), more preferably 2 to 8% (w/v) of trehalose and sucrose, respectively. When less than 1% (w/v) of trehalose or sucrose is used, there may be a problem of generating a large number of unidentified nanoparticle crystals, and the redissolution rate may also be slow.
The present invention is characterized by mixing and adding trehalose and sucrose as active ingredients of a freeze-dry protective agent, and the trehalose and the sucrose may be mixed in a volume ratio of preferably 1:0.05 to 5, more preferably 1:0.5 to 3, and even more preferably 1:0.5 to 2. In a preferred embodiment of the present invention, the effect was confirmed using a freeze-dry protective agent mixed in a 1:1 volume ratio.
The freeze-dry protective composition of the present invention may include a mixture of 1 to 10% (w/v) trehalose and sucrose mixed in a volume ratio of 1:0.05 to 5, and may include a mixture of trehalose and sucrose at the concentrations in a volume ratio of 1:0.1 to 3, more preferably 1:0.5 to 1.
The freeze-dry protective agent composition mixed with trehalose and sucrose of the present invention may reduce crystal formation that occurs during freeze-drying, rehydration, and redissolution of extracellular vesicles.
The trehalose and sucrose as the freeze-dry protective agent may be mixed with extracellular vesicles to be freeze-dried in a volume ratio of 1:0.5 to 5, preferably 1:0.5 to 3, more preferably 1:0.5 to 2.
The freeze-dry protective agent is mixed with the extracellular vesicles, frozen at −70 to −90° C., and freeze-dried in a freeze-dryer for 3 to 7 days to produce the freeze-dried extracellular vesicles.
The freeze-dry protective agent is diluted with PBS as a base and prepared, and may use sterilized distilled water for redissolution after freeze-drying. Through this process, the compounds constituting freeze-dried PBS together with extracellular vesicles are dissolved in distilled water again, and the extracellular vesicles may be preserved in PBS.
The extracellular vesicles to be freeze-dried of the present invention may be extracellular vesicles isolated from cells isolated from natural organisms or extracellular vesicles isolated from ingredients such as milk. In addition, the cells may be derived from any type of animal or plant, including humans and non-human mammals, and may be various types of immune cells, tumor cells, and stem cells, and preferably, the stem cells may be mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells.
The extracellular vesicles refer to vesicles released outside the cells and include microvesicles, apoptotic vesicles, exosomes, etc. Therefore, the present invention may include, without limitation, various extracellular vesicles capable of achieving the purpose by adding trehalose and sucrose as freeze-dry protective agents, and for example, the extracellular vesicles may be exosomes.
The exosomes of the present invention mean all vehicles (e.g., exosome-like vehicles) that have a nano-sized vesicle structure that is secreted from cells of various animals, plants, bacteria, fungi, and algae and released into an extracellular space, and have compositions similar to exosomes. In particular, the exosome of the present invention may be exosomes derived from stem cells.
In the present invention, the extracellular vesicles may be extracellular vesicles that exhibit therapeutically useful effects, and may be treated with the freeze-dry protective agent composition and the freeze-dry protective agent of the present invention during the freeze-drying process of these extracellular vesicles, so that unique characteristics, properties, and therapeutic effects of the extracellular vesicles may be maintained at equivalent levels or higher.
In the present invention, when freeze-drying the extracellular vesicles by adding trehalose and sucrose, it was confirmed that the formation of undesired and unidentified nanoparticle crystals was suppressed during the freeze-drying process, but the rehydration and dissolution rates thereof were significantly accelerated.
Furthermore, it was confirmed that there was no loss in the amount of total protein and the amount of total RNA contained in the extracellular vesicles, and the levels of miRNA exhibiting therapeutic effect were also maintained equally. In addition, it was confirmed that CD63, a marker for extracellular vesicles, also exhibited a similar or increased expression pattern compared to before freeze-drying.
The therapeutic efficacy of the extracellular vesicles, whose efficacy is maintained by the freeze-dry protective agent, may be a broad regenerative or immunomodulatory effect, which is the inherent therapeutic efficacy of extracellular vesicles, and for example, may be blood vessel regeneration capacity. The extracellular vesicles frozen by the freeze-dry protective agent of the present invention may be used as pharmaceutical compositions, cosmetic compositions, food compositions, and external skin compositions, and may be administered orally or parenterally.
The types of diseases requiring angiogenesis to which the freeze-dried extracellular vesicles of the present invention may be applied are not limited thereto, but may be at least one selected from the group consisting of burns, ulcers, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cardiovascular diseases, cerebrovascular diseases, and alopecia, particularly cardiovascular diseases or cerebrovascular diseases.
The pharmaceutical composition including the freeze-dried extracellular vesicles of the present invention may further include appropriate carriers, excipients, and diluents commonly used in the production of pharmaceutical compositions, in addition to the active ingredients. The pharmaceutical composition of the present invention may further include other pharmaceutically active ingredients or active mixtures.
The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods, respectively. The carrier, the excipient, and the diluent that may be included in the composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When the pharmaceutical composition is formulated, the formulation may be prepared by using diluents or excipients, such as a filler, an extender, a binder, a wetting agent, a disintegrating agent, and a surfactant, which are generally used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid formulations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Further, lubricants such as magnesium stearate and talc are used in addition to simple excipients.
Liquid formulations for oral administration may correspond to suspensions, oral liquids, emulsions, syrups, and the like, and may include various excipients, for example, a wetting agent, a sweetener, an aromatic agent, a preservative, and the like, in addition to water and liquid paraffin which are commonly used as simple diluents. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, and suppositories. As the non-aqueous solution and the suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base compound of the suppository, witepsol, macrogol, tween 61, cacao butter, laurinum, glycerogelatin, and the like may be used.
A preferable dose of the pharmaceutical composition of the present invention varies according to the condition and weight of a patient, the degree of a disease, a drug form, and the route and period of administration, but may be properly selected by those skilled in the art. The administration may be performed once a day or several times a day. The dose does not limit the scope of the present invention in any aspect.
The pharmaceutical composition of the present invention may be administered to mammals such as mice, rats, livestock, and humans through various routes. All methods of administration may be expected, and for example, the pharmaceutical composition may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
The definitions of terms for the excipients, binders, disintegrants, lubricants, flavor enhancers, flavoring agents, and the like of the present invention are described in literature known in the art and include those with the same or similar functions.
Further, the present invention provides a freeze-drying method of extracellular vesicles including 1) mixing a mixture of trehalose and sucrose, as freeze-dry protective agents, with extracellular vesicles: and 2) freeze-drying the mixture in step 1).
The description of the freeze-drying method may be applied in the same manner as the description of the freeze-dry protective composition and the freeze-dry protective agent described above.
In the freeze-drying method, the freeze-drying of step 2) may include freezing at −70 to −90° C. and then freeze-drying for 3 to 7 days. The freezing may be performed overnight, and the freeze-drying may be performed under a pressure of 1 to 7 mTorr, and may be performed in first, second, or third freeze-drying processes.
Duplicated contents are omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have the meanings commonly used in the art to which the present invention pertains.
Terms not defined otherwise in this specification have meanings commonly used in the technical art to which the present invention pertains.
Hereinafter, the present invention will be described in detail by Examples. However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.
Human bone marrow-derived stem cells were cultured to collect exosomes, which were extracellular vesicles (hereinafter, EV) to be used in an experiment. The cells were incubated at a concentration of 2.5×105 cells in a 100 mm culture dish (SPL, 20100), and incubated in a low-glucose Dulbecco's modified Eagle's medium (DMEM, Life Technologies Corporation, CA, USA) filtered to 0.2 μm and a medium of 10% fetal bovine serum (FBS, Life Technologies Corporation) and 1% antibiotics-antimycotics (Life Technologies Corporation). Subculture was performed every 80 to 90% from P2 to P6, and incubated in an incubator at 37° C. and 5% CO2.
Stem cells incubated under the same conditions after P6 were incubated for 5 days in a low-glucose DMEM, and a medium consisting of 10% exosome-depleted fetal bovine serum (System Biosciences, Palo Alto, CA, USA) and 1% antibiotics-antimycotics. Thereafter, the culture medium was harvested and centrifuged under conditions of 4° C., 2500 g, and 10 min, and only a supernatant was taken and filtered to 0.2 μm. The medium collected above was immediately subjected to tangential flow filtration (TFF), and subjected to concentration and diafiltration, respectively, and concentrated at about 10 times the starting volume. At this time, the membrane was used with a Minimate TFF 300K membrane (Pall Corporation, NY, USA). Exosomes were collected through the same process as above, and used in subsequent freeze-drying experiments.
To perform freeze-drying of EVs, EVs were collected as in Example 1, and then the size and concentration were measured by qNano (IZON Ltd, Christchurch, New Zealand) using tunable resistive pulse sensing technology. Thereafter, the freeze-dry protective agent and exosomes were mixed at a 1:1 volume ratio so that the final concentration was 1×1011 EVs/ml. As candidates for selecting a freeze-dry protective agent suitable for exosomes, trehalose (Sigma, St. Louis, MO, USA), mannitol (Sigma), DMSO (Sigma), and sucrose (Sigma) were used.
As an experimental group, mannitol 0 to 5% (w/v), DMSO 0 to 10% (w/v), and trehalose 0 to 4% (w/v) were set as a single-administered group; and a mixture of sucrose and trehalose at each concentration of 2 to 8% (w/v) mixed in a 1:1 volume ratio was used as the experimental group. After mixing, the mixture was kept overnight at −80° C. and then freeze-dried (−80° C., 5 mTorr) for 4 days. After freeze-drying was completed, the mixture was sealed and stored at room temperature. The sample stored above was subjected to a rehydration process for use in the experiment, and upon rehydration, deionized water filtered to 0.2 μm was added to the original volume of the mixture and kept at 37° C. for 30 minutes.
Exosomes were mixed while changing the freeze-dry protective agent to 0 to 10% DMSO, 0.1 to 5% mannitol, and 0.03 to 0.17% trehalose, and the results of visually confirming the frozen state and the state after rehydration were illustrated in
As illustrated in
As in Example 2, trehalose and mannitol were treated as a freezing protective agent and frozen, and then rehydrated, and the number and size of EVs obtained were confirmed through qNano measurement. qNano was a device for measuring the concentration and size of particles through tunable resistive pulse sensing technology. A nanopore used in the experiment was NP200, the radial stretch was 46.5 mm, a voltage of 0.66 mV was applied, and the concentration and size of EVs in the sample were measured using 0.075 ml of the sample. All the samples were analyzed by measuring at least 500 particles, and all of the samples were calibrated with CPC100 (100 mm, 1.4E+13 particles/ml, IZON Ltd). The measurement result was illustrated in
As illustrated in
It was known that a freeze-dry protective agent may form salts and crystals due to the presence of a PBS component, and that crystallization may be promoted under certain conditions. In order to confirm newly generated unidentified nanoparticles other than EVs as confirmed in Example 4, only PBS alone, and mannitol and trehalose diluted in PBS were freeze-dried without EVs, and whether nanoparticles were formed after freeze-drying was confirmed through XRD peak analysis. The internal structure was analyzed by measuring XRD under Cu radiation (45 kV×200 mA) using Smartlab (Rigaku, JP). The range of 20 was set to 2 to 45, and the analysis was performed by setting the step size to 0.05° and the dwell time to 2 sec, and the results were illustrated in
As illustrated in
As the XRD analysis result, it was assumed that the unspecified nanoparticles generated during freeze-drying of the trehalose/PBS solution were sodium chloride and phosphate, so that it was confirmed whether the sodium chloride and phosphate may be gradually dissolved over time. As a result of redissolving the sodium chloride and phosphate while shaking at room temperature, as confirmed in
When described these results together, it was confirmed that when trehalose was used as a freeze-dry protective agent at a low concentration, crystallized salts were generated during the freeze-drying process, which took a long time for redissolution.
In Example 5, when a low concentration of trehalose was used as a freeze-dry protective agent, a problem of generating salt crystals was confirmed, and thus, in order to solve the problem, the formation of nanoparticles was confirmed in an experimental group in which the concentration of trehalose was changed from 2 to 4%, and in an experimental group in which trehalose and sucrose, known as an amorphous freezing protective agent were mixed. 2 to 8% (w/v) of trehalose and sucrose were mixed at a 1:1 volume ratio and used as a freeze-dry protective agent experimental group. Using the experimental group, a freeze-dry protective agent and PBS were mixed and redissolved without EV, and qNano analysis and XRD analysis were performed in the same manner as Examples 4 and 5. The results were illustrated in
As illustrated in
In
As illustrated in
Through the results, it was confirmed that as the concentration increased, crystal generation and redissolution capacity increased, and in particular, the trehalose and sucrose mixture had a significantly low crystal generation rate and excellent redissolution rate during freeze-drying.
As these results, when freeze-drying EVs using a mixture of trehalose and sucrose as a freeze-dry protective agent, unidentified nanoparticle crystals were not formed or quickly redissolved, and thus, the mixture was suitable as a freeze-dry protective agent for an EV therapeutic agent required to be administered to patients by controlling the number of EVs.
Since it has been confirmed that the freeze-dry protective agent mixed with trehalose and sucrose was suitable as a freeze-dry protective agent for an EV therapeutic agent, when applied to freeze-drying of the MSC-EVs obtained in Example 1, the redissolution rate was further confirmed, and the results were illustrated in
The EVs were treated with the freeze-dry protective agent and then freeze-dried and redissolved, and the number of EV particles was confirmed, and the results were illustrated in
As illustrated in
When redissolved after freeze-drying, changes in EV size were confirmed, and the results were illustrated in
As illustrated in
In addition, the shape of EV particles in each experimental group was confirmed through Cryo-EM. The Fresh EV experimental group was able to be used immediately, but the freeze-dried experimental group was redissolved at 37° C. for 30 minutes and then used in the experiment. Before sampling, a Glow discharge system (PELCO easiGlow™, Ted pella) was used to make grids (Quantifoil, R1.2/1.3, 200 mesh, EMS) into a hydrophilic surface. 4 μl of each sample was placed on the grid and blot force 3 was applied for 1.5 seconds while maintaining 100% moisture at 4° C. Thereafter, the samples were frozen in liquid ethane using a Vitrobot Mark IV (FEI) method to vitrify the samples. Thereafter, analysis was performed at the Nanobioimaging center (Seoul national university, Korea) using a Talos L120C (FEI) microscope with gun type Lab6 at 120 kV. The results were illustrated in
As illustrated in
The results showed that in the freeze-dried group using PBS, nanoparticles were confirmed about twice greater than the initial freeze-dried EVs, and new nanoparticles out of the EV size distribution were identified, and thus there was a crystal formation problem as confirmed above. On the other hand, when the mixture of trehalose and sucrose was treated as the freeze-dry protective agent, there was no problem of crystal formation, and thus, it was confirmed that EVs may be obtained by redissolving after freeze-drying while preserving the number of EVs.
In order to be used as a freeze-dry protective agent for an EV therapeutic agent, it is important not only to obtain EVs without EV loss or production of other materials after freeze-drying and redissolution, but also not to affect the properties and functions of EVs to be freeze-dried. Accordingly, in EVs treated with the mixture of trehalose and sucrose as the freeze-dry protective agent and frozen and redissolved, it was confirmed whether the total RNA contained in EVs was restored without loss and whether changes in miRNA, which was an efficacy factor for EVs, were not induced, and changes in the expression of CD63 as an EV marker were confirmed.
Protein quantitative analysis was performed as follows: All the samples were centrifuged at 120000 g for 1 hour using an ultra-high-speed centrifuge (Beckman Coulter, CA, USA), and then the supernatant was discarded, and lysis was performed.
The lysis was performed using RIPA (Life Technologies Corporation), and proteins were quantified using BCA protein assay (Life Technologies Corporation).
Analysis of CD63, the EV marker, was confirmed through ELISA and was performed using the following method: ELISA was performed with a commercial kit according to the manual of a manufacturer. A CD63 (EH95RB, ThermoFisher Scientific, Inc, Waltham, MA, USA) ELISA kit included standard proteins, and thus the amounts of proteins and extracellular vesicles were determined based on the standard curve of the kit. Isolated Fresh EV and freeze-dried EVs were dispensed in the same amount (200 uL/well) of EVs and Standard in a 96-well microplate coated with a capture antibody without pretreatment, and then reacted at 4° C. The next day, a sandwich-type ELISA method was performed and the absorbance was measured using a microplate reader.
Analysis of the expression of miRNA, which was an efficacy factor of EV, was performed by qPCR and preceded as follows: RNA was extracted from EVs using Trizol™ according to the instructions of the manufacturer and RNA was quantified by a nanodrop. RNA was made into cDNA through a reverse transcription (RT) process, and real-time PCR was performed according to the manual of the manufacturer using Taqman probes suitable for miRNA and mRNA.
The results of confirming changes in EV protein amount, total RNA recovery rate, miRNA amount, and EV marker expression as described above were illustrated in
As illustrated in
As illustrated in
As illustrated in
EV was known to have blood vessel regeneration capacity and be able to be used as a therapeutic agent for various diseases requiring angiogenesis. An experiment was performed to determine whether EVs mixed with the freeze-dry protective agent of the present invention, and then frozen and redissolved, could maintain the inherent blood vessel regeneration capacity of EVs to an equivalent degree or improve the effect. The EV blood vessel regeneration capacity was confirmed through tube formation capacity and cell migration capacity experiments using HUVEC cells. More specifically, in order to see the effect of neovascularization by EVs obtained in Example 1, HUVECs attached on Matrigel were treated with EVs to confirm the tube formation effect. Specifically, HUVECs were cultured in an M199 medium (Gibco) supplemented with 20% FBS, 5 U/mL heparin, and 3 ng/mL bFGF. The cells were inoculated on a growth factor-reduced Matrigel Matrix (BD Bioscience, MA, USA) from u-Slides Angiogenesis (ibidi, Graefelfing, Germany) at a density of 1.0×104 cells, and the tube was formed for 7 hours in a humidifying chamber under a 37° C. and 5% CO2 environment. Images were taken on a phase-contrast microscope (Olympus), and the number of tubular structures was quantified in a microscope field (4× magnification) using ImageJ software.
For analysis of cell migration capacity, HUVECs were incubated in a M199 medium (Gibco) supplemented with 20% FBS, 5 U/mL heparin, and 3 ng/mL bFGF in a humidifying chamber with 37° C. and 5% CO2 environment. 1.0×105 cells were cultured in a 24-well plate to reach 100% confluency and then treated with MMC (2.5 ug/ml) to stop cell division. The cells were scratched vertically using a pipette tip and then washed with PBS. The scratches were imaged under a phase-contrast microscope (Olympus) and the corresponding regions were marked. The same area of the scratched wound was photographed again after 7 hours, and the degree of cell migration and wound closure was measured using ImageJ software. As freeze-dry protective agent experimental groups, an EV experimental group freeze-dried and rehydrated by treating a freeze-dry protective agent mixed with 0, 2, 4, and 8% (w/v) trehalose and sucrose (freeze-dried and rehydrated after treatment with PBS) in a 1:1 volume, Fresh EV, which was EV immediately after isolation without treatment with the freeze-dry protective agent, a positive control VEGF-treated group and a PBS-treated group. As a negative control, an experimental group in which HUVECs were treated with PBS was used. EVs were all treated at 5×108 EV/ml, and treated with VEGF at 100 ng/ml. The result of confirming angiogenesis capacity was illustrated in
As illustrated in
As described above, specific parts of the present invention have been described in detail, and it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0055116 | Apr 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2022/006105 | 4/28/2022 | WO |
