Freeze-dry protective agent for extracellular vesicles

Abstract

To provide a composition for freeze-dry protecting extracellular vesicles, capable of preserving the function and characteristics of extracellular vesicles, and an extracellular vesicle freeze-drying method using the same.SOLUTION: The composition for freeze-dry protecting extracellular vesicles comprises trehalose and sucrose, in which the trehalose and sucrose are each 1 to 10%(w / v) in the composition, and the trehalose and sucrose are mixed and contained at a volume ratio of 1:0.05 to 5.EFFECT: The trehalose and sucrose of the present invention 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for freeze-drying and protecting extracellular vesicles, which can maintain the functions and properties of extracellular vesicles, and a method for freeze-drying extracellular vesicles using the same. [Background technology]

[0002] Positive clinical results have been reported for the treatment of various diseases using stem cells, particularly mesenchymal stem cells (MSCs). However, stem cell therapy agents carry the risk of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders, and efficacy verification through clinical trials is still required. The paracrine effect of stem cells is known to induce the regeneration of surrounding skin cells and promote vascular regeneration, and extracellular vesicles (EVs) in particular are known to be the main effective factor in this paracrine effect. Extracellular vesicles are classified into exosomes and microvesicles based on their size. Exosomes range in diameter from 30 to 150 nm, while microvesicles range in size from 100 to 1,000 nm. Extracellular vesicles are portions of the cell membrane released into the bloodstream. They contain both proteins and nuclear components and are known to mediate intercellular communication. Using extracellular vesicles instead of stem cells not only minimizes side effects associated with stem cell use and increases safety, but also offers advantages in terms of biodistribution and production processes. However, despite the usefulness of extracellular vesicles, there is still a lack of research into techniques for stably maintaining and storing extracellular vesicles after isolating them, which are difficult to obtain, and into stable dosage forms. Therefore, there is a need for a novel preservation method for stably maintaining and storing extracellular vesicles that can be used as therapeutic substances for various diseases. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, while the present inventors were researching technologies for stably maintaining and storing extracellular vesicles, they discovered a novel freeze-drying protectant that can effectively alleviate problems that may arise during the freeze-drying process and preserve the inherent properties and functions of extracellular vesicles, thereby completing the present invention. Therefore, the present invention relates to a composition for freeze-protecting extracellular vesicles, which comprises trehalose and sucrose, and a method for freeze-drying extracellular vesicles using the same. [Means for solving the problem]

[0004] To achieve the above object, the present invention provides a composition for cryopreservation of extracellular vesicles, comprising trehalose and sucrose. The present invention also provides a lyoprotectant for extracellular vesicles, comprising trehalose and sucrose. The present invention also provides a composition for freeze-drying extracellular vesicles, comprising a mixture of trehalose and sucrose as a freeze-drying protectant; and extracellular vesicles. The present invention also provides a method for freeze-drying extracellular vesicles, comprising the steps of: 1) mixing extracellular vesicles with a mixture of trehalose and sucrose, which are cryoprotectants; and 2) freeze-drying the mixture obtained in step 1). [Effects of the Invention]

[0005] The trehalose and sucrose of the present invention are added to the lyophilization process of extracellular vesicles, and solve the problems of existing lyophilization protection agents, such as the formation of crystals and a decrease in resolubility, while maintaining the expression of specific markers and functional factors of extracellular vesicles and their therapeutic effects. Therefore, they can be used in a variety of ways in the production of various therapeutic agents using extracellular vesicles. [Brief explanation of the drawings]

[0006] [Figure 1]FIG. 1 shows the results of visual inspection of extracellular vesicles that were freeze-dried and rehydrated after adding DMSO, trehalose, or mannitol as a cryoprotectant. [Figure 2] Trehalose and mannitol were treated with a cryoprotectant, freeze-dried, and then rehydrated. The number (A) and size distribution (B) of extracellular vesicles (EVs) obtained by qNano measurement were confirmed. [Figure 3-AB] This figure shows the results of freeze-drying PBS alone without extracellular vesicles (EVs), mannitol diluted in PBS (A), and trehalose (B) alone, and confirming the formation of nanoparticles after freeze-drying through XRD peak analysis. [Figure 3-C] This figure shows the results of qNano analysis of unspecified nanoparticles produced in experimental groups in which 0.07% or 0.17% PBS and trehalose (T) were freeze-dried and then reconstituted (Lyo: freeze-dried experimental group; -80: experimental group frozen at -80°C and then reconstituted). [Figure 4-D] FIG. 1 shows the results of confirming the redissolution of unspecified nanoparticles formed after freeze-drying 0.07% trehalose (T) in PBS at room temperature for 48 hours. [Figure 4-A] This figure shows the results of qNano measurement of unspecified nanoparticles produced in the experimental group of trehalose (T) alone and the experimental group of trehalose and sucrose (T+S) mixed freeze-dried and reconstituted after freeze-drying without extracellular vesicles (EVs) (*p<0.05, **p<0.01, ***p<0.001). [Figure 4-B] This figure shows the results of confirming the redissolution and dissolution rate of unspecified nanoparticles produced in the experimental group of trehalose (T) alone and the experimental group of trehalose and sucrose (T+S) mixed freeze-dried and redissolved without extracellular vesicles (EVs) for 20 minutes. [Figure 4-C]This figure shows the results of XRD analysis of the crystals formed in the experimental group of trehalose (T) alone and the experimental group of trehalose and sucrose (T+S) mixed freeze-dried and redissolved after freeze-drying and redissolving without extracellular vesicles (EV). [Figure 5] This figure shows the results of confirming the number of extracellular vesicles (EV) particles in lyophilized and reconstituted EVs after treatment with a mixed lyoprotectant of 2-8% trehalose and sucrose. [Figure 6] This figure shows the results of examining the change in size distribution of extracellular vesicles (EVs) that were freeze-dried and rehydrated after treatment with a mixed lyoprotectant of trehalose and sucrose. [Figure 7] This figure shows the results of confirming the EV shape in each experimental group from cryo-EM data. [Figure 8] This figure shows the change in total protein content in lyophilized and rehydrated extracellular vesicles (EVs) after treatment with a 2-8% trehalose and sucrose lyoprotectant mixture (*p<0.05, **p<0.01, ***p<0.001). [Figure 9] This figure shows the results of examining changes in total RNA levels and miRNA expression levels associated with therapeutic effects in extracellular vesicles (EVs) that were treated with a 2-8% trehalose and sucrose mixed cryoprotectant and then lyophilized and rehydrated. [Figure 10] This figure shows the results of confirming the expression of the EV marker, CD63, in extracellular vesicles (EVs) that were treated with a 2-8% trehalose and sucrose mixed cryoprotectant (TS), followed by freeze-drying and rehydration (*p<0.05, **p<0.01, ***p<0.001). [Figure 11] This figure shows the results of confirming the vascular regeneration effects of freeze-dried and rehydrated extracellular vesicles (EVs) after treatment with PBS, VEGF, fresh EVs, and a mixed cryoprotectant (TS) of trehalose and sucrose, as measured by their tube-forming ability and cell migration ability (*p<0.05, **p<0.01, ***p<0.001). BEST MODE FOR CARRYING OUT THE INVENTION

[0007] The present invention provides a composition for freeze-drying and protecting extracellular vesicles, which comprises trehalose and sucrose, and a method for freeze-drying extracellular vesicles using the same. The composition for cryopreservation of extracellular vesicles according to the present invention solves the problems of existing cryopreservatives, such as crystal formation and reduced resolubility, and can maintain or increase the expression of markers and functional factors specific to extracellular vesicles and their therapeutic effects. The present invention will be described in detail below.

[0008] The present invention provides a cryoprotectant for extracellular vesicles, which can be characterized by containing trehalose and sucrose as active ingredients, and a composition for cryoprotection of extracellular vesicles containing trehalose and sucrose, and a cryoprotectant for extracellular vesicles containing trehalose and sucrose. Mixing trehalose and sucrose and processing the mixture into extracellular vesicles not only solves the problem of unidentified nanoparticles being formed when a single component, such as trehalose, mannitol, or sucrose, crystallizes with a salt in the PBS component during the freeze-drying process, but also increases the dissolution rate when the freeze-dried extracellular vesicles are redissolved. Thus, the present invention provides a composition for freeze-drying extracellular vesicles, comprising a mixture of trehalose and sucrose as cryoprotectants; and extracellular vesicles.

[0009] The trehalose and sucrose may be 1 to 10% (w / v), preferably 2 to 9% (w / v), and more preferably 2 to 8% (w / v). If less than 1% (w / v) of trehalose or sucrose is used, problems may arise such as the formation of a large number of unidentified nanoparticle crystals and the redissolution rate may be slow.

[0010] The present invention is characterized in that trehalose and sucrose are mixed and added as active ingredients of the lyoprotectant, and the trehalose and sucrose may be mixed at a volume ratio of preferably 1:0.05-5, more preferably 1:0.5-3, and even more preferably 1:0.5-2. In a preferred embodiment of the present invention, the effect was confirmed using a lyoprotectant mixed at a volume ratio of 1:1.

[0011] The freeze-protecting composition of the present invention may comprise a mixture of 1 to 10% (w / v) trehalose and sucrose in a volume ratio of 1:0.05 to 5, or may comprise a mixture of trehalose and sucrose at the above concentrations in a volume ratio of 1:0.1 to 3, more preferably 1:0.5 to 1.

[0012] The mixed lyoprotectant composition of trehalose and sucrose of the present invention can reduce crystal formation that occurs during the process of freeze-drying, rehydrating, and redissolving extracellular vesicles.

[0013] The cryoprotectants trehalose and sucrose may be mixed with the extracellular vesicles to be freeze-dried at a volume ratio of 1:0.5-5, preferably 1:0.5-3, more preferably 1:0.5-2.

[0014] The lyophilized extracellular vesicles can be produced by mixing the lyoprotectant with the extracellular vesicles, freezing them at −70 to −90° C., and lyophilizing them in a lyophilizer for 3 to 7 days.

[0015] The lyoprotectant is prepared by diluting it with PBS, and sterilized distilled water can be used for the reconstitution process after lyophilization. Through this process, the compounds constituting the PBS lyophilized together with the extracellular vesicles are redissolved in distilled water, allowing the extracellular vesicles to be preserved in PBS. The extracellular vesicles to be freeze-dried in the present invention may be extracellular vesicles isolated from cells isolated from natural organisms or extracellular vesicles isolated from components such as milk. The cells may be derived from any type of animal, including humans and non-human mammals, or plants, and may be various types of immune cells, tumor cells, or stem cells. Preferably, the stem cells may be mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells.

[0016] Extracellular vesicles refer to vesicles released to the outside of cells, and include microvesicles, apoptotic vesicles, exosomes, etc. Therefore, the present invention includes, without limitation, various extracellular vesicles that can achieve the purpose by adding trehalose and sucrose as cryoprotectants, and for example, the extracellular vesicles can be exosomes.

[0017] The exosomes of the present invention have nano-sized vesicle structures secreted into the extracellular space by cells of various animals, plants, bacteria, fungi, algae, etc., and include all vesicles with a composition similar to that of exosomes (e.g., exosome-like vesicles). In particular, the exosomes of the present invention may be exosomes derived from stem cells.

[0018] In the present invention, the extracellular vesicles may be extracellular vesicles that exhibit therapeutically useful effects, and by treating the lyoprotectant composition and lyoprotectant of the present invention during the freeze-drying process of such extracellular vesicles, the unique characteristics, properties, and therapeutic effects of the extracellular vesicles can be maintained at the same level or higher.

[0019] In the present invention, we confirmed that the addition of trehalose and sucrose to lyophilized extracellular vesicles not only suppressed the formation of undesired nanoparticle crystals during the lyophilization process, but also significantly accelerated their rehydration and dissolution rates. Furthermore, we confirmed that there was no loss in the total protein or RNA content of the extracellular vesicles, and that the therapeutic miRNA levels were maintained at the same level. Furthermore, we confirmed that the expression pattern of CD63, a marker for extracellular vesicles, was similar or even increased compared to before lyophilization.

[0020] The therapeutic efficacy of extracellular vesicles whose efficacy is maintained by the lyoprotectant can be a broad regenerative or immunomodulatory effect of the therapeutic efficacy inherent to extracellular vesicles, such as the ability to regenerate blood vessels. The extracellular vesicles frozen with the lyoprotectant of the present invention can be used as pharmaceutical compositions, cosmetic compositions, food compositions, and external skin care compositions, and can be administered orally or parenterally.

[0021] The types of diseases requiring angiogenesis for which the freeze-dried extracellular vesicles of the present invention can be utilized include, but are not limited to, one or more selected from the group consisting of burns, ulcers, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cardiovascular diseases, cerebrovascular diseases, and alopecia, and particularly cardiovascular diseases or cerebrovascular diseases.

[0022] The pharmaceutical composition containing the lyophilized extracellular vesicles of the present invention may further contain suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions in addition to the active ingredient. The pharmaceutical composition of the present invention may further contain other pharmaceutically active ingredients or active compounds.

[0023] The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions by conventional methods. Carriers, excipients, and diluents that can be contained in the compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0024] Oral liquid formulations include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases include Witepsol®, macrogol, Tween 61, cocoa powder, laurin, and glycerogelatin.

[0025] The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the administration route, and the duration of administration, but can be appropriately selected by those skilled in the art. Administration can be once a day or in multiple divided doses. The dosage does not limit the scope of the present invention in any way.

[0026] The pharmaceutical compositions of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc., by a variety of routes, including oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebroventricular injection, although any route of administration is contemplated.

[0027] The definitions of the terms for the excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, etc. used in the present invention are those described in documents known in the art, and include those having the same or similar functions.

[0028] The present invention also provides a method for freeze-drying extracellular vesicles, comprising the steps of: 1) mixing a mixture of trehalose and sucrose, which are freeze-drying protection agents, with extracellular vesicles; and 2) freeze-drying the mixture of step 1).

[0029] The above-described descriptions regarding the lyoprotectant composition and the lyoprotectant can be similarly applied to the present lyoprotection method.

[0030] In the freeze-drying method, the freeze-drying 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 step may be performed under a pressure of 1 to 7 mTorr, and one, two, or three freeze-drying processes may be performed.

[0031] In consideration of the complexity of this specification, redundant content will be omitted, and terms not otherwise specified in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0032] Terms used in this specification unless otherwise specified have the meanings commonly used in the technical field to which the present invention belongs.

[0033] The present invention will be described in more detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. DETAILED DESCRIPTION OF THE INVENTION

[0034] Example 1. Cell culture and extracellular vesicle recovery To collect exosomes, the extracellular vesicles (EVs) used in the experiments, human bone marrow-derived stem cells were cultured at 2.5 x 10 cells per 100 mm culture dish (SPL, 20100). 5 The cells were cultured at a concentration of 0.2 μm filtered low-glucose Dulbecco's modified Eagle's medium (DMEM, Life Technologies Corporation, CA, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies Corporation) and 1% antibiotics-antimycotics (Life Technologies Corporation). Subcultures were performed every 80–90% from P2 to P6 and cultured in an incubator at 37°C with 5% CO2. Stem cells cultured under the same conditions from P6 onward were cultured for 5 days in a medium consisting of low-glucose DMEM, 10% exosome-depleted fetal bovine serum (System Biosciences, Palo Alto, CA, USA), and 1% antibiotics / antimycotics. The culture medium was then harvested and centrifuged at 2500 g for 10 min at 4°C. The supernatant was then filtered through a 0.2 μm filter. The collected medium was immediately subjected to tangential flow filtration (TFF), followed by concentration and buffer exchange (diafiltration), resulting in a concentration of approximately 10-fold compared to the starting volume. A Minimate TFF 300K membrane (Pall Corporation, NY, USA) was used. Exosomes were collected using the above process and used for freeze-drying experiments.

[0035] Example 2. Freeze-drying process To freeze-dry EVs, EVs were collected as in Example 1, and their size and concentration were measured using qNano (IZON Ltd, Christchurch, New Zealand) using tunable resistive pulse sensing technology. 11 The lyoprotectants were mixed with exosomes at a volume ratio of 1:1 to achieve EVs / ml. Trehalose (Sigma, St. Louis, MO, USA), mannitol (Sigma), DMSO (Sigma), and sucrose (Sigma) were used as candidates to screen for suitable lyoprotectants for exosomes. The experimental groups were treated with mannitol (0-5% w / v), DMSO (0-10% w / v), and trehalose (0-4% w / v), respectively. Sucrose and trehalose were mixed at a 1:1 volume ratio at concentrations of 2-8% w / v, respectively. After mixing, the mixture was stored at -80°C overnight and then lyophilized (-80°C, 5 mTorr) for 4 days. After lyophilization, the containers were sealed and stored at room temperature. For use in experiments, the samples were rehydrated by adding 0.2 μm-filtered deionized water to the original mixture volume and then storing at 37°C for 30 minutes.

[0036] Example 3. Visual analysis after freeze-drying The cryoprotectants were varied from 0% to 10%, mannitol from 0.1% to 5%, and trehalose from 0.03% to 0.17%, and mixed with exosomes. The frozen state and the state after rehydration were visually inspected. The results are shown in Figure 1. As shown in Figure 1, in the case of trehalose and mannitol, visual observation revealed the presence of fine particles after freeze-drying, confirming the progress of freeze-drying. However, in the case of DMSO, a thin film-like substance formed after freeze-drying, confirming that DMSO is an unsuitable freeze-drying agent for EVs. In particular, the DMSO mixture was not dissolved in PBS, even with the naked eye, and large particles were present. On the other hand, the trehalose and mannitol mixture was visually confirmed to have redissolved well after rehydration.

[0037] Example 4. EV concentration and size analysis As in Example 2, trehalose and mannitol were treated as cryoprotectants, frozen, and rehydrated. The number and size of the EVs obtained were confirmed by qNano measurement. The qNano is a device that measures particle concentration and size using tunable resistive pulse sensing technology. The nanopore used in the experiment was an NP200 with a radial stretch of 46.5 mm. A voltage of 0.66 mV was applied, and EV concentration and size in the sample were measured using a 0.075 ml sample. All samples were analyzed through at least 500 particle measurements, and all were calibrated using CPC100 (100 mm, 1.4E+13 particles / ml, IZON Ltd). The measurement results are shown in Figure 2. However, DMSO-treated samples were difficult to dissolve in PBS, making qNano measurement impossible. As shown in Figure 2, even though the same number of EVs as in the Fresh EV group (in which qNano was measured immediately after EV isolation) were mixed with a lyoprotectant, rehydrated, and counted, an increase in the number of nanoparticles was observed in all trehalose and mannitol-treated groups, as well as in the PBS-treated group. This indicates that a large amount of unidentified nanoparticles are generated in addition to EVs. Analysis of the particle size distribution of qNano nanoparticles confirmed that the size of the unidentified nanoparticles was similar to that of EVs, ranging from 80 to 300 nm.

[0038] Example 5. XRD analysis of EVs after lyoprotectant treatment It is known that lyoprotectants can form crystals with salts due to the components of PBS, and crystallization can be promoted under certain conditions. To confirm the newly formed, unidentified nanoparticles outside of EVs, as confirmed in Example 4, PBS alone without EVs, and mannitol and trehalose diluted in PBS alone were lyophilized, and the formation of nanoparticles after lyophilization was confirmed by XRD peak analysis. The internal structure was analyzed by XRD using Cu radiation (45 kV x 200 mA) using a Smartlab (Rigaku, JP). The 2θ range was set to 2–45°, with a step size of 0.05° and a dwell time of 2 s. The results are shown in Figure 3. In the XRD peak analysis, the peaks at 22–23° and 27–28° correspond to sodium chloride crystals due to the PBS components, and the peak at 32–33° corresponds to phosphate crystals. As shown in Figure 3, XRD peak analysis of the freeze-dried experimental group (Figure 3A) after the addition of mannitol revealed the formation of multiple peaks in addition to salt crystals from the PBS components, indicating that the mannitol component itself also forms crystals. Mannitol is known as a crystalline cryoprotectant and has a total of three crystalline forms. β-mannitol crystals, one of the most representative crystal forms, are stable crystals that are known to be difficult to dissolve in water. On the other hand, in the case of trehalose (Figure 3B), it was confirmed that only peaks corresponding to sodium chloride crystals and phosphate crystals from the PBS components were formed. Figure 3C shows the results of qNano analysis, confirming the formation of unspecified nanoparticles when PBS or trehalose solution alone, without EV, was lyophilized and then reconstituted. In the experimental group in which the solution was frozen at -80°C without freezing and then reconstituted (labeled -80°C), all samples dissolved, confirming the absence of nanoparticle formation. No particles were detected in the qNano analysis. In contrast, the experimental groups containing low concentrations of trehalose, such as the 0.07% trehalose group (2 mM) and the 0.17% trehalose group (8 mM), formed more particles than when PBS alone was lyophilized. XRD analysis indicated that the unspecified nanoparticles generated during freeze-drying of the trehalose / PBS solution were presumed to be sodium chloride and phosphate, so we investigated whether they would gradually dissolve over time. When the solution was redissolved by shaking at room temperature, almost all of the salts were redissolved after about 48 hours, as shown in Figure 3D. Taking these results together, it was confirmed that when trehalose was used as a lyoprotectant at a low concentration, crystallized salts were generated during the lyophilization process, and this took a long time to redissolve.

[0039] Example 6. Experiment on reduction of crystallinity by treatment with lyoprotectants In Example 5, the use of low concentrations of trehalose as a lyoprotectant resulted in the formation of salt crystals. To address this issue, we varied the trehalose concentration from 2% to 4% in an experimental group, and mixed trehalose with sucrose, a known amorphous lyoprotectant, to determine whether nanoparticles would form. Trehalose and sucrose were mixed at a 1:1 volume ratio at 2% to 8% (w / v) and used as the lyoprotectant experimental group. The lyoprotectant was reconstituted by mixing it with PBS without EVs, and qNano analysis and XRD analysis were performed as in Examples 4 and 5. The results are shown in Figure 4. As shown in Figure 4A, qNano measurements immediately after freeze-drying and reconstitution (within 3 minutes) confirmed that some nanoparticles were measured in the 2% trehalose group compared to the PBS-only treatment group, whereas the 4% trehalose and 1:1 trehalose and sucrose mixture treatment groups were completely reconstituted and no nanoparticles were measured. Figure 4B shows the dissolution rate over time, and it was confirmed that the higher the concentration of the lyoprotectant, the faster the dissolution rate. In particular, the mixed solution of trehalose and sucrose achieved reconstitution within 5 minutes, demonstrating excellent dissolution ability. As shown in FIG. 4C, the XRD analysis peaks decreased as the concentration of the lyoprotectant increased, and it was confirmed that the mixed solution of trehalose and sucrose in particular had a very low degree of crystal formation. From the above results, it was confirmed that the crystallization and resolubility increase with increasing concentration, and in particular, the mixture of trehalose and sucrose exhibited a significantly low rate of crystallization during freeze-drying and an excellent resolubility rate. These results indicate that when EVs are freeze-dried using a mixture of trehalose and sucrose as a lyoprotectant, unidentified nanoparticle crystals do not form or are quickly redissolved, making the mixture suitable as a lyoprotectant for EV therapeutics that need to control the number of EVs and be administered to patients.

[0040] Example 7. Confirmation of the crystal formation inhibitory effect of a trehalose and sucrose mixed freeze-drying protectant Having confirmed that a trehalose and sucrose mixed lyoprotectant is suitable as a lyoprotectant for EV therapeutics, we applied it to the freeze-drying of MSC-EVs obtained in Example 1 and examined the resolubility rate. The results are shown in Figures 5 and 6. Experimental groups included an experimental group in which EVs were treated with a lyoprotectant made by mixing trehalose and sucrose at a volume ratio of 2%, 4%, and 8% (w / v) in a 1:1 ratio, freeze-dried, and resolubilized in the same manner as in Example 2; an experimental group in which EVs contained in PBS without a lyoprotectant were freeze-dried and resolubilized (0%); and fresh EVs, which were not freeze-dried and were immediately isolated. After treatment with a lyoprotectant, the cells were freeze-dried and reconstituted, and the number of EV particles was confirmed. The results are shown in Figure 5. As shown in Figure 5, after freeze-drying, the number of nanoparticles was slightly reduced compared to fresh EVs in the 2% trehalose and sucrose mixture group, although no statistically significant difference was observed. On the other hand, in the 4% and 8% experimental groups, the initial number of freeze-dried EVs was preserved at almost 100% and reconstituted. The change in size of EVs was confirmed when they were reconstituted after freeze-drying, and the results are shown in Figure 6 . As shown in Figure 6, the experimental group to which a lyoprotectant was added showed a size distribution similar to that of fresh EVs, but the experimental group to which PBS alone was added showed nanoparticles of slightly smaller size. The EV particle morphology of each experimental group was also confirmed via cryo-EM. Fresh EV experimental samples could be used as is, while lyophilized samples were resuspended at 37°C for 30 minutes before use. Prior to sampling, grids (Quantifoil, R1.2 / 1.3, 200 mesh, EMS) were pre-dissolved using a Glow discharge system (PELCO easiGlow™, Ted Pella). Four microliters of each sample was placed on the grid and subjected to blotting at 4°C under 100% humidity for 1.5 seconds at blotting force 3. The samples were then frozen in liquid ethane using the Vitrobot Mark IV (FEI) method to vitrify them. Analysis was then performed at the Nanobioimaging Center (Seoul National University, Korea) using a Talos L120C (FEI) microscope with a Lab6 gun type at 120 kV. The results are shown in Figure 7. As shown in Figure 7, in EVs freeze-dried and rehydrated using only PBS without the freeze-drying protection agent of the present invention, many fine particles presumed to be salts were observed in the background, whereas in EVs freeze-dried using a mixed freeze-drying protection agent of trehalose and sucrose, almost no fine particles were observed. These results suggest that the freeze-dried EVs using PBS contained nearly twice as many nanoparticles as the initial freeze-dried EVs, and that new nanoparticles outside the EV size distribution were identified, indicating the problem of crystal formation identified above. On the other hand, when the mixture of trehalose and sucrose was treated with a lyoprotectant, the problem of crystal formation did not occur, and the number of EVs was preserved, and they were able to be redissolved after freeze-drying and obtain EVs.

[0041] Example 8. Confirmation of the effect of a mixed lyoprotectant of trehalose and sucrose on EVs To utilize EVs as cryoprotectants for therapeutic EVs, it is important that they are obtained without loss of EVs or the generation of other substances after freeze-drying and reconstitution, as well as without affecting the properties and function of the EVs that were freeze-dried. Therefore, we treated a mixture of trehalose and sucrose with the cryoprotectant, and confirmed whether the total RNA contained in EVs was restored without loss in frozen and reconstituted EVs. We also confirmed whether they induced changes in miRNA, a factor in EV efficacy, and whether they changed the expression of the EV marker CD63. Protein quantitative analysis was performed as follows: all samples were centrifuged at 120,000 g for 1 hour in an ultra-high-speed centrifuge (Beckman Coulter, CA, USA), the supernatant was discarded, and lysis was performed. Lysis was performed using RIPA (Life Technologies Corporation), and protein was quantified using BCA protein assay (Life Technologies Corporation). Analysis of the EV marker CD63 was confirmed using ELISA. ELISA was performed using a commercially available kit according to the manufacturer's instructions. The CD63 (EH95RB, ThermoFisher Scientific, Inc., Waltham, MA, USA) ELISA kit contains a standard protein, so the amount of protein and extracellular vesicles was determined based on the kit's standard curve. Isolated fresh EVs and lyophilized EVs were dispensed into a 96-well microplate coated with a capture antibody at equal volumes (200 μL / well) along with the standard, without any pretreatment, and incubated at 4°C. The next day, sandwich ELISA was performed, and the absorbance was measured using a microplate reader. Analysis of miRNA expression, a factor in EV efficacy, was performed by qPCR as follows: RNA was extracted using Trizol™ according to the manufacturer's instructions and quantified using Nanodrop. RNA was then reverse-transcribed (RT) to produce cDNA, and real-time PCR was performed using TaqMan probes appropriate for each miRNA and mRNA according to the manufacturer's instructions. The results of confirming the changes in EV protein amount, total RNA recovery rate, miRNA amount, and EV marker expression are shown in Figures 8 to 10. As shown in Figure 8, the PBS-treated group showed a decrease in protein content of approximately 38% compared to Fresh EV, but the experimental group in which the mixture of trehalose and sucrose of the present invention was treated with a lyoprotectant showed less protein loss, and in particular, the 4% and 8% mixed groups showed protein content at levels similar to that of the Fresh EV group. As shown in Figure 9, the recovery rate of total RNA after lyophilization and reconstitution showed no statistically significant difference among all experimental groups, and the expression of therapeutically effective miRNAs was also uniformly expressed without significant difference among all experimental groups. As shown in Figure 10, there was no significant difference in the expression of the EV marker CD63 between the experimental groups.

[0042] Example 9. Verification of the revascularization ability of frozen-thawed EVs after cryoprotectant treatment EVs are known to have revascularization capabilities and can be used as therapeutic agents for various diseases requiring angiogenesis. Experiments were conducted to determine whether EVs mixed with the lyoprotectant of the present invention, frozen, and re-thawed, maintain or improve their inherent revascularization capabilities. The revascularization capabilities of EVs were confirmed through tube formation and cell migration experiments using HUVEC cells. More specifically, to demonstrate the angiogenesis growth effect of the EVs obtained in Example 1, HUVECs attached to Matrigel were treated with EVs to confirm their tube formation. Specifically, HUVECs were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF. Cells were cultured at a density of 1.0 × 10 4 Cells were seeded onto growth factor-reduced Matrigel Matrix (BD Bioscience, MA, USA) in μ-Slides Angiogenesis (ibidi, Graefelfing, Germany) at a density of 1000 μm and allowed to form tubes for 7 h in a humidified chamber at 37°C and 5% CO. Images were taken with a phase-contrast microscope (Olympus), and the number of tube-like structures was quantified per microscopic field (4x magnification) using ImageJ software. For cell migration assays, HUVECs were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF at 37°C in a 5% CO2 humidified chamber. 5Cells were cultured in 24-well plates to 100% densities and then treated with MMC (2.5 μg / ml) to stop cell division. Cells were scratched vertically with a pipette tip and washed with PBS. Images of the scratched area were taken with a phase-contrast microscope (Olympus) to indicate the location. The scratched area was photographed again 7 hours later, and the extent of cell migration and wound closure was measured using ImageJ software. The cryoprotectant experimental group consisted of EVs treated with a 1:1 volumetric mixture of trehalose and sucrose (0%, 2%, 4%, and 8% w / v, lyophilized and rehydrated), fresh EVs (immediately isolated without lyoprotectant treatment), and the positive control groups (VEGF-treated and PBS-treated groups) were used. The negative control group consisted of HUVECs treated with PBS. All EVs were 5 × 10 8 The cells were treated with EV / ml and VEGF at 100 ng / ml. The results of confirming angiogenic activity are shown in Figure 11. As shown in Figure 11, all EV-treated groups exhibited similar levels of revascularization as the VEGF-treated experimental group. In particular, the experimental group treated with a 1:1 mixture of trehalose and sucrose at concentrations of 2-4% and then frozen and re-thawed EVs exhibited superior revascularization. This result demonstrates that the therapeutic efficacy of EVs can be maintained and restored without loss when treated with the lyoprotectant of the present invention, lyophilized, and re-thawed.

[0043] Although the present invention has been described in detail above, it is obvious to those skilled in the art that the specific description is merely a preferred embodiment and does not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for lyophilization protection of extracellular vesicles comprising trehalose and sucrose.

2. The composition for cryopreservation of extracellular vesicles according to claim 1, wherein the trehalose and sucrose are each 1 to 10% (w / v).

3. The composition for freeze-drying and protecting extracellular vesicles according to claim 1, wherein the trehalose and sucrose are mixed in a volume ratio of 1:0.05-5.

4. The composition for freeze-drying and protecting extracellular vesicles according to claim 1, wherein the trehalose and sucrose reduce the formation of crystals during the freeze-drying and rehydration process of the extracellular vesicles.

5. The composition for cryopreservation of extracellular vesicles according to claim 1, wherein the composition is mixed with extracellular vesicles in a volume ratio of 1:0.5-5.

6. The composition for cryopreservation of extracellular vesicles according to claim 1, wherein the extracellular vesicles are exosomes or microvesicles.

7. The composition for cryopreservation of extracellular vesicles according to claim 1, wherein the extracellular vesicles are derived from immune cells, tumor cells or stem cells.

8. A cryoprotectant for extracellular vesicles, comprising trehalose and sucrose.

9. A composition for freeze-drying extracellular vesicles, comprising: a mixture of trehalose and sucrose as a freeze-drying protectant; and extracellular vesicles.

10. The composition for freeze-drying extracellular vesicles according to claim 9, wherein the extracellular vesicles have revascularization ability.

11. 1) mixing a mixture of lyoprotectants, trehalose and sucrose, with extracellular vesicles; and 2) A method for freeze-drying extracellular vesicles, comprising freeze-drying the mixture in step 1).

12. The method for freeze-drying extracellular vesicles according to claim 11, wherein step 2) comprises freezing the mixture of step 1) at -70 to -90°C and then freeze-drying it for 3 to 7 days.

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