Lipofreeze protective agent for extracellular vesicles

Abstract

The present invention relates to a composition for lyophilization protection of extracellular vesicles that can maintain the functions and properties of extracellular vesicles, and a method for lyophilization of extracellular vesicles using the same. The trehalose and sucrose of the present invention are added to the lyophilization process of extracellular vesicles to solve the problems of existing lyophilization protection agents, such as crystal formation and reduced resolubility, while maintaining the expression of markers and functional factors specific to extracellular vesicles and therapeutic effects, and therefore can be used in a variety of ways in the production of various therapeutic agents using extracellular vesicles.

Description

Technical Field

[0001] The present invention relates to a composition for freeze-drying protection of extracellular vesicles capable of maintaining the functions and characteristics of extracellular vesicles, and a method for freeze-drying extracellular vesicles using the same.

Background Art

[0002] In various diseases, positive clinical results have been reported for treatment methods using stem cells, particularly mesenchymal stem cells (MSCs). However, stem cell therapeutics have risks of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders as side effects, and there is still a need for validation of effectiveness through clinical trials. It is known that the paracrine effect of stem cells induces the regeneration of peripheral skin cells and the enhancement of angiogenesis ability. In particular, extracellular vesicles (EVs) are known as effective factors for the main paracrine effect. Extracellular vesicles are classified into exosomes and microvesicles according to size. Exosomes have a diameter of 30 to 150 nm, and microvesicles have a size of 100 to 1,000 nm. Extracellular vesicles are those in which a part of the cell membrane is released into the blood, contain both proteins and nuclear components, and are known to mediate cell-to-cell communication. Using extracellular vesicles instead of stem cells not only minimizes the side effects caused by the use of stem cells and enhances safety but is also advantageous in terms of biodistribution and production processes in vivo. However, despite the usefulness of extracellular vesicles, research on techniques for stably maintaining and storing extracellular vesicles after separation, which are difficult to obtain, and on stable dosage forms has not yet been achieved. Therefore, there is a need for a new storage method for stably maintaining and storing extracellular vesicles that can be used as therapeutic substances for various diseases.

Summary of the Invention

[0003] Therefore, while researching techniques for stably maintaining and storing extracellular vesicles, the inventors identified a novel freeze-drying protective agent that effectively improves the problems that can occur during the freeze-drying process and can preserve the unique properties and functions of extracellular vesicles, thus completing the present invention. Therefore, the present invention relates to a composition for freeze-drying protection of extracellular vesicles, comprising trehalose and sucrose, and a method for freeze-drying extracellular vesicles using the same. [Means for solving the problem]

[0004] To achieve the above objective, the present invention provides a composition for lyophilization protection of extracellular vesicles, comprising trehalose and sucrose. Furthermore, the present invention provides a lyophilization protective agent for extracellular vesicles, comprising trehalose and sucrose. Furthermore, the present invention provides a composition for lyophilization of extracellular vesicles, comprising a mixture of trehalose and sucrose as a lyophilization protective agent, and extracellular vesicles. Furthermore, the present invention provides a method for freeze-drying extracellular vesicles, comprising the steps of: 1) mixing a mixture of trehalose and sucrose, which are freeze-drying protective agents, with extracellular vesicles; and 2) freeze-drying the mixture from step 1). [Effects of the Invention]

[0005] The trehalose and sucrose in this invention are added to the freeze-drying process of extracellular vesicles, solving the problems of crystal formation and reduced re-dissolution ability that are present in existing freeze-drying protective agents, while maintaining the expression of unique markers, functional factors, and therapeutic effects of extracellular vesicles. Therefore, they can be widely used in the production of various therapeutic agents using extracellular vesicles. [Brief explanation of the drawing]

[0006] [Figure 1]This figure shows the results of visually inspecting extracellular vesicles after lyophilization and rehydration, with DMSO, trehalose, and mannitol added as lyophilization protective agents. [Figure 2] This figure shows the results of treating trehalose and mannitol with a cryoprotective agent, freeze-drying them, rehydrating them, and then examining the number (A) and size distribution (B) of extracellular vesicles (EVs) obtained by qNano measurement. [Figure 3-AB] This figure shows the results of lyophilizing PBS alone, mannitol diluted in PBS (A), and trehalose (B) without extracellular vesicles (EVs), and confirming nanoparticle formation after lyophilization via XRD peak analysis. [Figure 3-C] This figure shows the results of qNano analysis to identify unspecified nanoparticles generated in experimental groups where PBS and trehalose (T) were freeze-dried at 0.07% or 0.17% concentrations and then redissolved (Lyo: freeze-dried experimental group, -80: frozen at -80°C and then redissolved experimental group). [Figure 4-D] This figure shows the results of confirming the redissolution of unspecified nanoparticles formed after lyophilization of 0.07% PBS and trehalose (T) at room temperature for 48 hours. [Figure 4-A] This figure shows the results of qNano measurements to identify unspecified nanoparticles generated in the experimental group of trehalose (T) alone and the experimental group of trehalose and sucrose (T+S) mixed lyophilized and re-thawed 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 observing the redissolution and dissolution rate of unspecified nanoparticles generated after lyophilization and redissolution without extracellular vesicles (EVs) in experimental groups of trehalose (T) alone and trehalose and sucrose (T+S) mixed lyophilization and redissolution for 20 minutes. [Figure 4-C]This figure shows the results of XRD analysis of crystals formed in the experimental group using trehalose (T) alone and the experimental group using a mixture of trehalose and sucrose (T+S) after lyophilization and re-thawing without extracellular vesicles (EVs). [Figure 5] This figure shows the results of examining the number of extracellular vesicle (EV) particles in lyophilized and re-thawed extracellular vesicles (EVs) after treatment with a lyophilized protective agent containing 2-8% trehalose and sucrose. [Figure 6] This figure shows the results of examining the changes in the size distribution of extracellular vesicles (EVs) after treatment with a mixed freeze-drying protective agent of trehalose and sucrose, followed by freeze-drying and rehydration. [Figure 7] This figure shows the results of confirming the EV shape in each experimental group from the Cryo-EM data. [Figure 8] This figure shows the changes in total protein content in extracellular vesicles (EVs) after treatment with a lyophilized protective agent containing 2-8% trehalose and sucrose, followed by lyophilization and rehydration (*p<0.05, **p<0.01, ***p<0.001). [Figure 9] This figure shows the results of examining changes in total RNA content and miRNA expression levels, which are associated with therapeutic effect, in extracellular vesicles (EVs) after treatment with a lyophilized protective agent containing 2-8% trehalose and sucrose, followed by lyophilization and rehydration. [Figure 10] This figure shows the results of examining the expression of the EV marker CD63 in extracellular vesicles (EVs) that were treated with a 2-8% trehalose and sucrose mixed lyophilization protective agent (TS), followed by lyophilization and rehydration (*p<0.05, **p<0.01, ***p<0.001). [Figure 11] This figure shows the results of examining the vascular regeneration effect in extracellular vesicles (EVs) treated with PBS, VEGF, Fresh EV, and a mixed lyophilized protective agent (TS) of trehalose and sucrose, followed by lyophilization and rehydration, using tube formation 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 protection of extracellular vesicles containing trehalose and sucrose, and a method for freeze-drying extracellular vesicles using the same. The composition for freeze-drying protection of extracellular vesicles according to the present invention solves the problems of crystal formation and reduced redissolution ability, which are problems of existing freeze-drying protectants, and can maintain or increase the expression of markers and functional factors specific to extracellular vesicles and the therapeutic effect. Hereinafter, the present invention will be described in detail.

[0008] The present invention can be characterized in that it contains trehalose and sucrose as active ingredients as a cryoprotectant for extracellular vesicles, and provides a composition for freeze-drying protection of extracellular vesicles containing trehalose and sucrose, and a freeze-drying protectant for extracellular vesicles containing trehalose and sucrose. Thus, when trehalose and sucrose are mixed and treated with extracellular vesicles, single components such as trehalose, mannitol, and sucrose can not only solve the problem that unconfirmed nanoparticles formed by forming salts and crystals with PBS components are formed during the freeze-drying process, but also increase the dissolution rate when the freeze-dried extracellular vesicles are redissolved. Therefore, the present invention provides a composition for freeze-drying of extracellular vesicles, which contains a mixture of trehalose and sucrose as a cryoprotective substance; and extracellular vesicles.

[0009] The trehalose and sucrose may each be 1 to 10% (w / v) of trehalose and sucrose, preferably 2 to 9% (w / v), more preferably 2 to 8% (w / v). When using less than 1% (w / v) of trehalose or sucrose, there may be a problem that a large number of unconfirmed nanoparticle crystals occur, and the redissolution rate may also become slow.

[0010] The present invention is characterized in that trehalose and sucrose are mixed and added as active ingredients of a lyoprotectant. The trehalose and sucrose may preferably be mixed at a volume ratio of 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 lyoprotectant mixed at a volume ratio of 1:1.

[0011] The lyoprotectant composition of the present invention may contain a mixture of 1 to 10% (w / v) of trehalose and sucrose mixed at a volume ratio of 1:0.05 to 5, or may contain a mixture of trehalose and sucrose at the above concentrations mixed at 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 occurring during the processes of lyophilization, rehydration, and redissolution of extracellular vesicles.

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

[0014] The lyoprotectant is mixed with extracellular vesicles, frozen at -70 to -90 °C, and lyophilized in a lyophilizer for 3 to 7 days to produce lyophilized extracellular vesicles.

[0015] The lyoprotectant is diluted and produced based on PBS. After lyophilization, sterilized distilled water can be used in the process of redissolution. Through such a process, the compounds constituting the PBS lyophilized together with the extracellular vesicles can be redissolved in distilled water so that the extracellular vesicles can be stored in PBS. The extracellular vesicles subject to freeze-drying in this invention may be extracellular vesicles isolated from cells isolated from naturally occurring organisms or extracellular vesicles isolated from components such as milk. Furthermore, the cells may be derived from any type of animal, including humans and non-human mammals, or from 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 outside the cell, and include microvesicles, apoptotic vesicles, and exosomes. Therefore, the present invention can include a variety of extracellular vesicles without limitation in which trehalose and sucrose can be added as lyophilization protective agents to achieve its purpose, and for example, the extracellular vesicles can be exosomes.

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

[0018] In the present invention, the extracellular vesicles can be extracellular vesicles that exhibit therapeutically useful effects, and by treating such extracellular vesicles with the lyophilization protective agent composition and lyophilization protective agent of the present invention during the lyophilization process, the unique characteristics, properties, and therapeutic effects of the extracellular vesicles can be maintained at an equivalent or higher level.

[0019] In this invention, when trehalose and sucrose are added and extracellular vesicles are freeze-dried, it was confirmed that not only is the formation of unwanted, unidentified nanoparticle crystals suppressed during the freeze-drying process, but the rehydration and dissolution rates of these crystals are significantly accelerated. Furthermore, it was confirmed that there is no loss of total protein or total RNA content in the extracellular vesicles, and the miRNA levels that indicate therapeutic effects are also maintained at the same level. In addition, it was confirmed that CD63, a marker for extracellular vesicles, also shows a similar or increased expression pattern compared to before freeze-drying.

[0020] The therapeutic efficacy of extracellular vesicles, whose efficacy is maintained by the lyophilized protective agent, can be a broad range of regenerative or immunomodulatory effects inherent to the extracellular vesicles, such as vascular regeneration degrading ability. Extracellular vesicles frozen with the lyophilized protective agent of the present invention can be used as pharmaceutical compositions, cosmetic compositions, food compositions, or topical skin preparation compositions, and can be administered orally or parenterally.

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

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

[0023] The pharmaceutical compositions of the present invention can be prepared by conventional methods 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 injection solutions. Examples of carriers, excipients, and diluents that may be included 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, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, the compositions are prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. Such solid dosage forms are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0024] Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. Besides commonly used simple diluents such as water and liquid paraffin, they can contain various excipients, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include Witepsol®, macrogol, Tween 61, cocoa jelly, laurin jelly, and glycerol gelatin.

[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 route of administration, and the duration, but can be appropriately selected by those skilled in the art. The drug may be administered once a day or in multiple divided doses. The aforementioned 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, and humans via a variety of routes. While any method of administration is expected, they can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebral (intracerebroventricular) injection.

[0027] The definitions of terms such as excipients, binders, disintegrants, lubricants, flavoring agents, and fragrances described above in this invention include those described in literature known to the art and having the same or similar functions.

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

[0029] The explanation of this freeze-drying method can be similarly applied to the explanation of the freeze-drying protective composition and freeze-drying protective agent described above.

[0030] In the freeze-drying method described above, step 2) freeze-drying may include a step of freezing at -70 to -90°C followed by freeze-drying for 3 to 7 days. The freezing may be carried out overnight, the freeze-drying step may be carried out under a pressure of 1 to 7 mTorr, and a first, second, or third freeze-drying process may be carried out.

[0031] Repetitive information has been omitted in consideration of the complexity of this specification, and terms used herein, unless otherwise specified, have the meanings commonly used in the art to which the present invention pertains.

[0032] Unless otherwise specified herein, terms have the meanings commonly used in the art to which this invention pertains.

[0033] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples. [Modes for carrying out the invention]

[0034] Example 1. Cell culture and recovery of extracellular vesicles Human bone marrow-derived stem cells were cultured to collect exosomes, which are extracellular vesicles (EVs) used in the experiment. The cells were placed in a 100 mm culture dish (SPL, 20100) at a density of 2.5 x 10⁶. 5 Cells were cultured at the specified concentration in low-glucose Dulbecco's modified Eagle's medium (DMEM, Life Technologies Corporation, CA, USA) filtered through a 0.2 μm filter, along with 10% fetal bovine serum (FBS, Life Technologies Corporation) and 1% antibiotics-antimycotics (Life Technologies Corporation). Subculturing was performed every 80-90% from P2 to P6, and the cells were cultured in an incubator at 37°C with 5% CO2. Stem cells cultured under the same conditions as in P6 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, centrifuged at 4°C, 2500g, and 10min, and the supernatant was filtered through a 0.2μm filter. The recovered medium was immediately subjected to tangential flow filtration (TFF), followed by concentration and buffer exchange (diafiltration) to achieve a concentration of approximately 10 times the initial volume. A Minimate TFF 300K membrane (Pall Corporation, NY, USA) was used as the membrane. Exosomes were recovered using the above process and used for freeze-drying experiments.

[0035] Example 2. Freeze-drying treatment To freeze-dry the EVs, the EVs were collected in the same manner as in Example 1, and then their size and concentration were measured using a qNano (IZON Ltd, Christchurch, New Zealand) with tunable resistive pulse sensing technology. Subsequently, the final concentration was 1 x 10⁻⁶. 11 Lyophilized protective agents and exosomes were mixed in a 1:1 volume ratio to achieve an EVs / ml ratio. Trehalose (Sigma, St. Louis, MO, USA), mannitol (Sigma), DMSO (Sigma), and sucrose (Sigma) were used as candidate groups for selecting lyophilized protective agents suitable for exosomes. As experimental groups, concentrations of mannitol 0-5% (w / v), DMSO 0-10% (w / v), and trehalose 0-4% (w / v) were set as single-dose groups. For each group, a mixture of sucrose and trehalose at concentrations of 2-8% (w / v) in a 1:1 volume ratio was used as an experimental group. After mixing, the mixtures were incubated overnight at -80°C, followed by lyophilization for 4 days (-80°C, 5 mTorr). After lyophilization, the samples were sealed and stored at room temperature. The stored samples underwent a rehydration process for use in experiments. During rehydration, deionized water filtered through a 0.2 μm sieve was added to the original mixture in an amount equal to its volume, and the mixture was kept at 37°C for 30 minutes before use.

[0036] Example 3. Visual analysis after freeze-drying. Figure 1 shows the results of visually inspecting the frozen state and the state after rehydration after mixing with exosomes while varying the lyophilization protective agent content from DMSO 0% to 10%, mannitol 0.1% to 5%, and trehalose 0.03% to 0.17%. As shown in Figure 1, in the case of trehalose and mannitol, visual observation revealed the presence of fine particles after freeze-drying, indicating that freeze-drying had progressed. However, in the case of DMSO, a thin film-like substance formed after freeze-drying, confirming that DMSO is unsuitable as a freeze-drying agent for EV. In particular, the DMSO mixture was not dissolved in PBS even to the naked eye, and the presence of large particles was confirmed. On the other hand, the trehalose and mannitol mixture was confirmed to have redissolved well in all cases even after rehydration, as observed visually.

[0037] Example 4. Concentration and size analysis of EV Similar to Example 2, trehalose and mannitol were treated as cryoprotective agents, frozen, and then rehydrated. The number and size of EVs obtained by qNano measurement were then confirmed. qNano is a device that measures particle concentration and size through 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 the concentration and size of EVs in the sample were measured using 0.075 ml of sample. All samples were analyzed through at least 500 particle measurements, and all were calibrated using a CPC100 (100 mm, 1.4E+13 particles / ml, IZON Ltd). The measurement results are shown in Figure 2. On the other hand, qNano measurement was not possible for the DMSO-treated group because it was difficult to dissolve in PBS. As shown in Figure 2, despite mixing the same number of EVs with a lyophilized protective agent and rehydrating them, and then measuring the number of EVs, as in the Fresh EV group (an experimental group in which qNano was measured immediately after EV isolation), an increase in the number of nanoparticles was observed in all trehalose and mannitol-administered groups, as well as in the PBS-treated group. This means that a large amount of unidentified nanoparticles are generated in addition to the EVs, and particle size distribution analysis of the qNano nanoparticles confirmed that the size of the unidentified nanoparticles was similar to that of the EVs, in the range of 80-300 nm.

[0038] Example 5. XRD analysis of EV after freeze-drying protective agent treatment. It is known that freeze-drying protective agents can form salts and crystals with the components of PBS, and that crystallization can be promoted under certain conditions. To confirm newly formed, unidentified nanoparticles outside the extravasation zone (EV), as confirmed in Example 4, PBS alone, mannitol diluted in PBS, and trehalose alone were freeze-dried without EV, and the formation of nanoparticles after freeze-drying was confirmed by XRD peak analysis. XRD was measured using Cu radiation (45kV × 200mA) with Smartlab (Rigaku, JP) to analyze the internal structure. The 2θ range was set to 2~45, the step size to 0.05°, and the dwell time to 2 sec. The analysis was performed, and the results are shown in Figure 3. In the XRD peak analysis, the peaks at 22°~23° and 27°~28° represent sodium chloride crystals due to the PBS components, and the peak at 32°~33° represents phosphate crystals. As shown in Figure 3, XRD peak analysis of the freeze-dried experimental group (Figure 3A) to which mannitol was added showed the formation of multiple peaks in addition to the salt crystals due to the PBS component, indicating that the mannitol component itself generates its own crystals. Mannitol is known as a crystalline freeze-protective substance and has a total of three crystalline forms. Of these, β-mannitol crystals, one of the representative crystalline forms, are stable crystals and are known to be poorly soluble in water. On the other hand, in the case of trehalose (Figure 3B), it was confirmed that only peaks for sodium chloride crystals and phosphate crystals due to the PBS component were formed. Figure 3C shows the results of qNano analysis confirming that unspecified nanoparticles are formed when PBS or trehalose solution is freeze-dried without EV and then redissolved. In the experimental group where only freezing was performed without drying and freezing at -80°C (indicated as -80 in the figure), all samples were dissolved, and it was confirmed that no such nanoparticles were formed, and no particles were detected in the qNano measurement. On the other hand, in the experimental groups with low concentrations of trehalose added, the 0.07% trehalose group (2mM) and the 0.17% trehalose group (8mM) formed more particles than when PBS was freeze-dried alone. XRD analysis suggested that the unspecified nanoparticles generated during the freeze-drying of the trehalose / PBS solution were sodium chloride and phosphate. Therefore, we investigated whether these would gradually dissolve over time. When these were redissolved at room temperature while shaking, it was confirmed that almost all of the salts redissolved after approximately 48 hours, as can be seen from Figure 3D. In summary, these results confirm that when trehalose is used as a freeze-drying protective agent at low concentrations, crystallized salts are generated during the freeze-drying process, and these salts require a long time to redissolve.

[0039] Example 6. Experiment on crystallinity reduction due to freeze-drying protective agent treatment. In Example 5, it was confirmed that salt crystals formed when low concentrations of trehalose were used as a freeze-drying protective agent. To address this, experimental groups were created using trehalose concentrations ranging from 2% to 4%, and a mixture of trehalose and sucrose, known as an amorphous freeze-drying protective agent, to determine whether nanoparticles were formed. A mixture of 2% to 8% (w / v) trehalose and sucrose in a 1:1 volume ratio was used as the freeze-drying protective agent experimental group. Using these experimental groups, the freeze-drying protective agent was mixed with PBS without EV testing and redissolved. QNano analysis and XRD analysis were performed in the same manner as in Examples 4 and 5. The results are shown in Figure 4. As shown in Figure 4A, qNano measurements taken immediately after freeze-drying and redissolution (within 3 minutes) revealed that a small number of nanoparticles were detected in the 2% trehalose group compared to the PBS-only treatment group. In the 4% trehalose and 1:1 trehalose-sucrose mixture treatment groups, complete redissolution was confirmed, and no nanoparticles were measured. In Figure 4B, the dissolution rate with respect to redissolution time was examined, and it was confirmed that the dissolution rate tended to increase with higher concentrations of the freeze-drying protective agent. In particular, the mixed solution of trehalose and sucrose achieved redissolution within 5 minutes, demonstrating excellent solubility. As shown in Figure 4C, the XRD analysis peaks decreased as the concentration of the freeze-drying protective agent increased, and in particular, it was confirmed that the mixed solution of trehalose and sucrose had a very low degree of crystal formation. Through the results described above, we confirmed that crystal formation and redissolvability increased with increasing concentration, and that the mixture of trehalose and sucrose, in particular, showed a remarkably low crystal formation rate and excellent redissolvability during freeze-drying. These results indicate that when EVs are freeze-dried using a mixture of trehalose and sucrose as a freeze-drying protective agent, no unidentified nanoparticle crystals are formed, or they are rapidly redissolved. Therefore, the mixture is suitable as a freeze-drying protective agent for EV therapeutic agents that need to be administered to patients to control the number of EVs.

[0040] Example 7. Confirmation of the crystal formation inhibitory effect of a mixed freeze-dried protective agent of trehalose and sucrose. Having confirmed that a lyophilized protective agent of trehalose and sucrose is suitable as a lyophilized protective substance for EV therapeutic agents, we applied it to the lyophilization of MSC-EVs obtained in Example 1 and confirmed the redissolution rate, which is shown in Figures 5 and 6. The experimental groups included: an experimental group in which EVs were treated with a lyophilized protective agent consisting of trehalose and sucrose 2%, 4%, and 8% (w / v) mixed in a 1:1 volume ratio, in the same manner as in Example 2, lyophilized, and redissolved; an experimental group (0%) in which EVs contained in PBS were lyophilized and redissolved without a lyophilized protective agent; and Fresh EVs, which were EVs immediately after isolation and not lyophilized. After freeze-drying and protective treatment, the material was freeze-dried and re-dissolved, and the number of extravalent particles was confirmed. The results are shown in Figure 5. As shown in Figure 5, after freeze-drying and redissolution, no statistically significant difference was observed in the group with a 2% trehalose and sucrose mixture, but a slight decrease in the number of nanoparticles was confirmed compared to Fresh EV. On the other hand, in the 4% and 8% experimental groups, it was confirmed that almost 100% of the initial freeze-dried EV number was preserved upon redissolution. After freeze-drying, the change in EV size upon re-thawing was observed, and the results are shown in Figure 6. As shown in Figure 6, the experimental group treated with a freeze-drying protective agent showed a similar size distribution to Fresh EV, but the experimental group treated with PBS alone showed slightly smaller nanoparticles. Furthermore, the EV particle shape of each experimental group was confirmed via Cryo-EM. The fresh EV experimental group could be used as is, but the freeze-dried experimental group was re-dissolved at 37°C for 30 minutes before use in the experiment. Prior to sampling, a Glow discharge system (PELCO easiGlow®, Ted pella) was used to make the grids (Quantifoil, R1.2 / 1.3, 200 mesh, EMS) hydrophilic. 4 µl of each sample was placed on the grid, and a blot force 3 was applied for 1.5 seconds while maintaining 100% humidity at 4°C. Next, the samples were frozen using the Vitrobot Mark IV (FEI) method with liquid ethane to vitrify them. Subsequently, gun type Lab6 samples were analyzed at the Nanobioimaging Center (Seoul National University, Korea) using a Talos L120C (FEI) microscope at 120 kV. The results are shown in Figure 7. As shown in Figure 7, in extravitamin (EV) freeze-dried and rehydrated using only PBS without the freeze-drying protective agent of the present invention, numerous fine particles presumed to be salts were observed in the background. However, in EV using a mixed freeze-drying protective agent of trehalose and sucrose, it was confirmed that almost no fine particles were observed. The results described above indicate that in the freeze-dried group using PBS, nearly twice as many nanoparticles were observed compared to the initial freeze-dried extracellular matrix (EV), and new nanoparticles deviating from the EV size distribution were identified, suggesting the crystal formation problems identified earlier. On the other hand, when a mixture of trehalose and sucrose was treated with a freeze-drying protective agent, these crystal formation problems did not occur, and it was confirmed that the number of EVs could be preserved, redissolved after freeze-drying, and EVs could be obtained.

[0041] Example 8. Confirmation of the effect of a mixed freeze-dried protective agent of trehalose and sucrose on EV. For use as a freeze-drying protective agent for EV therapeutics, it is crucial that the EV is obtained without loss or generation of other substances after freeze-drying and re-thawing, and that the freeze-drying does not affect the properties and function of the EV being freeze-dried. Therefore, we treated EV with a mixture of trehalose and sucrose as a freeze-protective agent, and in the frozen and re-thawed EVs, we confirmed whether the total RNA contained in the EV was restored without loss, whether or not it induced changes in miRNAs, which are efficacy factors of EV, and confirmed changes in the expression of CD63, an EV marker. Protein quantification was performed using the following method: All samples were centrifuged at 120,000 g for 1 hour using an ultra-high-speed centrifuge (Beckman Coulter, CA, USA), the supernatant was discarded, and lysis was performed. Lysis was carried out using RIPA (Life Technologies Corporation), and proteins were quantified using the BCA protein assay (Life Technologies Corporation). CD63 analysis, an EV marker, was confirmed using ELISA and performed as follows: ELISA was performed using a commercially available kit according to the manufacturer's manual. Since the CD63 (EH95RB, ThermoFisher Scientific, Inc., Waltham, MA, USA) ELISA kit contains a standard protein, the amounts of protein and extracellular vesicles were determined based on the kit's standard curve. Separated fresh EVs and lyophilized EVs were dispensed in equal volumes (200 μL / well) with standard into 96-well microplates coated with capture antibody, without any pretreatment, and reacted at 4°C. The following day, the ELISA method was carried out using the sandwich method, and absorbance was measured using a microplate reader. miRNA expression analysis, a key efficacy factor for extravasation (EV), was performed using qPCR as follows: RNA was extracted using Trizol® according to the manufacturer's guidelines, and the RNA was quantified using nanodrop. The RNA was then converted into cDNA via reverse transcription (RT), and Real Time PCR was performed using Taqman probes appropriate for each miRNA and mRNA, according to the manufacturer's manual. The results of confirming the changes in EV protein quantity, total RNA restoration rate, miRNA quantity, and EV marker expression, as described above, 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. However, the experimental group treated with the trehalose and sucrose mixture of the present invention and the freeze-dried protective agent showed less protein reduction, and in particular, the 4% and 8% mixture groups measured protein content at levels similar to that of the Fresh EV group. As shown in Figure 9, after freeze-drying and re-dissolution, the total RNA recovery rate did not show any statistically significant differences in any of the experimental groups, and the expression of miRNAs with therapeutic efficacy was also uniformly expressed in all experimental groups without any significant differences. As shown in Figure 10, there were no significant differences in the expression of the EV marker CD63 among the experimental groups.

[0042] Example 9. Verification of vascular regeneration ability of EVs after freeze-drying and thawing treatment with a protective agent. EV is known to possess vascular regeneration capabilities and can be used as a therapeutic agent for various diseases requiring angiogenesis. Experiments were conducted to confirm whether EV, after being mixed with the lyophilized protective agent of the present invention, can maintain or improve the intrinsic vascular regeneration capabilities of EV after freezing and re-thawing. The vascular regeneration capabilities of EV were confirmed through tube formation and cell migration experiments using HUVEC cells. More specifically, to demonstrate the neovascularization effect of EV obtained in Example 1, HUVEC cells attached to Matrigel were treated with EV, and the tube formation effect was confirmed. Specifically, HUVEC cells were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF. Cells were 1.0 × 10⁶ 4 Cell density was measured using μ-Slides Angiogenesis (ibidi, Graefelfing, Germany) to inoculate Matrigel Matrix (BD Bioscience, MA, USA) with reduced growth factors, and the cells were inoculated into a humidified chamber at 37°C with 5% CO2 for 7 hours to form tubes. Images were taken using a phase-contrast microscope (Olympus), and the number of tubular structures was quantified in the microscopic field of view (4x magnification) using ImageJ software. For cell migration analysis, HUVEC cells were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF in a 37°C, 5% CO2 humidified chamber. 1.0 × 10 5Cells were cultured in 24-well plates to 100% capacity, then treated with MMC (2.5 ug / ml) to stop cell division. Cells were vertically scratched using a pipette tip and then washed with PBS. The scratched areas were imaged using a phase-contrast microscope (Olympus) and the affected areas were displayed. The same scratched area was imaged again after 7 hours, and the degree of wound closure was measured using ImageJ software to observe cell migration. For the lyophilized protective agent experiment, a lyophilized protective agent consisting of trehalose and sucrose 0 (lyophilized and rehydrated after PBS treatment), 2%, 4%, and 8% (w / v) in a 1:1 volume ratio was used. This included EV experimental groups (lyophilized and rehydrated), Fresh EVs (EVs isolated immediately after lyophilization without lyophilized protective agent treatment), positive control groups (VEGF-treated group and PBS-treated group). The negative control group was HUVEC treated with PBS. All EVs were 5 × 10⁴ 8 The sample was treated with EV / ml, and VEGF was added at 100 ng / ml. The results of the angiogenic activity assessment are shown in Figure 11. As shown in Figure 11, all EV-treated groups showed a similar level of vascular regeneration effect to the experimental group treated with VEGF. In particular, excellent vascular regeneration ability was confirmed in the experimental group treated with a 1:1 mixture of trehalose and sucrose at concentrations of 2-4%, followed by freezing, re-thawing, and re-thawing of EV. This result indicates that the therapeutic efficacy of EV can be maintained and restored without loss after treatment with the lyophilizing protective agent of the present invention, lyophilization, and re-thawing.

[0043] Having described in detail certain aspects of the present invention, it will be obvious to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for lyophilization protection of extracellular vesicles, comprising trehalose and sucrose, wherein the trehalose and sucrose are each present in an amount of 2-8% (w / v), and the trehalose and sucrose are mixed in a volume ratio of 1:0.05-5.

2. The trehalose and sucrose mentioned above reduce crystal formation during the freeze-drying and rehydration of extracellular vesicles, as described in claim 1, for freeze-drying protection of extracellular vesicles.

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

4. The lyophilization protection composition for extracellular vesicles according to claim 1, wherein the extracellular vesicles are exosomes or microvesicles.

5. The lyophilization protection composition for extracellular vesicles according to claim 1, wherein the extracellular vesicles are extracellular vesicles derived from immune cells, tumor cells, or stem cells.

6. A lyophilization protective agent for extracellular vesicles, comprising trehalose and sucrose, wherein the trehalose and sucrose are each present in an amount of 2-8% (w / v), and the trehalose and sucrose are mixed in a volume ratio of 1:0.05-5.

7. A lyophilization composition for extracellular vesicles, comprising a mixture of trehalose and sucrose as a lyophilization protective agent; and extracellular vesicles, wherein the trehalose and sucrose are each present in an amount of 2-8% (w / v), and the trehalose and sucrose are mixed in a volume ratio of 1:0.05-5.

8. The lyophilization composition for extracellular vesicles according to claim 7, wherein the extracellular vesicles have the ability to regenerate blood vessels.

9. 1) The step of mixing a mixture of trehalose and sucrose, which are freeze-drying protective agents, with extracellular vesicles; and 2) A method for freeze-drying extracellular vesicles, comprising the step of freeze-drying the mixture in step 1), wherein the trehalose and sucrose are each contained in an amount of 2-8% (w / v), and the trehalose and sucrose are mixed in a volume ratio of 1:0.05-5.

10. The method for freeze-drying extracellular vesicles according to claim 9, wherein step 2) includes freezing the mixture from step 1) at -70 to -90°C and then freeze-drying it for 3 to 7 days.

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