Pharmaceutical composition comprising freeze-thaw-activated platelet-derived extracellular vesicles of different sizes, preparation process thereof, and use in promoting wound healing of respiratory tract-related tissues and alleviating inflammatory response

Abstract

To provide a natural, non-synthetic, easy-to-use and easy-to-preserve inhalable formulation and a manufacturing method thereof.SOLUTION: The present disclosure provides a pharmaceutical composition including freeze-thaw-activated platelet-derived extracellular vesicles with different sizes, a preparation process thereof, and a use thereof for promoting wound healing of respiratory tract-related tissues and alleviating inflammatory response. The freeze-thaw-activated platelet-derived extracellular vesicles are processed by size exclusion chromatography (SEC) to have different particle sizes.SELECTED DRAWING: Figure 2B

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical compositions containing freeze-thaw activated platelet-derived extracellular vesicles of different sizes, methods for their preparation, and their use for promoting wound healing and alleviating inflammatory responses in tissues related to the airways. [Background technology]

[0002] Drug delivery via the respiratory system is called "inhalation therapy" and is primarily used to treat airway and lung diseases. Known features of common inhaled formulations include non-invasive therapy, rapid onset of action, minimal side effects, and low dosage.

[0003] The types of inhalation preparations currently in use can be divided into the following categories: 1. Metered-dose inhalers (MDIs), the most common type of inhaler, in which the medication is sprayed by pressurization when the medication button is pressed; 2. Dry powder inhalers (DPIs), which require a "fast and deep" inhalation, inhaling the medication into the trachea and preventing excess medication from remaining; and 3. Nebulizers, which atomize liquid inhalation solution medications into gas, making them convenient and effective for users with poor hand-mouth coordination or insufficient breathing power.

[0004] However, the preparations themselves are chemically synthesized drugs, biological products, or cell preparations, which require special storage conditions, are inconvenient to use, and often have side effects. Furthermore, chemically synthesized drugs, biological products, or cell preparations are often administered via injection, which is an invasive treatment. The administration of biological products or cell preparations is expensive. Some drugs have drug resistance issues, which necessitate the use of higher doses to control the disease. Chemically synthesized drugs, biological products, or cell preparations require long development times and are expensive.

[0005] To solve the above problems, those skilled in the art urgently need to develop natural, non-synthetic, easy-to-use and easy-to-store inhalation formulations and methods for their manufacture, which will benefit a wide range of people in need. Summary of the Invention

[0006] In view of this, an object of the present invention is to provide a pharmaceutical composition comprising freeze-thaw activated platelet-derived extracellular vesicles, which have different sizes as a result of being processed by size exclusion chromatography (SEC).

[0007] Another object of the present invention is to provide a method for preparing the pharmaceutical composition, comprising the steps of: (a) centrifuging a human whole blood sample, separating the plasma layer, and removing leukocytes and erythrocytes to obtain concentrated platelet-rich plasma (PRP); (b) centrifuging the concentrated platelet-rich plasma, collecting the precipitate, and suspending platelets in the precipitate with a solvent to obtain a platelet solution; (c) repeatedly freezing and thawing the platelet solution to obtain a freeze-thaw activated platelet solution; (d) centrifuging the freeze-thaw activated platelet solution and removing the supernatant to obtain the freeze-thaw activated platelet solution supernatant; and (e) filtering the freeze-thaw activated platelet solution supernatant to obtain the freeze-thaw activated platelet-derived extracellular vesicles, wherein the freeze-thaw activated platelet-derived extracellular vesicles are separated by size exclusion chromatography (SILC). They are processed by SEC (Separate Chromatography) and have different sizes.

[0008] In one embodiment of the present invention, the platelet-derived extracellular vesicles activated by freeze-thawing are 70 to 1 , 000nm in size.

[0009] In one embodiment of the present invention, the platelet-derived extracellular vesicles activated by freeze-thawing have a size of 35 to 400 nm.

[0010] In one embodiment of the present invention, the freeze-thaw activated platelet-derived extracellular vesicles have a size of 20 to 100 nm.

[0011] In one embodiment of the present invention, the freeze-thaw activated platelet-derived extracellular vesicles are further freeze-dried to prepare the pharmaceutical composition in an inhalable dosage form.

[0012] In one embodiment of the present invention, the freeze-thaw activated platelet-derived extracellular vesicles are further subjected to atomization to prepare the pharmaceutical composition in an inhalant dosage form.

[0013] In one embodiment of the present invention, the inhalant is a nebulizer, a dry powder inhaler (DPI) or a nebulizer.

[0014] Another object of the present invention is to provide the use of said pharmaceutical composition for preparing a medicament for promoting wound healing and alleviating inflammatory responses in tissues associated with the airways.

[0015] In one embodiment of the present invention, the pharmaceutical composition promotes wound healing in tissues associated with the airways by promoting cell migration and proliferation and reducing inflammation. [Effects of the Invention]

[0016] As described above, the present invention achieves the following technical effects through the results illustrated in the following examples: improving storage conditions and environmental issues; improving the problem of side effects caused by chemically synthesized drugs, biological products or cell products; improving the problem of invasive therapy; improving the problem of restrictions and regulations on the use of chemically synthesized drugs, biological products or cell products; improving the problem of high costs of biological products and cell products; and improving the problem of bioresistance of natural biological products produced by cells and blood derivatives.

[0017] The means and improvements that the present invention can use to solve the problems are as follows: Most chemically synthesized drugs, biological products, or cell preparations require special storage conditions, but the present invention does not require special storage conditions or environments; Most chemically synthesized drugs and biological products are synthetic and are suspected of having side effects, but the present invention is a natural biological product produced by cells and blood derivatives and has no side effects; Most chemically synthesized drugs, biological products, or cell preparations require invasive treatment, but the present invention provides treatment via inhalation, making it a non-invasive therapy.

[0018] While chemically synthesized drugs, biological products, or cell products are difficult to use and involve the risk of operational failure, the present invention is simple to use and does not pose operational failure issues. The present invention is a natural biological product produced by cells and blood derivatives, which can be mass-produced, reducing costs. The present invention is a natural biological product produced by cells and blood derivatives, which does not have the problem of biological resistance. The present invention can be used in combination with other natural biological products produced by cells and blood derivatives to enhance therapeutic effects.

[0019] The following further describes the embodiments of the present invention. The examples listed below are used to clearly explain the present invention and are not used to limit the scope of the present invention. Those skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is based on that defined in the appended claims. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows a comparison of the concentrations of platelet-derived extracellular vesicles of different sizes before and after nebulization. [Figure 2A] Figure 2A shows the ability of non-atomized platelet-derived extracellular vesicles to promote cell migration, and "Control" represents the control group. [Figure 2B] Figure 2B shows the ability of atomized platelet-derived extracellular vesicles to promote cell migration, and "Control" represents the control group. [Figure 2C] Figure 2C shows the ability of non-atomized platelet-derived extracellular vesicles to promote cell proliferation, and "Control" indicates the control group. [Figure 2D] Figure 2D shows the ability of atomized platelet-derived extracellular vesicles to promote cell proliferation, and "Control" represents the control group. [Figure 3] Figure 3 shows a comparison of the inflammatory index (IL-6) before and after atomization of platelet-derived extracellular vesicles of various sizes. [Figure 4]Figure 4 shows the ability of platelet-derived extracellular vesicles of various sizes to promote cell migration, with or without freeze-drying. Control represents the control group. [Figure 5] Figure 5 shows the ability of platelet-derived extracellular vesicles of various sizes to promote cell proliferation with or without freeze-drying, and "Control" represents the control group. [Figure 6] Figure 6 shows a comparison of the inflammation index (IL-6) between platelet-derived extracellular vesicles of various sizes that were freeze-dried and those that were not. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition Numerical values ​​used herein are approximate and all experimental data are expressed within a range of ±10%, preferably ±5%.

[0022] As used herein (particularly in the claims which follow), the terms "a," "the," "the," and similar terms should be understood to include the singular and plural forms unless the context clearly indicates otherwise.

[0023] According to the present invention, extracellular vesicles (EVs) are small membrane-bound particles secreted by cells, with a diameter of approximately 30 nm to 100 nm. , Extracellular vesicles (EVs) are lipid bilayer particles that are released from almost all cells and cannot replicate. They function as a medium for intercellular message transmission. They can transport large amounts of proteins, nucleic acids, lipids, metabolites, and even organelles from the original cell. Their sources include human organ cells, animal cells, plants, and microorganisms. Within the human body, various cells can produce exosomes, which are present in various biofluids, such as blood, saliva, amniotic fluid, blood, placenta, joint fluid, tumor cells, semen, sweat glands, urine, lymph, and cerebrospinal fluid.

[0024] Aerosol particles of different sizes are deposited in different parts of the respiratory tract; small sprays atomize the drug to 0.5-5 μm and can be deposited in the trachea and bronchi; larger particles may be deposited in the oro-nasal and pharyngeal regions; and particles that are too small may be deposited in the alveoli and even exhaled.

[0025] In addition, humidity increases the size of aerosol particles, which further reduces the number of aerosol particles deposited in the trachea and bronchi. Therefore, the present invention uses platelets as a source to prepare platelet-derived extracellular vesicles of different sizes, overcomes the problem that the membrane structure of extracellular vesicles is unstable and easily destroyed, and nebulizes them, thereby effectively meeting the needs of natural, non-synthetic, easy to use, and easy to store.

[0026] According to the present invention, tissues associated with the respiratory tract include, but are not limited to, the nasal mucosa, the trachea, and the bronchi.

[0027] The present invention will be further illustrated by the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention, which is set forth in the claims appended below.

[0028] Example 1. Comparison of the concentrations of platelet-derived extracellular vesicles of different sizes before and after nebulization The purpose of this example was to prepare and atomize platelet-derived extracellular vesicles of different sizes and to confirm that the membrane structures can stably pass through the atomization process.

[0029] Experimental steps of this example: 250 mL of human whole blood was placed in a blood bag containing an anticoagulant, and the whole blood was centrifuged (500-1 ,Human whole blood is separated into three layers by centrifugation at 200 g for 5 to 8 minutes (first centrifugation), which are, from top to bottom, the plasma layer, the buffy coat layer where white blood cells are located, and the red blood cell layer. The separation of blood cells into layers after centrifugation is well known to those skilled in the art, and the centrifugation conditions for the first centrifugation are 300 g to 1 , The platelets are centrifuged at 500 g for 3 to 10 minutes. After centrifugation, the platelets are distributed in the plasma layer and adjacent to the buffy coat layer. The plasma layer is aspirated using aseptic techniques, and white and red blood cells are completely removed. This results in highly concentrated platelet-rich plasma (PRP), also known as plasma layer solution.

[0030] Take one of the highly concentrated platelet-rich plasma (PRP) and centrifuge the plasma layer solution (1 , 000~2 , The platelets were then centrifuged at 500 g for 5 minutes (a second centrifugation) to precipitate the platelets and form a pellet. This pellet was the platelet layer, and the supernatant was the plasma and plasma protein layer. The supernatant was then completely removed to completely remove the plasma and plasma proteins. The platelets in the platelet layer were then suspended in isotonic sodium chloride solution injection (brand: Xin Dong, Taiwan Biotech Co., Ltd.) to a platelet concentration of 1 x 10 9 / mL (total 10-20mL) to obtain a pure platelet solution. The platelet solution is frozen at -80°C (24 hours), thawed at 37°C (15 minutes), and the freeze-thaw cycle is repeated three times to obtain a freeze-thaw activated platelet solution.

[0031] The platelet solution activated by freezing and thawing was centrifuged (10 , 000~15, The platelets were centrifuged at 1000 x g for 5-10 minutes (this was the third centrifugation), and the supernatant was removed to obtain the freeze-thaw activated platelet supernatant. The freeze-thaw activated platelet supernatant was filtered through a 0.45 μm syringe filter (PES material, 25 mm, model number C0000296, brand Labfil (R) ) and filtered Platelets and most impurities are filtered out, and freeze-thaw activated platelet-derived EVs are obtained.

[0032] A portion of the activated platelet-derived extracellular vesicles was taken and subjected to size exclusion chromatography (SEC) using different size exclusion columns (IZON, qEV10 column). The large size (70–100%) of the platelet-derived extracellular vesicles was then determined. , Three types of freeze-thaw activated platelet-derived extracellular vesicles are prepared: small (000 nm), medium (35-400 nm), and small (20-100 nm). Freeze-thaw activated platelet-derived extracellular vesicles without size exclusion chromatography are freeze-thaw activated platelet-derived extracellular vesicles without size separation.

[0033] Using an aerosol generator (model Pulmogine, brand HCmed), platelet-derived extracellular vesicles of various sizes and platelet-derived extracellular vesicles without size exclusion were nebulized to obtain multiple aerosols. The aerosol generator may be one that complies with the Medical Device Quality Management System (QMS) or is approved by the national health authority (e.g., FDA), such as the medical Pulmogine nebulizer (brand HCmed), the medical NEB 800 nebulizer (brand Microlife), or the medical TD-7001 nebulizer (brand Clever Check).

[0034] The aerosol generator used in this example has a 5 μm pore size and is primarily composed of two main bodies: the upper body is the reservoir, and the lower body is the main unit. The freeze-thaw activated platelet-derived EV solution is poured into the reservoir, the reservoir cap is attached, the reservoir is connected to the lower body, and the centrifuge tube is connected to the reservoir, and the recovered solution after atomization is collected. By pressing the start button on the main body, all of the freeze-thaw activated platelet-derived EVs can be atomized into aerosols.

[0035] The collected liquid after atomization is subjected to quantitative nanoparticle analysis using tunable resistive pulse sensing (TRPS, model number Exoid, brand IZON Science), and the degree of atomization (concentration after atomization ÷ concentration before atomization × 100%) is calculated.

[0036] The results of the experiment are shown in Table 1 and FIG.

[0037] [Table 1]

[0038] As can be seen from Table 1 and Figure 1, the degree of atomization of platelet-derived extracellular vesicles that are not separated by size is greater than that of large, medium, and small sizes.

[0039] Example 2. Healing tests using platelet-derived extracellular vesicles of different sizes The purpose of this example was to culture human nasal mucosa epithelial cells, add platelet-derived extracellular vesicles of different sizes (final concentration 5%), conduct healing tests (cell migration, cell proliferation), and observe whether or not there was an effect of atomization.

[0040] 2-1. Ability of non-atomized platelet-derived extracellular vesicles to promote cell migration Experimental steps: Prepare four types of platelet-derived extracellular vesicles according to the preparation method in Example 1.

[0041] Human nasal epithelial cells (brand: Promocell, product number: C-12620) were cultured in Dulbecco's modified Eagle medium (DMEM, Corning 10-013CV) containing 2% fetal bovine serum (FBS), and the culture medium was replaced with fresh medium as a backup.

[0042] A 24-well plate containing cell culture inserts was divided into three groups: untreated wells, wells containing unsorted platelet-derived extracellular vesicles, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, and wells containing small-sized platelet-derived extracellular vesicles. Each well was replicated three times. Human nasal epithelial cells were added to each group of wells, so that each well contained the same number of cells. These wells were then cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0043] The used medium and cell culture inserts for each group were removed and photographed under a microscope for recording. Then, each group was subjected to the following steps:

[0044] Untreated wells: Add DMEM culture medium (containing 2% FBS) and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0045] Unsorted platelet-derived extracellular vesicles: Mix unsorted platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for unsorted platelet-derived extracellular vesicles. Then, add the culture medium for unsorted platelet-derived extracellular vesicles to the wells containing the unsorted platelet-derived extracellular vesicles and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0046] Large-sized platelet-derived extracellular vesicles: Mix large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for large-sized platelet-derived extracellular vesicles. Then, add the culture medium for large-sized platelet-derived extracellular vesicles to the wells containing the large-sized platelet-derived extracellular vesicles and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0047] Medium-sized platelet-derived extracellular vesicles: Mix medium-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a medium-sized platelet-derived extracellular vesicle culture medium. Then, add the medium-sized platelet-derived extracellular vesicle culture medium to the wells containing the medium-sized platelet-derived extracellular vesicles and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0048] Small-sized platelet-derived extracellular vesicles: Mix small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for small-sized platelet-derived extracellular vesicles. Then, add the culture medium for small-sized platelet-derived extracellular vesicles to the wells containing the small-sized platelet-derived extracellular vesicles and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0049] The images are photographed and recorded under a microscope, and the area covered by the cell migration of each group is calculated. The results of the experiment are shown in Table 2 and Figure 2A.

[0050] [Table 2]

[0051] As can be seen from Table 2 and Figure 2A, the effect of medium-sized platelet-derived extracellular vesicles was the best in terms of the difference in the area of ​​cell migration that did not undergo the atomization process.

[0052] 2-2. Ability of atomized platelet-derived extracellular vesicles to promote cell migration Experimental steps: Prepare four types of platelet-derived extracellular vesicles according to the preparation method in Example 1.

[0053] Four types of platelet-derived extracellular vesicles were taken for each group, and the platelet-derived extracellular vesicles were subjected to the atomization process (as described in Example 1) using an aerosol generator (Pulmogine, brand HCmed) to obtain multiple aerosols for each group. 、 A centrifugal tube is used to collect the atomized aerosol, and the aerosol is collected in the centrifugal tube to become a recovered liquid for subsequent experiments.

[0054] The experiment is carried out using the same human nasal mucosa epithelial cells as in Example 2-1.

[0055] A 24-well plate containing cell culture inserts was divided into three groups: untreated wells, wells containing unsorted platelet-derived extracellular vesicles, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, and wells containing small-sized platelet-derived extracellular vesicles. Each well was treated three times. Human nasal epithelial cells were added to each group of wells, ensuring that each well contained the same number of cells. The wells were then cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0056] The old culture medium and cell culture inserts for each group were removed and photographed under a microscope for recording. Then, each group was subjected to the following steps:

[0057] Untreated wells: Add DMEM culture medium (containing 2% FBS) and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0058] Nebulized group of unsized platelet-derived extracellular vesicles: Mix the nebulized collection solution of unsized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of unsized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of unsized platelet-derived extracellular vesicles to the wells of the nebulized group of unsized platelet-derived extracellular vesicles and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0059] Large-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of large-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of large-sized platelet-derived extracellular vesicles to the wells of the large-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0060] Medium-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized medium-sized platelet-derived extracellular vesicles recovery solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the medium-sized platelet-derived extracellular vesicles nebulization culture medium. Then, add the medium-sized platelet-derived extracellular vesicles nebulized culture medium to the wells of the medium-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 6 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0061] Small-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of small-sized platelet-derived extracellular vesicles. The nebulized culture solution of small-sized platelet-derived extracellular vesicles was then added to the wells of the small-sized platelet-derived extracellular vesicles nebulization group and incubated for 6 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0062] The images are photographed and recorded under a microscope, and the area covered by the cell migration of each group is calculated. The results of the experiment are shown in Table 3 and Figure 2B.

[0063] [Table 3]

[0064] As can be seen from Table 3 and Figure 2B, the effect of small-sized platelet-derived extracellular vesicles was the most excellent in terms of the difference in the area of ​​cell migration after the atomization process.

[0065] 2-3. Ability of non-atomized platelet-derived extracellular vesicles to promote cell proliferation Experimental steps: Prepare four types of platelet-derived extracellular vesicles according to the preparation method in Example 1.

[0066] The same human nasal mucosa epithelial cells as in Example 2-1 are used to conduct experiments together with platelet-derived extracellular vesicles.

[0067] A 24-well plate was divided into three groups: untreated wells, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, and wells containing small-sized platelet-derived extracellular vesicles. Each well was treated three times. Human nasal epithelial cells were added to each group of wells, with the same number of cells per well. These were then cultured in a 37°C, 5% CO2 incubator for 24 hours. Each group then underwent the following steps:

[0068] Remove the used medium and perform the following steps: Untreated wells: DMEM culture medium (containing 2% FBS) is added to the untreated wells, and the cells are cultured in an incubator at 37°C and 5% carbon dioxide (CO 2 ) for 18 hours.

[0069] Large-sized platelet-derived extracellular vesicles: Mix large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for large-sized platelet-derived extracellular vesicles. Then, add the culture medium for large-sized platelet-derived extracellular vesicles to the wells containing the large-sized platelet-derived extracellular vesicles and continue culturing for 18 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0070] Medium-sized platelet-derived extracellular vesicles: Mix medium-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a medium-sized platelet-derived extracellular vesicle culture medium. Then, add the medium-sized platelet-derived extracellular vesicle culture medium to the wells containing the medium-sized platelet-derived extracellular vesicles and continue culturing for 18 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0071] Small-sized platelet-derived extracellular vesicles: Mix small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for small-sized platelet-derived extracellular vesicles. Then, add the culture medium for small-sized platelet-derived extracellular vesicles to the wells containing the small-sized platelet-derived extracellular vesicles and continue culturing for 18 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0072] Remove the old culture medium from each group. Add 90 μL of DMEM culture medium and 10 μL of CCK-8 buffer (DOJINDO, product number: CK04) to each well. Continue culturing for 3 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0073] Multimode microplate spectrophotometer (model number Varioskan LUX)TM , brand Thermo Scientific TM The analysis was performed using a CCK-8 buffer (CCK-8 buffer) and the absorbance was set at 450 nm. A regression curve between absorbance and cell number was established according to the CCK-8 buffer product instructions, and the absorbance was converted to the number of viable cells.

[0074] The results of the experiment are shown in Table 4 and Figure 2C.

[0075] [Table 4]

[0076] As can be seen from Table 4 and Figure 2C, in the case of cell proliferation that has not undergone the atomization process, it is clear that small-sized platelet-derived extracellular vesicles have the best cell proliferation effect.

[0077] 2-4. Ability of atomized platelet-derived extracellular vesicles to promote cell proliferation Experimental steps: Four types of platelet-derived extracellular vesicles were prepared and nebulized according to the preparation method in Example 1.

[0078] Four types of platelet-derived extracellular vesicles were taken for each group, and the platelet-derived extracellular vesicles were subjected to the atomization process (as described in Example 1) using an aerosol generator (Pulmogine, brand HCmed) to obtain multiple aerosols for each group. 、 A centrifugal tube is used to collect the atomized aerosol, and the aerosol is collected in the centrifugal tube to become a recovered liquid for subsequent experiments.

[0079] The same human nasal mucosa epithelial cells as in Example 2-1 are used to conduct experiments together with platelet-derived extracellular vesicles.

[0080] A 24-well plate was divided into three groups: untreated wells, wells containing large-sized, medium-sized, and small-sized platelet-derived extracellular vesicles, with each well being treated three times. Human nasal epithelial cells were added to each group of wells, with the same number of cells per well. These were then cultured in a 37°C, 5% CO2 incubator for 24 hours. Each group was then subjected to the following steps:

[0081] Remove the used medium and perform the following steps: Untreated wells: DMEM culture medium (containing 2% FBS) is added to the untreated wells, and the cells are cultured in an incubator at 37°C and 5% carbon dioxide (CO 2 ) for 18 hours.

[0082] Large-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of large-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of large-sized platelet-derived extracellular vesicles to the wells of the large-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 18 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0083] Medium-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized medium-sized platelet-derived extracellular vesicles recovery solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the medium-sized platelet-derived extracellular vesicles nebulization culture medium. Then, add the medium-sized platelet-derived extracellular vesicles nebulized culture medium to the wells of the medium-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 18 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0084] Small-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of small-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of small-sized platelet-derived extracellular vesicles to the wells of the small-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 18 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0085] Remove the old culture medium from each group. Add 90 μL of DMEM culture medium and 10 μL of CCK-8 buffer (DOJINDO, product number: CK04) to each well. Continue culturing for 3 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0086] Multimode microplate spectrophotometer (model number Varioskan LUX) TM , brand Thermo Scientific TM The analysis was performed using a CCK-8 buffer (CCK-8 buffer) and the absorbance was set at 450 nm. A regression curve between absorbance and cell number was established according to the CCK-8 buffer product instructions, and the absorbance was converted to the number of viable cells.

[0087] The results of the experiment are shown in Table 5 and Figure 2D.

[0088] [Table 5]

[0089] As can be seen from Table 5 and Figure 2D, atomized small-sized platelet-derived extracellular vesicles (20-100 nm) have the best effect on cell proliferation.

[0090] Example 3. Comparison of the ability of freeze and thaw activated platelet-derived extracellular vesicles (freeze and thaw activated platelet-derived extracellular vesicles) to alleviate inflammation with and without atomization Experimental steps: Four types of platelet-derived extracellular vesicle solutions were prepared as in Example 1, divided into two portions, and a portion of each was atomized.

[0091] Four types of platelet-derived extracellular vesicles were taken for each group, and the platelet-derived extracellular vesicles were subjected to the atomization process (as described in Example 1) using an aerosol generator (Pulmogine, brand HCmed) to obtain multiple aerosols for each group. 、 A centrifugal tube is used to collect the atomized aerosol, and the aerosol is collected in the centrifugal tube to become a recovered liquid for subsequent experiments.

[0092] The experiment is carried out using the same human nasal mucosa epithelial cells as in Example 2-1.

[0093] A 96-well plate was divided into three groups: untreated (original) wells, LPS control wells, wells containing unsized platelet-derived extracellular vesicles, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, wells containing small-sized platelet-derived extracellular vesicles, and wells containing unsized platelet-derived extracellular vesicles, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, and wells containing small-sized platelet-derived extracellular vesicles. Each well was then incubated three times. Human nasal epithelial cells were added to each group of wells, ensuring that each well contained the same number of cells. The wells were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0094] Remove the used medium. Suspend the cells in the LPS control wells and experimental wells in DMEM medium (containing 2% FBS) containing 5 μg / mL LPS. Suspend the cells in the untreated wells in DMEM medium (containing 2% FBS) without LPS. Incubate the wells at 37°C in a 5% CO2 incubator for 1 hour.

[0095] Remove the used medium and perform the following steps: DMEM culture medium (containing 2% FBS) is added to the untreated (Original) wells and the LPS control wells, and the cells are cultured in an incubator at 37°C and 5% carbon dioxide (CO2) for 24 hours.

[0096] Unsorted platelet-derived extracellular vesicles: Mix unsorted platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for unsorted platelet-derived extracellular vesicles. Then, add the culture medium for unsorted platelet-derived extracellular vesicles to the wells containing the unsorted platelet-derived extracellular vesicles and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0097] Large-sized platelet-derived extracellular vesicles: Mix large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a culture medium for large-sized platelet-derived extracellular vesicles. Then, add the culture medium for large-sized platelet-derived extracellular vesicles to the wells containing the large-sized platelet-derived extracellular vesicles and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0098] Medium-sized platelet-derived extracellular vesicles: Mix medium-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare a medium-sized platelet-derived extracellular vesicle culture medium (medium-sized EV medium). Then, add the medium-sized platelet-derived extracellular vesicle culture medium (medium-sized EV medium) to the well containing the medium-sized platelet-derived extracellular vesicles and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0099] Small-sized platelet-derived extracellular vesicles: Mix small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare small-sized platelet-derived extracellular vesicles culture medium (small-sized EV medium). Then, add the small-sized platelet-derived extracellular vesicles culture medium to the wells containing the small-sized platelet-derived extracellular vesicles and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0100] Nebulized group of unsized platelet-derived extracellular vesicles: Mix the nebulized collection solution of unsized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of unsized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of unsized platelet-derived extracellular vesicles to the wells of the nebulized group of unsized platelet-derived extracellular vesicles and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0101] Large-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of large-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of large-sized platelet-derived extracellular vesicles to the wells of the large-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0102] Medium-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized medium-sized platelet-derived extracellular vesicles recovery solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the medium-sized platelet-derived extracellular vesicles nebulization culture medium. Then, add the medium-sized platelet-derived extracellular vesicles nebulized culture medium to the wells of the medium-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0103] Small-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of small-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of small-sized platelet-derived extracellular vesicles to the wells of the small-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide (CO2).

[0104] The cell culture medium from each well was transferred to a microcentrifuge tube. The concentration of human IL-6 in the cell culture medium was measured using an ELISA kit (Human IL-6 ELISA Kit) (product number: ab178013, brand: Abcam). The measurement was then performed using a multimode microplate spectrophotometer (model number: Varioskan LUX). TM , brand Thermo Scientific TM ) and set the absorbance at 450 nm. Establish a regression curve between absorbance and IL-6 concentration according to the product instructions of the interleukin (IL-6) ELISA kit, and convert the absorbance to the IL-6 concentration.

[0105] The results of the experiment are shown in Table 6 and FIG.

[0106] [Table 6]

[0107] As can be seen from Table 6 and Figure 3, atomized small-sized platelet-derived extracellular vesicles (20-100 nm) have the best ability to alleviate inflammation.

[0108] Example 4. Healing studies using platelet-derived extracellular vesicles with and without freeze-drying 4-1. Comparison of the ability of freeze-dried and non-freezed platelet-derived extracellular vesicles to promote cell migration Experimental steps: Prepare four types of platelet-derived extracellular vesicles as in Example 1, and divide each into two portions.

[0109] A portion of each of the above four types of platelet-derived extracellular vesicles is taken and subjected to a freeze-drying process in a volume of 1 mL to prepare four types of freeze-thaw activated platelet-derived extracellular vesicles dry powder.

[0110] A portion of each of the four types of freeze-thawed activated platelet-derived extracellular vesicle dry powders was taken and redissolved in 1 mL of physiological saline to prepare four sets of reconstituted freeze-thawed activated platelet-derived extracellular vesicle dry powder solutions.

[0111] Using an aerosol generator (Pulmogine, brand HCmed), each group of platelet-derived extracellular vesicles was subjected to the atomization process (as described in Example 1) to obtain multiple aerosols. 、 A centrifugal tube is used to collect the atomized aerosol, and the aerosol is collected in the centrifugal tube to become a recovered liquid for subsequent experiments.

[0112] The experiment is carried out using the same human nasal mucosa epithelial cells as in Example 2-1.

[0113] A 24-well plate containing cell culture inserts was divided into three groups: control (untreated) wells, wells containing unsized platelet-derived extracellular vesicles (UN-sized), wells containing large-sized platelet-derived extracellular vesicles (UN-sized), wells containing medium-sized platelet-derived extracellular vesicles (M-sized), wells containing small-sized platelet-derived extracellular vesicles (M-sized), wells containing lyophilized platelet-derived extracellular vesicles (UN-sized), wells containing large-sized platelet-derived extracellular vesicles (M-sized), wells containing medium-sized platelet-derived extracellular vesicles (M-sized), and wells containing small-sized platelet-derived extracellular vesicles (M-sized), with each group being subjected to three replicates. Human nasal epithelial cells were added to each well, ensuring that each well contained the same number of cells. The cells were then cultured for 24 hours in an incubator at 37°C with 5% carbon dioxide (CO2).

[0114] The used medium and cell culture inserts for each group were removed and photographed under a microscope for recording. Then, each group was subjected to the following steps:

[0115] Control (untreated) wells: DMEM culture medium (containing 2% FBS) was added, and the wells were cultured in an incubator at 37°C and 5% carbon dioxide (CO 2 ) for 6 hours.

[0116] Nebulized group of unsized platelet-derived extracellular vesicles: Mix the nebulized collection solution of unsized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of unsized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of unsized platelet-derived extracellular vesicles to the wells of the nebulized group of unsized platelet-derived extracellular vesicles and continue culturing for 6 hours in an incubator at 37°C with 5% carbon dioxide.

[0117] Large-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of large-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of large-sized platelet-derived extracellular vesicles to the wells of the large-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 6 hours in an incubator at 37°C with 5% carbon dioxide.

[0118] Medium-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized medium-sized platelet-derived extracellular vesicles recovery solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the medium-sized platelet-derived extracellular vesicles nebulization culture medium. Then, add the medium-sized platelet-derived extracellular vesicles nebulized culture medium to the wells of the medium-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 6 hours in an incubator at 37°C with 5% carbon dioxide.

[0119] Small-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of small-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of small-sized platelet-derived extracellular vesicles to the wells of the small-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 6 hours in an incubator at 37°C with 5% carbon dioxide.

[0120] Lyophilized nebulized culture medium of unsized platelet-derived extracellular vesicles: After reconstitution of the dried powder of unsized platelet-derived extracellular vesicles, the nebulized culture medium was mixed with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to prepare the nebulized culture medium of unsized platelet-derived extracellular vesicles. The nebulized culture medium of unsized platelet-derived extracellular vesicles was then added to the wells of the nebulized culture medium of unsized platelet-derived extracellular vesicles and incubated in a 37°C, 5% carbon dioxide incubator for 6 hours.

[0121] Lyophilized nebulized large-sized platelet-derived extracellular vesicles: The nebulized collected solution after reconstitution of the dried powder of large-sized platelet-derived extracellular vesicles was mixed with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to prepare the nebulized culture medium of large-sized platelet-derived extracellular vesicles. The nebulized culture medium of large-sized platelet-derived extracellular vesicles was then added to the wells of the nebulized large-sized platelet-derived extracellular vesicles group and incubated in a 37°C, 5% carbon dioxide incubator for 6 hours.

[0122] Freeze-dried nebulized medium-sized platelet-derived extracellular vesicles: Mix the nebulized medium-sized platelet-derived extracellular vesicles (prepared by reconstitution) with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to prepare a nebulized medium-sized platelet-derived extracellular vesicle culture medium. Add the nebulized medium-sized platelet-derived extracellular vesicles culture medium to the wells of the nebulized medium-sized platelet-derived extracellular vesicles culture medium and incubate for 6 hours at 37°C in a 5% carbon dioxide incubator.

[0123] Freeze-dried nebulized small-sized platelet-derived extracellular vesicles: The nebulized recovery solution obtained by redissolving the dried powder of small-sized platelet-derived extracellular vesicles was mixed with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the freeze-dried nebulized culture solution of small-sized platelet-derived extracellular vesicles. The freeze-dried nebulized culture solution of small-sized platelet-derived extracellular vesicles was then added to the wells of the freeze-dried nebulized small-sized platelet-derived extracellular vesicles group and incubated for 6 hours in a 37°C, 5% carbon dioxide incubator.

[0124] The images are photographed and recorded under a microscope, and the area covered by cell migration for each group is calculated.

[0125] The results of the experiment are shown in Table 7 and FIG.

[0126] [Table 7]

[0127] As can be seen from Table 7 and Figure 4, freeze-dried and atomized small-sized platelet-derived extracellular vesicles have the best ability to promote cell migration.

[0128] 4-2. Comparison of the cell proliferation-promoting ability of freeze-dried and non-freezed platelet-derived extracellular vesicles Experimental steps: Prepare four types of platelet-derived extracellular vesicles as in Example 1, and divide each into two portions.

[0129] A portion of each of the above four types of platelet-derived extracellular vesicles is taken and subjected to a freeze-drying process in a volume of 1 mL to prepare four types of freeze-thaw activated platelet-derived extracellular vesicles dry powder.

[0130] A portion of each of the four types of freeze-thawed activated platelet-derived extracellular vesicle dry powders was taken and redissolved in 1 mL of physiological saline to prepare four sets of reconstituted freeze-thawed activated platelet-derived extracellular vesicle dry powder solutions.

[0131] Using an aerosol generator (Pulmogine, brand HCmed), each group of platelet-derived extracellular vesicles was subjected to the atomization process (as described in Example 1) to obtain multiple aerosols. 、 A centrifugal tube is used to collect the atomized aerosol, and the aerosol is collected in the centrifugal tube to become a recovered liquid for subsequent experiments.

[0132] The experiment is carried out using the same human nasal mucosa epithelial cells as in Example 2-1.

[0133] A 24-well plate was divided into three groups: a control group (untreated), wells containing unsized platelet-derived extracellular vesicles, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, wells containing small-sized platelet-derived extracellular vesicles, and wells containing lyophilized unsized platelet-derived extracellular vesicles, wells containing large-sized platelet-derived extracellular vesicles, wells containing medium-sized platelet-derived extracellular vesicles, and wells containing small-sized platelet-derived extracellular vesicles. Each well was then incubated three times. Human nasal epithelial cells were added to each group of wells, ensuring that each well contained the same number of cells. The wells were then cultured at 37°C in a 5% CO2 incubator for 24 hours. Each group then underwent the following steps:

[0134] Remove the used medium and perform the following steps: Control (untreated) wells: DMEM culture medium (containing 2% FBS) was added, and the wells were cultured in an incubator at 37°C and 5% carbon dioxide for 18 hours.

[0135] Nebulized group of unsized platelet-derived extracellular vesicles: Mix the nebulized collection solution of unsized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of unsized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of unsized platelet-derived extracellular vesicles to the wells of the nebulized group of unsized platelet-derived extracellular vesicles and continue culturing for 18 hours in an incubator at 37°C with 5% carbon dioxide.

[0136] Large-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of large-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of large-sized platelet-derived extracellular vesicles to the wells of the large-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 18 hours in an incubator at 37°C with 5% carbon dioxide.

[0137] Medium-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized medium-sized platelet-derived extracellular vesicles recovery solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the medium-sized platelet-derived extracellular vesicles nebulization culture medium. Then, add the medium-sized platelet-derived extracellular vesicles nebulized culture medium to the wells of the medium-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 18 hours in an incubator at 37°C with 5% carbon dioxide.

[0138] Small-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of small-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of small-sized platelet-derived extracellular vesicles to the wells of the small-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 18 hours in an incubator at 37°C with 5% carbon dioxide.

[0139] Lyophilized nebulized culture medium of unsized platelet-derived extracellular vesicles: The nebulized culture medium of unsized platelet-derived extracellular vesicles was prepared by redissolving the dried powder of unsized platelet-derived extracellular vesicles and mixing it with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19. The nebulized culture medium of unsized platelet-derived extracellular vesicles was then added to the wells of the nebulized culture medium of unsized platelet-derived extracellular vesicles and incubated in an incubator at 37°C with 5% carbon dioxide for 18 hours.

[0140] Freeze-dried nebulized large-sized platelet-derived extracellular vesicles: The nebulized recovery solution after redissolving the dried powder of large-sized platelet-derived extracellular vesicles was mixed with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized large-sized platelet-derived extracellular vesicles culture medium. The nebulized large-sized platelet-derived extracellular vesicles culture medium was then added to the wells of the nebulized large-sized platelet-derived extracellular vesicles culture medium and incubated for 18 hours in a 37°C, 5% carbon dioxide incubator.

[0141] Freeze-dried nebulized medium-sized platelet-derived extracellular vesicles: Mix the nebulized medium-sized platelet-derived extracellular vesicles (prepared by reconstitution) with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to prepare a nebulized medium-sized platelet-derived extracellular vesicle culture medium. The nebulized medium-sized platelet-derived extracellular vesicles culture medium was then added to the wells of the nebulized medium-sized platelet-derived extracellular vesicles culture medium and incubated for 18 hours in a 37°C, 5% carbon dioxide incubator.

[0142] Freeze-dried nebulized small-sized platelet-derived extracellular vesicles: The nebulized collected solution after reconstitution of the dried powder of small-sized platelet-derived extracellular vesicles was mixed with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the freeze-dried nebulized culture medium of small-sized platelet-derived extracellular vesicles. The freeze-dried nebulized culture medium of small-sized platelet-derived extracellular vesicles was then added to the wells of the freeze-dried nebulized small-sized platelet-derived extracellular vesicles group and incubated for 18 hours in a 37°C, 5% carbon dioxide incubator.

[0143] Remove the old culture medium from each group. Add 90 μL of DMEM culture medium and 10 μL of CCK-8 buffer (DOJINDO, product number: CK04) to each well. Continue culturing for 3 hours in an incubator at 37°C with 5% carbon dioxide.

[0144] Multimode microplate spectrophotometer (model number Varioskan LUX) TM , brand Thermo Scientific TM The analysis was performed using a CCK-8 buffer (CCK-8 buffer) and the absorbance was set at 450 nm. A regression curve between absorbance and cell number was established according to the CCK-8 buffer product instructions, and the absorbance was converted to the number of viable cells.

[0145] The experimental results are shown in Table 8 and Figure 5. As can be seen from the results, freeze-dried and atomized platelet-derived extracellular vesicles have an improved ability to promote cell proliferation compared to atomization alone.

[0146] [Table 8]

[0147] Example 5. Comparison of the ability of freeze-dried and non-freeze-dried platelet-derived extracellular vesicles to alleviate inflammation Experimental steps: Prepare four types of platelet-derived extracellular vesicles as in Example 1, and divide each into two portions.

[0148] A portion of each of the above four types of platelet-derived extracellular vesicles is taken and subjected to a freeze-drying process in a volume of 1 mL to prepare four types of freeze-thaw activated platelet-derived extracellular vesicles dry powder.

[0149] A portion of each of the four types of freeze-thawed activated platelet-derived extracellular vesicle dry powders was taken and redissolved in 1 mL of physiological saline to prepare four sets of reconstituted freeze-thawed activated platelet-derived extracellular vesicle dry powder solutions.

[0150] Using an aerosol generator (Pulmogine, brand HCmed), each group of platelet-derived extracellular vesicles was subjected to the atomization process (as described in Example 1) to obtain multiple aerosols. 、 A centrifugal tube is used to collect the atomized aerosol, and the aerosol is collected in the centrifugal tube to become a recovered liquid for subsequent experiments.

[0151] The experiment is carried out using the same human nasal mucosa epithelial cells as in Example 2-1.

[0152] A 96-well plate was divided into three groups: untreated (original) wells, LPS control wells, wells containing unsized, atomized platelet-derived extracellular vesicles, wells containing large, medium, and small platelet-derived extracellular vesicles, and wells containing unsized, lyophilized, atomized platelet-derived extracellular vesicles, wells containing large, medium, and small platelet-derived extracellular vesicles. Each well was then incubated three times. Human nasal epithelial cells were added to each group of wells, ensuring that each well contained the same number of cells. The wells were then incubated at 37°C in a 5% carbon dioxide incubator for 24 hours.

[0153] Remove the used medium. Suspend the cells in the LPS control wells and experimental wells in DMEM medium (containing 2% FBS) containing 5 μg / mL LPS. Suspend the cells in the untreated wells in DMEM medium (containing 2% FBS) without LPS. Incubate the wells at 37°C in a 5% carbon dioxide incubator for 1 hour.

[0154] Remove the used medium and perform the following steps: Untreated (original) wells: DMEM culture medium (containing 2% FBS) was added, and the wells were cultured in an incubator at 37°C and 5% carbon dioxide for 24 hours.

[0155] Nebulized group of unsized platelet-derived extracellular vesicles: Mix the nebulized collection solution of unsized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of unsized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of unsized platelet-derived extracellular vesicles to the wells of the nebulized group of unsized platelet-derived extracellular vesicles and continue culturing for 24 hours in an incubator at 37°C with 5% carbon dioxide.

[0156] Large-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of large-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of large-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of large-sized platelet-derived extracellular vesicles to the wells of the large-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 24 hours in an incubator at 37°C with 5% carbon dioxide.

[0157] Medium-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized medium-sized platelet-derived extracellular vesicles recovery solution with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the medium-sized platelet-derived extracellular vesicles nebulization culture medium. Then, add the medium-sized platelet-derived extracellular vesicles nebulized culture medium to the wells of the medium-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 24 hours in an incubator at 37°C and 5% carbon dioxide.

[0158] Small-sized platelet-derived extracellular vesicles nebulization group: Mix the nebulized collection solution of small-sized platelet-derived extracellular vesicles with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture solution of small-sized platelet-derived extracellular vesicles. Then, add the nebulized culture solution of small-sized platelet-derived extracellular vesicles to the wells of the small-sized platelet-derived extracellular vesicles nebulization group and continue culturing for 24 hours in an incubator at 37°C with 5% carbon dioxide.

[0159] Lyophilized nebulized culture medium of unsized platelet-derived extracellular vesicles: After redissolving the dried powder of unsized platelet-derived extracellular vesicles, the nebulized culture medium was mixed with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture medium of unsized platelet-derived extracellular vesicles. The nebulized culture medium of unsized platelet-derived extracellular vesicles was then added to the wells of the nebulized culture medium of unsized platelet-derived extracellular vesicles and incubated in an incubator at 37°C with 5% carbon dioxide for 24 hours.

[0160] Lyophilized nebulized large-sized platelet-derived extracellular vesicles: The nebulized collected solution after reconstitution of the dried powder of large-sized platelet-derived extracellular vesicles was mixed with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the nebulized culture medium of large-sized platelet-derived extracellular vesicles. The nebulized culture medium of large-sized platelet-derived extracellular vesicles was then added to the wells of the nebulized large-sized platelet-derived extracellular vesicles group and incubated for 24 hours in a 37°C, 5% carbon dioxide incubator.

[0161] Freeze-dried nebulized medium-sized platelet-derived extracellular vesicles: Mix the nebulized medium-sized platelet-derived extracellular vesicles (prepared by reconstitution) with DMEM culture medium (containing 2% FBS) at a volume ratio of 1:19 to prepare a nebulized medium-sized platelet-derived extracellular vesicle culture medium. Add the nebulized medium-sized platelet-derived extracellular vesicles culture medium to the wells containing the nebulized medium-sized platelet-derived extracellular vesicles and incubate for 24 hours at 37°C in a 5% CO2 incubator.

[0162] Freeze-dried nebulized small-sized platelet-derived extracellular vesicles: The nebulized recovery solution after reconstitution of the dried powder of small-sized platelet-derived extracellular vesicles was mixed with DMEM culture medium (containing 2% FBS) (volume ratio 1:19) to prepare the freeze-dried nebulized culture solution of small-sized platelet-derived extracellular vesicles. The freeze-dried nebulized culture solution of small-sized platelet-derived extracellular vesicles was then added to the wells of the freeze-dried nebulized small-sized platelet-derived extracellular vesicles group and incubated for 24 hours in a 37°C, 5% carbon dioxide incubator.

[0163] The cell culture medium from each well was transferred to a microcentrifuge tube. The concentration of human IL-6 in the cell culture medium was measured using a Human IL-6 ELISA Kit (product number: ab178013, brand: Abcam). The measurement was then performed using a multimode microplate spectrophotometer (product number: Varioskan LUX). TM , brand Thermo Scientific TM) and set the absorbance at 450 nm. Establish a regression curve between absorbance and IL-6 concentration according to the product instructions of the interleukin (IL-6) ELISA kit, and convert the absorbance to the IL-6 concentration.

[0164] The results of the experiment are shown in Table 9 and FIG.

[0165] [Table 9]

[0166] As described above, the present invention achieves the following technical effects through the results illustrated in the above examples: improving storage conditions and environmental issues; improving the problem of side effects caused by chemically synthesized drugs, biological products or cell products; improving the problem of invasive therapy; improving the problem of restrictions and regulations on the use of chemically synthesized drugs, biological products or cell products; improving the problem of high costs of biological products and cell products; and improving the problem of bioresistance of natural biological products produced by cells and blood derivatives.

[0167] The means and improvements that the present invention can solve the problems are as follows: While many chemically synthesized drugs, biological products, or cell preparations require special storage conditions, the present invention does not require special storage conditions or environments. Most chemically synthesized drugs and biological products are synthetic and are suspected of having side effects, while the present invention is a natural biological product produced by cells and blood derivatives, which has no side effects. Many chemically synthesized drugs, biological products, or cell preparations require invasive treatment; the present invention provides treatment via inhalation, making it a non-invasive therapy. While chemically synthesized drugs, biological products, or cell preparations are difficult to use and carry the risk of operational failure, the present invention is simple to use and does not have the problem of operational failure. The present invention is a natural biological product produced by cells and blood derivatives, which can be mass-produced, reducing costs. The present invention is a natural biological product produced by cells and blood derivatives, which does not have the problem of bioresistance. The present invention can be used in combination with other natural biological products produced by cells and blood derivatives to enhance the therapeutic effects.

[0168] The above is merely illustrative and not limiting, and any modifications or variations equivalent thereto that do not depart from the spirit and scope of the present invention should be included within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising freeze-thaw activated platelet-derived extracellular vesicles, the freeze-thaw activated platelet-derived extracellular vesicles having different sizes as a result of being processed by size exclusion chromatography (SEC).

2. The pharmaceutical composition of claim 1, wherein the freeze-thaw activated platelet-derived extracellular vesicles have a size of 70 to 1000 nm.

3. The pharmaceutical composition of claim 1, wherein the freeze-thaw activated platelet-derived extracellular vesicles have a size of 35 to 400 nm.

4. The pharmaceutical composition of claim 1, wherein the freeze-thaw activated platelet-derived extracellular vesicles have a size of 20 to 100 nm.

5. The pharmaceutical composition of claim 1, wherein the freeze-thaw activated platelet-derived extracellular vesicles are further freeze-dried to prepare the pharmaceutical composition in an inhalant dosage form.

6. The pharmaceutical composition according to claim 1 or 5, wherein the freeze-thaw activated platelet-derived extracellular vesicles are further subjected to atomization to make the pharmaceutical composition into an inhalant dosage form.

7. The pharmaceutical composition of claim 6, wherein the inhalant is a nebulizer, a dry powder inhaler (DPI), or a nebulizer.

8. 10. A method for preparing the pharmaceutical composition of claim 1, comprising: (a) centrifuging a sample of human whole blood to separate the plasma layer and remove white blood cells and red blood cells to obtain concentrated platelet-rich plasma (PRP); (b) centrifuging the concentrated platelet-rich plasma, collecting the precipitate, and suspending the platelets in the precipitate with a solvent to obtain a platelet solution; (c) repeatedly subjecting the platelet solution to a freeze-thaw treatment to obtain a freeze-thaw activated platelet solution; (d) centrifuging the freeze-thaw activated platelet solution and removing the supernatant to obtain the freeze-thaw activated platelet solution supernatant; (e) filtering the supernatant of the freeze-thaw activated platelet solution to obtain the freeze-thaw activated platelet-derived extracellular vesicles; The freeze-thaw activated platelet-derived extracellular vesicles are processed by size exclusion chromatography (SEC) to have different sizes.

9. The method of claim 8, wherein the freeze-thaw activated platelet-derived extracellular vesicles have a size of 70 to 1000 nm.

10. The method of claim 8, wherein the freeze-thaw activated platelet-derived extracellular vesicles have a size of 35 to 400 nm.

11. The method of claim 8, wherein the freeze-thaw activated platelet-derived extracellular vesicles have a size of 20 to 100 nm.

12. The method according to claim 8, wherein the freeze-thaw activated platelet-derived extracellular vesicles are further freeze-dried to prepare the pharmaceutical composition in an inhalable dosage form.

13. The method according to claim 8 or 12, wherein the freeze-thaw activated platelet-derived extracellular vesicles are further subjected to atomization to prepare the pharmaceutical composition in an inhalant dosage form.

14. 14. The method of claim 13, wherein the inhalant is a nebulizer, a dry powder inhaler (DPI), or a nebulizer.

15. 10. Use of the pharmaceutical composition according to claim 1 for preparing a medicament for promoting wound healing and alleviating inflammatory responses in tissues associated with the airways.

16. 16. The use of the pharmaceutical composition according to claim 15, wherein the pharmaceutical composition promotes wound healing of tissues associated with the airways by promoting cell migration and proliferation and reducing inflammation.

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