Synthetic exosome as well as preparation method and application thereof

Abstract

The invention provides a synthetic exosome as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing a dry cell membrane with a buffer solution containing an active substance and miRNA, and ultrasonically mixing uniformly; and performing high-pressure homogenization and extrusion to obtain the synthetic exosome. Compared with a combination of phospholipid and cholesterol, the exosome obtained by taking the cell membrane as the raw material has higher stability; and compared with a wet membrane, the exosome formed by using the dry cell membrane has better entrapment performance.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a synthetic exosome and a preparation method and application thereof. Background Art

[0002] Exosomes are extracellular lipid nanovesicles with a size of approximately 30-150 nm. They are actively secreted by exocytosis in most living cells and exist in all body fluids, such as plasma, urine, serum, saliva, and cerebrospinal fluid (CSF).

[0003] Exosomes contain lipid bilayer structures and carry typical transmembrane proteins and receptors, adhesion molecules, lipid raft-associated proteins, and immunomodulatory molecules. A variety of proteins and nucleic acids can be found within the exosome lumen, varying depending on the cell type. As key messengers of intercellular communication, exosomes deliver bioactive molecules to recipient cells, thereby participating in a variety of pathophysiological processes, including tumors, cardiovascular disease, and neurodegenerative diseases. A series of studies have reported that tumor-derived exosomes play important roles in tumor progression, immune regulation, and metastasis prognosis. There is also growing evidence that exosomes can be used for early cancer detection, prognosis, and therapeutic guidance.

[0004] Exosomes can transport a variety of cargo types, including DNA, RNA, lipids, metabolites, and proteins. To date, the ExoCarta Exosome Database (http: / / www.exocarta.org) has collected 9,769 proteins, 3,408 mRNAs, 2,838 miRNAs, and 1,116 lipids identified in exosomes from diverse cell types and multiple organisms. Although the cell of origin determines the types of proteins secreted by exosomes, 80% of exosome proteins are highly conserved across different cells. Several proteins have been used as exosome biomarkers, including ALIX, TSG101, heat shock proteins, and the tetraspanins CD63, CD9, and CD81. These transmembrane proteins are targeted and selectively transported into recipient cells by exosomes.

[0005] Exosomes have multiple uses, but they are a highly heterogeneous mixture with a complex purification process and are easily mixed with non-exosomal components. Naturally derived cellular exosomes face unresolved challenges in purification, quality control, and storage. Furthermore, exosomes have low yields and high extraction costs, which restricts research on the biological mechanisms of exosomes and their large-scale application in the health field. Summary of the Invention

[0006] The present invention provides a synthetic exosome and its preparation method and application to solve the problems existing in the related technologies. The technical solution is as follows:

[0007] In a first aspect, the present invention provides a method for preparing synthetic exosomes, comprising the following steps:

[0008] The dried cell membrane is mixed with a buffer containing active substances and miRNA, and ultrasonically mixed; the synthetic exosomes are obtained after high-pressure homogenization and extrusion.

[0009] In one embodiment, the dry cell membrane is prepared by extraction:

[0010] The cells are broken and the cell membranes are collected; the cell membranes are dispersed using anhydrous ethanol, and the ethanol is evaporated to obtain the dry cell membranes.

[0011] In one embodiment, the stem cell membrane is one or a combination of two or more cell membranes of HEK293F, umbilical cord mesenchymal stem cells, plant cells, or yeast cells.

[0012] In one embodiment, the active substance includes one or a combination of two or more of superoxide dismutase, glutathione peroxidase, collagen, fibronectin, elastin, vitronectin, platelet-derived growth factor, tranexamic acid, and glutathione.

[0013] In one embodiment, the miRNA comprises one or a combination of two or more of miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130 or miR-145.

[0014] In one embodiment, the cells are disrupted using ultrasound; and the cells are collected by centrifugation at a temperature of 10,000-13,000 rpm for 10-40 min.

[0015] In one embodiment, anhydrous ethanol is used for dispersion, and ultrasound is applied at 40-60 Hz for 3-5 minutes.

[0016] In one embodiment, evaporating ethanol is performed at 48-52° C. and a rotation speed of 90-180 rpm / min.

[0017] In one embodiment, the ultrasonic mixing conditions are: ultrasonication at 40-60 Hz for 4-5 min.

[0018] In one embodiment, the high-pressure homogenization conditions are: high-pressure homogenization is performed 7-10 times at a high-pressure homogenizer pressure of 600-700 bar and a flow rate of 35-45 L / h.

[0019] In one embodiment, a 0.1 μm pore size filter membrane is used for extrusion, a pressure reducing valve does not exceed 200 bar, and the extrusion is cycled 7-10 times.

[0020] In a second aspect, an embodiment of the present application provides a synthetic exosome, wherein the synthetic exosome is prepared by any of the above-described methods for preparing synthetic exosomes.

[0021] In one embodiment, the particle size of the synthetic exosomes is 30-150 nm.

[0022] In a third aspect, an embodiment of the present application provides an application of synthetic exosomes, wherein the synthetic exosomes are used for drug delivery.

[0023] The advantages or beneficial effects of the above technical solution include at least:

[0024] The present application discloses a method for preparing synthetic exosomes, in which dry cell membranes are directly mixed with a buffer containing active substances and miRNA to prepare exosomes. Compared with a combination of phospholipids and cholesterol, the exosomes obtained using cell membranes as raw materials are more stable; and compared with wet membranes, the exosomes formed using dry cell membranes have better encapsulation properties.

[0025] The synthetic exosomes of the present application have excellent stability and encapsulation performance, and the particle size is the same as that of natural exosomes. They can be used for drug delivery, achieving transdermal administration and in vivo delivery.

[0026] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Transmission electron micrographs of the exosomes prepared in Example 1 before and after storage at different temperatures for 6 months. The left image is the image before storage, the middle image is the image after storage at 5°C ± 3°C for 6 months, and the right image is the image after storage at 25°C ± 2°C for 6 months. Humidity: 60% RH ± 5% RH.

[0028] Figure 2 Transmission electron micrographs of exosomes prepared in Comparative Example 1 before and after storage at different temperatures for 6 months; the left image is the electron micrograph before storage, the middle image is the electron micrograph after storage at 5°C ± 3°C for 6 months, and the right image is the electron micrograph after storage at 25°C ± 2°C for 6 months; humidity: 60% RH ± 5% RH;

[0029] Figure 3Transmission electron micrographs of exosomes prepared in Comparative Example 2 before and after storage at different temperatures for 6 months; the left image is the electron micrograph before storage, the middle image is the electron micrograph after storage at 5°C ± 3°C for 6 months, and the right image is the electron micrograph after storage at 25°C ± 2°C for 6 months; humidity: 60% RH ± 5% RH;

[0030] Figure 4 Transmission electron micrographs of exosomes prepared in Comparative Example 3 before and after storage at different temperatures for 6 months; the left image is the electron micrograph before storage, the middle image is the electron micrograph after storage at 5°C ± 3°C for 6 months, and the right image is the electron micrograph after storage at 25°C ± 2°C for 6 months; humidity: 60% RH ± 5% RH;

[0031] Figure 5 The particle size distribution of exosomes prepared in Example 1 after storage at different temperatures for 6 months; the left figure is 5°C ± 3°C, the right figure is 25°C ± 2°C; the humidity is 60% RH ± 5% RH;

[0032] Figure 6 Particle size distribution of exosomes prepared in Comparative Example 1 after storage at different temperatures for 6 months; the left figure is 5°C ± 3°C, the right figure is 25°C ± 2°C; humidity is 60% RH ± 5% RH;

[0033] Figure 7 Particle size distribution of exosomes prepared in Comparative Example 2 after storage at different temperatures for 6 months; the left figure is 5°C ± 3°C, the right figure is 25°C ± 2°C; humidity is 60% RH ± 5% RH;

[0034] Figure 8 Particle size distribution of exosomes prepared in Comparative Example 3 after storage at different temperatures for 6 months; the left figure is 5°C ± 3°C, the right figure is 25°C ± 2°C; humidity is 60% RH ± 5% RH;

[0035] Figure 9 These are photos of the appearance of exosomes prepared in Example 1, Example 2, and Comparative Examples 1-3 after being stored at different temperatures for 6 months; the left picture is at 5°C ± 3°C, the right picture is at 25°C ± 2°C, and the humidity is 60% RH ± 5% RH.

[0036] Figure 10 Transmission electron microscopy image and particle size distribution diagram of exosomes prepared in Example 2. DETAILED DESCRIPTION

[0037] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the description is considered to be illustrative and non-restrictive in nature.

[0038] Exosomes are highly heterogeneous mixtures, making purification complex and prone to contamination with non-exosomal components. Naturally derived exosomes present unresolved challenges in purification, quality control, and storage. Furthermore, the high production and extraction costs of exosomes hinder research on their biological mechanisms and their large-scale application in healthcare. Therefore, there is a need for synthetic exosomes with a cell membrane nanovesicle structure.

[0039] In addition, active substances such as SOD (superoxide dismutase) are difficult to store for a long time in ordinary dosage forms, which limits its application; however, the use of synthetic nanovesicles to encapsulate SOD can enable SOD to be stably stored in the product.

[0040] Therefore, the present application provides a method for preparing synthetic exosomes, comprising the following steps:

[0041] The dried cell membrane is mixed with a buffer containing active substances and miRNA, and ultrasonically mixed; the synthetic exosomes are obtained after high-pressure homogenization and extrusion.

[0042] Compared with the combination of phospholipids and cholesterol, cell membranes are used as raw materials. Phospholipids and cholesterol need to be assembled into a phospholipid bilayer first. After the assembled phospholipid bilayer is encapsulated, its membrane stability depends on the assembled phospholipid bilayer. However, under external factors, the stability of the phospholipid bilayer is easily affected, resulting in poor overall stability of the formed exosomes. Moreover, the phospholipid bilayer formed by phospholipids and cholesterol does not have the same membrane structure and membrane component composition as natural exosomes, which leads to compatibility and effects in subsequent applications. Using cell membranes as the membrane layer of exosomes is a natural component. On the one hand, it has the same membrane structure and membrane component composition ratio as natural exosomes, can realize most of the functions of the cell membrane, and has excellent compatibility. On the other hand, the cell membrane is a structure that ensures a relatively stable intracellular environment and has strong stability. Therefore, the exosomes prepared using cell membranes have higher stability.

[0043] Using dry cell membranes, compared to wet membranes, exosomes have better encapsulation performance. The wet membrane area is approximately 1.5 times that of the dry membrane area, demonstrating the excellent water absorption and expansion properties of stem cell membranes. When encapsulating exosomes, the dry membrane has a higher encapsulation capacity when compared to wet membranes of the same area. In particular, the stem cell membranes automatically curl during absorption and expansion, facilitating the self-assembly of the capsule.

[0044] In one embodiment, the dry cell membrane is prepared by extraction:

[0045] The cells are broken and the cell membranes are collected; the cell membranes are dispersed using anhydrous ethanol, and the ethanol is evaporated to obtain the dry cell membranes.

[0046] The dry cell membrane is extracted using cells, so that the dry cell membrane retains the same membrane structure and membrane component composition ratio as the cells, so that the prepared synthetic exosomes are similar to the natural exosome membrane structure and composition.

[0047] In one embodiment, the stem cell membrane is one or a combination of two or more cell membranes of HEK293F, umbilical cord mesenchymal stem cells, plant cells, or yeast cells.

[0048] The dried cell membranes of the present application can be derived from a variety of different cells and are suitable for preparing exosomes with various membrane characteristics, making the preparation method of the present application widely applicable.

[0049] In one embodiment, the active substance includes one or a combination of two or more of superoxide dismutase, glutathione peroxidase, collagen, fibronectin, elastin, vitronectin, platelet-derived growth factor, tranexamic acid, and glutathione.

[0050] Active substances have poor stability and are easily inactivated during storage, which limits the use of active substances. For example, SOD is a superoxide dismutase, a type of metalloenzyme that is widely present in animals, plants, microorganisms and other organisms. SOD is a superoxide anion (O2 - ) as a substrate, catalyzing the dismutation reaction of superoxide anions. The mechanism of action is as follows: SOD dismutates superoxide anion radicals into hydrogen peroxide and oxygen, which are then converted into water and oxygen by catalase (CAT), thereby eliminating superoxide anion radicals. Therefore, SOD is difficult to store for a long time in common dosage forms, limiting its application.

[0051] In one embodiment, the miRNA comprises one or a combination of two or more of miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130 or miR-145.

[0052] MiRNA plays a key role in gene regulation and biological function, and different miRNAs have cell and tissue specificity. The above-mentioned miRNA in this application can directly regulate the biological activity of cell membranes and maintain the stability of cell membranes, which helps to stabilize exosomes and preserve them for a long time.

[0053] In one embodiment, the cells are disrupted using ultrasound; and the cells are collected by centrifugation at a temperature of 10,000-13,000 rpm for 10-40 min.

[0054] The physical methods of ultrasonic disruption and centrifugal separation will not change the biochemical properties of the cell membrane, maintain the original activity of the cell membrane, and the preparation method is simple, which helps to reduce the cost of exosome preparation.

[0055] In one embodiment, anhydrous ethanol is used for dispersion, and ultrasound is applied at 40-60 Hz for 3-5 minutes.

[0056] In one embodiment, evaporating ethanol is performed at 48-52° C. and a rotation speed of 90-180 rpm / min.

[0057] During the aforementioned dispersion, sonication, and membrane formation processes, the resulting stem cell membranes are relatively uniform in size, resulting in ideal stem cell membranes. The choice of organic solvent is crucial, considering factors such as volatility and toxicity. Anhydrous ethanol is a preferred choice to avoid residual solvent that may affect the efficacy and safety of exosomes.

[0058] In one embodiment, the ultrasonic mixing conditions are: ultrasonication at 40-60 Hz for 4-5 min.

[0059] In one embodiment, the high-pressure homogenization conditions are: high-pressure homogenization is performed 7-10 times at a high-pressure homogenizer pressure of 600-700 bar and a flow rate of 35-45 L / h.

[0060] In one embodiment, a 0.1 μm pore size filter membrane is used for extrusion, a pressure reducing valve does not exceed 200 bar, and the extrusion is cycled 7-10 times.

[0061] The above homogenization and extrusion are used to optimize the properties of exosomes such as particle size and encapsulation efficiency to ensure the stability and encapsulation effect of exosomes.

[0062] The embodiments of the present application provide a synthetic exosome, wherein the synthetic exosome is prepared by any of the above-described methods for preparing synthetic exosomes.

[0063] In one embodiment, the particle size of the synthetic exosomes is 30-150 nm.

[0064] An embodiment of the present application provides an application of synthetic exosomes, wherein the synthetic exosomes are used for drug delivery.

[0065] The following is a further description with reference to specific embodiments.

[0066] Example 1

[0067] Step 1: Cell Culture

[0068] Remove the HEK293F cell cryovial from liquid nitrogen and quickly thaw it in a 37°C water bath until no crystals remain. Wipe the outer wall of the cryovial with 75% alcohol. Transfer the cells from the cryovial to a 15ml centrifuge tube containing 5ml of complete culture medium and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, resuspend the pellet in 5ml of complete culture medium, inoculate it into a T25 culture flask, and incubate it in a 37°C, 5% CO2 incubator. The next day, switch to fresh complete culture medium and continue culturing. If the cell density exceeds 80%, subculture can be performed. Collect the cells, centrifuge at 1000rpm for 8-10 minutes, discard the supernatant, add 1-2ml of culture medium, and vortex to mix. Divide the cell suspension into a new dish or flask containing 8ml of culture medium at a ratio of 1:2 to 1:5.

[0069] Step 2: Cell membrane extraction

[0070] When cells have grown to cover 80% of the flask, collect the suspension from the T25 flask into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, and wash the pellet three times with 50 ml of PBS buffer. Disrupt the cells using an ultrasonic cell disruptor, and then collect the cell membranes by centrifugation at 12000 rpm for 30 minutes.

[0071] The extracted cell membrane was evenly dispersed by adding anhydrous ethanol under 50 Hz ultrasound for 3-5 min; rotary evaporation was performed at 50° C. and a rotation speed of 150 rpm / min until the solution in the rotary evaporation bottle became a thin film and set aside.

[0072] Step 3:

[0073] 10 ml of a mixed solution consisting of SOD solution (enzyme activity 150,000 U / ml), miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130a, and miR-145 (1 pg / ml) was added to the prepared film, and the film was shaken continuously under 50 Hz ultrasound for 4-5 minutes. High-pressure homogenization was performed 8 times at a flow rate of 40 L / h in a high-pressure homogenizer at 600-700 bar. The film was then extruded 10 times using a 0.1 μm pore size filter membrane of a pneumatic liposome extruder with a pressure reducing valve not exceeding 200 bar to prepare the synthetic exosomes.

[0074] Example 2

[0075] Step 1: Cell Culture

[0076] Remove the HEK293F cell cryovial from liquid nitrogen and quickly thaw it in a 37°C water bath until no crystals remain. Wipe the outer wall of the cryovial with 75% alcohol. Transfer the cells from the cryovial to a 15ml centrifuge tube containing 5ml of complete culture medium and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, resuspend the pellet in 5ml of complete culture medium, inoculate it into a T25 culture flask, and incubate it in a 37°C, 5% CO2 incubator. The next day, switch to fresh complete culture medium and continue culturing. If the cell density exceeds 80%, subculture can be performed. Collect the cells, centrifuge at 1000rpm for 8-10 minutes, discard the supernatant, add 1-2ml of culture medium, and vortex to mix. Divide the cell suspension into a new dish or flask containing 8ml of culture medium at a ratio of 1:2 to 1:5.

[0077] Step 2: Cell membrane extraction

[0078] When cells have grown to cover 80% of the flask, collect the suspension from the T25 flask into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, and wash the pellet three times with 50 ml of PBS buffer. Disrupt the cells using an ultrasonic cell disruptor, and then collect the cell membranes by centrifugation at 12000 rpm for 30 minutes.

[0079] The extracted cell membrane was evenly dispersed by adding anhydrous ethanol under 50 Hz ultrasound for 3-5 min; rotary evaporation was performed at 50° C. and a rotation speed of 150 rpm / min until the solution in the rotary evaporation bottle became a thin film and set aside.

[0080] Step 3:

[0081] 10 ml of a mixed solution consisting of 10% tranexamic acid aqueous solution, miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130a, and miR-145 (1 pg / ml) was added to the prepared film, and the film was shaken continuously under 50 Hz ultrasound for 4-5 minutes. High-pressure homogenization was performed 10 times in a high-pressure homogenizer at 600-700 bar and a flow rate of 35 L / h. The film was then extruded 10 times in a pneumatic liposome extruder with a 0.1 μm pore size filter membrane and a pressure reducing valve not exceeding 200 bar to prepare the synthetic exosomes.

[0082] Comparative Example 1

[0083] Compared with Example 1, the first and second steps were the same, only 10 ml of SOD solution (enzyme activity 150,000 U / ml) was added in the third step, and no miRNA was added. The other steps were the same as in Example 1.

[0084] Comparative Example 2

[0085] Step 1: Cell Culture

[0086] Remove the HEK293F cell cryovial from liquid nitrogen and quickly thaw it in a 37°C water bath until no crystals remain. Wipe the outside of the cryovial with 75% alcohol. Transfer the cells from the cryovial to a 15ml centrifuge tube containing 5ml of complete culture medium and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, resuspend the pellet in 5ml of complete culture medium, and inoculate it into a T25 culture flask. Incubate in a 37°C, 5% CO2 incubator. The next day, switch to fresh complete culture medium and continue culturing. If the cell density exceeds 80%, subculture is possible. Collect the cells, centrifuge at 1000rpm for 8-10 minutes, discard the supernatant, add 1-2ml of culture medium, and then vortex to mix. Divide the cell suspension into a fresh dish or flask containing 8ml of culture medium at a ratio of 1:2 to 1:5.

[0087] Step 2: Cell membrane extraction

[0088] When cells have grown to cover 80% of the flask, collect the suspension from the T25 flask into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, and wash the pellet three times with 50 ml of PBS buffer. Disrupt the cells using an ultrasonic cell disruptor, and then collect the cell membranes by centrifugation at 12000 rpm for 30 minutes.

[0089] Step 2:

[0090] 10 ml of a mixed solution consisting of SOD solution (enzyme activity 150,000 U / ml), miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130a, and miR-145 (1 pg / ml) was added to the prepared film, and the film was shaken continuously under 50 Hz ultrasound for 4-5 min. High-pressure homogenization was performed three times at a flow rate of 40 L / h in a high-pressure homogenizer at 600-700 bar. The film was extruded 10 times through a 0.1 μm pore size filter membrane of a pneumatic liposome extruder with a pressure reducing valve not exceeding 200 bar to obtain exosomes.

[0091] Comparative Example 3

[0092] A mixture of phospholipids and cholesterol in a mass ratio of 8:2 was added with anhydrous ethanol under 50 Hz ultrasonic conditions for 3-5 minutes to disperse evenly; rotary evaporation was performed at 40°C and a rotation speed of 150 rpm / min until the solution in the rotary evaporation flask formed a thin film, which was then set aside; the phospholipids were a mixture of lecithin and sphingomyelin, wherein the mass ratio of lecithin to sphingomyelin was 1:1;

[0093] 10 ml of a mixed solution consisting of SOD solution (enzyme activity 150,000 U / ml), miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130a, and miR-145 (1 pg / ml) was added to the prepared film, and the film was shaken continuously under 50 Hz ultrasound for 4-5 minutes. High-pressure homogenization was performed three times at a flow rate of 40 L / h in a high-pressure homogenizer at 600-700 bar. The film was then extruded 10 times using a 0.1 μm pore filter membrane in a pneumatic liposome extruder with a pressure reducing valve not exceeding 200 bar to prepare the synthetic exosomes.

[0094] Stability testing:

[0095] The exosomes prepared in Example 1 and Comparative Examples 1-3 were placed at 5°C ± 3°C, 25°C ± 2°C, and 60% RH ± 5% RH, respectively. The appearance, odor, and SOD activity of the samples were tested at 6 months. The appearance, odor, and SOD activity of the exosomes at 5°C ± 3°C are shown in Table 1; the appearance, odor, and SOD activity of the exosomes at 25°C ± 2°C are shown in Table 2.

[0096] Appearance Figure 9 As shown, the upper row is the appearance of the product treated in a 5°C ± 3°C environment, the lower row is the appearance of the product treated in a 25°C ± 2°C environment, and from left to right are the appearance pictures of the products of Example 2, Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3;

[0097] Transmission electron microscopy images Figure 1-Figure 4 As shown, Figure 1 For the product of Example 1, Figure 2 For the product of Comparative Example 1, Figure 3 For the product of Comparative Example 2, Figure 4 For the product of Comparative Example 3, Figure 1-Figure 4 The left picture is an electron microscope picture before storage, the middle picture is the appearance of the product after being treated in a 5℃±3℃ environment for 6 months; the right picture is the appearance of the product after being treated in a 25℃±2℃ environment for 6 months;

[0098] Particle size distribution Figure 5-8 As shown, Figure 5 For the product of Example 1, Figure 6 For the product of Comparative Example 1, Figure 7 For the product of Comparative Example 2, Figure 8 For the product of Comparative Example 3, Figure 6-Figure 8 In the figure, the left picture shows the particle size distribution of the product treated in a 5℃±3℃ environment; the right picture shows the particle size distribution of the product treated in a 25℃±2℃ environment.

[0099] The transmission electron microscope image and particle size distribution diagram of the product prepared in Example 2 are as follows: Figure 10shown.

[0100] Table 1

[0101]

[0102]

[0103] Table 2

[0104] Group Appearance odor Enzyme activity (10,000 U / ml) Example 1 Uniform light green liquid No special smell 11.9 Comparative Example 1 Uniform appearance No special smell 8.1 Comparative Example 2 Layered, with a small amount of precipitation No special smell 7.8 Comparative Example 3 Layered, with a small amount of aggregated precipitation Has a special smell 4.5

[0105] According to the stability analysis, the exosomes of Example 1 of the present application have good stability. After being stored at low temperature and 25°C for 6 months, the appearance does not change, and the enzyme activity decreases slightly, indicating that the stability of the exosomes provides a protective environment for SOD. In contrast, in Comparative Example 1, where no miRNA is added, the enzyme activity decreases significantly. This is because the stability of the exosomes deteriorates due to the lack of miRNA protection. In Comparative Example 2, where wet cell membranes are used, the stability of the exosomes is poor, and the enzyme activity decreases significantly, especially when stored at 25°C, the enzyme activity is only 52% of the original. The stability of the exosomes prepared in Example 1 is significantly better than that of the exosomes in Comparative Examples 1-3. In practice, the exosomes of Example 1 can be stored at 2-8°C for 2 years, while natural exosomes can only be stored at 2-8°C for 1 month.

[0106] Transmission electron microscopy images also show that the exosomes prepared in the examples of this application did not undergo significant structural changes after long-term storage at different temperatures, indicating good exosome stability. In contrast, the exosomes prepared in the comparative examples showed significant differences in structure after long-term storage at high and low temperatures, indicating poor temperature stability.

[0107] Encapsulation efficiency test

[0108] The encapsulation efficiency was determined using dialysis. 1.0 ml of the exosomes from Example 1 and Comparative Examples 1-3 were added to a dialysis bag. 250 ml of PBS was used as the release medium, and the mixture was stirred at 100 rpm at room temperature. 2 ml of the release medium was withdrawn at different time points, and 2 ml of the release medium at the same temperature was replenished after each withdrawal. The withdrawn samples were filtered through a 0.22 μm filter and analyzed according to chromatographic conditions. The drug concentration C was calculated, and a dialysis equilibrium time curve was plotted with drug concentration C as the ordinate and sampling time t as the abscissa to determine the dialysis equilibrium time.

[0109] At the same time, 0.5 ml of the samples of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were accurately pipetted, and the phospholipase was destroyed with 2% Triton X-100. According to the provisions of the Chinese Pharmacopoeia, the SOD content was determined by HPLC using a C18 chromatographic column, with a mobile phase A of pure water, a mobile phase B of methanol, and a mobile phase C of a phosphate mixed solution for gradient elution, and an absorption wavelength of 220 nm.

[0110] The encapsulation efficiency and drug loading of the synthesized exosomes were calculated using the following formula: Encapsulation efficiency = (1-We / W0) × 100%, and drug loading = (W0-We) / (W0+W) × 100%. Where W0 is the drug content added to the synthesized exosomes, and W is the mass of the stem cell membrane added to the synthesized exosomes. Encapsulation efficiency and drug loading data are shown in Table 3.

[0111] Table 3

[0112] Group Encapsulation efficiency (%) Drug loading (%) Example 1 65.2 86.7 Comparative Example 1 48.3 56.9 Comparative Example 2 50.9 61.6 Comparative Example 3 32.1 37.6

[0113] From the data in Table 3, the encapsulation efficiency of the present application is 65.2% and the drug loading capacity is 86.7%, while the encapsulation efficiency of Comparative Example 1 is less than 50%. In particular, Comparative Example 2 uses a wet membrane, and the encapsulation efficiency is only 50.9% and the drug loading capacity is 61.6%, which are significantly lower than the encapsulation efficiency and drug loading capacity of Example 1, indicating that the dry cell membrane has a higher encapsulation efficiency and drug loading capacity than the wet cell membrane; and Comparative Example 3 uses a composition of phospholipids and cholesterol as a liposome membrane, and its encapsulation efficiency and drug loading capacity are also lower than those of Example 1.

[0114] In summary, the present application provides a method for preparing synthetic exosomes, in which dry cell membranes are directly mixed with a buffer containing active substances and miRNA to prepare exosomes. After storage at 25°C for 6 months, the appearance of the exosomes does not change significantly, the encapsulation efficiency can reach 65%, and the drug loading capacity can reach 86%. The exosomes have high stability and better encapsulation performance.

[0115] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described may be combined in any appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless they are mutually inconsistent.

[0116] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0117] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this application, and such modifications or substitutions should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A method for preparing synthetic exosomes, characterized in that: The following steps are involved: The dried cell membrane is mixed with a buffer containing active substances and miRNA, and ultrasonically mixed; the synthetic exosomes are obtained after high-pressure homogenization and extrusion.

2. The method for preparing synthetic exosomes according to claim 1, wherein The dry cell membrane is prepared by extraction: The cells are broken and the cell membranes are collected; the cell membranes are dispersed using anhydrous ethanol, and the ethanol is evaporated to obtain the dry cell membranes.

3. The method for preparing synthetic exosomes according to claim 1 or 2, characterized in that: The stem cell membrane is one or a combination of two or more cell membranes of HEK293F, umbilical cord mesenchymal stem cells, plant cells or yeast cells.

4. The method for preparing synthetic exosomes according to claim 1 or 2, characterized in that: The active substance includes one or a combination of two or more of superoxide dismutase, glutathione peroxidase, collagen, fibronectin, elastin, vitronectin, platelet-derived growth factor, tranexamic acid, and glutathione.

5. The method for preparing synthetic exosomes according to claim 1 or 2, characterized in that: The miRNA comprises one or a combination of two or more of miR-29a-3p, miRNA-181a, miR-24, miR-21-3p, miR-21-5p, miRNA-542-3p, miR-146a, miR-130 or miR-145.

6. The method for preparing synthetic exosomes according to claim 2, wherein: The cells were disrupted using ultrasound; the cells were collected by centrifugation at 10,000-13,000 rpm for 10-40 min; Use anhydrous ethanol to disperse and ultrasonicate at 40-60 Hz for 3-5 minutes; Evaporation of ethanol was performed at 48-52°C and a rotation speed of 90-180 rpm / min.

7. The method for preparing synthetic exosomes according to claim 1 or 2, characterized in that: The conditions for ultrasonic mixing were: ultrasonication at 40-60 Hz for 4-5 min; The conditions for high-pressure homogenization are: high-pressure homogenizer 600-700 bar, 35-45 L / h flow rate, high-pressure homogenization 7-10 times; Use 0.1um pore size filter membrane for extrusion, the pressure reducing valve does not exceed 200bar, and the extrusion cycle is 7-10 times.

8. A synthetic exosome, characterized in that The synthetic exosomes are prepared by the method for preparing synthetic exosomes according to any one of claims 1 to 8.

9. The synthetic exosome according to claim 8, characterized in that The particle size of the synthetic exosomes is 30-150 nm.

10. An application of synthetic exosomes, characterized in that, The synthetic exosomes are used for drug delivery.

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