An extracellular vesicle protectant and its application

Abstract

The present invention provides an extracellular vesicle protectant and its application. The extracellular vesicle protectant has a simple composition, including a buffer system, a saccharide substance, and a surfactant. It can be used as a cryopreservation solution or a lyophilization protectant, and has a wide applicable temperature range. The protectant can reduce the storage and transportation cost of exosomes and expand the applicable range of exosomes. The exosome lyophilized powder prepared by using the above protectant and exosomes can be stably stored for a long time. The lyophilized powder can be realized on a large-scale lyophilization production device, making it possible to industrially produce exosomes on a large scale.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosomes, and particularly relates to an extracellular vesicle protectant and its application. Background Art

[0002] Exosomes are small membrane vesicles secreted by cells, with a diameter of about 30 - 150 nanometers, and have attracted extensive attention in biomedical research in recent years. It has been found that exosomes play various functions in various normal physiological processes and disease processes. Thanks to their low immunogenicity and targeting properties, they show good clinical application potential in disease treatment.

[0003] However, there is little research on the storage conditions of exosomes, and there is a lack of standardized exosome storage conditions. For the commonly used -80°C condition, the storage stability of exosomes varies greatly in different preservation buffers. Some studies have shown ( A etal., Identification of storage conditions stabilizing extracellular vesicles preparations. J Extracell Vesicles. 2022 Jun;11(6):e12238. doi:10.1002 / jev2.12238. PMID: 35716060; PMCID: PMC9206228.), that the concentration of exosomes in buffers with poor protection effect drops by more than half at 1.5 months, and the preservation effect is not ideal. In addition, the -80°C storage condition is relatively harsh, which poses a great challenge for transportation and long-term storage, severely restricting the application of exosomes in clinical practice. Therefore, there is an urgent need to develop a stable formulation that enables high-concentration exosomes to be stored and transported at refrigerated (2 - 8°C) or room temperature and can withstand multiple freeze-thaw cycles. Summary of the Invention

[0004] To make up for the deficiencies of the prior art, the purpose of the present invention is to provide a preservation preparation for extracellular vesicles that is not only suitable for storage at -80°C and 2 - 8°C in solution form, but also suitable for lyophilization.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides an extracellular vesicle protectant.

[0007] Furthermore, the protectant consists of a buffer system and a lyophilization stabilizer, and the lyophilization stabilizer includes a surfactant and / or a saccharide substance.

[0008] Further, the buffer system includes Tris-NaCl buffer, PBS buffer, citrate buffer, histidine buffer, phosphate buffer, and NaCl solvent.

[0009] Further, the concentrations of Tris and NaCl in the Tris-NaCl buffer are 20 mM and 137 mM, respectively.

[0010] Further, the buffer system provides a solution pH of 6.0 - 8.0.

[0011] Preferably, the buffer system is PBS buffer.

[0012] Preferably, the PBS buffer provides a solution pH of 7.3.

[0013] Further, the surfactant includes Tween, DMSO, and F68.

[0014] Preferably, the surfactant is F68.

[0015] Preferably, the final concentration of F68 in the buffer system is 0.005%.

[0016] Further, the saccharide substances include trehalose, sucrose, polysucrose, glucose, dextran, lactose, raffinose, fructose, and maltose.

[0017] Preferably, the saccharide substance is sucrose.

[0018] Preferably, the final concentration of sucrose in the buffer system is 1.5%.

[0019] Further, the cryoprotectant is a combination of PBS buffer, F68, and sucrose.

[0020] Further, the cryoprotectant is a combination of PBS buffer and F68.

[0021] Preferably, the final concentrations of F68 and sucrose in the PBS buffer are 0.005% and 1.5%, respectively.

[0022] The term "extracellular vesicle" should be understood to refer to any type of vesicle that can be obtained from cells in any form, such as microvesicles (e.g., any vesicle shed from the plasma membrane of a cell), exosomes (e.g., any vesicle derived from the endolysosomal pathway), apoptotic bodies (e.g., that can be obtained from apoptotic cells), microparticles (which can be derived from, for example, platelets), ectosomes (which can be derived from, for example, neutrophils and monocytes in serum), prostasomes (e.g., that can be obtained from prostate cancer cells) or cardiosomes (e.g., that can be obtained from heart cells), etc. The size of extracellular vesicles can vary widely, but extracellular vesicles generally have a hydrodynamic diameter in the nanometer range, i.e., a diameter less than 1000 nm. Obviously, extracellular vesicles can be derived from any cell type in vivo, ex vivo and in vitro. Preferred extracellular vesicles include exosomes and microvesicles, but other extracellular vesicles can also be advantageous in various situations. In addition, the term should also be understood to refer to extracellular vesicle mimics, cell membrane-based vesicles obtained by, for example, membrane extrusion, sonication or other techniques. It will be apparent to those skilled in the art that when describing the medical and scientific uses and applications of extracellular vesicles, the present invention generally relates to a plurality of extracellular vesicles, i.e., a population of extracellular vesicles that can contain thousands, millions, billions or even trillions of extracellular vesicles. In a specific embodiment of the present invention, the extracellular vesicles are exosomes.

[0023] The cells of the present invention or the cells that can secrete extracellular vesicles mentioned hereinafter include, but are not limited to, primary cells, cell lines, cells present in multicellular organisms, or basically any other type of cell source. The cells of the present invention include cells that produce extracellular vesicles in vivo. The cells according to the present invention can be selected from a wide range of cells and cell lines, such as mesenchymal stem cells or stromal cells (which can be obtained from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissues, placenta, dental buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells and more specifically amniotic epithelial cells that optionally express various early markers, myeloid-derived suppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. Particularly concerned cell lines include human umbilical vein endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular endothelial cells or lymphatic endothelial cells, red blood cells, erythroid progenitor cells, chondrocytes, MSCs from different sources, amniotic cells, amniotic epithelial (AE) cells, any cells obtained by amniocentesis or from the placenta, airway epithelial cells or alveolar epithelial cells, fibroblasts, endothelial cells, etc. In addition, immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DC) are also within the scope of the present invention, and basically any type of cell that can produce extracellular vesicles is also included herein. Generally, extracellular vesicles can basically be derived from any cell source, whether it is a primary cell source or an immortalized cell line. The extracellular vesicle source cells can be any embryonic, fetal, and adult somatic stem cell types, including induced pluripotent stem cells (iPSC) and other stem cells derived by any method. When treating neurological diseases, it may be considered to utilize, for example, primary nerve cells, astrocytes, oligodendrocytes, microglia, and neural progenitor cells as source cells. For the patient to be treated, the cells can essentially be allogeneic, autologous, or even xenogeneic, that is, the cells can be from the patient himself or from unrelated, matched, or unmatched donors.

[0024] In some embodiments, the term "exosome" refers to a phospholipid bilayer vesicle body secreted by cells, with a diameter of 30 to 150 nm, carrying various nucleic acids, proteins, lipids, and metabolites of the mother cell on the membrane and within the vesicle cavity, and serving as a medium for intercellular communication. Exosomes can be secreted by all types of cells and have been found in plasma, urine, semen, saliva, bronchial fluid, cerebrospinal fluid, breast milk, serum, amniotic fluid, synovial fluid, tears, lymph fluid, bile, and gastric acid. The formation of exosomes begins with the invagination of the cell membrane to form early endosomes. These early endosomes can exchange substances with other organelles or fuse with different endosomes to further form intracellular multivesicular bodies. The multivesicular bodies then fuse with the cell membrane and are released extracellularly to form exosomes. Different cells secrete exosomes carrying different components. Exosomes are highly heterogeneous, manifested by differences in their size, contents, functions, and cell sources. The microenvironment and the inherent biological characteristics of the mother cell may affect the content and biomarkers of exosomes. Exosomes may contain membrane proteins, cell membrane and nuclear proteins, extracellular matrix proteins, metabolites, and nucleic acids, namely mRNA, non-coding RNA, and DNA, etc. The effects of exosomes on recipient cells may vary depending on the expression of cell surface receptors. This functional heterogeneity may result in one group of exosomes inducing cell survival, another group inducing apoptosis, and another group inducing immune regulation in different target cell types, etc. At the same time, exosomes derived from different organs and tissues exhibit different characteristics. The combination of the above various characteristics makes exosomes potentially more complex and heterogeneous. After being released extracellularly, exosomes move in the tissue fluid to adjacent or distant recipient cells. After being absorbed by the recipient cells, the exosomes release their contents through material exchange to achieve the exchange of substances and signals. This transfer of substances and information can directly act on the signal molecules on the surface of the recipient cell membrane through transmembrane proteins on the exosomes, activating intracellular signal cascades; or it can fuse with the cell membrane of the recipient cell to deliver the contents of the exosomes into the recipient cell; or enter the recipient cell through endocytosis. As a communication pathway between cells, the functions of exosomes affect a wide range of physiological processes, such as immune responses, tissue repair, stem cell maintenance, cardiovascular diseases, neurodegeneration, cancer, and inflammation and other pathological processes.

[0025] In some embodiments, the exosomes are derived from at least one of hematopoietic stem cells, mesenchymal stem cells, and natural killer cells. The present invention does not limit the sources of exosomes to which the provided exosome protectant is applicable. Exosomes secreted by any cell or derived from any body fluid can be protected using the above protectant.

[0026] In some embodiments, the term "buffer system" refers to components present in a composition or solution that can provide resistance against significant changes in pH caused by strong acids or bases. The buffer system can comprise a single reagent or more than one reagent, such as a weak acid and its conjugate base. The buffer system provides resistance against significant pH changes by interacting with strong acids or strong bases in the composition or solution, thereby at least partially preventing significant changes in the pH of the composition or solution. In a specific embodiment of the present invention, the buffer system used is PBS buffer, which provides a solution pH of 7.3. It is a pharmaceutical excipient with an osmotic pressure similar to that of physiological saline and is suitable for intravenous injection.

[0027] In some embodiments, the term "sugar" includes monosaccharides and oligosaccharides. A monosaccharide is a monomeric carbohydrate that cannot be hydrolyzed by an acid, including monosaccharides and their derivatives, such as amino sugars. Sugars generally have a D conformation. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid. An oligosaccharide is a carbohydrate composed of more than one monomeric sugar unit, which are linked by one or more glycosidic bonds and can be branched or linear. The monomeric sugar units in an oligosaccharide can be the same or different. Depending on the number of monomeric sugar units, oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, and so on. Unlike polysaccharides, monosaccharides and oligosaccharides are water-soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose.

[0028] In some embodiments, the term "surfactant" is a class of compounds that can significantly reduce the surface tension or interfacial tension between two liquids, between a liquid and a gas, or between a liquid and a solid. Surfactants have a wide range of applications in daily life and industrial production. They have multiple functions such as emulsification, dispersion, solubilization, foaming, and detergency, and some specific types of surfactants also have antibacterial effects. There are various types of surfactants, including but not limited to Tween, DMSO, or F68. In a specific embodiment of the present invention, the surfactant F68 is selected as an exosome protectant, which plays a very good protective role during the storage of exosomes.

[0029] In some embodiments, the term "protectant" refers to a pharmaceutically acceptable excipient that can protect unstable active ingredients (such as exosomes) from destabilizing conditions during the lyophilization process, subsequent storage, and reconstitution processes. Protectants include but are not limited to sugars, polyols (such as sugar alcohols), amino acids, or surfactants. In a specific embodiment, the protectant can be selected from sugars such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine, galactosamine, N-methylglucosamine ("Meglumine"), polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine or mixtures thereof.

[0030] In some embodiments, the main factors affecting the denaturation of exosomes during preservation are: protein aggregation caused by physical (non-covalent) interactions or chemical aggregation (covalent); oxidation reactions; hydrolysis, etc. Although the freeze-dried bioactive factors contain a very small amount of water, hydrolysis still occurs during preservation. Therefore, in order to prevent the denaturation or inactivation of exosomes during freeze-drying and preservation, effective protectants need to be added to stabilize the activity of exosomes. There are a wide variety of freeze-drying protectants with complex mechanisms. If the protectant is added improperly, it not only cannot effectively protect exosomes, but may even have a greater negative impact such as toxicity in the later use of exosome lyophilized powder. Moreover, the concentration of the added protectant also plays a very important role in the protection effect of the protectant. Generally, within a certain concentration range, the protection effect of the protectant increases with the increase in concentration. When a certain concentration is reached, the protection effect reaches the maximum value. After that, if the concentration of the protectant is further increased, the protection effect of the protectant will no longer increase significantly, and an excessively high concentration may even cause the denaturation of exosomes during the freeze-drying process. However, the protection mechanisms of different protectants are different, and the concentrations at which the maximum protection effect is presented are also different. In actual use, the comprehensive effects of multiple protectants need to be considered. In the specific embodiments of the present invention, a general-purpose exosome protectant is provided, which is composed of surfactant F68 and PBS buffer, or composed of surfactant F68, sucrose and PBS buffer; wherein, the final concentrations of F68 and sucrose in the PBS buffer are 0.005% and 1.5% respectively.

[0031] The second aspect of the present invention provides a preparation of extracellular vesicles.

[0032] Furthermore, the preparation includes the protectant described in the first aspect of the present invention and extracellular vesicles.

[0033] Furthermore, the types of the extracellular vesicle preparation include solution, lyophilized powder, suspension, gel or nanoparticles.

[0034] In some embodiments, the extracellular vesicle preparation includes an exosome preparation. In addition to the common solution and lyophilized powder, the types of exosome preparations also include several other forms, including but not limited to: activated exosomes (this type of exosome is to suspend exosomes in a liquid matrix to maintain their activity during storage, transportation, and use), exosomes loaded with microspheres (using microspheres to load exosomes to achieve a sustained release effect of exosomes in vivo), exosomes prepared with gels, exosomes complexed with specific carriers (such as complexed with liposomes, nanoparticles, etc.). It should be noted that in the case where the extracellular vesicle protectant is disclosed, those skilled in the art can use any method to assemble the protectant disclosed in the present invention and extracellular vesicles into any preparation type; therefore, any type of extracellular vesicle preparation made using the protectant disclosed in the present invention is within the protection scope of the present invention.

[0035] The third aspect of the present invention provides any one of the following applications.

[0036] Furthermore, the application includes:

[0037] 1) The application of the protectant described in the first aspect of the present invention in the preparation of extracellular vesicle preparations and drugs containing extracellular vesicles;

[0038] 2) The application of the protectant described in the first aspect of the present invention in the storage of extracellular vesicles;

[0039] 3) The application of the extracellular vesicle preparation described in the first aspect of the present invention in the preparation of drugs or drug carriers, and cosmetics.

[0040] The fourth aspect of the present invention provides a method for preparing a freeze-dried preparation of extracellular vesicles.

[0041] Furthermore, the method includes: freeze-drying a mixed system containing extracellular vesicles and the protectant described in the first aspect of the present invention.

[0042] Furthermore, the extracellular vesicles described in the first, second, third, and fourth aspects of the present invention are exosomes.

[0043] Preferably, the cell source of exosomes is mesenchymal stem cells.

[0044] In some embodiments, the term "mesenchymal stem cell" refers to an important member of the stem cell family. It is derived from the mesoderm in the early stage of development, belongs to pluripotent stem cells, was initially discovered in the bone marrow, and has characteristics such as multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation, and self-renewal. Mesenchymal stem cells include bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, skeletal mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreatic mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc. Among them, the term "umbilical cord mesenchymal stem cell" refers to mesenchymal stem cells derived from the umbilical cord. The term "adipose mesenchymal stem cell" refers to mesenchymal stem cells derived from adipose tissue.

[0045] The present invention has the following advantages and effects compared with the prior art:

[0046] (1) The protective agent of the present invention has simpler raw materials.

[0047] (2) The protective agent of the present invention is a general-purpose protective agent, compatible with storage at -80°C and 2-8°C in solution form, and is also suitable for lyophilization.

[0048] (3) The PBS, F68, and sucrose selected for the new protective agent are all pharmaceutical excipients, and the osmotic pressure is similar to that of physiological saline, making it suitable for intravenous injection.

[0049] (4) The new protective agent can effectively protect high-concentration exosomes, and has good protective effects on high-concentration exosomes of 5×10 10 particles / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0051] Figure 1 It is the freeze-dried morphology diagram when the prescription is 1-101 ml / bottle;

[0052] Figure 2 It is the freeze-dried morphology diagram when the prescription is 11-121 ml / bottle;

[0053] Figure 3 It is the freeze-dried morphology diagram when the prescription is 1-102 ml / bottle. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The technical solution of the present invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0055] Example 1 Screening of Exosome Preparation Formulations

[0056] The screening scheme for the exosome preparation formulations of the present invention is shown in Table 1 below. A total of 10 groups of formulations are set. Among them, groups 1, 2, and 3 mainly screen the buffer systems; groups 4, 5, 6, and 7 mainly screen suitable cryoprotectants through single-factor experiments and judge whether to add F68 and albumin; groups 8, 9, and 10 mainly judge whether there is an additive effect between variables through multi-factor combinations. Group 11 is Comparative Example 1 (refer to Patent CN202310822228), and Group 12 is Comparative Example 2 (refer to Patent CN202311665550).

[0057] Table 1 Screening Scheme for Exosome Preparation Formulations

[0058]

[0059]

[0060] Example 2 Investigation of the Storage Stability and Freeze-Thaw Stability of Exosome Solution at -80°C, 2-8°C, and 37°C

[0061] 1. Experimental Materials

[0062] (1) Method for generating MSCs (mesenchymal stem cell) exosomes: The exosome cell culture supernatant is the remaining liquid obtained by removing stem cells and all other cell components from the stem cell culture medium, including the culture medium, cytokines, and exosomes.

[0063] (2) Culture method of MSCs (mesenchymal stem cell) exosomes: Pre-culture overnight in a fixed-bed, detect the biosafety of the pre-culture medium, resuscitate 300-500 million cells required for the fixed-bed, resuscitate the required amount of seed cells and make them into a 50-100 ml cell suspension. Open the lid of the sampling hole, add the cell suspension into the tank body, and start the batch culture mode. Parameter settings: Temperature: 37 °C, pH: 7.2, DO: 50%, Aeration rate: 100 ml / min, Rotation speed: 550 rpm. After 24 h - 72 h, sample and detect GLU, LAC, AMM, LDH, sample and observe the free cells and count the number of free cells, stain with crystal violet on the sampling strip, observe the cell adhesion state, DO curve. When the cell adhesion state is observed to be good, open the fixed-bed circulation mode. Parameter settings: Temperature: 37 °C, pH: 7.2, DO: 50%, Aeration rate: 50 ml / min, Rotation speed: 550 rpm, Circulation flow rate: 10 ml / min, Sampling circulation flow rate: 10 ml / min.

[0064] (3) Supernatant collection: Collect the supernatant after starting the circulation mode. Judge the exosome yield and the number of medium replacements outside the fixed-bed by detecting NTA. Shut down the circulation mode, use the emptying mode to discharge the supernatant inside the tank body. After the supernatant collection is completed, according to the collected amount, open the filling mode to supplement the complete medium until the fixed-bed circulation liquid level is reached, and start the circulation mode to continue the culture. Judge the culture cycle according to the NTA detection data.

[0065] 2. Experimental methods

[0066] (1) Exosome preparation: The supernatant of the cell culture solution of the same batch of MSC cells is clarified and filtered through multiple stages, and then concentrated by a 300KD hollow fiber. The buffer solutions are respectively replaced with three stock solutions of normal saline, PBS, and 20 mM Tris + 137 mM NaCl. The exosome concentration in the stock solution > 5×10 10 particles / ml.

[0067] (2) According to the design of the screening scheme in Table 1, dilute the corresponding exosome stock solution with the corresponding buffer solution to a theoretical concentration of 5×10 10particles / ml; and corresponding cryoprotectant components were added. By adding F68 stock solution (1000×), sucrose stock solution (30×), trehalose stock solution (50×), and albumin stock solution (100×) respectively, the final concentrations of F68, sucrose, trehalose, and albumin were 0.005%, 1.5%, 25 mM, and 0.2% respectively. Groups 1, 2, and 3 were directly dispensed into 5-ml vials without adding cryoprotectants, with a dispensing specification of 1 ml / vial and 8 vials per group. Group 4 was added with F68 stock solution (1000×) at a ratio of 1:1000 and then dispensed into 5-ml vials, with a dispensing specification of 1 ml / vial and a total of 10 vials. Groups 5 to 10 were similar to Group 4. After adding the corresponding cryoprotectant components, they were dispensed into 5-ml vials, with a dispensing specification of 1 ml / vial and 10 vials per group. The preparation method for Group 11 was: The stock solution stored in physiological saline was replaced with a solution of 10 mM PBS + 0.05% F68 + 8% trehalose, and the concentration was adjusted to 5×10 10 particles / ml, dispensed into 5-ml vials, with a dispensing specification of 1 ml / vial and 10 vials per group. The preparation method for Group 12 was: The stock solution stored in physiological saline was replaced with a solution of 10 mM PB + 100 mg / ml trehalose, and the concentration was adjusted to 5×10 10 particles / ml, dispensed into 5-ml vials, with a dispensing specification of 1 ml / vial and 10 vials per group. The stoppers of the vials of all groups were capped under aseptic conditions.

[0068] (3) Stability study protocol for storage at -80°C, 2-8°C, and 37°C

[0069] The prepared samples were placed into a -80°C refrigerator, a 2-8°C refrigerator, and a 37°C incubator respectively for relevant stability experiments. The protocol is shown in Table 2. One vial of each of the 12 samples with different formulations formed a set of samples. Four sets of samples were placed at -80°C and 2-8°C respectively. One set of samples was taken out at each time point, and the concentration, particle size, and potential of exosomes were detected by NTA.

[0070] Table 2 Stability protocol for exosome liquid storage

[0071] Investigation items 0h 2 weeks 4 weeks -80℃ Concentration, particle size, potential Concentration, particle size, potential Concentration, particle size, potential 2-8℃ Concentration, particle size, potential Concentration, particle size, potential Concentration, particle size, potential

[0072] (4) Stability study protocol for freeze-thaw of exosome solution

[0073] The stability study protocol for freeze-thaw of exosome solution is shown in Table 3. One set of samples at 0 h was continuously freeze-thawed 5 times, frozen at -80°C and thawed at room temperature, with a freeze-thaw frequency of 1 day / time. Samples were taken after 1, 3, and 5 freeze-thaws, and the concentration, particle size, and potential of exosomes were detected by NTA.

[0074] Table 3 Stability protocol for freeze-thaw of exosome liquid

[0075]

[0076] 3. Experimental Results

[0077] The exosome solution was stored at -80°C for 2 weeks or 4 weeks, and the summary of the detection data for each formulation is shown in Table 4. Comparing Formulation 1, Formulation 2, and Formulation 3, it was found that the exosome concentration recovery rate in the PBS buffer was higher than that in the normal saline and Tris buffer groups, and there were no significant changes in particle size and Zeta potential. Comparing Formulation 4, Formulation 5, Formulation 6, and Formulation 7, it was found that the concentration recovery rates were similar, and the protective effects of each protective agent on exosomes were similar. The concentration recovery rates of Formulation 3 and Formulation 10 were very low, indicating that Tris buffer at pH 8.0 was not suitable for storing exosomes at -80°C. As a control, the concentration recovery rates of Formulation 11 and Formulation 12 were low, and at the same time, the particle size increased significantly, which was not suitable for storing exosomes at -80°C.

[0078] Table 4 Stability of Exosome Solution Stored at -80°C

[0079]

[0080]

[0081] The exosome solution was stored at 2 - 8°C for 2 weeks, and the summary of the detection data for each formulation is shown in Table 5. Comparing Formulation 1, Formulation 2, and Formulation 3, it was found that the exosome concentration recovery rate in the PBS buffer group was higher than that in the normal saline and Tris buffer groups, and there were no significant changes in particle size and Zeta potential. Comparing Formulation 4, Formulation 5, Formulation 6, and Formulation 7, it was found that the concentration recovery rates of Formulation 4, Formulation 5, and Formulation 6 were higher, indicating that F68, trehalose, and sucrose had better protective effects on exosomes. The concentration recovery rates of Formulation 3 and Formulation 10 were very low, indicating that Tris buffer at pH 8.0 was not suitable for storing exosomes at 2 - 8°C. The concentration recovery rate of Formulation 12 was low, which was not suitable for storing exosomes at 2 - 8°C.

[0082] Table 5 Stability of Exosome Solution Stored at 2 - 8°C

[0083]

[0084]

[0085] The summary of the detection data for the freeze-thaw stability of the exosome solution is shown in Table 6. The results showed that the concentration recovery rates of the PBS group after 3 and 5 freeze-thaw cycles were higher than those of the normal saline group; in addition, the recovery rates of Formulation 4 and Formulation 8 after 3 and 5 freeze-thaw cycles were both >80%, indicating that the addition of F68 and sucrose in PBS played a good protective role on exosomes during the freeze-thaw process; finally, the recovery rates of Formulation 11 and Formulation 12 after 5 freeze-thaw cycles were >80%, indicating that trehalose played a good protective role on exosomes during the freeze-thaw process.

[0086] Table 6 Summary of Detection Data for Freeze-Thaw Stability of Exosome Solution

[0087]

[0088] 4. Conclusions

[0089] Considering the results of the preservation of exosome solution at -80°C, 2 - 8°C, and freeze-thaw stability, PBS buffer is superior to normal saline and Tris buffer; Formulations No. 4 and No. 8 are slightly superior to other formulations, suitable for stable preservation at -80°C and 2 - 8°C, and can withstand 5 freeze-thaw cycles.

[0090] Example 3 Freeze-dried Preservation Stability of Exosomes

[0091] 1. Experimental Materials

[0092] (1) Method for generating MSCs (mesenchymal stem cell) exosomes: The exosome cell culture supernatant is the remaining liquid obtained by removing stem cells and all other cell components from the stem cell culture medium, including the culture medium, cytokines, and exosomes.

[0093] (2) Culture method of MSCs (mesenchymal stem cell) exosomes: Pre-culture in a fixed-bed overnight, detect the biosafety of the pre-culture medium, resuscitate 300 - 500 million cells required for the fixed-bed, prepare a 50 - 100 ml cell suspension after resuscitating the required amount of seed cells, open the sampling hole lid, add the cell suspension into the tank body, start the batch culture mode, parameter settings: temperature: 37°C, pH: 7.2, DO: 50%, ventilation volume: 100 ml / min, rotation speed: 550 rpm. After 24 h - 72 h, sample and detect GLU, LAC, AMM, LDH, sample and observe the free cells and count the number of free cells, take the sampling strip and stain with crystal violet to observe the cell adhesion state and the DO curve. When observing that the cell adhesion state is good, open the fixed-bed circulation mode, parameter settings: temperature: 37°C, pH: 7.2, DO: 50%, ventilation volume: 50 ml / min, rotation speed: 550 rpm, circulation flow rate: 10 ml / min, sampling circulation flow rate: 10 ml / min.

[0094] (3) Supernatant collection: Collect the supernatant after starting the circulation mode. Judge the exosome yield and the number of medium replacements in the fixed-bed by detecting NTA, stop the circulation mode, use the emptying mode to drain the supernatant inside the tank body. After the supernatant collection is completed, according to the collected volume, open the filling mode to supplement the complete culture medium until reaching the fixed-bed circulation liquid level and start the circulation mode to continue the culture, and judge the culture cycle according to the NTA detection data.

[0095] 2. Experimental Methods

[0096] (1) Exosome preparation: The supernatant of the same batch of MSC cell culture medium as in Example 2 was subjected to multi-stage clarification and filtration, and then concentrated by 300KD hollow fiber. The buffer was replaced with three stock solutions of normal saline, PBS, and 20 mM Tris + 137 mM NaCl respectively, and the exosome concentration in the stock solution > 5×10 10 particles / ml.

[0097] (2) According to the design of the screening scheme in Table 1, the corresponding exosome stock solution was diluted with the corresponding buffer to a theoretical concentration of 5×10 10 particles / ml; and the corresponding protective agent components were added. By adding the F68 stock solution (1000×), sucrose stock solution (30×), trehalose stock solution (50×), and albumin stock solution (100×) respectively, the final concentrations of F68, sucrose, trehalose, and albumin were 0.005%, 1.5%, 25 mM, and 0.2% respectively. Groups 1, 2, and 3 were directly dispensed into 5 ml vials without adding protective agents, with a dispensing specification of 1 ml / vial, 8 vials in each group. Group 4 was added with the F68 stock solution (1000×) at a ratio of 1:1000 and then dispensed into 5 ml vials, with a dispensing specification of 1 ml / vial, a total of 10 vials. Other groups were similar to Group 4. After adding the corresponding protective agent components, they were dispensed into 5 ml vials, with a dispensing specification of 1 ml / vial, 10 vials in each group. The vials were stoppered and freeze-dried, and crimped after freeze-drying.

[0098] (3) Research plan for the stability of exosome lyophilized powder stored at 2 - 8°C

[0099] The prepared samples were respectively placed in a 2 - 8°C refrigerator for relevant stability experiments, and the plan is shown in Table 7. One bottle of each of the 10 groups of samples with different prescriptions formed a set of samples. Three sets of samples were placed at 2 - 8°C. One set of samples was taken out at each time point, and the concentration, particle size, and potential of exosomes were detected by NTA.

[0100] Table 7 Research plan for the stability of exosome lyophilized powder stored at 2 - 8°C

[0101]

[0102] 3. Experimental results

[0103] (1) Evaluation of exosome freeze-drying effect

[0104] The exosomes were freeze-dried twice. One time was 1 ml per vial, and the appearance after freeze-drying is shown in Figure 1 ; the other time was 2 ml per vial, and the freeze-dried form is shown in Figure 2, in terms of appearance, the overall performance of the 1 ml specification is better than that of the 2 ml specification. In addition, it was found that for the groups with sucrose and trehalose added, the lyophilization failed at 2 ml / bottle, while it showed a compact block shape at 1 ml / bottle. Whether it is 1 ml / bottle or 2 ml / bottle, the overall shapes of Formulations 2, 3, 4, and 7 are better.

[0105] (2) Reconstitution effect of exosomes freeze-dried at 1 ml / bottle, and storage stability at 2 - 8 °C and 25 °C

[0106] The detection data of the reconstitution of exosomes freeze-dried at 1 ml / bottle after reconstitution and the freeze-dried powder stored at 2 - 8 °C for 2 weeks are shown in Table 8. The results show that the concentration recovery rates of Formulations 4, 8, 11, and 12 are significantly better than those of other groups.

[0107] Table 8 Detection data of the reconstitution effect of exosomes freeze-dried at 1 ml / bottle and the storage stability of the freeze-dried powder at 2 - 8 °C

[0108]

[0109] The detection data of exosomes freeze-dried at 1 ml / bottle before freeze-drying and the freeze-dried powder stored at 25 °C for 4 weeks are shown in Table 9. The results show that the concentration recovery rate of Formulation 4 is better than those of Formulations 11 and 12.

[0110] Table 9 Detection data of the reconstitution effect of exosomes freeze-dried at 1 ml / bottle and the storage stability of the freeze-dried powder at 25 °C

[0111]

[0112] (3) Reconstitution effect of exosomes freeze-dried at 2 ml / bottle

[0113] The detection data of the reconstitution of exosomes freeze-dried at 2 ml / bottle after reconstitution are shown in Table 10. The results show that the concentration recovery rates of Formulations 2, 4, 5, 7, and 8 after reconstitution are significantly better than those of other groups; there are no significant differences in particle size and zeta potential.

[0114] Table 10 Detection data of the reconstitution effect of exosomes freeze-dried at 2 ml / bottle

[0115]

[0116]

[0117] 4. Experimental conclusions

[0118] Considering the morphology of exosomes after freeze-drying, the compatibility of the freeze-drying process, the concentration recovery rate after reconstitution, the storage stability of the freeze-dried powder at 2 - 8 °C, and the storage stability of the freeze-dried powder at 25 °C, Formulation 4 is the optimal prescription for exosome freeze-dried preparations.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An exosome protective agent, characterized in that The protective agent consists of PBS buffer and F68, wherein the PBS buffer provides a solution pH of 7.3, and the final concentration of the F68 in the buffer system is 0.005%.

2. The protective agent according to claim 1, characterized in that The protective agent also includes sucrose, and the final concentration of the sucrose in the buffer system is 1.5%.

3. An exosome preparation, characterized in that The preparation comprises the protective agent according to any one of claims 1 to 2 and exosomes.

4. The exosome preparation according to claim 3, characterized in that The exosome preparation types include solutions, lyophilized powders, suspensions, gels or nanoparticles.

5. Any of the following applications, characterized in that: The applications include: 1) Use of the protective agent according to any one of claims 1 to 2 in the preparation of exosome preparations and exosome-containing drugs; 2) Use of the protective agent according to any one of claims 1 to 2 in exosome storage; 3) Use of the exosome preparation according to any one of claims 3 to 4 in the preparation of drugs, drug carriers, or cosmetics.

6. A method for preparing a lyophilized exosome preparation, characterized in that: The method comprises: freeze-drying a mixed system comprising exosomes and the protective agent according to any one of claims 1 to 2.

7. The protective agent according to any one of claims 1-2, the exosome preparation according to any one of claims 3-4, the use according to claim 5 or the method according to claim 6, characterized in that: The cell source of the exosomes is mesenchymal stem cells.

Images