Exosome freeze-drying protective agent and exosome preparation

Abstract

The invention belongs to the field of biological medicine, and provides an exosome freeze-drying protective agent and an exosome preparation. The exosome freeze-drying protective solution comprises the following freeze-drying protective agents in percentage by weight: 2%-4% of trehalose, 4%-6% of mannitol and 0.01%-0.02% of poloxamer 188. A buffer solution of the exosome freeze-drying protection solution is a compound electrolyte injection, a Tris buffer solution or a phosphate buffer solution. According to the freeze-drying protective agent provided by the invention, the prepared freeze-drying preparation can have a good form, a good protection effect is provided for positive particles in the exosome freeze-drying process under the condition that the dosage of each component is relatively small, and the integrity and biological activity of the exosome after freeze-drying are effectively maintained; and the exosome can be stably preserved at 2-10 DEG C. The exosome freeze-drying procedure provided by the invention is simple and time-saving, and sample collapse in the freeze-drying process can be avoided, so that a final product with a good appearance is obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an exosome freeze-dried protective agent and an exosome preparation prepared therefrom. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Exosomes are nanoscale double-layer vesicles that are actively released by cells after the fusion of intracellular multivesicular bodies (MVBs) with cell membranes. The size ranges from 30 nm to 160 nm. Exosomes contain nucleic acids, proteins, lipids and other bioactive molecules derived from parental cells, and can play a variety of roles, including promoting intercellular communication, transmitting genetic information, regulating biological activities and regulating immune responses. Exosomes have different physiological processes, including immune response, tissue repair, stem cell maintenance, central nervous system communication, and participate in pathological processes such as cardiovascular disease, neurodegeneration, cancer, and various types of inflammation. As a "cell-free" therapy, exosome therapy has the advantages of avoiding the risk of uncontrolled differentiation or reaction of implanted cells, the ability to target specific tissues, higher safety and lower immunogenicity.

[0004] In order for exosomes to be widely used in clinical settings, many obstacles still need to be overcome. In addition to the reproducibility of exosome isolation and purification, storage stability is also a huge challenge. Although studies have shown that storage conditions have an impact on the function of exosomes, there is still no established standard for the storage conditions of exosomes. The International Extracellular Vesicles Association recommends that exosomes be stored in phosphate-buffered saline (PBS) at -80°C, but exosomes preserved under these conditions have a short shelf life and high transportation costs, which is not conducive to the expansion of exosomes and commercialization. As more and more clinical results confirm the effectiveness of exosomes in clinical applications, how to preserve and transport exosomes, so as to effectively maintain the biological activity and structural stability of exosomes, get rid of the dependence on cold chain supply, and enhance clinical applicability, has become an urgent problem to be solved.

[0005] Freeze drying (lyophilization) is an attractive method commonly used in the pharmaceutical industry, including the drying of unstable drugs, the production of long-term stable vaccine formulations, the preservation of nucleic acid drugs, and the development of microparticle and nanoparticle systems for drug delivery. It improves the stability and shelf life of various products by removing water from drug formulations, and can maintain the integrity of complex biological structures during long-term storage. During the freeze-drying process, exosomes are first rapidly frozen to a cryogenic state. This process can quickly fix their structure and biological activity and reduce the damage to the membrane structure caused by ice crystal formation. Subsequently, the temperature is slowly raised and the water is removed under a vacuum environment, avoiding the thermal stress and chemical changes that may be caused by traditional drying methods, thereby maximizing the preservation of the integrity and functional activity of the exosomes.

[0006] The components of the exosome lyophilized liquid usually include exosomes and buffer. At the same time, adding a suitable lyophilization protectant can effectively maintain the morphology of the lyophilized product and the structure and function of the exosomes after lyophilization. Although there are currently multiple patents that apply lyophilization technology to the preservation of exosomes, most of them have complex formulations, time-consuming lyophilization procedures, and high preparation costs for large-scale production. Therefore, it is necessary to develop an exosome lyophilization system with streamlined lyophilized preparation components and a simple and time-saving lyophilization procedure. Summary of the invention

[0007] In view of the problems of complex ingredients and high cost of current exosome freeze-dried preparations, the present invention provides an exosome freeze-dried protective agent with simple ingredients and small amount used during the freeze-drying process.

[0008] Another object of the present invention is to provide an exosome preparation prepared by the above-mentioned exosome lyophilization protectant.

[0009] To achieve the above purpose, the present invention adopts the following technical solution.

[0010] An exosome freeze-dried protective solution comprises the following contents of freeze-dried protective agents: 2%-4% trehalose, 4%-6% mannitol and 0.01%-0.02% poloxamer 188.

[0011] The buffer of the exosome freeze-dried protection solution is a compound electrolyte injection solution, a Tris buffer or a phosphate buffer.

[0012] The pH of the buffer is 7.0-7.6, preferably 7.2-7.4. The concentration of the Tris buffer is 15mM-20mM, and the concentration of the phosphate buffer is 10mM-20mM. The Tris buffer is preferably Tris-HCl buffer.

[0013] The preparation method of the above-mentioned exosome lyophilized protective solution can adopt conventional methods in the art, such as dissolving the lyophilized protective agent in a buffer solution, or preparing the lyophilized protective agent into a mother solution and then mixing it with a buffer solution.

[0014] A use of the above exosome freeze-dried protection solution in the preparation of an exosome freeze-dried preparation.

[0015] A method for preparing a lyophilized exosome preparation comprises the following steps: (1) mixing the exosomes with an appropriate solvent, and then adding trehalose, mannitol, and poloxamer 188 to a concentration of 2%-4%, 4%-8%, and 0.01%-0.02%, respectively, to obtain a lyophilized exosome solution; (2) The exosome freeze-dried material liquid is freeze-dried according to the steps of pre-freezing-freeze drying-sublimation drying-freeze drying-analytical drying to obtain an exosome freeze-dried preparation.

[0016] In step (2), the pre-freezing temperature is -35°C, the first freeze-drying temperature is -40°C~-45°C, and the time is 10h-15h; the sublimation drying temperature is -15°C~-18°C, and the time is 3.5h-5h; the second freeze-drying temperature is 2°C~8°C, and the time is 2h-5h; the analytical drying temperature is 35°C~40°C, and the time is 2h-10h.

[0017] A lyophilized exosome preparation prepared by the above method.

[0018] The present invention does not limit the sources of the exosome lyoprotectant and the exosome lyo-drying equipment, and exosomes secreted by any cells or exosomes derived from any body fluids can be used to prepare lyophilized preparations using the above-mentioned exosome lyoprotectant and lyo-drying procedure.

[0019] The present invention has the following advantages: The exosome lyophilization protectant provided by the present invention uses compound electrolyte injection as a buffer system, which is different from the phosphate buffered saline (PBS) used in most technologies, and can reduce the loss of exosomes during the lyophilization process; the lyophilization protectant provided by the present invention can make the prepared lyophilized preparation have a good morphology, provide good protection for the positive particles in the lyophilization process of the exosomes when the amount of each component is small, effectively maintain the integrity and biological activity of the lyophilized exosomes, and can stably store the exosomes at 2°C-10°C. The exosome freeze-drying procedure determined by the present invention is simple and time-saving, and can avoid sample collapse during the lyophilization process, thereby obtaining a final product with a good appearance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the appearance of the lyophilized exosome preparations obtained using lyophilization protectants of different formulations; Figure 2 are transmission electron microscopy images of reconstituted exosome freeze-dried powder in the Control group (A) and E-2 group (B); Figure 3This is a comparison of the animal wound model before and after treatment. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0022] Example 1 Preparation of lyophilized exosome preparation 1. Screening of exosome freeze-dried protective agent concentration The exosomes of umbilical cord mesenchymal stem cells were obtained by differential centrifugation and resuspended in PBS (pH 7.4, 10 mM) to obtain 2.83 × 10 11 P / mL exosome suspension.

[0023] A 50% (w / v) trehalose mother solution, a 25% (w / v) mannitol mother solution and a 0.5% (w / v) poloxamer 188 mother solution were prepared, and filtered through a 0.22 μm pore size filter membrane for later use.

[0024] According to the formula in Table 1, the exosome suspension, trehalose mother solution, mannitol mother solution and poloxamer 188 mother solution were mixed, and the compound electrolyte injection solution was added to make the total volume 1 mL and the final exosome concentration 2.5×10 10 P / mL, and then the exosome freeze-dried preparation was obtained by the same freeze-drying procedure.

[0025] Table 1 Screening of freeze-drying protective agents The lyophilized preparations obtained from different formulas were immediately reconstituted with 1 mL of compound electrolyte injection, and the following parameters of the lyophilized exosome preparations were detected and calculated using a nanoflow cytometer: particle concentration, total number of CD9 and CD63 positive particles, and positive particle yield (total number of positive particles = total positive rate of positive particles × total number of particles, positive particle yield = total number of positive particles after lyophilization / total number of positive particles before lyophilization*100%).

[0026] The appearance of each exosome lyophilized preparation is as follows Figure 1 As shown in the figure: except for the Control group which showed collapse and incomplete freeze-dried morphology, the other groups achieved good freeze-dried forming, indicating that the freeze-drying protectant has a certain shaping and protective effect on the freeze-drying process.

[0027] The changes in the number of particles and positive particles before and after lyophilization are shown in Table 2. The results showed that the particle concentration, total number of positive particles and positive particle yield of the Control group without adding lyoprotectant after lyophilization were lower than those of the protectant group. The positive particles of E-1 and E-2 groups increased slightly after lyophilization, which was a difference within a reasonable range of variation. The overall change was not large, indicating that suitable lyoprotectants can protect exosomes and their positive particles during the lyophilization process. Among the different protectants, trehalose has a high glass transition temperature and low hygroscopicity. It can form a stable glassy structure during the lyophilization process to prevent the biological products from denaturing and inactivating during freezing and drying. It also has good hydration ability, further enhancing its anti-freeze dehydration ability; mannitol is used as a filler to provide a support structure for the active ingredient and inhibit the crystallization of sodium chloride in the buffer; poloxamer 188, as a non-ionic surfactant, can stabilize the protein in the lyophilized preparation. The appropriate concentration ranges are 2-4%, 4-6% and 0.02%, respectively. When the above concentration ranges are exceeded, the yield of positive particles decreases significantly.

[0028] Table 2 Changes in the number of particles before and after freeze-drying in freeze-dried preparations obtained with different formulations 2. Freeze-drying program screening The above two optimal combinations, E-1 group and E-2 group, were freeze-dried liquids prepared with lyoprotectant formulas, with a final volume of 1 mL. The freezing onset temperature (freezing point) and melting temperature were measured using differential scanning calorimetry (DSC), and the collapse temperature of the exosome freeze-dried liquid during the freeze-drying process was determined using a cryomicroscope.

[0029] Table 3 Freezing start temperature, collapse temperature and melting temperature of different formulations According to the sample quantity and test results, the freezing point was determined to be around -18 to -20 °C. The collapse temperature of the control group was -34.65±0.778 °C, the collapse temperature of the E-1 group was -37.93±3.111 °C, the collapse temperature of the E-2 group was -36.4±0.354 °C, and the overall collapse temperature was around -35 °C. The freeze-drying program parameters optimized according to the above parameters are shown in Table 4.

[0030] Table 4 Exosome freeze-drying process parameters The two freeze-drying programs in Table 4 were used to freeze-dry the Control group, E-1 group and E-2 group. Both programs could achieve good freeze-drying effects. The freeze-drying sublimation time of program 2 was longer, which could ensure that the ice crystals or movable water inside the exosome freeze-dried liquid were completely sublimated, and the skeleton was basically stable, which could ensure that the subsequent programs would not collapse. Therefore, program 2 was determined to be the best freeze-drying program.

[0031] Immediately after freeze-drying, the freeze-dried preparation obtained in procedure 2 was reconstituted with 1 mL of compound electrolyte injection solution. The morphology of the freeze-dried and reconstituted exosomes was then observed using a transmission electron microscope, and the particle concentration, number of positive particles, and particle size distribution before and after freeze-drying were detected using a nanoflow cytometer.

[0032] Table 5 Exosome characteristics before and after freeze-drying The particle concentration, number of positive particles and particle size distribution before and after freeze-drying detected by nanoflow cytometry are shown in Table 5: the particle size of each group did not change much before and after freeze-drying; although the positive rate of the Control group did not change much before and after freeze-drying, the particle concentration and the number of positive particles decreased significantly; the positive rate and the number of positive particles of the E-1 group decreased after freeze-drying; the number of positive particles and the positive rate of the E-2 group increased after freeze-drying.

[0033] Transmission electron microscopy was used to observe the morphology of freeze-dried exosomes in the control group and E-2 group with freeze-dried protective agent formulations. Figure 2 As shown in the figure. The morphology of some exosome particles in the control group changed, and there was leakage of contents after membrane rupture (where the arrow points to), while the exosomes using the E-2 group formula as a lyophilization protectant after lyophilization, most of them can maintain a complete and clear structure, in a cup shape, and with less background impurities, indicating that the lyophilization process is destructive to the structure of exosome particles. Appropriate lyophilization protectants can play a structural protective role in the lyophilization process.

[0034] 3. Stability of freeze-dried preparations The three batches of exosomes were freeze-dried using the lyophilization protectant of group E-2 according to the freeze-drying procedure in Table 4 and stored in the dark at 2-10°C for 1 month. The particle concentration and particle size distribution were tested before freeze-drying, after freeze-drying and for 1 month of storage to evaluate the storage stability of the freeze-dried preparation.

[0035] Table 6 Stability of three batches of freeze-dried preparations Note: * indicates compared with before freeze-drying; △ Compared with freeze-dried.

[0036] The results are shown in Table 6. The results of the samples of exosome freeze-dried powder stored at 2-10°C for 1 month were compared with those of the samples immediately reconstituted after freeze-drying (0 month). The results showed that after the freeze-dried powder was stored for 1 month, the particle concentration increased slightly compared with before freeze-drying and when it was immediately reconstituted after freeze-drying, but the results were not significant (P=0.12, P=0.20). The particle size was detected and analyzed, and it was found that the particle size was slightly reduced, which was significantly reduced compared with the exosome particle size at 0 month. Considering that the nano-scale particle components such as exosomes contained in the supernatant are uncertain, the mean particle size distribution is within the range of less than 200nm, and there is a particle size peak within this range.

[0037] Example 2 Preparation of lyophilized exosome preparation Different lyoprotectant solutions were prepared according to the formula in Table 7, and then mixed with the umbilical cord-derived mesenchymal stem cell exosome suspension isolated from the same batch. The final volume of each group was 1 mL, and lyophilized according to the procedure in Table 4.

[0038] Table 7 Formulas of different lyophilized exosome preparations The lyophilized preparations obtained with different formulas were immediately reconstituted with 1 mL of the original solvent, and the lyophilization protection effects of each group of lyoprotectants were compared by detecting and calculating the particle concentration, particle size distribution, number of positive particles and yield of positive particles before and after lyophilization of exosomes.

[0039] Table 8 Exosome characteristics of different formulations before and after freeze-drying From the results in Table 8, it can be seen that compared with Comparative Examples 1-3, the amount of each component of the lyophilization protectant in the formula of E-2 is the smallest and the protection effect on the positive particles of exosomes during the lyophilization process is the best. In addition, compared with Comparative Example 1, the compound electrolyte injection solution used in E-2 as a solvent has an advantage in reducing the loss of exosomes during the lyophilization process, while ensuring that the particle size distribution does not change significantly.

[0040] Application Example 1: Repair of wound surface by freeze-dried exosome preparation 1. Animal Modeling The mice were injected with sodium pentobarbital for intraperitoneal anesthesia. After anesthesia, the hair on the back of the rats was shaved, the surrounding skin was disinfected, and the rats were fixed in a prone position. A 1 cm × 1 cm square full-thickness skin defect was prepared on the back of the rats until the muscle layer was exposed. The wound surface of each mouse was photographed and the corresponding ear number was recorded to obtain a skin trauma mouse model.

[0041] 2. Experimental Grouping and Treatment The mouse models were randomly divided into four groups: Group 1: control group, without any treatment; Group 2: Fresh exosome suspension was sprinkled on the skin wound on the back of mice; Group 3: Control group exosome freeze-dried powder, reconstituted with PBS and then sprinkled on the back skin wound of mice; Group 4: The exosome freeze-dried powder obtained from group E-2 was reconstituted with compound electrolytes and then sprinkled on the back skin wound of mice; A layer of vaseline gauze was covered on the wound surface of the back skin of each group of rats, and after pressing with a cotton ball, a medical self-adhesive bandage was wrapped for one week. After the animals recovered, they were returned to the cage for continued feeding and fed with double-antibiotic water after surgery. The mice were changed every three days according to the above method. Regular observation was carried out to prevent the bandage from falling off or being bitten or torn off by the mice. Photos were taken on the 0th day, the 7th day, and the 14th day.

[0042] The results are as follows Figure 3 As shown, the exosome freeze-dried powder obtained from the E-2 group freeze-dried protectant showed good wound repair effect, which was equivalent to that of fresh exosome suspension.

[0043] Example 3 Preparation of lyophilized exosome preparation Different lyoprotectant solutions were prepared according to the formula in Table 9, and then mixed with the exosome suspension of adipose-derived mesenchymal stem cells isolated from the same batch. The final volume of each group was 1 mL, and lyophilized according to the procedure in Table 4.

[0044] Table 9 Formulas of different lyophilized exosome preparations The lyophilized preparations obtained with different formulas were immediately reconstituted with 1 mL of the original solvent, and the lyophilization protection effects of each group of lyoprotectants were compared by detecting and calculating the particle concentration, particle size distribution, number of positive particles and yield of positive particles before and after lyophilization of exosomes.

[0045] Table 10 Exosome characteristics of different formulations before and after freeze-drying From the results in Table 10, it can be seen that the particle concentration of the freeze-dried preparations obtained by the three groups of freeze-dried protective agents after reconstitution was lower than that before freeze-drying, but within a reasonable range of variation, without causing serious loss of the number of positive particles, and the particle size distribution did not change significantly. The compound electrolyte injection, Tris buffer and PB buffer can show good stability under freezing conditions, thereby effectively maintaining the stability of the solvent pH during the freeze-drying process of exosomes, thereby ensuring the integrity and biological activity of the freeze-dried exosomes.

[0046] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. An exosome freeze-dried protection solution, characterized in that: The invention comprises the following contents of freeze-drying protective agents: 2%-4% trehalose, 4%-6% mannitol and 0.01%-0.02% poloxamer 188.

2. The exosome freeze-dried protective solution according to claim 1, characterized in that: The buffer of the exosome freeze-dried protection solution is a compound electrolyte injection solution, a Tris buffer or a phosphate buffer.

3. The exosome freeze-dried protective solution according to claim 2, characterized in that: The pH of the buffer solution is 7.0-7.6; the concentration of the Tris buffer solution is 15mM-20mM; and the concentration of the phosphate buffer solution is 10mM-20mM.

4. The exosome freeze-dried protective solution according to claim 2, characterized in that: The pH of the buffer solution is 7.2-7.4; the Tris buffer solution is Tris-HCl buffer solution.

5. Use of the exosome freeze-dried protection solution according to any one of claims 1 to 4 in the preparation of an exosome freeze-dried preparation.

6. A method for preparing a lyophilized exosome preparation, characterized in that: The following steps are involved: (1) mixing the exosomes with an appropriate solvent, and then adding trehalose, mannitol, and poloxamer 188 to a concentration of 2%-4%, 4%-8%, and 0.01%-0.02%, respectively, to obtain a lyophilized exosome solution; (2) The exosome freeze-dried material liquid is freeze-dried according to the steps of pre-freezing-freeze drying-sublimation drying-freeze drying-analytical drying to obtain an exosome freeze-dried preparation.

7. The preparation method according to claim 6, characterized in that: In step (2), the pre-freezing temperature is -35°C, the first freeze-drying temperature is -40°C~-45°C, and the time is 10h-15h; the sublimation drying temperature is -15°C~-18°C, and the time is 3.5h-5h; the second freeze-drying temperature is 2°C~8°C, and the time is 2h-5h; the analytical drying temperature is 35°C~40°C, and the time is 2h-10h.

8. A lyophilized exosome preparation obtained according to the preparation method of claim 6 or 7.

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