Freeze-drying protective agent for mesenchymal stem cell exosome and use thereof

Abstract

The present invention belongs to the technical field of exosome freeze-drying protection. Disclosed is a freeze-drying protective agent for a mesenchymal stem cell exosome. The freeze-drying protective agent consists of 0.1-0.2 v / v% of ceramide, lysine and 0.8-1.5 v / v% of glycerol, wherein after the exosome freeze-drying protective agent and an exosome are mixed, the final concentration of lysine is 50-80 mM. The freeze-drying protective agent can maintain the morphology integrity of the exosome, avoid exosome aggregation, and can well maintain the biological functions of the exosome, and therefore can be applied to exosome freeze-drying technology, and has wide application value.

Description

A mesenchymal stem cell exosome freeze-dried protective agent and its application Technical Field

[0001] The present invention belongs to the technical field of exosome freeze-drying protection, and more specifically, relates to a mesenchymal stem cell exosome freeze-drying protectant and its application. Background Art

[0002] Exosomes are small membrane vesicles (30-150 nm) containing complex RNA and proteins. Today, they specifically refer to disc-shaped vesicles with a diameter of 40-100 nm. They were first discovered in sheep reticulocytes in 1983 and named "exosomes" by Johnstone in 1987. Exosomes are secreted by a variety of cells under both normal and pathological conditions. They primarily originate from multivesicular bodies formed by the invagination of intracellular lysosomal microparticles. These vesicles are released into the extracellular matrix after the outer membrane of the multivesicular body fuses with the cell membrane. All cultured cell types can secrete exosomes, and exosomes are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. Extensive research on their biological origin, composition and transport, intercellular signaling, and distribution in body fluids has revealed that exosomes possess diverse functions. The functions of exosomes depend on the cell type from which they originate, and they can participate in various aspects of the immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion.

[0003] The storage of exosomes is crucial. Without qualified storage techniques, long-term storage is impossible. Existing studies have found that -80°C is the optimal temperature for long-term storage, effectively inhibiting the loss of bioactive proteins (including exosomes) at this temperature. However, under these storage conditions, the properties of exosomes still change to some extent, and stable transport and storage of exosomes is difficult to achieve at this temperature. Lyophilization is commonly used to preserve bioactive compounds and is now being used for the storage of exosomes. Exosome lyoprotectants reported include trehalose, mannitol, and other sugars, alcohols, and combinations of other substances. However, various studies have found that the lyoprotectants used for specific exosome types vary widely, and known lyoprotectants do not adequately preserve the morphological integrity of exosomes, significantly impacting their biological functions.

[0004] Exosomes, derived from the endocrine system of mesenchymal stem cells, are rich in over 100 extracellular matrix proteins, mRNA, and multiple growth factors. Clinically, stem cell exosomes are primarily used to treat burns, scalds, and skin ulcers, and to regenerate healthy skin. In skincare, they primarily repair damaged skin, such as those caused by trauma or aging, comprehensively conditioning and improving skin texture, restoring a youthful, healthy state. Mesenchymal stem cell exosomes are currently a hot topic in dermatology research. However, existing technologies lack a lyoprotectant specifically for mesenchymal stem cell exosomes. Technical issues

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned problems existing in the prior art. First, a freeze-dried protective agent for mesenchymal stem cell exosomes is provided.

[0006] The second object of the present invention is to provide the use of the above-mentioned lyoprotectant.

[0007] The third object of the present invention is to provide a freeze-dried preparation of mesenchymal stem cell exosomes. Technical Solutions

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A mesenchymal stem cell exosome lyoprotectant is composed of 0.1-0.2 v / v % ceramide, lysine and 0.8-1.5 v / v % glycerol, wherein after the exosome lyoprotectant and exosomes are mixed, the final concentration of lysine is 50-80 mM.

[0010] Preferably, the mesenchymal stem cell exosome lyoprotectant consists of 0.18 v / v% ceramide, lysine and 1.3 v / v% glycerol, wherein after the exosome lyoprotectant and exosomes are mixed, the final concentration of lysine is 70 mM.

[0011] The present invention also protects the use of the above-mentioned lyoprotectant in the preparation of lyophilized exosome preparations.

[0012] The present invention also protects the use of the above-mentioned lyoprotectant in improving the morphology of exosomes.

[0013] Preferably, improving the morphology of exosomes refers to increasing the dispersion of exosomes and / or reducing exosome membrane fusion.

[0014] The present invention also provides a mesenchymal stem cell exosome freeze-dried preparation containing the above-mentioned mesenchymal stem cell exosome freeze-dried protective agent. Beneficial effects

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a mesenchymal stem cell exosome lyoprotectant, which is composed of 0.1-0.2 v / v% ceramide, lysine and 0.8-1.5 v / v% glycerol. After the exosome lyoprotectant and exosomes are mixed, the final concentration of lysine is 50-80 mM. The lyoprotectant can maintain the morphological integrity of exosomes, prevent exosome aggregation, and well maintain the biological function of exosomes. It can be used in exosome freeze-drying technology and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is an electron microscopic image of exosomes; the scale bar on the left is 200 nm, and the scale bar on the right is 600 nm;

[0018] Figure 2 is an analysis of exosome particle size;

[0019] Figure 3 shows the wound repair experiment of exosomes. Best Mode for Carrying Out the Invention

[0020] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0021] 1. Preparation of Mesenchymal Stem Cell Exosomes

[0022] The operation is as follows:

[0023] (1) Collect 10 8 The supernatant of mesenchymal stem cells was centrifuged at 3000g and the supernatant was collected;

[0024] (2) Take the supernatant from (1), centrifuge at 3000 g to remove cell debris, and collect the supernatant;

[0025] (3) Take the supernatant from (2), centrifuge at 10,000 g to remove protein, collect the supernatant, and filter the supernatant with a 0.22 μm filter membrane;

[0026] (4) Take the supernatant after filtration in (3) and ultracentrifuge at 100,000 g for 1.5 h. Discard the supernatant, resuspend the precipitate with PBS, and collect the resuspended liquid.

[0027] (5) The resuspension in (4) was taken to detect the exosome content of mesenchymal stem cell exosomes. The electron microscopy was shown in Figure 1, and the NTA particle size analysis was shown in Figure 2. The exosome protein content was determined by the BCA method to be 0.28 mg / ml.

[0028] 2. Mixing of exosomes and lyoprotectants

[0029] 2 ml of the exosomes prepared above were mixed with a protective agent to obtain a freeze-dried exosome mixture. The protective agent consisted of ceramide, lysine, and glycerol. The specific composition of the protective agent in the freeze-dried exosome mixture for each example and comparative example is shown in Table 1.

[0030]

[0031] 3. Freeze-drying procedure

[0032] The exosome mixture to be freeze-dried was placed in liquid nitrogen and quickly frozen at -80°C overnight, and then vacuum-freeze-dried using a freeze dryer. The freeze-drying conditions were: cold trap temperature -50 to -55°C, vacuum pressure 5 to 10 Pa, and treatment for 24 hours.

[0033] After freeze-drying, the product is in powder form and stored sealed at 4°C.

[0034] 4. Effects of Lyoprotectants on Exosomes

[0035] 4.1. Morphology of exosomes

[0036] Redissolve the freeze-dried exosomes by adding pre-chilled PBS solution and vortexing, and observe the morphology of the reconstituted exosomes.

[0037]

[0038] Table 2 lists the changes in exosome morphology in each group. As can be seen from Table 2, in the absence of a lyoprotectant, the exosomes in the blank control group aggregated in large quantities, had poor dispersion, destroyed the exosome membrane structure, and produced a large amount of membrane fusion. The amount of lysine in Comparative Example 1 was small, while the amount of lysine in Comparative Example 2 was large. From the results of Comparative Examples 1 and 2, it can be seen that lysine at a certain concentration plays a major role in protecting the integrity of the exosome morphology. Comparative Example 3 lacks ceramide, which is mainly combined with lysine to assist lysine in further reducing the aggregation and membrane fusion of exosomes. Glycerol in the lyoprotectant of the present invention plays a role in preventing lattice formation, thereby avoiding large-scale damage to the exosome membrane structure. Comparative Example 4 lacks glycerol. Therefore, compared with exosome aggregation, the phenomenon of exosome membrane structure fusion is more obvious.

[0039] 4.2 Biological Functions of Exosomes

[0040] A. Anti-inflammatory effect

[0041] 1. Experimental animals: male SPF 6-week-old mice.

[0042] 2. Experimental Design: Forty healthy mice with intact skin were selected and divided into 10 groups. After conventional anesthesia, the abdominal hair was removed, and a 2 cm × 2 cm area was removed. The reconstituted exosomes from each group listed in Table 1 were then applied to the depilated skin. After 24 hours, the skin where the exosomes were applied was washed with warm water. The skin of the mice that had been treated with exosomes in the above experiments was removed and homogenized for the detection of immune-inflammatory factors. This detection was performed using existing kits. The results are shown in Table 3.

[0043]

[0044] As can be seen from Table 3, in the absence of a lyoprotectant, the blank control group had a higher inflammatory factor value; the amount of lysine in Comparative Example 1 was less, while the amount of lysine in Comparative Example 2 was more. From the results of Comparative Examples 1 and 2, it can be seen that lysine at a certain concentration can further reduce the inflammatory response; Comparative Example 3 lacked ceramide, and its inflammatory factor value was higher, which also indirectly shows that the addition of ceramide played a role in reducing the inflammatory response; Comparative Example 4 lacked glycerol, and its effect on inflammatory factors was less than that of Comparative Example 3.

[0045] Therefore, the lyoprotectant of the present invention can not only protect the integrity of the morphology of exosomes, but also further reduce the inflammatory response and the content of inflammatory factors, and also assist in playing an anti-inflammatory role.

[0046] B. Wound repair

[0047] To investigate the wound repair ability of the reconstituted exosomes described in this example, mice were exposed to chemical stimulation. DNFB (dinitrofluorobenzene) is a compound commonly used in laboratory research to induce skin allergic reactions and can cause chemical damage to the skin. BALB / c mice were randomly divided into a DNFB positive control (Model), a DNFB plus exosomes (Gel), and a control (Normal) group, with six mice in each group. The mice were provided with a standard diet and water and maintained under standard conditions to acclimate to the experimental environment. DNFB was prepared with carbomer into a gel formulation at an appropriate dose (0.25-0.5%). DNFB was applied to the back of the mice once on days 1, 4, 7, and 10 of the experiment. The exosome group received the drug twice daily starting on day 5.

[0048] The results showed that on the 7th day, the skin lesion area of ​​the Gel group was significantly larger than that of the positive control group. On the 15th day, the wound in the Gel group was basically completely healed, while the positive control group still had a significantly larger wound area. This shows that the application of the reconstituted exosomes of Example 3 of the present invention still has a significant wound healing effect, showing a stable biological function.

[0049] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A mesenchymal stem cell exosome freeze-dried protective agent, characterized in that: It is composed of 0.1-0.2 v / v % ceramide, lysine and 0.8-1.5 v / v % glycerol, wherein after the exosome lyoprotectant and the exosomes are mixed, the final concentration of lysine is 50-80 mM.

2. The mesenchymal stem cell exosome freeze-dried protective agent according to claim 1, characterized in that It is composed of 0.18 v / v % ceramide, lysine and 1.3 v / v % glycerol, wherein the final concentration of lysine is 70 mM after the exosome lyoprotectant and exosomes are mixed.

3. Use of the lyoprotectant according to claim 1 or 2 in the preparation of a lyophilized exosome preparation.

4. Use of the lyoprotectant according to claim 1 or 2 in improving the morphology of exosomes.

5. The use according to claim 4, characterized in that: Improving the exosome morphology refers to increasing the degree of exosome dispersion and / or reducing exosome membrane fusion.

6. A mesenchymal stem cell exosome freeze-dried preparation, characterized in that: Contains the mesenchymal stem cell exosome freeze-dried protective agent according to claim 1 or 2.

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