Preparation method of exosome freeze-dried powder

Abstract

The invention discloses a preparation method of exosome freeze-dried powder, and belongs to the technical field of preparation of exosome freeze-dried powder. The freeze-drying protective agent comprises active components and a solution for dissolving the active components, the active components comprise trehalose, polyvinylpyrrolidone, compound amino acid, taurine and hydroxyethyl starch, and the solution is a PBS solution. When the exosome freeze-dried powder is prepared, the unfrozen exosome stock solution and the freeze-drying protective agent are mixed, then the mixed solution is placed in a freeze-drying machine, and freeze-drying is carried out under a set freezing procedure. By adopting the cryoprotectant and the freeze-drying method, the exosome can be effectively protected from being damaged by external force in the freeze-drying process, and the biological activity of the exosome can be effectively guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of lyophilized exosome powder, and particularly relates to a method for preparing lyophilized exosome powder. Background Art

[0002] Exosomes are tiny vesicles that can be secreted by most cells in the body. They have a lipid bilayer membrane and a diameter of about 30~150nm. They are physiologically active substances secreted by cells during physiological activities. The main effective ingredients include protein substances and miRNA nucleic acid substances. Exosomes are widely present and distributed in various body fluids, carrying and transmitting important signal molecules, forming a new cell-to-cell information transmission system, affecting the physiological state of cells, and widely participating in various biological processes such as immune response, antigen presentation, and cell differentiation. Each cell is a factory for producing and transmitting molecules, which are transmitted to the required parts of the body at a specific time through the vesicles around the cells. Exosomes can bind to the cell membrane, directly enter the cell to release the signals they carry to affect the receptor cells, and at the same time improve the microenvironment around the cells, playing a role in treating and repairing cell damage in the form of vesicles. Therefore, at this stage, exosomes are also used in the healing of wounds such as diabetic foot, with advantages that other ingredients cannot match, such as no immunogenicity, rejection reaction, good tolerance, and high effectiveness.

[0003] The selection of lyoprotectants requires many strategies. Currently, the most commonly used ingredient in lyoprotectants is DMSO, but DMSO is highly toxic to proteins. Lyoprotectants are divided into permeable and non-permeable protectants, and their theories are also different. Trehalose and polyvinylpyrrolidone (PVP) are currently the most popular lyoprotectant ingredients. They are essentially non-permeable protectants that can reduce the formation of ice crystals during the freeze-drying process. Trehalose has a high glass transition temperature (Tg), and the freeze-dried sample can still maintain a glassy state for a long time at room temperature. In addition, trehalose, as a reducing disaccharide, can bind to cell membranes or other macromolecules in the body through hydrogen bonds, greatly improving the tolerance of mesenchymal stem cell exosomes to processes such as drying and dehydration, and avoiding damage to the outer membrane of the exosomes. Polyvinylpyrrolidone (PVP) is a non-ionic polymer compound that is widely used in medicine, industry, etc. and has the highest degree of research. Similar to trehalose, PVP also has a high Tg. Combined with its unique advantages, it can prevent the leakage of freeze-dried contents and the damage of the outer membrane during hydration and is widely used in the freeze-drying process. The preparation of mesenchymal stem cell freeze-dried powder uses trehalose, mannitol, PVP40, etc. as intracellular and extracellular protective agents. At low temperatures, intracellular proteins and biofilms can withstand drying without being destroyed, and their activity can still be restored after rehydration.

[0004] The shelf life of currently stored exosomes is relatively short, and the storage conditions are relatively harsh. Generally, they can only be stored for about half a year under -80°C freezing conditions, which greatly limits the application of exosomes. Therefore, how to conveniently store exosomes and maintain their biological activity and stability is an urgent problem to be solved. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for preparing exosome freeze-dried powder to obtain a freeze-dried powder with excellent physical and chemical properties, good appearance, good resolubility, no degradation of active factor effective substances and good cosmetic effect.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing lyophilized exosome powder, comprising the following steps: S1: thawing the exosome stock solution, and then mixing the exosomes with a lyophilization protective agent to obtain a mixed solution; the lyophilization protective agent includes an active component and a solution for dissolving the active component; the active component includes trehalose, polyvinyl pyrrolidone, compound amino acids, taurine and hydroxyethyl starch, and the solution is a PBS solution; S2: Place the mixed solution into a freeze dryer and freeze dry it according to the following conditions to obtain;

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, each 1 mL of the mixture contains 1×10 9 ~5×10 9 Exosomes.

[0009] Furthermore, the pre-freezing to freeze-thaw cycle stage in S2 is repeated twice before entering the freeze-drying stage.

[0010] Furthermore, the mass ratio of trehalose, polyvinyl pyrrolidone, compound amino acid, taurine and hydroxyethyl starch is 180-220:450-550:3-8:3-8:80-120.

[0011] Furthermore, the mass ratio of trehalose, polyvinyl pyrrolidone, compound amino acid, taurine and hydroxyethyl starch is 200:500:5:5:100.

[0012] Furthermore, the concentration of the active component in the solution is 0.20-0.21 g / mL.

[0013] Furthermore, the polyvinyl pyrrolidone is polyvinyl pyrrolidone-40; and the compound amino acid is 18AA.

[0014] Further, the lyophilized protective agent is prepared by the following steps: Dissolve the active components in the solution, mix well and store at 4°C to obtain the product.

[0015] The beneficial effects of the present invention are: 1. When using the lyoprotectant in the present invention to prepare the lyophilized exosome powder, the exosomes are first pre-frozen by pre-freezing, annealing, freeze-thaw cycles and other treatments; wherein, pre-freezing can quickly reduce the temperature of the exosomes and reduce the activity of the water molecules inside them, thereby slowing down or preventing the degradation of the bioactive substances; and pre-freezing can reduce the formation of ice crystals in the exosomes and avoid the damage of the ice crystals to the exosome structure. Annealing can optimize the structure of the ice crystals, making them more uniform and fine, thereby reducing the damage to the exosome structure. At the same time, annealing can change the morphology and size distribution of the ice crystals, making them easier to sublimate during the drying process, which helps to shorten the drying time and improve the drying efficiency.

[0016] 2. The lyoprotectants in this application include trehalose and polyvinyl pyrrolidone (PVP). Trehalose has excellent lyoprotective effects, can prevent ice crystal formation, increase glass transition temperature, reduce hygroscopicity, and has good chemical stability and biocompatibility. It can form a protective film on the cell surface to prevent biomolecules from being damaged during the lyophilization process. Polyvinyl pyrrolidone, as a polymer compound, can form a protective film covering the surface of biomolecules during the lyophilization process. The protective film can maintain the shape and integrity of biomolecules, prevent them from deformation or rupture during the lyophilization process, thereby avoiding denaturation or inactivation of biomolecules.

[0017] 3. Compound amino acids are also added to the freeze-drying protective agent. The amino acid components in the compound amino acids can inhibit the pH changes caused by the crystallization of phosphate buffered saline (PBS solution) during the freeze-drying process, thereby preventing protein denaturation; and the compound amino acids and PVP can play a synergistic role in the freeze-drying process. PVP can form a protective film to prevent the denaturation of biological molecules, while the compound amino acids can provide the necessary nutritional support and osmotic pressure regulation, which helps to maintain the stability of the structure and function of biological molecules.

[0018] 4. Taurine and hydroxyethyl starch are also added to the freeze-drying protective agent. Taurine has a stabilizing effect on the cell membrane, which can reduce the cell membrane damage caused by water evaporation and temperature reduction during the freeze-drying process, thereby ensuring the biological activity of biological molecules; in addition, taurine has antioxidant properties, which can eliminate the oxidative free radicals accumulated during the freeze-drying process, thereby reducing the damage to cells caused by oxidative stress, thereby extending the shelf life and ensuring the activity of biological molecules. Hydroxyethyl starch is a non-permeable cryoprotectant that can form a protective film on the outside of the cell. This protective film can reduce the formation and growth of ice crystals, thereby preventing the cell from rupturing due to mechanical damage of ice crystals during the freezing process; and the protective film formed can prevent external harmful substances from entering the cell, thereby protecting the biological molecules from damage by these harmful substances, thereby ensuring the activity of the biological molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of lyophilized exosome powder on the proliferation of HSFs; Figure 2 This is the result of the scratch test of lyophilized exosome powder. DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0021] Example 1 A freeze-dried protective agent comprises an active component and a solution for dissolving the active component; wherein the active component comprises trehalose powder, polyvinylpyrrolidone-40 (PVP40), compound amino acid 18AA, taurine and hydroxyethyl starch, and the solution is a 0.6×PBS solution.

[0022] The lyophilized protective agent in this embodiment is prepared by the following steps: Under sterile conditions, 200 mg of trehalose powder, 500 mg of PVP40 powder, 5 mg of compound amino acid 18AA, 5 mg of taurine and 100 mg of hydroxyethyl starch were weighed on an electronic balance, and these materials were dissolved in 4 mL of 00.6×PBS solution, stirred and mixed, and stored at 4°C.

[0023] Example 2 A freeze-dried protective agent comprises an active component and a solution for dissolving the active component; wherein the active component comprises trehalose powder, polyvinylpyrrolidone-40 (PVP40), compound amino acid 18AA, taurine and hydroxyethyl starch, and the solution is a 0.6×PBS solution.

[0024] The lyophilized protective agent in this embodiment is prepared by the following steps: Under sterile conditions, 180 mg of trehalose powder, 550 mg of PVP40 powder, 3 mg of compound amino acid 18AA, 8 mg of taurine and 80 mg of hydroxyethyl starch were weighed on an electronic balance, and these materials were dissolved in 4 mL of 00.6×PBS solution, stirred and mixed, and stored at 4°C.

[0025] Example 3 A freeze-dried protective agent comprises an active component and a solution for dissolving the active component; wherein the active component comprises trehalose powder, polyvinylpyrrolidone-40 (PVP40), compound amino acid 18AA, taurine and hydroxyethyl starch, and the solution is a 0.6×PBS solution.

[0026] The lyophilized protective agent in this embodiment is prepared by the following steps: Under sterile conditions, 220 mg of trehalose powder, 450 mg of PVP40 powder, 8 mg of compound amino acid 18AA, 3 mg of taurine and 120 mg of hydroxyethyl starch were weighed on an electronic balance, and these materials were dissolved in 4 mL of 00.6×PBS solution, stirred and mixed, and stored at 4°C.

[0027] Experimental Example 1 A method for preparing lyophilized exosome powder comprises the following steps: S1: Thaw the exosome stock solution at 4°C, then add the lyophilization protective agent prepared in Example 1 to the thawed exosomes to obtain a mixed solution. Each 1 mL of the mixed solution contains about 1×10 9 exosomes; S2: Take 3 mL of the mixed solution, put it into a freeze-dried bottle, seal the freeze-dried bottle with a sealing film, and freeze it at -80°C overnight; S3: putting the mixed solution treated in S2 into a freeze dryer, setting the freezing parameters according to the freezing conditions in the table below, and freeze drying the mixed solution;

[0028] The cold trap temperature of the freeze dryer is -50°C and the absolute pressure is 6.3 Pa; the pre-freezing end section is 1-4, and the final end section is 9. After freezing, the sample is in powder form. After the sealing film is removed, it is placed in a dryer and sealed at 4°C to prevent the exosome freeze-dried powder from absorbing moisture and light.

[0029] Experimental Example 2 A method for preparing lyophilized exosome powder comprises the following steps: S1: Thaw the exosome stock solution at 4°C, then add the lyophilization protective agent prepared in Example 1 to the thawed exosomes to obtain a mixed solution, wherein each 1 mL of the mixed solution contains about 5×10 9 exosomes; S2: Take 3 mL of the mixed solution, put it into a freeze-dried bottle, seal the freeze-dried bottle with a sealing film, and freeze it at -80°C overnight; S3: putting the mixed solution treated in S2 into a freeze dryer, setting the freezing parameters according to the freezing conditions in the table below, and freeze drying the mixed solution;

[0030] The cold trap temperature of the freeze dryer is -50°C and the absolute pressure is 6.3 Pa; the pre-freezing end section is 1-4, and the final end section is 9. After freezing, the sample is in powder form. After the sealing film is removed, it is placed in a dryer and sealed at 4°C to prevent the exosome freeze-dried powder from absorbing moisture and light.

[0031] Results Analysis The HSFs proliferation experiment was used to detect the effect of the exosome freeze-dried powder prepared in the above experimental example on the proliferation of HSFs. The results are as follows Figure 1 As shown in the figure, the number of particles is 1×10 9 (Experimental Example 1), 5×10 9 The exosome freeze-dried powder of (Experimental Example 2) was used to treat the normally cultured HSFs. On the first day, there was no statistical difference between the control group, Experimental Example 1, and Experimental Example 2 (P>0.05). On the third and fifth days, compared with the control group, both Experimental Example 1 and Experimental Example 2 could promote the proliferation of HSFs in vitro (P<0.01), and on the fifth day, there was a significant difference between Experimental Example 1 and Experimental Example 2 (P<0.05).

[0032] Under sterile conditions, take a 6-well plate and mark the position and number with a marker pen, and draw 5 vertical lines evenly on the bottom of each well. 5 The cells were inoculated at a density of 1 mL / well in a 6-well plate, with each well containing 1 mL of culture medium, and cultured until the cell confluence reached 90%; then the medium was replaced with serum-free medium and cultured for 12 hours. A 200 μl pipette tip was used to draw a straight line from top to bottom in the middle of each well perpendicular to the marked line to scratch the cells. The observation time was set at 0 h, 8 h, and 16 h. The same part was photographed under an inverted microscope to record the migration of cells on both sides of the scratch.

[0033] The experiment was divided into groups according to the protein content of the lyophilized exosomes. The results of the scratch test were as follows: Figure 2 As shown in the figure, after incubation for 8h and 16h, compared with the control group, both Experimental Example 1 and Experimental Example 2 can promote the migration of HSFs (P<0.05), promote the movement of HSFs to both sides of the scratch, and the migration promotion rate of exosome freeze-dried powder is concentration-dependent (P<0.05).

[0034] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing lyophilized exosome powder, characterized in that: The following steps are involved: S1: thawing the exosome stock solution, and then mixing the exosomes with a lyophilization protective agent to obtain a mixed solution; the lyophilization protective agent includes an active component and a solution for dissolving the active component; the active component includes trehalose, polyvinyl pyrrolidone, compound amino acids, taurine and hydroxyethyl starch, and the solution is a PBS solution; S2: Place the mixed solution into a freeze dryer and freeze dry it according to the following conditions to obtain; 2. The preparation method according to claim 1, characterized in that: Each 1 mL of the mixture contains 1×10 9 ~5×10 9 Exosomes.

3. The preparation method according to claim 1, characterized in that: The pre-freezing to freeze-thaw cycle stage in S2 is repeated twice before entering the freeze-drying stage.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the trehalose, polyvinyl pyrrolidone, compound amino acid, taurine and hydroxyethyl starch is 180-220:450-550:3-8:3-8:80-120.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the trehalose, polyvinyl pyrrolidone, compound amino acid, taurine and hydroxyethyl starch is 200:500:5:5:

100.

6. The preparation method according to claim 5, characterized in that: The concentration of the active component in the solution is 0.20-0.21 g / mL.

7. The preparation method according to claim 4 or 5, characterized in that: The polyvinyl pyrrolidone is polyvinyl pyrrolidone-40; the compound amino acid is 18AA.

8. The preparation method according to claim 1, characterized in that: The lyophilized protective agent is prepared by the following steps: Dissolve the active components in the solution, mix well and store at 4°C to obtain the product.

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