A method for preparing lyophilized exosome powder

Abstract

The present invention discloses a method for preparing lyophilized exosome powder, which belongs to the technical field of preparation of lyophilized exosome powder. The lyophilization protectant in the present invention includes an active component and a solution for dissolving the active component, wherein the active component includes trehalose, polyvinyl pyrrolidone, compound amino acids, taurine and hydroxyethyl starch, and the solution is a PBS solution. When preparing lyophilized exosome powder, the thawed exosome stock solution is mixed with the lyophilization protectant, and then the mixture is placed in a freeze dryer and freeze-dried under a set freezing program. The use of the cryoprotectant and freeze-drying method in this application can effectively protect the exosomes from external damage during the freeze-drying process, and can effectively ensure the biological activity of the exosomes.

Description

Technical Field

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

[0002] Exosomes are tiny vesicles secreted by most cells in the body. They have a lipid bilayer membrane and are approximately 30-150 nm in diameter. They are bioactive substances secreted by cells during physiological activities. Their main active ingredients include proteins and miRNAs. Exosomes are widely present and distributed in various body fluids. They carry and transmit important signaling molecules, forming a novel cell-to-cell communication system, influencing cellular physiological states and participating in various biological processes such as immune response, antigen presentation, and cell differentiation. Each cell is a factory for the production and delivery of molecules, which are delivered to the desired parts of the body at specific times through vesicles surrounding the cell. Exosomes can bind to cell membranes, directly enter the cell, release the signals they carry, affect recipient cells, and improve the surrounding microenvironment. In their vesicular form, they play a therapeutic and repairing role in cellular damage. Therefore, exosomes are currently being used in wound healing, such as diabetic foot ulcers, offering unmatched advantages over other ingredients, including non-immunogenicity, rejection, good tolerability, and high efficacy.

[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 exosome outer membrane. Polyvinylpyrrolidone (PVP) is a highly researched non-ionic polymer widely used in medicine and industry. Similar to trehalose, PVP also has a high Tg. Combined with its unique advantages, PVP can prevent leakage of freeze-dried contents and damage to the outer membrane during hydration, making it widely used in freeze-drying processes. Mesenchymal stem cell freeze-dried powder is prepared using trehalose, mannitol, and PVP40 as intracellular and extracellular protective agents. At low temperatures, intracellular proteins and biofilms withstand drying without damage, and their activity is restored upon rehydration.

[0004] Currently, exosomes have a short shelf life and require stringent storage conditions. Typically, they can only be stored at -80°C for about six months, significantly limiting their potential applications. Therefore, finding a way to conveniently preserve exosomes while maintaining their biological activity and stability is an urgent challenge. Summary of the Invention

[0005] In response to 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 solubility, no degradation of active factors and effective substances, and good cosmetic effects.

[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:

[0007] 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;

[0008] S2: Place the mixed solution into a freeze dryer and freeze-dry it according to the following conditions to obtain;

[0009]

[0010] On the basis of the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, each 1 mL of the mixture contains 1×10 9 ~5×10 9 Exosomes.

[0012] Furthermore, the pre-freezing to freeze-thaw cycle stage in S2 is repeated twice before entering the freeze-drying stage.

[0013] 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.

[0014] Furthermore, the mass ratio of trehalose, polyvinyl pyrrolidone, compound amino acid, taurine and hydroxyethyl starch is 200:500:5:5:100.

[0015] Furthermore, the concentration of the active component in the solution is 0.20-0.21 g / mL.

[0016] Furthermore, the polyvinyl pyrrolidone is polyvinyl pyrrolidone-40; and the compound amino acid is 18AA.

[0017] Further, the freeze-dried protective agent is prepared by the following steps:

[0018] Dissolve the active ingredients in the solution, mix well and store at 4°C to obtain the product.

[0019] The beneficial effects of the present invention are:

[0020] 1. When using the lyoprotectant of the present invention to prepare lyophilized exosome powder, the exosomes are first pre-frozen through pre-freezing, annealing, and freeze-thaw cycles. Pre-freezing can rapidly lower the temperature of the exosomes, reducing the activity of water molecules within them, thereby slowing or preventing the degradation of bioactive substances. Furthermore, pre-freezing can reduce the formation of ice crystals within the exosomes, preventing ice crystal damage to the exosome structure. Annealing can optimize the structure of the ice crystals, making them more uniform and smaller, thereby reducing damage to the exosome structure. Furthermore, annealing can change the morphology and size distribution of the ice crystals, making them more likely to sublime during the drying process, which helps shorten drying time and improve drying efficiency.

[0021] 2. The lyoprotectants in this application include trehalose and polyvinylpyrrolidone (PVP). Trehalose has excellent lyoprotective effects, preventing ice crystal formation, increasing the glass transition temperature, reducing hygroscopicity, and exhibiting good chemical stability and biocompatibility. It can form a protective film on the cell surface, preventing biomolecules from being damaged during the lyophilization process. Polyvinylpyrrolidone, as a polymer compound, can form a protective film covering the surface of biomolecules during the lyophilization process. This protective film can maintain the shape and integrity of biomolecules, preventing them from deforming or rupturing during the lyophilization process, thereby preventing denaturation or inactivation of the biomolecules.

[0022] 3. Compound amino acids are also added to the freeze-drying protectant. 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; moreover, 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.

[0023] 4. Taurine and hydroxyethyl starch are also added to the freeze-drying protective agent. Taurine has a stabilizing effect on cell membranes, which can reduce cell membrane damage caused by water evaporation and temperature reduction during the freeze-drying process, thereby ensuring the biological activity of biomolecules. In addition, taurine has antioxidant properties and can eliminate 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 biomolecules. Hydroxyethyl starch is a non-permeable cryoprotectant that can form a protective film on the outside of cells. This protective film can reduce the formation and growth of ice crystals, thereby preventing cells from rupturing due to mechanical damage from ice crystals during the freezing process. In addition, the protective film formed can prevent external harmful substances from entering the cells, thereby protecting biomolecules from damage by these harmful substances and ensuring the activity of biomolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of lyophilized exosome powder on the proliferation of HSFs;

[0025] Figure 2 The figure shows the results of the scratch test of lyophilized exosome powder. DETAILED DESCRIPTION

[0026] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0027] Example 1

[0028] 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.

[0029] The lyoprotectant in this embodiment was prepared by the following steps:

[0030] 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 0.6× PBS solution, stirred and mixed, and stored at 4°C.

[0031] Example 2

[0032] 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.

[0033] The lyoprotectant in this embodiment was prepared by the following steps:

[0034] 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. These materials were dissolved in 4 mL of 0.6× PBS solution, stirred and mixed, and stored at 4°C.

[0035] Example 3

[0036] 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.

[0037] The lyoprotectant in this embodiment was prepared by the following steps:

[0038] 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. These materials were dissolved in 4 mL of 0.6× PBS solution, stirred and mixed, and stored at 4°C.

[0039] Experimental Example 1

[0040] A method for preparing lyophilized exosome powder comprises the following steps:

[0041] 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;

[0042] S2: Take 3 mL of the mixed solution, put it into a freeze-dried bottle, seal the freeze-dried bottle with sealing film, and freeze it at -80°C overnight;

[0043] S3: Place the mixed solution treated in S2 into a freeze dryer, set the freezing parameters according to the freezing conditions in the table below, and freeze-dry the mixed solution;

[0044]

[0045] The freeze dryer's cold trap temperature was -50°C and the absolute pressure was 6.3 Pa. The pre-freezing end segments were 1 to 4, and the final end segment was 9. After freezing, the sample was in powder form. After removing the sealing film, it was placed in a desiccator and stored sealed at 4°C to prevent the exosome freeze-dried powder from absorbing moisture and being exposed to light.

[0046] Experimental Example 2

[0047] A method for preparing lyophilized exosome powder comprises the following steps:

[0048] 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 5×10 9 exosomes;

[0049] S2: Take 3 mL of the mixed solution, put it into a freeze-dried bottle, seal the freeze-dried bottle with sealing film, and freeze it at -80°C overnight;

[0050] S3: Place the mixed solution treated in S2 into a freeze dryer, set the freezing parameters according to the freezing conditions in the table below, and freeze-dry the mixed solution;

[0051]

[0052] The freeze dryer's cold trap temperature was -50°C and the absolute pressure was 6.3 Pa. The pre-freezing end segments were 1 to 4, and the final end segment was 9. After freezing, the sample was in powder form. After removing the sealing film, it was placed in a desiccator and stored sealed at 4°C to prevent the exosome freeze-dried powder from absorbing moisture and being exposed to light.

[0053] Result Analysis

[0054] The HSFs proliferation experiment was used to test 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 experiment was divided into groups according to the protein content of the exosome freeze-dried powder hydration solution. As can be seen from the figure, the number of particles is 1×10 9 (Experimental Example 1), 5×10 9 When the lyophilized exosome powder of (Experimental Example 2) was used to treat normally cultured HSFs, there was no statistical difference between the control group, Experimental Example 1, and Experimental Example 2 on the first day (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 there was a significant difference between Experimental Example 1 and Experimental Example 2 on the fifth day (P<0.05).

[0055] 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. 5Cells were seeded at a density of 1 mL / well in a 6-well plate, with 1 mL of culture medium per well. Culture was continued until 90% cell confluency was achieved. The culture medium was then replaced with serum-free medium for an additional 12 hours. A 200 μL pipette tip was used to scratch the cells by drawing a line perpendicular to the marked line from top to bottom in the center of each well. Observation was performed at 0, 8, and 16 hours. The same area was photographed under an inverted microscope to record cell migration on both sides of the scratch.

[0056] 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 8 and 16 hours, compared with the control group, Experimental Examples 1 and 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 the exosome freeze-dried powder is concentration-dependent (P < 0.05).

[0057] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by 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 lyoprotectant to obtain a mixed solution; the lyoprotectant comprises an active component and a solution for dissolving the active component; the active component comprises trehalose, polyvinyl pyrrolidone, compound amino acids, taurine and hydroxyethyl starch, and the solution is a PBS solution; the mass ratio of the trehalose, polyvinyl pyrrolidone, compound amino acids, taurine and hydroxyethyl starch is 180-220:450-550:3-8:3-8:80-120; the polyvinyl pyrrolidone is polyvinyl pyrrolidone-40; and the compound amino acid is 18AA; S2: placing the mixed solution into a freeze dryer and freeze-drying it under the following conditions to obtain the product; wherein the pre-freezing to freeze-thaw cycle stage is repeated twice before entering the freeze-drying stage; 2. The preparation method according to claim 1, wherein: Each 1 mL of the mixture contains 1×10 9 ~5×10 9 Exosomes.

3. The preparation method according to claim 1, wherein: The mass ratio of the trehalose, polyvinyl pyrrolidone, compound amino acid, taurine and hydroxyethyl starch is 200:500:5:5:

100.

4. The preparation method according to claim 1, wherein: The concentration of the active component in the solution is 0.20-0.21 g / mL.

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

Images