CN110897993A - Preparation method and application of exosome freeze-dried powder - Google Patents
Preparation method and application of exosome freeze-dried powder Download PDFInfo
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- CN110897993A CN110897993A CN201811072844.1A CN201811072844A CN110897993A CN 110897993 A CN110897993 A CN 110897993A CN 201811072844 A CN201811072844 A CN 201811072844A CN 110897993 A CN110897993 A CN 110897993A
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- exosome
- dried powder
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0216—Solid or semisolid forms
- A61K8/022—Powders; Compacted Powders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/345—Alcohols containing more than one hydroxy group
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
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- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
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Abstract
The invention discloses a preparation method of exosome freeze-dried powder, which comprises the steps of mixing exosome extracting solution, a protective agent, an additive and ultrapure water, uniformly stirring to obtain a mixed solution, sequentially pre-freezing the mixed solution, strengthening pre-freezing, pre-vacuumizing, sublimation drying and analysis drying to obtain exosome freeze-dried powder, wherein the protein activity of the obtained exosome freeze-dried powder can be kept above 90%, the exosome freeze-dried powder can be refrigerated or stored at room temperature for 1-2 years, the whole production cycle only needs 15-25 hours, the requirement of storage conditions is greatly improved, and meanwhile, the transportation is facilitated; the exosome freeze-dried powder disclosed by the invention has important effects in promoting angiogenesis, promoting cell regeneration and regulating immunity; the exosome freeze-dried powder is applied to the beauty industry, effectively reacts with skin cells, promotes the nutrition metabolism of epithelial cells, reduces the damage of the skin caused by ultraviolet rays, free radicals and the like, reduces the generation of melanin, promotes the proliferation of collagen cells in the dermis, and has the effects of smoothing fine wrinkles, delaying skin aging and whitening the skin.
Description
Technical Field
The invention belongs to the technical field of biological medicine preparation, and particularly relates to a preparation method and application of exosome freeze-dried powder.
Background
Exosomes (Exo) are vesicle-like bodies secreted from cells to the outside of cells, have the diameter of 30 nm-150 nm and a typical lipid bilayer membrane structure, exist in cell culture supernatant, plasma, serum, saliva, urine and other biological fluids, play an important role in various physiological and pathological processes of a body, and are a hotspot problem in the research of the biomedical field in recent years. At present, stem cells are widely applied to the fields of tissue repair and the like. Compared with stem cells, the stem cell exosome serving as the paracrine of the stem cells has more stable biological characteristics, and researches show that the exosome can still maintain the biological function after being stored at the temperature of 80 ℃ below zero for 2 years. Recent researches find that the exosome derived from the stem cells can effectively transport bioactive substances such as mRNA, micro RNA and protein, has important biological functions of reducing apoptosis, relieving inflammatory reaction, promoting angiogenesis, inhibiting fibrosis, improving tissue repair potential and the like, has good clinical application prospect in the aspect of regulating and controlling tissue regeneration, and is currently and rarely applied to the field of biological cosmetology in China.
The quality guarantee period of the exosome extract stored in a liquid state is very short, the storage condition is very harsh, the exosome extract can be stored for about 2 days at 4 ℃ generally and can be stored for about half a year at-20 ℃ under refrigeration, and the application of exosomes is greatly limited. Therefore, how to conveniently store exosomes, maintain the bioactivity and stability of exosomes and facilitate transportation is an urgent problem to be solved.
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of exosome freeze-dried powder, which is used for preparing exosome freeze-dried powder, so that the bioactivity and stability of exosome are improved, the exosome freeze-dried powder can be stored for a long time at refrigeration or normal temperature, and is convenient to transport;
the second purpose of the invention is to provide the application of the exosome freeze-dried powder, and the exosome freeze-dried powder is applied to the beauty industry to realize the effects of whitening and resisting aging.
The technical scheme adopted by the invention is that the exosome freeze-dried powder preparation method comprises the steps of mixing exosome extracting solution, a protective agent, an additive and ultrapure water, uniformly stirring to obtain a mixed solution, and sequentially carrying out pre-freezing, reinforced pre-freezing, pre-vacuumizing, sublimation drying and desorption drying on the mixed solution to obtain the exosome freeze-dried powder.
The invention is also characterized in that:
the mixed solution is prepared from the following components in parts by weight: 40-50 parts of exosome extracting solution, 8-10 parts of protective agent, 0-2 parts of additive and 38-52 parts of ultrapure water.
The protective agent is polyhydric alcohol; the additive is at least one of polymer and sugar.
The polyalcohol is at least one of sorbitol and mannitol.
The polymer is at least one of dextran and polyethylene glycol; the sugar is at least one of lactose and trehalose.
The pre-freezing temperature is-40 to-50 ℃, and the freezing time is 2 to 3 hours.
The temperature for enhancing pre-freezing is-40 to-50 ℃, and the freezing time is 1 to 2 hours.
The pre-vacuum temperature is-40 to-50 ℃, the time is 0.5 to 1 hour, and the vacuum degree is 15 Pa.
The sublimation drying comprises three stages, wherein,
the temperature of the first stage is-15 ℃ to-10 ℃, the time is 6-8 h, and the vacuum degree is 15 Pa;
the temperature of the second stage is-5 ℃ to 0 ℃, the time is 1 to 3 hours, and the vacuum degree is 5 Pa;
the temperature of the third stage is 0-10 ℃, the time is 1-2 h, and the vacuum degree is 0.1 Pa;
the temperature of the desorption drying is 30-40 ℃, the time is 3-6 h, and the vacuum degree is 20 Pa.
The invention adopts another technical scheme that the application of the exosome freeze-dried powder is implemented according to the following steps:
step 1, preparing hyaluronic acid stock solution:
weighing 0.1-0.2 part of sodium hyaluronate, 0.05-0.1 part of hydrolyzed sodium hyaluronate and 99.5-99.85 parts of ultrapure water, fully stirring and dissolving at 80-85 ℃, cooling to below 35 ℃, filling, sterilizing with high-pressure steam at 121 ℃ for 5-20 min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 20-30 parts of the hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.5-1 part of the exosome freeze-dried powder, shaking the mixture until the hyaluronic acid stock solution is completely dissolved to obtain an exosome essence, and introducing the exosome essence into a face through a microneedle or directly coating the exosome essence on the face.
The invention has the beneficial effects that:
1. according to the preparation method of the exosome freeze-dried powder, the protective agent and the exosome extracting solution are mixed and then sequentially subjected to pre-freezing, reinforced pre-freezing, pre-vacuumizing, sublimation drying and analysis drying treatment, the protein activity of the obtained exosome freeze-dried powder can be kept above 90%, the exosome freeze-dried powder can be refrigerated or stored at room temperature for 1-2 years, the whole production period only needs 15-25 hours, the requirement of the storage condition is greatly improved, and meanwhile, the transportation is facilitated;
2. the exosome freeze-dried powder has important functions in the aspects of promoting angiogenesis, promoting cell regeneration and regulating immunity; the exosome freeze-dried powder is applied to the beauty industry, effectively reacts with skin cells, promotes the nutrition metabolism of epithelial cells, reduces the damage of the skin caused by ultraviolet rays, free radicals and the like, reduces the generation of melanin, promotes the proliferation of collagen cells in the dermis, and has the effects of smoothing fine wrinkles, delaying skin aging and whitening the skin.
Drawings
FIG. 1 is a graph of the lyophilization profile provided in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The technical scheme adopted by the invention is that the exosome freeze-dried powder preparation method comprises the following steps of mixing exosome extracting solution, a protective agent, an additive and ultrapure water, and uniformly stirring to obtain a mixed solution, wherein the mixed solution is prepared from the following components in parts by weight: 40-50 parts of exosome extracting solution, 8-10 parts of protective agent, 0-2 parts of additive and 38-52 parts of ultrapure water;
wherein the protective agent is polyhydric alcohol; the additive is at least one of polymer and sugar;
the polyalcohol is at least one of sorbitol and mannitol;
the polymer is at least one of dextran and polyethylene glycol;
the sugar is at least one of lactose and trehalose;
pre-freezing the mixed solution in sequence: the temperature is-40 to-50 ℃, and the freezing time is 2 to 3 hours; strengthening pre-freezing: the temperature is-40 to-50 ℃, and the freezing time is 1 to 2 hours; pre-vacuumizing: the temperature is-40 to-50 ℃, the time is 0.5 to 1 hour, and the vacuum degree is 15 Pa; sublimation drying: the method comprises three stages, wherein the temperature of the first stage is-15 ℃ to-10 ℃, the time is 6-8 h, and the vacuum degree is 15 Pa; the temperature of the second stage is-5 ℃ to 0 ℃, the time is 1 to 3 hours, and the vacuum degree is 5 Pa; the temperature of the third stage is 0-10 ℃, the time is 1-2 h, and the vacuum degree is 0.1 Pa; and (3) resolving and drying: obtaining exosome freeze-dried powder at the temperature of 30-40 ℃, the time of 3-6 h and the vacuum degree of 20 Pa;
the application of the exosome freeze-dried powder is specifically implemented according to the following steps:
step 1, preparing hyaluronic acid stock solution:
weighing 0.1-0.2 part of sodium hyaluronate, 0.05-0.1 part of hydrolyzed sodium hyaluronate and 99.5-99.85 parts of ultrapure water, fully stirring and dissolving at 80-85 ℃, cooling to below 35 ℃, filling, sterilizing with high-pressure steam at 121 ℃ for 5-20 min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 20-30 parts of the hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.5-1 part of the exosome freeze-dried powder, shaking the mixture until the hyaluronic acid stock solution is completely dissolved to obtain an exosome essence, and introducing the exosome essence into a face through a microneedle or directly coating the exosome essence on the face.
The exosome is an important carrier for intercellular information exchange, and the characteristics of the exosome are related to cells from which the exosome is derived, so that the function of the umbilical cord mesenchymal stem cell exosome is closely related to the function of the stem cell thereof, and the umbilical cord mesenchymal stem cell exosome can influence the development of skin cells to a young and healthy state. The high-activity exosome has important functions in promoting angiogenesis, promoting cell regeneration and immunoregulation, can effectively react with skin cells, promote the nutrition metabolism of epithelial cells, prevent the skin from being damaged by ultraviolet rays, free radicals and the like, reduce the generation of melanin, promote the proliferation of collagen cells in the dermis layer, accelerate the repair of the skin after operation, smooth fine wrinkles of the general skin, delay the skin aging, whiten the skin and the like. The umbilical cord mesenchymal stem cell exosome extracting solution is obtained by culturing umbilical cord mesenchymal stem cells, collecting culture solution, and then separating and purifying the culture solution.
The freeze-drying process of exosomes is a multi-step process, and various effects such as low temperature, freezing and dehydration can be generated, so that the freezing conditions need to be controlled effectively and strictly;
the freezing effect includes:
1. in the freeze-drying process of the exosome extracting solution, the concentration of the solution is rapidly increased due to continuous crystallization, and when the concentration of the solution is changed, the ion concentration is increased, so that the chemical reaction is promoted;
2. during the freeze-drying process, the mixed solution containing the exosomes generates a large amount of ice-water interfaces, and the bioactive factors can be adsorbed on the interfaces, so that the natural structures of the bioactive factors can be damaged, and the exosomes are denatured;
3. during the process of freeze-drying the mixed liquor of the exosomes, the pH value of the mixed liquor can be changed, so that the exosomes are subjected to physical aggregation and chemical denaturation.
Dehydration effect:
a phenomenon in which a part of the bound water is removed during lyophilization. Removal of bound water can destroy the native structure of the exosomes, resulting in denaturation of the exosomes. This is because exosomes rich in bound water are exposed to a depleted aqueous environment during lyophilization, converting protons to charged carboxylic acid groups, disrupting the charge balance in the bioactive factors, and the reduction in charge density may promote hydrophobic interactions between exosomes, thereby allowing exosomes to aggregate.
The freeze-drying process of the invention adopts multiple steps, wherein, in the pre-freezing stage, the exosome exists in a state of being dissolved in the mixed liquid (material), ice is generated by supercooling the material because the temperature is reduced all the time, in the reinforcing pre-freezing stage, the material can continuously separate out ice crystals along a balanced melting line, the remained unfrozen mixed liquid around the ice crystals is reduced along with the temperature, the concentration is continuously increased until the remained moisture in the material can not be crystallized, the material reaches the maximum freezing concentration state at the moment, and the higher concentration surrounds the ice crystals in an amorphous state to form a glass body embedded with the ice crystals. The vitreous body is a state that a substance exists in an amorphous state, the viscosity of the vitreous body is extremely high, the mobility of molecules is almost zero, the diffusion coefficient of the amorphous structure is very low, so that the molecular motion and molecular denaturation reaction in the structure are very weak, and adverse chemical reactions can be inhibited, so that the stability of the composite bioactive factor is improved. In addition, through the pre-freezing and pre-freezing strengthening stages of the embodiment of the invention, the obtained glass body has uniform crystallization and proper crystal nucleus number, which is beneficial to maintaining a smooth sublimation channel in the subsequent sublimation drying process and accelerating the sublimation speed;
the sublimation drying stage is divided into three temperature gradients for heating, because with the sublimation of moisture, the concentration of the exosome is increased, the glass transition temperature is also increased, through properly gradually increasing the temperature, the sublimation can be accelerated to be carried out, energy can be saved, and meanwhile, the vacuum pump is used for vacuumizing, so that the free water frozen into ice is directly sublimated into steam, and more than 90% of the initial moisture is removed.
In the sublimation drying stage, if the temperature is too high, the exosome can be softened, collapsed and even sprayed, so that the freeze-drying failure is caused; if the temperature is too low, not only is an overhigh requirement on a refrigerating system provided, but also the speed of the sublimation process is greatly reduced, and time and energy are wasted;
the vacuum degree of the sublimation drying stage is also very important to control, and the vacuum degree of the sublimation drying stage adopts three gradients, so that proper convection heat transfer can be formed, the surface of the material can be always in a pressure state of uniform-speed drying, and the drying speed is improved while the requirements of different sublimation drying stages on the vacuum degree are ensured.
And in the desorption drying stage, the temperature is raised again, the material is heated at a higher temperature, part of the combined water is desorbed, and the heat is absorbed and evaporated into steam to further remove the water.
Even if the freeze drying is successfully completed, the key is to ensure the stability of the exosome freeze-dried powder in the subsequent storage process, so the selection and the proportion of the protective agent are particularly important;
the main factors influencing the degeneration of exosomes during storage are: protein aggregation due to physical (non-covalent) interactions or chemical agglutination (covalent); carrying out oxidation reaction; hydrolysis, etc., which occurs during storage, although the lyophilized bioactive factor contains a very small amount of water;
therefore, in order to prevent the exosomes from being denatured or inactivated during the freeze-drying and storage processes, an effective protective agent needs to be added to stabilize the activity of the exosomes. The freeze-drying protective agent has various types and complex mechanisms, and if the protective agent is improperly added, the exosome cannot be effectively protected, and even the great negative effects such as toxicity and the like can be generated in later-stage use of the exosome freeze-dried powder. In addition, the added concentration of the protective agent plays a very important role in the protective effect of the protective agent, generally, in a certain concentration range, the protective effect of the protective agent is increased along with the increase of the concentration, when the concentration reaches a certain concentration, the protective effect reaches the maximum value, and then the concentration of the protective agent is increased, the protective effect of the protective agent is not obviously increased any more, and the exosome is denatured even in the freeze drying process due to the excessively high concentration; however, different protective agents have different protective mechanisms and different concentrations of the protective agent exhibiting the maximum protective effect, and the comprehensive effect of various protective agents needs to be considered in actual use.
Sucrose and trehalose are used as non-reducing disaccharides, are very stable, can form a special protective film on the surface of cells under severe conditions of high temperature, high cold, drying and dehydration and the like, and effectively protect the structure of bioactive factors from being damaged, so that the activity of the bioactive factors is maintained.
Dextran is the polymerization product of the dehydration of several glucose molecules. The dextran 40 is low molecular weight, and the molecular weight range is 25000-50000. The dextran 40 can inhibit the growth of ice crystal, so the dextran can effectively protect bioactive factors.
Polyethylene glycol (PEG) can prevent the exosome from being denatured in the freeze-drying process of the bioactive factor.
The formula of the protective agent disclosed by the invention not only can play a good protection role on exosomes in the whole freeze-drying process, but also can play an inhibiting role on denaturation of exosomes in the storage period, so that the storage condition of exosomes is greatly improved by the formula of the protective agent disclosed by the embodiment of the invention, the exosomes can be stored for 1-2 years in a refrigeration or normal temperature, and the storage cost of exosomes is effectively reduced.
The freeze drying equipment used in this example was a freeze dryer.
Example 1
The preparation method of the exosome freeze-dried powder comprises the following steps:
step 1, weighing the following raw materials: mixing and uniformly stirring 40 parts of exosome extracting solution, 8 parts of sorbitol, 1 part of dextran and 51 parts of ultrapure water to obtain a mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-10 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 7h, continuously heating to 0 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 1h, then continuously heating to 10 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 1.5h, finally carrying out resolution drying, namely heating to 40 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 4h to obtain the exosome freeze-dried powder.
FIG. 1 is the graph of the lyophilization curve of example 1, as shown in FIG. 1, in the pre-freezing stage, the exosomes exist in the state of being dissolved in the mixed solution (material), the initial temperature of the material is 20 ℃, and after 2.5h of pre-freezing, the material is cooled to-45 ℃ to form the vitreous body. Then through strengthening the prefreezing stage, the crystallization of the vitreous body is more uniform, the crystal nucleus quantity is suitable, in the sublimation drying stage, heat the above-mentioned vitreous body in three stages, make its temperature rise from minus 45 deg.C to 10 deg.C, vacuumize with the vacuum pump at the same time, make free water frozen into ice sublimate into water vapor directly therein, make more than 90% of the original moisture removed;
in the desorption drying stage, the material is heated at a higher temperature by raising the temperature from 10 ℃ to 40 ℃ to desorb part of the bound water, and then endothermically evaporated to steam to further remove water.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.1 part of sodium hyaluronate, 0.05 part of hydrolyzed sodium hyaluronate and 99.85 parts of ultrapure water, fully stirring and dissolving at 80 ℃, cooling to 25 ℃, filling into 6 mL/piece, sterilizing with high-pressure steam at 121 ℃ for 5min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 6mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.2g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
Example 2
Step 1, weighing the following raw materials: 45 parts of exosome extracting solution, 9 parts of mannitol, 1 part of lactose and 39 parts of ultrapure water are mixed and uniformly stirred to obtain mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-15 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 6h, continuously heating to-5 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 2h, then continuously heating to 8 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 1h, finally carrying out resolution drying, namely heating to 35 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 3h to obtain the exosome freeze-dried powder.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.2 part of sodium hyaluronate, 0.1 part of hydrolyzed sodium hyaluronate and 99.5 parts of ultrapure water, fully stirring and dissolving at 82 ℃, cooling to 25 ℃, filling into 4 mL/piece, and sterilizing with high-pressure steam at 121 ℃ for 5min to obtain a hyaluronic acid stock solution;
and 2, weighing 4mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.1g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
Example 3
Step 1, weighing the following raw materials: mixing and uniformly stirring 50 parts of exosome extracting solution, 2 parts of sorbitol, 8 parts of mannitol and 38 parts of ultrapure water to obtain a mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-12 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 8h, continuously heating to-3 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 3h, then continuously heating to 0 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 2h, finally carrying out resolution drying, namely heating to 30 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 6h to obtain the exosome freeze-dried powder.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.15 part of sodium hyaluronate, 0.08 part of hydrolyzed sodium hyaluronate and 99.6 parts of ultrapure water, fully stirring and dissolving at 85 ℃, cooling to 25 ℃, filling into 10 mL/piece, sterilizing with high-pressure steam at 121 ℃ for 20min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 10mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.4g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
Example 4
Step 1, weighing the following raw materials: 45 parts of exosome extracting solution, 9 parts of mannitol, 1 part of lactose and 39 parts of ultrapure water are mixed and uniformly stirred to obtain mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-15 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 6h, continuously heating to-5 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 2h, then continuously heating to 8 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 1h, finally carrying out resolution drying, namely heating to 35 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 3h to obtain the exosome freeze-dried powder.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.2 part of sodium hyaluronate, 0.1 part of hydrolyzed sodium hyaluronate and 99.5 parts of ultrapure water, fully stirring and dissolving at 82 ℃, cooling to 25 ℃, filling into 8 mL/piece, sterilizing with high-pressure steam at 121 ℃ for 15min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 6mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.25g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
Example 5
Step 1, weighing the following raw materials: mixing and uniformly stirring 50 parts of exosome extracting solution, 2 parts of sorbitol, 8 parts of mannitol, 1 part of dextran, 1 part of polyethylene glycol and 45 parts of ultrapure water to obtain a mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-12 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 8h, continuously heating to-3 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 3h, then continuously heating to 0 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 2h, finally carrying out resolution drying, namely heating to 30 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 6h to obtain the exosome freeze-dried powder.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.15 part of sodium hyaluronate, 0.08 part of hydrolyzed sodium hyaluronate and 99.6 parts of ultrapure water, fully stirring and dissolving at 85 ℃, cooling to 25 ℃, filling into 6 mL/piece, sterilizing with high-pressure steam at 121 ℃ for 5min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 6mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.2g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
Example 6
Step 1, weighing the following raw materials: mixing and uniformly stirring 50 parts of exosome extracting solution, 8 parts of mannitol, 0.5 part of polyethylene glycol, 1 part of trehalose and 50 parts of ultrapure water to obtain a mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-12 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 8h, continuously heating to-3 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 3h, then continuously heating to 0 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 2h, finally carrying out resolution drying, namely heating to 30 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 6h to obtain the exosome freeze-dried powder.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.15 part of sodium hyaluronate, 0.05 part of hydrolyzed sodium hyaluronate and 99.7 parts of ultrapure water, fully stirring and dissolving at 85 ℃, cooling to 25 ℃, filling into 6 mL/piece, sterilizing with high-pressure steam at 121 ℃ for 5min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 6mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.2g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
Example 7
Step 1, weighing the following raw materials: mixing and uniformly stirring 50 parts of exosome extracting solution, 2 parts of sorbitol, 8 parts of mannitol, 1 part of lactose, 1 part of trehalose and 45 parts of ultrapure water to obtain a mixed solution;
and 3, continuously carrying out sublimation drying on the frozen mixture obtained in the step 2, firstly reducing the temperature to-12 ℃, keeping the vacuum degree at 15Pa, keeping the temperature for 8h, continuously heating to-3 ℃, reducing the vacuum degree to 5Pa, keeping the temperature for 3h, then continuously heating to 0 ℃, reducing the vacuum degree to 0.1Pa, keeping the temperature for 2h, finally carrying out resolution drying, namely heating to 30 ℃, increasing the vacuum degree to 20Pa, and keeping the temperature for 6h to obtain the exosome freeze-dried powder.
Preparing the exosome freeze-dried powder obtained in the step 3 of the preparation method of the exosome freeze-dried powder into exosome essence:
step 1, preparing hyaluronic acid stock solution:
weighing 0.15 part of sodium hyaluronate, 0.05 part of hydrolyzed sodium hyaluronate and 99.7 parts of ultrapure water, fully stirring and dissolving at 85 ℃, cooling to 25 ℃, filling into 6 mL/piece, sterilizing with high-pressure steam at 121 ℃ for 5min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 6mL of hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.2g of exosome freeze-dried powder, stirring until the hyaluronic acid stock solution is completely dissolved to obtain exosome essence, and introducing or directly smearing the exosome essence on the face through a microneedle.
The use effect experiment of the exosome essence comprises the following steps:
the use test of the exosome essence obtained in the above example 1 was carried out, and the use method thereof was as follows: 30 volunteers of 23-43 years old are selected, and the exosome essence is applied to the face in the morning and evening every day and is continuously used for 15 days.
As a result: the volunteers are smeared with the exosome essence for 15 days, the acne is reduced, the acne marks are reduced, the skin is smoother, finer and smoother, and is whitened, fine wrinkles and dry wrinkles around the eyes are obviously reduced, and the facial skin is fuller, fair and moist.
In summary, the preparation method of the exosome freeze-dried powder comprises the steps of mixing a protective agent and an exosome extracting solution, and then sequentially carrying out pre-freezing, enhanced pre-freezing, pre-vacuumizing, sublimation drying and resolution drying treatment, wherein the protein activity of the obtained exosome freeze-dried powder can be kept above 90%, the exosome freeze-dried powder can be refrigerated or stored at room temperature for 1-2 years, the whole production period only needs 15-25 hours, and the requirement of storage conditions is greatly improved, and meanwhile, the exosome freeze-dried powder is convenient to transport; the application of the exosome freeze-dried powder plays an important role in promoting angiogenesis, promoting cell regeneration and regulating immunity; the exosome freeze-dried powder is applied to the beauty industry, effectively reacts with skin cells, promotes the nutrition metabolism of epithelial cells, reduces the damage of the skin caused by ultraviolet rays, free radicals and the like, reduces the generation of melanin, promotes the proliferation of collagen cells in the dermis, and has the effects of smoothing fine wrinkles, delaying skin aging and whitening the skin.
Claims (10)
1. The preparation method of the exosome freeze-dried powder is characterized by mixing exosome extracting solution, a protective agent, an additive and ultrapure water, uniformly stirring to obtain a mixed solution, and sequentially pre-freezing, strengthening pre-freezing, pre-vacuumizing, sublimation drying and desorption drying the mixed solution to obtain the exosome freeze-dried powder.
2. The preparation method of exosome freeze-dried powder according to claim 1, wherein the mixed solution is prepared from the following components in parts by weight: 40-50 parts of exosome extracting solution, 8-10 parts of protective agent, 0-2 parts of additive and 38-52 parts of ultrapure water.
3. A method of preparing exosome freeze-dried powder according to claim 1 or 2, characterized in that the protective agent is a polyol; the additive is at least one of polymer and sugar.
4. A method of preparing exosome freeze-dried powder according to claim 3, wherein the polyalcohol is at least one of sorbitol and mannitol.
5. A method of preparing an exosome freeze-dried powder according to claim 3, wherein the polymer is at least one of dextran and polyethylene glycol; the sugar is at least one of lactose and trehalose.
6. A preparation method of exosome freeze-dried powder according to any one of claims 1 to 5, characterized in that the pre-freezing temperature is-40 to-50 ℃, and the freezing time is 2 to 3 hours.
7. A preparation method of exosome freeze-dried powder according to any one of claims 1 to 5, characterized in that the temperature for enhancing pre-freezing is-40 to-50 ℃, and the freezing time is 1 to 2 hours.
8. A method for preparing exosome freeze-dried powder according to any one of claims 1 to 5, characterized in that the pre-vacuum temperature is-40 to-50 ℃, the time is 0.5 to 1 hour, and the vacuum degree is 15 Pa.
9. A method of preparing exosome freeze-dried powder according to any one of claims 1-5, characterized in that the sublimation drying comprises three stages, wherein,
the temperature of the first stage is-15 ℃ to-10 ℃, the time is 6-8 h, and the vacuum degree is 15 Pa;
the temperature of the second stage is-5 ℃ to 0 ℃, the time is 1 to 3 hours, and the vacuum degree is 5 Pa;
the temperature of the third stage is 0-10 ℃, the time is 1-2 h, and the vacuum degree is 0.1 Pa;
the temperature of the desorption drying is 30-40 ℃, the time is 3-6 h, and the vacuum degree is 20 Pa.
10. An exosome freeze-dried powder prepared by the preparation method according to any one of claims 1 to 9, which is characterized by being implemented according to the following steps:
step 1, preparing hyaluronic acid stock solution:
weighing 0.1-0.2 part of sodium hyaluronate, 0.05-0.1 part of hydrolyzed sodium hyaluronate and 99.5-99.85 parts of ultrapure water, fully stirring and dissolving at 80-85 ℃, cooling to below 35 ℃, filling, sterilizing with high-pressure steam at 121 ℃ for 5-20 min, and cooling to obtain a hyaluronic acid stock solution;
and 2, weighing 20-30 parts of the hyaluronic acid stock solution obtained in the step 1, pouring the hyaluronic acid stock solution into 0.5-1 part of the exosome freeze-dried powder, shaking the mixture until the hyaluronic acid stock solution is completely dissolved to obtain an exosome essence, and introducing the exosome essence into a face through a microneedle or directly coating the exosome essence on the face.
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2018
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