Exosome freeze-dried powder as well as preparation method and application thereof

Abstract

The invention relates to the technical field of freeze-dried preparations, and provides exosome freeze-dried powder as well as a preparation method and application thereof. The exosome freeze-drying preparation is prepared by mixing an exosome solution and a freeze-drying protection solution and then freeze-drying the mixture, the concentration of the exosome solution in the mixed solution is 0.1-10% V / V, and the freeze-drying protection solution comprises 0.4-4% W / V of saccharides and 0.25-4% W / V of alcohols; the saccharide is trehalose, cane sugar or lactose; the alcohol is mannitol, sorbitol or a combination of mannitol and polyethylene glycol; and the solvent is sterilized water for injection or a PBS buffer solution. The exosome freeze-dried powder is stable in activity, small in irritation, good in safety and good in fibroblast proliferation promoting capacity, when the exosome freeze-dried powder is applied to supporting essence or masks in the medical beauty industry, the exosome activity of the exosome freeze-dried powder is still kept stable, and the application prospect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of freeze-dried preparations, and in particular to an active and stable exosome freeze-dried preparation and a preparation method and application thereof. Background Art

[0002] Exosomes are a type of extracellular vesicles (EVs), and they are the smallest in size. EVs are a type of extracellular body that can be secreted by almost all types of cells, with a lipid bilayer membrane structure derived from endocytosis and containing mother cell components. According to their size, source, function, etc., EVs can be divided into three categories: exosomes (30-150nm), ectosomes (100nm-1μm) and apoptotic bodies (50nm-5μm). Structurally, exosomes have a typical lipid bilayer membrane structure, surrounded by a water-soluble medium, which contains a variety of biological molecules, including nucleic acids, peptides, lipids, proteins, etc. At present, nearly 100,000 proteins and more than 1,000 lipids have been recorded in exosomes. These components play an important role in intercellular and intracellular communication, and have the characteristics of transmitting genetic information and regulating physiological functions. Exosomes exist in a variety of biological fluids, including blood, urine, saliva, breast milk, amniotic fluid, synovium, cerebrospinal fluid, tears, etc. The function of exosomes depends on the cell type from which they are derived, and they can participate in the body's immune response, antigen presentation, cell migration, cell differentiation, tumor invasion, etc.

[0003] With the expansion of exosome applications, how to preserve exosomes safely and effectively has become very important. The current consensus on the storage of exosomes is that exosomes can be stably stored at 2-8°C for a short period of time; in order to maintain the structural integrity and biological activity of exosomes, they must be stored at low temperatures. If stored at -20°C for about 1 month, their activity will change significantly; they can only be stored at -80°C for 3-6 months, and they cannot be repeatedly frozen and thawed. Exosomes have low stability, are easily inactivated, have high storage requirements, and have a short stable storage time. These shortcomings affect the transportation, storage, and application of exosomes.

[0004] Freeze-drying technology provides a new option for the preservation of exosomes. The freeze-drying process is carried out at low temperatures, which will not denature or lose the biological activity of heat-sensitive substances such as proteins and microorganisms. At the same time, the growth of microorganisms and the action of enzymes cannot be carried out, so the original properties can be maintained. At present, there are literatures and patents that prepare exosomes as freeze-dried powder. In some existing patents or patent application schemes, "A preparation method and use method of freeze-dried powder of human mesenchymal stem cell exosomes" (publication number CN107245472A) uses a single component mannitol as a protective agent; "A method for preparing freeze-dried powder of human amniotic mesenchymal stem cell exosomes" (publication number CN104488850A) and "Exosome freeze-dried powder from human adipose stem cells and its preparation method and application" (publication number CN110193025A) both use a single component trehalose as a protective agent for exosomes. Other patent applications use composite protective agents such as platelet-rich factor, albumin, glycine, lysine, glycerol, gelatin hydrolysate, arginine, sucrose, sorbitol, ceramide, etc. The single-component or multi-component freeze-drying protective agents used in these patents cannot effectively guarantee the integrity and biological function of exosomes before and after freeze-drying, and still have defects and deficiencies. Summary of the invention

[0005] The present invention provides an exosome freeze-dried powder and a preparation method and application thereof, so as to alleviate the problem in the prior art that the freeze-dried protectant cannot effectively maintain the integrity and biological activity of the exosomes after freeze-drying; the freeze-dried powder of the present invention has stable activity, small irritation reaction, and a simple production process, which is easy to implement.

[0006] In a first aspect of the present invention, there is provided an exosome lyophilized powder, which is prepared by mixing an exosome solution and a lyophilization protection solution and then freeze-drying the mixture. The exosome solution is 0.1%~10%V / V, and the lyophilization protection solution includes 0.4%~4%W / V of sugars and 0.25%~4%W / V of alcohols, based on the concentration of each substance in the mixed solution; the sugar is any one of trehalose, sucrose or lactose; the alcohol is mannitol, sorbitol or a combination of mannitol and polyethylene glycol; and the solvent is sterile water for injection or PBS buffer.

[0007] Preferably, the sugar is trehalose, the alcohol is a combination of mannitol and polyethylene glycol, the polyethylene glycol is PEG4000, and the solvent is sterile water for injection.

[0008] Optionally, the freeze-drying protective solution also includes 0.1% to 0.5% W / V of protein substances.

[0009] Optionally, the protein substance includes one or more of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

[0010] Preferably, the protein substance is human serum albumin.

[0011] Optionally, the exosomes in the exosome solution are mesenchymal stem cell exosomes.

[0012] A second aspect of the present invention provides a method for preparing the above-mentioned lyophilized exosome powder, comprising the following steps: A. Weigh the raw materials of each component of the freeze-dried protective solution according to the proportion of each substance; B. dissolving the above raw materials in a solvent, filtering and sterilizing through a 0.22 μm filter membrane to obtain a lyophilized protective agent solution; C. taking the exosome solution and mixing it evenly with the lyophilization protective agent solution to obtain a pre-lyophilization solution; D. The pre-lyophilized solution is dispensed into sterile vials, and after vacuum freeze-drying, lyophilized exosome powder is obtained.

[0013] Optionally, the vacuum freeze drying in step D comprises: D1. Pre-freezing: temperature is -30~-50℃, setting time is 1~3h, duration is 2~5h, target vacuum degree is 1000mbar; D2. Pre-vacuum: 0.15mbar, alarm vacuum: 0.8mbar, pre-vacuum duration: 60s; D3. Sublimation drying: temperature is -10~-25℃, setting time is 1~4h, duration is 18~28h, target vacuum degree is 0.1~0.15mbar; D4. Analytical drying: temperature is 20~25℃, set time is 3~8h, duration is 8~20h, target vacuum degree: 0~0.1mbar.

[0014] In a third aspect of the present invention, an exosome essence or an exosome mask is provided, comprising the above-mentioned lyophilized exosome powder or the lyophilized exosome powder prepared by the above-mentioned preparation method, a reconstitution solvent, a moisturizer and a skin conditioner.

[0015] Optionally, the moisturizer includes 0.1% to 2% W / V propylene glycol and 0.5% to 5% W / V glycerin, the skin conditioner includes 0.1% to 0.5% W / V sodium hyaluronate and 1% to 3% W / V niacinamide, and the reconstitution solvent is water for injection.

[0016] A fourth aspect of the present invention provides a method for preparing the above-mentioned exosome essence or exosome mask, comprising: 1) Preparing an essence stock solution or a facial mask stock solution: weighing a moisturizer and a skin conditioning agent, adding them to a re-dissolving solvent, stirring and dissolving, and obtaining an essence stock solution or a facial mask stock solution; 2) Pour the essence liquid or mask liquid into the exosome freeze-dried powder, shake until completely dissolved, and obtain the exosome essence liquid or exosome mask.

[0017] A fifth aspect of the present invention provides use of the above-mentioned lyophilized exosome powder or the lyophilized exosome powder prepared by the above-mentioned preparation method in the preparation of skin care products or cosmetics.

[0018] The solution components and preparation ratios of the exosome freeze-dried powder of the present invention have been developed and optimized, and can effectively maintain the integrity and biological activity of the exosomes after freeze-drying; the prepared exosome freeze-dried powder has stable activity, and does not contain irritating raw materials such as preservatives, has low irritation and good safety; and has a good ability to promote fibroblast proliferation. The exosome freeze-dried powder is applied to the medical beauty industry, for example, to prepare exosome essence or facial mask. It is determined that the exosome activity remains stable. It can be seen that the exosome freeze-dried powder of the present invention has good effects on skin anti-wrinkle and anti-aging, and has good application prospects. More specifically, it has been verified through experiments that when the exosomes are freeze-dried using the exosome freeze-dried protective solution of the present invention, the particle size and concentration of the exosomes do not change significantly before and after freeze-drying, indicating that the freeze-dried protective solution of the present invention can effectively reduce the aggregation or rupture of exosomes and the reduction in the number of particles during the freeze-drying process; the surface specific marker proteins (CD9, CD63, CD81) of the exosomes before and after freeze-drying were detected, and each marker protein was significantly expressed, indicating that the composition of the molecular markers on the surface of the exosomes did not change significantly after freeze-drying; the freeze-dried exosomes were tested for fibroblast proliferation activity, and compared with the exosome fresh stock solution group, the proliferation-promoting activity of the freeze-dried powder and the exosome fresh stock solution was consistent, indicating that the exosome freeze-dried powder protective solution of the present invention can effectively protect the biological activity of the exosomes during the freeze-drying process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the morphology of exosome freeze-dried powder.

[0020] Figure 2 Transmission electron microscopy image of lyophilized exosome powder.

[0021] Figure 3 This is a graph showing the particle size change rate of exosomes before and after freeze-drying.

[0022] Figure 4 This is the concentration recovery rate diagram of exosomes before and after freeze-drying.

[0023] Figure 5 Schematic diagram of fluorescence detection of marker proteins.

[0024] Figure 6 This is a graph showing the cell proliferation rate of HDF cells after 72 hours.

[0025] Figure 7 This is a detection diagram of exosome essence promoting HDF cell proliferation. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific implementation modes. It should be understood that the given embodiments are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0027] The exosome lyophilized powder of the present invention is prepared by mixing an exosome solution and a lyophilization protection solution and then freeze-drying. It is made from raw materials containing the following components: 0.1%~10% V / V of exosome solution, 0.4%~4% W / V of sugars, and 0.25%~4% W / V of alcohols. The concentration of the above ingredients is measured by their working concentration in the solvent, which refers to the proportion of a single component. For example, when the alcohol is a combination of two alcohol substances, such as a combination of mannitol and polyethylene glycol, the proportion refers to 0.25%~4% W / V mannitol and 0.25%~4% W / V polyethylene glycol. In some schemes, 0.1%~0.5% W / V of protein substances are also added. During the preparation process, the solvent uses any one of sterile water for injection, PBS buffer or physiological saline.

[0028] Specifically, for the components of the above-mentioned freeze-dried protective solution, the sugars include one or more of trehalose, sucrose, lactose, and glucose; the alcohols include one or more of mannitol, sorbitol, and polyethylene glycol; and the protein substances include one or more of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

[0029] The solvent is any one of sterile water for injection, PBS buffer or physiological saline. Preferably, the solvent is sterile water for injection or PBS buffer. When the exosome solution and freeze-dried protective solution are actually prepared, the addition of different components and proportions will cause changes in the pH and osmotic pressure of the mixed solution, which requires close attention and appropriate adjustment of the prescription. The characteristics of the PBS buffer can also be used to adjust the osmotic pressure, pH, etc. of the buffer solution.

[0030] In a preferred embodiment, the sugar is any one of trehalose, sucrose or lactose, and more preferably, the sugar is trehalose. The alcohol is preferably mannitol, sorbitol or a combination of mannitol and polyethylene glycol, and is also preferably any one of mannitol and polyethylene glycol; more specifically, the polyethylene glycol uses PEG4000; more preferably, the alcohol is a combination of mannitol and polyethylene glycol; the protein substance is preferably human serum albumin.

[0031] A more preferred scheme is to use trehalose, mannitol, and PEG4000, the solvent is sterile water for injection, and the component ratio is 10% v / v exosomes, 2% w / v trehalose, 4% w / v mannitol, and 0.5% w / v PEG4000 (the solvent is sterile water for injection) based on the final concentration of the pre-freezing solution.

[0032] The mechanism and effect of each component of the above-mentioned freeze-dried protective solution: Trehalose, as a non-reducing disaccharide (disaccharide), is widely used as a stabilizer and protectant for protein drugs, vaccines, and liposomes. It is used as an exosome freeze-drying protectant. On the one hand, it can reduce the glass transition temperature in the exosome membrane, reduce ice formation during freezing, and prevent protein aggregation, thereby reducing the rupture or aggregation of exosomes during freeze-drying. On the other hand, under the low temperature and dehydration conditions of the freeze-drying process, trehalose can act as a cryoprotectant and dehydration protectant to form a special protective film on the cell surface to protect the cell structure from being destroyed, thereby maintaining the activity of bioactive factors.

[0033] Mannitol is non-hygroscopic, dries quickly, and has good chemical stability. It can not only serve as an excellent excipient in the freeze-drying process to provide a support structure for the active components, but also doubles as a freeze-drying protectant to protect biological molecules from damage caused by freezing and drying stress, ensuring the integrity and activity of biological products. It can also stabilize proteins in freeze-dried formulas to prevent protein aggregation and denaturation. In addition, as an effective osmotic agent, mannitol helps control the osmotic pressure in the preparation and maintain the stability and efficacy of biological preparations during storage and use.

[0034] Polyethylene glycol (PEG) series products are non-toxic, non-irritating, and have good water solubility. They are a polymer protective agent that can prevent protein degradation and denaturation during the freeze-drying process of bioactive factors.

[0035] Human serum albumin (HSA) is derived from the human body, has no immunogenicity, and no toxic side effects. Serum albumin is a classic high-quality protein stabilizer that protects most proteins during the freeze-drying process.

[0036] The exosomes in the exosome solution are preferably derived from natural exosomes, more preferably, derived from umbilical cord mesenchymal stem cell exosomes.

[0037] Exosomes derived from human umbilical cord mesenchymal stem cells have low immunogenicity and have a variety of important physiological functions. They carry some information factors of human umbilical cord mesenchymal stem cells (mainly regenerative factors and a variety of bioactive factors) and can mediate cell-to-cell material transfer and information conduction. In the field of dermatology, exosomes from human umbilical cord mesenchymal stem cells can deliver growth factors and signal factors to promote fibroblast proliferation and migration. In addition, it can also regulate the formation and maintenance of the stratum corneum, thereby maintaining the health of the skin. Finally, it can also participate in the skin repair and regeneration process, promote the synthesis of type I collagen and elastin and the reconstruction of the extracellular matrix, inhibit cell apoptosis and cell peroxidation, and play an anti-aging role, prevent skin dullness, and inhibit the formation of wrinkles.

[0038] For the preparation method of exosome lyophilized powder, some schemes include the following steps: A. Weighing: weigh the raw materials according to the following weight percentages: 0.4%~4% W / V for carbohydrates, 0.25%~4% W / V for alcohols, and 0.1%~0.5% W / V for proteins; B. Fully dissolve the above raw materials in the solvent, filter and sterilize through a 0.22 μm filter membrane to obtain a lyophilized protective agent solution; C. Take 0.1%~10% V / V of the exosome solution and mix it evenly with the lyophilization protectant solution to serve as the pre-lyophilization solution; D. The pre-lyophilized solution is dispensed into sterile vials, and vacuum freeze-dried to obtain exosome lyophilized powder.

[0039] Wherein, the vacuum freezing method specifically comprises: D1, the temperature of the pre-freezing stage is -30~-50℃, the setting time is 1~3h, the duration is 2~5h, and the target vacuum degree is 1000mbar; D2, pre-vacuum: 0.15mbar, alarm vacuum: 0.8mbar, pre-vacuum duration: 60s; D3, the temperature of the sublimation drying stage is -10~-25℃, the setting time is 1~4h, the duration is 18~28h, and the target vacuum degree is 0.1~0.15mbar; D4, the temperature of the analytical drying stage is 20~25℃, the set time is 3~8h, the duration is 8~20h, and the target vacuum degree is 0~0.1 mbar.

[0040] Preferably, the specific method of vacuum freeze drying in step D comprises: The freezing control temperature is -50°C, the setting time is 2h, the duration is 3h, and the target vacuum degree is 1000mbar; The pre-vacuuming: 0.15mbar, the alarm vacuum: 0.8mbar, the pre-vacuuming duration: 60s; The sublimation drying temperature is -25°C, the set time is 2h, the duration is 22h, and the target vacuum degree is 0.15mbar; The temperature of the analytical drying is 25° C., the set time is 6 hours, the duration is 8 hours, and the target vacuum degree is ultimate vacuum.

[0041] Another aspect of the present invention provides the use of lyophilized exosome powder in the preparation of skin care products or cosmetics. The lyophilized exosome powder can be used to prepare exosome essence or facial mask, etc., for wrinkle removal, anti-aging, improving skin firmness, improving skin gloss, the ingredients are safe and effective, and the application prospects are good.

[0042] Based on the exosome freeze-dried powder of the present invention, an exosome essence or an exosome mask can be obtained. In some embodiments, the exosome essence or exosome mask ingredients include exosome freeze-dried powder, a reconstitution solvent, a moisturizer, and a skin conditioner. More specifically, in some embodiments, the moisturizer includes 0.1%~2% W / V propylene glycol and 0.5%~5% W / V glycerol, the skin conditioner includes 0.1%~0.5% W / V sodium hyaluronate and 1%~3% W / V niacinamide, and the reconstitution solvent is water for injection.

[0043] In some embodiments, the specific preparation method includes: Step 1: Prepare essence / mask stock solution: Weigh 0.1%~0.5% W / V sodium hyaluronate, 0.1%~2% W / V propylene glycol, 0.5%~5% W / V glycerin, and 1%~3% W / V niacinamide, add to solvent water for injection, stir thoroughly to dissolve, and obtain essence / mask stock solution.

[0044] Step 2: Weigh the essence / mask stock solution obtained in step 1, pour it into the exosome freeze-dried powder, shake until completely dissolved, and obtain the exosome essence or exosome mask. It can be applied directly to the face or introduced through microneedles, roller needles, water light needles, or applied to the face through mask paper. Example

[0045] 1. Preparation of Lyophilized Exosome Powder Preparation of freeze-dried protective solution: According to the prescription composition of the embodiment and comparative example in Table 1, weigh different freeze-dried protective agents and add them into the solvent, dissolve and mix them evenly, and filter them through a 0.22 μm filter membrane to obtain a clear solution.

[0046] Mixing of exosomes with freeze-dried protective solution: According to the prescription composition of the embodiment and comparative example in Table 1, take the exosome solution, add it to the freeze-dried protective solution, mix evenly, and dispense it into 10ml vials at 2ml / bottle. Taking Example 8 as an example, take 10ml of purified exosomes, add 2g trehalose, 4g mannitol, 0.5g polyethylene glycol 4000, and 90ml of sterile water for injection, mix well and dispense it into 10ml freeze-dried vials, 2ml / bottle. Then the rubber stopper is half-pressed, and the freeze-dried powder is made through pre-freezing, sublimation drying, analytical drying and other procedures, vacuum pressurized, and then out of the box. The specific program parameters are shown in Table 2 below.

[0047] Table 1

[0048]

[0049] Comparative Example 3 is a control group set up with reference to patents CN104488850A and CN 110193025 A.

[0050] Comparative Example 5 is a control group set up with reference to patent CN107245472A.

[0051] Comparative Example 6 is a control group set up with reference to the formula of 8% mannitol and 2% sorbitol in Example 3 of Patent CN110897993A. However, the osmotic pressure of the auxiliary material solution was measured to be 569 mOsmol / kg after actual preparation, which is significantly higher than the normal blood osmotic pressure (285-310 mOsmol / kg). Therefore, the formula was adjusted to 4% mannitol and 1% sorbitol.

[0052] Comparative Example 7 is a control group set with reference to patent CN112261871A. Comparative Example 8 is a control group set with reference to patent CN110339338A.

[0053] In addition, we also tried different proportions of prescriptions with reference to other patents. Among them, referring to Example 6 in CN110897993A (prescription: 1% trehalose, 8% mannitol, 0.5% polyethylene glycol), the osmotic pressure was measured to be 639mOsmol / kg when preparing the auxiliary material solution, which was obviously too high. Using this prescription for freeze-dried powder preparation would obviously destroy the activity of exosomes and the actual application effect. Therefore, this scheme was excluded and no subsequent freeze-drying experiments were performed.

[0054] Table 2 Freeze drying procedure

[0055] 2. Morphological observation of freeze-dried samples After freeze-drying, the appearance of the freeze-dried powders of the embodiment and the comparative example was observed. The specific results are shown in Table 3.

[0056] Table 3 Appearance observation of freeze-dried samples

[0057] The exosome freeze-dried powder of Examples 1-10 of the present invention has a uniform white powder cake appearance. Although the edges of Example 9 are slightly shrunk, the appearance is slightly affected, and subsequent experiments can be carried out. It is preliminarily evaluated that the freeze-drying effect of Examples 1-10 is good. Except for Comparative Examples 5, 6, and 8, the freeze-drying protection schemes of the preparations of Comparative Examples 1-4, 7, or 9 have adverse changes in texture and volume after freeze-drying. It is preliminarily evaluated that the freeze-drying protection effect is poor, and the changes in texture and volume also make the freeze-dried powder have great obstacles and difficulties in the subsequent practical application in daily chemical products (such as cosmetics, skin care products), medicines and other fields.

[0058] Attached Figure 1 This is the appearance of the freeze-dried powder prepared in Example 8.

[0059] 3. Osmotic pressure and pH characterization The samples of the embodiment and the comparative example before and after lyophilization were characterized by osmotic pressure and pH, and the specific test results are shown in Table 4. Reconstitution process of lyophilized exosome powder: 2 ml of sterile water for injection was added to the lyophilized vial, shaken to dissolve and clarify, and then the osmotic pressure and pH of the lyophilized powder reconstituted solution were measured.

[0060] Table 4 Osmotic pressure and pH measurement results

[0061]

[0062] It is known that the molar osmotic pressure concentration of human blood is between 285 and 310 mOsmol / kg, and the average physiological osmotic pressure is about 297.5 mOsmol / kg. Similarly, during the freeze-drying process, the appropriate osmotic pressure can maintain the good morphology and integrity of the exosomes, prevent them from rupturing or losing water, and ensure their activity. When the osmotic pressure value is maintained at 270-330 mOsmol / kg, it is better for the exosomes to maintain their morphology and function. Exosomes are relatively stable in a neutral environment, and the stability of the exosomes is better when the pH of the freeze-dried powder is 5.0-8.0.

[0063] The results show that the osmotic pressure and pH did not change significantly before and after freeze-drying, indicating that the freeze-drying process of the present invention has little effect on the osmotic pressure and pH of the solution. Except for the high osmotic pressure result of Comparative Example 4, the osmotic pressure and pH measurement results of the other embodiments and comparative examples are within the normal range.

[0064] 4. NTA detection of lyophilized exosomes The particle size and concentration of the exosomes before and after freeze-drying in the examples and comparative examples were measured by particle size analysis (NTA). The specific test results are shown in Table 5 and Figure 3 and Figure 4 .

[0065] Table 5 Particle size and concentration measurement results

[0066] From the results of particle size and concentration determination, it can be seen that the particle size and concentration of the samples in the examples did not change significantly before and after freeze-drying, the particle size change before and after freeze-drying was within 5%, and the concentration recovery rate (by number of particles) was above 85%. In the comparative examples, the particle size generally increased after freeze-drying, suggesting that there may be aggregation of exosomes, and the concentration decreased significantly or increased abnormally. In other words, from the perspective of particle size and concentration changes, the freeze-drying effect of the formulation schemes of comparative examples 1-9 is poor, and the consistency of particle size or concentration after freeze-drying cannot be guaranteed, and the activity and efficacy of exosomes after freeze-drying are questionable. The freeze-drying protective solution of the present invention can keep the exosomes in their original structure without aggregation.

[0067] 5. Transmission electron microscopy observation of lyophilized exosomes The morphology of exosomes in the lyophilized powder of Example 8 was observed using a transmission electron microscope. The specific process was as follows: 2 ml of sterile water for injection was taken to reconstitute the lyophilized powder, and then the reconstituted lyophilized powder solution was observed using a transmission electron microscope. The results are shown in the attached Figure 2 It can be seen from the transmission electron microscopy image that the freeze-dried exosomes of the present invention present a typical "cup-and-disc" morphology and maintain good integrity, which can fully prove the existence of exosomes in the freeze-dried powder.

[0068] 6. Characterization of marker proteins of exosome lyophilized powder The exosome surface marker proteins (CD9, CD63, CD81) of the freeze-dried powder of the example were detected. The specific process is: take 2 ml of sterile injection water, re-dissolve the freeze-dried powder, and then take the exosome sample for fluorescence labeling and nanoflow detection. The schematic diagram of the fluorescence detection of the marker proteins of the samples of Example 8 and Example 9 is attached. Figure 5 , all data results are shown in Table 6.

[0069] Table 6 Positive rate of exosome surface marker protein expression

[0070]

[0071] As shown in Table 6, before and after freeze-drying, CD9, CD63, and CD81 exosome specific marker proteins were all significantly expressed in the exosome samples of the embodiment, and the positive rates before and after freeze-drying were relatively close. However, in the samples of Comparative Example 9 (PBS control group), the positive rate of exosome surface protein expression after freeze-drying was significantly reduced compared with that before freeze-drying.

[0072] 7. Detection of biological activity of lyophilized exosomes The CCK-8 method was used to detect the effect of the lyophilized exosome powder of the example on the growth of human dermal fibroblasts (HDF cells) for 72 hours, and compared with the PBS control group and comparative example 9 (PBS lyophilized group) not treated with exosomes to detect the biological activity of the lyophilized exosome powder in promoting cell proliferation.

[0073] Experimental methods: 1. Inoculate HDF cells into a 96-well plate, with 3000 cells per well. After the cells have grown for 24 hours, discard the culture medium and replace it with 1.0×1010 Particle / ml exosome solution.

[0074] 2. The cells were divided into different groups. One group was added with 100ul PBS (control group), one group was added with 100ul non-lyophilized exosomes (fresh stock solution group), and the rest were added with 100ul lyophilized powder solution of exosomes in Example / Comparison (lyophilized powder was dissolved with sterile water for injection), with four parallel groups in each group.

[0075] 3. At 72 hours after treatment, the growth of human dermal fibroblasts was detected using the CCK-8 method. The growth of human fibroblasts without exosomes (control group) was used as a benchmark to calculate the proliferation-promoting effect of fresh exosome solution and freeze-dried powder of the example on cells. The calculation formula is as follows:

[0076] The experimental results are as attached Figure 5 As shown, the blank PBS control group could not promote the proliferation of fibroblasts, while the exosome fresh stock solution group and the freeze-dried powder example group both had good cell proliferation promoting activity. The freeze-dried exosome powder of Example 8 had the best proliferation promoting effect, while the proliferation promoting activity of Comparative Example 9 was significantly lower.

[0077] Table 7 Cell proliferation rate promoted by lyophilized exosomes

[0078] 8. Preparation of Exosome Essence / Mask Step 1: Prepare essence / mask stock solution: Essence stock solution: Weigh 0.25% W / V sodium hyaluronate, 0.75% W / V propylene glycol, and 0.5% W / V glycerol, add to water for injection, stir thoroughly to dissolve, and obtain the essence stock solution.

[0079] Mask stock solution: Weigh 0.18% W / V sodium hyaluronate, 1.35% W / V propylene glycol, and 0.9% W / V glycerin, add to water for injection, stir thoroughly to dissolve, and obtain the mask stock solution.

[0080] Step 2: Prepare exosome serum / mask Exosome essence: Measure 4 ml of the essence obtained in step 1, pour it into 1 part of the exosome freeze-dried powder prepared in Example 8, shake until completely dissolved, and obtain exosome essence, which can be introduced through microneedles or directly applied to the face.

[0081] Exosome mask: Weigh 20 ml of the mask stock solution obtained in step 1, pour it into 1 part of the exosome freeze-dried powder prepared in Example 8, shake until completely dissolved, and obtain the exosome mask solution, which can be applied to the face with mask paper.

[0082] The osmotic pressure, pH, particle size, concentration and exosome surface marker proteins (CD9, CD63, CD81) of the exosome freeze-dried powder and the essence / mask before and after preparation were characterized to observe the effect of the essence / mask prescription on the exosome freeze-dried powder. The results are shown in Table 8.

[0083] Table 8 Characterization of lyophilized exosome powder, essence and mask

[0084] The results show that the osmotic pressure and pH value of the reconstituted freeze-dried powder, its essence and mask are within the normal range, the particle size of the essence and mask has no significant change, and the concentration is close to the theoretical concentration. CD9, CD63, CD81, these exosome-specific marker proteins are all significantly expressed. From the above characterization results, it can be seen that the essence / mask prescription has no significant effect on the exosome freeze-dried powder.

[0085] 9. Detection of exosome bioactivity of serum The CCK-8 method was used to detect the effects of exosome essence, lyophilized powder and original solution on the growth of human dermal fibroblasts (HDF cells) at 0 / 24h / 48h / 72h, and the biological activity of exosome essence in promoting cell proliferation was detected by comparing with the auxiliary solution control group without exosome treatment.

[0086] Test subjects: Exosome essence, lyophilized powder and original solution.

[0087] Experimental methods: 1. Inoculate HDF cells into a 96-well plate, with 3000 cells per well. After the cells have grown for 24 hours, discard the culture medium and replace it with 3.0E+09 particles / ml of exosome solution.

[0088] 2. The cells were divided into exosome essence group, exosome freeze-dried preparation group and exosome stock solution group. Each group had a control group. The essence control group was added with 100ul of essence auxiliary material solution, the freeze-dried preparation control group was added with 100ul of freeze-dried auxiliary material solution, and the exosome stock solution control group was added with 100ul of PBS dilution solution. Each group had eight parallels.

[0089] 3. The CCK-8 method was used to detect the growth of human dermal fibroblasts with and without the addition of exosomes at 0h, 24h, 48h, and 72h after treatment.

[0090] Activity test results are attached Figure 7 The experimental results showed that compared with their respective control groups, the exosome essence group, the exosome freeze-dried preparation group and the exosome stock solution group all had significant cell proliferation promoting activity, and the cell proliferation curves of the three exosome groups were consistent, indicating that the exosome essence had good pro-proliferation activity.

[0091] In summary, the present invention mixes the exosome stock solution with the freeze-dried protective solution and then sequentially undergoes pre-freezing, pre-vacuuming, primary drying, and analytical drying treatment. The particle size and concentration of the obtained exosome freeze-dried powder are not significantly different from those of the exosome stock solution. The exosome-specific proteins CD9, CD63, and CD81 are all significantly expressed, and the activity of promoting fibroblast proliferation is consistent with that of the exosome stock solution. The application of exosome freeze-dried powder, after preparing it into essence and mask, the activity remains stable. The application of exosome freeze-dried powder in the beauty industry can be used for wrinkle removal, anti-aging, improving skin firmness, and improving skin glossiness, which is safe and effective.

[0092] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. An exosome lyophilized powder, which is prepared by mixing an exosome solution and a lyophilization protection solution and then freeze-drying, characterized in that: Based on the concentration of each substance in the mixed solution, the exosome solution is 0.1%~10%V / V, the lyophilization protection solution includes 0.4%~4%W / V of sugars and 0.25%~4%W / V of alcohols; the sugars are any one of trehalose, sucrose or lactose; the alcohols are mannitol, sorbitol or a combination of mannitol and polyethylene glycol; the solvent is sterile water for injection or PBS buffer.

2. The exosome freeze-dried powder according to claim 1, characterized in that The freeze-dried protective solution also includes 0.1% to 0.5% W / V of protein substances; the protein substances include one or more of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

3. The exosome freeze-dried powder according to claim 1, characterized in that The sugar is trehalose, the alcohol is a combination of mannitol and polyethylene glycol, and the solvent is sterile water for injection.

4. The exosome freeze-dried powder according to claim 1, characterized in that The exosomes in the exosome solution are mesenchymal stem cell exosomes.

5. The method for preparing the lyophilized exosome powder according to any one of claims 1 to 4, characterized in that: The following steps are involved: A. Weigh the raw materials of each component of the freeze-dried protective solution according to the proportion of each substance; B. dissolving the above raw materials in a solvent, filtering and sterilizing through a 0.22 μm filter membrane to obtain a lyophilized protective agent solution; C. taking the exosome solution and mixing it evenly with the lyophilization protective agent solution to obtain a pre-lyophilization solution; D. The pre-lyophilized solution is dispensed into sterile vials, and after vacuum freeze-drying, lyophilized exosome powder is obtained.

6. The preparation method according to claim 5, characterized in that: The vacuum freeze drying described in step D comprises: D1. Pre-freezing: temperature is -30~-50℃, setting time is 1~3h, duration is 2~5h, target vacuum degree is 1000mbar; D2. Pre-vacuum: 0.15mbar, alarm vacuum: 0.8mbar, pre-vacuum duration: 60s; D3. Sublimation drying: temperature is -10~-25℃, setting time is 1~4h, duration is 18~28h, target vacuum degree is 0.1~0.15mbar; D4. Analytical drying: temperature is 20~25℃, set time is 3~8h, duration is 8~20h, target vacuum degree: 0~0.1mbar.

7. An exosome essence or exosome mask, characterized in that: It comprises the lyophilized exosome powder according to any one of claims 1 to 4 or the lyophilized exosome powder prepared by the preparation method according to claim 5 or 6, a reconstitution solvent, a moisturizer and a skin conditioner.

8. The exosome essence or exosome mask according to claim 7, characterized in that: The moisturizer includes 0.1% to 2% W / V propylene glycol and 0.5% to 5% W / V glycerin, the skin conditioner includes 0.1% to 0.5% W / V sodium hyaluronate and 1% to 3% W / V niacinamide, and the reconstitution solvent is water for injection.

9. The method for preparing the exosome essence or exosome mask according to claim 7 or 8, characterized in that: include: 1) Preparing an essence stock solution or a facial mask stock solution: weighing a moisturizer and a skin conditioner, adding them to a re-dissolving solvent, stirring and dissolving, and obtaining an essence stock solution or a facial mask stock solution; 2) Pour the essence solution or mask solution into the exosome freeze-dried powder and shake until it is completely dissolved to obtain the exosome essence solution or exosome mask.

10. Use of the lyophilized exosome powder according to any one of claims 1 to 4 or the lyophilized exosome powder prepared by the preparation method according to claim 5 or 6 in the preparation of skin care products or cosmetics.

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