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

Abstract

The invention belongs to the technical field of exosome freeze-dried powder, and discloses exosome freeze-dried powder and a preparation method thereof, the exosome freeze-dried powder contains mesenchymal stem cell exosome, trehalose, sodium hyaluronate, hydroxypropyl-beta-cyclodextrin, glutathione, ceramide and fructo-oligosaccharide. According to the exosome freeze-dried powder, hydroxypropyl-beta-cyclodextrin, glutathione, ceramide and fructo-oligosaccharide are added, the membrane structure integrity of the exosome is effectively protected, the retention rate of active substances in the exosome freeze-dried powder is high, and therefore the effects of resisting inflammation, regulating cell metabolism, promoting tissue repair and the like of the exosome can be fully exerted. In addition, glutathione can directly remove skin free radicals and inhibit oxidative stress injury, and has an anti-oxidation effect; ceramide can also supplement skin lipid barrier, enhance the water locking capacity and enhance the moisturizing effect of sodium hyaluronate; fructo-oligosaccharide can also regulate skin micro-ecological balance, and has anti-inflammatory and bacteriostatic effects.

Description

Technical Field

[0001] This invention relates to the field of exosome lyophilized powder technology, and more specifically, to an exosome lyophilized powder, its preparation method, and its application. Background Technology

[0002] Exosome freeze-dried powder is a powdered biological product made from exosomes secreted by cells as the core raw material, which is separated, purified and freeze-dried at low temperature. The main components include exosomes (active substances include growth factors, proteins, nucleic acids and lipids), trehalose, sodium hyaluronate, etc. It is mainly used in the fields of facial anti-aging, fading of pigmentation and acne marks, tissue damage repair and immune regulation.

[0003] The core of the exosome lyophilized powder's function is that exosomes act as "cell messengers," delivering bioactive substances to target cells through two mechanisms: fusion of the lipid layer with the target cell and receptor-mediated endocytosis. This regulates gene expression and metabolic function in target cells, promotes the expression of related genes, thereby modulating immune responses, inhibiting inflammation, and promoting related cell proliferation and tissue repair.

[0004] Currently, the main components of exosome lyophilized powder are mesenchymal stem cell exosomes, trehalose, and sodium hyaluronate. Although it has anti-inflammatory and repair effects, its efficacy is limited due to the low retention rate of active substances. The low retention rate of active substances in exosome lyophilized powder is due to two main reasons. First, the exosome membrane structure is fragile, and the components of the lyophilized powder form solid ice crystals with sharp edges under low-temperature freezing conditions. These crystals can physically puncture and damage the exosome membrane, causing sudden changes in external osmotic pressure and friction between membrane lipids after exosome aggregation, leading to membrane rupture and leakage of active substances. Second, exosomes are prone to membrane lipid oxidation and protein denaturation during storage, resulting in the loss of activity of some active substances. Summary of the Invention

[0005] This invention provides an exosome lyophilized powder with high activity retention and high stability.

[0006] Another object of the present invention is to provide a method for preparing exosome lyophilized powder.

[0007] Another object of the present invention is to provide the application of the exosome lyophilized powder in the preparation of cosmetics.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A lyophilized exosome powder contains mesenchymal stem cell exosomes, trehalose, sodium hyaluronate, hydroxypropyl-β-cyclodextrin, glutathione, ceramide, and fructooligosaccharides.

[0009] The exosome lyophilized powder of this invention incorporates hydroxypropyl-β-cyclodextrin, glutathione, ceramide, and fructooligosaccharides. Ceramide can intercalate within the exosome lipid bilayer, repairing damaged membranes, enhancing membrane stability, reducing the damage to membrane permeability caused by low temperatures, and maintaining membrane structural integrity. Fructooligosaccharides can regulate the system's osmotic pressure, reduce ice crystal formation, and alleviate the physical puncture damage to vesicle structures caused by ice crystals. Hydroxypropyl-β-cyclodextrin can form hydrophobic cavities to encapsulate the exosome membrane, preventing membrane rupture caused by friction between membrane lipids due to exosome aggregation. Glutathione, as an antioxidant, can prevent lipid oxidation and protein denaturation, thereby preventing exosome membrane rupture. All these components effectively protect the structural integrity of the exosome membrane, and the lyophilized exosome powder retains a high rate of active substances, thus fully leveraging the anti-inflammatory, cell metabolism-regulating, and tissue-repairing effects of exosomes.

[0010] In addition, glutathione can directly scavenge free radicals in the skin and inhibit oxidative stress damage, thus having antioxidant effects; ceramides can replenish the skin's lipid barrier, enhance water-locking ability, and enhance the moisturizing effect of sodium hyaluronate; and fructooligosaccharides can regulate the skin's microecological balance, thus having antibacterial and anti-inflammatory effects.

[0011] Preferably, the exosome lyophilized powder also contains human serum albumin and platelet-rich growth factor lyophilized powder, vitamin E, and ginsenoside Rh2.

[0012] The components of the exosome lyophilized powder of the present invention include: (1) Platelet-rich lyophilized powder containing multiple growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), which can directly stimulate the proliferation of human dermal fibroblasts and collagen synthesis, accelerate the repair of damaged skin barrier, and form a synergistic effect with growth factors EGF and bFGF in exosomes to enhance the repair efficacy of exosome lyophilized powder; (2) Vitamin E and glutathione form an antioxidant network, which not only prevents lipid oxidation and protein denaturation, thus preventing exosome membrane rupture, but also enhances antioxidant effects; (3) Ginsenosides can scavenge free radicals, inhibit the generation of reactive oxygen species, inhibit the oxidative degradation of EGF, bFGF and platelet-rich growth factor in exosomes, and prolong the storage stability of freeze-dried powder; they can also inhibit the activity of matrix metalloproteinases (MMPs), reduce collagen loss, delay the formation of skin wrinkles and sagging, and work synergistically with ceramides and sodium hyaluronate to achieve the dual effects of "anti-aging + water-locking and moisturizing".

[0013] Preferably, the content of epidermal growth factor (EGF) in the mesenchymal stem cell exosomes in the exosome lyophilized powder is ≥90 pg / mL, and the content of basic fibroblast growth factor (bFGF) is ≥70 pg / mL.

[0014] Preferably, the mass fractions of each component in the exosome lyophilized powder are: 40-65 portions of mesenchymal stem cell exosomes, Trehalose 8-12 parts, 3-6 parts sodium hyaluronate 5-8 parts of hydroxypropyl-β-cyclodextrin Glutathione 1-3 parts, Ceramide 0.8~1.5 parts, 5-8 parts of fructooligosaccharides.

[0015] More preferably, the mass fractions of each component of the exosome lyophilized powder are: Human serum albumin 2-5 parts, 3-5 parts of platelet-rich growth factor lyophilized powder Vitamin E 0.5-1.5 parts, Ginsenoside Rh2 1-2 parts.

[0016] This invention also provides a method for preparing the exosome lyophilized powder according to any one of the above claims, comprising the following steps: S1. Exosome preparation: Mesenchymal stem cells were cultured and purified to obtain stem cell exosomes; S2. Take two portions of PBS buffer. Add trehalose, hydroxypropyl-β-cyclodextrin, glutathione, and fructooligosaccharide to one portion of PBS buffer and dissolve to obtain solution A. Add sodium hyaluronate and ceramide to the other portion of PBS buffer and dissolve to obtain solution B. S3. Add exosomes to solution A in proportion and incubate, then slowly add solution B and stir to obtain exosome lyophilized solution; S4. The exosome lyophilized solution is pre-frozen at -45~-35℃ for 2.5~3.5 h, deeply pre-frozen at -85~-75℃ for 10~12 h, and then annealed twice to obtain exosome lyophilized powder. The second annealing operation is to raise the temperature to -12~-8℃ and hold for 1.5~2.5 h, and then lower the temperature to -45~-35℃ and hold for 50~70 min. The preparation method provided by this invention can produce the above-mentioned exosome lyophilized powder. In the preparation method: S4 breaks down the conventional operation step of "directly freezing the exosome lyophilized liquid at -80℃" into two steps: pre-freezing at -45~-35℃ and deep pre-freezing at -85~-75℃, to avoid the formation of large ice crystals in the exosome lyophilized liquid that puncture the exosome membrane; secondary annealing can effectively eliminate ice crystal residue and avoid puncturing the exosome membrane.

[0017] Preferably, during the S1 culture process, 0.05-0.15% ascorbic acid is added to the culture medium.

[0018] This invention adds 0.05-0.15% ascorbic acid to the culture medium to promote the secretion of growth factors EGF and bFGF in exosomes.

[0019] Preferably, the S1 purification process involves three-stage centrifugation, specifically: the supernatant obtained from the culture is centrifuged sequentially at 2500-3500g in the first stage, 8000-10000g in the second stage, the supernatant after centrifugation is mixed with sucrose buffer, and finally centrifuged in the third stage at 100000-130000g, followed by washing and resuspending to obtain mesenchymal stem cell exosomes.

[0020] The S1 purification method described in this invention is as follows: the supernatant obtained from culture is centrifuged sequentially at 2500-3500g and 2-6℃ for 8-12 min to remove cell debris, centrifuged at 8000-10000g and 2-6℃ for 25-35 min to remove microvesicles, and then the supernatant is mixed with 25-35% (w / v) sucrose buffer at a volume ratio of (2.5-3.5):1 and centrifuged at 100000-130000g and 2-6℃ for 100-140 min.

[0021] The specific washing and resuspending operations in S1 of this invention are as follows: After three-stage centrifugation, the intermediate milky white exosome layer is collected, washed twice with PBS buffer, centrifuged at 90,000-110,000g at 2-6℃ for 50-70 min, the precipitate is collected and resuspended to a concentration of 5×10⁻⁶. 10 ~8×10 10 per mL.

[0022] Preferably, the S2 process for preparing solution B employs intermittent ultrasonic dissolution with the following parameters: ultrasonic power of 80~120W, power of 35~45Hz, duration of a single ultrasonic session of 4~6min, pause duration of 0.8~1.2min, and total duration of ultrasonic dissolution of 12~18min.

[0023] In the other PBS buffer solution described in S2 of this invention, platelet-rich growth factor is first added to dissolve the PBS, followed by the addition of ginsenoside Rh2, ceramide, and sodium hyaluronate. The ultrasonic power is 80-120W, the frequency is 35-45Hz, the duration of a single ultrasonic session is 4-6 minutes, the pause time is 0.8-1.2 minutes, and the total duration of ultrasonic sessions is 12-18 minutes, resulting in solution B.

[0024] The final concentration of exosomes in the exosome lyophilization solution described in S3 of this invention is 2 × 10⁻⁶. 10 ~5×10 10 per mL.

[0025] The present invention also provides the use of the exosome lyophilized powder described in any of the above claims in the preparation of cosmetics. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments.

[0027] The platelet-rich growth factor lyophilized powder described in this invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model PRP-001 platelet-rich growth factor lyophilized powder.

[0028] Example 1 A lyophilized exosome powder contains the following components in parts by weight: The composition of the mesenchymal stem cell exosomes consisted of 50 parts, trehalose 10 parts, sodium hyaluronate 5 parts, hydroxypropyl-β-cyclodextrin 6 parts, glutathione 2 parts, ceramide 1.0 part, fructooligosaccharides 6 parts, human serum albumin 3 parts, platelet-rich growth factor lyophilized powder 4 parts, vitamin E 1.0 part, and ginsenoside Rh2 1.5 parts. The epidermal growth factor (EGF) content in the mesenchymal stem cell exosomes was 100 pg / mL, and the basic fibroblast growth factor (bFGF) content was 80 pg / mL.

[0029] The method for preparing the exosome lyophilized powder includes the following steps: S1. Exosome Preparation: Mesenchymal stem cells are cultured and purified to obtain mesenchymal stem cell exosomes, specifically including the following operations: S11. Mesenchymal stem cell culture: P4 generation human umbilical cord mesenchymal stem cells were seeded in DMEM / F12 serum-free medium (pH 7.25) containing 2% B27 additive and 0.1% ascorbic acid, and cultured at 37℃ and 5% CO2 until confluence reached 83%. The medium was replaced with fresh medium and cultured for 60 h. The supernatant was collected.

[0030] S12. Mesenchymal stem cell purification: The collected supernatant was subjected to three stages of centrifugation, washing, and resuspending to obtain mesenchymal stem cell exosomes, wherein: The specific procedure for three-stage centrifugation is as follows: First-stage centrifugation at 3000g and 4℃ for 10 min to remove cell debris; second-stage centrifugation at 9000g and 4℃ for 30 min to remove microvesicles; then the supernatant is mixed with 30% (w / v) sucrose buffer at a volume ratio of 3.0:1 and centrifuged at 120000g and 4℃ for 120 min. The specific washing and resuspending procedures are as follows: After three-stage centrifugation, collect the intermediate milky white exosome layer, wash twice with PBS buffer, centrifuge at 100,000g and 4℃ for 60 min, collect the precipitate and resuspend to a concentration of 6.5 × 10⁻⁶. 10 per mL.

[0031] S2. Take two portions of PBS buffer. Add trehalose, hydroxypropyl-β-cyclodextrin, glutathione, fructooligosaccharides, vitamin E, and human serum albumin to one portion of the PBS buffer. Stir at 300 rpm for 30 min at 4°C to dissolve. Filter through a 0.22 μm filter membrane to obtain solution A. First, add platelet-rich growth factor to the other portion of the PBS buffer to dissolve, and then add sodium hyaluronate, ceramide, and ginsenoside Rh2 to dissolve to obtain solution B. In the preparation of solution B in S2, intermittent ultrasonic dissolution is used with the following parameters: ultrasonic power of 100 W, power of 40 Hz, single ultrasonic duration of 5 min, pause duration of 1.0 min, and total ultrasonic duration of 15 min.

[0032] S3. Add exosomes to solution A according to the specified ratio and incubate at 4℃ for 1 hour. Then slowly add solution B and stir at 150 rpm and 4℃ for 30 minutes to obtain a lyophilized exosome solution with an exosome concentration of 3.5 × 10⁻⁶. 10 cells / mL; S4. The exosome lyophilized solution was dispensed into sterile vials, pre-frozen at -40℃ for 3.5 h and then pre-frozen at -80℃ for 11 h. It was then transferred to a freeze dryer and the two annealing steps were repeated twice. Finally, the vacuum degree of the freeze dryer was reduced to 13 Pa, and the temperature was increased to -10℃ at a rate of 1℃ / h and held for 13 h. Then the temperature was increased to 25℃ at a rate of 2℃ / h and held for 9 h until the moisture content was ≤3%. After vacuum sealing, nitrogen gas with a purity of ≥99.99% was introduced and sealed to obtain exosome lyophilized powder. The secondary annealing operation involves heating to -10℃ and holding for 2.0 hours, then cooling to -40℃ and holding for 60 minutes.

[0033] Example 2 This embodiment is the second embodiment of the present invention. Unlike embodiment 1, the exosome lyophilized powder contains the following components in parts by weight: 40 parts of mesenchymal stem cell exosomes, 8 parts of trehalose, 6 parts of sodium hyaluronate, 5 parts of hydroxypropyl-β-cyclodextrin, 3 parts of glutathione, 0.8 parts of ceramide, 5 parts of fructooligosaccharides, 2 parts of human serum albumin, 3 parts of lyophilized platelet-rich growth factor powder, 0.5 parts of vitamin E, and 1 part of ginsenoside Rh2.

[0034] Example 3 This embodiment is the third embodiment of the present invention. Unlike embodiment 1, the exosome lyophilized powder contains the following components in parts by weight: Mesenchymal stem cell exosomes 65 parts, trehalose 12 parts, sodium hyaluronate 3 parts, hydroxypropyl-β-cyclodextrin 8 parts, glutathione 1 part, ceramide 1.5 parts, fructooligosaccharides 8 parts, human serum albumin 5 parts, platelet-rich growth factor lyophilized powder 5 parts, vitamin E 1.5 parts, ginsenoside Rh2 2 parts.

[0035] Example 4 This embodiment is the fourth embodiment of the present invention. Unlike embodiment 1, the components of the exosome lyophilized powder do not contain human serum albumin and platelet-rich growth factor lyophilized powder, vitamin E and ginsenoside Rh2.

[0036] In the preparation method of the exosome lyophilized powder, S2. Take two portions of PBS buffer. Add trehalose, hydroxypropyl-β-cyclodextrin, glutathione and fructooligosaccharide to one portion of the PBS buffer, stir at 300 rpm for 30 min at 4°C to dissolve, filter through a 0.22 μm filter membrane to obtain solution A. Add sodium hyaluronate and ceramide to the other portion of the PBS buffer to dissolve to obtain solution B. In the preparation of solution B in S2, intermittent ultrasonic dissolution is used with the following parameters: ultrasonic power of 100 W, power of 40 Hz, single ultrasonic duration of 5 min, pause duration of 1.0 min, and total ultrasonic duration of 15 min.

[0037] Example 5 This embodiment is the fifth embodiment of the present invention. The difference from embodiment 1 is that in the preparation method of the exosome lyophilized powder, the three-stage centrifugation in S12 is as follows: first-stage centrifugation at 2500g and 2℃ for 8 min to remove cell debris; second-stage centrifugation at 8000g and 2℃ for 25 min to remove microvesicles; then the supernatant is mixed with 25% (w / v) sucrose buffer at a volume ratio of 2.5:1 and centrifuged at 100000g and 2℃ for 100 min.

[0038] Example 6 This embodiment is the sixth embodiment of the present invention. Unlike embodiment 1, in the preparation method of the exosome lyophilized powder, the three-stage centrifugation in S12 is specifically performed as follows: first-stage centrifugation at 3500g and 6℃ for 12min to remove cell debris; second-stage centrifugation at 10000g and 6℃ for 35min to remove microvesicles; then the supernatant is mixed with 35% (w / v) sucrose buffer at a volume ratio of 3.5:1 and centrifuged at 130000g and 6℃ for 140min.

[0039] Example 7 This embodiment is the 7th embodiment of the present invention. Unlike embodiment 1, in the preparation method of the exosome lyophilized powder, ascorbic acid is not added to the DMEM / F12 serum-free culture medium in S11; the content of epidermal growth factor EGF in the mesenchymal stem cell exosomes is 95 pg / mL, and the content of basic fibroblast growth factor bFGF is 74 pg / mL.

[0040] Example 8 This embodiment is the 8th embodiment of the present invention. Unlike embodiment 1, in the preparation method of the exosome lyophilized powder, intermittent ultrasonic dissolution was not used in the preparation of solution B in S2. Instead, platelet-rich growth factor was first added to PBS buffer to dissolve the exosomes, and then sodium hyaluronate, ceramide and ginsenoside Rh2 were added. The mixture was stirred at 300 rpm for 30 min to dissolve the exosomes and obtain solution B.

[0041] Example 9 This embodiment is the 9th embodiment of the present invention. The difference from embodiment 1 is that in the preparation method of the exosome lyophilized powder, the pre-freezing operation in S4 is: pre-freezing at -45℃ for 2.5h and deep pre-freezing at -85℃ for 10h; the secondary annealing operation is to raise the temperature to -12℃ and hold for 1.5h, and then lower the temperature to -45℃ and hold for 50min.

[0042] Example 10 This embodiment is the 10th embodiment of the present invention. Unlike embodiment 1, in the preparation method of the exosome lyophilized powder, the pre-freezing operation in S4 is: pre-freezing at -35℃ for 3.5 h and deep pre-freezing at -75℃ for 12 h; the secondary annealing operation is to raise the temperature to -8℃ and hold for 2.5 h, and then lower the temperature to -35℃ and hold for 70 min.

[0043] Example 11 This embodiment is the 11th embodiment of the present invention. Unlike embodiment 1, the exosome lyophilized powder contains the following components in parts by weight: The mesenchymal stem cell exosomes contained 40 parts, trehalose 8 parts, sodium hyaluronate 6 parts, hydroxypropyl-β-cyclodextrin 5 parts, glutathione 3 parts, ceramide 0.8 parts, and fructooligosaccharides 5 parts; the epidermal growth factor (EGF) content in the mesenchymal stem cell exosomes was 91 pg / mL, and the basic fibroblast growth factor (bFGF) content was 70 pg / mL.

[0044] In the method for preparing exosome lyophilized powder: S11 DMEM / F12 serum-free medium does not contain ascorbic acid.

[0045] The specific operation of the three-stage centrifugation in S12 is as follows: First stage centrifugation at 2500g and 2℃ for 8 min to remove cell debris; second stage centrifugation at 8000g and 2℃ for 25 min to remove microvesicles; then the supernatant is mixed with 25% (w / v) sucrose buffer at a volume ratio of 2.5:1 and centrifuged at 100000g and 2℃ for 100 min. The specific washing and resuspending procedures are as follows: After three-stage centrifugation, collect the intermediate milky white exosome layer, wash twice with PBS buffer, centrifuge at 90,000g and 2℃ for 50 min, collect the precipitate and resuspend to a concentration of 5×10⁻⁶. 10 10 10 per mL.

[0046] In S2, two portions of PBS buffer were taken. Trehalose, hydroxypropyl-β-cyclodextrin, glutathione, and fructooligosaccharides were added to one portion of the PBS buffer and stirred at 300 rpm for 30 min at 4°C to dissolve the solution. The solution was then filtered through a 0.22 μm filter to obtain solution A. Sodium hyaluronate and ceramide were added to the other portion of the PBS buffer and dissolved to obtain solution B. In the preparation of solution B in S2, intermittent ultrasonic dissolution was used with the following parameters: ultrasonic power of 80 W, power of 35 Hz, single ultrasonic duration of 4 min, pause duration of 0.8 min, and total ultrasonic duration of 12 min.

[0047] Exosome concentration in S3 was 2 × 10⁻⁶ 10 per mL.

[0048] The pre-freezing operation in S4 is as follows: pre-freeze at -35℃ for 3.5 h and deep pre-freeze at -75℃ for 12 h; the secondary annealing operation is to raise the temperature to -8℃ and hold for 2.5 h, and then lower the temperature to -35℃ and hold for 70 min.

[0049] Example 12 This embodiment is the 12th embodiment of the present invention. Unlike embodiment 1, the exosome lyophilized powder contains the following components in parts by weight: The mesenchymal stem cell exosomes contained 65 parts, trehalose 12 parts, sodium hyaluronate 3 parts, hydroxypropyl-β-cyclodextrin 8 parts, glutathione 1 part, ceramide 1.5 parts, and fructooligosaccharides 8 parts; the epidermal growth factor (EGF) content in the mesenchymal stem cell exosomes was 90 pg / mL, and the basic fibroblast growth factor (bFGF) content was 71 pg / mL.

[0050] In the method for preparing exosome lyophilized powder: S11 DMEM / F12 serum-free medium does not contain ascorbic acid.

[0051] The specific procedure for the three-stage centrifugation in S12 is as follows: First stage centrifugation at 3500g and 6℃ for 12min to remove cell debris; second stage centrifugation at 10000g and 6℃ for 35min to remove microvesicles; then the supernatant is mixed with 35% (w / v) sucrose buffer at a volume ratio of 3.5:1 and centrifuged at 130000g and 6℃ for 140min. The specific washing and resuspending procedures are as follows: After three-stage centrifugation, collect the intermediate milky white exosome layer, wash twice with PBS buffer, centrifuge at 110,000g and 6℃ for 70 min, collect the precipitate and resuspend to a concentration of 8×10⁻⁶. 10 per mL.

[0052] In S2, two portions of PBS buffer were taken. Trehalose, hydroxypropyl-β-cyclodextrin, glutathione, and fructooligosaccharides were added to one portion of the PBS buffer and stirred at 300 rpm for 30 min at 4°C to dissolve the solution. The solution was then filtered through a 0.22 μm filter to obtain solution A. Sodium hyaluronate and ceramide were added to the other portion of the PBS buffer and dissolved to obtain solution B. In the preparation of solution B in S2, intermittent ultrasonic dissolution was used with the following parameters: ultrasonic power of 120 W, power of 45 Hz, single ultrasonic duration of 6 min, pause duration of 1.2 min, and total ultrasonic duration of 18 min.

[0053] Exosome concentration in S3 was 5 × 10⁻⁶ 10 per mL.

[0054] The pre-freezing operation in S4 is as follows: pre-freeze at -45℃ for 2.5 hours and deep pre-freeze at -85℃ for 10 hours; the secondary annealing operation is to raise the temperature to -12℃ and hold for 1.5 hours, and then lower the temperature to -45℃ and hold for 50 minutes.

[0055] Comparative Example 1 This comparative example is the first comparative example of the present invention. Unlike Example 4, the exosome lyophilized powder does not contain hydroxypropyl-β-cyclodextrin.

[0056] Comparative Example 2 This comparative example is the second comparative example of the present invention. Unlike Example 4, the exosome lyophilized powder does not contain glutathione.

[0057] Comparative Example 3 This comparative example is the third comparative example of the present invention. Unlike Example 4, the exosome lyophilized powder contains hydroxypropyl-β-cyclodextrin, glutathione, ceramide, and fructooligosaccharides.

[0058] Comparative Example 4 This comparative example is the fourth comparative example of the present invention. Unlike Example 7, in the preparation process of the exosome lyophilized powder, S2 added trehalose, hydroxypropyl-β-cyclodextrin, glutathione, fructooligosaccharides, sodium hyaluronate and ceramide to PBS buffer and dissolved them to obtain solution B. S3. Add mesenchymal stem cell exosomes to solution B and incubate at 4°C for 1 hour to obtain exosome lyophilized solution.

[0059] Comparative Example 5 This comparative example is the fifth comparative example of the present invention. Unlike Example 7, the exosome lyophilized solution in S4 was directly quick-frozen at -80°C for 12 hours.

[0060] Comparative Example 6 This comparative example is the sixth comparative example of the present invention. Unlike Example 7, there is no secondary annealing step in S4.

[0061] Comparative Example 7 This comparative example is the 7th comparative example of the present invention. Unlike Example 7, in S4, the exosome lyophilized liquid was directly quick-frozen at -80°C for 12 hours, and there was no secondary annealing step.

[0062] Performance testing: The specific method for detecting the exosome activity retention rate in exosome lyophilized powder is as follows: (1) Reagent selection: EGF and bFGF dedicated ELISA kits (adapted for human growth factor detection) are used. (2) Sample processing: Take the exosome solutions before lyophilization and the exosome solutions after lyophilization and reconstituted for each example and comparative example, respectively, and dilute and incubate the samples; the reconstitution conditions are 2 mL of sterile water to reconstitute 2 mL of dispensed lyophilized powder, and let stand at room temperature for 30 s; (3) Detection procedure: Add enzyme-labeled antibody, wash and perform color development reaction, and then read the absorbance (OD value) at a wavelength of 450 nm using a Thermo Multiskan FC microplate reader. (4) Calculation method: The activity concentrations of EGF and bFGF before and after freeze-drying are calculated based on the standard curve. The activity retention rate = (activity concentration after freeze-drying / activity concentration before freeze-drying) × 100%. The average value of the detection results of the two growth factors is taken as the final activity retention rate.

[0063] The results of the exosome activity retention rate detection in the exosome lyophilized powders obtained in Examples 1-12 and Comparative Examples 1-7 are shown in Table 1.

[0064] The stability test of exosome lyophilized powder was conducted using the following method: (1) Sample preparation: The exosome lyophilized powder (2 mL / bottle) obtained in Example 4 was divided into two groups. (2) Do not place in a sealed environment at 4℃ (simulating long-term storage) or a room temperature environment at 25℃ (simulating accelerated storage); (2) Testing cycle: Samples were taken and tested at 0 months (initial state), 3 months, 6 months, 9 months, 12 months (4℃ group) and 0 months, 3 months, 6 months (25℃ group); (3) Detection indicators and methods: Activity retention rate: Same as the method for "Detection of Exosome Activity Retention Rate" (ELISA method); Particle size change rate: The particle size distribution of exosomes was detected using a nanoparticle tracking analyzer (Malvern Zetasizer Nano ZS). Particle size change rate = (average particle size after storage - initial average particle size) / initial average particle size × 100%.

[0065] The stability tests of the freeze-dried powders obtained in Examples 1, 4, 3, and 7 are shown in Table 2.

[0066] The specific detection method for the effect of exosome lyophilized powder on cell proliferation is as follows: (1) Cell culture: HDF cells were seeded in 96-well plates at a density of 5×10⁶ cells / well. 3 Cells / well were incubated at 37°C in a 5% CO2 incubator for 24 hours. (2) Group treatment: The experimental group was given different dilutions of the lyophilized powder obtained from the examples or comparative examples (final concentration containing 1×10⁻⁶ exosomes). 10 (number / mL), the control group was added with an equal volume of sterile PBS buffer, and cultured for another 24 hours; (3) Detection procedure: Add 10 μL of CCK-8 reagent to each well, incubate for 4 h, and then read the OD value at a wavelength of 450 nm using an ELISA reader; (4) Calculation method of cell proliferation rate: Proliferation rate = (OD value of experimental group / OD value of control group) × 100%.

[0067] The results of the exosome activity retention rate detection in the exosome lyophilized powders obtained in Examples 1-12 and Comparative Examples 1-7 are shown in Table 1.

[0068] The antioxidant effect of exosome lyophilized powder is specifically tested using the following methods: (1) Reagent preparation: Prepare a 0.1 mmol / L LPPH ethanol solution, and use it immediately after preparation; (2) Reaction system: Take the lyophilized powder reconstituted solution (exosome concentration 1×10⁻⁶) 10 Add 1 mL of (1 / mL) DPPH solution to 2 mL of the solution and incubate in the dark for 30 min. (3) Blank control: Use ethanol instead of the reconstituted solution and incubate under the same conditions; (4) Detection procedure: The absorbance was measured at a wavelength of 517 nm using an ultraviolet spectrophotometer (sample A). The absorbance of the blank control group was blank A. (5) Calculation method: DPPH free radical scavenging rate = (1 - A sample / A blank) × 100%.

[0069] The results of the exosome activity retention rate detection in the exosome lyophilized powders obtained in Examples 1-12 and Comparative Examples 1-7 are shown in Table 1.

[0070] The anti-inflammatory effect of exosome lyophilized powder was specifically tested using the following methods: (1) Cell induction: HDF cells were seeded in 6-well plates and cultured until the confluence reached 70%. 1 μg / mL LPS (lipopolysaccharide) was added to induce an inflammatory response for 24 h. (2) Group treatment: The experimental group was given a lyophilized powder reconstituted solution (exosome concentration 1×10⁻⁶). 10 (number / mL), the control group was added with an equal volume of sterile PBS and cultured for another 24 hours; (3) Detection procedure: Collect cell culture supernatant, use IL-6 ELISA kit, follow the instructions, read OD value with microplate reader, and calculate IL-6 concentration; (4) Calculation method: IL-6 inhibition rate = (IL-6 concentration in control group - IL-6 concentration in experimental group) / IL-6 concentration in control group × 100%.

[0071] The results of the exosome activity retention rate detection in the exosome lyophilized powders obtained in Examples 1-12 and Comparative Examples 1-7 are shown in Table 1.

[0072] Table 1. Performance testing of exosome lyophilized powders obtained in Examples 1-12 and Comparative Examples 1-7

[0073] Table 2. Stability testing of lyophilized powders obtained in Examples 1, 4, 3, and 7.

[0074] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection. Any non-substantial changes or substitutions made by those skilled in the art based on the present invention will still fall within the scope of patent protection.

Claims

1. An exosome lyophilized powder comprising mesenchymal stem cell exosomes, trehalose, sodium hyaluronate, characterized in that, It also contains hydroxypropyl-beta-cyclodextrin, glutathione, ceramide and fructooligosaccharide.

2. The exosome lyophilized powder according to claim 1, characterized in that, It also contains human serum albumin and platelet-rich growth factor lyophilized powder, vitamin E and ginsenoside Rh2.

3. The exosome lyophilized powder of claim 1, wherein, The content of epidermal growth factor EGF in the mesenchymal stem cell exosome is ≥90 pg / mL, and the content of basic fibroblast growth factor bFGF is ≥70 pg / mL.

4. The exosome lyophilized powder of claim 1, wherein, The mass parts of the components are: Mesenchymal stem cell exosome 40-65 parts, Trehalose 8-12 parts, Sodium hyaluronate 3-6 parts, Hydroxypropyl-beta-cyclodextrin 5-8 parts, Glutathione 1-3 parts, Ceramide 0.8-1.5 parts, Fructooligosaccharide 5-8 parts.

5. The exosome lyophilized powder of claim 2, wherein, The mass parts of the components are: Human serum albumin 2-5 parts, Platelet-rich growth factor lyophilized powder 3-5 parts, Vitamin E 0.5-1.5 parts, Ginsenoside Rh2 1-2 parts.

6. The method of preparing the exosome lyophilized powder according to any one of claims 1 to 5, characterized in that, It comprises the following steps: S1. Exosome preparation: mesenchymal stem cells are cultured and purified to obtain stem cell exosomes; S2. Take 2 parts of PBS buffer, one part of which is added with trehalose, hydroxypropyl-beta-cyclodextrin, glutathione and fructooligosaccharide to dissolve and obtain solution A, and the other part is added with sodium hyaluronate and ceramide to dissolve and obtain solution B; S3. Add mesenchymal stem cell exosomes to solution A for incubation, then slowly add solution B, and stir to obtain exosome lyophilized liquid; S4. The exosome lyophilized liquid is pre-frozen at -45 to -35℃ for 2.5-3.5 h, deeply pre-frozen at -85 to -75℃ for 10-12 h, and twice annealed to obtain exosome lyophilized powder, and the twice annealing operation is to warm up to -12 to -8℃ for 1.5-2.5 h, and then cool down to -45 to -35℃ for 50-70 min.

7. The method of claim 6, wherein the lyophilized exosome powder is prepared by, During the S1 culture process, 0.05-0.15% ascorbic acid is added to the culture medium.

8. The method of claim 6, wherein the exosome lyophilized powder is prepared by, During the S1 purification process, three-stage centrifugation is carried out, specifically: the supernatant obtained by culture is centrifuged at 2500-3500g for the first stage, 8000-10000g for the second stage, and then the supernatant after centrifugation is mixed with sucrose buffer, and finally centrifuged for three times at 100000-130000g, and then washed and resuspended to obtain mesenchymal stem cell exosomes.

9. The method of claim 6, wherein the lyophilized exosome powder is prepared by, During the preparation of solution B in S2, intermittent ultrasonic dissolution is used, and the parameters are: ultrasonic power 80-120 W, power 35-45 Hz, single ultrasonic time 4-6 min, pause time 0.8-1.2 min, and total ultrasonic time 12-18 min.

10. Use of the exosome lyophilized powder of any one of claims 1-5 in the preparation of cosmetics.

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