A type of deer placental stem cell exosome and its application in cosmetics

Abstract

This invention relates to the field of tissue engineering technology, specifically disclosing a deer placental stem cell exosome and its application in cosmetics. The deer placental stem cell exosome preparation includes the following steps: cleaning, mincing, enzymatically hydrolyzing, centrifuging, washing, and culturing sika deer placental tissue to obtain deer placental mesenchymal stem cells; culturing the deer placental mesenchymal stem cells in 3D and collecting the culture medium; centrifuging, filtering, and tangentially filtering the culture medium to obtain a purified exosome solution; adjusting the pH of the purified exosome solution and adding Mal-PGA solution for coupling modification to obtain a polyglutamic acid-exosome solution; adding a freeze-drying protectant to the polyglutamic acid-exosome solution and then drying to obtain the deer placental stem cell exosome. The prepared deer placental stem cell exosome can be used as an active ingredient in the preparation of cosmetics with moisturizing, repairing, soothing, and skin-improving effects.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, and more specifically, to a deer placental stem cell exosome and its application in cosmetics. Background Technology

[0002] Deer placenta, as an animal-derived bioactive raw material with high development value, contains abundant proteins, peptides, and various bioactive components. Deer placenta-derived stem cells typically possess strong proliferative capacity and good stemness maintenance ability, and their secreted exosomes are rich in growth factors, miRNAs, and other active substances, showing promising application prospects in skin care, tissue repair, and the development of bioactive products. However, current technologies for the preparation and application of deer placenta stem cell exosomes still have the following shortcomings: 1. Limitations of the preparation process: In the existing technology, stem cell culture systems mostly rely on culture media containing animal serum, which not only increases the risk of exogenous impurities, but may also lead to potential pollution problems, and is not conducive to the quality control of subsequent exosome products, making it difficult to meet the needs of large-scale production.

[0003] 2. Exosome stability issues: Natural exosomes have poor stability and are prone to aggregation, rupture, or loss of active ingredients during storage, transportation, and application. Especially in complex cosmetic or biological agent systems, the membrane structure of exosomes is easily damaged, leading to a significant decrease in their biological activity and thus affecting their efficacy.

[0004] 3. Lack of efficient purification methods: Traditional exosome purification methods, such as ultracentrifugation, are prone to causing damage, aggregation and reduced activity of exosomes. In addition, they are complicated to operate and time-consuming, which cannot meet the needs of large-scale production.

[0005] 4. Loss of activity during freeze-drying: Existing freeze-drying protection technologies often use a single protectant, which leads to structural damage and loss of activity of exosomes during freeze-drying, affecting their application efficacy and stability in cosmetics.

[0006] Therefore, there is an urgent need to develop a deer placental stem cell exosome that can not only improve the yield and purity of exosomes, but also effectively solve problems such as their stability, structural protection and biological activity.

[0007] Based on the above statements, this invention proposes a deer placental stem cell exosome and its application in cosmetics. Summary of the Invention

[0008] To address the problems of low isolation and culture efficiency, limited yield, unsatisfactory purification effect, insufficient stability, and difficulty in formulation of deer placental stem cell exosomes in existing technologies, this invention provides deer placental stem cell exosomes and their application in cosmetics.

[0009] In a first aspect, the present invention provides a deer placental stem cell exosome, which adopts the following technical solution: The method for preparing deer placental stem cell exosomes includes the following steps: S1. Collect placental tissue from artificially bred sika deer, clean it, cut it into tissue blocks, perform stepwise enzymatic digestion, collect the digestion liquid, filter, centrifuge, wash it, spread the precipitated cells on the bottom of a culture flask coated with gelatin, add modified primary culture medium, invert and adhere to the culture for 2-4 hours, then invert and perform primary culture. When the cell confluence reaches 60-70%, perform passage culture to obtain deer placental mesenchymal stem cells. S2. Deer placental mesenchymal stem cells were seeded into a modified complete culture medium for 3D cell culture. After the culture was completed, the cells were washed and replaced with serum-free culture medium for exosome collection for further culture. After the culture was completed, the culture medium was collected, centrifuged, and filtered to obtain cell supernatant. S3. The cell supernatant is concentrated and washed using a tangential flow filtration system to obtain a purified exosome solution. S4. Adjust the pH of the exosome purification solution to 7.8-8.2 with sodium bicarbonate solution, add Mal-PGA solution, stir and react. After the reaction is complete, wash and filter to obtain polyglutamic acid-exosome solution. S5. Add the freeze-drying protectant to the polyglutamic acid-exosome solution, mix well, and then dry to obtain deer placental stem cell exosomes.

[0010] Preferably, the washing step S1 refers to rinsing with PBS buffer 3-5 times.

[0011] Preferably, the stepwise enzymatic digestion process in step S1 refers to: primary digestion: adding 0.1-0.2 wt% hyaluronidase and shaking digesting at 36-38℃ for 20-30 min; secondary digestion: adding a mixed enzyme solution of 0.1-0.3 wt% collagenase type I and 0.1-0.15 wt% neutral protease and continuing digestion for 70-90 min.

[0012] Preferably, in step S1, the filtration, centrifugation, and washing process refers to: filtering through a 200-mesh sieve, collecting the filtrate, centrifuging at 1500-2000 rpm for 5-10 minutes, and washing the precipitated cells 2-3 times with PBS buffer.

[0013] Preferably, the modified primary culture medium in step S1 is a DMEM / F12 medium supplemented with 10-15% (v / v) exosome-free fetal bovine serum, 1-2 ng / mL bFGF, 5-10 ng / mL EGF, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 ng / mL deer antler polypeptide extract.

[0014] Preferably, the modified complete culture medium in step S2 is a DMEM / F12 medium supplemented with 10 g / L dextran microcarriers, 10-20 mg / L recombinant human insulin, 10-20 mg / L recombinant human transferrin, 5-10 μg / L sodium selenite, 2-3 μg / mL recombinant collagen, 10-20 ng / mL bFGF, and 5-10 ng / mL EGF.

[0015] Preferably, the washing step S2 refers to rinsing with PBS buffer 2-4 times.

[0016] Preferably, in step S2, centrifugation and filtration refer to: centrifuging for 10-20 minutes at a temperature of 0-4℃ and a rotation speed of 2000-3000 rpm, and then filtering the supernatant sequentially using filter membranes with pore sizes of 0.45μm and 0.22μm.

[0017] Preferably, the concentration and filtration process in step S3 refers to: passing the cell supernatant through a hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa, concentrating it under the conditions of transmembrane pressure of 1-1.5 bar and circulation flow rate of 200-300 mL / min, and when the volume of the concentrated solution is reduced to 1 / 10-1 / 15 of the original volume, adding PBS buffer for filtration, and repeating the filtration 2-3 times.

[0018] Preferably, the method for preparing the Mal-PGA solution in step S4 includes the following steps: A1. Mix polyglutamic acid and MES buffer evenly, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir to react, and after the reaction is complete, the activated solution is obtained. A2. Under stirring, N-(2-aminoethyl)maleimide solution was added to the activation solution and stirred to react. After the reaction was completed, it was dialyzed and dried to obtain Mal-PGA. Mal-PGA was dissolved in PBS buffer and filtered to obtain Mal-PGA solution.

[0019] Preferably, in step A1, the mass ratio of polyglutamic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and MES buffer is 1:0.3-0.4:0.2-0.3:12-15.

[0020] Preferably, the concentration of the MES buffer in step A1 is 0.1-0.2 mol / L and the pH is 5.5-6.0.

[0021] Preferably, the stirring reaction in step A1 refers to stirring the reaction at room temperature and a speed of 300-500 rpm for 2-4 hours.

[0022] Preferably, in step A2, the volume ratio of N-(2-aminoethyl)maleimide solution to activation solution is 1:5-6.

[0023] Preferably, the mass fraction of the N-(2-aminoethyl)maleimide solution in step A2 is 2-3%.

[0024] Preferably, in step A2, the stirring speed of adding the N-(2-aminoethyl)maleimide solution is 200-300 rpm.

[0025] Preferably, the stirring reaction in step A2 refers to stirring the reaction for 6-8 hours under the conditions of room temperature, protection from light, and a rotation speed of 300-400 rpm.

[0026] Preferably, in step A2, the dialysis and drying process refers to: placing the reaction solution into a dialysis bag with a molecular weight cutoff of 3-5 kDa, dialyzing it in deionized water for 36-48 hours, changing the water every 5-6 hours, and after dialysis, freeze-drying the solution in the bag at a temperature of -45°C to -55°C and a vacuum degree of ≤10 Pa for 24-48 hours.

[0027] Preferably, in step A2, filtration refers to filtration using a 0.22μm microporous membrane.

[0028] Preferably, the mass fraction of the Mal-PGA solution in step A2 is 3-5%.

[0029] Preferably, the volume ratio of exosome purification solution to Mal-PGA solution in step S4 is 10-15:1.

[0030] Preferably, the stirring reaction in step S4 refers to stirring the reaction for 2-3 hours under the conditions of avoiding light, temperature of 0-4℃, and rotation speed of 100-150rpm.

[0031] Preferably, the washing and filtration process in step S4 refers to: passing the reaction solution through a tangential flow filtration system with a molecular weight cutoff of 100 kDa, washing and filtration with PBS buffer under the conditions of transmembrane pressure of 0.3-0.8 bar and circulation flow rate of 150-200 mL / min, and repeating the washing and filtration process 2-3 times.

[0032] Preferably, in step S5, the volume ratio of the freeze-drying protectant to the polyglutamic acid-exosome solution is 1:5-8.

[0033] Preferably, the freeze-drying protectant in step S5 is composed of a mixture of 8-12% trehalose aqueous solution, 2-4% mannitol aqueous solution, and 1-2% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:2-4:1-3.

[0034] Preferably, the drying in step S5 refers to: pre-freezing in an ultra-low temperature freezer at a temperature of -75℃ to -85℃ for 4-6 hours, then freeze-drying in a freeze dryer at a cold trap temperature of -50℃ to -55℃ and a vacuum degree of ≤10Pa for 12-14 hours, then raising the temperature to -20℃ for freeze-drying for 12-14 hours, and finally raising the temperature to 0℃ for freeze-drying for 12-14 hours.

[0035] Secondly, the present invention provides the application of deer placental stem cell exosomes in the preparation of cosmetics with moisturizing, repairing, soothing and improving skin condition.

[0036] In summary, the present invention has the following beneficial effects: 1. This invention uses PBS buffer to wash deer placental tissue, followed by stepwise enzymatic digestion with hyaluronidase, collagenase type I, and neutral protease. This effectively reduces the impact of residual blood, oxidative stress products, and impurities in the placental tissue on the cell separation process, improving cell release efficiency and primary cell viability. Simultaneously, the combination of gelatin-coated adherent culture and a modified primary culture medium containing bFGF, EGF, and deer antler polypeptide extract promotes the adherent growth and expansion of deer placental mesenchymal stem cells, thus enabling the stable acquisition of highly active, uniformly sized deer placental mesenchymal stem cells suitable for subsequent culture and exosome isolation.

[0037] 2. In this invention, deer placental mesenchymal stem cells are seeded in a modified complete culture medium for 3D culture, and then replaced with serum-free culture medium in the later stage of culture. This can reduce the interference of exogenous vesicles from serum sources while improving the cell secretion activity of the cell culture system, thereby improving the yield, uniformity and biological activity of exosomes. At the same time, the pretreatment method of combining centrifugation and graded filtration can effectively remove cell debris, large particulate impurities and apoptotic bodies, providing a high-quality supernatant for subsequent exosome purification.

[0038] 3. This invention employs a tangential flow filtration system to concentrate and wash the cell supernatant, enabling the enrichment and purification of exosomes under relatively mild conditions. This avoids vesicle structure damage, aggregation, or reduced activity caused by traditional high-speed centrifugation methods, thus better preserving the integrity and biological function of exosomes. Furthermore, by coupling Mal-PGA with exosomes, this invention introduces a polyglutamic acid molecular layer onto the surface of exosomes, thereby improving the dispersion stability, hydrophilicity, and system compatibility of exosomes. This also enhances their compatibility stability and skin application performance in cosmetic systems.

[0039] 4. This invention, by adding a freeze-drying protectant composed of trehalose aqueous solution, mannitol aqueous solution, and polyvinylpyrrolidone aqueous solution to a polyglutamic acid-exosome solution and employing a staged temperature-increasing freeze-drying process, can effectively reduce the impact of ice crystal damage, osmotic pressure fluctuations, and interfacial inactivation on the structure and activity of exosomes during freeze-drying. This results in deer placental stem cell exosomes with good resolubility, storage stability, and ease of transportation. Furthermore, when applied to cosmetics, these deer placental stem cell exosomes exhibit good compatibility with conventional basic raw materials, making them suitable for preparing various cosmetic formulations such as serums, lotions, creams, and masks, demonstrating promising prospects for industrial application. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the embodiments.

[0041] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0042] The key raw materials used in this invention are sourced from the following sources: Polyglutamic acid: provided by Hebei Tuohai Biotechnology Co., Ltd. PBS buffer: Model: 10010023, provided by Zhejiang Lianshuo Biotechnology Co., Ltd. Exosome-free fetal bovine serum: Product No.: 40139ES50, provided by Yisheng Biotechnology Co., Ltd.; Deer antler polypeptide extract: Brand: Fuhe, provided by Shandong Fuhe Biotechnology Co., Ltd.; Recombinant collagen: Brand: Langbowan, RHC content: 30%, provided by Hubei Langbowan Biomedical Co., Ltd. Hyaluronidase: Brand: YHSF, Model: YHSF02, provided by Shaanxi Yihe Shuofang Health Industry Co., Ltd. Collagenase type I: Brand: sinnhu, Product No.: HC04194, provided by Shanghai sinnhu Biotechnology Co., Ltd. Neutral protease: Catalog number: 04071725, provided by Shenzhen Lefu Biotechnology Co., Ltd.; Dextran microcarrier: Brand: Zhongke Kangde, Product No.: MC-2001-Dex, provided by Jilin Zhongke Kangde Technology Co., Ltd. DMEM / F12 culture medium: Brand: AMEKO, Model: FB8162, provided by Shanghai Lusen Biotechnology Co., Ltd. Polyvinylpyrrolidone: Brand: Yingruida, CAS No.: 9003-39-8, provided by Nantong Yingruida Biotechnology Co., Ltd.

[0043] Examples 1-3 provide a deer placental stem cell exosome.

[0044] Example 1 The method for preparing Mal-PGA solution includes the following steps: A1. Control the mass ratio of polyglutamic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and MES buffer to 1:0.3:0.2:12. Mix polyglutamic acid and MES buffer (concentration of 0.1 mol / L, pH 5.5) evenly, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir the mixture at room temperature and 300 rpm for 4 hours. After the reaction is completed, the activated solution is obtained. A2. Controlling the volume ratio of N-(2-aminoethyl)maleimide solution to activation solution to 1:5, N-(2-aminoethyl)maleimide solution was added to activation solution at 200 rpm. The mixture was stirred at room temperature, protected from light, and at 300 rpm for 8 hours. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed in deionized water for 36 hours, with the water changed every 5 hours. After dialysis, the solution in the bag was freeze-dried at -45℃ and vacuum ≤10 Pa for 48 hours to obtain Mal-PGA. Mal-PGA was dissolved in PBS buffer and filtered using a 0.22 μm microporous membrane to obtain a 3% (w / w) Mal-PGA solution. The method for preparing deer placental stem cell exosomes includes the following steps: S1. Collect placental tissue from artificially bred sika deer, wash three times with PBS buffer, and cut into 2mm pieces. 3 For the tissue blocks, first add 4 times the volume of 0.1wt% hyaluronidase and digest at 36℃ with shaking for 30 min; then add a mixed enzyme solution of 0.1wt% collagenase type I and 0.1wt% neutral protease, and continue digestion at 36℃ for 90 min. Collect the digestion solution, filter through a 200-mesh sieve, collect the filtrate, and centrifuge at 1500 rpm for 10 min. Wash the precipitated cells twice with PBS buffer, and then centrifuge the precipitated cells at 1×10⁻⁶ ppm. 5 Seeds were inoculated at a density of 0.2 mL / cm² at the bottom of gelatin-coated culture flasks. 2To determine the bottom area, add 0.1% (w / v) gelatin aqueous solution and let stand at 37℃ for 50 min. Add modified primary culture medium (DMEM / F12 medium supplemented with 10% (v / v) exosome-free fetal bovine serum, 1 ng / mL bFGF, 5 ng / mL EGF, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 ng / mL deer antler polypeptide extract). Incubate at 37℃, 5% CO2 for 2 h inverted adherence culture, then invert. When cell confluence reaches 60%, add digestion solution (0.02 wt% containing 0.25 wt% trypsin). Digest the cells with EDTA solution for 3 min, observing cell morphology changes during this period. When the cells become rounded and the intercellular spaces increase, add twice the volume of complete culture medium (DMEM / F12 medium supplemented with 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to terminate the digestion reaction. Collect the culture medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and dilute the cell suspension with complete culture medium to 3 × 10⁻⁶. 4 At a density of cells / mL, at 5 × 10⁻⁶ cells / mL per bottle. 5 One cell was seeded into a new T75 culture flask containing 10 mL of complete culture medium and placed in an incubator at 37°C and 5% CO2 for passage culture. Fresh complete culture medium was replaced every 2 days. When the cell confluence reached 80%, deer placental mesenchymal stem cells were obtained. S2, Deer placental mesenchymal stem cells were prepared at a rate of 2×10⁻⁶. 5 Cells were seeded at a density of cells / mL in modified complete medium (DMEM / F12 medium supplemented with 10 g / L dextran microcarriers, 10 mg / L recombinant human insulin, 10 mg / L recombinant human transferrin, 5 μg / L sodium selenite, 2 μg / mL recombinant collagen, 10 ng / mL bFGF, and 5 ng / mL EGF) and cultured for 3D at 37°C, 5% CO2, and 80 rpm. The medium was replaced with fresh medium every 48 h for 5 days. After culturing, the cells were washed twice with PBS buffer and replaced with serum-free medium for exosome collection. The cells were then cultured under the same conditions for another 48 h. After culturing, the culture medium was collected and centrifuged at 0°C and 2000 rpm for 20 min. The supernatant was filtered through 0.45 μm and 0.22 μm pore sizes to obtain the cell supernatant. S3. The cell supernatant was concentrated by passing it through a hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa, under the conditions of transmembrane pressure of 1 bar and circulation flow rate of 200 mL / min. When the volume of the concentrated solution was reduced to 1 / 10 of the original volume, an equal volume of PBS buffer was added for washing and filtration. The washing and filtration were repeated twice to obtain the exosome purification solution. S4. Control the volume ratio of exosome purification solution to Mal-PGA solution to 10:1. Adjust the pH of exosome purification solution to 7.8 with 1M sodium bicarbonate solution. Add Mal-PGA solution and stir for 3 hours under the conditions of avoiding light, temperature 0℃, and speed 100 rpm. After the reaction is completed, pass the reaction solution through a tangential flow filtration system with a molecular weight cutoff of 100 kDa. Under the conditions of transmembrane pressure 0.3 bar and circulation flow rate 150 mL / min, wash the solution with 10 times the volume of PBS buffer. Repeat the washing and filtration twice to obtain polyglutamic acid-exosome solution. S5. Controlling the volume ratio of the freeze-drying protectant and the polyglutamic acid-exosome solution to 1:5, the freeze-drying protectant (composed of 8% trehalose aqueous solution, 2% mannitol aqueous solution and 1% polyvinylpyrrolidone aqueous solution mixed in a volume ratio of 5:2:1) was added to the polyglutamic acid-exosome solution. After mixing evenly, it was pre-frozen in an ultra-low temperature freezer at -75℃ for 6 hours, then freeze-dried in a freeze dryer at a cold trap temperature of -50℃ and a vacuum degree of ≤10Pa for 14 hours, then heated to -20℃ for 12 hours, and finally heated to 0℃ for 12 hours to obtain deer placental stem cell exosomes.

[0045] Example 2 The method for preparing Mal-PGA solution includes the following steps: A1. Control the mass ratio of polyglutamic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and MES buffer to 1:0.35:0.25:13.5. Mix polyglutamic acid and MES buffer (concentration of 0.15 mol / L, pH 5.5) evenly, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir the mixture at room temperature and 400 rpm for 3 hours. After the reaction is completed, the activated solution is obtained. A2. Controlling the volume ratio of N-(2-aminoethyl)maleimide solution to activation solution to 1:5.5, N-(2-aminoethyl)maleimide solution was added to activation solution at 250 rpm. The mixture was stirred at room temperature, protected from light, and at 350 rpm for 7 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 4 kDa and dialyzed in deionized water for 40 h, with the water changed every 5.5 h. After dialysis, the solution in the bag was freeze-dried at -50℃ and vacuum ≤10 Pa for 40 h to obtain Mal-PGA. Mal-PGA was dissolved in PBS buffer and filtered using a 0.22 μm microporous membrane to obtain a 4% (w / w) Mal-PGA solution. The method for preparing deer placental stem cell exosomes includes the following steps: S1. Collect placental tissue from artificially bred sika deer, wash four times with PBS buffer, and cut into 2mm pieces. 3 For the tissue blocks, first add 5 times the volume of 0.15wt% hyaluronidase and digest at 37°C with shaking for 25 min; then add a mixed enzyme solution of 0.2wt% collagenase type I and 0.13wt% neutral protease, and continue digestion at 37°C for 80 min. Collect the digestion solution, filter through a 200-mesh sieve, collect the filtrate, and centrifuge at 1800 rpm for 8 min. Wash the precipitated cells three times with PBS buffer, and then centrifuge the precipitated cells at 1×10⁻⁶ ppm. 5 Seeds were inoculated at a density of 0.2 mL / cm² at the bottom of gelatin-coated culture flasks. 2 To determine the bottom area, add 0.1% (w / v) gelatin aqueous solution and let stand at 37℃ for 50 min. Add modified primary culture medium (DMEM / F12 medium supplemented with 13% (v / v) exosome-free fetal bovine serum, 1.5 ng / mL bFGF, 8 ng / mL EGF, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 ng / mL deer antler polypeptide extract), and incubate at 37℃ with 5%... After inverting and adhering to the culture vessel in a CO2 incubator for 3 hours, the cells were inverted and cultured until they reached 65% confluence. A digestion solution (0.02 wt% EDTA solution containing 0.25 wt% trypsin) was added, and the cells were digested for 4 minutes, observing cell morphology changes during this time. When the cells became rounded and the intercellular spaces increased, three times the volume of complete culture medium (DMEM / F12 medium supplemented with 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) was added to terminate the digestion reaction. The culture medium was collected, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell suspension was diluted with complete culture medium to 3 × 10⁶ cells / mL. 4 At a density of cells / mL, at 6×10⁶ cells / mL per bottle. 5 One cell was seeded into a new T75 culture flask containing 13 mL of complete culture medium and placed in an incubator at 37°C and 5% CO2 for passage culture. Fresh complete culture medium was replaced every 2.5 days. When the cell confluence reached 85%, deer placental mesenchymal stem cells were obtained. S2, Deer placental mesenchymal stem cells were prepared at a rate of 2×10⁻⁶. 5Cells were seeded at a density of cells / mL in modified complete medium (DMEM / F12 medium supplemented with 10 g / L dextran microcarriers, 15 mg / L recombinant human insulin, 15 mg / L recombinant human transferrin, 8 μg / L sodium selenite, 2.5 μg / mL recombinant collagen, 15 ng / mL bFGF, and 8 ng / mL EGF) and cultured for 3D at 37°C, 5% CO2, and 80 rpm. The medium was replaced with fresh medium every 48 h for 5 days. After culturing, the cells were washed three times with PBS buffer and replaced with serum-free medium for exosome collection. The cells were then cultured under the same conditions for another 48 h. After culturing, the culture medium was collected and centrifuged at 2500 rpm for 15 min at 2°C. The supernatant was filtered through 0.45 μm and 0.22 μm pore sizes to obtain the cell supernatant. S3. The cell supernatant was concentrated by passing it through a hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa under the conditions of transmembrane pressure of 1.3 bar and circulation flow rate of 250 mL / min. When the volume of the concentrated solution was reduced to 1 / 12 of the original volume, an equal volume of PBS buffer was added for washing and filtration. The washing and filtration were repeated 3 times to obtain the exosome purification solution. S4. Control the volume ratio of exosome purification solution to Mal-PGA solution to 12.5:1. Adjust the pH of exosome purification solution to 8 with 1M sodium bicarbonate solution. Add Mal-PGA solution and stir for 2.5 h under the conditions of avoiding light, temperature 2℃, and speed 130 rpm. After the reaction is completed, pass the reaction solution through a tangential flow filtration system with a molecular weight cutoff of 100 kDa. Under the conditions of transmembrane pressure 0.5 bar and circulation flow rate 170 mL / min, wash the solution with 10 times the volume of PBS buffer. Repeat the washing and filtration 3 times to obtain polyglutamic acid-exosome solution. S5. Controlling the volume ratio of the freeze-drying protectant and the polyglutamic acid-exosome solution to 1:6.5, the freeze-drying protectant (composed of a mixture of 10% trehalose aqueous solution, 3% mannitol aqueous solution, and 1.5% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:3:2) was added to the polyglutamic acid-exosome solution. After mixing evenly, the mixture was pre-frozen in an ultra-low temperature freezer at -80℃ for 5 hours, then freeze-dried in a freeze dryer at a cold trap temperature of -53℃ and a vacuum degree ≤10Pa for 13 hours, then heated to -20℃ for 13 hours, and finally heated to 0℃ for 13 hours to obtain deer placental stem cell exosomes.

[0046] Example 3 The method for preparing Mal-PGA solution includes the following steps: A1. Controlling the mass ratio of polyglutamic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and MES buffer to 1:0.4:0.3:15, polyglutamic acid and MES buffer (concentration 0.2mol / L, pH 6.0) were mixed evenly, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added. The mixture was stirred at room temperature and 500 rpm for 2 hours. After the reaction was completed, the activated solution was obtained. A2. Controlling the volume ratio of N-(2-aminoethyl)maleimide solution to activation solution to 1:6, N-(2-aminoethyl)maleimide solution was added to activation solution at 300 rpm. The mixture was stirred at room temperature, protected from light, and at 400 rpm for 6 hours. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 5 kDa and dialyzed in deionized water for 48 hours, with the water changed every 6 hours. After dialysis, the solution in the bag was freeze-dried at -55℃ and a vacuum of ≤10 Pa for 36 hours to obtain Mal-PGA. Mal-PGA was dissolved in PBS buffer and filtered using a 0.22 μm microporous membrane to obtain a 5% (w / w) Mal-PGA solution. The method for preparing deer placental stem cell exosomes includes the following steps: S1. Collect placental tissue from artificially bred sika deer, wash 5 times with PBS buffer, and cut into 2mm pieces. 3 For the tissue blocks, first add 6 times the volume of 0.2wt% hyaluronidase and digest at 38℃ with shaking for 20 min; then add a mixed enzyme solution of 0.3wt% collagenase type I and 0.15wt% neutral protease, and continue digestion at 38℃ for 70 min. Collect the digestion solution, filter it through a 200-mesh sieve, collect the filtrate, and centrifuge at 2000 rpm for 5 min. Wash the precipitated cells three times with PBS buffer, and then centrifuge the precipitated cells at 1×10⁻⁶ ppm. 5 Seeds were inoculated at a density of 0.2 mL / cm² at the bottom of gelatin-coated culture flasks. 2To determine the bottom area, add 0.1% (w / v) gelatin aqueous solution and let stand at 37℃ for 50 min. Add modified primary culture medium (DMEM / F12 medium supplemented with 15% (v / v) exosome-free fetal bovine serum, 2 ng / mL bFGF, 10 ng / mL EGF, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 ng / mL deer antler polypeptide extract). Incubate at 37℃ and 5% CO2 for 4 h inverted adherence culture, then invert the culture. When the cell confluence reaches 65%, add digestion solution (0.02 wt% containing 0.25 wt% trypsin). Digest the cells with EDTA solution for 5 min, observing cell morphology changes during this period. When the cells become rounded and the intercellular spaces increase, add 3 times the volume of complete culture medium (DMEM / F12 medium supplemented with 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to terminate the digestion reaction. Collect the culture medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and dilute the cell suspension with complete culture medium to 3 × 10⁻⁶. 4 At a density of cells / mL, at 7×10⁻⁶ cells / mL per bottle. 5 One cell was seeded into a new T75 culture flask containing 15 mL of complete culture medium and placed in an incubator at 37°C and 5% CO2 for passage culture. Fresh complete culture medium was replaced every 3 days. When the cell confluence reached 90%, deer placental mesenchymal stem cells were obtained. S2, Deer placental mesenchymal stem cells were prepared at a rate of 2×10⁻⁶. 5 Cells were seeded at a density of cells / mL in modified complete medium (DMEM / F12 medium supplemented with 10 g / L dextran microcarriers, 20 mg / L recombinant human insulin, 20 mg / L recombinant human transferrin, 10 μg / L sodium selenite, 3 μg / mL recombinant collagen, 20 ng / mL bFGF, and 10 ng / mL EGF) and cultured for 3D at 37°C, 5% CO2, and 80 rpm. The medium was replaced with fresh medium every 48 h for 5 days. After culturing, the cells were washed 4 times with PBS buffer and replaced with serum-free medium for exosome collection. The cells were then cultured under the same conditions for another 48 h. After culturing, the culture medium was collected and centrifuged at 4°C and 3000 rpm for 10 min. The supernatant was filtered through 0.45 μm and 0.22 μm pore sizes to obtain the cell supernatant. S3. The cell supernatant was concentrated by passing it through a hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa under the conditions of transmembrane pressure of 1.5 bar and circulation flow rate of 300 mL / min. When the volume of the concentrated solution was reduced to 1 / 15 of the original volume, an equal volume of PBS buffer was added for washing and filtration. The washing and filtration were repeated 3 times to obtain the exosome purification solution. S4. Control the volume ratio of exosome purification solution to Mal-PGA solution to 15:1. Adjust the pH of exosome purification solution to 8.2 with 1M sodium bicarbonate solution. Add Mal-PGA solution and stir for 2 hours under the conditions of avoiding light, temperature 4℃, and speed 150 rpm. After the reaction is completed, pass the reaction solution through a tangential flow filtration system with a molecular weight cutoff of 100 kDa. Under the conditions of transmembrane pressure 0.8 bar and circulation flow rate 200 mL / min, wash the solution with 10 times the volume of PBS buffer. Repeat the washing and filtration 3 times to obtain polyglutamic acid-exosome solution. S5. Controlling the volume ratio of the freeze-drying protectant and the polyglutamic acid-exosome solution to 1:8, the freeze-drying protectant (composed of a mixture of 12% trehalose aqueous solution, 4% mannitol aqueous solution and 2% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:4:3) was added to the polyglutamic acid-exosome solution. After mixing evenly, the mixture was pre-frozen in an ultra-low temperature freezer at -85℃ for 4 hours, then freeze-dried in a freeze dryer at a cold trap temperature of -55℃ and a vacuum degree of ≤10Pa for 12 hours, then freeze-dried at -20℃ for 14 hours, and finally freeze-dried at 0℃ for 14 hours to obtain deer placental stem cell exosomes.

[0047] To verify the comprehensive performance of deer placental stem cell exosomes in Examples 1-3 of the present invention, the inventors set up Comparative Examples 1-6, as follows: Comparative Example 1 The difference between this comparative example and Example 1 is that in the original step S1, a single-step enzymatic digestion process was used, while the remaining steps and raw materials were the same as in Example 1. S1. Collect placental tissue from artificially bred sika deer, wash three times with PBS buffer, and cut into 2mm pieces. 3 For the tissue blocks, first add 4 times the volume of a mixed enzyme solution of 0.1 wt% hyaluronidase, 0.1 wt% collagenase type I, and 0.1 wt% neutral protease. Digest at 36°C with shaking for 120 min. Collect the digestion solution, filter through a 200-mesh sieve, collect the filtrate, and centrifuge at 1500 rpm for 10 min. Wash the precipitated cells twice with PBS buffer. Then, use 1×10⁻⁶ cells per 100 ml solution to digest the cells. 5 Seeds were inoculated at a density of 0.2 mL / cm² at the bottom of gelatin-coated culture flasks. 2To determine the bottom area, add 0.1% (w / v) gelatin aqueous solution and let stand at 37℃ for 50 min. Add modified primary culture medium (DMEM / F12 medium supplemented with 10% (v / v) exosome-free fetal bovine serum, 1 ng / mL bFGF, 5 ng / mL EGF, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 ng / mL deer antler polypeptide extract). Incubate at 37℃, 5% CO2 for 2 h inverted adherence culture, then invert. When cell confluence reaches 60%, add digestion solution (0.02 wt% containing 0.25 wt% trypsin). Digest the cells with EDTA solution for 3 min, observing cell morphology changes during this period. When the cells become rounded and the intercellular spaces increase, add twice the volume of complete culture medium (DMEM / F12 medium supplemented with 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to terminate the digestion reaction. Collect the culture medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, and dilute the cell suspension with complete culture medium to 3 × 10⁻⁶. 4 At a density of cells / mL, at 5 × 10⁻⁶ cells / mL per bottle. 5 One cell was seeded into a new T75 culture flask containing 10 mL of complete culture medium and placed in an incubator at 37°C and 5% CO2 for passage culture. Fresh complete culture medium was replaced every 2 days. When the cell confluence reached 80%, deer placental mesenchymal stem cells were obtained.

[0048] Comparative Example 2 The difference between this comparative example and Example 1 is that in the original step S3, the ultracentrifugation method is used to replace the tangential flow filtration system, while the remaining steps and raw materials are the same as in Example 1. S3. Centrifuge the cell supernatant at 4℃ and 10000×g for 30 min. Take the supernatant and ultracentrifuge at 4℃ and 100000×g for 70 min. Resuspend the precipitate in PBS buffer at 4℃ and ultracentrifuge again at 4℃ and 100000×g for 70 min to obtain exosome precipitate. Resuspend the exosome precipitate in PBS buffer to obtain exosome purification solution.

[0049] Comparative Example 3 The difference between this comparative example and Example 1 is that the Mal-PGA solution is replaced by an equal mass of the activation solution in step A1, while the remaining steps and raw materials are the same as in Example 1. S4. Control the volume ratio of exosome purification solution to activation solution to 10:1. Adjust the pH of exosome purification solution to 7.8 with 1M sodium bicarbonate solution. Add activation solution and stir for 3 hours under the conditions of avoiding light, temperature 0℃, and speed 100rpm. After the reaction is completed, pass the reaction solution through a tangential flow filtration system with a molecular weight cutoff of 100kDa. Under the conditions of transmembrane pressure 0.3bar and circulation flow rate 150mL / min, wash the solution with 10 times the volume of PBS buffer. Repeat the washing and filtration twice to obtain polyglutamic acid-exosome solution.

[0050] Comparative Example 4 The difference between this comparative example and Example 1 is that step S4 is deleted, while the remaining steps and raw materials are the same as in Example 1. S4. Control the volume ratio of freeze-drying protectant to exosome purification solution to 1:5. Add the freeze-drying protectant (composed of 8% trehalose aqueous solution, 2% mannitol aqueous solution and 1% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:2:1) to the exosome purification solution. After mixing evenly, pre-freeze in an ultra-low temperature freezer at -75℃ for 6 hours. Then freeze-dry in a freeze dryer at a cold trap temperature of -50℃ and a vacuum degree of ≤10Pa for 14 hours. Then raise the temperature to -20℃ and freeze-dry for 12 hours. Finally, raise the temperature to 0℃ and freeze-dry for 12 hours to obtain deer placental stem cell exosomes.

[0051] Comparative Example 5 The difference between this comparative example and Example 1 is that in the original step S5, the freeze-drying protectant was composed of an 8% trehalose aqueous solution and a 1% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:3. The remaining steps and raw materials were the same as in Example 1. S5. Control the volume ratio of the freeze-drying protectant and the polyglutamic acid-exosome solution to 1:5. Add the freeze-drying protectant (composed of 8% trehalose aqueous solution and 1% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:3) to the polyglutamic acid-exosome solution, mix evenly, pre-freeze in an ultra-low temperature freezer at -75℃ for 6 hours, then freeze-dry in a freeze dryer at a cold trap temperature of -50℃ and a vacuum degree ≤10Pa for 14 hours, then raise the temperature to -20℃ for 12 hours, and finally raise the temperature to 0℃ for 12 hours to obtain deer placental stem cell exosomes.

[0052] Comparative Example 6 The difference between this comparative example and Example 1 is that in the original step S5, the freeze-drying protectant was composed of an 8% trehalose aqueous solution and a 2% mannitol aqueous solution in a volume ratio of 5:3. The remaining steps and raw materials were the same as in Example 1. S5. Control the volume ratio of the freeze-drying protectant and the polyglutamic acid-exosome solution to 1:5. Add the freeze-drying protectant (composed of 8% trehalose aqueous solution and 2% mannitol aqueous solution in a volume ratio of 5:3) to the polyglutamic acid-exosome solution, mix evenly, pre-freeze in an ultra-low temperature freezer at -75℃ for 6 hours, then freeze-dry in a freeze dryer at a cold trap temperature of -50℃ and a vacuum degree ≤10Pa for 14 hours, then raise the temperature to -20℃ for 12 hours, and finally raise the temperature to 0℃ for 12 hours to obtain deer placental stem cell exosomes.

[0053] Performance testing 1. Detection of exosome particle number and concentration (nanoparticle tracking analysis, NTA) Weigh out the PBS buffer, filter it through a 0.22 μm microporous membrane, and prepare a 0.01 mol / L PBS buffer with a pH of 7.4. Store the prepared buffer at 4°C.

[0054] Use sterile, light-protected centrifuge tubes to set up sample groups and blank control groups. Each group should have 3 technical replicates and 3 parallel duplicates. The sample volumes are as follows: Sample group: Deer placental stem cell exosome samples prepared in Examples 1-3 and Comparative Examples 1-6 were taken, and after the particle concentration was determined by NTA prediction, they were diluted with 0.01 mol / L PBS buffer to a final particle concentration of (3 ± 0.5) × 10⁻⁶. 8 Particles / mL, after vortexing and mixing, take 10 μL for NTA analysis; Blank control group: 1000 μL of 0.01 mol / L PBS buffer; After adding the sample, let the centrifuge tube stand at room temperature in the dark for 5 minutes to equilibrate, avoiding shaking during this period. After equilibration, transfer 500 μL of sample from each centrifuge tube to a sterile transparent sample cell (one duplicate tube per cell). Set the NanoSight NS300 nanoparticle tracking analyzer to detection mode and set the detection parameters as follows: detection time 60 s, repeated detection 3 times, camera sensitivity set to 14, temperature controlled at 25℃. After calibrating the zero point, measure the particle number concentration and particle size distribution of each cell in sequence and record the data. During the measurement, ensure that the sample cell is free of air bubbles and impurities.

[0055] 2. Exosome yield The exosome yield was calculated based on the nanoparticle number concentration measured by nanoparticle tracking analysis (NTA) and the initial number of cultured cells. The specific calculation formula is as follows: Exosome yield (×10 9 particles / 10 6 cells) = C × V / N, where: C is the exosome particle number concentration measured by NTA (×10)9 particles / mL); V is the reconstitution volume of exosomes (mL); N is the number of deer placental mesenchymal stem cells used in the exosome collection phase (×10). 6 cells).

[0056] 3. Exosome functional activity assay (in vitro cell uptake experiment) (1) Fluorescent labeling and purification: Take equal volumes of the reconstituted exosome solutions from each group, add PKH67 green fluorescent dye, and incubate for 5 min at room temperature in the dark. Then add an equal volume of 1% (v / v) BSA solution to terminate the staining reaction. Transfer the mixture into an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa for centrifugation and elution. Repeat 3 times to completely remove free dye and obtain fluorescently labeled exosomes.

[0057] (2) Cell co-incubation: Target cells were incubated at a concentration of 1×10⁻⁶. 5 Seeds were inoculated at a density of 1 exosome per well in laser confocal microscopy culture dishes and cultured until adherent. The original culture medium was discarded, and serum-free culture medium containing fluorescently labeled exosomes was added. The dishes were then incubated in a 37°C, 5% CO2 incubator in the dark for 8 hours.

[0058] (3) Observation and quantification: The distribution and intensity of green fluorescent spots in cells were observed using a laser confocal microscope (CLSM). Cells from each group after co-incubation were collected simultaneously, and the mean fluorescence intensity (MFI) and percentage of positive cells were quantitatively detected by flow cytometry (FACS).

[0059] The test results are shown in Table 1 below: Table 1: Performance test data of deer placental stem cell exosomes in Examples 1-3 and Comparative Examples 1-6 As shown in Table 1, the deer placental stem cell exosomes prepared in Examples 1-3 of this invention are significantly superior to those in Comparative Examples 1-6 in terms of particle number concentration, exosome yield, particle size uniformity, and in vitro cell uptake activity.

[0060] The data from Example 1 and Comparative Example 1 show that the single-step enzymatic digestion process used in Comparative Example 1 resulted in insufficient digestion of placental tissue and poor removal of impurities, which in turn reduced cell release efficiency and primary cell activity, leading to a decrease in exosome production, poor particle size distribution, and weakened cell uptake capacity.

[0061] The data from Example 1 and Comparative Example 2 show that the use of ultracentrifugation to purify exosomes in Comparative Example 2 makes the exosomes more prone to structural damage and aggregation during the separation process, which in turn reduces the yield and purity of exosomes and reduces their functional activity.

[0062] As shown by the data from Example 1 and Comparative Example 3, when Comparative Example 3 used the activation solution prepared in step A1 to replace the Mal-PGA solution for coupling modification, the activation solution lacked maleimide (Mal) groups and could not covalently bind with the thiol groups (-SH) on the exosome membrane proteins. As a result, the polyglutamic acid molecules failed to be effectively and stably modified onto the exosome surface, and the particle concentration, yield and cellular uptake activity decreased significantly.

[0063] As shown by the data from Example 1 and Comparative Example 4, Comparative Example 4 directly freeze-drying unmodified exosomes resulted in insufficient dispersion stability and system compatibility of exosomes, which weakened their interaction with target cells and reduced their internalization and uptake capacity.

[0064] The data from Example 1 and Comparative Examples 5 and 6 show that Comparative Example 5 uses a combination of trehalose aqueous solution and polyvinylpyrrolidone aqueous solution, and Comparative Example 6 uses a combination of trehalose aqueous solution and mannitol aqueous solution. Neither of these methods can form an effective glassy framework to support the exosome structure during freeze-drying, resulting in exosome aggregation and larger particles after reconstitution, thereby reducing their biological activity and cellular uptake activity.

[0065] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A type of deer placental stem cell exosome, characterized in that, The method for preparing deer placental stem cell exosomes includes the following steps: S1. Collect placental tissue from artificially bred sika deer, clean it, cut it into tissue blocks, perform stepwise enzymatic digestion, collect the digestion liquid, filter, centrifuge, wash it, spread the precipitated cells on the bottom of a culture flask coated with gelatin, add modified primary culture medium, invert and adhere to the culture for 2-4 hours, then invert and perform primary culture. When the cell confluence reaches 60-70%, perform passage culture to obtain deer placental mesenchymal stem cells. S2. Deer placental mesenchymal stem cells were seeded into a modified complete culture medium for 3D cell culture. After the culture was completed, the cells were washed and replaced with serum-free culture medium for exosome collection for further culture. After the culture was completed, the culture medium was collected, centrifuged, and filtered to obtain cell supernatant. S3. The cell supernatant is concentrated and washed using a tangential flow filtration system to obtain a purified exosome solution. S4. Adjust the pH of the exosome purification solution to 7.8-8.2 with sodium bicarbonate solution, add Mal-PGA solution, stir and react. After the reaction is complete, wash and filter to obtain polyglutamic acid-exosome solution. S5. Add the freeze-drying protectant to the polyglutamic acid-exosome solution, mix well, and then dry to obtain deer placental stem cell exosomes.

2. The deer placental stem cell exosomes according to claim 1, characterized in that, The stepwise enzymatic digestion process in step S1 refers to: primary digestion: adding 0.1-0.2 wt% hyaluronidase and shaking digesting at 36-38℃ for 20-30 min; secondary digestion: adding a mixed enzyme solution of 0.1-0.3 wt% collagenase type I and 0.1-0.15 wt% neutral protease and continuing digestion at 36-38℃ for 70-90 min.

3. The deer placental stem cell exosomes according to claim 1, characterized in that, In step S1, the modified primary culture medium is a DMEM / F12 medium supplemented with 10-15% (v / v) exosome-free fetal bovine serum, 1-2 ng / mL bFGF, 5-10 ng / mL EGF, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 ng / mL deer antler polypeptide extract.

4. The deer placental stem cell exosomes according to claim 1, characterized in that, In step S2, the modified complete culture medium is a DMEM / F12 medium supplemented with 10 g / L dextran microcarriers, 10-20 mg / L recombinant human insulin, 10-20 mg / L recombinant human transferrin, 5-10 μg / L sodium selenite, 2-3 μg / mL recombinant collagen, 10-20 ng / mL bFGF, and 5-10 ng / mL EGF.

5. The deer placental stem cell exosomes according to claim 1, characterized in that, The concentration and filtration process in step S3 refers to: passing the cell supernatant through a tangential flow filtration system with a molecular weight cutoff of 100 kDa, concentrating it under the conditions of a transmembrane pressure of 1-1.5 bar and a circulation flow rate of 200-300 mL / min; when the volume of the concentrated solution is reduced to 1 / 10-1 / 15 of the original volume, adding PBS buffer for filtration, and repeating the filtration process 2-3 times.

6. The deer placental stem cell exosomes according to claim 1, characterized in that, The preparation method of the Mal-PGA solution in step S4 includes the following steps: A1. Mix polyglutamic acid and MES buffer evenly, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir to react, and after the reaction is complete, the activated solution is obtained. A2. Under stirring, N-(2-aminoethyl)maleimide solution was added to the activation solution and stirred to react. After the reaction was completed, it was dialyzed and dried to obtain Mal-PGA. Mal-PGA was dissolved in PBS buffer and filtered to obtain Mal-PGA solution.

7. The deer placental stem cell exosomes according to claim 6, characterized in that, In step A1, the mass ratio of polyglutamic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and MES buffer is 1:0.3-0.4:0.2-0.3:12-15.

8. The deer placental stem cell exosomes according to claim 6, characterized in that, In step A2, the volume ratio of N-(2-aminoethyl)maleimide solution to activation solution is 1:5-6.

9. The deer placental stem cell exosomes according to claim 1, characterized in that, In step S5, the freeze-drying protectant is composed of a mixture of 8-12% trehalose aqueous solution, 2-4% mannitol aqueous solution, and 1-2% polyvinylpyrrolidone aqueous solution in a volume ratio of 5:2-4:1-3.

10. The use of deer placental stem cell exosomes according to any one of claims 1-9 in the preparation of cosmetics having moisturizing, repairing, soothing and improving skin condition.

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