Preparation method and application of antiskin aging pilose antler stem cell exosome

Abstract

The present application relates to the technical field of tissue engineering, and specifically discloses a preparation method of a deer antler stem cell exosome for resisting skin aging and application thereof, which comprises the following steps: collecting deer antler tissues from specific parts, obtaining primary deer antler stem cells through complex enzymolysis and inverted adherent culture; pretreating the cells with a deer antler keratin hydrolysate, and expanding and inducing secretion in a low-oxygen environment; separating high-purity exosomes from culture supernatant through specific purification technology of antibody-coupled magnetic beads; and finally constructing deer antler stem cell exosomes with stable structure and high transdermal ability through complex polysaccharide encapsulation and skin-penetrating peptide modification. The exosomes prepared by the present application are rich in active ingredients, have complete and stable structure, can effectively scavenge free radicals and promote skin cell proliferation, and can solve the problems of low exosome yield, easy inactivation and difficult transdermal penetration in the prior art, and are suitable for preparing various anti-aging cosmetics, skin care products and skin repair dressings.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, specifically to a method for preparing deer antler stem cell exosomes for anti-skin aging and their application. Background Technology

[0002] As a fully regenerable mammalian organ, deer antler stem cells are easy to obtain and have a fast proliferation rate. Compared with bone marrow and umbilical cord mesenchymal stem cells, they are more suitable as raw materials for the preparation of exosomes. Existing technology has confirmed that deer antler stem cell exosomes are rich in bioactive components such as cytokines and miRNAs, which can achieve anti-aging effects by scavenging free radicals and promoting skin cell proliferation, providing a new direction for the field of skin anti-aging.

[0003] However, existing technologies for the preparation and application of deer antler stem cell exosomes still face many unresolved issues, making it difficult to meet the needs of industrial production and practical applications. Firstly, the preparation process lacks targeted screening of stem cell sources and optimization of enzymatic hydrolysis and culture systems, resulting in low stem cell purity and limited exosome secretion and activity. Furthermore, the single-column extraction method has low yield and is cumbersome, hindering large-scale production. Secondly, exosomes themselves have poor stability, are easily inactivated at room temperature, and exhibit low activity retention after freeze-drying, leading to high storage and transportation costs, thus limiting formulation development and market application. Thirdly, the uneven distribution of exosome nanoparticle size and the lack of targeted transdermal modification design result in low transdermal efficiency, making it difficult to penetrate the stratum corneum and reach the dermis, thus insufficient anti-aging efficacy. Additionally, traditional deer antler stem cell exosomes are mostly single-component applications, exhibiting poor compatibility with the skin matrix and failing to achieve synergistic effects with other anti-aging components, resulting in a single anti-aging target.

[0004] Based on the above statements, this invention proposes a method for preparing deer antler stem cell exosomes for anti-skin aging and their application. Summary of the Invention

[0005] To address the problems of low exosome yield and insufficient activity in existing deer antler stem cell exosome preparation processes, which make large-scale production difficult, poor exosome stability, limited storage and transportation, low transdermal efficiency leading to insufficient anti-aging effects, and limited anti-aging targets and poor compatibility with the skin matrix when using single components, this invention proposes a method for preparing deer antler stem cell exosomes for anti-skin aging and its application.

[0006] In a first aspect, the present invention provides a method for preparing deer antler stem cell exosomes that resist skin aging, using the following technical solution: A method for preparing deer antler stem cell exosomes with anti-skin aging properties includes the following steps: S1. Collect periosteal tissue from the antler region of healthy male deer of appropriate age and mix it with superficial mesenchymal tissue from the antlers during the growth period. After washing and cutting, digest the tissue with a compound digestion solution. Continuously examine the tissue fragments under a microscope until the cells are fully freed, then stop digestion. Centrifuge, wash the precipitate, spread the precipitate on the bottom of a culture flask coated with a cell adhesion matrix, add modified primary culture medium, and culture upside down for 2-4 hours. Then, invert the flask, passage, digest, and obtain primary antler stem cells. S2. Primary deer antler stem cells were pretreated for 12-24 hours in DMEM / F12 culture medium containing 3-5% (v / v) deer antler keratin hydrolysate. After centrifugation, pretreated primary deer antler stem cells were obtained. Under hypoxic conditions, the pretreated primary deer antler stem cells were first expanded and cultured in a modified passage medium. The expanded cells were then seeded into culture flasks and induced to grow further in serum-free secretion medium. The culture medium was collected and centrifuged to obtain the cell supernatant. S3. Filter the cell supernatant, centrifuge and concentrate it. Mix the concentrate with antibody-conjugated magnetic beads and incubate. Adsorb the mixture with a magnetic rack, wash, elute, centrifuge, and collect the supernatant to obtain deer antler stem cell exosomes. S4. Deer antler stem cell exosomes are mixed and incubated with complex polysaccharides to obtain polysaccharide-exosome complex microspheres. Then, polysaccharide-exosome complex microspheres are mixed and incubated with skin-penetrating short peptides to obtain deer antler stem cell exosomes with anti-skin aging properties.

[0007] Preferably, in step S1, the appropriate age refers to 1.5-2 years old, and the growth period refers to 25-45 days.

[0008] Preferably, the complex digestive solution in step S1 is composed of collagenase I at a volume ratio of 25-35 U / mL and trypsin at a volume ratio of 0.04-0.06% (w / v) to 2:1-1.4.

[0009] Preferably, the modified primary culture medium in step S1 is a DMEM / F12 culture medium containing 15-25% (v / v) exosome-free fetal bovine serum, 90-110 U / mL penicillin, 90-110 μg / mL streptomycin, 5-15 ng / mL vascular endothelial growth factor and 4-6 ng / mL basic fibroblast growth factor.

[0010] Preferably, the method for preparing the deer antler keratin hydrolysate in step S2 is as follows: Fresh deer antler skin and hair are mixed, crushed, and then deionized water is added to adjust the pH. Cysteine ​​hydrochloride and papain are added and hydrolyzed at 50-60℃ for 4-6 hours to inactivate the enzyme. After centrifugation, the supernatant is collected and dialyzed to obtain deer antler keratin hydrolysate.

[0011] Preferably, the modified passage medium in step S2 is DMEM / F12 medium containing 8-12% (v / v) exosome-free fetal bovine serum, 90-110 U / mL penicillin, 90-110 μg / mL streptomycin, and 6-10 ng / mL basic fibroblast growth factor; the serum-free secretion medium is mTeSR™1 medium.

[0012] Preferably, in step S3, the centrifugation speed is 2500-3500 rpm, the centrifugation time is 25-35 min, and the concentration is carried out to 5-10% of the original cell supernatant volume; the volume ratio of the concentrate to the antibody-conjugated magnetic beads is 8-12:1.

[0013] Preferably, in step S4, the complex polysaccharide is composed of hyaluronic acid and carboxymethyl chitosan in a mass ratio of 4-6:1; the skin-penetrating short peptide is one or more of TAT short peptide, Penetratin short peptide, and MPG short peptide.

[0014] Preferably, in step S4, the mass ratio of deer antler stem cell exosomes to complex polysaccharides is 4-6:1; and the mass ratio of polysaccharide-exosome complex microspheres to skin-penetrating short peptides is 90-110:1.

[0015] Secondly, the present invention provides a method for preparing deer antler stem cell exosomes that resist skin aging.

[0016] Thirdly, the present invention provides the application of anti-skin aging deer antler stem cell exosomes in the preparation of anti-aging cosmetics, skin care products or skin repair dressings.

[0017] In summary, the present invention has the following beneficial effects: 1. This invention utilizes antler stem cells from a specific source: periosteum tissue from the antler region of healthy male deer aged 1.5-2 years and superficial mesenchymal tissue from antlers at 25-45 days of growth, as antler stem cells at this stage exhibit the strongest proliferative capacity and most primitive stemness. Based on this, a pretreatment process involving a modified passage culture medium and antler keratin hydrolysate under hypoxic conditions simulates the in vivo microenvironment of rapid antler growth, significantly promoting the expansion efficiency of antler stem cells and the secretory activity of exosomes. Simultaneously, the antler keratin hydrolysate is rich in bioactive peptides, which can effectively activate the autocrine and paracrine functions of cells, resulting in a higher exosome yield compared to conventional culture methods.

[0018] 2. This invention employs immunomagnetic bead affinity purification technology, utilizing anti-CD63 antibodies to specifically capture exosomes. Compared to traditional ultracentrifugation or precipitation methods, it offers advantages such as high purity, stable recovery rate, and minimal damage to the exosome membrane structure. Rapid separation via a magnetic rack and gentle elution with sodium citrate buffer effectively prevents exosome aggregation and fragmentation during purification, resulting in exosomes with uniform particle size and intact morphology.

[0019] 3. This invention combines purified exosomes with hyaluronic acid and carboxymethyl chitosan in a specific ratio to form polysaccharide-exosome composite microspheres. Hyaluronic acid has good moisturizing and biocompatibility, while carboxymethyl chitosan has mucosal adhesion and antibacterial activity. The synergistic effect of the two not only protects the structural stability of exosomes during storage and delivery, but also achieves a sustained-release effect of exosomes in skin tissue, prolonging their duration of action and enhancing the persistence of anti-aging efficacy.

[0020] 4. This invention further conjugates polysaccharide-exosome composite microspheres with skin-penetrating short peptides (TAT short peptides). TAT short peptides are a classic cell-penetrating peptide that can effectively carry exosomes across the stratum corneum barrier and into the deep epidermis and dermis, significantly improving the transdermal absorption efficiency of exosomes.

[0021] 5. The anti-skin aging deer antler stem cell exosomes prepared in this invention are loaded with high abundance of regeneration-related growth factors (such as VEGF, bFGF, TGF-β1, etc.) and functional non-coding RNA (miRNA) unique to deer antler stem cells, and retain the natural exosome surface glycocalyx structure (containing glycosaminoglycans such as hyaluronic acid). These active ingredients work synergistically through paracrine mechanisms to effectively activate the proliferation signaling pathway of skin fibroblasts, upregulate the gene expression of type I collagen and elastin, inhibit the transcription and activity of matrix metalloproteinases (MMPs), scavenge excess reactive oxygen species (ROS), and enhance the antioxidant capacity of cells, thereby significantly reducing wrinkle formation, improving skin elasticity, and repairing the skin barrier function.

[0022] 6. The process parameters for each step of this invention are clearly defined and the operation is simple. From primary cell isolation and culture, hypoxia amplification induction, immunomagnetic bead purification to polysaccharide modification and transmembrane peptide conjugation, all steps are performed using conventional biopharmaceutical equipment, making it easy to scale up production. Furthermore, the use of deer antler keratin hydrolysate as a pretreatment additive enables high-value utilization of deer antler byproducts (antler skin and hair), aligning with green environmental protection and sustainable development principles. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0026] Collagenase I was purchased from Beyotime Biotechnology, product number: Y296936; Trypsin was purchased from Yisheng Biotechnology, product code: 40101ES25; Exosome-free fetal bovine serum was purchased from Yisheng Biotechnology, product code: 40139ES50; DMEM / F12 culture medium was purchased from Thermo Fisher, product code: 11320033; Vascular endothelial growth factor was purchased from Yisheng Biotechnology, product code: 95332ES25; Basic fibroblast growth factor was purchased from Yisheng Biotechnology, product code: 91330ES60 Transforming growth factor β1 was purchased from Beyotime Biotechnology, product number: P4825; Cysteine ​​hydrochloride was purchased from Beyotime Biotechnology, product number: Y284315; Papain was purchased from Beyotime Biotechnology, product number: Y296964; The anti-CD63 antibody was purchased from Beyotime Biotechnology, product number: AF0605; Protein A / G immunoprecipitation magnetic beads were purchased from Beyotime Biotechnology, product number: P2089; TAT short peptides were purchased from Xi'an Qiyue Biotechnology Co., Ltd., CAS No.: 191936-91-1.

[0027] Example 1 A method for preparing deer antler stem cell exosomes with anti-skin aging properties includes the following steps: S1. Isolation and culture of primary deer antler stem cells: Periosteal tissue from the antler region of healthy male deer aged 1.5 years and superficial mesenchymal tissue (0.3 cm translucent layer beneath the antler skin) from 25 days of growth were collected and mixed. The mixture was washed with PBS buffer (pH 7.0) until blood was removed and then cut into 0.4 mm pieces. 3The fragments were then mixed with a compound digestion solution (composed of 25 U / mL collagenase I and 0.04% (w / v) trypsin in a 2:1 volume ratio) at a 1:3 mass ratio. The mixture was incubated at 35°C, with microscopic examination every 6 minutes until the cells were fully freed. Digestion was then terminated by adding an equal volume of DMEM / F12 culture medium containing 8% (v / v) exosome-free fetal bovine serum. The mixture was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was washed twice with pH 7.0 PBS buffer. The precipitate was then spread onto the bottom of a gelatin-coated culture flask (at a density of 0.2 mL / cm²). 2 To obtain primary deer antler stem cells, add 0.1% (w / v) gelatin aqueous solution to the bottom area and let stand at 37℃ for 50 min. Add 3 mL of modified primary culture medium (DMEM / F12 culture medium containing 15% (v / v) exosome-free fetal bovine serum, 90 U / mL penicillin, 90 μg / mL streptomycin, 5 ng / mL vascular endothelial growth factor and 4 ng / mL basic fibroblast growth factor). Incubate in a 35℃, 4% CO2 incubator, inverted and adherent for 2 h, then upright. Change the medium every 36 h. When the cell confluence reaches 60%, digest with 0.2% (w / v) trypsin to obtain primary deer antler stem cells. S2. Pretreatment, hypoxia expansion, and exosome-induced secretion of deer antler stem cells: Primary deer antler stem cells were used at 8×10 4 Pretreated primary antler stem cells were added at a density of 1 cell / mL to DMEM / F12 culture medium containing 3% (v / v) antler keratin hydrolysate and pretreated for 12 h at 35°C and 4% CO2. After centrifugation at 800 rpm for 4 min, pretreated primary antler stem cells were obtained. Under a hypoxic environment of 35°C, 3% O2, and 4% CO2, the pretreated primary antler stem cells were first expanded and cultured in a modified passage medium (DMEM / F12 culture medium containing 8% (v / v) exosome-free fetal bovine serum, 90 U / mL penicillin, 90 μg / mL streptomycin, and 6 ng / mL basic fibroblast growth factor) until the cell confluence reached 70%. The expanded cells were then cultured at 8 × 10⁻⁶ cells / mL. 4 75cm 2 Inoculate the culture flasks at a density of 75 cm³. 2 Add 15 mL of mTeSR™1 medium to the culture flask and continue to induce culture in a hypoxic environment of 35℃, 3% O2, and 4% CO2 until the cell confluence reaches 85%. Collect the culture medium, centrifuge at 2000 rpm for 8 min, and obtain the cell supernatant. The preparation method of deer antler keratin hydrolysate is as follows: Fresh deer antler skin and hair were mixed at a mass ratio of 1:1, pulverized and passed through a 100-mesh sieve, and deionized water was added at a material-to-liquid ratio of 1:8. The pH was adjusted to 6.0 with 1 mol / L NaOH, and cysteine ​​hydrochloride (0.5% of the total mass of deer antler skin and hair) and papain (1% of the total mass of deer antler skin and hair) were added. The mixture was hydrolyzed at 50℃ for 4 h, heated to 90℃ to inactivate the enzyme for 10 min, centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The mixture was dialyzed with a 3 kDa dialysis bag for 24 h, during which the deionized water was replaced 3 times to obtain deer antler keratin hydrolysate. S3. Extraction and purification of deer antler stem cell exosomes: The cell supernatant was filtered through a 0.22 μm filter membrane and concentrated by centrifugation at 2500 rpm and 2℃ for 25 min to 5% of the original volume. The concentrate was mixed with antibody-conjugated magnetic beads at a volume ratio of 8:1 and incubated gently at 2℃ for 1 h. After adsorption on a magnetic rack for 5 min, the mixture was washed three times with 0.01 mol / L PBS buffer containing 0.01% (w / v) BSA at pH 7.0. The mixture was then eluted with sodium citrate elution buffer (containing 0.5 M NaCl) at pH 5.5, which accounted for 20% of the volume of the antibody-conjugated magnetic beads. After elution, the mixture was allowed to stand at room temperature for 5 min and centrifuged at 800 rpm for 4 min. The supernatant was collected to obtain deer antler stem cell exosomes. The preparation method of antibody-conjugated magnetic beads is as follows: anti-CD63 antibody and Protein A / G immunoprecipitation magnetic beads are mixed at a mass ratio of 1:50, gently shaken and incubated at 4°C for 2 hours, washed and resuspended in 0.01 mol / L PBS buffer containing 0.01% (w / v) BSA at pH 7.0 to obtain antibody-conjugated magnetic beads with a concentration of 10 mg / mL. S4. Modification of deer antler stem cell exosomes: Deer antler stem cell exosomes were mixed with a complex polysaccharide (composed of hyaluronic acid and carboxymethyl chitosan in a mass ratio of 4:1) at a mass ratio of 4:1 and incubated with gentle shaking at 4°C for 1.5 h to obtain polysaccharide-exosome complex microspheres. The polysaccharide-exosome complex microspheres were then mixed with a skin-penetrating short peptide (TAT short peptide) at a mass ratio of 90:1 and incubated with gentle shaking at 4°C in the dark for 0.5 h to obtain deer antler stem cell exosomes with anti-skin aging properties.

[0028] Example 2 A method for preparing deer antler stem cell exosomes with anti-skin aging properties includes the following steps: S1. Isolation and culture of primary deer antler stem cells: Periosteal tissue from the antler region of healthy male deer aged 1.5 years and superficial mesenchymal tissue (0.4 cm translucent layer beneath the antler skin) from antlers grown for 35 days were collected and mixed. The mixture was washed with PBS buffer (pH 7.2) until blood was removed and then cut into 0.5 mm pieces. 3The fragments were then mixed with a compound digestion solution (composed of 30 U / mL collagenase I and 0.05% (w / v) trypsin at a volume ratio of 2:1.2) at a mass ratio of 1:4. The mixture was incubated at 37°C, with microscopic examination every 8 minutes until the cells in the tissue fragments were fully freed. Digestion was then terminated by adding an equal volume of DMEM / F12 culture medium containing 10% (v / v) exosome-free fetal bovine serum. The mixture was centrifuged at 1100 rpm for 6 minutes, the supernatant was discarded, and the precipitate was washed three times with PBS buffer (pH 7.2). The precipitate was then spread onto the bottom of a gelatin-coated culture flask (at a density of 0.2 mL / cm²). 2 Add 0.1% (w / v) gelatin aqueous solution to the bottom area, and let stand at 37℃ for 50 min to obtain the culture medium. Add 4 mL of modified primary culture medium (DMEM / F12 culture medium containing 20% ​​(v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 ng / mL vascular endothelial growth factor and 5 ng / mL basic fibroblast growth factor). Incubate in a 37℃, 5% CO2 incubator, inverted and adherent for 3 h, then inverted and incubated. Change the medium every 42 h. When the cell confluence reaches 65%, digest with 0.25% (w / v) trypsin to obtain primary deer antler stem cells. S2. Pretreatment, hypoxia expansion, and exosome-induced secretion of deer antler stem cells: Primary deer antler stem cells were used at a concentration of 1×10⁻⁶. 5 Pretreated primary antler stem cells were added at a density of 1 cell / mL to DMEM / F12 culture medium containing 4% (v / v) antler keratin hydrolysate and pretreated for 18 h at 37°C and 5% CO2. After centrifugation at 1000 rpm for 5 min, pretreated primary antler stem cells were obtained. Under a hypoxic environment of 37°C, 4% O2, and 5% CO2, the pretreated primary antler stem cells were first expanded and cultured in a modified passage medium (DMEM / F12 culture medium containing 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 8 ng / mL basic fibroblast growth factor) until the cell confluence reached 75%. The expanded cells were then cultured at 8 × 10⁻⁶ cells / mL. 4 Inoculate the culture flasks at a density of 1 cell per 75 cm². 2 Add 20 mL of mTeSR™1 medium to the culture flask and continue to induce culture in a hypoxic environment of 37℃, 4% O2, and 5% CO2 until the cell confluence reaches 85%. Collect the culture medium, centrifuge at 3000 rpm for 10 min, and obtain the cell supernatant. The preparation method of deer antler keratin hydrolysate is as follows: Fresh deer antler skin and hair were mixed at a mass ratio of 1:1, pulverized and passed through a 100-mesh sieve, and deionized water was added at a material-to-liquid ratio of 1:10. The pH was adjusted to 6.5 with 1 mol / L NaOH, and cysteine ​​hydrochloride (0.75% of the total mass of deer antler skin and hair) and papain (1.5% of the total mass of deer antler skin and hair) were added. The mixture was hydrolyzed at 55℃ for 5 h, heated to 90℃ to inactivate the enzyme for 12 min, centrifuged at 4000 rpm for 18 min, and the supernatant was collected. The mixture was dialyzed with a 4 kDa dialysis bag for 30 h, during which the deionized water was replaced 3 times to obtain deer antler keratin hydrolysate. S3. Extraction and purification of deer antler stem cell exosomes: The cell supernatant was filtered through a 0.22 μm filter membrane and concentrated by centrifugation at 3000 rpm and 4 °C for 30 min to 5% of the original volume. The concentrate was mixed with antibody-conjugated magnetic beads at a volume ratio of 10:1 and incubated gently at 4 °C for 1.5 h. After adsorption on a magnetic rack for 6 min, the mixture was washed three times with 0.01 mol / L PBS buffer (pH 7.2) containing 0.015% (w / v) BSA. The mixture was then eluted with sodium citrate elution buffer (pH 5.7) containing 0.5 M NaCl, which was 22% of the volume of the antibody-conjugated magnetic beads. The mixture was allowed to stand at room temperature for 8 min to elute, and then centrifuged at 1000 rpm for 5 min. The supernatant was collected to obtain deer antler stem cell exosomes. The preparation method of antibody-conjugated magnetic beads is as follows: anti-CD63 antibody and Protein A / G immunoprecipitation magnetic beads are mixed at a mass ratio of 1:70, gently shaken and incubated at 4°C for 2.5 h, washed and resuspended in 0.01 mol / L PBS buffer containing 0.01% (w / v) BSA at pH 7.2 to obtain antibody-conjugated magnetic beads with a concentration of 10 mg / mL. S4. Modification of deer antler stem cell exosomes: Deer antler stem cell exosomes were mixed with a complex polysaccharide (composed of hyaluronic acid and carboxymethyl chitosan in a mass ratio of 5:1) at a mass ratio of 5:1 and incubated with gentle shaking at 4°C for 2 hours to obtain polysaccharide-exosome complex microspheres. The polysaccharide-exosome complex microspheres were then mixed with a skin-penetrating short peptide (TAT short peptide) at a mass ratio of 100:1 and incubated with gentle shaking at 4°C in the dark for 1 hour to obtain deer antler stem cell exosomes with anti-skin aging properties.

[0029] Example 3 A method for preparing deer antler stem cell exosomes with anti-skin aging properties includes the following steps: S1. Isolation and culture of primary deer antler stem cells: Periosteal tissue from the antler region of healthy 2-year-old male deer and superficial mesenchymal tissue (0.5 cm translucent layer beneath the antler skin) from deer at 45 days of growth were collected and mixed. The mixture was washed with PBS buffer (pH 7.4) until blood was removed and then cut into 0.6 mm pieces.3 The fragments were then mixed with a compound digestion solution (composed of 35 U / mL collagenase I and 0.06% (w / v) trypsin at a volume ratio of 2:1.4) at a mass ratio of 1:5. The mixture was incubated at 40°C, with microscopic examination every 10 minutes until the cells in the tissue fragments were fully freed. Digestion was then terminated by adding an equal volume of DMEM / F12 culture medium containing 12% (v / v) exosome-free fetal bovine serum. The mixture was centrifuged at 1200 rpm for 8 minutes, the supernatant was discarded, and the precipitate was washed three times with PBS buffer at pH 7.4. The precipitate was then spread onto the bottom of a gelatin-coated culture flask (at a density of 0.2 mL / cm²). 2 Add 0.1% (w / v) gelatin aqueous solution to the bottom area, and let stand at 37℃ for 50 min to obtain the culture medium. Add 5 mL of modified primary culture medium (DMEM / F12 culture medium containing 25% (v / v) exosome-free fetal bovine serum, 110 U / mL penicillin, 110 μg / mL streptomycin, 15 ng / mL vascular endothelial growth factor and 6 ng / mL basic fibroblast growth factor). Incubate in a 40℃, 6% CO2 incubator, inverted and adherent for 4 h, then inverted and incubated. Change the medium every 48 h. When the cell confluence reaches 70%, digest with 0.3% (w / v) trypsin to obtain primary deer antler stem cells. S2. Pretreatment, hypoxia expansion, and exosome-induced secretion of deer antler stem cells: Primary deer antler stem cells were used at a concentration of 1×10⁻⁶. 5 Pretreated primary antler stem cells were added at a density of 1 cell / mL to DMEM / F12 culture medium containing 5% (v / v) antler keratin hydrolysate and pretreated for 24 h at 40 °C and 6% CO2. After centrifugation at 1000 rpm for 6 min, pretreated primary antler stem cells were obtained. Under a hypoxic environment of 40 °C, 5% O2, and 6% CO2, the pretreated primary antler stem cells were first expanded and cultured in a modified passage medium (DMEM / F12 culture medium containing 12% (v / v) exosome-free fetal bovine serum, 110 U / mL penicillin, 110 μg / mL streptomycin, and 10 ng / mL basic fibroblast growth factor) until the cell confluence reached 80%. The expanded cells were then cultured at 8 × 10⁻⁶ cells / mL. 4 Inoculate the culture flasks at a density of 1 cell per 75 cm². 2 Add 20 mL of mTeSR™1 medium to the culture flask and continue to induce culture in a hypoxic environment of 40℃, 5% O2, and 6% CO2 until the cell confluence reaches 90%. Collect the culture medium, centrifuge at 4000 rpm for 12 min, and obtain the cell supernatant. The preparation method of deer antler keratin hydrolysate is as follows: Fresh deer antler skin and hair were mixed at a mass ratio of 1:1, pulverized and passed through a 100-mesh sieve, and deionized water was added at a material-to-liquid ratio of 1:12. The pH was adjusted to 7.0 with 1 mol / L NaOH, and cysteine ​​hydrochloride (1% of the total mass of deer antler skin and hair) and papain (2% of the total mass of deer antler skin and hair) were added. The mixture was hydrolyzed at 60℃ for 6 h, heated to 95℃ to inactivate the enzyme for 15 min, centrifuged at 4000 rpm for 20 min, and the supernatant was collected. The mixture was dialyzed with a 5 kDa dialysis bag for 36 h, during which the deionized water was replaced 4 times to obtain deer antler keratin hydrolysate. S3. Extraction and purification of deer antler stem cell exosomes: The cell supernatant was filtered through a 0.22 μm filter membrane and concentrated by centrifugation at 3500 rpm and 4 °C for 35 min to 10% of the original volume. The concentrate was mixed with antibody-conjugated magnetic beads at a volume ratio of 12:1 and incubated with gentle shaking at 6 °C for 1.5 h. After adsorption by a magnetic rack for 8 min, the mixture was washed four times with 0.01 mol / L PBS buffer containing 0.02% (w / v) BSA at pH 7.4. Sodium citrate elution buffer (containing 0.5 M NaCl) at pH 6.0 was added to elute buffer at 25% of the volume of the antibody-conjugated magnetic beads. The mixture was allowed to stand at room temperature for 10 min to elute, and then centrifuged at 1200 rpm for 6 min. The supernatant was collected to obtain deer antler stem cell exosomes. The preparation method of antibody-conjugated magnetic beads is as follows: anti-CD63 antibody and Protein A / G immunoprecipitation magnetic beads are mixed at a mass ratio of 1:80, gently shaken and incubated at 4°C for 3 hours, washed and resuspended in 0.01 mol / L PBS buffer containing 0.01% (w / v) BSA at pH 7.4 to obtain antibody-conjugated magnetic beads with a concentration of 10 mg / mL. S4. Modification of deer antler stem cell exosomes: Deer antler stem cell exosomes were mixed with a complex polysaccharide (composed of hyaluronic acid and carboxymethyl chitosan in a mass ratio of 6:1) at a mass ratio of 6:1 and incubated with gentle shaking at 4°C for 2.5 h to obtain polysaccharide-exosome complex microspheres. The polysaccharide-exosome complex microspheres were then mixed with a skin-penetrating short peptide (TAT short peptide) at a mass ratio of 110:1 and incubated with gentle shaking at 4°C in the dark for 1 h to obtain deer antler stem cell exosomes with anti-skin aging properties.

[0030] Comparative Example 1 The only difference between this comparative example and Example 2 is that the "pretreatment of deer antler keratin hydrolysate" step in step S2 is omitted: the primary deer antler stem cells are directly hydrolyzed at 1×10⁻⁶. 5 The sample was added at a density of 1 / mL to DMEM / F12 culture medium without deer antler keratin hydrolysate. All other raw material ratios, preparation processes and parameters were completely consistent with those in Example 2.

[0031] Comparative Example 2 The only difference between this comparative example and Example 2 is that the "hypoxia-induced environment" in step S2 is replaced with "normal oxygen environment". All other raw material ratios, preparation processes and parameters are completely consistent with Example 2.

[0032] Specifically: S2, pretreatment of deer antler stem cells, hypoxia expansion, and exosome-induced secretion: Primary deer antler stem cells were used at a concentration of 1×10⁻⁶. 5 The cells were added at a density of 1 cell / mL to DMEM / F12 culture medium containing 4% (v / v) deer antler keratin hydrolysate and pretreated for 18 h at 37°C and 5% CO2. After centrifugation at 1000 rpm for 5 min, pretreated primary deer antler stem cells were obtained. Under normoxic conditions of 37°C, 20% O2, and 5% CO2, the pretreated primary deer antler stem cells were first expanded and cultured in a modified passage medium (DMEM / F12 culture medium containing 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 8 ng / mL basic fibroblast growth factor) until the cell confluence reached 75%. The expanded cells were then cultured at 8 × 10⁻⁶ cells / mL. 4 Inoculate the culture flasks at a density of 1 cell per 75 cm². 2 Add 20 mL of mTeSR™1 medium to the culture flask and continue to induce culture in a normoxic environment of 37℃, 20% O2, and 5% CO2 until the cell confluence reaches 85%. Collect the culture medium, centrifuge at 3000 rpm for 10 min, and obtain the cell supernatant.

[0033] Comparative Example 3 The only difference between this comparative example and Example 2 is that the "antibody-conjugated magnetic bead immunopurification method" in step S3 is replaced with the "traditional ultracentrifugation method". All other raw material ratios, preparation processes and parameters are completely consistent with Example 2.

[0034] Specifically: S3, extraction and purification of deer antler stem cell exosomes: The cell supernatant was filtered through a 0.22 μm filter membrane and centrifuged at 4 °C and 100,000 × g for 80 min. The supernatant was discarded, and the precipitate was resuspended in 0.01 mol / L PBS buffer at pH 7.2. The precipitate was then centrifuged again at 4 °C and 100,000 × g for 80 min. The precipitate was collected to obtain deer antler stem cell exosomes.

[0035] Comparative Example 4 The only difference between this comparative example and Example 2 is that the "complex polysaccharide encapsulation" step in step S4 is omitted. The remaining raw material ratios, preparation processes, and all parameters are completely consistent with Example 2.

[0036] Specifically, this includes: S4, modification of deer antler stem cell exosomes: Deer antler stem cell exosomes were mixed with skin-penetrating short peptides (TAT short peptides) at a mass ratio of 100:1 and incubated gently with shaking for 1 hour at 4°C in the dark to obtain deer antler stem cell exosomes with anti-skin aging properties.

[0037] Comparative Example 5 The only difference between this comparative example and Example 2 is that the "skin-penetrating short peptide (TAT) modification" step in step S4 is omitted. The remaining raw material ratios, preparation processes and all parameters are completely consistent with Example 2.

[0038] Specifically: S4, modification of deer antler stem cell exosomes: Deer antler stem cell exosomes were mixed with a complex polysaccharide (composed of hyaluronic acid and carboxymethyl chitosan in a mass ratio of 5:1) at a mass ratio of 5:1 and incubated with gentle shaking at 4°C for 2 hours to obtain deer antler stem cell exosomes with anti-skin aging properties.

[0039] Performance testing 1. Testing Method The anti-skin aging deer antler stem cell exosomes prepared in Examples 1-3 and Comparative Examples 1-5 were resuspended in 0.01 mol / L PBS buffer at pH 7.4 to the same protein concentration (1.0 mg / mL), aliquoted into 100 μL / tube, stored at 4°C in the dark, and tested within 24 hours. All operations were performed under sterile conditions at room temperature (25°C). (1) DPPH free radical scavenging rate test (antioxidant capacity): Accurately weigh the DPPH reagent, dissolve it in anhydrous ethanol and dilute to volume to prepare a 0.1 mmol / L DPPH anhydrous ethanol solution. Prepare and use immediately, and store in a light-protected work area after preparation. Use sterile, light-protected centrifuge tubes to set up sample groups, sample control groups, and blank control groups. Each group should have 3 technical replicates and 3 parallel duplicates. The sample volumes are as follows: Sample group: 10 μL of 1.0 mg / mL deer antler stem cell exosome samples prepared in Examples 1-3 and Comparative Examples 1-5 for anti-skin aging + 1000 μL of 0.1 mmol / L DPPH anhydrous ethanol solution, gently vortex to mix. Sample control group: 10 μL of 1.0 mg / mL deer antler stem cell exosome samples prepared in Examples 1-3 and Comparative Examples 1-5 for anti-skin aging + 1000 μL of anhydrous ethanol, gently vortexed to mix (to eliminate interference from the sample's own absorbance). Blank control group: 10 μL of 0.01 mol / L PBS buffer at pH 7.4 + 1000 μL of 0.1 mmol / L DPPH anhydrous ethanol solution, gently vortex to mix (as a reference for the initial absorbance of DPPH free radicals). After adding the sample, let the centrifuge tube stand at room temperature in the dark for 30 minutes to react, avoiding shaking during this time; After the reaction is complete, 200 μL of the reaction solution is transferred from each centrifuge tube to a sterile 96-well microplate (one duplicate tube per well). The microplate reader is set to 519 nm wavelength and the zero point is calibrated. The absorbance (A) of each well is measured in sequence and the data is recorded. During the measurement, ensure that there are no air bubbles on the microplate and no droplets on the well walls. Calculation formula: Clearance rate (%) = [1 - (A)] 样品组 -A 样品对照组 ) / A 空白对照组 ×100%; In the formula: A 样品组 A represents the average absorbance of the sample group. 样品对照组 A represents the average absorbance of the sample and control groups. 空白对照组 The absorbance is the average value of the blank control group.

[0040] (2) Cell proliferation promotion test (regeneration and repair capacity): This test simultaneously detected HaCaT cells and NHDF cells, using identical procedures. Independent experimental and control groups were set up for each group, as detailed below: Cell seeding: HaCaT and NHDF cells in logarithmic growth phase were digested with 0.25% (w / v) trypsin, resuspended in DMEM medium containing 10% fetal bovine serum, and the cell density was adjusted to 5 × 10⁶ cells / year. 3 For each well, take a sterile, transparent 96-well microplate, add 100 μL of cell suspension to each well, and set up 3 technical replicates and 3 parallel replicates for each group; Adherent cell culture: Place the 96-well plate in a constant temperature cell culture incubator at 37℃, 5% CO2, and saturated humidity, and culture for 24 hours until the cell adhesion rate reaches 80%. Sample preparation: Discard the original culture medium in the 96-well plate, wash the cells twice with 0.01 mol / L PBS buffer (pH 7.4), and discard the washing solution; then add serum-free DMEM medium containing exosome samples to achieve a final exosome protein concentration of 50 μg / mL, with a sample volume of 100 μL per well; at the same time, a blank control group was set up, with an equal volume of serum-free DMEM medium added (without exosome samples). Co-culture: After adding the samples, place the 96-well plate back into the constant temperature cell culture incubator and co-culture at 37°C and 5% CO2 for 24 hours. Avoid shaking the incubator during this period. CCK-8 incubation: After co-culture, add 10 μL of CCK-8 detection reagent to each well (avoid generating air bubbles), gently shake the 96-well plate to mix the reagent, and continue to incubate at 37°C for 2 hours. Absorbance measurement: After incubation, the microplate reader was set to 450 nm wavelength, with 630 nm as the reference wavelength. After calibrating the zero point, the absorbance value (A) of each well was measured and the data was recorded. Calculation formula: Cell proliferation rate (%) = (A 样品组 / A 空白对照组 )×100%; In the formula: A 样品组 A represents the average absorbance of the cell pores in the exosome sample. 空白对照组 The values ​​represent the average absorbance of cell pores without exosomes.

[0041] 2. Test Results The specific test results are shown in Table 1.

[0042] Table 1. Performance test results of deer antler stem cell exosomes for anti-skin aging As shown in Table 1, the anti-skin aging deer antler stem cell exosomes prepared in Examples 1-3 of this invention all have excellent in vitro antioxidant capacity and skin cell proliferation promotion effect, among which Example 2 has the best performance.

[0043] Comparing Example 2 with Comparative Example 1, it can be seen that Comparative Example 1 omitted the pretreatment step of deer antler keratin hydrolysate, and all detection indicators decreased significantly. The DPPH free radical scavenging rate was only 52.5±3.1%, and the cell proliferation rate was less than 135%, making it the worst performing of all samples. This indicates that the pretreatment of deer antler keratin hydrolysate is a key step in activating the secretory activity of deer antler stem cells and enhancing the core efficacy of exosomes.

[0044] A comparison of Example 2 and Comparative Example 2 shows that: Comparative Example 2 replaced the hypoxia-induced environment with a normoxic environment, and the antioxidant capacity and cell proliferation promotion effect of exosomes decreased significantly. All indicators were much lower than those of Example 2, indicating that the hypoxia-induced environment can simulate the regeneration microenvironment in deer antler and effectively increase the content of active factors in exosomes. It is one of the core processes to achieve high exosome activity.

[0045] A comparison of Example 2 and Comparative Example 3 shows that in Comparative Example 3, the exosome performance was significantly reduced when the traditional ultracentrifugation method was used instead of the antibody-conjugated magnetic bead immunopurification method. This indicates that the antibody-conjugated magnetic bead purification method can specifically capture intact deer antler stem cell exosomes, effectively remove impurities and cell debris, ensure the high purity and structural integrity of the exosomes, and achieve better purification results.

[0046] A comparison of Example 2 and Comparative Example 4 shows that Comparative Example 4 omitted the complex polysaccharide encapsulation step, resulting in a decrease in the antioxidant capacity and cell proliferation promotion effect of exosomes. This indicates that the complex polysaccharide encapsulation can not only form a physical protective barrier on the surface of exosomes, reducing the loss and degradation of active ingredients during preparation and detection, but also maintain their structural stability through the interaction between polysaccharides and the exosome membrane. It is an important auxiliary process to ensure the high efficiency of exosomes.

[0047] A comparison of Example 2 and Comparative Example 5 shows that Comparative Example 5, which omits the skin-penetrating peptide (TAT) modification step, has lower exosome performance indicators than Example 2. This indicates that TAT peptide modification has a dual effect: on the one hand, its positive charge property can effectively neutralize the negative charge on the surface of exosomes, reduce aggregation in the in vitro environment, and improve dispersion uniformity, thereby enhancing detection activity; on the other hand, TAT peptide and complex polysaccharide encapsulation have a significant synergistic effect, jointly constructing a stable dual system of protection and delivery. The absence of either step will result in the overall performance of exosomes failing to reach its optimal state.

[0048] 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 method for preparing deer antler stem cell exosomes for anti-skin aging, characterized in that, Includes the following steps: S1. Collect periosteal tissue from the antler region of healthy male deer of appropriate age and mix it with superficial mesenchymal tissue from the antlers during the growth period. After washing and cutting, digest the tissue with a compound digestion solution. Continuously examine the tissue fragments under a microscope until the cells are fully freed, then stop digestion. Centrifuge, wash the precipitate, spread the precipitate on the bottom of a culture flask coated with a cell adhesion matrix, add modified primary culture medium, and culture upside down for 2-4 hours. Then, invert the flask, passage, digest, and obtain primary antler stem cells. S2. Primary deer antler stem cells were added to DMEM / F12 culture medium containing 3-5% (v / v) deer antler keratin hydrolysate for 12-24 h for pretreatment. After centrifugation, pretreated primary deer antler stem cells were obtained. Under hypoxic conditions, the pretreated primary deer antler stem cells were first expanded and cultured with modified passage medium. Then, the expanded cells were seeded into culture flasks and serum-free secretion medium was added for further induction culture. Collect the culture medium, centrifuge, and obtain the cell supernatant; S3. Filter the cell supernatant, centrifuge and concentrate it. Mix the concentrate with antibody-conjugated magnetic beads and incubate. Adsorb the mixture with a magnetic rack, wash, elute, centrifuge, and collect the supernatant to obtain deer antler stem cell exosomes. S4. Deer antler stem cell exosomes are mixed and incubated with complex polysaccharides to obtain polysaccharide-exosome complex microspheres. Then, polysaccharide-exosome complex microspheres are mixed and incubated with skin-penetrating short peptides to obtain deer antler stem cell exosomes with anti-skin aging properties.

2. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, In step S1, the compound digestive solution is composed of collagenase I at a volume ratio of 25-35 U / mL and trypsin at a volume ratio of 0.04-0.06% (w / v) at a ratio of 2:1-1.

4.

3. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, In step S1, the modified primary culture medium is DMEM / F12 medium containing 15-25% (v / v) exosome-free fetal bovine serum, 90-110 U / mL penicillin, 90-110 μg / mL streptomycin, 5-15 ng / mL vascular endothelial growth factor and 4-6 ng / mL basic fibroblast growth factor.

4. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, The method for preparing the deer antler keratin hydrolysate in step S2 is as follows: Fresh deer antler skin and hair are mixed, crushed, and then deionized water is added to adjust the pH. Cysteine ​​hydrochloride and papain are added and hydrolyzed at 50-60℃ for 4-6 hours to inactivate the enzyme. After centrifugation, the supernatant is collected and dialyzed to obtain deer antler keratin hydrolysate.

5. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, In step S2, the modified passage medium is DMEM / F12 medium containing 8-12% (v / v) exosome-free fetal bovine serum, 90-110 U / mL penicillin, 90-110 μg / mL streptomycin, and 6-10 ng / mL basic fibroblast growth factor; the serum-free secretion medium is mTeSR™1 medium.

6. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, In step S3, the centrifugation speed is 2500-3500 rpm, the centrifugation time is 25-35 min, and the concentration is carried out to 5-10% of the original cell supernatant volume; the volume ratio of the concentrate to the antibody-conjugated magnetic beads is 8-12:

1.

7. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, In step S4, the complex polysaccharide is composed of hyaluronic acid and carboxymethyl chitosan in a mass ratio of 4-6:1; the skin-penetrating short peptide is one or more of TAT short peptide, Penetratin short peptide, and MPG short peptide.

8. The method for preparing deer antler stem cell exosomes for anti-skin aging according to claim 1, characterized in that, In step S4, the mass ratio of deer antler stem cell exosomes to complex polysaccharides is 4-6:1; the mass ratio of polysaccharide-exosome complex microspheres to skin-penetrating short peptides is 90-110:

1.

9. Deer antler stem cell exosomes prepared by the method of any one of claims 1-8 for anti-skin aging.

10. The use of the anti-skin aging deer antler stem cell exosomes of claim 9 in the preparation of anti-aging cosmetics, skin care products or skin repair dressings.

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