Stem cell exosome composition for skin disease repair and anti-aging and preparation method of stem cell exosome composition

Abstract

The invention belongs to the technical field, and discloses a stem cell exosome composition for skin disease repair and anti-aging and a preparation method thereof, the product adopts the combined application of MSC and ADSC exosomes, the composition has a complementary effect in the aspects of promoting skin repair and anti-aging, the MSC exosome enhances skin regeneration, and the ADSC exosomes enhance skin regeneration. The ADSC exosome has better anti-inflammatory and anti-aging effects, the nanocapsule carrier can improve the stability of the exosome, can optimize the skin permeability of the exosome and prolong the action time of the exosome, and by adding skin barrier repair components (such as squalene and ceramide), the exosome can repair the skin, and meanwhile, the skin barrier repair effect of the exosome is improved. The self-repairing capability of the skin can be enhanced, and the skin barrier function is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin repair and anti-aging, and specifically relates to a stem cell exosome composition for skin disease repair and anti-aging and a preparation method thereof. Background Art

[0002] The treatment of skin aging, damage, and other skin conditions such as trauma, acne scars, and hyperpigmentation has always been a crucial topic in the skincare field. In recent years, stem cell exosomes have garnered widespread attention due to their prominent roles in cell repair, anti-inflammatory effects, angiogenesis, and anti-aging. However, existing exosomes still have significant room for improvement in terms of stability, targeting, and personalized customization. Summary of the Invention

[0003] To overcome the above technical problems, the present invention provides a stem cell exosome composition for skin disease repair and anti-aging and a preparation method thereof.

[0004] The present invention adopts the following technical solutions: A stem cell exosome composition for skin disease repair and anti-aging, comprising: Stem cell exosomes 20-50% MSC exosomes promote skin cell proliferation, angiogenesis, and wound healing; ADSC exosomes fight aging, reduce skin inflammation, and accelerate skin repair; iPSC exosomes enhance skin tissue regeneration and are particularly suitable for deep skin repair. Nanocapsule carrier system 5-15% Poly(lactic-co-glycolic acid) nanoparticles are biodegradable and have good drug-loading capacity and sustained-release effects; phospholipids serve as auxiliary ingredients to improve the skin permeability of exosomes; Skin barrier repair ingredients 5-10% Squalene, helps restore the skin barrier, reduces water loss, and enhances skin lubrication; ceramide, repairs the skin barrier, improves the skin's ability to lock in moisture, and reduces skin dryness and irritation; glycerin, enhances skin hydration; Anti-aging ingredients 5-8% Vitamin C promotes collagen synthesis and reduces free radical damage; Hepatocyte Growth Factor promotes skin cell proliferation and repair; Peptides: promote skin repair and reduce wrinkles; Excipients 1-5% Sodium hyaluronate, strong moisturizing, maintains skin elasticity and enhances skin comfort; Aloe vera extract, soothes and repairs, reduces skin irritation and inflammation; Replenish water to 100% solvent.

[0005] The present invention also discloses a method for preparing a stem cell exosome composition for skin disease repair and anti-aging, comprising: Step 1: Extraction of stem cell exosomes 1. Cell culture: Select appropriate stem cell sources, such as MSCs, ADSCs, and iPSCs, for expansion and culture; Serum-free medium was used to reduce exosome contamination and cultured to the logarithmic growth phase; 2. Exosome extraction: Exosomes were extracted using differential centrifugation or ultrafiltration; The size, morphology, and purity of exosomes were confirmed by nanoparticle tracking analysis and transmission electron microscopy; Step 2: Preparation of nanocapsule carriers 1. Preparation of PLGA nanocapsules: Exosomes are encapsulated in PLGA nanoparticles using solvent evaporation or self-assembly methods to ensure the stability of exosomes; Optimize the size and surface charge of nanoparticles through particle size analysis and Zeta potential testing to improve their penetration and drug loading capacity; Step 3: Mixing and homogenizing ingredients 1. Mix the extracted exosomes with nanocapsule carriers, skin barrier repair ingredients, and anti-aging ingredients in appropriate proportions; 2. Use a high shear homogenizer to homogenize the mixture so that all ingredients are evenly dispersed; 3. Further processing by freeze drying or lyophilization to ensure formulation stability; Step 4: Quality Control and Product Testing 1. Exosome activity testing: Testing the stability and activity of key growth factors and miRNAs in exosomes through miRNA and protein analysis; 2. Skin permeability test: A skin model is used to test the permeability of exosomes to ensure that they can reach deep into the skin; 3. Stability test: Test the stability and shelf life of the composition through environmental conditions such as temperature and light; Step 5: Packaging and Storage Sterile packaging, refrigerated at 2-8°C, and stored away from light to maintain the activity and stability of exosomes.

[0006] Compared with the prior art, the present invention has the following beneficial effects: The combined application of MSC and ADSC exosomes has complementary effects in promoting skin repair and anti-aging. MSC exosomes enhance skin regeneration, while ADSC exosomes have better anti-inflammatory and anti-aging effects. Nanocapsule carriers can not only improve the stability of exosomes, but also optimize their skin permeability and prolong the duration of action of exosomes. By adding skin barrier repair ingredients (such as squalene and ceramide), exosomes can not only repair the skin, but also enhance the skin's self-repair ability and improve skin barrier function.

[0007] Precise formulation and personalized customization: Using technologies such as genomics and skin biomarker analysis to achieve personalized customization, ensuring that the effect of exosomes can maximize the adaptation to the skin condition of each user.

[0008] Surface modification technology is used to fix specific targeting molecules (such as targeting peptides or antibodies) on the surface of exosomes to achieve targeted delivery of exosomes. DETAILED DESCRIPTION

[0009] The following embodiments of the present invention are described in detail. Unless otherwise specified, the raw materials and equipment used are commercially available or commonly used in the art. The methods in the embodiments, unless otherwise specified, are conventional methods in the art. The embodiments described below are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0010] Preparation of stem cell exosome composition for skin disease repair and anti-aging Laboratory equipment: 1. Centrifuge (adjustable speed, maximum speed at least 10,000 rpm) 2. Ultrafiltration system (with molecular weight cut-off membrane, 10kDa and 30kDa optional) 3. Clean bench 4. High-speed refrigerated centrifuge (with 4°C temperature control) 5. Nanoparticle Tracking Analyzer (NTA) 6. Transmission Electron Microscopy (TEM) 7. High shear homogenizer 8. Freeze Dryer 9. Small stirrer and magnetic stirrer 10. Laser particle size analyzer 11. Zeta potential measuring instrument 12. pH meter Materials needed: 1. Source of stem cells: MSC cells (mesenchymal stem cells) ADSC cells (adipose-derived stem cells) iPSC cells (induced pluripotent stem cells) 2. Culture medium: Serum-free MSC culture medium Serum-free ADSC culture medium Serum-free iPSC culture medium 3. Exosome extraction reagent: Glass beads, centrifuge tubes, PBS (Phosphate Buffer Saline) ExoQuick (Exosome Precipitation Reagent) Centrifugal filters (30kDa, 50kDa) 4. Nanoparticle preparation materials: PLGA (Poly-lactic-co-glycolic acid) Lecithin Organic solvents (such as chloroform, dichloromethane) 5. Skin repair and anti-aging ingredients: Squalene, ceramide, glycerol Vitamin C, HGF, copper peptide, tetrapeptide Sodium hyaluronate, aloe vera extract 6. Auxiliary solvents: Distilled water PBS (Phosphate Buffer Saline) Step 1: Extraction of stem cell exosomes Objective: Extract exosomes from MSC, ADSC, and iPSC cells as key ingredients for skin repair and anti-aging.

[0011] 1.1 Cell culture MSC, ADSC, and iPSC cells need to be cultured in serum-free medium suitable for their proliferation.

[0012] Culture conditions: Temperature: 37°C CO2 concentration: 5% Cell density: Cultured to 70-80% confluence.

[0013] 1.2 Exosome extraction Cell harvesting: After the cells reach the desired density, remove the culture medium and rinse once with PBS.

[0014] Digest the cells with 0.25% trypsin (0.1% EDTA) for 3-5 minutes.

[0015] After stopping the digestion reaction, add culture medium and centrifuge the cells (1,000 rpm for 5 minutes), remove the supernatant, and retain the cell pellet.

[0016] Exosome extraction: The cell culture medium was centrifuged (3,000 rpm, 10 minutes) to remove cell debris.

[0017] Centrifuge again (10,000 rpm, 30 min) to remove cell debris.

[0018] The exosome extraction method was the ExoQuick exosome precipitation reagent method. ExoQuick was added according to the instructions (ratio 1:5) and allowed to stand at room temperature for 1 hour.

[0019] The precipitate was collected by centrifugation at 12,000 rpm (30 min) to obtain crude exosomes.

[0020] The exosome pellet was washed twice with PBS to remove impurities.

[0021] Exosome purification: The crude exosomes were filtered through a 30 kDa ultrafiltration membrane (5,000 rpm, 10 min) to remove small molecular impurities.

[0022] The concentration and size of exosomes were analyzed by nanoparticle tracking analyzer (NTA).

[0023] Transmission electron microscopy (TEM) was used to observe the morphology of exosomes.

[0024] Step 2: Preparation of nanocapsule carriers Objective: To encapsulate exosomes in nanocapsules to improve their stability and skin permeability.

[0025] 2.1 Preparation of PLGA nanocapsules Preparation of PLGA nanoparticles by solvent evaporation method: Prepare PLGA solution: dissolve PLGA (100 mg) in chloroform (5 ml), add lecithin (10 mg), and stir well.

[0026] The PLGA solution was slowly added dropwise to a solution containing PBS (10 ml) to form an emulsion.

[0027] A stable emulsion was formed by high-speed stirring (10,000 rpm, 5 min).

[0028] The chloroform was removed by rotary evaporation to obtain PLGA nanoparticles.

[0029] Surface charge optimization: The nanoparticles were resuspended in PBS and the pH was adjusted to 7.4 to ensure stability.

[0030] The surface charge of the nanoparticles was measured by Zeta potential measurement to ensure their suitability for skin penetration.

[0031] 2.2 Exosome embedding Purified exosomes (20 mg) were added to the PLGA nanoparticle solution.

[0032] Homogenization was performed using a high shear homogenizer (20,000 rpm, 5 min) to ensure that the exosomes were evenly distributed within the nanoparticles.

[0033] The cells were freeze-dried to obtain a stable exosome-PLGA nanocapsule complex.

[0034] Step 3: Adding repair and anti-aging ingredients Objective: To add skin repair and anti-aging ingredients to the exosome-nanocapsule system to enhance the effect of the complex.

[0035] 3.1 Dissolution and mixing of skin repair and anti-aging ingredients Squalene (1%), ceramide (2%), glycerol (5%) and vitamin C (1%) were dissolved in distilled water or appropriate amount of ethanol, respectively.

[0036] HGF (hepatocyte growth factor) (0.5%), copper peptide (1%) and tetrapeptide (1%) were added to the solution and mixed well.

[0037] Sodium Hyaluronate (1%) and Aloe Vera Extract (3%) are added as moisturizing and soothing factors.

[0038] 3.2 Ingredient mixing The repair and anti-aging ingredient solution is slowly added into the exosome-nanocapsule system.

[0039] The mixture was stirred using a magnetic stirrer (300 rpm, 4 °C) for 3 h to ensure that the ingredients were thoroughly mixed.

[0040] Step 4: Testing and storage of final formulation 4.1 Quality Control The particle size of the nanoparticles was measured using a laser particle size analyzer (the optimized particle size should be in the range of 100-200 nm).

[0041] Transmission electron microscopy (TEM) was used to verify the morphology of the exosome-nanocapsule complexes.

[0042] Measure pH to ensure the formula is stable and suitable for skin use (pH: 6.5-7.5).

[0043] 4.2 Storage The prepared composition is filled into sterile glass bottles or tubes and sealed.

[0044] Store at 4°C in a refrigerated place away from light to maintain exosome activity.

[0045] Step 5: Packaging and Application After the final product is packaged, sterility testing and stability testing are carried out to ensure the stability and efficacy of the product within its shelf life.

[0046] Product usage effect data Experimental design Test subjects: 90 volunteers, age range: 25-60 years old, both men and women, skin types including dry, oily, and combination.

[0047] Use period: 12 weeks Experimental groups: Experimental group: used a skin repair composition containing stem cell exosomes and repair and anti-aging ingredients.

[0048] Control group: Use commercially available conventional anti-aging / skin repair cream.

[0049] Evaluation Metrics 1. Skin elasticity (using a surface skin analyzer, unit: % change) 2. Wrinkle depth (using a wrinkle meter, unit: mm) 3. Skin hydration (using a skin hydration meter, unit: skin hydration%) 4. Skin brightness and evenness (assessed by colorimeter and skin optical analyzer) 5. Pigmentation and spot area (measured by skin imaging) Experimental results 1. Skin elasticity (unit: % change) Data analysis: After 12 weeks, the experimental group showed a 45% increase in skin elasticity, significantly higher than the 18% increase in the control group. This suggests that the combination of exosomes and anti-aging ingredients can more effectively improve skin elasticity.

[0050] 2. Wrinkle depth (unit: mm) Data analysis: The experimental group's wrinkle depth decreased by 0.65mm over 12 weeks, while the control group's reduction was only 0.35mm. The wrinkle improvement effect in the experimental group was significantly greater than that in the control group, indicating that the composition has a significant anti-aging effect.

[0051] 3. Skin hydration (unit: skin hydration%) Data analysis: Skin hydration in the experimental group increased by 35% over 12 weeks, while in the control group it only increased by 16%. This indicates that the composition can significantly improve skin hydration and maintain long-term skin moisture and smoothness.

[0052] 4. Skin brightness and evenness (unit: color difference ΔE) Data Analysis: The experimental group showed significant improvement in skin brightness and evenness over 12 weeks, with the color difference value decreasing from 14.2 to 7.6, while the control group saw a decrease to 10.5. This result demonstrates that the exosome composition not only improves skin hydration but also significantly enhances skin brightness and evenness, resulting in a noticeable whitening effect.

[0053] 5. Pigmentation and spot area (unit: spot area change %) Data Analysis: The experimental group showed a 64.7% reduction in pigmented area over 12 weeks, while the control group saw a 21.0% reduction. This significant reduction in pigmentation suggests the product has a strong pigmentation-inhibiting effect.

[0054] in conclusion 1. Skin elasticity: The experimental group showed a more significant improvement in skin elasticity compared to the control group, proving that the exosome complex can significantly improve skin firmness.

[0055] 2. Wrinkle improvement: The rate of reduction in wrinkle depth in the experimental group was significantly higher than that in the control group, indicating that the product has an effective anti-aging effect.

[0056] 3. Hydration: The hydration of the skin in the experimental group was significantly improved, and the dryness and roughness of the skin were improved.

[0057] 4. Skin brightness and evenness: Exosome complex helps to improve skin brightness, reduce spots and pigmentation, and present an even and smooth effect.

[0058] 5. Pigmentation: The experimental group showed a significant reduction in the area of ​​pigmentation, demonstrating its significant effect in improving uneven skin tone and inhibiting melanin.

[0059] In summary, the exosome composition has obvious skin repair and anti-aging effects, and can significantly improve skin elasticity, wrinkle depth, hydration, brightness uniformity and pigmentation.

[0060] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the above embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A stem cell exosome composition for skin disease repair and anti-aging, comprising: Stem cell exosomes 20-50% MSC exosomes promote skin cell proliferation, angiogenesis, and wound healing; ADSC exosomes fight aging, reduce skin inflammation, and accelerate skin repair; iPSC exosomes enhance skin tissue regeneration and are particularly suitable for deep skin repair. Nanocapsule carrier system 5-15% Poly(lactic-co-glycolic acid) nanoparticles are biodegradable and have good drug-loading capacity and sustained-release effects; phospholipids serve as auxiliary ingredients to improve the skin permeability of exosomes; Skin barrier repair ingredients 5-10% Squalene, helps restore the skin barrier, reduces water loss, and enhances skin lubrication; ceramide, repairs the skin barrier, improves the skin's ability to lock in moisture, and reduces skin dryness and irritation; glycerin, enhances skin hydration; Anti-aging ingredients 5-8% Vitamin C promotes collagen synthesis and reduces free radical damage; Hepatocyte Growth Factor promotes skin cell proliferation and repair; Peptides: promote skin repair and reduce wrinkles; Excipients 1-5% Sodium hyaluronate, strong moisturizing, maintains skin elasticity and enhances skin comfort; Aloe vera extract, soothes and repairs, reduces skin irritation and inflammation; Replenish water to 100% solvent.

2. The method for preparing a stem cell exosome composition for skin disease repair and anti-aging according to claim 1, comprising: Step 1: Extraction of stem cell exosomes 1. Cell culture: Select appropriate stem cell sources, such as MSCs, ADSCs, and iPSCs, for expansion and culture; Serum-free medium was used to reduce exosome contamination and cultured to the logarithmic growth phase; 2. Exosome extraction: Exosomes were extracted using differential centrifugation or ultrafiltration; The size, morphology, and purity of exosomes were confirmed by nanoparticle tracking analysis and transmission electron microscopy; Step 2: Preparation of nanocapsule carriers 1. Preparation of PLGA nanocapsules: Exosomes are encapsulated in PLGA nanoparticles using solvent evaporation or self-assembly methods to ensure the stability of exosomes; Optimize the size and surface charge of nanoparticles through particle size analysis and Zeta potential testing to improve their penetration and drug loading capacity; Step 3: Mixing and homogenizing ingredients 1. Mix the extracted exosomes with nanocapsule carriers, skin barrier repair ingredients, and anti-aging ingredients in appropriate proportions; 2. Use a high shear homogenizer to homogenize the mixture so that all ingredients are evenly dispersed; 3. Further processing by freeze drying or lyophilization to ensure formulation stability; Step 4: Quality Control and Product Testing 1. Exosome activity testing: Testing the stability and activity of key growth factors and miRNAs in exosomes through miRNA and protein analysis; 2. Skin permeability test: A skin model is used to test the permeability of exosomes to ensure that they can reach deep into the skin; 3. Stability test: Test the stability and shelf life of the composition through environmental conditions such as temperature and light; Step 5: Packaging and Storage Sterile packaging, refrigerated at 2-8°C, and stored away from light to maintain the activity and stability of exosomes.

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