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CN-121971358-A - Anti-hair loss repair composition based on mesenchymal stem cell exosomes and preparation method thereof

CN121971358ACN 121971358 ACN121971358 ACN 121971358ACN-121971358-A

Abstract

The invention discloses an anti-hair loss repair composition based on mesenchymal stem cell exosomes and a preparation method thereof, and relates to the technical fields of biotechnology, regenerative medicine and cosmetics, wherein the composition comprises human umbilical mesenchymal stem cell-derived exosomes, pharmaceutically or cosmetically acceptable carriers, scalp barrier repair agents, hair follicle nutrition enhancers and targeted affinity enhancers; the preparation method of the composition comprises five steps of exosome preparation and identification, functional ingredient premixing, precise mixing of exosome and basic liquid, stabilization treatment, sterilization split charging and preservation, wherein exosome is prepared by specific generation human umbilical mesenchymal stem cell induction culture and combined purification, and a whole-process sterile and precise mixing and stabilization process is optimized by cooperation of multiple functional components, so that the problems of insufficient activity, poor targeting, unstable storage and the like of the existing product are solved, scalp irritation is avoided, and the dual effects of anti-drop and scalp restoration can be stably exerted for a long time.

Inventors

  • WANG DONGWEI
  • YIN FENGLAN
  • LIU JINGJING

Assignees

  • 北京福瑞天使国际医疗科技有限公司

Dates

Publication Date
20260505
Application Date
20260209

Claims (10)

  1. 1. An anti-hair loss repair composition based on mesenchymal stem cell exosomes is characterized by comprising exosomes derived from human umbilical mesenchymal stem cells, a pharmaceutically or cosmetically acceptable carrier, a scalp barrier repair agent, a hair follicle nutrition enhancer and a targeting affinity enhancer; The exosomes are at a concentration of 1×10 8 parts/mL to 1×10 11 parts/mL; The exosome is prepared by culturing 3 rd generation to 5 th generation human umbilical cord mesenchymal stem cells to 70% to 80% fusion degree in a serum-free culture medium, replacing the culture medium with an induction culture medium containing 10ng/mL to 50ng/mL VEGF and 5ng/mL to 20ng/mL FGF2, culturing for 48 hours to 72 hours in a 37 ℃ and 5% CO 2 environment, collecting the conditioned medium, and separating and purifying by combining an ultracentrifugation method with size exclusion chromatography; the pH of the carrier is from 6.0 to 7.4.
  2. 2. The composition of claim 1, wherein the scalp barrier repair agent is 0.05% to 0.3% ceramide NP or ceramide AP, the hair follicle nutritional enhancer is 0.01% to 0.1% folic acid and 0.005% to 0.02% biotin, and the targeting affinity enhancer is 0.001% to 0.01% RGD peptide.
  3. 3. The composition for preventing and repairing alopecia based on mesenchymal stem cell exosomes according to claim 1, further comprising one or more of 0.01-0.5% of chondroitin sulfate or sodium hyaluronate as exosomes delivery and stabilizer, wherein the molecular weight of sodium hyaluronate is less than 100kDa and the purity is not less than 98%, the purity of chondroitin sulfate is not less than 98%, 0.1-2% of caffeine or 0.5-3% of nicotinamide as hair follicle microcirculation promoter, 0.05-1% of ectoin or 0.1-2% of panthenol as antioxidant and anti-inflammatory agent, 1-8% of pumpkin seed oil extract or adenosine as 5 alpha-reductase inhibitor, and the purity of pumpkin seed oil extract is not less than 95%.
  4. 4. The composition for preventing and repairing alopecia based on mesenchymal stem cell exosomes according to claim 1, wherein the carrier is a buffer solution or an aqueous polyol solution, wherein the buffer solution is PBS or HEPES, and the aqueous polyol solution is a mixture of propylene glycol, glycerin and water.
  5. 5. The composition for preventing and repairing alopecia based on mesenchymal stem cell exosomes according to claim 1, wherein the particle size of the exosomes is 30-150 nm, and the ultracentrifugation method sequentially comprises removal of cell debris by centrifugation of 2000g, removal of microbubbles by centrifugation of 10000g, and precipitation of exosomes by centrifugation of 120000 g.
  6. 6. A method for preparing an anti-hair loss repair composition based on mesenchymal stem cell exosomes, the method being used for preparing an anti-hair loss repair composition based on mesenchymal stem cell exosomes according to any one of claims 1-5, the method comprising: S1, preparing and identifying exosomes, namely culturing 3 rd generation to 5 th generation human umbilical cord mesenchymal stem cells to 70-80% fusion degree in a serum-free culture medium, replacing the culture medium with an induction culture medium containing 10-50 ng/mL VEGF and 5-20 ng/mL FGF2, culturing the culture medium in a CO 2 environment at 37 ℃ for 48-72 hours, collecting the conditioned medium, purifying the exosomes by ultrafiltration concentration, ultracentrifugation and size exclusion chromatography, and identifying the exosomes to be qualified by nanoparticle tracking analysis and protein immunoblotting; s2, premixing functional components, namely dispersing exosome delivery and stabilizing agent, hair follicle microcirculation promoter, antioxidant and anti-inflammatory agent, 5 alpha-reductase inhibitor, scalp barrier repairing agent, hair follicle nutrition enhancer and targeting affinity enhancer in a carrier according to a proportion, and stirring until the exosome delivery and stabilizing agent, the hair follicle microcirculation promoter, the antioxidant and anti-inflammatory agent, the 5 alpha-reductase inhibitor, the scalp barrier repairing agent, the hair follicle nutrition enhancer and the targeting affinity enhancer are completely dissolved to form a base solution; s3, accurately mixing the exosomes with the base liquid, namely mixing the exosomes obtained in the S1 with the base liquid obtained in the S2 through microfluidic equipment in an environment of 4 ℃, and monitoring the particle size of the exosomes after mixing; s4, stabilizing treatment, namely adding trehalose into the mixed system, stirring and dissolving, and then performing gradient cooling and prefreezing; And S5, sterilizing, sub-packaging and preserving, namely filtering and sterilizing by adopting a sterile filter membrane, sub-packaging into a light-resistant container under the sterile condition, and sealing and preserving.
  7. 7. The method for preparing the anti-hair loss repair composition based on mesenchymal stem cell exosomes according to claim 6, wherein the ultrafiltration concentration in the step S1 adopts a 100kDa ultrafiltration membrane, the concentration multiple is 2-3 times, the parameters of ultracentrifugation are that cell debris is removed by centrifugation of 2000g for 10-15 minutes, microbubbles are removed by centrifugation of 10000g for 30-40 minutes, exosomes are precipitated by centrifugation of 120000g for 60-80 minutes, the precipitate is washed with PBS or HEPES for 2 times, and after each washing, 120000g is centrifuged for 30 minutes, the precipitate is resuspended with the PBS or HEPES, the target elution peak is collected after purification by size exclusion chromatography, the marker proteins detected by the protein immunoblotting method are CD63, CD81 and TSG101, the exosomes have a particle size of 30-150 nanometers and a concentration of 1X10 8 parts/mL to 1X10 11 parts/mL, and the protein immunoblotting is positive.
  8. 8. The method for preparing the composition for preventing and repairing alopecia based on mesenchymal stem cell exosomes according to claim 6, wherein in the step S2, the stirring container is a sterile container, the stirring rotation speed is 300-500 rpm, the stirring time is 2-4 hours, the stirring environment is normal temperature, the basic solution is cooled to 4 ℃ after the stirring is completed and is kept for 30 minutes, the solution is clear and has no insoluble particles visible to naked eyes, the mixing flow rate ratio of exosomes to the basic solution in the step S3 is 1-10, and the particle size monitoring adopts online nanoparticle tracking analysis, so that the particle size is ensured to be maintained within a range of 30-150 nanometers.
  9. 9. The method for preparing the anti-hair loss repair composition based on mesenchymal stem cell exosomes, which is characterized in that the adding amount of the trehalose in the step S4 is 0.5-2%, the gradient cooling pre-freezing process is that the composition is placed at 4 ℃ for 2 hours, -20 ℃ for 4 hours, -80 ℃ for more than 2 hours, and after the pre-freezing is completed, the composition is slowly thawed in a4 ℃ environment, and the thawing time is 4-6 hours, and intense shaking is forbidden during the thawing.
  10. 10. The method for preparing the composition for preventing and repairing alopecia based on mesenchymal stem cell exosomes, which is characterized in that the pore diameter of the sterile filter membrane in the step S5 is 0.22 μm, the light-resistant container is a brown sterile ampoule or a brown bottle, the specification of the brown sterile ampoule is 1-2 mL, the specification of the brown bottle is 10-50 mL, and the preservation condition is-20 ℃ or 4 ℃ and the light-resistant storage is carried out.

Description

Anti-hair loss repair composition based on mesenchymal stem cell exosomes and preparation method thereof Technical Field The invention relates to the technical fields of biotechnology, regenerative medicine and cosmetics, in particular to an anti-hair loss repair composition based on mesenchymal stem cell exosomes and a preparation method thereof. Background With the increase of life pressure, irregular work and rest and the influence of environmental factors, the problem of alopecia becomes a common problem which puzzles various crowds, not only affects personal images, but also possibly brings psychological burden, so that the development of safe and effective alopecia-preventing repair products has important market value and application prospect. At present, the mesenchymal stem cell exosome has good functions of promoting cell repair, resisting inflammation and nutrition delivery, is widely applied to the field of alopecia prevention, has wide sources of human umbilical cord mesenchymal stem cell exosome, high safety and no immunological rejection risk, becomes the preference of an active component for alopecia prevention, and meanwhile, various functional components are often adopted to be matched with exosome to prepare an alopecia prevention composition in the prior art, but how to promote exosome activity, optimize the synergistic effect of the components and the preparation process becomes a key problem to be solved in the research and development process of the current alopecia prevention product. In the prior related technology for preventing alopecia, the exosomes are prepared in a non-induction culture mode, the purification method is single, the concentration of the exosomes is unstable, the activity is low, the hair follicle repairing effect is difficult to fully play, meanwhile, the problem of unreasonable component collocation and poor cooperativity often exists in the anti-alopecia composition, the multiple requirements of scalp barrier repairing, hair follicle nutrition supplementing, alopecia inhibiting and the like cannot be met by a single component, the anti-alopecia effect is limited, in addition, the traditional preparation technology lacks standardized aseptic control, the exosomes and functional components are unevenly mixed, effective stabilization treatment measures are not available, the exosomes are easy to gather and inactivate, the storage period of the product is short, the large-scale production is difficult to realize, meanwhile, the scalp irritation is easy to be caused due to the fact that the carrier pH is not adapted to the scalp environment, and the long-term use requirement cannot be met. Disclosure of Invention The invention aims to make up the defects of the prior art, provides an anti-hair loss repair composition based on mesenchymal stem cell exosomes and a preparation method thereof, and combines ultrafiltration concentration, ultracentrifugation and size exclusion chromatography combined purification through specific generation human umbilical cord mesenchymal stem cell induction culture, the high-activity exosomes are prepared, and are matched with various functional components such as scalp barrier repairing agent, hair follicle nutrition enhancer and the like, so that the whole-process sterile, microfluidic and precise mixing and trehalose and gradient temperature change stabilizing process are optimized, and the alopecia preventing repairing composition is constructed. The scheme effectively solves the problems of insufficient exosome activity, uneven mixing, easy inactivation during storage, scalp irritation and other pain points of the traditional product, has strong exosome targeting, can repair scalp barriers, supplement hair follicle nutrition, inhibit the activity of related enzymes of alopecia, has high product stability and good activity retention rate, has no scalp irritation, can stably exert the synergistic effect of anti-drop and repair for a long time, and is suitable for large-scale production and daily scalp care application. In order to solve the technical problems, the invention provides the following technical proposal that on one hand, the composition for preventing and repairing alopecia based on mesenchymal stem cell exosomes comprises exosomes derived from human umbilical cord mesenchymal stem cells, a pharmaceutically or cosmetically acceptable carrier, a scalp barrier repairing agent, a hair follicle nutrition enhancer and a targeting affinity enhancer; The exosomes are at a concentration of 1×10 8 parts/mL to 1×10 11 parts/mL; The exosome is prepared by culturing 3 rd generation to 5 th generation human umbilical cord mesenchymal stem cells to 70% to 80% fusion degree in a serum-free culture medium, replacing the culture medium with an induction culture medium containing 10ng/mL to 50ng/mL VEGF and 5ng/mL to 20ng/mL FGF2, culturing for 48 hours to 72 hours in a 37 ℃ and 5% CO 2 environment, collecting the conditioned medium, and se