Exosome product as well as preparation method and application thereof

Abstract

The invention discloses an exosome product as well as a preparation method and application thereof, and belongs to the technical field of exosome products. The exosome product is prepared from the following raw materials: adipose-derived stem cell exosome, an exosome gel carrier, fibroblast liquid, collagen and elastin. Wherein adipose-derived stem cell exosomes and plant-derived exosome vesicles are used as entrapment substances, polyvinyl alcohol hydrogel is used as a carrier, fibroblast liquid, collagen and elastin are used as auxiliary materials, proliferation and differentiation of various cells such as fibroblasts and epithelial cells are promoted, the content of the collagen is increased, and therefore skin repair and maintenance are promoted. The exosome product disclosed by the invention is suitable for various skin types, and has potential help for people needing skin repair, anti-aging and skin quality improvement.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome products, and specifically relates to an exosome product and a preparation method and application thereof. Background Art

[0002] Exosomes are membrane vesicles released into the extracellular matrix after the fusion of intracellular multivesicular bodies with the cell membrane. They are tiny vesicles with a diameter of 30-100nm, which contain substances such as proteins, mRNA and microRNA. Exosomes exist naturally in body fluids, including blood, saliva, urine and breast milk. The secreted exosomes enter body fluids such as blood, saliva, urine and breast milk, and reach other cells and tissues through the circulatory system, producing remote regulatory effects. Extracellular vesicles regulate normal physiological processes, including angiogenesis, antigen presentation, apoptosis, coagulation, cell homeostasis, inflammation, and cell differentiation. Extracellular vesicles play an important role in stem cell maintenance, tissue repair and immune regulation.

[0003] Clinically, skin aging is manifested as wrinkles, abnormal pigmentation, loss of elasticity, thinning of the epidermis, and increased roughness of the epidermis. With age, stem cell function is impaired, and the ability to maintain epidermal tissue homeostasis and repair damaged tissue is weakened, leading to skin aging. Therefore, a series of skin care products have emerged. Before the human skin shows serious aging symptoms, the use of skin care products containing stem cell exosomes to intervene in human skin aging is a very important means. Exosomes have the following effects on the skin, ultimately improving the skin condition in an all-round way: ①. Promote base cell regeneration and repair; ②. Promote blood vessel regeneration and repair; ③. Promote collagen secretion and recombination.

[0004] In the existing technology, research focuses on exosomes from animals, but their preparation process is complicated, costly, and low in yield; while exosomes from plants have the outstanding advantages of large amount of raw materials and low cost, and have the possibility of large-scale preparation. More importantly, they have higher biocompatibility and safety factor. Plant exosomes have been less studied than cells, but they also play a vital role in promoting cell communication. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an exosome product and its preparation method and application, taking plant exosomes combined with a small amount of adipose stem cell exosomes as the research object, and through the mutual cooperation with other components, the purpose of improving the activity of exosomes and mass reproduction is achieved, and at the same time, the proliferation and differentiation of various cells such as fibroblasts and epithelial cells are promoted, and the collagen content is increased, thereby promoting skin repair and maintenance.

[0006] In a first aspect, the present invention provides an exosome product, comprising the following raw materials in parts by weight: 1-5 parts of adipose stem cell exosomes, 2-15 parts of exosome gel carriers, 15-50 parts of fibroblast fluid, 0.01-0.2 parts of collagen, and 0.02-0.1 parts of elastin;

[0007] The exosome gel carrier is a double-layer composite structure, with polyvinyl alcohol hydrogel on the outside and plant exosome vesicles on the inside.

[0008] Furthermore, the plant source of the plant exosome vesicles is Codonopsis pilosula, Notopterygium wilfordii or Basella alba.

[0009] Furthermore, the plant exosome vesicles are prepared by the following method:

[0010] Perform gradient centrifugation at 300-800 g, 1000-2000 g, and 2000-3000 g at 3-5 °C to obtain the supernatant;

[0011] The supernatant was centrifuged at 3000-5000 g and resuspended to obtain plant exosome vesicles.

[0012] Furthermore, the adipose stem cell exosomes are prepared by the following method:

[0013] The third generation cells were obtained by subculturing, cultured at a constant temperature in serum-free DMEM medium, and irradiated with near-infrared light;

[0014] The supernatant was collected and centrifuged at 3000-5000 g and 10000-18000 g, respectively. After incubation at 3-5° C. overnight, the precipitate was obtained to obtain the product.

[0015] Furthermore, the exosome product comprises the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 15 parts of exosome gel carriers, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin.

[0016] Furthermore, the preparation method of the polyvinyl alcohol hydrogel is:

[0017] The polyvinyl alcohol powder is mixed with deionized water, heated and stirred until transparent, and allowed to stand to obtain the product.

[0018] Furthermore, the mass ratio of the polyvinyl alcohol hydrogel to the plant exosome vesicles is 5-20:3-10.

[0019] In a second aspect, the present invention discloses a method for preparing an exosome product, comprising the following steps:

[0020] Prepare adipose stem cell exosomes and plant exosome vesicles respectively;

[0021] The plant exosome vesicles are placed in a buffer solution, and adipose stem cell exosomes are added under the condition of blue light irradiation to obtain a first product;

[0022] The polyvinyl alcohol powder is mixed with deionized water, heated and stirred until transparent, and the first product is added during cooling to obtain the second product;

[0023] The second product is mixed with fibroblast fluid, collagen and elastin, and then freeze-dried to obtain an exosome product.

[0024] On the third aspect, based on a general technical concept, the present invention discloses an application of an exosome preparation in the preparation of a hair growth product, which can effectively promote the proliferation of hair follicle cell vitality and stimulate new hair growth.

[0025] In a fourth aspect, based on a general technical concept, the present invention discloses an application of an exosome preparation in the preparation of a skin care product.

[0026] Furthermore, the preparation of skin care products includes: preparing skin care masks, skin care creams, skin care lotions, skin care essence water, etc., which are prepared by adding specific additives in this field.

[0027] The above technical solution provided by the embodiment of the present application has at least the following advantages compared with the prior art:

[0028] The exosome product disclosed in the present invention uses exosome vesicles extracted from plants and adipose stem cell exosomes for the first time, and is applicable to a variety of medical fields, such as wound healing, plastic surgery, bone and cartilage repair, cardiovascular disease treatment, etc. Among them, the product combines adipose stem cell exosomes, exosome gel carriers, fibroblast fluid, collagen, elastin and other components, which have complementary effects in tissue repair and regeneration and can enhance the therapeutic effect.

[0029] Among them, adipose stem cell exosomes contain a variety of growth factors and cytokines, which play a major role in promoting the repair and regeneration of damaged tissues. As a double-layer composite structure, the exosome gel carrier can provide a sustained-release effect, prolong the time of exosomes at the site of action, and improve their bioavailability. Plant exosome vesicles can enhance targeting, allowing therapeutic ingredients to reach the target tissue more effectively. Polyvinyl alcohol hydrogel has good biocompatibility, reducing adverse reactions and rejection to the human body. The addition of collagen and elastin not only helps cell attachment and growth, but also improves the mechanical strength and elasticity of the product, which is particularly important for tissue engineering applications. Fibroblast fluid may contain factors that promote cell proliferation and differentiation, which helps accelerate the tissue regeneration process.

[0030] In the preparation method of the present invention, different light exposures can respectively affect cell behavior and cell membrane permeability, thereby promoting exosome secretion and improving exosome activity. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0033] In a first aspect, the present application provides an exosome product, comprising the following raw materials in parts by weight: 1-5 parts of adipose stem cell exosomes, 2-15 parts of exosome gel carriers, 15-50 parts of fibroblast fluid, 0.01-0.2 parts of collagen, and 0.02-0.1 parts of elastin;

[0034] The exosome gel carrier is a double-layer composite structure, with polyvinyl alcohol hydrogel on the outside and plant exosome vesicles on the inside.

[0035] In the present invention, adipose stem cell exosomes are stem cells derived from adipose tissue and have multidirectional differentiation potential, containing a variety of growth factors, cytokines, proteins and nucleic acids, etc., which play an important role in promoting tissue repair and regeneration. Adipose stem cell exosomes can regulate immune response and reduce inflammation, which is very beneficial for treating inflammatory diseases and promoting wound healing. The present invention uses adipose stem cell exosomes in combination with plant-derived exosomes, containing different bioactive molecules, such as growth factors, proteins, lipids and RNA, etc., which can complement each other and enhance the therapeutic effect. Plant exosomes have the characteristics of regulating immune response, and combined with adipose stem cell exosomes can more effectively control inflammation and promote wound healing. At the same time, plant exosomes can help adipose stem cell exosomes be better absorbed by cells and improve their bioavailability in vivo. And the combined use of plant and animal exosomes can stimulate cross-species synergistic effects and produce therapeutic effects that single-source exosomes do not have.

[0036] Exosome vesicles derived from medicinal plants are rich in various bioactive lipids, proteins, RNA and other components, and have significant regulatory effects in skin repair, immune regulation, anti-inflammatory and anti-infection, and regenerative medicine. In addition, plant exosomes have the advantages of small size and strong tissue penetration, and can maintain good physical and chemical stability at different pH and temperatures. Plant exosomes have both the morphology and characteristics of nanocarriers, and show significant advantages in biocompatibility, stability, in vivo distribution, extended half-life and cell internalization. Among them, plant exosomes are derived from Tibetan Codonopsis, Notopterygium or Basella, which have multiple biological activities, such as anti-inflammatory, antioxidant, and cell regeneration, and are beneficial to skin health. Chinese medicine compositions have the functions of conditioning skin, balancing oils, and improving skin quality. Combined with modern skin care technology, they can improve the efficacy of products. Fibroblast fluid contains a variety of growth factors and cytokines, which can promote the proliferation and repair of skin cells, help improve skin elasticity and reduce wrinkles. Collagen and elastin are the main structural proteins of the skin. Supplementing these two proteins can help maintain the elasticity and firmness of the skin. Due to the use of natural plant extracts and bioactive ingredients, the product of the present invention has higher safety and a wide range of applications compared to chemically synthesized substances. The exosome product is suitable for a variety of skin types and has potential help for people who need skin repair, anti-aging, and skin quality improvement. By combining the effects of multiple ingredients, it helps to maintain the health of the skin and prevent skin problems. At the same time, the plant sources of the present invention include traditional Chinese medicine (basella, i.e. tofu vegetable), Tibetan medicine (Tibetan Codonopsis) and Qiang medicine (qianghuo). When these three medicinal ingredients are combined, their active ingredients interact with each other to produce a synergistic effect, which can enhance the overall effect of the product.

[0037] In some optional embodiments of the present invention, the plant exosome vesicles are derived from at least one of Codonopsis pilosula, Notopterygium wilfordii and Basella alba, preferably a composition of Codonopsis pilosula, Notopterygium wilfordii and Basella alba mixed in a mass ratio of 1:2:10.

[0038] In some optional embodiments of the present invention, the plant exosome vesicles are prepared by the following method:

[0039] Perform gradient centrifugation at 300-800 g (10-30 min), 1000-2000 g (10-30 min), and 2000-3000 g (5-15 min) at 3-5 °C to obtain the supernatant;

[0040] The supernatant was centrifuged at 3000-5000 g (10-30 min) and then resuspended to obtain plant exosome vesicles.

[0041] In the above scheme, the use of ultracentrifugation to prepare plant exosome vesicles has at least the following advantages: ① High purity. Through gradient centrifugation steps at different speeds, impurities such as dead cells, cell debris, microvesicles and plant fibers can be effectively removed, and relatively pure exosomes can be finally obtained. ② Good repeatability. Ultracentrifugation is a mature laboratory technology with standardized operating steps and good repeatability, making the experimental results of different laboratories and different time points comparable. ③ Maintaining biological activity. Centrifugation under low temperature conditions helps to maintain the biologically active components in the exosomes and avoid damage to the active substances caused by high temperature.

[0042] Preferably, the plant exosome vesicles are prepared by ultracentrifugation, comprising the following steps:

[0043] At 4°C, gradient centrifugation was performed at 500g (10min), 1000g (20min) and 2000g (10min) respectively. After removing dead cells, cell debris, microvesicles and plant fibers, the precipitate obtained after centrifugation at 5000g for 10min was the exosome. Specifically, 500g centrifugation can initially remove larger cells and cell debris, reducing interference in subsequent steps. 1000g centrifugation can further remove smaller cell debris and some vesicles, making the exosomes more concentrated. 2000g centrifugation can remove most microvesicles and other smaller particles. 5000g centrifugation can separate exosomes from other smaller vesicles and proteins under extremely high centrifugal force to obtain a relatively pure exosome precipitate.

[0044] In a preferred embodiment of the present invention, the adipose stem cell exosomes are prepared by the following method:

[0045] The third generation cells were obtained by subculturing, cultured at a constant temperature in serum-free DMEM medium, and irradiated with near-infrared light;

[0046] The supernatant was collected and centrifuged at 3000-5000 g (10-30 min) and 10000-18000 g (30-120 min), respectively. After being kept at 3-5° C. overnight, the precipitate was obtained.

[0047] In this scheme, the use of third-generation cells for culture can ensure the purity and vitality of the cells, reduce genetic variation and potential tumor-derived risks. The use of serum-free DMEM culture medium can avoid the interference of unknown components in serum, reduce the risk of immunogenicity and pathogen transmission, and improve the safety of the product. Near-infrared light irradiation can promote cell metabolism and exosome secretion, increase the yield and quality of exosomes. By step-by-step centrifugation (3000-5000g and 10000-18000g), exosomes of different sizes and densities can be effectively separated to reduce damage to exosomes. Overnight at 3-5°C can maintain the stability and activity of exosomes and reduce the degradation of bioactive components. The present invention can obtain exosomes of higher purity, reduce impurities, and improve the quality of the product by optimizing the centrifugation step. Preferably, the wavelength of the near-infrared light is 780-1000nm, the power is 1-5W, and the irradiation time is 1-2h. Preferably, the constant temperature culture time is 24-48h.

[0048] In a preferred embodiment of the present invention, the exosome product comprises the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 15 parts of exosome gel carrier, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin.

[0049] In some optional embodiments of the present invention, the preparation method of the polyvinyl alcohol hydrogel is:

[0050] After mixing polyvinyl alcohol powder with deionized water, heating and stirring until transparent, and standing to obtain

[0051] Preferably, the mass ratio of polyvinyl alcohol to deionized water is 5-15:50, the weight average molecular weight of polyvinyl alcohol is 40000-110000, the alcoholysis degree is 99%, and the heating and stirring temperature is 90-100° C., and the time is 1-3 hours.

[0052] In some optional embodiments of the present invention, the mass ratio of the polyvinyl alcohol hydrogel to the plant exosome vesicle is 5 to 20:3 to 10. Preferably, the mass ratio of the polyvinyl alcohol hydrogel to the plant exosome vesicle is 20:3.

[0053] In the second aspect, based on a general technical concept, the present invention discloses a method for preparing an exosome product, comprising the following steps:

[0054] Prepare adipose stem cell exosomes and plant exosome vesicles respectively;

[0055] The plant exosome vesicles are placed in a buffer solution, and adipose stem cell exosomes are added under the condition of blue light irradiation to obtain a first product;

[0056] The polyvinyl alcohol powder is mixed with deionized water, heated and stirred until transparent, and the first product is added during cooling to obtain the second product;

[0057] The second product is mixed with fibroblast fluid, collagen and elastin, and then freeze-dried to obtain an exosome product.

[0058] In the above scheme, encapsulating plant-derived exosomes and adipose stem cell exosomes in PVA hydrogel can integrate the bioactive components of exosomes from different sources, exert a synergistic effect, and enhance the therapeutic effect. Among them, blue light irradiation can help activate or modify the components in exosomes, increase their bioactivity or improve their interaction with cells. Using polyvinyl alcohol (PVA) hydrogel as a carrier can form a sustained-release system, prolong the action time of exosomes in the body, and protect exosomes from the influence of the external environment, improving their stability during storage and transportation.

[0059] Preferably, the wavelength of blue light is 455nm and the light intensity is 4.28mW / cm 2 The irradiation time is 30 to 60 minutes.

[0060] In a third aspect, based on a general technical concept, the present invention discloses an application of an exosome preparation in the preparation of a hair growth product.

[0061] In a fourth aspect, based on a general technical concept, the present invention discloses an application of an exosome preparation in the preparation of a skin care product.

[0062] Specifically, the present invention can prepare a skin care lotion, the ingredients of which include exosome products, moisturizers, emulsifiers, propylene glycol, glycerin, and the like.

[0063] Specifically, the present invention can prepare a skin care cream, the ingredients of which include an exosome product and a thickener, a moisturizer, an emulsifier, a solvent, and the like.

[0064] Specifically, the present invention can prepare a skin care essence water, the ingredients of which include exosome products and moisturizing ingredients, water, plant essence, small molecule hyaluronic acid and pH regulator, etc.

[0065] Specifically, the present invention can prepare a hair growth shampoo, the ingredients of which include exosome products, surfactants (foaming agents), conditioners, organic acids (acid-base regulating agents) and other auxiliary ingredients.

[0066] The principles and features of the present invention are described below in conjunction with the examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0067] Example 1

[0068] This embodiment provides an exosome product, comprising the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 15 parts of exosome gel carrier, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin. In the exosome gel carrier, the mass ratio of polyvinyl alcohol hydrogel to plant exosome vesicles is 20:3.

[0069] This embodiment provides a method for preparing an exosome product, comprising the following steps:

[0070] S1: Preparation of adipose-derived stem cell exosomes

[0071] Take 12 mL of adipose tissue, rinse repeatedly with PBS buffer, and then digest with 12 mL of 0.2% type I collagenase until it becomes a paste; centrifuge at 4°C and 300g for 5 min, discard the supernatant; resuspend the precipitate in culture medium and inoculate it into a culture bottle, place it at 37°C and 5% CO 2 Culture in an incubator. Change the medium for the first time after 2 days, and change the medium every other day thereafter. Subculture when the cell confluence reaches 80%.

[0072] The third generation cells were obtained by subculture, cultured in serum-free DMEM medium at a constant temperature for 24 h, and irradiated with near-infrared light (wavelength of 780 nm, power of 5 W) for 60 min.

[0073] The supernatant was collected, centrifuged at 5000 g (10 min) and 10000 g (120 min), respectively, and the precipitate was obtained after being incubated at 4° C. overnight.

[0074] S2: Preparation of plant exosome vesicles

[0075] Weigh fresh Tibetan Codonopsis pilosula, Notopterygium wilfordii and Basella alba in a mass ratio of 1:2:10, crush and mix in water, and perform gradient centrifugation at 500g (10min), 1000g (20min) and 2000g (10min) at 4°C to obtain the supernatant;

[0076] The supernatant was centrifuged at 5000 g (10 min) and resuspended to obtain plant exosome vesicles.

[0077] S3: The plant exosome vesicles were placed in PBS buffer and exposed to blue light (blue light with a wavelength of 455 nm and a light intensity of 4.28 mW / cm 2 Adipose stem cell exosomes were added under irradiation conditions of 50 °C and 100 °C for 24 h (irradiation time was 60 min) to obtain the first product.

[0078] S4: 10 g of polyvinyl alcohol with a molecular weight of 88,000 is mixed with 50 g of deionized water, heated to 90° C. and stirred until transparent, and the first product is added during the cooling process to obtain the second product.

[0079] S5: The second product is mixed with fibroblast fluid, collagen and elastin, and freeze-dried at -30°C for 24 hours to obtain an exosome product.

[0080] Example 2

[0081] This embodiment provides an exosome product, comprising the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 10 parts of exosome gel carrier, 15 parts of fibroblast fluid, 0.2 parts of collagen and 0.05 parts of elastin. In the exosome gel carrier, the mass ratio of polyvinyl alcohol hydrogel to plant exosome vesicles is 20:3.

[0082] This embodiment provides a method for preparing an exosome product, comprising the following steps:

[0083] S1: Preparation of adipose-derived stem cell exosomes

[0084] Take 12 mL of adipose tissue, rinse repeatedly with PBS buffer, and then digest with 12 mL of 0.2% type I collagenase until it becomes a paste; centrifuge at 4°C and 300g for 5 min, discard the supernatant; resuspend the precipitate in culture medium and inoculate it into a culture bottle, place it at 37°C and 5% CO 2 Culture in an incubator. Change the medium for the first time after 2 days, and change the medium every other day thereafter. Subculture when the cell confluence reaches 80%.

[0085] The third generation cells were obtained by subculture, cultured in serum-free DMEM medium at a constant temperature for 24 h, and irradiated with near-infrared light (wavelength of 780 nm, power of 5 W) for 60 min.

[0086] The supernatant was collected, centrifuged at 3000 g (10 min) and 10000 g (120 min), respectively, and the precipitate was obtained after being kept at 5° C. overnight.

[0087] S2: Preparation of plant exosome vesicles

[0088] Weigh fresh Basella alba, crush it and mix it with water, and perform gradient centrifugation at 500g (10min), 1000g (20min) and 2500g (10min) at 4°C to obtain the supernatant;

[0089] The supernatant was centrifuged at 5000 g (10 min) and resuspended to obtain plant exosome vesicles.

[0090] S3: The plant exosome vesicles were placed in PBS buffer and exposed to blue light (blue light with a wavelength of 455 nm and a light intensity of 4.28 mW / cm 2 Adipose stem cell exosomes were added under irradiation conditions (irradiation time was 45 min) to obtain the first product.

[0091] S4: 12.5 g of polyvinyl alcohol with a molecular weight of 88,000 is mixed with 50 g of deionized water, heated to 90° C. and stirred until transparent, and the first product is added during the cooling process to obtain the second product.

[0092] S5: The second product is mixed with fibroblast fluid, collagen and elastin, and freeze-dried at -30°C for 24 hours to obtain an exosome product.

[0093] Example 3

[0094] This embodiment provides an exosome product, comprising the following raw materials in parts by weight: 5 parts of adipose stem cell exosomes, 10 parts of exosome gel carrier, 20 parts of fibroblast fluid, 0.1 parts of collagen and 0.1 parts of elastin. In the exosome gel carrier, the mass ratio of polyvinyl alcohol hydrogel to plant exosome vesicles is 20:5.

[0095] This embodiment provides a method for preparing an exosome product, comprising the following steps:

[0096] S1: Preparation of adipose-derived stem cell exosomes

[0097] Take 12 mL of adipose tissue, rinse repeatedly with PBS buffer, and then digest with 12 mL of 0.2% type I collagenase until it becomes a paste; centrifuge at 4°C and 300g for 5 min, discard the supernatant; resuspend the precipitate in culture medium and inoculate it into a culture bottle, place it at 37°C and 5% CO 2 Culture in an incubator. Change the medium for the first time after 2 days, and change the medium every other day thereafter. Subculture when the cell confluence reaches 80%.

[0098] The third generation cells were obtained by subculture, cultured in serum-free DMEM medium at a constant temperature for 24 h, and irradiated with near-infrared light (wavelength of 780 nm, power of 5 W) for 60 min.

[0099] The supernatant was collected, centrifuged at 5000 g (10 min) and 15000 g (60 min), respectively, and the precipitate was obtained after being kept at 5° C. overnight.

[0100] S2: Preparation of plant exosome vesicles

[0101] Weigh fresh Tibetan Codonopsis pilosula and Basella alba in a mass ratio of 1:10, crush and mix in water, and perform gradient centrifugation at 800g (10min), 2000g (20min) and 3000g (10min) at 4°C to obtain the supernatant;

[0102] The supernatant was centrifuged at 3000 g (20 min) and resuspended to obtain plant exosome vesicles.

[0103] S3: The plant exosome vesicles were placed in PBS buffer and exposed to blue light (blue light with a wavelength of 455 nm and a light intensity of 4.28 mW / cm 2 Adipose stem cell exosomes were added under irradiation conditions (irradiation time was 30 min) to obtain the first product.

[0104] S4: 8 g of polyvinyl alcohol with a molecular weight of 88,000 is mixed with 50 g of deionized water, heated to 90° C. and stirred until transparent, and the first product is added during the cooling process to obtain the second product.

[0105] S5: The second product is mixed with fibroblast fluid, collagen and elastin, and freeze-dried at -30°C for 24 hours to obtain an exosome product.

[0106] Comparative Example 1

[0107] Different from Example 1, this comparative example provides an exosome product, comprising the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin.

[0108] Comparative Example 2

[0109] Different from Example 1, this comparative example provides an exosome product, comprising the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 15 parts of exosome gel carriers, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin.

[0110] Among them, the exosome gel carrier is polyvinyl alcohol hydrogel, which does not include plant exosome vesicles.

[0111] Comparative Example 3

[0112] Different from Example 1, this comparative example provides an exosome product, including the following raw materials in parts by weight: 3 parts of mesenchymal stem cell exosomes, 15 parts of exosome gel carrier, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin. In the exosome gel carrier, the mass ratio of polyvinyl alcohol hydrogel to plant exosome vesicles is 20:3. Among them, mesenchymal stem cell exosomes are commercially available products, derived from Qiyue Biology, mesenchymal stem cell (MSC) exosomes, and exosomes secreted by mutant HIF-1α modified BMSCs.

[0113] Comparative Example 4

[0114] Different from Example 1, this comparative example provides a method for preparing an exosome product, comprising the following steps:

[0115] S1: Preparation of adipose-derived stem cell exosomes

[0116] Take 12 mL of adipose tissue, rinse repeatedly with PBS buffer, and then digest with 12 mL of 0.2% type I collagenase until it becomes a paste; centrifuge at 4°C and 300g for 5 min, discard the supernatant; resuspend the precipitate in culture medium and inoculate it into a culture bottle, place it at 37°C and 5% CO 2 Culture in an incubator. Change the medium for the first time after 2 days, and change the medium every other day thereafter. Subculture when the cell confluence reaches 80%.

[0117] The third generation cells were obtained by subculturing, and placed in serum-free DMEM medium at constant temperature for 24 hours. The supernatant was collected, centrifuged at 3000 (10 min) and 15000g (120 min), respectively, and the precipitate was obtained after overnight at 4°C.

[0118] S2: Preparation of plant exosome vesicles

[0119] Weigh fresh Tibetan Codonopsis pilosula, Notopterygium wilfordii and Basella alba in a mass ratio of 1:2:10, crush and mix in water, and perform gradient centrifugation at 500g (10min), 1000g (20min) and 2000g (10min) at 4°C to obtain the supernatant;

[0120] The supernatant was centrifuged at 5000 g (10 min) and resuspended to obtain plant exosome vesicles.

[0121] S3: Place the plant exosome vesicles in PBS buffer, add adipose stem cell exosomes, and obtain the first product.

[0122] S4: 10 g of polyvinyl alcohol with a molecular weight of 88,000 is mixed with 50 g of deionized water, heated to 90° C. and stirred until transparent, and the first product is added during the cooling process to obtain the second product.

[0123] S5: The second product is mixed with fibroblast fluid, collagen and elastin, and freeze-dried at -30°C for 24 hours to obtain an exosome product.

[0124] Comparative Example 5

[0125] Different from Example 1, this comparative example provides a method for preparing an exosome product, comprising the following steps:

[0126] S1: Preparation of adipose-derived stem cell exosomes

[0127] Take 12 mL of adipose tissue, rinse repeatedly with PBS buffer, and then digest with 12 mL of 0.2% type I collagenase until it becomes a paste; centrifuge at 4°C and 300g for 5 min, discard the supernatant; resuspend the precipitate in culture medium and inoculate it into a culture bottle, place it at 37°C and 5% CO 2 Culture in an incubator. Change the medium for the first time after 2 days, and change the medium every other day thereafter. Subculture when the cell confluence reaches 80%.

[0128] The third generation cells were obtained by subculture, cultured in serum-free DMEM medium at a constant temperature for 24 h, and irradiated with near-infrared light (wavelength of 780 nm, power of 5 W) for 60 min.

[0129] The supernatant was collected, centrifuged at 5000 g (10 min) and 10000 g (120 min), respectively, and the precipitate was obtained after being incubated at 4° C. overnight.

[0130] S2: Preparation of plant exosome vesicles

[0131] Weigh fresh Tibetan Codonopsis pilosula, Notopterygium wilfordii and Basella alba in a mass ratio of 1:2:10, crush and mix in water, and perform gradient centrifugation at 500g (10min), 1000g (20min) and 2000g (10min) at 4°C to obtain the supernatant;

[0132] The supernatant was centrifuged at 5000 g (10 min) and resuspended to obtain plant exosome vesicles.

[0133] S3: Place the plant exosome vesicles in PBS buffer, add adipose stem cell exosomes and fibroblast fluid, collagen and elastin to obtain the first product.

[0134] S4: Mix 10 g of polyvinyl alcohol with a molecular weight of 88,000 with 50 g of deionized water, heat to 90°C and stir until transparent, add the first product during cooling, and freeze-dry at -30°C for 24 hours to obtain the exosome product.

[0135] Test Example 1

[0136] The exosome products prepared in Example 1 and Comparative Examples 1-5 were used for cell proliferation experiments. Specifically, rat dermal fibroblasts were cultured at 7*10 4 The cells were seeded into 12-well plates at a density of 100 μl and incubated at 37°C with 5% CO 2 Incubate in an incubator for 12 hours. Add the products obtained in Example 1 and Comparative Example 15 respectively and incubate for 1, 3, and 5 days, and replace the culture medium every other day. Use PBS as a blank control. Digest the cells with 0.25% trypsin to prepare a single cell suspension, mix the cell suspension with 0.4% trypan blue staining solution at a ratio of 9:1 and stain, count the cells under a microscope with a cell counting plate, and calculate the growth rate (%) of rat dermal fibroblasts.

[0137] Growth rate (%) = (number of rat dermal fibroblasts after incubation on day n - number of rat dermal fibroblasts at the beginning) / number of rat dermal fibroblasts at the beginning * 100%. The test results are shown in Table 1.

[0138] Table 1

[0139]

[0140]

[0141] As can be seen from Table 1, the exosome product prepared by the present invention has the best rat dermal fibroblast proliferation effect, and its proliferation rate is the fastest, which can promote the repair and regeneration of skin fibroblasts in a short time. At the same time, it can not only promote the proliferation of skin cells, but also provide nutrition for the skin, delay skin aging, promote skin wound repair, and have lower drug side effects and more significant efficacy. The reason may be that the growth factors, cytokines and other bioactive molecules in the exosomes of adipose stem cells can promote the proliferation of fibroblasts. For example, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF), etc., may all stimulate the growth of fibroblasts. At the same time, the plant exosome vesicles loaded therein have specific plant hormones and secondary metabolites, which can synergize with the molecules in the exosomes of adipose stem cells to promote cell proliferation, differentiation and tissue repair.

[0142] Test Example 2

[0143] 100 female subjects aged 15-50 were selected and a 2×2 cm mark was made on the inner side of the left and right forearms. 2 Test area, 2 mg sample / cm 2The samples prepared in the examples and comparative examples were evenly applied to the test area, with a total of 8 test areas. Among them, the blank control group was sterile pure water, and the elastic parameters (elasticity of the rebound part / elasticity of the stretching part, the ratio of the elastic properties of the two processes) and firming parameters (area composed of maximum stretching amount and time) of the test area and the blank control area before and after application were tested using a skin moisture tester to evaluate the skin improvement effect. The experimental results are shown in Table 2.

[0144] Table 2

[0145]

[0146]

[0147] The closer the elasticity parameter is to 1, the better the skin elasticity is, the smaller the firmness parameter is, the firmer the skin is, and the test of the same subject is completed by the same measurement personnel, and the test time is 0 weeks, 2 weeks, 4 weeks, and 6 weeks. It can be seen from Table 2 that the product prepared by strictly adopting the formula and production process of the present invention can not only achieve the effect of firming the skin and restoring the skin elasticity in a short time, but also maintain the skin elasticity for a long time, that is to say, the product of the present invention can be further used to produce skin care products to improve skin aging, and can also be used to produce anti-aging products to avoid skin aging from the root and maintain skin stability.

[0148] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. An exosome product, characterized in that: The invention comprises the following raw materials in parts by weight: 1-5 parts of adipose stem cell exosomes, 2-15 parts of exosome gel carrier, 15-50 parts of fibroblast fluid, 0.01-0.2 parts of collagen and 0.02-0.1 parts of elastin; The exosome gel carrier is a double-layer composite structure, with polyvinyl alcohol hydrogel on the outside and plant exosome vesicles on the inside.

2. The exosome preparation according to claim 1, characterized in that The plant exosome vesicles are derived from at least one of Codonopsis pilosula, Notopterygium wilfordii and Basella alba.

3. The exosome preparation according to claim 2, characterized in that The plant exosome vesicles are prepared by the following method: Perform gradient centrifugation at 300-800 g, 1000-2000 g, and 2000-3000 g at 3-5 °C to obtain the supernatant; The supernatant was centrifuged at 3000-5000 g and resuspended to obtain plant exosome vesicles.

4. The exosome preparation according to claim 1, characterized in that The adipose stem cell exosomes are prepared by the following method: The third generation cells were obtained by subculturing, cultured at a constant temperature in serum-free DMEM medium, and irradiated with near-infrared light; The supernatant was collected and centrifuged at 3000-5000 g and 10000-18000 g, respectively. After being incubated at 3-5° C. overnight, the precipitate was obtained to obtain the product.

5. The exosome product according to any one of claims 1 to 4, characterized in that The exosome product comprises the following raw materials in parts by weight: 3 parts of adipose stem cell exosomes, 15 parts of exosome gel carriers, 20 parts of fibroblast fluid, 0.2 parts of collagen and 0.1 parts of elastin.

6. The exosome preparation according to claim 1, characterized in that The preparation method of the polyvinyl alcohol hydrogel is: The polyvinyl alcohol powder is mixed with deionized water, heated and stirred until transparent, and allowed to stand to obtain the product.

7. The exosome preparation according to claim 1, characterized in that The mass ratio of the polyvinyl alcohol hydrogel to the plant exosome vesicles is 5-20:3-10.

8. A method for preparing an exosome product according to any one of claims 1 to 7, characterized in that: The following steps are involved: Prepare adipose stem cell exosomes and plant exosome vesicles respectively; The plant exosome vesicles are placed in a buffer solution, and adipose stem cell exosomes are added under blue light irradiation to obtain a first product; The polyvinyl alcohol powder is mixed with deionized water, heated and stirred until transparent, and the first product is added during cooling to obtain the second product; The second product is mixed with fibroblast fluid, collagen and elastin, and then freeze-dried to obtain an exosome product.

9. Use of the exosome preparation according to any one of claims 1 to 7 in the preparation of hair growth products.

10. Use of the exosome preparation according to any one of claims 1 to 7 in the preparation of skin care products.

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