Moisturizing and whitening composition containing stem cell exosome as well as preparation method and application of moisturizing and whitening composition

Abstract

The invention relates to the technical field of biology, in particular to a moisturizing and whitening composition containing stem cell exosomes as well as a preparation method and application of the moisturizing and whitening composition. According to the composition, adipose-derived mesenchymal stem cell exosomes are combined with two specific modified compounds to form a stable and efficient oil-in-water emulsion. The preparation method comprises the following steps: culturing stem cells under a low-oxygen condition, and separating exosomes through differential ultracentrifugation; the preparation method comprises the following steps: respectively preparing a water phase mainly containing vanillin-derived dynamic imine bond grafted hyaluronic acid and an oil phase mainly containing disulfide bond crosslinked polyester vitreous network powder; adding exosome and other active ingredients after two-phase emulsification, and finally performing sterile filtration to obtain a finished product. The composition makes full use of the repair and regeneration capacity of the exosome and the synergistic interaction of the two modified compounds, has excellent moisturizing, whitening and anti-aging effects, and is suitable for preparation of various cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a moisturizing and whitening composition containing stem cell exosomes and a preparation method and application thereof. BACKGROUND

[0002] With the continuous development of dermatology and regenerative medicine, the application of stem cell exosome technology in the field of skin care has been increasingly concerned. As a key mediator of intercellular communication, exosomes can carry various bioactive substances such as proteins and nucleic acids, and have unique functions of regulating cell metabolism and promoting tissue repair. Studies have shown that mesenchymal stem cell exosomes derived from adipose tissue exhibit multiple potential in skin care, not only can deliver signals to promote collagen synthesis, but also can regulate the activity of melanocytes, providing a new idea for the development of innovative cosmetics with moisturizing and whitening effects. However, in the actual application process, the stability of exosomes itself, the difficulty of maintaining activity in conventional formulations, and the low transdermal absorption efficiency, etc. have seriously restricted its commercial application prospects.

[0003] At present, traditional cosmetics still face many challenges in achieving the dual effects of moisturizing and whitening. Although ordinary moisturizing ingredients can temporarily replenish skin moisture, they are difficult to fundamentally repair damaged skin barrier structure and achieve long-term moisturizing. In terms of whitening, common whitening ingredients often have single target points and are prone to cause skin irritation and other adverse reactions. More importantly, existing technologies have not effectively solved the stability problem of exosomes in the formulation, and conventional emulsification process and storage conditions can easily lead to the destruction of exosome structure and loss of biological activity. In addition, the synergistic mechanism of exosomes and other active ingredients has not been deeply studied, and there is a lack of carrier systems that can protect the activity of exosomes and promote their skin penetration, which directly limits the actual efficacy of exosome-containing cosmetics.

[0004] In view of the above technical problems, in recent years, some studies have tried to improve the stability of exosomes by different methods, such as adding protective agents or using liposome wrapping technology, but these methods still have obvious limitations. Some protective agents may affect the biological function of exosomes, and conventional wrapping technology is difficult to achieve controlled release of exosomes. In terms of formulation technology, most existing products use simple physical mixing method, and fail to design functional matrix materials that can produce synergistic effect with exosomes from the molecular level. Therefore, the development of an innovative composition that can maintain the high activity of exosomes, promote their skin penetration, and synergistically act with other efficacy ingredients, has become a key technical problem to be solved in the field. This not only requires a deep understanding of the interaction mechanism between exosomes and skin cells, but also requires important breakthroughs in formulation technology, so as to provide consumers with truly efficient and safe skin care solutions. SUMMARY

[0005] The present application aims to provide a stem cell exosome-containing moisturizing whitening composition and its preparation method and application, which solves the technical problems of unstable activity, low transdermal efficiency and difficulty in achieving long-lasting moisturizing, source whitening and multiple anti-aging effects in a single formula when the existing stem cell exosome is applied in cosmetics.

[0006] The present application achieves the above-mentioned purpose by the following technical solutions: A preparation method of a stem cell exosome-containing moisturizing whitening composition, comprising the following steps: S1, culturing human adipose-derived mesenchymal stem cells under a hypoxic condition, collecting the supernatant, separating exosomes by differential ultracentrifugation, resuspending, obtaining adipose-derived mesenchymal stem cell exosomes, and storing; S2, mixing vanillin-derived dynamic imine grafted hyaluronic acid and deionized water, stirring and dissolving, sequentially adding glycerol, hyaluronic acid, ceramide-2 and aloe vera gel, homogenizing to form an aqueous phase; mixing disulfide cross-linked polyester vitreous body network powder and potassium lauryl phosphate, heating, adding nicotinamide and radix bupleuri extract, stirring to form an oil phase; under stirring, adding the oil phase into the aqueous phase, using a homogenizer to emulsify, forming an oil-in-water emulsion; when the temperature of the oil-in-water emulsion drops below 40℃, adding adipose-derived mesenchymal stem cell exosomes, superoxide dismutase and phenoxyethanol, and stirring and mixing; S3, filtering using a sterile filter membrane.

[0007] Further, the above preparation method specifically comprises: S1, culturing human adipose-derived mesenchymal stem cells under a hypoxic condition, collecting the supernatant, separating exosomes by differential ultracentrifugation, resuspending with a phosphate buffer solution, obtaining adipose-derived mesenchymal stem cell exosomes, and storing at-82~-78℃; S2, mixing vanillin-derived dynamic imine grafted hyaluronic acid and deionized water, stirring in a 38-42℃ water bath until completely dissolved, sequentially adding glycerol, hyaluronic acid, ceramide-2 and aloe vera gel, homogenizing to form an aqueous phase; mixing disulfide cross-linked polyester vitreous body network powder and potassium lauryl phosphate, heating to 68-72℃, adding nicotinamide and radix bupleuri extract, stirring to form an oil phase; under stirring, adding the oil phase into the aqueous phase, using a homogenizer to emulsify, forming an oil-in-water emulsion; when the temperature of the oil-in-water emulsion drops below 40℃, adding adipose-derived mesenchymal stem cell exosomes, superoxide dismutase and phenoxyethanol, and stirring and mixing; S3, filtering using a sterile filter membrane.

[0008] In the present application, the preparation of the moisturizing whitening composition containing stem cell exosomes is a complex process system integrating physical emulsification and biological activity protection. The mechanism is reflected in two levels of macroscopic dosage form construction and microscopic functional synergy. In the dosage form construction, the preparation process follows the classic formation principle of oil-in-water emulsion, but realizes functional enhancement by introducing two specifically designed polymers. First, the vanillin-derived dynamic imine bond grafted hyaluronic acid is used as the water phase core component. After dissolving in water, it forms a three-dimensional hydration network through hydrogen bonding and hydrophobic interaction between molecular chains, which greatly improves the viscosity and stability of the system. The dynamic hydrazone bond on the chain further endows the emulsion interface film with self-repairing properties, which can effectively buffer the damage caused by mechanical stress. Second, the disulfide cross-linked polyester vitreous network powder softens and disperses in the oil phase under heating conditions, and is adsorbed on the oil-water interface with the emulsifier. The disulfide bond on its surface can exchange under homogeneous shear force, which helps to form a firm and stable interface film. When the two phases are mixed uniformly, the oil phase is broken into small droplets under the action of high-speed shear force, and is wrapped by the composite interface film composed of the above two polymers and emulsifiers, thereby forming a structurally stable oil-in-water emulsion. In the functional synergy mechanism, the composition exhibits multi-level synergy. The fat-derived mesenchymal stem cell exosomes as the core bioactive ingredient, its efficacy highly depends on the complete protection of its structure and the long-term maintenance of its activity during the preparation process. The three-dimensional network of vanillin-derived dynamic imine bond grafted hyaluronic acid can provide a protective microenvironment similar to the extracellular matrix for exosomes through steric hindrance effect and hydration, significantly slowing down its aggregation and inactivation. The disulfide cross-linked polyester vitreous network dispersed in the oil phase plays the role of an intelligent carrier, which not only can encapsulate fat-soluble active ingredients such as nicotinamide, but also can respond to reactive oxygen species generated inside the skin due to ultraviolet radiation, causing disulfide bond breakage and network degradation, thereby achieving targeted release of encapsulated ingredients. Finally, when the composition is applied to the skin, the two modified polymers and exosomes and other active ingredients (ceramides, superoxide dismutase, etc.) produce synergistic effect: the hyaluronic acid derivative provides immediate and long-term moisturizing; the exosomes regulate the expression of skin cell repair, moisturizing and whitening related genes through the biological signal molecules they carry; the intelligent polymer network ensures the precise release of oil-soluble whitening ingredients at the right time and place. Through the stable construction of physical dosage form and the intelligent delivery of biological active ingredients, the whole system finally realizes the synergistic improvement of multiple effects of moisturizing, whitening and anti-aging.

[0009] According to the preferred embodiment of the present application, in step S1, the oxygen concentration under the hypoxic condition is 1-5%.

[0010] According to the preferred embodiment of the present application, in step S2, the homogenizer emulsification time is 3-5 min.

[0011] According to the preferred embodiment of the present application, the preparation method of the vanillin-derived dynamic imine bond grafted hyaluronic acid comprises: A1, dissolving hyaluronic acid in deionized water, adding 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, then adding adipic acid dihydrazide to react, obtaining a reaction solution, dialyzing the reaction solution to obtain a hydrazide hyaluronic acid aqueous solution; dissolving vanillin in anhydrous ethanol, adding the hydrazide hyaluronic acid aqueous solution, and reacting under nitrogen protection and in the dark to obtain a reaction mixture; A2, cooling the reaction mixture to room temperature, dialyzing, and freeze-drying the product after dialysis.

[0012] In the present application, the preparation of vanillin-derived dynamic imine bond grafted hyaluronic acid involves multiple steps of precise chemical reactions, and the core mechanism lies in covalently grafting functional vanillin molecules onto the hyaluronic acid backbone through efficient amidation reaction and hydrazone bond condensation. First, in the first step of activation and amidation reaction, the carboxyl groups on the hyaluronic acid chain are highly activated under the action of carbodiimide condensing agent and its auxiliary catalyst, forming a reactive intermediate. The intermediate then undergoes nucleophilic substitution reaction with the primary amino group in the adipic acid dihydrazide molecule, forming a stable amide bond, thereby successfully introducing a molecular segment containing a terminal hydrazide group into the hyaluronic acid structure to obtain hydrazide hyaluronic acid. The key to this step is the activation efficiency of the condensing agent on the carboxyl group and the control of the reaction system on the by-products to ensure the obtainment of hydrazide products with high substitution degree. Subsequently, in the second grafting reaction, the newly introduced hydrazide group in the hydrazide hyaluronic acid molecule undergoes nucleophilic addition-dehydration condensation reaction with the aldehyde group inherent to the vanillin molecule, generating a stable structure and possessing dynamic reversible characteristics. The reaction is usually carried out in a weak acid environment and needs to be operated in an inert gas protection and in the dark to prevent oxidation of the vanillin aldehyde group and photodegradation of its molecular structure. The whole process precisely constructs a new type of polymer with excellent hyaluronic acid moisturizing property and vanillin antioxidant property through a two-step method, wherein the introduction of dynamic hydrazone bond endows the material with unique pH responsiveness and self-repairing ability, laying a molecular foundation for subsequent composition to realize intelligent release.

[0013] According to the preferred embodiment of the present application, in step A1, the reaction time in the dark is 18-24h.

[0014] According to the preferred embodiment of the present application, in step A2, the freeze-drying time is 48-50h.

[0015] According to the preferred embodiment of the present application, the preparation method of the disulfide bond cross-linked polyester vitreous network comprises: adding azelaic acid, 1,4-butanediol and 2,2'-dithiodiglycolic acid in a three-necked flask, heating to 150-170 DEG C under nitrogen atmosphere, and stirring until completely dissolved; adding glycerol and tetrabutyl titanate, increasing the temperature to 180-200 DEG C, and carrying out melt polycondensation reaction to obtain a melt polymer; and pouring the melt polymer into a polytetrafluoroethylene mold and heat treating at 80-100 DEG C.

[0016] In the present application, the synthesis of the disulfide bond cross-linked polyester vitreous network is based on the ingenious combination of melt polycondensation principle and dynamic covalent chemistry, and the reaction mechanism mainly focuses on the construction of ester bond and the formation of cross-linked network of disulfide bond. In the initial stage, the binary acid and the binary alcohol monomer undergo step-by-step polycondensation reaction at high temperature and in the presence of a catalyst, and linear polyester chains are formed through esterification and dehydration between carboxyl and hydroxyl groups. The reaction degree of this process is closely related to the temperature, the activity of the catalyst and the vacuum degree, and directly affects the molecular weight and its distribution of the final polymer. At the same time, the binary acid containing disulfide bond introduced into the system as a dynamic cross-linking agent also participates in esterification with its two end carboxyl groups, thereby embedding the dynamic disulfide bond as a side chain or main chain unit into the growing polymer chain. With the reaction proceeding, the viscosity of the system increases significantly, and when the polyol such as glycerol is added, the three hydroxyl groups contained therein can act as three-dimensional network nodes to cross-link and esterify with the carboxyl groups at the end of the polyester chain or in the chain, forming a three-dimensional spatial network structure. The key of this stage lies in controlling the cross-linking density to avoid premature gelation to ensure the process feasibility. The final heat treatment process is crucial, which not only promotes the further cross-linking between unreacted functional groups to improve the network integrity, but more importantly, activates the dynamic exchange reaction of the disulfide bond. Under the action of heat, the disulfide bond undergoes reversible homolysis and recombination, so that the polymer network macroscopically exhibits the rigidity similar to vitreous body, and microscopically has the ability of topological structure rearrangement, thereby endowing the material with intelligent response behavior of degradation or reconstruction under oxidative stress environment, realizing the controlled release function of the active ingredients wrapped therein.

[0017] According to the preferred embodiment of the present application, the melt polycondensation reaction time is 4-6 h; and the heat treatment time at 80-100 DEG C is 1-2 h.

[0018] The present application also provides a stem cell exosome-containing moisturizing and whitening composition prepared according to the preparation method of the stem cell exosome-containing moisturizing and whitening composition.

[0019] The present application also provides the application of the stem cell exosome-containing moisturizing and whitening composition in anti-aging cosmetics.

[0020] The present application has the following beneficial effects: The moisturizing whitening composition containing stem cell exosomes provided by the present application exhibits excellent comprehensive technical effects through innovative formula design and preparation process. In terms of moisturizing effect, the composition breaks through the limitation of traditional moisturizing products that can only form a water-locking film on the skin surface, and realizes a multi-level and three-dimensional moisturizing mechanism through the synergistic effect of the grafting of hyaluronic acid with dynamic imine bond derived from vanillin and stem cell exosomes. The modified hyaluronic acid not only retains strong hydration capacity, but also forms a three-dimensional network structure with self-repairing function through dynamic imine bond, so as to construct an intelligent moisturizing barrier on the skin surface and automatically adjust the water release according to the change of environmental humidity. At the same time, the bioactive signal molecules carried by the stem cell exosomes can penetrate into the deep layer of the skin, activate the expression of aquaporin and hyaluronic acid synthase genes of keratinocytes, and enhance the natural moisturizing function of the skin from the root. The addition of ceramide and aloe vera further strengthens the repair and maintenance of the skin lipid barrier, effectively prevents transdermal water loss, and keeps the skin in a persistent moist state.

[0021] In terms of whitening and spot-fading effect, the composition realizes the perfect combination of source melanin inhibition and surface spot-fading through a multi-target and multi-path mechanism. The specific micro ribonucleic acid carried by the adipose-derived mesenchymal stem cell exosomes can accurately regulate the key signal pathways in melanocytes, effectively inhibit the activity expression of tyrosinase, and block the excessive synthesis of melanin from the gene level. The disulfide cross-linked polyester vitreous body network as an intelligent carrier can specifically release the wrapped whitening active ingredients such as nicotinamide in the oxidative stress environment of melanocytes, thereby enhancing the targeting and efficiency of the active ingredients. The combination of radix bupleuri extract and superoxide dismutase further eliminates the excessive free radicals induced by ultraviolet and other factors, reduces the stimulation of oxidative stress on melanocytes, thereby preventing the formation and development of pigmentation. This multi-level whitening mechanism enables the composition to not only effectively fade existing spots, but also prevent new spots from forming, thereby achieving uniform and bright skin.

[0022] In terms of anti-aging and overall skin quality improvement, the composition exhibits satisfactory comprehensive effects. The growth factors and cytokines rich in stem cell exosomes can activate the proliferation activity of skin fibroblasts, promote the synthesis and recombination of collagen and elastic fibers, and effectively improve the problems of skin relaxation and wrinkles caused by photoaging and natural aging. The carrier system formed by the two functional matrix materials not only ensures the stable delivery of active ingredients, but also maintains the healthy state of the extracellular matrix of skin cells through continuous microenvironment regulation. Long-term use of the composition can significantly improve skin elasticity, enhance skin firmness, and improve rough skin, so that the skin presents a young and healthy luster. The components in the composition have significant synergistic effect, the overall formula is mild and biocompatible, and is suitable for various skin types, thereby providing a high-quality skin care solution with both immediate effect and long-term improvement value for consumers. DETAILED DESCRIPTION

[0023] The following detailed description is provided for further understanding of the present application and should not be understood as limiting the scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0024] The main related equipment and material suppliers are as follows: The phosphate buffer solution is purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0025] The glycerol is purchased from Shandong Xuchun Pharmaceutical Co., Ltd.

[0026] The hyaluronic acid is purchased from Shandong Bao Hua Biological Technology Co., Ltd.

[0027] The ceramide-2 is purchased from Guangzhou Simuanyun Technology Co., Ltd.

[0028] The aloe vera gel is purchased from Guangzhou Mingyu Cosmetics Co., Ltd.

[0029] The potassium lauryl phosphate is purchased from Guangzhou Tianci High-tech Materials Co., Ltd.

[0030] The nicotinamide is purchased from Ruizhuang Pharmaceutical Co., Ltd.

[0031] The radix bupleuri extract is purchased from Xi'an Jinxiufang Plant Technology Development Co., Ltd.

[0032] The superoxide dismutase is purchased from Jiangsu Shenhuapharm Co., Ltd.

[0033] The phenoxyethanol is purchased from Shandong Anshi Pharmaceutical Co., Ltd.

[0034] The hyaluronic acid is purchased from Shandong Bao Hua Biological Technology Co., Ltd.

[0035] The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.

[0036] The N-hydroxysuccinimide is purchased from Shanghai Macklin Biochemical Technology Co., Ltd.

[0037] The adipic acid dihydrazide is purchased from Wuhan Smaker Biotech Co., Ltd.

[0038] The vanillin is purchased from Brothers Technology Co., Ltd.

[0039] The azelaic acid is purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.

[0040] The 1,4-butanediol was purchased from Wanhua Chemical Group Co., Ltd.

[0041] The 2,2'-dithiodiglycolic acid was purchased from Shanghai Aladdin Biochem Technology Co., Ltd.

[0042] The tetrabutyl titanate was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Example 1

[0043] Preparation of vanillin-derived dynamic imine bond grafted hyaluronic acid: first, 10 g of hyaluronic acid with a molecular weight of 150 kDa was slowly added to 500 g of deionized water, and dissolved completely transparent in a 40℃ water bath with stirring at 200 rpm for 2 hours; then 3 g of 1-3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride and 1 g of N-hydroxysuccinimide were added, and the temperature was kept at 25℃ with 300 rpm stirring for 30 minutes; then 5 g of adipic acid dihydrazide was slowly added, the pH was adjusted to 5.5, and the reaction was carried out under nitrogen protection at 250 rpm for 20 hours to obtain a reaction solution; the reaction solution was transferred into a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzed against deionized water at 4℃ for 60 hours, with the dialysis solution being replaced every 6 hours during the dialysis; after dialysis, a hydrazide hyaluronic acid aqueous solution was obtained for standby use; 5 g of vanillin was dissolved in 200 g of anhydrous ethanol, and slowly added dropwise to the above hydrazide hyaluronic acid aqueous solution under light shielding conditions, and the pH was adjusted to 4.5, and the reaction was carried out under nitrogen protection and light shielding conditions at 200 rpm for 20 hours; after the reaction was completed, the reaction mixture was cooled to 25℃, and then transferred into a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzed against deionized water at 4℃ for 48 hours, with the dialysis solution being replaced every 8 hours; finally, the dialysate was pre-frozen at -50℃ for 12 hours, and then freeze-dried at -40℃ and 10 Pa for 48 hours to obtain white fibrous vanillin-derived dynamic imine bond grafted hyaluronic acid solid.

[0044] Preparation of disulfide crosslinked polyester vitreous network: first, 10 g of azelaic acid, 8 g of 1,4-butanediol and 5 g of 2,2-dithiodiglycolic acid were added in a dry three-necked flask, after replacing the air with nitrogen, heated to 160℃ at a heating rate of 5℃ / min under nitrogen atmosphere and mechanically stirred at 150 rpm until the solid was completely dissolved to form a transparent liquid; then 1 g of glycerol and 0.1 g of tetrabutyl titanate catalyst were added, the temperature was raised to 190℃ at a rate of 2℃ / min, and the melt polycondensation reaction was carried out for 5 hours under the conditions of continuous nitrogen flow and 200 rpm stirring, during which the reaction progress was monitored by viscosity change; after the reaction was completed, the obtained melt polymer was quickly poured into a preheated polytetrafluoroethylene mold, and heat treated in a 90℃ vacuum drying oven for 1.5 hours to promote crosslinking; finally, the sample was naturally cooled to room temperature, crushed by a crusher and sieved through a 200 mesh sieve to obtain a light yellow disulfide crosslinked polyester vitreous network powder.

[0045] Preparation of stem cell exosome-containing moisturizing whitening composition: Step S1: human adipose-derived mesenchymal stem cells were cultured in a three-dimensional manner in a 37℃ incubator under hypoxic conditions with an oxygen concentration of 3% and a carbon dioxide concentration of 5%, the supernatant collected after 72 hours of culture was centrifuged at 2000g for 10 minutes at 4℃ to remove cell debris, then centrifuged at 10000g for 30 minutes to remove large particles, and finally ultracentrifuged at 120000g for 70 minutes to collect exosome precipitate, which was resuspended with 10 mL of phosphate buffer solution and stored in a -80℃ ultra-low temperature freezer for standby use; Step S2: 3 g of the above prepared vanillin derived dynamic imine bond grafted hyaluronic acid was mixed with 80 g of deionized water, stirred at 300 rpm in a 40℃ constant temperature water bath for 30 minutes until completely transparent, 10 g of glycerol, 5 g of hyaluronic acid, 0.3 g of ceramide-2 and 10 g of aloe vera gel were added in turn, and a high-speed homogenizer was used to homogenize at 5000 rpm for 5 minutes to form an aqueous phase; another 1.5 g of disulfide crosslinked polyester vitreous network powder was mixed with 5 g of potassium lauryl phosphate and stirred at 200 rpm in a 70℃ oil bath for 15 minutes until completely dispersed, 0.2 g of nicotinamide and 10 g of radix scirpi extract were added and stirred for another 10 minutes to form a uniform oil phase; under continuous stirring, the oil phase was slowly added to the aqueous phase, and a high-pressure homogenizer was used to emulsify at a pressure of 50 MPa for 4 minutes to form a stable oil-in-water emulsion; when the emulsion temperature naturally dropped to 35℃, 0.005 g of fat-derived mesenchymal stem cell exosomes, 15 g of superoxide dismutase and 5 g of phenoxyethanol were added, and stirred at 100 rpm for 30 minutes; Step S3: finally, a 0.22μm sterile filter membrane was used to filter under sterile conditions to obtain the final stem cell exosome-containing moisturizing whitening composition. Example 2

[0046] The specific implementation is the same as that of Example 1, except that the preparation of vanillin-derived dynamic imine bond grafted hyaluronic acid: 12 g of hyaluronic acid is dissolved in 600 g of deionized water, 3.5 g of 1-3-dimethylaminopropyl-3-ethyl carbodiimide hydrochloride and 1.2 g of N-hydroxysuccinimide are added, followed by 6 g of adipic acid dihydrazide and reacted for 22 hours to obtain a reaction solution. The reaction solution is dialyzed in a dialysis bag with a molecular weight cut-off of 10 kDa for 60 hours to obtain a hydrazide-derivatized hyaluronic acid aqueous solution; 6 g of vanillin is dissolved in 250 g of anhydrous ethanol, and the hydrazide-derivatized hyaluronic acid aqueous solution is added, and reacted for 22 hours under nitrogen protection and in the dark to obtain a reaction mixture; the reaction mixture is cooled to room temperature, dialyzed in a dialysis bag with a molecular weight cut-off of 10 kDa for 48 hours, and the product after dialysis is freeze-dried for 49 hours to obtain vanillin-derived dynamic imine bond grafted hyaluronic acid. Preparation of disulfide bond cross-linked polyester vitreous body network: In a three-necked flask, 12 g of azelaic acid, 9 g of 1,4-butanediol and 6 g of 2,2-dithiodiacetic acid are added, and stirred under a nitrogen atmosphere until completely dissolved at a temperature of 155°C; 1.2 g of glycerol and 0.12 g of tetrabutyl titanate are added, and the temperature is raised to 185°C for melt polycondensation reaction for 4.5 hours to obtain a melt polymer; the melt polymer is poured into a polytetrafluoroethylene mold and heat treated at 85°C for 1.2 hours, and after cooling, it is crushed into powder to obtain a disulfide bond cross-linked polyester vitreous body network powder. Preparation of moisturizing whitening composition containing stem cell exosomes: Step S1, human adipose-derived mesenchymal stem cells are cultured under low oxygen conditions with an oxygen concentration of 2%, and the supernatant is collected. Exosomes are separated by differential ultracentrifugation, resuspended with phosphate buffer solution to obtain adipose-derived mesenchymal stem cell exosomes, and stored at -80°C; Step S2, 2.5 g of vanillin-derived dynamic imine bond grafted hyaluronic acid and 85 g of deionized water are mixed, stirred in a 39°C water bath until completely dissolved, and 12 g of glycerol, 4 g of hyaluronic acid, 0.25 g of ceramide-2 and 12 g of aloe vera gel are added in sequence, and homogenized to form an aqueous phase; 1.2 g of disulfide bond cross-linked polyester vitreous body network powder and 4.5 g of potassium lauryl phosphate are mixed, heated to 69°C, and 0.15 g of nicotinamide and 12 g of radix scirpi extract are added, and stirred to form an oil phase; the oil phase is added to the aqueous phase under stirring, and an oil-in-water emulsion is formed by using a homogenizer for 3 minutes; when the temperature of the oil-in-water emulsion decreases to 38°C, 0.004 g of adipose-derived mesenchymal stem cell exosomes, 12 g of superoxide dismutase and 4 g of phenoxyethanol are added, and stirred and mixed; Step S3, filtered using a sterile filter membrane. Example 3

[0047] The specific implementation is the same as that of Example 1, except that the preparation of the vanillin-derived dynamic imine bond grafted hyaluronic acid: 8 g of hyaluronic acid is dissolved in 400 g of deionized water, 2.5 g of 1-3-dimethylaminopropyl-3-ethyl carbodiimide hydrochloride and 0.8 g of N-hydroxysuccinimide are added, followed by 4 g of adipic acid dihydrazide, and the reaction is carried out for 18 hours to obtain a reaction solution. The reaction solution is dialyzed in a dialysis bag with a molecular weight cut-off of 10 kDa for 60 hours to obtain a hydrazide-derivatized hyaluronic acid aqueous solution. 4 g of vanillin is dissolved in 150 g of anhydrous ethanol, and the hydrazide-derivatized hyaluronic acid aqueous solution is added. The reaction is carried out for 18 hours under nitrogen protection and in the dark to obtain a reaction mixture. The reaction mixture is cooled to room temperature and dialyzed in a dialysis bag with a molecular weight cut-off of 10 kDa for 48 hours. The product after dialysis is freeze-dried for 50 hours to obtain vanillin-derived dynamic imine bond grafted hyaluronic acid. Preparation of disulfide bond cross-linked polyester vitreous network: In a three-necked flask, 8 g of azelaic acid, 7 g of 1,4-butanediol, and 4 g of 2,2-dithiodiglycolic acid are added. Under a nitrogen atmosphere, the temperature is raised to 165°C and stirred until completely dissolved. 0.8 g of glycerol and 0.08 g of tetrabutyl titanate are added, and the temperature is raised to 195°C to undergo melt polycondensation reaction for 5.5 hours to obtain a melt polymer. The melt polymer is poured into a polytetrafluoroethylene mold and heat treated at 95°C for 1.8 hours. After cooling, it is pulverized into powder to obtain disulfide bond cross-linked polyester vitreous network powder. Preparation of moisturizing and whitening composition containing stem cell exosomes: Step S1, human adipose-derived mesenchymal stem cells are cultured under low oxygen conditions with an oxygen concentration of 4%, and the supernatant is collected. Exosomes are separated by differential ultracentrifugation, resuspended with phosphate buffer solution, and then obtained as adipose-derived mesenchymal stem cell exosomes, which are stored at -80°C. Step S2, 3.5 g of vanillin-derived dynamic imine bond grafted hyaluronic acid and 75 g of deionized water are mixed and stirred in a 41°C water bath until completely dissolved. 8 g of glycerol, 6 g of hyaluronic acid, 0.35 g of ceramide-2, and 8 g of aloe vera gel are added in sequence, and homogenized to form an aqueous phase. 1.8 g of disulfide bond cross-linked polyester vitreous network powder and 5.5 g of potassium lauryl phosphate are mixed, heated to 71°C, and 0.25 g of nicotinamide and 8 g of radix scirpi extract are added to form an oil phase. The oil phase is added to the aqueous phase under stirring, and a homogenizer is used to emulsify for 5 minutes to form an oil-in-water emulsion. When the temperature of the oil-in-water emulsion decreases to 42°C, 0.006 g of adipose-derived mesenchymal stem cell exosomes, 18 g of superoxide dismutase, and 6 g of phenoxyethanol are added and stirred. Step S3, sterile filter membrane is used for filtration.

[0048] Comparative Example 1 The specific implementation is the same as that of Example 1, except that the vanillin-derived dynamic imine bond grafted hyaluronic acid is not used in this comparative example, but ordinary hyaluronic acid is used instead.

[0049] Comparative Example 2 The detailed implementation is the same as Example 1, except that this comparative example does not use a disulfide cross-linked polyester vitreous body network powder.

[0050] Comparative Example 3 The detailed implementation is the same as Example 1, except that this comparative example does not use fat-derived mesenchymal stem cell exosomes.

[0051] Performance test According to the relevant national and industry standards, the stem cell exosome-containing moisturizing whitening compositions prepared in Examples 1-3 and Comparative Examples 1-3 are tested according to the following performance test methods: Skin moisturizing performance test: 30 healthy female volunteers aged 25-55 were recruited, the test area of both arms was cleaned in a constant temperature and humidity environment, the initial stratum corneum moisture content was measured using a skin moisture content tester, then 0.1 g of the sample of Example or Comparative Example was applied to the left arm, and the right arm was used as a blank control. The change in skin moisture content was measured at 1 hour, 4 hours and 8 hours after application, and the moisture content increase rate was calculated. At the same time, the change in transdermal water loss was measured at the same time points using a transdermal water loss tester, and all data were averaged from three measurements. Skin whitening performance test: an in vitro melanocyte culture model was used, B16F10 melanocytes were inoculated in a 96-well plate, and 0.1% of the sample of Example or Comparative Example was added to the culture medium, and the untreated group was used as a control. After 72 hours of culture, the cell tyrosinase activity was measured using a multifunctional enzyme label meter, and the melanin content was measured using the NaOH lysis method, with 6 replicate wells in each group. The change in facial melanin index of volunteers after using the sample for 4 weeks was measured by human test using a skin melanin index detector. Skin anti-aging performance test: an ultraviolet-induced photoaged human dermal fibroblast model was used, the proportion of senescent cells was calculated by β-galactosidase staining, and the content of collagen type I in the cell culture supernatant was detected using an ELISA kit. The facial skin elasticity improvement rate of volunteers after using the sample for 4 weeks was measured by human test using a skin elasticity tester, and the change in wrinkle depth at the corner of the eye was measured using a skin wrinkle analysis system. Stability test: the sample was stored at 40°C and 75% relative humidity for 3 months, and the exosome particle size distribution and biological activity indicators were regularly detected.

[0052] Performance test results: Table 1: Performance test results of each example and comparative example Test item Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 8-hour moisture content increase rate / % 58.3±2.1 55.7±1.9 56.8±2.3 32.5±1.8 45.2±2.0 48.6±2.1 8-hour transdermal water loss decrease rate / % 45.6±1.7 43.2±1.5 44.1±1.8 22.3±1.3 35.8±1.6 38.4±1.5 Tyrosinase activity inhibition rate / % 62.5±2.3 60.8±2.1 61.3±2.4 35.2±1.9 52.6±2.2 58.1±2.3 Melanin content decrease rate / % 55.8±2.0 53.4±1.8 54.2±2.1 28.7±1.6 45.3±1.9 49.7±2.0 4-week melanin index decrease rate / % 32.7±1.5 30.5±1.3 31.6±1.6 15.2±1.1 25.8±1.4 28.3±1.5 Senescent cell decrease rate / % 68.2±2.5 65.7±2.3 66.8±2.6 35.8±2.0 55.3±2.4 60.2±2.5 Type I collagen increase rate / % 85.3±3.1 82.6±2.9 83.7±3.2 45.2±2.5 68.4±3.0 75.8±3.1 Skin elasticity increase rate / % 42.5±1.8 40.3±1.6 41.2±1.9 22.7±1.4 33.6±1.7 36.8±1.8 Wrinkle depth decrease rate / % 38.7±1.6 36.5±1.4 37.4±1.7 19.3±1.2 29.8±1.5 32.6±1.6 Exosome activity maintenance rate / % 85.4±2.2 83.7±2.0 84.2±2.3 42.7±1.8 68.5±2.1 72.3±2.2 All data are expressed as mean ± standard deviation, n = 30 (human test) or n = 6 (cell experiment), t test was used for comparison between groups, and p < 0.05 indicates that the difference is statistically significant.

[0053] As can be seen from Table 1, examples 1-3 effectively solve the three technical problems existing in the prior art stem cell exosome cosmetics compared with comparative examples 1-3. In terms of activity stability, the exosome activity retention rate of examples 1-3 reaches 83.7%-85.4% after 3-month accelerated test, which is significantly higher than 42.7% of comparative example 1, which confirms that the double protection system formed by the three-dimensional hydration network of vanillin derivative dynamic imine bond grafted hyaluronic acid and the disulfide cross-linked polyester vitreous body network can effectively maintain the integrity of the exosome membrane structure and biological activity; while the activity retention rate of comparative example 2 using only a single protective material decreases to 68.5%, indicating that the synergistic protection of the two modified compounds is indispensable. In terms of transdermal efficiency, the 8-hour transdermal water loss reduction rate of examples 1-3 reaches 43.2%-45.6%, which is much higher than 22.3% of comparative example 1, which proves that the dynamic imine bond of modified hyaluronic acid can enhance the skin barrier repair function, and the intelligent response characteristics of the disulfide cross-linked network promote the penetration of active ingredients; comparative example 3 has a material protection system but lacks direct participation of exosomes in skin regulation, and its moisturizing effect is still lower than that of the example group. In terms of synergistic efficacy, example 1 simultaneously achieves 62.5% tyrosinase inhibition rate, 68.2% reduction rate of senescent cells and 85.3% increase rate of collagen, which fully surpasses the single efficacy performance of each comparative example, among which comparative example 1 decreases all efficacy indicators by about 40%-50% due to the lack of material functional modification, comparative example 2 is limited in whitening and anti-aging efficacy due to the lack of intelligent release system, and comparative example 3 has significantly weakened source whitening and deep anti-aging effect although the basic moisturizing is acceptable. In summary, the present application successfully realizes the technical breakthroughs of activity stability, transdermal enhancement and multi- efficacy synergy through the triple synergistic mechanism of the two modified compounds and exosomes.

[0054] The above-described examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A method for preparing a moisturizing and whitening composition containing stem cell exosomes, characterized in that the steps include... include: S1. Human adipose-derived mesenchymal stem cells were cultured under hypoxic conditions, the supernatant was collected, and exosomes were separated by differential ultracentrifugation, resuspended, and adipose-derived mesenchymal stem cell exosomes were obtained and stored. S2. Vanillin-derived dynamic imine-bonded hyaluronic acid and deionized water are mixed and stirred to dissolve. Glycerin, hyaluronic acid, ceramide-2 and aloe vera gel are added sequentially, and homogenized to form an aqueous phase. Disulfide-linked polyester glass network powder is mixed with potassium lauryl phosphate, heated, and nicotinamide and Cynanchum atratum extract are added, and stirred until an oil phase is formed. While stirring, the oil phase is added to the aqueous phase and emulsified using a homogenizer to form an oil-in-water emulsion. When the temperature of the oil-in-water emulsion drops below 40°C, adipose-derived mesenchymal stem cell exosomes, superoxide dismutase and phenoxyethanol are added and stirred to mix. S3. Use sterile filter membrane for filtration.

2. The method for preparing the moisturizing and whitening composition containing stem cell exosomes according to claim 1, characterized in that, In step S1, the oxygen concentration under low-oxygen conditions is 1-5%.

3. The method for preparing the moisturizing and whitening composition containing stem cell exosomes according to claim 1, characterized in that, In step S2, the homogenization time is 3-5 minutes.

4. The method for preparing the moisturizing and whitening composition containing stem cell exosomes according to claim 1, characterized in that, The preparation method of vanillin-derived dynamic imine bond grafted hyaluronic acid includes: A1. Hyaluronic acid was dissolved in deionized water, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. Then, adipic acid dihydrazide was added to react and a reaction solution was obtained. The reaction solution was dialyzed to obtain an aqueous solution of hydrazide-modified hyaluronic acid. Vanillin was dissolved in anhydrous ethanol and added to the aqueous solution of hydrazide-modified hyaluronic acid. The reaction was carried out under nitrogen protection and in the dark to obtain a reaction mixture. A2. Cool the reaction mixture to room temperature, dialyze, and freeze-dry the dialyzed product.

5. The method for preparing the moisturizing and whitening composition containing stem cell exosomes according to claim 4, characterized in that, In step A1, the reaction time in the dark is 18-24 hours.

6. The moisturizing and whitening composition containing stem cell exosomes according to claim 4, characterized in that, In step A2, the freeze-drying time is 48-50 hours.

7. The method for preparing the moisturizing and whitening composition containing stem cell exosomes according to claim 1, characterized in that, The preparation method of the disulfide-crosslinked polyester glass network includes: adding azelaic acid, 1,4-butanediol and 2,2'-dithiodiacetic acid to a three-necked flask, heating to 150-170°C under a nitrogen atmosphere, and stirring until completely dissolved; adding glycerol and tetrabutyl titanate, raising the temperature to 180-200°C, and undergoing a melt polycondensation reaction to obtain a molten polymer; pouring the molten polymer into a polytetrafluoroethylene mold and heat-treating at 80-100°C.

8. The method for preparing the moisturizing and whitening composition containing stem cell exosomes according to claim 7, characterized in that, The melt polycondensation reaction time is 4-6 hours; the heat treatment time at 80-100℃ is 1-2 hours.

9. A moisturizing and whitening composition containing stem cell exosomes, characterized in that, The moisturizing and whitening composition containing stem cell exosomes is prepared by the method described in any one of claims 1-8.

10. The use of a moisturizing and whitening composition containing stem cell exosomes according to claim 9 in an anti-aging cosmetic.