Exosome composition, preparation method and application

Abstract

The invention provides an exosome composition, a preparation method and application, and belongs to the technical field of cosmetics. Comprises, by weight, 5%-20% of exosome, 2%-4% of tranexamic acid, 0.1%-0.3% of sodium hyaluronate, 0.05%-0.15% of citric acid, 2%-4% of nicotinamide, 1%-3% of VC ethyl ether, 0.004%-0.006% of nonapeptide-1 and the balance sterile water for injection. The exosome is combined with other substances with the whitening effect to achieve a synergistic effect, a better whitening effect is achieved, other components have the effect of maintaining the appearance form and activity of the exosome, and the situation that the synergistic whitening effect between the exosome and other substances becomes poor due to the fact that the exosome is inactivated or changed in form is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to an exosome composition, a preparation method and an application thereof. Background Art

[0002] Stem cell exosomes are tiny vesicles with a diameter of 30-100nm secreted by the paracrine mechanism during the process of stem cell culture. They contain a variety of biological information such as proteins, RNA, miRNA, DNA, dsDNA, etc. In addition, they are also rich in EGF, VEGF, bFGF, HGF, IGF and other active factors. As a carrier of information transmission between cells, stem cell exosomes play an important role in human health. Studies have shown that stem cell exosomes can participate in the body's immune regulation, promote cell proliferation, inhibit cell apoptosis, repair tissue damage, etc. In the field of medical beauty, stem cell exosomes can promote the healing of burns and scalds, promote the synthesis of collagen, delay skin aging, and repair inflammation, red blood streaks and other problem skin. Therefore, they have good application prospects in the field of medical beauty.

[0003] At present, the applications of exosomes in the field of medical beauty are mostly reported in the areas of skin acne mark repair, skin scar repair, wound healing, and skin anti-aging. However, there are relatively few reports on the skin whitening effect of exosomes. Even though there are a small number of articles / patents reporting that exosomes have a relatively ideal effect in skin whitening, in actual application, the real whitening effect is not satisfactory. Theoretically, exosomes do not contain related substances that can directly inhibit the production of melanin / dilute melanin, but the growth factors such as EGF, VEGF, bFGF contained in exosomes can accelerate cell metabolism and proliferation and differentiation. The RNA, miRNA and other biological information contained in them can accelerate the signal transduction between cells and activate cell functions. In actual application, the real whitening effect is not satisfactory because of the insufficient activity of exosomes and the failure of exosomes during transportation and storage, such as positive surface antigens, which greatly attenuates the efficacy. Summary of the invention

[0004] To solve the above problems, the present invention provides an exosome composition, a preparation method and an application, wherein exosomes are used in combination with other substances having whitening effects to achieve a synergistic effect and achieve a better whitening effect. The other ingredients have the function of maintaining the appearance, morphology and activity of the exosomes, thereby avoiding the inactivation of the exosomes or changes in morphology, which leads to a deterioration of the synergistic whitening effect with other substances.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides an exosome composition, comprising, by mass percentage: exosomes: 5-20%, tranexamic acid: 2-4%, sodium hyaluronate: 0.1-0.3%, citric acid: 0.05-0.15%, niacinamide: 2-4%, VC ethyl ether: 1-3%, nonapeptide-1: 0.004-0.006%, and adding sterile water for injection to 100%.

[0006] Further, tranexamic acid: 3%, sodium hyaluronate: 0.2%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, nonapeptide-1: 0.005%.

[0007] Furthermore, the exosomes are umbilical cord mesenchymal stem cell exosomes.

[0008] On the other hand, the present invention provides a method for preparing the above-mentioned exosome composition, comprising: dissolving sodium hyaluronate in sterile water for injection to obtain solution A; weighing nicotinamide, VC ethyl ether, and nonapeptide-1, and adding citric acid to A to dissolve to obtain solution B; adding tranexamic acid and exosomes to solution B, and adding sterile water for injection, mixing evenly to obtain solution C; and adjusting the pH value of the solution to 6.0-7.4 with citric acid.

[0009] Furthermore, before adding tranexamic acid and exosomes, solution B was filtered using a filter with a pore size of 0.22 um.

[0010] Furthermore, the protein concentration of the prepared exosome composition is 3-5 mg / ml, and the exosome concentration is not less than 2.5×10 11 Particles / ml.

[0011] Furthermore, the method for preparing exosomes is as follows: screening umbilical cord tissue that has passed the virus and infectious disease tests, crushing it, and extracting primary cells; screening primary cells with good growth conditions, and passage and amplifying them; collecting the supernatant of P3 and P4 stem cell cultures in a sterile container, enriching and collecting the exosomes, and storing them in a -80°C refrigerator after sterilization.

[0012] Furthermore, the protein concentration in the obtained exosomes was 20-40 mg / ml, and the exosome concentration was (0.8-1.5)×10 12 Particles / ml.

[0013] Furthermore, the exosome composition can be stored at 25°C for up to 3 months; the exosome composition can be stored at 2-8°C for up to 6 months; and the exosome composition can be stored at below -20°C for at least 12 months.

[0014] On the other hand, an embodiment of the present invention also provides an application of the exosome composition in the preparation of cosmetics.

[0015] The beneficial effects brought by the technical solution provided by the embodiment of the present invention include: The present invention limits the components and contents. Under the premise of ensuring that the activity of exosomes is not affected, other types of components are added to achieve that the exosome composition can be stored for up to 3 months at 25°C; up to 6 months at 2-8°C; and at least 12 months at below -20°C. Specifically, nicotinamide, tranexamic acid and nonapeptide-1 are commonly used whitening components, and VC ethyl ether is a commonly used antioxidant component. When they are mixed with exosomes, the influence of each component on the activity of exosomes needs to be considered. It is found through experiments that when only tranexamic acid is added, the activity of exosomes is reduced, mainly The reason is that tranexamic acid affects the morphology of exosomes and limits the release of growth factors. In order to solve the above problems, the amount of tranexamic acid added is reduced, but the small amount of tranexamic acid added affects the whitening effect. In order to improve the whitening effect, niacinamide is added, but the addition of niacinamide only reduces the effect on the activity of exosomes. By adding nonapeptide-1, the activity of exosomes is activated to offset the effects of tranexamic acid and niacinamide. VC ethyl ether is added to improve the stability of the composition. The exosome composition prepared in this application has high activity, and the average spot lightening rate seven days after surgery is 19.7%, which is much higher than the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 The morphological diagram of exosomes provided in the embodiment of the present invention, a is group A1, b is group B1; Figure 2 A statistical diagram of the number of exosomes provided in an embodiment of the present invention, a is group A1, b is group B1; Figure 3 A diagram of the detection of exosome surface antigens provided in an embodiment of the present invention; Figure 4 The natural light image and brown spot image of the face of a volunteer after using the A1 group product provided in the embodiment of the present invention; Figure 5 The natural light image and brown spot image of the face of a volunteer after using the A2 group product provided in the embodiment of the present invention; Figure 6The natural light image and brown spot image of the face of a volunteer after using the B1 group product provided in the embodiment of the present invention; Figure 7 This is a picture of local brown spots on volunteers after using different products provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] An embodiment of the present invention provides an exosome composition, which comprises, by mass percentage: exosomes: 5-20%, tranexamic acid: 2-4%, sodium hyaluronate: 0.1-0.3%, citric acid: 0.05-0.15%, niacinamide: 2-4%, VC ethyl ether: 1-3%, nonapeptide-1: 0.004-0.006%, and sterile water for injection is added to 100%.

[0020] Tranexamic acid and sterile water for injection are injection grade, with endotoxin content ≤0.05EU / ml; sodium hyaluronate, niacinamide, VC ethyl ether, and nonapeptide-1 are medical grade, with endotoxin content ≤1EU / ml; citric acid is pharmaceutical grade, with endotoxin content ≤1EU / ml. Sodium hyaluronate was purchased from McLean.

[0021] In the prior art, exosomes are used for whitening, but the whitening effect is not satisfactory. The main reason is that other components affect the activity of exosomes, such as the surface morphology and surface antigen positivity, which greatly attenuates the whitening effect; secondly, the whitening effect requires exosomes to be combined with whitening, antioxidant, moisturizing and other ingredients. If a certain type of component is removed or its concentration is reduced only for the activity of exosomes, it will not be able to cooperate with the exosomes to achieve the effect of whitening and synergistic. In order to solve this problem, the present application limits the components and contents. Under the premise of ensuring that the activity of exosomes is not affected, other types of components are added to achieve the exosome composition. It can be stored for up to 3 months at 25°C; up to 6 months at 2-8°C; at least 12 months below -20°C. Specifically, niacinamide, tranexamic acid and nonapeptide-1 are commonly used whitening components, and VC ethyl ether is a commonly used antioxidant component. When they are mixed with exosomes, the influence of each component on the activity of exosomes needs to be considered. Experiments have found that when only tranexamic acid is added, the activity of exosomes is reduced. The main reason is that tranexamic acid will affect the morphology of exosomes and limit the release of growth factors. In order to solve the above problems, the amount of tranexamic acid added is reduced, but the reduction in the amount of tranexamic acid added will affect the whitening effect. In order to improve the whitening effect, niacinamide is added, but the addition of niacinamide only reduces the effect on the activity of exosomes. By adding nonapeptide-1, the activity of exosomes is activated to offset the effects of tranexamic acid and niacinamide. VC ethyl ether is added to improve the stability of the composition. The exosome composition prepared in the present application has high activity, and the average spot lightening rate seven days after surgery is 19.7%, which is much higher than the prior art.

[0022] Specifically, tranexamic acid: 3%, sodium hyaluronate: 0.2%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, and nonapeptide-1: 0.005%.

[0023] Preferably, the exosomes are umbilical cord mesenchymal stem cell exosomes.

[0024] The present invention also provides a method for preparing the above-mentioned exosome composition, comprising: S1: dissolve sodium hyaluronate in sterile water for injection to obtain solution A. Specifically, weigh sodium hyaluronate according to the mass ratio, dissolve it in 50 ml of water for injection, and heat in a water bath at 40° C. to assist dissolution to obtain solution A.

[0025] S2: Niacinamide, VC ethyl ether and nonapeptide-1 are weighed according to the mass ratio, and citric acid is added to A to dissolve to obtain solution B.

[0026] S3 added tranexamic acid and exosomes to solution B, and added sterile water for injection, and mixed them evenly to obtain solution C. Specifically, before adding tranexamic acid and exosomes, solution B was filtered using a filter with a pore size of 0.22 um (manufacturer: Hangzhou Kebet, item number: A50BBDN66PC2222A1P) to remove bacteria and endotoxins, and then tranexamic acid and exosome solutions were added, and water for injection was added to 100%, and mixed evenly to obtain solution C.

[0027] S4 uses citric acid to adjust the pH value of the solution to 6.0-7.4.

[0028] Measure the pH value of solution C and make it within the range of 6-7.4. If the pH value of the solution is higher, the pH value of the solution can be adjusted with sterilized citric acid solution.

[0029] The final solution C had a protein concentration of 2-6 mg / ml and an exosome concentration of (1-3)×10 11 Particles / ml, pH value is in the range of 6-7.4.

[0030] Specifically, the acquisition of exosomes derived from umbilical cord mesenchymal stem cells includes: 1. Culture of Umbilical Cord Mesenchymal Stem Cells (1) Screen the umbilical cord tissue that has passed the virus and infectious disease tests, break it, and extract primary cells; (2) Passaging and expansion of stem cells: Select primary cells with good growth conditions and pass them on and expand them using culture medium without antibiotics, phenol red, or serum; (3) Collection of stem cell supernatant: Collect the P3 and P4 stem cell culture supernatant in a sterile container.

[0031] 2. Obtaining stem cell exosomes: (1) The "stem cell culture supernatant" collected in step 1 was used as the raw material, and the exosomes were enriched and collected using the FTVE10 3D FloTrix® vivaEXO Exosome Harvesting System, and then sterilized and stored in a -80°C refrigerator; (2) The protein concentration of the obtained exosome concentrated solution is 20-40 mg / ml, and the exosome concentration is (0.8-1.5)×10 12 Particles / ml.

[0032] The prepared exosome composition can be stored at 25°C for up to 3 months, at 2-8°C for up to 6 months, and at below -20°C for at least 12 months.

[0033] The embodiments of the present invention also protect the use of the above-mentioned exosome composition in the preparation of cosmetics.

[0034] In order to better illustrate the embodiments of the present invention, the present invention is further described in detail below through specific examples.

[0035] Example

[0036] (1) This example provides exosome compositions of different components.

[0037] Group A is the control group, including: Component A1: Take the concentrated exosome solution and dilute it with 80% water for injection to obtain an exosome solution with a concentration of 20%.

[0038] Component A2: Exosomes: 0%, tranexamic acid: 3%, sodium hyaluronate: 0.1%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, nonapeptide-1: 0.005%, and sterile water for injection added to 100%.

[0039] A3 component: exosomes: 20%, tranexamic acid: 6%, sodium hyaluronate: 0.1%, citric acid: 0.1%, VC ethyl ether: 2%, nonapeptide-1: 0.005%, and sterile water for injection added to 100%.

[0040] A4 component: exosomes: 20%, tranexamic acid: 3%, sodium hyaluronate: 0.1%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, sterile water for injection added to 100%.

[0041] A5 component: exosomes: 20%, sodium hyaluronate: 0.1%, citric acid: 0.1%, niacinamide: 6%, VC ethyl ether: 2%, nonapeptide-1: 0.005%, and sterile water for injection added to 100%.

[0042] Group B is the experimental group, including: Component B1: Exosomes: 20%, tranexamic acid: 3%, sodium hyaluronate: 0.1%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, nonapeptide-1: 0.005%, and sterile water for injection added to 100%.

[0043] Component B2: Exosomes: 5%, tranexamic acid: 2%, sodium hyaluronate: 0.1%, citric acid: 0.05%, niacinamide: 2%, VC ethyl ether: 1%, nonapeptide-1: 0.004%, and sterile water for injection added to 100%.

[0044] B3 component: exosomes: 10%, tranexamic acid: 3%, sodium hyaluronate: 0.2%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, nonapeptide-1: 0.005%, and sterile water for injection added to 100%.

[0045] B4 component: Exosomes: 20%, tranexamic acid: 4%, sodium hyaluronate: 0.3%, citric acid: 0.15%, niacinamide: 4%, VC ethyl ether: 3%, nonapeptide-1: 0.006%, and sterile water for injection added to 100%.

[0046] (2) Compare the performance of different components.

[0047] For illustration, in the examples, only the A1 and A2 groups were subjected to exosome morphology observation, exosome quantity counting, surface antigen detection, and stability testing, and the A1-A4 and B1-B4 groups were subjected to whitening efficacy testing.

[0048] (I) Exosome morphology: Figure 1 As shown in a and 1b, it can be observed under an electron microscope that the exosomes in group B1 are still vesicle-like structures, indicating that the introduction of other raw materials will not affect the appearance of the exosomes.

[0049] (II) Exosome quantity: Figure 2 As shown in a and 2b, the amount of exosome concentrated solution added to the samples of groups A1 and B1 was the same as 20%. The NTA test results of group A1 showed that the number of exosome particles was 1.2×10 11 Particles / ml; NTA test results of group B1 showed that the number of exosome particles was 3.3×10 11 Particles / ml, it can be seen that the introduction of other raw materials into the exosome composition proposed in the present invention will not cause a significant change in the number of exosomes.

[0050] (III) Detection of exosome surface antigens: Western Blot was used to detect the antigen expression on the surface of exosomes in samples of groups A1 and B1. Detection antigens: CD9, CD63, TSG101; There are three parallel samples in groups A1 and B1, namely A11, A12, A13, B11, B12, B13 Experimental conditions: sample concentration 4ug / ul; add 5×SDS-PAGE protein loading buffer, 95℃ metal bath, 5min, vortex to mix, centrifuge; sample volume 10ul; separation gel uses 100V voltage, electrophoresis 20min, concentration gel uses 130V voltage, electrophoresis 50min; after transfer, primary antibody is incubated at 4℃ overnight, secondary antibody is decolorized and incubated on a shaker at 37℃ for 1.5h, TBST quick wash 3 times and then developed.

[0051] like Figure 3As shown in the figure, it can be seen from the Western Blot test results that the expression of exosome surface antigens CD9, CD63, and TSG101 in both groups A and B samples is positive, and the other raw materials introduced into group B samples do not affect the expression of antigen markers on the exosome surface.

[0052] (IV) Solution stability test: Taking groups A1 and B1 as examples, whether the addition of other raw materials will affect the stability of the solution, the following experiment was designed: group A1 was used as the control group, and samples of groups A1 and B1 were stored at 25°C (room temperature), 2-8°C, -20°C, and -80°C respectively; the observation time intervals were 0 day, 1 month, 3 months, 6 months, 9 months, and 12 months; the detection indicators were mainly appearance, pH value, and exosome surface antigen expression. The test results are shown in Table 1. Table 1 Solution stability test record

[0053] For samples in group B1 at 25℃ and 2-8℃, VC ethyl ether will undergo a slight oxidation reaction, causing the solution to turn yellow. At -20℃ and -80℃, there are no abnormalities in the appearance, pH value, and expression of surface antigen markers of samples in group B. Sample 1 in group B1 can be stored for a long time at -20℃ and -80℃.

[0054] (V) Whitening efficacy test: In this embodiment, whitening efficacy tests were performed on both the experimental group and the control group.

[0055] The test method is: (1) Recruit healthy women aged 18-45 years with facial skin problems such as pigmentation, dark spots, and dull skin, 5 people per group; (2) The method of use is to use a rolling needle to break the skin and introduce it. The rolling needle length is 0.5 mm, and the sample volume is 4 ml; (3) After the operation, apply a cold compress to the face to fade the redness. Avoid getting wet for 24 hours after the operation and protect yourself from the sun for one month after the operation. (4) Use a skin detector to record the original skin condition of the volunteer before the operation, and record the skin condition after 7 days.

[0056] The test results are shown in Table 2.

[0057] Table 2 Data on the change of the area and depth of the spots of volunteers in each embodiment

[0058] By comparing the data in Table 2, it can be seen that the area and depth of the spots of B1-B4 are reduced by at least 18%, while the area and depth of the spots of A1-A5 are reduced by no more than 10%. The whitening effect of the technical solution provided in the embodiment of the present invention is much higher than that of the control group.

[0059] Take groups A1, A2 and B1 as an example. Figure 4 As shown in Figures 5, 6 and 7, after using the product for 7 days, the skin color of the volunteers using group A1 samples did not change much, and the instrument showed that the average pigmentation rate of the volunteers was 2%; the ultraviolet pigmentation of the volunteers using group A2 samples was lightened, but the changes in the skin were not very obvious when observed under natural light. The instrument showed that the average pigmentation rate of the volunteers in group A2 was 7.2%; the skin color of the volunteers using group B1 samples became whiter and brighter, and the ultraviolet pigmentation was lightened. The effect was most obvious on the 7th day after surgery. The instrument showed that the average pigmentation rate of the volunteers in group B1 samples was 19.7%.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An exosome composition, characterized in that: Calculated by mass percentage, it includes: exosomes: 5-20%, tranexamic acid: 2-4%, sodium hyaluronate: 0.1-0.3%, citric acid: 0.05-0.15%, niacinamide: 2-4%, VC ethyl ether: 1-3%, nonapeptide-1: 0.004-0.006%, and sterile water for injection is added to 100%.

2. The exosome composition according to claim 1, characterized in that Tranexamic acid: 3%, sodium hyaluronate: 0.2%, citric acid: 0.1%, niacinamide: 3%, VC ethyl ether: 2%, nonapeptide-1: 0.005%.

3. The exosome composition according to claim 1, characterized in that The exosomes are umbilical cord mesenchymal stem cell exosomes.

4. The method for preparing the exosome composition according to any one of claims 1 to 3, characterized in that: Dissolve sodium hyaluronate in sterile water for injection to obtain solution A; Weigh nicotinamide, VC ethyl ether, and nonapeptide-1, add citric acid to solution A to dissolve, and obtain solution B; Add tranexamic acid and exosomes to solution B, add sterile water for injection, and mix well to obtain solution C; Use citric acid to adjust the pH value of the solution to 6.0-7.

4.

5. The preparation method according to claim 4, characterized in that: Before adding tranexamic acid and exosomes, solution B was filtered using a filter with a pore size of 0.22 μm.

6. The preparation method according to claim 5, characterized in that: The protein concentration of the prepared exosome composition is 3-5 mg / ml, and the exosome concentration is not less than 2.5×10 11 Particles / ml.

7. The preparation method according to claim 4, characterized in that: The preparation method of the exosomes is: Screen the umbilical cord tissue that has passed the virus and infectious disease tests, break it up, and extract primary cells; Screen primary cells with good growth conditions, and subculture and expand them; The supernatants of stem cell cultures at generations P3 and P4 were collected in sterile containers, exosomes were enriched and collected, and stored in a -80°C refrigerator after sterilization.

8. The preparation method according to claim 7, characterized in that: The protein concentration in the obtained exosomes was 20-40 mg / ml, and the exosome concentration was (0.8-1.5)×10 12 Particles / ml.

9. Use of the exosome composition according to any one of claims 1 to 3 in the preparation of cosmetics.

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