Whitening and anti-aging extracellular vesicle compound as well as preparation method and application thereof

Abstract

The invention provides a whitening and anti-aging extracellular vesicle compound as well as a preparation method and application thereof. The extracellular vesicle compound comprises extracellular vesicles derived from mesenchymal stem cells and active ingredients loaded in the extracellular vesicles, the active component comprises any one or a combination of at least two of retinoic acid, arbutin, panthenol, nicotinamide, ectoine, vitamine, hyaluronic acid, vitamin C, resveratrol or retinol; the ratio of the extracellular vesicles to the active ingredients is (1-10) * 10 < 8 >: (0.01-1) mg. The skin-care active ingredients are entrapped in the extracellular vesicles derived from the mesenchymal stem cells, so that the delivery efficiency of the skin-care active ingredients can be remarkably improved, the direct irritation of free components to the skin can be reduced, the generation of matrix metalloproteinase-1 (MMP-1) and melanin can be effectively inhibited, the light aging process of the skin can be delayed, and the skin-care composition has excellent whitening and anti-aging effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a whitening and anti-aging extracellular vesicle complex, a preparation method and an application thereof. Background Art

[0002] Skin aging is a complex biological process driven by the combined effects of endogenous and exogenous factors, involving multiple mechanisms such as oxidative stress, collagen degradation, and decreased barrier function. Finding solutions that can improve skin quality and slow aging is a key focus in this field. While traditional anti-aging ingredients (such as retinol and vitamin C) have demonstrated efficacy, they still face technical bottlenecks such as low transdermal absorption, photosensitivity instability (such as retinol), and high irritation (such as retinoic acid).

[0003] Extracellular vesicles carry abundant protein molecules, lipid molecules and microRNA, and have antioxidant, anti-inflammatory and collagen production-promoting properties. They have potential application prospects in wound healing, scar prevention, photodamage prevention, skin regeneration, anti-wrinkle and anti-aging, and drug carriers.

[0004] Therefore, encapsulating anti-aging ingredients based on the multi-dimensional biological characteristics of extracellular vesicles and developing a carrier system for extracellular vesicle-complex functional ingredients are of great significance for expanding the application of extracellular vesicles in the field of functional skin care. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a whitening and anti-aging extracellular vesicle complex and its preparation method and application. The extracellular vesicle complex provided by the present invention can significantly delay the photoaging process of the skin and has excellent whitening and anti-aging effects.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an extracellular vesicle complex for whitening and anti-aging, wherein the extracellular vesicle complex comprises extracellular vesicles derived from mesenchymal stem cells and an active ingredient loaded in the extracellular vesicles;

[0008] The active ingredient includes any one or a combination of at least two of retinoic acid, arbutin, panthenol, niacinamide, ectoine, phosphodiesterone, hyaluronic acid, vitamin C, resveratrol or retinol; the ratio of the extracellular vesicles to the active ingredient is (1-10)×10 8 Pieces: (0.01-1) mg.

[0009] The specific point values ​​in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0010] The specific point values ​​in (0.01-1) can be 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.5, 0.8 or 1, etc.

[0011] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0012] Mesenchymal stem cell-derived extracellular vesicles (EVs) carry a rich supply of proteins, lipids, and microRNAs, exhibiting antioxidant, anti-inflammatory, and collagen-promoting properties. This invention encapsulates skincare active ingredients within EVs derived from mesenchymal stem cells. The synergistic combination of the active ingredients and the EV contents significantly improves the delivery efficiency of the active ingredients, reduces the direct irritation of free components to the skin, effectively inhibits the production of matrix metalloproteinase-1 (MMP-1) and melanin, and slows the progression of photoaging, resulting in excellent whitening and anti-aging effects.

[0013] The average particle size of the extracellular vesicle complex of the present invention is 120-180 nm. Retinoic acid, resveratrol, and retinol are loaded into the hydrophobic layer of the extracellular vesicle, and arbutin, panthenol, niacinamide, ectoine, phosphoinosin, hyaluronic acid, and vitamin C are loaded into the hydrophilic inner cavity of the extracellular vesicle.

[0014] Preferably, the active ingredients include retinoic acid, arbutin and panthenol.

[0015] The present invention preferably encapsulates retinoic acid, arbutin and panthenol in extracellular vesicles. The synergistic combination of fat-soluble and water-soluble active components can better inhibit the production of matrix metalloproteinase-1 (MMP-1) and melanin, delay the photoaging process of the skin, and have significant whitening and anti-aging effects.

[0016] Preferably, the mass ratio of retinoic acid, arbutin and panthenol is (0.5-2):(0.8-3):(0.2-2).

[0017] The specific point values ​​in (0.5-2) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.7 or 2, etc.

[0018] The specific point values ​​in (0.8-3) can be 0.8, 0.9, 1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0019] The specific point values ​​in (0.2-2) can be 0.2, 0.3, 0.4, 0.5, 0.8, 1, 1.3, 1.5, 1.7 or 2, etc.

[0020] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0021] In the present invention, when the retinoic acid, arbutin and panthenol are within the above-mentioned ratio range, the whitening and anti-aging effects are more significant.

[0022] Preferably, the mesenchymal stem cells include any one or a combination of at least two of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells or synovial mesenchymal stem cells.

[0023] In a second aspect, the present invention provides a method for preparing the extracellular vesicle complex as described in the first aspect, the method comprising:

[0024] Mesenchymal stem cells are cultured, cell culture fluid is collected, and extracellular vesicles are extracted; the extracellular vesicles are mixed with active ingredients and ultrasonicated to obtain the extracellular vesicle complex.

[0025] Preferably, the method for extracting extracellular vesicles comprises ultracentrifugation and / or membrane filtration.

[0026] Preferably, the ultracentrifugation method comprises subjecting the cell culture fluid to a first centrifugation, a second centrifugation, and a third centrifugation in sequence, collecting the supernatant, and then subjecting the cell culture fluid to a fourth centrifugation to collect the precipitate to obtain the extracellular vesicles.

[0027] The first centrifugation speed is 200-400g, for example, it can be 200g, 220g, 250g, 270g, 300g, 330g, 350g, 380g or 400g; the time is 5-15min, for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min.

[0028] The second centrifugation speed is 1000-3000g, for example, it can be 1000g, 1200g, 1500g, 1700g, 2000g, 2300g, 2500g, 2800g or 3000g; the time is 15-25min, for example, it can be 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min or 25min.

[0029] The rotation speed of the third centrifugation is 9000-11000g, for example, it can be 9000g, 9200g, 9500g, 9700g, 10000g, 10300g, 10500g, 10800g or 11000g; the time is 20-40min, for example, it can be 20min, 22min, 25min, 27min, 30min, 33min, 35min, 38min or 40min.

[0030] The fourth centrifugation speed is 140000-160000g, for example, it can be 140000g, 142000g, 145000g, 147000g, 150000g, 153000g, 155000g, 158000g or 160000g, etc.; the time is 80-100min, for example, it can be 80min, 82min, 85min, 87min, 90min, 93min, 95min, 98min or 100min, etc.

[0031] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0032] Preferably, the ultrasonic power is 100-300W, for example, it can be 100W, 120W, 150W, 170W, 200W, 230W, 250W, 280W or 300W, etc.; the ultrasonic time is 2-5s, for example, it can be 2s, 2.2s, 2.5s, 2.7s, 3s, 3.3s, 3.5s, 3.8s, 4s, 4.5s or 5s, etc.; the interval is 1-3s, for example, it can be 1s, 1.2s, 1.5s, 1.7s, 2s, 2.3s, 2.5s, 2.8s or 3s, etc.

[0033] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0034] In a third aspect, the present invention provides use of the extracellular vesicle complex as described in the first aspect in cosmetics.

[0035] Preferably, the cosmetics include any one of lotion, emulsion, cream, essence, spray, mask or sunscreen.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention encapsulates skin care active ingredients in extracellular vesicles derived from mesenchymal stem cells, which can significantly improve the delivery efficiency of skin care active ingredients, reduce the direct irritation of free components to the skin, effectively inhibit the production of matrix metalloproteinase-1 (MMP-1) and melanin, delay the process of skin photoaging, and have excellent whitening and anti-aging effects. DETAILED DESCRIPTION

[0038] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0039] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0040] The sources of materials used in the following specific embodiments are as follows:

[0041] Retinoic acid, arbutin, panthenol, resveratrol, niacinamide, ectoine, retinol, hyaluronic acid were purchased from Shanghai MacLean Biotechnology Co., Ltd.

[0042] Example 1

[0043] This embodiment provides a whitening and anti-aging extracellular vesicle complex, the preparation method of which is as follows:

[0044] (1) Umbilical cord mesenchymal stem cells were expanded and cultured in a cell culture incubator at 37°C and 5% CO2 to the fourth passage. The serum-free DMEM medium was then replaced and cultured in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cell culture medium was then collected. The cell culture medium was centrifuged at 300g for 10 minutes to remove cell debris and the supernatant was collected; the cell culture medium was centrifuged at 2000g for 20 minutes to remove apoptotic bodies and the supernatant was collected; the cell culture medium was centrifuged at 10,000g for 30 minutes to remove large vesicles and the supernatant was collected; the cell culture medium was centrifuged at 150,000g for 90 minutes to collect the precipitate and obtain extracellular vesicles derived from umbilical cord mesenchymal stem cells.

[0045] (2) Resuspend the extracellular vesicle pellet in 1 mL of PBS buffer and take 2×10 8 The extracellular vesicles derived from umbilical cord mesenchymal stem cells were mixed with 100 μg of retinoic acid, 200 μg of arbutin, and 100 μg of panthenol, and ultrasonically treated at a power of 200 W, with a working period of 3 seconds and a rest period of 2 seconds to obtain the extracellular vesicle complex.

[0046] Example 2

[0047] This embodiment provides a whitening and anti-aging extracellular vesicle complex, the preparation method of which is as follows:

[0048] (1) Umbilical cord mesenchymal stem cells were expanded and cultured in a cell culture incubator at 37°C and 5% CO2 to the fourth generation. The serum-free DMEM medium was then replaced and cultured in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cell culture medium was then collected. The cell culture medium was centrifuged at 200g for 15 minutes to remove cell debris and the supernatant was collected; the cell culture medium was centrifuged at 1000g for 25 minutes to remove apoptotic bodies and the supernatant was collected; the cell culture medium was centrifuged at 9000g for 40 minutes to remove large vesicles and the supernatant was collected; the cell culture medium was centrifuged at 140,000g for 100 minutes to collect the precipitate and obtain extracellular vesicles derived from umbilical cord mesenchymal stem cells.

[0049] (2) Resuspend the extracellular vesicle pellet in 1 mL of PBS buffer and take 1×10 8 The extracellular vesicles derived from umbilical cord mesenchymal stem cells were mixed with 50 μg retinoic acid, 300 μg arbutin, and 20 μg ubiquinol, and ultrasonically treated at a power of 100 W, with a working period of 2 seconds and a rest period of 1 second to obtain the extracellular vesicle complex.

[0050] Example 3

[0051] This embodiment provides a whitening and anti-aging extracellular vesicle complex, the preparation method of which is as follows:

[0052] (1) Umbilical cord mesenchymal stem cells were expanded and cultured in a cell culture incubator at 37°C and 5% CO2 to the fourth generation. The serum-free DMEM medium was then replaced and cultured in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cell culture medium was then collected. The cell culture medium was centrifuged at 400g for 5 minutes to remove cell debris and the supernatant was collected; the cell culture medium was centrifuged at 3000g for 15 minutes to remove apoptotic bodies and the supernatant was collected; the cell culture medium was centrifuged at 11000g for 20 minutes to remove large vesicles and the supernatant was collected; the cell culture medium was centrifuged at 160000g for 80 minutes to collect the precipitate and obtain extracellular vesicles derived from umbilical cord mesenchymal stem cells.

[0053] (2) Resuspend the extracellular vesicle pellet in 1 mL of PBS buffer and take 1×10 8 The extracellular vesicles derived from umbilical cord mesenchymal stem cells were mixed with 200 μg retinoic acid, 80 μg arbutin, and 200 μg ubiquinol, and ultrasonically treated at a power of 300 W, with a working period of 5 seconds and a rest period of 3 seconds to obtain the extracellular vesicle complex.

[0054] Example 4

[0055] This embodiment provides an extracellular vesicle complex for whitening and anti-aging, which differs from Example 1 only in that "100 μg retinoic acid, 200 μg arbutin, 100 μg panthenol" are replaced with "100 μg resveratrol, 200 μg niacinamide, 100 μg ectoine", and the other steps and raw materials remain unchanged.

[0056] Example 5

[0057] This embodiment provides an extracellular vesicle complex for whitening and anti-aging, which differs from Example 1 only in that "100 μg retinoic acid, 200 μg arbutin, 100 μg panthenol" are replaced with "100 μg retinol, 200 μg hyaluronic acid, 100 μg hyaluronic acid", and the other steps and raw materials remain unchanged.

[0058] Example 6

[0059] This embodiment provides an extracellular vesicle complex for whitening and anti-aging, which differs from Example 1 only in that "100 μg retinoic acid, 200 μg arbutin, 100 μg panthenol" are replaced with "30 μg retinol, 70 μg hyaluronic acid, 300 μg hyaluronic acid", and the other steps and raw materials remain unchanged.

[0060] Example 7

[0061] This embodiment provides an extracellular vesicle complex for whitening and anti-aging, which differs from embodiment 1 only in that retinoic acid is not added, and its reduced amount is proportionally distributed to arbutin and panthenol, and the remaining steps and raw materials remain unchanged.

[0062] Example 8

[0063] This embodiment provides an extracellular vesicle complex for whitening and anti-aging, which differs from embodiment 1 only in that arbutin is not added, and its reduced amount is proportionally distributed to retinoic acid and panthenol, and the remaining steps and raw materials remain unchanged.

[0064] Example 9

[0065] This example provides an extracellular vesicle complex for whitening and anti-aging. The only difference from Example 1 is that no panthenol is added, and its reduced amount is proportionally distributed to retinoic acid and arbutin. The other steps and raw materials remain unchanged.

[0066] Test Example 1

[0067] Human immortalized keratinocytes (HaCaT) in logarithmic growth phase were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to prepare 5×10 4100 μL of cell suspension was added to each well of a 96-well cell culture plate and placed in a 37°C cell culture incubator for adherent culture. After the cells were completely adhered, the original culture medium was discarded and 100 μL of fresh DMEM complete culture medium was added to each well, wherein 2×10 cells were added to each well. 8 The extracellular vesicle complexes provided in Examples 1-9 were used as the experimental group, DMEM complete medium without any additional substances was used as the control group 1, and 100 μg retinoic acid + 200 μg arbutin + 100 μg ubiquinol were added to each well as the control group 2. The cells were incubated in a 37°C cell culture incubator for 48 h. The supernatant was then removed, the cells were washed twice with PBS buffer, 100 μL PBS buffer was added to each well, and UV (50 mJ / cm 2 ) for 90 seconds. After replacing with fresh DMEM complete medium, the cells were cultured in a 37°C cell culture incubator for another 24 hours. The cell culture medium was then centrifuged at 1000 g for 10 minutes, and the supernatant was collected. MMP-1 levels in the cell supernatants of the different treatment groups were measured using an ELISA kit (Beijing Solebold Technology Co., Ltd., Cat. No. SEKP-0047).

[0068] The test results, shown in Table 1, demonstrate that the extracellular vesicle complex provided by the present invention effectively inhibits MMP-1 expression, slowing the progression of photoaging, and demonstrating excellent anti-aging effects. Encapsulation of skincare active ingredients within extracellular vesicles enhances their anti-aging effects, with retinoic acid, arbutin, and panthenol synergistically inhibiting MMP-1 expression.

[0069] Table 1

[0070] Test samples MMP-1 content (ng / mL) Example 1 735 Example 2 1008 Example 3 1157 Example 4 1515 Example 5 1627 Example 6 1771 Example 7 2072 Example 8 1847 Example 9 2204 Control group 1 3889 Control group 2 3217

[0071] Test Example 2

[0072] Mouse melanoma cells (B16) in the logarithmic growth phase were cultured in a DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) for 5×10 5 100 μL of cell suspension was added to each well of a 96-well cell culture plate and placed in a 37°C cell culture incubator for adherent culture. After the cells were completely adhered, the original culture medium was discarded and 100 μL of fresh DMEM complete culture medium was added to each well, wherein 2×10 cells were added to each well. 8The extracellular vesicle complexes provided in Examples 1-9 were used as the experimental group, DMEM complete medium without any additional substances was used as control group 1, and 100 μg retinoic acid, 200 μg arbutin, and 100 μg ubiquinol were added to each well as control group 2. The cells were incubated in a 37°C cell culture incubator for 72 hours. The supernatant was then discarded, and the cells were treated with a 1M NaOH solution containing 10% dimethyl sulfoxide at 90°C for 2 hours. The absorbance was measured at 405 nm, and the protein content was determined by the BCA method. The relative melanin content of the cells was calculated as follows: relative melanin content (%) = [(absorbance of melanin content in the test sample group / absorbance of protein concentration in the test sample group) / (absorbance of melanin content in control group 1 / absorbance of protein concentration in control group 1)] × 100%.

[0073] The test results, shown in Table 2, demonstrate that the extracellular vesicle complex provided by the present invention effectively inhibits melanin production, slows the progression of photoaging, and exhibits excellent anti-aging effects. Encapsulation of skincare active ingredients within extracellular vesicles enhances their anti-aging effects, with retinoic acid, arbutin, and panthenol synergistically inhibiting melanin production.

[0074] Table 2

[0075] Test samples Relative content of melanin (%) Example 1 43 Example 2 37 Example 3 40 Example 4 63 Example 5 61 Example 6 68 Example 7 59 Example 8 72 Example 9 55 Control group 2 85

[0076] The present invention uses the above-described embodiments to illustrate a whitening and anti-aging extracellular vesicle complex, its preparation method, and its application. However, the present invention is not limited to the above-described embodiments, and it does not mean that the present invention must rely on the above-described embodiments for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0077] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0078] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A whitening and anti-aging extracellular vesicle complex, characterized in that: The extracellular vesicle complex comprises extracellular vesicles derived from mesenchymal stem cells and active ingredients loaded in the extracellular vesicles; The active ingredient includes any one or a combination of at least two of retinoic acid, arbutin, panthenol, niacinamide, ectoine, phosphodiesterone, hyaluronic acid, vitamin C, resveratrol or retinol; the ratio of the extracellular vesicles to the active ingredient is (1-10)×10 8 Pieces: (0.01-1) mg.

2. The extracellular vesicle complex according to claim 1, characterized in that The active ingredients include retinoic acid, arbutin and panthenol.

3. The extracellular vesicle complex according to claim 2, characterized in that The mass ratio of the retinoic acid, arbutin and panthenol is (0.5-2):(0.8-3):(0.2-2).

4. The extracellular vesicle complex according to any one of claims 1 to 3, characterized in that The mesenchymal stem cells include any one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells or synovial mesenchymal stem cells, or a combination of at least two of them.

5. The method for preparing the extracellular vesicle complex according to any one of claims 1 to 4, characterized in that: The method comprises: Mesenchymal stem cells are cultured, cell culture fluid is collected, and extracellular vesicles are extracted; the extracellular vesicles are mixed with active ingredients and ultrasonicated to obtain the extracellular vesicle complex.

6. The method according to claim 5, characterized in that The method for extracting extracellular vesicles includes ultracentrifugation and / or membrane filtration.

7. The method according to claim 6, characterized in that The ultracentrifugation method comprises sequentially subjecting the cell culture fluid to a first centrifugation, a second centrifugation, and a third centrifugation, collecting the supernatant, and then subjecting the cell culture fluid to a fourth centrifugation to collect the precipitate to obtain the extracellular vesicles; The first centrifugation is performed at a speed of 200-400 g for 5-15 min. The second centrifugation is performed at a speed of 1000-3000 g for 15-25 min. The third centrifugation is performed at a speed of 9000-11000 g for 20-40 min. The fourth centrifugation is performed at a speed of 140,000-160,000 g and for a time of 80-100 min.

8. The method according to any one of claims 5 to 7, characterized in that The power of the ultrasound is 100-300W, and the ultrasound time is 2-5s on and 1-3s off.

9. Use of the extracellular vesicle complex according to any one of claims 1 to 4 in cosmetics.

10. The use according to claim 9, characterized in that The cosmetics include any one of lotion, emulsion, cream, essence, spray, mask or sunscreen.