Anti-aging fusion vesicle as well as preparation method and application thereof

Abstract

The invention provides an anti-aging fusion vesicle as well as a preparation method and application thereof. The fusion vesicle comprises a fusion product of extracellular vesicles derived from mesenchymal stem cells and lipidosome, the liposome comprises a carrier formed by assembling phospholipid, cholesterol and an amphiphilic polymer, a fat-soluble active matter loaded on a hydrophobic layer of the carrier and a water-soluble active matter loaded on a hydrophilic inner cavity of the carrier, the fat-soluble active matter comprises any one or a combination of at least two of retinoic acid, resveratrol or retinol; the water-soluble active matter comprises any one or a combination of at least two of arbutin, panthenol, nicotinamide, ectoine, glassine, hyaluronic acid or vitamin C. The fusion vesicle provided by the invention can significantly improve the delivery efficiency of active skin care components, reduce the direct irritation of free components to skin, effectively inhibit the expression quantity of matrix metalloproteinase-1 and delay the photoaging process of skin, and has an excellent anti-aging effect.

Description

Technical Field

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

[0002] Aging is an inevitable natural process of life. Finding ways to delay or even reverse skin aging has become a research hotspot in the fields of medical aesthetics and anti-aging. Dysfunction of the epidermis and fibroblasts, as well as changes in the composition and content of the extracellular matrix, are common pathophysiological manifestations of skin aging.

[0003] Stem cell-derived extracellular vesicles (EVs) have a strong ability to promote tissue repair and regeneration, and have potential for application in anti-aging treatments. As an alternative to cell-free therapies, stem cell-derived EVs can avoid the risks of immune rejection, stemness maintenance, cellular senescence, and tumorigenesis associated with stem cell transplantation. They not only promote the repair of sensitive and aging skin and accelerate skin regeneration, but also possess multiple functions such as anti-aging and immune regulation. However, the limited encapsulation rate of EVs for active ingredients (retinoic acid, retinol, arbutin, etc.) has hampered their application in cosmetics and medical aesthetics.

[0004] The structural components of traditional liposomes are similar to those of the stratum corneum cell membrane, which improves their biocompatibility. However, they tend to aggregate during transdermal penetration, preventing them from penetrating deep into the skin. Furthermore, the phospholipids in their structure are easily oxidized, affecting their stability.

[0005] Therefore, it is necessary to combine the beneficial effects of extracellular vesicles and liposomes to develop a composite effective ingredient carrier system. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide an anti-aging fusion vesicle and its preparation method and application. The fusion vesicle provided by the present invention can delay the photoaging process of the skin and has excellent anti-aging effect.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an anti-aging fusion vesicle, comprising a fusion product of an extracellular vesicle derived from a mesenchymal stem cell and a liposome; the liposome comprises a carrier assembled from phospholipids, cholesterol, and an amphiphilic polymer, a fat-soluble active substance loaded on the hydrophobic layer of the carrier, and a water-soluble active substance loaded on the hydrophilic inner cavity of the carrier;

[0009] The fat-soluble active ingredient includes any one of retinoic acid, resveratrol or retinol, or a combination of at least two of them; the water-soluble active ingredient includes any one of arbutin, panthenol, niacinamide, ectoine, phosphocyanine, hyaluronic acid or vitamin C, or a combination of at least two of them.

[0010] The fusion vesicles provided by the present invention simultaneously encapsulate fat-soluble and water-soluble active skin care components, enabling the combined application of multiple components, significantly improving the delivery efficiency of active skin care components, and reducing the direct irritation of free components to the skin; the extracellular vesicles derived from mesenchymal stem cells carry rich protein molecules, lipid molecules, and microRNA, which, combined with active skin care components, can effectively inhibit the expression of matrix metalloproteinase-1 (MMP-1), delay the process of skin photoaging, and have excellent anti-aging effects. The average particle size of the anti-aging fusion vesicles in the present invention is 100-200nm.

[0011] Preferably, the ratio of the number of extracellular vesicles to liposomes is (1-5):(1-10).

[0012] The specific point values ​​in (1-5) can be 1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8, 3, 3.5, 4, 4.5 or 5, etc.

[0013] The specific point values ​​in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0014] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0015] In the present invention, the extracellular vesicles and liposomes are fused within the above-mentioned ratio range, and the anti-aging effect is more significant.

[0016] Preferably, the mass ratio of phospholipid, cholesterol and amphiphilic polymer in the carrier is (140-160):(20-30):(5-15).

[0017] The specific point values ​​in (140-160) can be 140, 142, 145, 147, 150, 153, 155, 158 or 160, etc.

[0018] The specific point values ​​in (20-30) can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.

[0019] The specific point values ​​in (5-15) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, etc.

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

[0021] Preferably, the mass ratio of the carrier to the fat-soluble active ingredient is 1:(0.01-0.1).

[0022] The specific point values ​​in (0.01-0.1) can be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1, etc.

[0023] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0024] Preferably, the mass ratio of the carrier to the water-soluble active ingredient is 1:(0.1-5).

[0025] The specific point values ​​in (0.1-5) can be 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0026] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0027] Preferably, the amphiphilic polymer includes any one of distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol or dilauroylphosphatidylethanolamine-polyethylene glycol, or a combination of at least two thereof.

[0028] Preferably, the number average molecular weight of the polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol or dilauroylphosphatidylethanolamine-polyethylene glycol is independently 1000-6000, for example, it can be 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500 or 6000, etc.

[0029] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0030] 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.

[0031] Preferably, the fat-soluble active ingredient is retinoic acid, and the water-soluble active ingredients are arbutin and panthenol.

[0032] The present invention preferably encapsulates retinoic acid, arbutin and panthenol in fusion vesicles, and the fat-soluble and water-soluble active components are synergistically compatible, which has a better matrix metalloproteinase-1 (MMP-1) inhibitory effect, delays the skin photoaging process, and has a significant anti-aging effect.

[0033] Preferably, the mass ratio of retinoic acid, arbutin and panthenol is (0.02-0.06):(0.5-1):(1-3).

[0034] The specific point values ​​in (0.02-0.06) can be 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055 or 0.06, etc.

[0035] The specific point value in (0.5-1) can be 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1, etc.

[0036] The specific point values ​​in (1-3) can be 1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0037] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0038] In the present invention, when retinoic acid, arbutin and panthenol are within the above-mentioned ratio range, the anti-aging effect is more significant.

[0039] Preferably, the phospholipid comprises any one of egg yolk lecithin, soybean lecithin or hydrogenated soybean lecithin, or a combination of at least two thereof.

[0040] In a second aspect, the present invention provides a method for preparing the fusogenic vesicle according to the first aspect, characterized in that the method comprises:

[0041] (1) Cultivating mesenchymal stem cells, collecting cell culture fluid, and extracting extracellular vesicles;

[0042] Phospholipids, cholesterol, amphiphilic polymers, fat-soluble active substances and organic solvents are mixed and dissolved, and the organic solvent is evaporated to remove the organic solvent to form a film; an aqueous solution of the water-soluble active substance is added, and ultrasonication is performed to prepare liposomes;

[0043] (2) The extracellular vesicles and liposomes are mixed, sonicated, incubated, freeze-thawed, and extruded through a polycarbonate membrane to obtain the fusion vesicles.

[0044] Preferably, in step (1), the method for extracting extracellular vesicles comprises ultracentrifugation and / or membrane filtration.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0051] Preferably, the organic solvent includes any one of ethanol, acetonitrile, chloroform or methanol, or a combination of at least two of them.

[0052] Preferably, the concentration of the water-soluble active ingredient in the aqueous solution of the water-soluble active ingredient is 100-1000 mg / L, for example, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L or 1000 mg / L, etc.

[0053] Preferably, in step (1), the ultrasonic power is 100-300 W, for example, it can be 100 W, 120 W, 150 W, 170 W, 200 W, 230 W, 250 W, 280 W or 300 W, etc.; the ultrasonic time is 2-5 s, for example, it can be 2 s, 2.2 s, 2.5 s, 2.7 s, 3 s, 3.3 s, 3.5 s, 3.8 s, 4 s, 4.5 s or 5 s, etc.; the interval is 1-3 s, for example, it can be 1 s, 1.2 s, 1.5 s, 1.7 s, 2 s, 2.3 s, 2.5 s, 2.8 s or 3 s, etc.

[0054] Preferably, in step (1), the number of ultrasounds is 15-25 times, for example, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times or 25 times.

[0055] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0056] Preferably, in step (2), the ultrasonic time is 20-40s, for example, 20s, 22s, 25s, 27s, 30s, 33s, 35s, 38s or 40s, etc.; the number of ultrasonic times is 2-5 times, for example, 2 times, 3 times, 4 times or 5 times, etc.

[0057] Preferably, the incubation temperature is 30-40°C, for example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C; the incubation time is 20-40min, for example, it can be 20min, 22min, 25min, 27min, 30min, 33min, 35min, 38min or 40min.

[0058] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0059] Preferably, the freeze-thaw cycle process is to first freeze with liquid nitrogen and then thaw.

[0060] Preferably, the freezing time is 4-7 min, for example, it can be 4 min, 4.2 min, 4.5 min, 4.7 min, 5 min, 5.3 min, 5.5 min, 5.8 min, 6 min, 6.5 min or 7 min.

[0061] Preferably, the thawing temperature is 25-35°C, for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C; the thawing time is 10-20 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0062] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0063] Preferably, the number of freeze-thaw cycles is 3-5 times, for example, 3 times, 4 times or 5 times.

[0064] Preferably, the size of the polycarbonate film is 200-800 nm, for example, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm or 800 nm.

[0065] Preferably, the polycarbonate film is extruded 20-50 times, for example, 20 times, 22 times, 25 times, 27 times, 30 times, 33 times, 35 times, 38 times, 40 times, 45 times or 50 times.

[0066] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0067] In a third aspect, the present invention provides a use of the fusogenic vesicles described in the first aspect in cosmetics, wherein the cosmetics include any one of lotion, emulsion, cream, essence, spray, mask or sunscreen.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] The fusion vesicles provided by the present invention simultaneously encapsulate fat-soluble and water-soluble active skin care components, which can realize the combined application of multiple components, significantly improve the delivery efficiency of active skin care components, and reduce the direct irritation of free components to the skin; the extracellular vesicles derived from mesenchymal stem cells carry rich protein molecules, lipid molecules and microRNA, which can effectively inhibit the expression of matrix metalloproteinase-1 (MMP-1) in combination with active skin care components, delay the process of skin photoaging, and have excellent anti-aging effects. DETAILED DESCRIPTION

[0070] 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.

[0071] 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.

[0072] The sources of materials used in the following specific embodiments are as follows:

[0073] Egg yolk lecithin, soybean lecithin, and hydrogenated soybean lecithin were purchased from Avituo Pharmaceutical Technology Co., Ltd.; distearoyl phosphatidylethanolamine-polyethylene glycol 2000 was purchased from Genzyme; cholesterol, retinoic acid, arbutin, panthenol, resveratrol, niacinamide, ectoine, retinol, hyaluronic acid, and hyaluronic acid were purchased from Shanghai MacLean Biotechnology Co., Ltd.

[0074] Example 1

[0075] This embodiment provides an anti-aging fusogenic vesicle, the preparation method of which is as follows:

[0076] (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.

[0077] (2) 150 μg egg yolk lecithin, 25 μg cholesterol, 10 μg distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 4 μg retinoic acid were mixed and dissolved in 100 mL of ethanol, and then the organic solvent was evaporated at 40°C to form a film. 80 μg arbutin and 200 μg panthenol were mixed and dissolved in 1 mL of water, and then added to the prepared film. The liposomes were then sonicated at 200 W for 3 s on, 2 s off, and 20 times to obtain liposomes.

[0078] (3) Extracellular vesicles and liposomes were resuspended in 1 mL of PBS buffer, and then 2 × 10 8 umbilical cord mesenchymal stem cell-derived extracellular vesicles and 5×10 8The liposomes were mixed, sonicated at a power of 200 W for 30 seconds, sonicated three times, and then incubated at 35°C for 30 minutes; then frozen in liquid nitrogen for 5 minutes, thawed at 30°C for 15 minutes, and frozen and thawed four times; finally, extruded through a 600 nm polycarbonate membrane 40 times to obtain the fusion vesicles.

[0079] Example 2

[0080] This embodiment provides an anti-aging fusogenic vesicle, the preparation method of which is as follows:

[0081] (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.

[0082] (2) 140 μg of soybean lecithin, 20 μg of cholesterol, 5 μg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 2 μg of retinoic acid were mixed and dissolved in 100 mL of acetonitrile, and then the organic solvent was evaporated at 37°C to form a thin film. 50 μg of arbutin and 100 μg of panthenol were mixed and dissolved in 1 mL of water, and then the prepared film was added and sonicated at 100 W power for 5 seconds on, 3 seconds off, and 15 times to obtain liposomes.

[0083] (3) Extracellular vesicles and liposomes were resuspended in 1 mL of PBS buffer, and then 2 × 10 8 umbilical cord mesenchymal stem cell-derived extracellular vesicles and 1×10 8 The liposomes were mixed, sonicated at a power of 200 W for 20 seconds, sonicated 5 times, and then incubated at 30° C. for 40 minutes; then frozen in liquid nitrogen for 4 minutes, thawed at 25° C. for 20 minutes, and frozen and thawed 5 times; finally, extruded through a 200 nm polycarbonate membrane 20 times to obtain the fusion vesicles.

[0084] Example 3

[0085] This embodiment provides an anti-aging fusogenic vesicle, the preparation method of which is as follows:

[0086] (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.

[0087] (2) 160 μg of hydrogenated soybean lecithin, 30 μg of cholesterol, 15 μg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 6 μg of retinoic acid were mixed and dissolved in 100 mL of chloroform, and then the organic solvent was evaporated at 42°C to form a thin film. 100 μg of arbutin and 300 μg of panthenol were mixed and dissolved in 1 mL of water, and then the prepared film was added and sonicated at 300 W power for 2 s on, 1 s off, and 25 times to obtain liposomes.

[0088] (3) Extracellular vesicles and liposomes were resuspended in 1 mL of PBS buffer, and then 2 × 10 8 umbilical cord mesenchymal stem cell-derived extracellular vesicles and 1×10 9 The liposomes were mixed, sonicated at a power of 200 W for 40 seconds, sonicated twice, and then incubated at 40° C. for 20 minutes; then frozen in liquid nitrogen for 7 minutes, thawed at 35° C. for 10 minutes, and frozen and thawed three times; finally, extruded through an 800 nm polycarbonate membrane 50 times to obtain the fusion vesicles.

[0089] Example 4

[0090] This embodiment provides an anti-aging fusion vesicle, which differs from Example 1 only in that: in step (2), "4 μg retinoic acid", "80 μg arbutin" and "200 μg panthenol" are replaced by "4 μg resveratrol", "80 μg niacinamide" and "200 μg ectoine", respectively, and the remaining steps and raw materials remain unchanged.

[0091] Example 5

[0092] This embodiment provides an anti-aging fusion vesicle, which differs from Example 1 only in that: in step (2), "4 μg retinoic acid", "80 μg arbutin" and "200 μg panthenol" are replaced by "4 μg retinol", "80 μg hyaluronic acid" and "200 μg hyaluronic acid", respectively, and the remaining steps and raw materials remain unchanged.

[0093] Example 6

[0094] This embodiment provides an anti-aging fusion vesicle, which differs from Example 1 only in that: in step (2), "4 μg retinoic acid", "80 μg arbutin", and "200 μg panthenol" are replaced by "10 μg retinoic acid", "194 μg arbutin", and "80 μg panthenol", respectively, and the remaining steps and raw materials remain unchanged.

[0095] Example 7

[0096] This embodiment provides an anti-aging fusion vesicle, which differs from embodiment 1 only in that: in step (3), "2×10 8 umbilical cord mesenchymal stem cell-derived extracellular vesicles and 5×10 8 liposomes" is replaced by "0.5×10 8 Umbilical cord mesenchymal stem cell-derived extracellular vesicles and 11×10 8 The rest of the steps and raw materials remained unchanged.

[0097] Example 8

[0098] This embodiment provides an anti-aging fusion vesicle, which differs from embodiment 1 only in that: in step (2), 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.

[0099] Example 9

[0100] This embodiment provides an anti-aging fusion vesicle, which differs from embodiment 1 only in that: in step (2), no panthenol is added, and its reduced amount is proportionally distributed to retinoic acid and arbutin, and the remaining steps and raw materials remain unchanged.

[0101] Comparative Example 1

[0102] This comparative example provides an anti-aging fusion vesicle, which differs from Example 1 only in that: in step (2), 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.

[0103] Comparative Example 2

[0104] This comparative example provides an extracellular vesicle complex, and the preparation method thereof is as follows:

[0105] (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.

[0106] (2) The extracellular vesicle pellet was resuspended in 1 mL of PBS buffer and 2 × 10 8 The extracellular vesicles derived from umbilical cord mesenchymal stem cells were mixed with 4 μg retinoic acid, 80 μg arbutin, and 200 μg ubiquinol, 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.

[0107] Comparative Example 3

[0108] This comparative example provides a liposome, and its preparation method is as follows:

[0109] 150 μg of egg yolk lecithin, 25 μg of cholesterol, 10 μg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 4 μg of retinoic acid were dissolved in 100 mL of ethanol, and the organic solvent was evaporated at 40°C to form a thin film. 80 μg of arbutin and 200 μg of panthenol were dissolved in 1 mL of water, and then added to the prepared film. The liposomes were sonicated at 200 W for 3 seconds on, 2 seconds off, and 20 cycles of sonication to obtain liposomes.

[0110] Test Case

[0111] 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 4 100 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 vesicles provided in Examples 1-9 or Comparative Examples 1-2 or the liposomes provided in Comparative Example 3 were used as the experimental group, and DMEM complete medium without any additional substances was used as the control group 1, and 4 μg retinoic acid + 80 μg arbutin + 200 μg panthenol were added to each well as the control group 2. The cells were incubated in a 37°C cell culture incubator for 48 hours. The supernatant was then removed, and 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).

[0112] The test results, shown in Table 1, show that mesenchymal stem cell-derived extracellular vesicles (EVs) carry a rich array of proteins, lipids, and microRNAs. These VEs, when fused with liposomes, effectively inhibit MMP-1 expression, delaying the progression of photoaging and demonstrating excellent anti-aging effects. Retinoic acid, arbutin, and panthenol exhibit synergistic effects, achieving optimal anti-aging effects when combined together. Compared to single vesicle or liposome encapsulation, these VEssicles exhibit significant anti-aging benefits.

[0113] Table 1

[0114] Test samples MMP-1 content (ng / mL) Example 1 726 Example 2 955 Example 3 989 Example 4 1497 Example 5 1508 Example 6 1206 Example 7 1297 Example 8 1886 Example 9 1906 Comparative Example 1 1997 Comparative Example 2 1396 Comparative Example 3 1516 Control group 1 3987 Control group 2 3801

[0115] The present invention uses the above-described embodiments to illustrate the anti-aging fusogenic vesicles, their preparation methods, and applications. However, the present invention is not limited to these embodiments, nor does it necessarily rely on these 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.

[0116] 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.

[0117] 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. An anti-aging fusion vesicle, characterized in that: The fusion vesicle comprises a fusion product of an extracellular vesicle derived from a mesenchymal stem cell and a liposome; the liposome comprises a carrier formed by assembling phospholipids, cholesterol and an amphiphilic polymer, as well as a fat-soluble active substance loaded on the hydrophobic layer of the carrier and a water-soluble active substance loaded on the hydrophilic inner cavity of the carrier; The fat-soluble active ingredient includes any one of retinoic acid, resveratrol or retinol, or a combination of at least two of them; the water-soluble active ingredient includes any one of arbutin, panthenol, niacinamide, ectoine, phosphocyanine, hyaluronic acid or vitamin C, or a combination of at least two of them.

2. The fusion vesicle according to claim 1, characterized in that The ratio of the number of extracellular vesicles to liposomes is (1-5):(1-10); Preferably, the mass ratio of phospholipid, cholesterol and amphiphilic polymer in the carrier is (140-160):(20-30):(5-15); Preferably, the mass ratio of the carrier to the fat-soluble active ingredient is 1:(0.01-0.1); Preferably, the mass ratio of the carrier to the water-soluble active ingredient is 1:(0.1-5).

3. The fusion vesicle according to claim 1 or 2, characterized in that The amphiphilic polymer includes any one or a combination of at least two of distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol or dilauroylphosphatidylethanolamine-polyethylene glycol; Preferably, the number average molecular weight of the polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol or dilauroylphosphatidylethanolamine-polyethylene glycol is independently 1000-6000.

4. The fusion vesicle 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 fusion vesicle according to any one of claims 1 to 4, characterized in that The fat-soluble active ingredient is retinoic acid, and the water-soluble active ingredients are arbutin and panthenol; Preferably, the mass ratio of retinoic acid, arbutin and panthenol is (0.02-0.06):(0.5-1):(1-3).

6. The fusion vesicle according to any one of claims 1 to 5, characterized in that The phospholipids include any one of egg yolk lecithin, soybean lecithin or hydrogenated soybean lecithin, or a combination of at least two of them.

7. The method for preparing fusogenic vesicles according to any one of claims 1 to 6, characterized in that: The method comprises: (1) Cultivating mesenchymal stem cells, collecting cell culture fluid, and extracting extracellular vesicles; Phospholipids, cholesterol, amphiphilic polymers, fat-soluble active substances and organic solvents are mixed and dissolved, and the organic solvent is evaporated to remove the organic solvent to form a film; an aqueous solution of the water-soluble active substance is added, and ultrasonication is performed to prepare liposomes; (2) The extracellular vesicles and liposomes are mixed, sonicated, incubated, freeze-thawed, and extruded through a polycarbonate membrane to obtain the fusion vesicles.

8. The method according to claim 7, characterized in that In step (1), the method for extracting extracellular vesicles includes ultracentrifugation and / or membrane filtration; Preferably, 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 140000-160000 g for 80-100 min; Preferably, the organic solvent comprises any one or a combination of at least two of ethanol, acetonitrile, chloroform or methanol; Preferably, the concentration of the water-soluble active substance in the aqueous solution of the water-soluble active substance is 100-1000 mg / L; Preferably, in step (1), the ultrasonic power is 100-300W, and the ultrasonic time is 2-5s on and 1-3s off; Preferably, in step (1), the number of ultrasounds is 15-25 times.

9. The method according to claim 7 or 8, characterized in that In step (2), the ultrasonic time is 20-40 seconds, and the number of ultrasonic times is 2-5 times; Preferably, the incubation temperature is 30-40°C and the incubation time is 20-40 minutes; Preferably, the freeze-thaw cycle process is specifically to first freeze with liquid nitrogen and then thaw; Preferably, the freezing time is 4-7 minutes; Preferably, the thawing temperature is 25-35°C and the thawing time is 10-20 minutes; Preferably, the number of freeze-thaw cycles is 3-5 times; Preferably, the size of the polycarbonate film is 200-800 nm; Preferably, the polycarbonate film is extruded 20-50 times.

10. Use of the fusogenic vesicle according to any one of claims 1 to 6 in cosmetics, characterized in that: The cosmetics include any one of lotion, emulsion, cream, essence, spray, mask or sunscreen.