Preparation method and application of artificial synthetic exosome with anti-aging effect

Abstract

The application relates to a preparation method and application of artificially synthesized exosomes with anti-aging effects, and belongs to the fields of cosmetics and biotechnology. The artificially synthesized exosomes are prepared from components of hydrogenated lecithin, plant sphingosine, cholesterol, ceramide, squalane, sodium stearoyl glutamate, a surface modification structure, a polyhydric alcohol, active ingredients and water; the preparation method comprises the preparation of an alcohol phase, the preparation of an aqueous phase, the preparation of a mixed solution and high-pressure homogenization treatment. The artificially synthesized exosomes prepared by the preparation method have good stability and high anti-aging activity.

Description

Technical Field

[0001] The present invention relates to the fields of cosmetics and biotechnology, and in particular to a preparation method and application of artificially synthesized exosomes with anti-aging efficacy. Background Art

[0002] Exosomes are nanoscale extracellular vesicles widely present in organisms, typically ranging in diameter from 30 to 150 nm. They are secreted by a variety of cell types, including human, animal, and plant cells. Natural exosomes play an important role in intercellular communication, immune regulation, tissue repair, and disease development by carrying bioactive molecules such as proteins, lipids, mRNA, and miRNA.

[0003] Recent studies have demonstrated that human exosomes possess significant skincare benefits, including anti-aging, antioxidant benefits, skin repair, and barrier function enhancement, thus demonstrating broad application prospects in cosmetics, biopharmaceuticals, and other fields. However, obtaining human exosomes relies on extraction from stem cells or body fluids (such as blood and urine), which presents challenges such as low yield, complex isolation and purification processes, and difficulties in storage and transportation. Furthermore, ethical concerns have limited their large-scale application.

[0004] In comparison, although plant-derived exosomes avoid ethical issues and are easy to produce on a large scale, they are generally weaker than human-derived exosomes in terms of biological activity, targeting and functionality, and are difficult to meet the performance requirements of high-end skin care products or medical products.

[0005] To overcome these bottlenecks, research has attempted to develop synthetic exosomes that mimic the structure and function of natural exosomes. Currently, two main approaches exist: one involves chemically or physically modifying stem cells to induce them to secrete exosomes with specific functions; the other involves encapsulating active ingredients in human cell membranes or other biomimetic materials to form biomimetic vesicles with specific delivery capabilities. While these approaches achieve a degree of mimicking exosome function, they still face challenges such as high cost, complex preparation processes, and instability.

[0006] Therefore, there is an urgent need for a new method for preparing artificially synthesized exosomes that is efficient, low-cost, scalable, and stable. Summary of the Invention

[0007] To solve the above technical problems, the present application successfully constructs a kind of structure stable, function excellent, good biocompatibility exosome-like system based on scientific proportioning of multiple active ingredients and combining with specific high-pressure homogenization process. The process not only effectively simulates the core characteristics of natural exosomes, but also significantly improves its application potential in skin care and anti-aging, while avoiding the ethical problems and high cost barriers existing in traditional methods, and has good industrialization prospect.

[0008] In a first aspect, the present application provides a preparation method of artificial synthetic exosomes.

[0009] A preparation method of artificial synthetic exosomes, the artificial synthetic exosomes are prepared from components of hydrogenated lecithin, phytosphingosine, cholesterol, ceramide, squalane, sodium stearoyl glutamate, surface modification structure, polyol, active ingredient and water;

[0010] The preparation method comprises the following steps:

[0011] (1) Preparation of alcohol phase: hydrogenated lecithin, phytosphingosine, cholesterol, ceramide, squalane, sodium stearoyl glutamate, surface modification structure and polyol are mixed to obtain alcohol phase;

[0012] (2) Preparation of water phase: active ingredient and water are mixed to obtain water phase;

[0013] (3) Preparation of mixed solution: the water phase obtained in step (2) is added to the alcohol phase obtained in step (1), stirred, homogenized to obtain a mixed solution;

[0014] (4) Ultrasonic, high-pressure homogenization or extrusion through membrane treatment: the mixed solution obtained in step (3) is subjected to ultrasonic, high-pressure homogenization or extrusion through membrane treatment to obtain the artificial synthetic exosomes;

[0015] The surface modification structure comprises a targeting peptide; the targeting peptide comprises a cyclo-tetrapeptide-24 aminocyclohexane formate;

[0016] The weight ratio of hydrogenated lecithin to phytosphingosine is (3-7):(0.03-0.1);

[0017] The weight ratio of hydrogenated lecithin to cholesterol is (3-7):(0.3-1);

[0018] The weight ratio of hydrogenated lecithin to ceramide is (3-7):(0.005-0.04);

[0019] The weight ratio of hydrogenated lecithin to squalane is (3-7):(0.05-0.2);

[0020] The weight ratio of the hydrogenated lecithin to sodium stearoyl glutamate is (3-7): (0.2-0.8);

[0021] The weight ratio of the hydrogenated lecithin to the polyol is (3-7): (7-30);

[0022] The weight ratio of the hydrogenated lecithin to the surface modification structure is (3-7): (0.001-0.05);

[0023] The weight ratio of the polyol to the water is (7-30): (19.2-85.1);

[0024] The weight ratio of the active ingredient to water is (0.61-51.52): (19.2-85.1).

[0025] In some embodiments, the weight ratio of hydrogenated lecithin to phytosphingosine is 7:0.1 to 4:0.03. In some embodiments, the weight ratio of hydrogenated lecithin to phytosphingosine is 7:0.1, 3:0.03, or 4:0.03.

[0026] In some embodiments, the weight ratio of hydrogenated lecithin to cholesterol is 3:0.6-4:0.3. In some embodiments, the weight ratio of hydrogenated lecithin to cholesterol is 3:0.6, 5:0.8, 7:1, 4:0.4 or 4:0.3.

[0027] In some embodiments, the weight ratio of hydrogenated lecithin to ceramide is 3:0.02 to 3:0.005. In some embodiments, the weight ratio of hydrogenated lecithin to ceramide is 5:0.03, 3:0.02, 7:0.04, 4:0.01, or 3:0.005.

[0028] In some embodiments, the weight ratio of hydrogenated lecithin to squalane is 7:0.2 to 3:0.05. In some embodiments, the weight ratio of hydrogenated lecithin to squalane is 7:0.2, 5:0.1, or 3:0.05.

[0029] In some embodiments, the weight ratio of hydrogenated lecithin to sodium stearoyl glutamate is 3:0.6 to 5:0.2. In some embodiments, the weight ratio of hydrogenated lecithin to sodium stearoyl glutamate is 3:0.6, 7:0.8, 4:0.4, or 5:0.2.

[0030] In some embodiments, the weight ratio of the hydrogenated lecithin to the polyol is 3:30 to 5:7. In some embodiments, the weight ratio of the hydrogenated lecithin to the polyol is 3:30, 4:15, 3:10, 7:20, or 5:7.

[0031] In some embodiments, the weight ratio of the hydrogenated lecithin to the surface modification structure is 7:0.05 to 3:0.001. In some embodiments, the weight ratio of the hydrogenated lecithin to the surface modification structure is 7:0.05, 5:0.01, 4:0.002, or 3:0.001.

[0032] In some embodiments, the weight ratio of the polyol to the water is 10:85.0 to 20:19.2. In some embodiments, the weight ratio of the polyol to the water is 10:85.0885 to 20:19.2905. In some embodiments, the weight ratio of the polyol to the water is 10:85.0885, 7:55.594, 7:45.609, 15:79.327, 15:59.966, 30:39.9585, or 20:19.2905.

[0033] In some embodiments, the weight ratio of the active ingredient to water is 0.61:85.0 to 51.5:19.2. In some embodiments, the weight ratio of the active ingredient to water is 0.6105:85.0885 to 51.5195:19.2905. In some embodiments, the weight ratio of the active ingredient to water is 0.6105:85.0885, 0.751:79.327, 20.212:59.966, 25.7555:39.9585, 31.216:55.594, 41.201:45.609, or 51.5195:19.2905.

[0034] In some embodiments, the active ingredient comprises at least one of arginine / lysine polypeptide solution, PDRN, miRNA, mixed amino acids, and nicotinamide adenine dinucleotide.

[0035] In some embodiments, the mixed amino acids include at least one of alanine, histidine hydrochloride, arginine, serine, proline, glutamic acid, threonine, valine, leucine, isoleucine, glycine, phenylalanine, taurine, and allantoin. In some embodiments, the mixed amino acids include alanine, histidine hydrochloride, arginine, serine, proline, glutamic acid, threonine, valine, leucine, isoleucine, glycine, phenylalanine, taurine, and allantoin. In some embodiments, the mixed amino acids include alanine, histidine hydrochloride, arginine, serine, proline, glutamic acid, threonine, valine, leucine, isoleucine, glycine, phenylalanine, taurine, and allantoin, and the components are mixed in equal mass ratios.

[0036] In some embodiments, the polyol is selected from at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol.

[0037] In some embodiments, the content of hydrogenated lecithin is 3 wt%-7 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of hydrogenated lecithin is 3 wt%, 4 wt%, 5 wt%, 6 wt% or 7 wt% based on the total weight of the artificially synthesized exosomes.

[0038] In some embodiments, the content of phytosphingosine is 0.03 wt%-0.1 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of phytosphingosine is 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt% or 0.1 wt% based on the total weight of the artificially synthesized exosomes.

[0039] In some embodiments, the cholesterol content is 0.3 wt%-1 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the cholesterol content is 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt% or 1 wt% based on the total weight of the artificially synthesized exosomes.

[0040] In some embodiments, the ceramide content is 0.005 wt%-0.04 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the ceramide content is 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.03 wt% or 0.04 wt% based on the total weight of the artificially synthesized exosomes.

[0041] In some embodiments, the content of squalane is 0.05 wt%-0.2 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of squalane is 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.15 wt% or 0.2 wt% based on the total weight of the artificially synthesized exosomes.

[0042] In some embodiments, the content of sodium stearoyl glutamate is 0.2 wt%-0.8 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of sodium stearoyl glutamate is 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt% or 0.8 wt% based on the total weight of the artificially synthesized exosomes.

[0043] In some embodiments, the content of the polyol is 7 wt % to 30 wt % based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of the polyol is 7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %, 13 wt %, 14 wt %, 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt % or 30 wt % based on the total weight of the artificially synthesized exosomes.

[0044] In some embodiments, the content of the surface modification structure is 0.001 wt%-0.05 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of the surface modification structure is 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.004 wt%, 0.005 wt%, 0.006 wt%, 0.007 wt%, 0.008 wt%, 0.009 wt%, 0.01 wt%, 0.015 wt%, 0.02 wt%, 0.025 wt%, 0.03 wt%, 0.035 wt%, 0.04 wt%, 0.045 wt%, or 0.05 wt% based on the total weight of the artificially synthesized exosomes.

[0045] In some embodiments, the active ingredient is present in an amount of 0.61 wt % to 51.52 wt % based on the total weight of the artificially synthesized exosomes. In some embodiments, the active ingredient is present in an amount of 0.61 wt % to 51.52 wt % based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of the active ingredient is 0.61 wt%, 0.65 wt%, 0.70 wt%, 0.75 wt%, 0.751wt%, 1 wt%, 1.5 wt%, 1.6 wt%, 0.6105wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 21 wt%, 21.2 wt%, 20.212wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 25.7555wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 31.216wt%, 33 wt%, 34 wt%, 35 wt%, 40 wt%, 41 wt%, 41.201 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 50 wt%, 51 wt%, 51.5195wt% or 51.52wt%.

[0046] In some embodiments, the water content is 19.2 wt %-85.1 wt % based on the total weight of the artificially synthesized exosomes. In some embodiments, the water content, calculated based on the total weight of the artificial exosomes, is 19.2 wt%, 19.2905 wt%, 19.5 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 39 wt%, 39.9585 wt%, 40 wt%, 45 wt%, 45.5 wt%, 45.609 wt%, 50 wt%, 54 wt%, 55.594 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 58.5 wt%, 59.966 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 79 wt%, 79.5 wt%, 79.327 wt%, 80 wt%, 81 wt%, 82 wt%, 83 wt%, 84 wt%, 85.0885 wt% or 85.1 wt%.

[0047] In some embodiments, the content of the arginine / lysine polypeptide solution is 0-50 wt %, calculated based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of the arginine / lysine polypeptide solution is 0 wt %, 5 wt %, 10 wt %, 15 wt %, 20 wt %, 25 wt %, 30 wt %, 35 wt %, 40 wt %, 45 wt %, or 50 wt %, calculated based on the total weight of the artificially synthesized exosomes.

[0048] In some embodiments, the content of PDRN is 0-1 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of PDRN is 0.1 wt%-1 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of PDRN is 0.1 wt%, 0.5 wt% or 1 wt% based on the total weight of the artificially synthesized exosomes.

[0049] In some embodiments, the miRNA is present in an amount of 0 wt% to 0.002 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the miRNA is present in an amount of 0.0005 wt% to 0.002 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the miRNA is present in an amount of 0.0005 wt%, 0.001 wt%, 0.0015 wt%, or 0.002 wt% based on the total weight of the artificially synthesized exosomes.

[0050] In some embodiments, the content of the mixed amino acids is 0 wt% to 0.018 wt% based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of the mixed amino acids is 0 wt%, 0.005 wt%, 0.01 wt%, 0.015 wt%, 0.016 wt%, 0.017 wt% or 0.018 wt% based on the total weight of the artificially synthesized exosomes.

[0051] In some embodiments, the content of nicotinamide adenine dinucleotide is 0.1 wt % to 0.5 wt % based on the total weight of the artificially synthesized exosomes. In some embodiments, the content of nicotinamide adenine dinucleotide is 0.1 wt %, 0.125 wt %, 0.15 wt %, 0.2 wt %, 0.25 wt %, 0.3 wt %, 0.35 wt %, 0.4 wt %, 0.45 wt % or 0.5 wt % based on the total weight of the artificially synthesized exosomes.

[0052] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 3wt%-7wt%, the content of the phytosphingosine is 0.03wt%-0.1wt%, the content of the cholesterol is 0.3wt%-1wt%, the content of the ceramide is 0.005wt%-0.04wt%, the content of the squalane is 0.05wt%-0.2wt%, the content of the sodium stearoyl glutamate is 0.2wt%-0.8wt%, the content of the polyol is 7wt%-30wt%, the content of the surface modification structure is 0.001wt%-0.05wt%, the content of the active ingredient is 0.61wt%-51.52wt%, and the content of water is 19.2wt%-85.1wt%.

[0053] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 3wt%-7wt%, the content of the phytosphingosine is 0.03wt%-0.1wt%, the content of the cholesterol is 0.3wt%-1wt%, the content of the ceramide is 0.005wt%-0.04wt%, the content of the squalane is 0.05wt%-0.2wt%, the content of the sodium stearoyl glutamate is 0.2wt%-0.8wt%, the content of the polyol is 7wt%-30wt%, the content of the surface modification structure is 0.001wt%-0.05wt%, the content of the active ingredient is 0.61wt%-51.52wt%, and the balance is water.

[0054] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 3wt%-7wt%, the content of the phytosphingosine is 0.03wt%-0.1wt%, the content of the cholesterol is 0.3wt%-1wt%, the content of the ceramide is 0.005wt%-0.04wt%, the content of the squalane is 0.05wt%-0.2wt%, the content of the sodium stearoyl glutamate is 0.2wt%-0.8wt%, the content of the polyol is 7wt%-30wt%, the content of the surface modification structure is 0.001wt%-0.05wt%, the content of the active ingredient is 0.6105wt%-51.5195wt%, and the balance is water.

[0055] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 4wt%, the content of the phytosphingosine is 0.03wt%, the content of the cholesterol is 0.3wt%, the content of the ceramide is 0.01wt%, the content of the squalane is 0.08wt%, the content of the sodium stearoyl glutamate is 0.4wt%, the content of the polyol is 15wt%, the content of the surface modification structure is 0.002wt%, the content of the active ingredient is 20.212wt%, and the balance is water.

[0056] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 3wt%, the content of the phytosphingosine is 0.03wt%, the content of the cholesterol is 0.6wt%, the content of the ceramide is 0.005wt%, the content of the squalane is 0.05wt%, the content of the sodium stearoyl glutamate is 0.6wt%, the content of the polyol is 30wt%, the content of the surface modification structure is 0.001wt%, the content of the active ingredient is 25.7555wt%, and the balance is water.

[0057] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 5wt%, the content of the phytosphingosine is 0.05wt%, the content of the cholesterol is 0.8wt%, the content of the ceramide is 0.03wt%, the content of the squalane is 0.1wt%, the content of the sodium stearoyl glutamate is 0.2wt%, the content of the polyol is 7wt%, the content of the surface modification structure is 0.01wt%, the content of the active ingredient is 31.216wt%, and the balance is water.

[0058] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 7wt%, the content of the phytosphingosine is 0.1wt%, the content of the cholesterol is 1wt%, the content of the ceramide is 0.04wt%, the content of the squalane is 0.2wt%, the content of the sodium stearoyl glutamate is 0.8wt%, the content of the polyol is 20wt%, the content of the surface modification structure is 0.05wt%, the content of the active ingredient is 51.5195wt%, and the balance is water.

[0059] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 4wt%, the content of the phytosphingosine is 0.03wt%, the content of the cholesterol is 0.4wt%, the content of the ceramide is 0.01wt%, the content of the squalane is 0.08wt%, the content of the sodium stearoyl glutamate is 0.4wt%, the content of the polyol is 15wt%, the content of the surface modification structure is 0.002wt%, the content of the active ingredient is 0.751wt%, and the balance is water.

[0060] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 3wt%, the content of the phytosphingosine is 0.03wt%, the content of the cholesterol is 0.6wt%, the content of the ceramide is 0.02wt%, the content of the squalane is 0.05wt%, the content of the sodium stearoyl glutamate is 0.6wt%, the content of the polyol is 10wt%, the content of the surface modification structure is 0.001wt%, the content of the active ingredient is 0.6105wt%, and the balance is water.

[0061] In some embodiments, based on the total weight of the artificially synthesized exosomes, the content of the hydrogenated lecithin is 5wt%, the content of the phytosphingosine is 0.05wt%, the content of the cholesterol is 0.8wt%, the content of the ceramide is 0.03wt%, the content of the squalane is 0.1wt%, the content of the sodium stearoyl glutamate is 0.2wt%, the content of the polyol is 7wt%, the content of the surface modification structure is 0.01wt%, the content of the active ingredient is 41.201wt%, and the balance is water.

[0062] In some embodiments, the pressure of the high-pressure homogenization in step (4) is 1500 bar to 2000 bar. In some embodiments, the pressure of the high-pressure homogenization in step (4) is 1500 bar, 1600 bar, 1700 bar, 1800 bar, 1900 bar or 2000 bar.

[0063] In some embodiments, the number of cycles of high pressure homogenization in step (4) is 4-6 times. In some embodiments, the number of cycles of high pressure homogenization in step (4) is 4 times, 5 times or 6 times.

[0064] In some embodiments, the temperature of the high-pressure homogenization in step (4) is 10° C. to 25° C. In some embodiments, the temperature of the high-pressure homogenization in step (4) is 10° C., 15° C., 20° C., or 25° C.

[0065] In a second aspect, the present invention provides an artificially synthesized exosome.

[0066] An artificially synthesized exosome, comprising the artificially synthesized exosome prepared by the preparation method of the first aspect.

[0067] In a third aspect, the present invention provides an artificially synthesized exosome prepared by the aforementioned preparation method or an application of the aforementioned artificially synthesized exosome.

[0068] A use of the artificially synthesized exosomes prepared by the preparation method of the first aspect or the artificially synthesized exosomes of the second aspect in the preparation of cosmetics.

[0069] In some embodiments, the cosmetic is used for anti-aging.

[0070] In a fourth aspect, the present invention provides a cosmetic.

[0071] A cosmetic comprising the artificially synthesized exosomes prepared by the preparation method of the first aspect or the artificially synthesized exosomes of the second aspect.

[0072] In some embodiments, the cosmetic is used for anti-aging.

[0073] Beneficial effects

[0074] Compared with the prior art, a certain embodiment of the present invention has at least one of the following beneficial effects:

[0075] (1) The artificially synthesized exosomes provided by the present invention have a good inhibitory effect on β-galactosidase, a high effect on increasing the elastin content, and a high effect on increasing the elastin content. In addition, the anti-aging effect does not change significantly during the stable placement process, and has excellent anti-aging effect, stability and safety.

[0076] (2) Compared with other targeting peptides, the cyclic tetrapeptide-24 aminocyclohexanecarboxylate provided by the present invention is more conducive to improving the β-galactosidase inhibition effect of the obtained artificially synthesized exosomes, increasing the elastin content and the effect of enhancing the elastin content, and has good anti-aging effects and unexpected technical effects.

[0077] (3) Compared with other phospholipids (such as lecithin or cephalin), the use of hydrogenated lecithin in the present invention is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0078] (4) Compared with other stabilizers (such as cetearyl alcohol polyether-2 and sodium lauryl aminopropionate), the use of sodium stearoyl glutamate in the present invention is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0079] (5) Compared with other stabilizers (such as caprylic / capric / stearic triglyceride and caprylic / capric triglyceride), the use of squalane in the present invention is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0080] (6) Compared with the method without phytosphingosine or the method with other sphingolipids, the method of the present invention using phytosphingosine is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has an unexpected technical effect.

[0081] Terminology

[0082] In the description of the present invention, “plurality” means two or more, unless otherwise clearly defined.

[0083] The term "room temperature" or "normal temperature" means ambient temperature, which refers to a temperature between about 10°C and about 35°C, about 10°C and about 30°C, or about 20°C and 30°C, or about 25°C.

[0084] The term "wt%" means percent by weight.

[0085] The term "rpm" means revolutions per minute.

[0086] The term "v / v" means a volume ratio.

[0087] The term "synthetic exosomes" refers to nanovesicles with a lipid bilayer membrane structure that are manufactured through non-biological synthetic pathways (such as self-assembly, template methods, microfluidics technology, cell membrane biomimetic, etc.). The resulting nanovesicles have the same or similar key physicochemical properties (such as size, morphology, membrane structure) and biological functions (such as efficient drug loading, cell targeting, and crossing biological barriers) as natural exosomes. The physicochemical properties of the resulting nanovesicles may be the same as or different from those of natural exosomes, aiming to selectively mimic their key properties to achieve similar application goals (such as targeted delivery, immune escape, biocompatibility, etc.).

[0088] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0089] In the following description, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number having a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% is expressly disclosed, where "+ / -" means plus or minus. DETAILED DESCRIPTION

[0090] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0091] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0092] 1. The sources of some of the reagents used in the embodiments or comparative examples of the present invention are as follows:

[0093] Arginine / lysine peptide solution: Trade name: EPITENSIVE™ EVO; Manufacturer: LipoTrue Science & Biotechnologies.

[0094] PDRN: INCI name: Sodium DNA; List of Used Chemical Raw Materials (2021 Edition) serial number: 00383; Trade name: PDRN-C; Manufacturer: Shanghai Huiwen Biotechnology Co., Ltd.

[0095] miRNA: Trade name: miRITA-129™; Manufacturer: Xi'an Alink Biotechnology Co., Ltd.

[0096] Mixed amino acids: alanine, histidine hydrochloride, arginine, serine, proline, glutamic acid, threonine, valine, leucine, isoleucine, glycine, phenylalanine, taurine and allantoin are mixed in equal mass ratios to obtain a mixed amino acid.

[0097] Nicotinamide adenine dinucleotide: CAS number is 53-84-9.

[0098] Cyclic tetrapeptide-24-aminocyclohexanecarboxylate: Shenzhen Weiqi Technology Co., Ltd.

[0099] Examples 1-4: Artificial synthesis of exosomes

[0100] Recipe: See Table 1.

[0101] Table 1: Artificially synthesized exosome formula

[0102]

[0103] Preparation method:

[0104] (1) Preparation of alcohol phase: hydrogenated lecithin, phytosphingosine, cholesterol, ceramide, squalane, sodium stearoyl glutamate, surface modification structure and polyol are mixed to obtain an alcohol phase;

[0105] (2) Preparation of aqueous phase: Mix the active ingredient and water to obtain an aqueous phase;

[0106] (3) Preparation of a mixed solution: adding the aqueous phase obtained in step (2) to the alcohol phase obtained in step (1), stirring, and homogenizing to obtain a mixed solution;

[0107] (4) High-pressure homogenization: The mixed solution obtained in step (3) was subjected to high-pressure homogenization (pressure 1500 bar, 5 cycles, temperature controlled at 25°C) to obtain the artificially synthesized exosomes.

[0108] Example 5-Example 6: Investigation of high-pressure homogenization pressure conditions

[0109] Example 5: The only difference from Example 1 is that the high-pressure homogenization pressure is adjusted to 1800 bar, and the other conditions are the same as Example 1.

[0110] Example 6: The only difference from Example 1 is that the high-pressure homogenization pressure is adjusted to 2000 bar, and the other conditions are the same as Example 1.

[0111] Example 7-Example 8: Investigation of the number of high-pressure homogenization cycles

[0112] Example 7: The only difference from Example 1 is that the number of high-pressure homogenization cycles is adjusted to 4 times, and the other conditions are the same as Example 1.

[0113] Example 8: The only difference from Example 1 is that the number of high-pressure homogenization cycles is adjusted to 6 times, and the other conditions are the same as Example 1.

[0114] Example 9-Example 11: Artificial synthesis of exosomes

[0115] Recipe: See Table 2.

[0116] Table 2: Examples 9-11 Artificially synthesized exosome formula

[0117]

[0118] Preparation method: The difference from the preparation method of Example 1 to Example 4 is that the temperature of the high-pressure homogenization in step (4) is adjusted to 10°C~20°C (Example 9 adopts 10°C, Example 10 adopts 15°C, and Example 11 adopts 20°C). In addition, if the weight portion of a certain component is 0, it is not added in the preparation method. The other conditions are the same as the preparation method of Example 1 to Example 4.

[0119] Comparative Example 1-Comparative Example 2: Investigation of Bionic Shell Composition

[0120] Comparative Example 1: The only difference from Example 7 is that hydrogenated lecithin is replaced by lecithin, and the other conditions are the same as Example 7.

[0121] Comparative Example 2: The only difference from Example 7 is that hydrogenated lecithin is replaced by cephalin, and the other conditions are the same as Example 7.

[0122] Comparative Example 3-Comparative Example 4: Investigation of Bionic Shell Composition

[0123] Comparative Example 3: The only difference from Example 7 is that sodium stearoyl glutamate is replaced with cetearyl alcohol polyether-2, and the other conditions are the same as Example 7.

[0124] Comparative Example 4: The only difference from Example 7 is that sodium stearoyl glutamate is replaced by sodium lauryl aminopropionate, and the other conditions are the same as Example 7.

[0125] Comparative Example 5-Comparative Example 6: Investigation of Bionic Shell Composition

[0126] Comparative Example 5: The only difference from Example 7 is that squalane is replaced by caprylic / capric / stearic triglyceride, and the other conditions are the same as Example 7.

[0127] Comparative Example 6: The only difference from Example 7 is that squalane is replaced by caprylic / capric triglyceride, and the other conditions are the same as Example 7.

[0128] Comparative Example 7-Comparative Example 8: Investigation of Bionic Shell Composition

[0129] Comparative Example 7: The only difference from Example 7 is that phytosphingosine is replaced by ceramide, that is, Comparative Example 7 does not contain phytosphingosine, and the other conditions are the same as Example 7.

[0130] Comparative Example 8: The only difference from Example 7 is that phytosphingosine is replaced by acetylphytosphingosine, and the other conditions are the same as Example 7.

[0131] Comparative Example 9-Comparative Example 10: Investigation of surface modification structure

[0132] Comparative Example 9: The only difference from Example 7 is that cyclotetrapeptide-24 aminocyclohexanecarboxylate is replaced by palmitoyl pentapeptide-3, and the other conditions are the same as Example 7.

[0133] Comparative Example 10: The only difference from Example 7 is that cyclotetrapeptide-24 aminocyclohexanecarboxylate is replaced by acetyl hexapeptide-8, and the other conditions are the same as Example 7.

[0134] Example 12: Preparation of aqueous essence

[0135] Recipe: See Table 3.

[0136] Table 3:

[0137]

[0138] Preparation method:

[0139] 1. Heat water to 85°C, add p-hydroxyacetophenone and 3 / 5 of the formula amount of 1,3-butanediol and stir until fully dissolved, then cool to below 60°C (e.g. 50°C) to obtain phase A;

[0140] 2. Premix the remaining amount of 1,3-butanediol and transparent xanthan gum, then add them to Phase A at a temperature below 60°C (e.g., 50°C) and stir until fully swollen, free of fish-eye particles, and uniform. Continue cooling to below 40°C (e.g., 35°C) to obtain Phase B.

[0141] 3. Add the artificially synthesized exosomes obtained in Example 1 and 1,2-pentanediol to phase B at a temperature below 40°C (e.g., 35°C), stir thoroughly, and then add water until the total content of each component is 100 wt%, to obtain the product of Example 12.

[0142] Test Example 1: Cytotoxicity Test

[0143] The cytotoxic effects of the synthetic exosomes obtained in each example on human skin cells were tested using human epidermal HaCaT cells and human dermal NHDFs cells. Stable cells from normal cultures were digested, counted, and plated in 96-well plates at 10,000 cells per well. After 24 hours, a monolayer was formed. Culture medium containing various concentrations of the test substance was added and cultured for another 48 hours. The cells were then treated with a CCK-8 kit and the absorbance (OD) at 450 nm was measured using a microplate reader. Negative controls were prepared using human epidermal HaCaT cells or human dermal NHDFs cells cultured in culture medium without the test substance for 48 hours. Relative cell viability was calculated using the following formula.

[0144] Relative cell activity = (OD value of sample group - background OD value) / (OD value of negative control - background OD value) ☓ 100%

[0145] The results are shown in Table 4.

[0146] Table 4: Relative cell viability

[0147]

[0148] Conclusion: The relative activity of the sample groups was higher than that of the negative control, indicating that the artificially synthesized exosomes provided by the present invention have good safety.

[0149] Test Example 2: β-galactosidase inhibition test

[0150] 1. Test Purpose

[0151] β-Galactosidase (SA-β-Gal) is a commonly used marker of cellular senescence, exhibiting increased activity during cellular senescence. By measuring the activity level of SA-β-Gal in cells, the degree of cellular senescence can be assessed. This test, based on a heat-damaged human fibroblast model, uses SA-β-Gal staining to detect changes in SA-β-Gal expression in human fibroblasts to assess whether the test sample can withstand heat damage.

[0152] 2. Test items

[0153] β-Galactosidase detection in heat-damaged human fibroblasts

[0154] 3. Test materials

[0155] 3.1 Test System

[0156] Human fibroblasts.

[0157] 3.2 Main Reagents

[0158] Low-glucose DMEM culture medium (Pneusai), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), β-galactosidase staining kit (Biyuntian), Geldanamycin (HSP inhibitor Biyuntian).

[0159] 3.3 Main equipment

[0160] CO2 incubator (Thermo, 160i), biological safety cabinet (Sujing Antai, BSC-1604ⅡA2), inverted fluorescence microscope (Keyence BZ-X810), and microplate reader (Tecan, Spark).

[0161] 3.4 Sample Information

[0162] The artificially synthesized exosomes obtained in Examples 1 to 11 and Comparative Examples 1 to 10.

[0163] 4. Test Method

[0164] 4.1 Grouping: The specific settings of the experimental groups are shown in Table 5.

[0165] Table 5: Experimental groups

[0166]

[0167] 4.2 Operation steps:

[0168] 1) Cell seeding: According to the appropriate seeding density (8x10 4 Cells were seeded into 24-well plates (100 μl / well) and incubated overnight in an incubator (37°C, 5% CO2).

[0169] 2) Administration: Add 1 mL of culture medium containing 0.1 wt% sample according to Table 5.

[0170] 3) Staining: Stain according to the instructions of the β-galactosidase staining kit

[0171] 4) Result analysis: SA-β-Gal positive cells were quantitatively analyzed using Image Pro Plus software.

[0172] 5. Test results

[0173] The positive cell rates are shown in Table 6.

[0174] Table 6: Summary of SA-β-Gal positive cell rate results

[0175]

[0176] in conclusion:

[0177] (1) Compared with other targeting peptides, the cyclic tetrapeptide-24 aminocyclohexanecarboxylate provided by the present invention is more conducive to improving the β-galactosidase inhibitory effect of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0178] Test Example 3: Stability Investigation

[0179] The artificially synthesized exosomes obtained in Comparative Examples 1 to 8 and Examples 1 to 11 were sealed and placed at 45°C ± 2°C / 75% RH for 1 month. The obtained accelerated samples were tested for β-galactosidase inhibition according to Test Example 1. The results are shown in Table 7.

[0180] Table 7: Summary of SA-B-Gal positive cell rate results of accelerated samples

[0181]

[0182] The data for day 0 are shown in Table 6.

[0183] in conclusion:

[0184] (1) Compared with other phospholipids (such as lecithin or cephalin), the use of hydrogenated lecithin in the present invention is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0185] (2) Compared with other stabilizers (such as cetearyl alcohol polyether-2 and sodium lauryl aminopropionate), the use of sodium stearoyl glutamate in the present invention is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0186] (3) Compared with other stabilizers (such as caprylic / capric / stearic triglyceride and caprylic / capric triglyceride), the use of squalane in the present invention is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0187] (4) Compared with the method without phytosphingosine or the method with other sphingolipids, the method of the present invention using phytosphingosine is more conducive to improving the stability of the obtained artificially synthesized exosomes, and has an unexpected technical effect.

[0188] Test Example 4: Anti-wrinkle and anti-aging efficacy test: Elastin content

[0189] 1. Preparation of working fluid:

[0190] (1) The blank control group was a blank control, in which only an equal amount of complete culture medium was added;

[0191] (2) TGF-β1 solution: 10 μg / mL TGF-β1 (purchased from GenScript, model Z03411) was diluted to 100 ng / mL using complete culture medium to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0192] (3) Preparation of sample solution: The artificially synthesized exosomes of the example or comparative example were diluted with complete culture medium to a sample solution with a concentration of 1 wt%.

[0193] (4) The complete culture medium is DMEM (purchased from Gibco, model number 10567014), which includes DMEM, low glucose, GlutaMAXTM supplement, and pyruvate.

[0194] 2. The specific steps are as follows:

[0195] 1) Cell plating: The cell density is 5×10 4 Human dermal fibroblasts (HDFs) were seeded into 96-well plates at a density of 100 μl / mL. The inoculated cell culture plates were placed in an incubator and cultured for 24 h (5% CO2, 37°C).

[0196] 2) Drug administration: The drug administration groups are divided into the following ways:

[0197] Blank control: After culturing the cells for 24 h, remove the supernatant, add 200 μL of complete culture medium, mix well, and place in an incubator for 24 h±1 h. Collect the cell culture supernatant for ELISA detection.

[0198] Test sample group: After 24 h of cell culture, the supernatant was removed, 200 μL of 1% test sample was added, mixed, and placed in an incubator for 24 h ± 1 h. The cell culture supernatant was collected for ELISA detection.

[0199] Positive control group: After culturing cells for 24 h, the supernatant was removed and 200 μL of 100 ng / ml TGF-β1 solution was added and mixed. The cells were placed in an incubator for 24 h±1 h and the cell culture supernatant was collected for ELISA detection.

[0200] 3) ELISA assay: The cell culture supernatant collected in step 2) was subjected to ELISA assay, and the elastin expression upregulation rate was calculated according to the following formula:

[0201] Elastin expression upregulation rate (%) = (TC) / C☓100%

[0202] Wherein, T represents the elastin content of the sample group, and C represents the elastin content of the blank control group.

[0203] The results are shown in Table 8.

[0204] Table 8: Elastin content test results

[0205]

[0206] in conclusion:

[0207] (1) As shown in Table 8, the artificially synthesized exosomes provided by the present invention have a good effect of increasing the elastin content and have good anti-aging effects.

[0208] (2) Compared with other targeting polypeptides, the targeting polypeptide (cyclotetrapeptide-24-aminocyclohexanecarboxylate) provided by the present invention is more conducive to improving the effect of the obtained artificially synthesized exosomes on increasing the elastin content, and has unexpected technical effects.

[0209] Test Example 5: Anti-wrinkle and firming effects

[0210] The products obtained in Example 12 were respectively used to evaluate their anti-wrinkle and firming efficacy. The specific procedures were as follows:

[0211] Subject information

[0212] A total of 33 subjects were included, with 31 valid subjects, including 0 males and 31 females, aged 24-42 years, with an average age of 34.52±4.31 years.

[0213] All enrolled subjects met the following inclusion and exclusion criteria:

[0214] 1.1 Subject inclusion criteria

[0215] 1.1.1 Healthy women or men aged 18-60 years;

[0216] 1.1.2 Facial skin problems such as redness, sensitivity, and wrinkles;

[0217] 1.1.3 People with sensitive skin (as measured by the Sensitive Skin Questionnaire and lactic acid stinging score ≥ 3 points);

[0218] 1.1.4 The applicant has not participated in any other clinical studies in the past two months, and the test area has not undergone any skin treatment, cosmetic surgery, or other tests that may affect the test results;

[0219] 1.1.5 Those who can understand the trial process, voluntarily participate in the trial and sign the written informed consent.

[0220] 1.2 Subject Exclusion Criteria

[0221] 1.2.1 Pregnant or breastfeeding women or those planning to get pregnant in the near future;

[0222] 1.2.2 Those with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, severe acne, or other chronic systemic diseases;

[0223] 1.2.3 Those who have taken oral or topical corticosteroids or other anti-inflammatory drugs in the past month;

[0224] 1.2.4 Those with severe allergies;

[0225] 1.2.5 Those who have used tretinoin preparations or undergone chemical peels, laser treatments, pulsed light treatments, or other cosmetic treatments on the test area within the past three months;

[0226] 1.2.6 Those who are inevitably exposed to sunlight for a long time;

[0227] 1.2.7 Participants in other clinical trials within the past two months;

[0228] 1.2.8 Other patients deemed unsuitable for trial participation by clinical assessment.

[0229] Test equipment

[0230] Facial image capture system VISIA7 (Canfield, USA)

[0231] Cutometer dual MPA580 skin elasticity test probe (Courage+Khazaka, Germany)

[0232] 3. Test Environment

[0233] Test environment: temperature 21±1℃; humidity 50±10%.

[0234] 4. Test Methods

[0235] 4.1 Sample Usage Method and Frequency

[0236] After cleansing in the morning and evening, take an appropriate amount of product on the palm of your hand, apply evenly on the face, and gently massage until absorbed.

[0237] 4.2 Test Items

[0238] 4.2.1 Instrument testing

[0239] Table 9: Description of instrument test sites, time points and parameters

[0240]

[0241] 4.3 Test steps

[0242] 1) Subjects were recruited as required and signed written informed consent. Prior to enrollment, subjects were asked a series of questions regarding their medical history and health status based on inclusion and exclusion criteria. A lactic acid sting test was then performed, and subjects meeting the criteria were screened for participation in this testing project.

[0243] 2) Participants should cleanse their faces with cleanser, rinse thoroughly with water, and then dry their faces with a non-dandruff absorbent tissue. After cleansing, they should enter a constant temperature and humidity chamber and remain in the chamber for 30 minutes. During this stabilization period, they should refrain from eating or drinking, expose their faces, remain relaxed, and avoid touching the test area.

[0244] 3) After the break, complete the test of instruments such as Cutometer dual MPA580.

[0245] 4) Return visits will be conducted after 7 and 28 days of use of the sample. Participants will complete a self-assessment based on their own circumstances and truthfully fill out questionnaires related to the sample.

[0246] 4.4 Data Analysis

[0247] Statistical analysis software was used for data analysis. Measurement data were expressed as mean ± standard deviation and tested for normal distribution. Before-after comparisons were performed using a paired t-test; otherwise, a two-sample rank sum test was used. Random data were compared before-after using a two-sample rank sum test. All statistical analyses were two-tailed, with a significance level of α = 0.05.

[0248] 4.5 Basis for test conclusions

[0249] 4.5.1 Claims of anti-wrinkle efficacy: Using self-control before and after, observe the average wrinkle area per person. If any indicator test value at any visit time point after using the sample is better than the baseline value before use, and the result is significantly different ( P <0.05), the test sample is considered to have anti-wrinkle effect.

[0250] 4.5.2 Claims of firming efficacy: Using self-control before and after, observe the skin firmness F4 value. If any index test value at any visit time point after using the sample is better than the baseline value before use, and the result is significantly different ( P <0.05), the test sample is considered to have a firming effect.

[0251] 5 Results

[0252] The results are shown in Tables 10 and 11.

[0253] Table 10: Descriptive statistics of skin firmness F4 value test

[0254]

[0255] Table 11: Descriptive statistics of average wrinkle area per person (unit: mm) 2 )

[0256]

[0257] Conclusion: From the results in Tables 10 and 11, it can be seen that the cosmetics containing the artificially synthesized exosomes provided by the present invention have good anti-wrinkle and firming effects.

[0258] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. A method for preparing artificially synthesized exosomes, characterized in that: The artificially synthesized exosomes are prepared from components of hydrogenated lecithin, phytosphingosine, cholesterol, ceramide, squalane, sodium stearoyl glutamate, a surface modification structure, polyols, an active ingredient, and water; The preparation method comprises the following steps: (1) Preparation of alcohol phase: hydrogenated lecithin, phytosphingosine, cholesterol, ceramide, squalane, sodium stearoyl glutamate, surface modification structure and polyol are mixed to obtain an alcohol phase; (2) Preparation of aqueous phase: Mix the active ingredient and water to obtain an aqueous phase; (3) Preparation of a mixed solution: adding the aqueous phase obtained in step (2) to the alcohol phase obtained in step (1), stirring, and homogenizing to obtain a mixed solution; (4) Ultrasonication, high-pressure homogenization or membrane extrusion treatment: The mixed solution obtained in step (3) is subjected to ultrasonication, high-pressure homogenization or membrane extrusion treatment to obtain the artificially synthesized exosomes; Wherein, the surface modification structure includes a targeting peptide; the targeting peptide includes cyclotetrapeptide-24 aminocyclohexanecarboxylate; The weight ratio of the hydrogenated lecithin to phytosphingosine is (3-7): (0.03-0.1); The weight ratio of hydrogenated lecithin to cholesterol is (3-7): (0.3-1); The weight ratio of the hydrogenated lecithin to ceramide is (3-7): (0.005-0.04); The weight ratio of hydrogenated lecithin to squalane is (3-7): (0.05-0.2); The weight ratio of the hydrogenated lecithin to sodium stearoyl glutamate is (3-7): (0.2-0.8); The weight ratio of the hydrogenated lecithin to the polyol is (3-7): (7-30); The weight ratio of the hydrogenated lecithin to the surface modification structure is (3-7): (0.001-0.05); The weight ratio of the polyol to the water is (7-30): (19.2-85.1); and The weight ratio of the active ingredient to water is (0.61-51.52): (19.2-85.1).

2. The preparation method according to claim 1, wherein the active ingredient comprises at least one of arginine / lysine polypeptide solution, PDRN, miRNA, mixed amino acids, and nicotinamide adenine dinucleotide.

3. The preparation method according to any one of claims 1 to 2, wherein the polyol is selected from at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol.

4. The preparation method according to any one of claims 1-2, wherein, based on the total weight of the artificially synthesized exosomes, the content of hydrogenated lecithin is 3 wt%-7 wt%, the content of phytosphingosine is 0.03 wt%-0.1 wt%, the content of cholesterol is 0.3 wt%-1 wt%, the content of ceramide is 0.005 wt%-0.04 wt%, the content of squalane is 0.05 wt%-0.2 wt%, the content of sodium stearoyl glutamate is 0.2 wt%-0.8 wt%, the content of the polyol is 7 wt%-30 wt%, the content of the surface modification structure is 0.001 wt%-0.05 wt%, the content of the active ingredient is 0.61 wt%-51.52 wt%, and the content of water is 19.2 wt%-85.1 wt%.

5. The preparation method according to any one of claims 1-2, wherein the pressure of the high-pressure homogenization in step (4) is 1500-2000 bar; The number of cycles of high pressure homogenization in step (4) is 4-6 times; and The temperature of the high-pressure homogenization in step (4) is 10°C-25°C.

6. An artificially synthesized exosome, characterized in that: The invention relates to artificially synthesized exosomes prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the artificially synthesized exosomes prepared by the preparation method according to any one of claims 1 to 5 or the artificially synthesized exosomes according to claim 6 in the preparation of anti-aging cosmetics.

8. A cosmetic, characterized in that: Including the artificially synthesized exosomes prepared by the preparation method according to any one of claims 1 to 5 or the artificially synthesized exosomes according to claim 6. The cosmetic according to claim 8 , which is used for anti-aging.

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