A method for preparing and applying synthetic exosomes with soothing effects

Abstract

This invention belongs to the fields of medical devices, medical or cosmetic formulations, biopharmaceutical manufacturing, and pharmaceutical preparation manufacturing. Specifically, it relates to a synthetic exosome comprising a membrane structure, an active ingredient, a polyol, and water. The membrane structure includes phospholipids, membrane structure modifiers, membrane flow stabilizers, membrane stability enhancers, emulsifiers, and surface modification structures. The synthetic exosome provided by this invention exhibits excellent soothing efficacy and stability.

Description

Technical Field

[0001] This invention relates to the fields of medical devices, medical or cosmetic preparations, biopharmaceutical manufacturing technology, and formulation manufacturing technology, specifically to a method for preparing and applying synthetic exosomes with soothing effects. Background Technology

[0002] Exosomes are vesicle-like structures secreted by cells, ranging in size from 30 to 150 nm. Their core function is as "intercellular messengers"—carrying bioactive substances such as nucleic acids (mRNA, miRNA), proteins, and lipids to facilitate intercellular signal transduction and substance transport. Based on this characteristic, exosomes have shown great potential for application in areas such as drug delivery (e.g., targeted tumor drug delivery, gene therapy), skin repair (e.g., anti-aging and barrier repair in the medical aesthetics field), and disease diagnosis (e.g., biomarker carriers).

[0003] However, the industrial application of natural exosomes faces insurmountable bottlenecks: extremely low yields, high purification difficulty, poor batch stability, and uncontrollable targeting.

[0004] To address the shortcomings of natural exosomes, the synthesis of artificial exosomes (also known as "biomimetic exosomes") has become a key focus of technological research and development. By mimicking the "lipid bilayer vesicle structure" of natural exosomes, artificially designed materials (such as natural lipids, biodegradable polymers, and cell membrane biomimetic components) are used as carriers to load active substances on demand and modify targeting molecules, achieving the industrialization goals of "controllable yield, adjustable composition, and precise targeting." Currently, the synthesis of artificial exosomes has become a core research direction in fields such as biomedicine and aesthetic medicine, but existing technologies still face two key technical challenges that urgently need to be overcome:

[0005] 1. Poor stability in particle size and appearance.

[0006] Artificial exosome products must remain clear and transparent, with a slight bluish tint, and free from stratification, precipitation, and flocculent matter. Microscopically, the particle size should be maintained between 30-150 nm (matching the in vivo circulation characteristics of natural exosomes), and the polydispersity index (PDI) should be <0.3. However, products prepared using existing technologies are prone to deterioration during storage (such as refrigeration at 4°C, room temperature storage, and fluctuations in cold chain transportation), including decreased clarity and turbidity, stratification and precipitation, or the formation of flocculent matter. If stratification / turbidity occurs, it not only affects the user experience but also leads to uneven release of active substances (e.g., growth factors encapsulated in precipitates cannot be absorbed by the skin), resulting in reduced or ineffective efficacy. Furthermore, the deterioration in macroscopic appearance directly reflects internal structural instability.

[0007] 2. Targeted peptides are prone to detachment.

[0008] Temperature can accelerate bond breakage, leading to the shedding of the target peptide. This shedding directly results in the loss of the "precise delivery" value of synthetic exosomes.

[0009] Therefore, there is still an urgent need for a stable synthetic exosome product. Summary of the Invention

[0010] To address the aforementioned technical problems, the present invention provides the following technical solutions.

[0011] In a first aspect, the present invention provides a synthetic exosome.

[0012] A synthetic exosome includes a membrane structure, an active ingredient, a polyol, and water; said membrane structure includes phospholipids, membrane structure modifiers, membrane flow stabilizers, membrane stability enhancers, emulsifiers, and surface modification structures.

[0013] The phospholipids include at least one of stearoyl lecithin, myristoyl lecithin, or phosphatidylcholine;

[0014] The membrane structure modifier includes at least one of dihydrosphingosine or phytosphingosine phosphate;

[0015] The membrane fluidity stabilizer includes at least one of sitosterol or hydrogenated cholesterol;

[0016] The membrane stability enhancer includes squalane;

[0017] The emulsifier includes sodium stearoyl glutamate;

[0018] The surface-modified structure includes a targeting peptide; the targeting peptide includes cyclotetrapeptide-24-aminocyclohexanecarboxylate.

[0019] In some embodiments, the weight ratio of the phospholipid and the membrane structure modifier is (2~8):(0.4~0.8), wherein the 2~8 of the phospholipid in this weight ratio can be any real number from 2 to 8, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8; and the 0.4~0.8 of the membrane structure modifier in this weight ratio can be any real number from 0.4 to 0.8, such as 0.4, 0.5, 0.6, 0.7 or 0.8.

[0020] In some embodiments, the weight ratio of the phospholipid to the membrane flow stabilizer is (2~8):(0.8~1.2), wherein the 2~8 of the phospholipid in this weight ratio can be any real number from 2 to 8, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8; and the 0.8~1.2 of the membrane flow stabilizer in this weight ratio can be any real number from 0.8 to 1.2, such as 0.8, 0.9, 1.0, 1.1 or 1.2.

[0021] In some embodiments, the weight ratio of the phospholipid to the membrane stability enhancer is (2~8):(0.3~0.6), wherein the 2~8 of the phospholipid in this weight ratio can be any real number from 2 to 8, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8; and the 0.3~0.6 of the membrane stability enhancer in this weight ratio can be any real number from 0.3 to 0.6, such as 0.3, 0.4, 0.5 or 0.6.

[0022] In some embodiments, the weight ratio of phospholipid to emulsifier is (2~8):(0.2~0.8), wherein the 2~8 of the phospholipid in this weight ratio can be any real number from 2 to 8, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8; and the 0.2~0.8 of the emulsifier in this weight ratio can be any real number from 0.2 to 0.8, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8.

[0023] In some embodiments, the weight ratio of the phospholipid to the surface-modified structure is (2-8): (0.001~0.05), wherein the 2~8 in the weight ratio of the phospholipid can be any real number from 2 to 8, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8; and the 0.001~0.05 in the weight ratio of the surface-modified structure can be any real number from 0.001 to 0.05, such as 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04 or 0.05.

[0024] In some embodiments, the weight ratio of the phospholipid to the polyol is (2-8):(8~30), wherein the 2~8 of the phospholipid in this weight ratio can be any real number from 2 to 8, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8; and the 8~30 of the polyol in this weight ratio can be any real number from 8 to 30, such as 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30.

[0025] In some embodiments, the weight ratio of the polyol to the water is (8~30):(56~85.1), wherein the 8~30 of the polyol in this weight ratio can be any real number from 8 to 30, such as 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30, and the 56~85.1 of the water in this weight ratio can be any real number from 56 to 85.1, such as 56, 56.344, 57, 58, 59, 60, 65, 66, 67, 68, 68.673, 69, 70, 75.385, 80, 82, 82.989, 83, 84, 84.098, 84.189, 84.198, 84.489, 84.5, 85 or 85.098.

[0026] In some embodiments, the weight ratio of the active ingredient to water is (1~5):(56~85.1). In this weight ratio, the active ingredient 1 to 5 can be any integer from 1 to 5, such as 1, 1.5, 1.81, 2.101, 2.101, 2.11, 2.201, 2.206, 2.207, 2.5, 3, 3.205, 3.312, 3.5, 4, 4.5 or 5. In this weight ratio, the water 56 to 85.1 can be any real number from 56 to 85.1, such as 56, 56.344, 57, 58, 59, 60, 65, 66, 67, 68, 68.673, 69, 70, 75.385, 80, 82, 82.989, 83, 84, 84.098, 84.189, 84.198, 84.489, 84.5, 85 or 85.098.

[0027] In some embodiments, the active ingredient includes at least one of PDRN, mixed amino acids, miRNA, siRNA, and SOD.

[0028] In some embodiments, the polyol is selected from at least one of 1,3-butanediol, 1,3-propanediol, glycerol, 1,2-hexanediol, and ethoxydiethylene glycol.

[0029] In some embodiments, the phospholipid content is 2 wt% to 8 wt% based on the total weight of the synthetic exosomes. In some embodiments, the phospholipid content is 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, or 8 wt%, or any value within a range of any two of these values, based on the total weight of the synthetic exosomes.

[0030] In some embodiments, the content of the membrane structure modifier is 0.4 wt% to 0.8 wt% based on the total weight of the synthetic exosomes. In some embodiments, the content of the membrane structure modifier is 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, or 0.8 wt% based on the total weight of the synthetic exosomes, or any value within a range of any two of these values.

[0031] In some embodiments, the content of the membrane fluidity stabilizer is 0.8 wt% to 1.2 wt% based on the total weight of the synthetic exosomes. In some embodiments, the content of the membrane fluidity stabilizer is 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, or 1.2 wt% based on the total weight of the synthetic exosomes, or any value within a range of any two of these values.

[0032] In some embodiments, the content of the membrane stability enhancer is 0.3 wt% to 0.6 wt% based on the total weight of the synthetic exosomes. In some embodiments, the content of the membrane stability enhancer is 0.3 wt%, 0.4 wt%, 0.5 wt%, or 0.6 wt% based on the total weight of the synthetic exosomes, or any value within a range of any two of these values.

[0033] In some embodiments, the emulsifier content is 0.2 wt% to 0.8 wt% based on the total weight of the synthetic exosomes. In some embodiments, the emulsifier content 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 synthetic exosomes, or any value within a range of any two of these values.

[0034] In some embodiments, the content of the surface-modified structure is 0.001 wt% to 0.05 wt% based on the total weight of the synthetic exosomes. In some embodiments, the content of the surface-modified structure is 0.001 wt%, 0.002 wt%, 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, or 0.05 wt%, or any value within a range of any two of these values, based on the total weight of the synthetic exosomes.

[0035] In some embodiments, the polyol content is 8 wt% to 30 wt% based on the total weight of the synthetic exosomes. In some embodiments, the polyol content is 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 20 wt%, 25 wt%, or 30 wt%, or any value within a range of any two of these values, based on the total weight of the synthetic exosomes.

[0036] In some embodiments, the water content, calculated based on the total weight of the synthetic exosomes, is 56 wt% to 85.1 wt%. In some embodiments, the water content, calculated based on the total weight of the synthetic exosomes, is 56 wt%, 56.344 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 68.673 wt%, 69 wt%, 70 wt%, 75.385 wt%, 80 wt%, 82 wt%, 82.989 wt%, 83 wt%, 84 wt%, 84.098 wt%, 84.189 wt%, 84.198 wt%, 84.489 wt%, 84.5 wt%, 85 wt%, or 85.098 wt%, or any value within a range of any two of these values.

[0037] In some embodiments, the content of the active ingredient, calculated based on the total weight of the synthetic exosomes, is 1 wt% to 5 wt%. In some embodiments, the content of the active ingredient, calculated based on the total weight of the synthetic exosomes, is 1 wt%, 1.5 wt%, 1.81 wt%, 2.101 wt%, 2.101 wt%, 2.11 wt%, 2.201 wt%, 2.206 wt%, 2.207 wt%, 2.5 wt%, 3 wt%, 3.205 wt%, 3.312 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%.

[0038] In some embodiments, based on the total weight of the synthetic exosomes, the content of the active ingredient is 1 wt% to 5 wt%, the content of the phospholipid is 2 wt% to 8 wt%, the content of the membrane structure modifier is 0.4 wt% to 0.8 wt%, the content of the membrane flow stabilizer is 0.8 wt% to 1.2 wt%, the content of the membrane stability enhancer is 0.3 wt% to 0.6 wt%, the content of the emulsifier is 0.2 wt% to 0.8 wt%, the content of the surface modification structure is 0.001 wt% to 0.05 wt%, the content of the polyol is 8 wt% to 30 wt%, and the content of water is 56 wt% to 85.1 wt%.

[0039] In some embodiments, the total content of the active ingredient, polyol, water, phospholipid, membrane structure modifier, membrane fluidity stabilizer, membrane stability enhancer, emulsifier, and surface modification structure is 100 wt%, calculated based on the total weight of the synthetic exosomes.

[0040] In some embodiments, based on the total weight of the synthetic exosomes, the content of the active ingredient is 1 wt% to 5 wt%, the content of the phospholipid is 2 wt% to 8 wt%, the content of the membrane structure modifier is 0.4 wt% to 0.8 wt%, the content of the membrane flow stabilizer is 0.8 wt% to 1.2 wt%, the content of the membrane stability enhancer is 0.3 wt% to 0.6 wt%, the content of the emulsifier is 0.2 wt% to 0.8 wt%, the content of the surface modification structure is 0.001 wt% to 0.05 wt%, the content of the polyol is 8 wt% to 30 wt%, and the balance is water.

[0041] In some embodiments, based on the total weight of the synthetic exosomes, the content of the active ingredient is 1.8 wt% to 3.5 wt%, the content of the phospholipid is 2 wt% to 8 wt%, the content of the membrane structure modifier is 0.4 wt% to 0.8 wt%, the content of the membrane flow stabilizer is 0.8 wt% to 1.2 wt%, the content of the membrane stability enhancer is 0.3 wt% to 0.6 wt%, the content of the emulsifier is 0.2 wt% to 0.8 wt%, the content of the surface modification structure is 0.001 wt% to 0.05 wt%, the content of the polyol is 8 wt% to 30 wt%, and the balance is water.

[0042] In some embodiments, based on the total weight of the synthetic exosomes, the content of the active ingredient is 1.81 wt% to 3.4 wt%, the content of the phospholipid is 2 wt% to 8 wt%, the content of the membrane structure modifier is 0.4 wt% to 0.8 wt%, the content of the membrane flow stabilizer is 0.8 wt% to 1.2 wt%, the content of the membrane stability enhancer is 0.3 wt% to 0.6 wt%, the content of the emulsifier is 0.2 wt% to 0.8 wt%, the content of the surface modification structure is 0.001 wt% to 0.05 wt%, the content of the polyol is 8 wt% to 30 wt%, the content of the active ingredient is 1.81 wt% to 3.4 wt%, and the balance is water.

[0043] In some embodiments, based on the total weight of the synthetic exosomes, the content of the active ingredient is 1.81 wt% to 3.31 wt%, the content of the phospholipid is 2 wt% to 8 wt%, the content of the membrane structure modifier is 0.4 wt% to 0.8 wt%, the content of the membrane flow stabilizer is 0.8 wt% to 1.2 wt%, the content of the membrane stability enhancer is 0.3 wt% to 0.6 wt%, the content of the emulsifier is 0.2 wt% to 0.8 wt%, the content of the surface modification structure is 0.001 wt% to 0.05 wt%, the content of the polyol is 8 wt% to 30 wt%, and the balance is water.

[0044] In a second aspect, the present invention provides a method for preparing the artificially synthesized exosomes described in the first aspect.

[0045] A method for preparing the synthetic exosomes described in the first aspect, comprising the following steps:

[0046] (1) Preparation of alcohol phase: Phospholipids, membrane structure modifiers, membrane flow stabilizers, membrane stability enhancers, emulsifiers, surface modification structures and polyols are mixed to obtain alcohol phase;

[0047] (2) Preparation of the aqueous phase: The active ingredient is mixed with water to obtain the aqueous phase;

[0048] (3) Preparation of mixed solution: Add the aqueous phase obtained in step (2) to the alcohol phase obtained in step (1), stir, homogenize, and obtain a mixed solution;

[0049] (4) Ultrasonic, high-pressure homogenization or extrusion membrane treatment: The mixed solution obtained in step (3) is subjected to ultrasonic, high-pressure homogenization or extrusion membrane treatment to obtain the artificially synthesized exosomes.

[0050] In some embodiments, the pressure of high-pressure homogenization in step (4) is 1500 bar - 2000 bar. In some embodiments, the pressure of high-pressure homogenization in step (4) is 1500 bar, 1600 bar, 1700 bar, 1800 bar, 1900 bar, 2000 bar, or any value within a range of any two of these values.

[0051] In some embodiments, the high-pressure homogenization in step (4) is repeated 4-6 times. In some embodiments, the high-pressure homogenization in step (4) is repeated 4 times, 5 times, or 6 times.

[0052] In some embodiments, the high-pressure homogenization temperature in step (4) is 10°C-25°C. In some embodiments, the high-pressure homogenization temperature in step (4) is 10°C, 15°C, 20°C, 25°C, or any value within a range of any two of these values.

[0053] Thirdly, the present invention provides an application of artificially synthesized exosomes obtained by the preparation method described in the first aspect or the second aspect.

[0054] The use of a synthetic exosome as described in the first aspect or prepared by the method described in the second aspect in the preparation of an anti-inflammatory pharmaceutical product.

[0055] In some embodiments, the formulation product includes a medical device or a cosmetic.

[0056] In some embodiments, the medical device includes medical dressings.

[0057] In some embodiments, the cosmetic is a functional cosmetic. In some embodiments, the cosmetic is used for anti-inflammatory or soothing purposes.

[0058] Fourthly, the present invention provides a formulation product.

[0059] A pharmaceutical product comprising the synthetic exosomes described in the first aspect or the synthetic exosomes prepared by the preparation method described in the second aspect.

[0060] In some embodiments, the formulation product is used for anti-inflammatory or soothing purposes.

[0061] In some embodiments, the formulation product includes a medical device or a cosmetic.

[0062] In some embodiments, the medical device includes medical dressings.

[0063] In some embodiments, the cosmetic is a functional cosmetic.

[0064] Beneficial effects

[0065] Compared with the prior art, at least one of the following beneficial effects is present in a certain embodiment of the present invention:

[0066] (1) Compared with other phospholipids (such as lecithin or hydrogenated lecithin), the present invention preferably uses stearoyl lecithin as the phospholipid, which is more conducive to improving the stability of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0067] (2) Compared with other membrane structure regulators (such as phytosphingosine or sphingosine), the present invention preferably uses dihydrosphingosine as a structure regulator, which is more conducive to improving the stability of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0068] (3) Compared with other membrane fluidity stabilizers (such as cholesterol or stigmasterol), the present invention preferably uses sitosterol as a membrane fluidity stabilizer, which is more conducive to improving the stability of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0069] (4) The present invention uses stearoyl lecithin as phospholipid, dihydrosphingosine as a structure regulator, and sitosterol as a membrane fluidity stabilizer. The three substances work together to improve the stability of the obtained artificially synthesized exosomes, resulting in unexpected technical effects.

[0070] (5) The artificially synthesized exosomes provided by the present invention have excellent inflammatory factor inhibition rate and excellent soothing and anti-inflammatory effects.

[0071] (6) The exosome membrane structure provided by the present invention has a synergistic effect among the components in the membrane structure, which greatly improves the stability of the soothing and anti-inflammatory effects of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0072] (7) Compared with other surface modification structures, using the cyclic tetrapeptide-24-aminocyclohexane carboxylate provided by the present invention as a surface modification structure is more conducive to improving the soothing and anti-inflammatory effects of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0073] (8) Compared with other surface modification structures, the present invention uses cyclic tetrapeptide-24-aminocyclohexane carboxylate for surface modification, and the resulting synthetic exosomes have better stability in terms of soothing and anti-inflammatory effects. The possible reason is that, compared with other surface modification structures, the connection stability of the membrane structure formed by the specific surface modification structure (cyclic tetrapeptide-24-aminocyclohexane carboxylate) provided by the present invention and the specific component provided by the present invention is better, and it is less likely to break and fall off during stable placement.

[0074] Terminology Explanation

[0075] In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0076] The terms “room temperature” or “normal temperature” refer to ambient temperature, which is approximately 10°C to approximately 35°C, approximately 10°C to approximately 30°C, or approximately 20°C to 30°C, or approximately 25°C.

[0077] The term "wt%" indicates a weight percentage.

[0078] The term "rpm" stands for the unit of rotational speed, "revolutions per minute".

[0079] The term "v / v" indicates a volume ratio.

[0080] The term "synthetic exosome" refers to a type of nanovesicle with a lipid bilayer membrane structure manufactured through non-biosynthetic pathways (such as self-assembly, template method, microfluidic technology, cell membrane biomimicry, etc.). The resulting nanovesicles possess the same or similar key physicochemical properties (such as size, morphology, membrane structure) and biological functions (such as efficient drug delivery, cell targeting, and crossing biological barriers) as natural exosomes. The physicochemical properties of the resulting nanovesicles may be the same as or not completely identical to those of natural exosomes, with the aim of selectively mimicking their key properties to achieve similar application goals (such as targeted delivery, immune escape, biocompatibility, etc.).

[0081] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0082] In the following content, all figures disclosed herein, whether or not they use the words "approximately" or "about," are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a figure with a value of N is disclosed, any figure with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction. Detailed Implementation

[0083] 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 provide a more detailed description of the present invention.

[0084] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0085] I. The sources of some of the reagents used in the various embodiments or comparative examples of this invention are as follows:

[0086] (1) PDRN: INCI name: Sodium DNA; Catalogue of Chemical Raw Materials Used (2021 Edition) No.: 00383;

[0087] (2) miRNA: Trade name: Xiumiyin; Manufacturer: Guangzhou Qingnang Biotechnology Co., Ltd.

[0088] (3) SOD: Trade name: Oxygen Shield; Manufacturer: Guangzhou Qingnang Biotechnology Co., Ltd.

[0089] (4) Mixed amino acids: Alanine, histidine hydrochloride, arginine, serine, proline, glutamic acid, threonine, valine, leucine, isoleucine, glycine, phenylalanine, taurine and allantoin are mixed in equal weight ratio to obtain mixed amino acids.

[0090] (5) Cyclotetrapeptide-24-aminocyclohexane carbamate: Shenzhen Vikitech Co., Ltd.

[0091] (6) Sitosterol: β-sitosterol.

[0092] (7) Cyclopeptide-5: Merck Cyclopeptide-5.

[0093] (8) Cyclic peptides: .

[0094] II. In vitro fibroblast inflammatory factor content test

[0095] 1. Definition / Abbreviation

[0096] DPBS: Duchenne phosphate buffer;

[0097] FBS: Fetal bovine serum;

[0098] DMSO: Dimethyl sulfoxide;

[0099] LPS: Lipopolysaccharide;

[0100] DXM: Dexamethasone.

[0101] 2. Principle

[0102] When skin is stimulated by external factors, it ultimately produces an irritation response through various pathways. When cells are stimulated, they secrete factors such as IL-1α, IL-1β, IL-6, COX-2, and TNF-α, causing skin irritation. After determining the safe concentration of the test sample, this experiment used an LPS-stimulated fibroblast model to evaluate the anti-inflammatory efficacy based on an in vitro anti-inflammatory efficacy assessment model. The anti-inflammatory efficacy of the test sample was evaluated by detecting changes in the levels of relevant inflammatory factors after the sample acted on fibroblasts.

[0103] 3. Procedure

[0104] 3.1 Equipment and Consumables: CO2 incubator, biosafety cabinet, PCR instrument, real-time PCR instrument, autoclave, ELISA reader, cell counter, 12-well cell culture plate, pipette.

[0105] 3.2 Solution reagents:

[0106] (1) Complete culture medium: containing 89 wt% DMEM medium (low sugar, containing pyruvate) (Gibco; catalog number: 11885076), 10 wt% fetal bovine serum and 1 wt% penicillin-streptomycin mixture (Gibco, the penicillin-streptomycin mixture contains 10,000 units / mL penicillin and 10,000 μg / mL streptomycin, catalog number: 15140-122);

[0107] (2) LPS solution: Weigh a certain amount of LPS (lipopolysaccharide) and dissolve it in phosphate buffer to prepare a 1 mg / mL stock solution. Filter the solution through a 0.22 μm filter to remove bacteria and store it at -20℃. Avoid repeated freezing.

[0108] (3) DXM stock solution: Weigh a certain amount of dexamethasone and dissolve it in DMSO (dimethyl sulfoxide) to prepare a stock solution of 500 mg / ml. Filter it with a 0.22 μm filter to remove bacteria and store it at -20℃. Avoid repeated freezing and storage.

[0109] (4) Positive control: Take a certain amount of DXM stock solution and dilute it with complete culture medium to prepare a working solution of 400 μg / mL. Prepare and use immediately.

[0110] 4. Experimental Design

[0111] See Table 1.

[0112] Table 1:

[0113] ;

[0114] 5. Experimental Methods

[0115] (1) Sample processing

[0116] Water-soluble samples are diluted with cell culture medium to create multi-level concentration gradients; oil-soluble samples are first diluted with DMSO or other organic solvents, then diluted with cell culture medium to create multi-level concentration gradients, and the presence of any precipitates is checked. Samples are first diluted to a high concentration, sterilized through a sterile filter, and then diluted to the working concentration.

[0117] (2) Experimental steps

[0118] a. Cell passage culture

[0119] The cell revival, passage, and cryopreservation procedures are performed to revive and culture the cells.

[0120] b. Cell proliferation rate assay

[0121] In cytotoxicity experiments, appropriate sample concentrations are selected based on the toxicity results to determine the levels of inflammatory factors in cells.

[0122] c. Cell seeding plate

[0123] Cells in the logarithmic growth phase were selected, digested with trypsin, and collected to form a cell suspension. Cells were then counted using a cell counter. The cells were diluted with cell culture medium to a suitable seeding density (70%–80% confluence 24 h after seeding) and seeded into 12-well plates at a volume of 1 mL per well. After seeding, the plates were incubated in a CO2 incubator for 24 h ± 2 h.

[0124] d. Cell sample loading

[0125] Discard the culture medium from the cell culture plate and proceed with the drug administration. Add complete culture medium to the blank wells, add complete culture medium containing 1 μg / mL LPS and different concentrations of the test substance to the test substance wells, add complete culture medium containing 10 μg / mL LPS / 10 μg / mL LPS to the model group wells, and add complete culture medium containing 10 μg / mL LPS and 400 μg / mL DXM to the positive control wells. Use 1 mL of complete culture medium per well in a 12-well plate. Incubate in a CO2 incubator for 4 h ± 15 min.

[0126] e. Detection of inflammatory factor levels

[0127] 1. RNA extraction

[0128] RNA was extracted according to the kit (manufacturer: RNA-easy Isolation Reagent, Novizan), and its concentration was detected and quantified.

[0129] 2. Reverse transcription

[0130] RNA reverse transcription to cDNA: Prepare the reverse transcription reaction system according to Table 2.

[0131] Table 2:

[0132] ;

[0133] 3. qPCR detection

[0134] Prepare the mixture in the qPCR tubes, with the ratios shown in Table 3.

[0135] Table 3:

[0136] ;

[0137] RNA volume = Corrected RNA concentration (ng / μL) / Mass of RNA (ng)

[0138] The Chinese names of the reagents in Table 3 correspond to the English names of the reagents in the reagent kits:

[0139] ChamQ Universal SYBR qPCR Master Mix;

[0140] Primer 1: Primer 1;

[0141] Primer 2: Primer 2;

[0142] Template DNA / cDNA: Template DNA / cDNA;

[0143] Double-distilled water: ddH2O.

[0144] Perform the qPCR reaction according to the procedure in Table 4.

[0145] Table 4:

[0146] ;

[0147] 6. Result Calculation

[0148] Calculate according to the formula

[0149]

[0150] Among them: internal reference genes: refer to genes used as internal references in molecular experiments. Their expression is relatively constant in various tissues and cells. The internal reference gene for TNF-α is β-actin, and the internal reference gene for IL-6 is β-actin.

[0151] NC average: refers to the average ΔCT value of the model group, which is used to eliminate minor errors that may be caused by the experimental operation itself.

[0152] Fold expression: refers to the relative fold increase in gene expression, which directly reflects the expression level of the target gene in the experimental group.

[0153] Example 1: Artificial Synthesis of Exosomes

[0154] Formula: See Table 5.

[0155] Table 5: Formulation of Synthetic Exosomes

[0156] ;

[0157] Preparation method:

[0158] (1) Preparation of alcohol phase: Phospholipids, membrane structure modifiers, membrane flow stabilizers, membrane stability enhancers, emulsifiers, surface modification structures and polyols are mixed to obtain alcohol phase;

[0159] (2) Preparation of the aqueous phase: The active ingredient is mixed with water to obtain the aqueous phase;

[0160] (3) Preparation of mixed solution: Add the aqueous phase obtained in step (2) to the alcohol phase obtained in step (1), stir, homogenize, and obtain a mixed solution;

[0161] (4) Ultrasonic, high-pressure homogenization or extrusion membrane treatment: The mixed solution obtained in step (3) is subjected to high-pressure homogenization (pressure 1500 bar, 5 cycles, temperature controlled at 25°C) to obtain the artificially synthesized exosomes.

[0162] Comparative Examples 1-2: Phospholipid Investigation

[0163] Comparative Example 1: The only difference from Example 1 is that stearoyl lecithin is replaced with lecithin, and the other conditions are the same as in Example 1.

[0164] Comparative Example 2: The only difference from Example 1 is that stearoyl lecithin is replaced with hydrogenated lecithin, and the other conditions are the same as in Example 1.

[0165] Comparative Examples 3-4: Investigation of Membrane Structure Modifiers

[0166] Comparative Example 3: The only difference from Example 1 is that dihydrosphingosine is replaced with phytosphingosine, and the other conditions are the same as in Example 1.

[0167] Comparative Example 4: The only difference from Example 1 is that dihydrosphingosine is replaced with sphingosine, and the other conditions are the same as in Example 1.

[0168] Comparative Examples 5-6: Investigation of Membrane Flow Stabilizers

[0169] Comparative Example 5: The only difference from Example 1 is that sitosterol is replaced with cholesterol, and the other conditions are the same as in Example 1.

[0170] Comparative Example 6: The only difference from Example 1 is that sitosterol is replaced with stigmasterol, and the other conditions are the same as in Example 1.

[0171] Comparative Examples 7-9: Investigation of Surface Modification Structures

[0172] Comparative Example 7: The only difference from Example 1 is that cyclic tetrapeptide-24-aminocyclohexane carbamate is replaced with cyclic peptide-5, and the other conditions are the same as in Example 1.

[0173] Comparative Example 8: The only difference from Example 1 is that cyclic tetrapeptide-24-aminocyclohexane carbamate is replaced with a cyclic peptide, and the other conditions are the same as in Example 1.

[0174] Comparative Example 9: The only difference from Example 1 is that no surface modification structure is added; all other conditions are the same as in Example 1.

[0175] Examples 2 to 9:

[0176] Formula: See Tables 6 and 7.

[0177] Table 6: Formulation of Synthetic Exosomes

[0178] ;

[0179] Table 7: Formulation of Artificially Synthetic Exosomes

[0180] ;

[0181] Preparation method: Refer to Example 1.

[0182] Application Example 1: Preparation of Aqueous Essence

[0183] The artificially synthesized exosomes obtained from the above examples and comparative examples were prepared according to the formulation in Table 8 and the following preparation method to obtain aqueous extracts of Examples 1A to 9A and Comparative Examples 1A to 9A (the numbers of the aqueous extracts correspond to the numbers of the examples or comparative examples, for example, Example 1A corresponds to the artificially synthesized exosomes of Example 1).

[0184] Formula: See Table 8.

[0185] Table 8:

[0186] ;

[0187] Preparation method:

[0188] 1. Heat water to 85°C, add p-hydroxyacetophenone and three-fifths 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;

[0189] 2. After premixing the remaining amount of 1,3-butanediol with transparent xanthan gum, stir and add it to phase A at a temperature below 60°C (e.g., 50°C). Stir until fully swollen, free of fish-eye particles, and thoroughly homogenized. Continue to cool to a temperature below 40°C (e.g., 35°C) to obtain phase B.

[0190] 3. Add the artificially synthesized exosomes and 1,2-pentanediol obtained in Example 1 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 100wt% to obtain the water-based essence product.

[0191] Experimental Example 1: Stability Study

[0192] The synthetic exosomes obtained in the above examples or comparative examples were taken respectively, and the average particle size (0-day average particle size) and appearance of the synthetic exosomes were detected. Then, each sample was placed at 45℃±2℃ / 75%RH for 3 months, and the average particle size, polydispersity index (PDI) and appearance of the synthetic exosomes were detected. The results are shown in Tables 9 and 10.

[0193] Table 9: Results of Particle Size and Particle Size Stability

[0194] ;

[0195] Table 10: Results of stability of appearance traits

[0196] ;

[0197] Conclusion: The results in Tables 9 and 10 show that:

[0198] (1) Compared with other phospholipids (such as lecithin or hydrogenated lecithin), the present invention preferably uses stearoyl lecithin as the phospholipid, which is more conducive to improving the stability of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0199] (2) Compared with other membrane structure regulators (such as phytosphingosine or sphingosine), the present invention preferably uses dihydrosphingosine as a structure regulator, which is more conducive to improving the stability of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0200] (3) Compared with other membrane fluidity stabilizers (such as cholesterol or stigmasterol), the present invention preferably uses sitosterol as a membrane fluidity stabilizer, which is more conducive to improving the stability of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0201] (4) The present invention uses stearoyl lecithin as phospholipid, dihydrosphingosine as a structure regulator, and sitosterol as a membrane fluidity stabilizer. The three substances work together to improve the stability of the obtained artificially synthesized exosomes, resulting in unexpected technical effects.

[0202] Experimental Example 2: Anti-inflammatory and soothing effects (in vitro fibroblast inflammatory factor inhibition assay)

[0203] The above-mentioned examples, comparative examples, and their stability samples were tested in vitro at a test concentration of 0.1 wt% (based on the composition) to determine the content of inflammatory factors in fibroblasts. The inhibition rate was calculated, and the results are shown in Table 11.

[0204] Table 11: Inhibition rate of inflammatory factors in in vitro fibroblasts

[0205] ;

[0206] in conclusion:

[0207] (1) The artificially synthesized exosomes provided by the present invention have excellent inflammatory factor inhibition rate and excellent soothing and anti-inflammatory effects.

[0208] (2) The exosome membrane structure provided by the present invention has a synergistic effect among the components in the membrane structure, which greatly improves the stability of the soothing and anti-inflammatory effects of the obtained artificially synthesized exosomes and has unexpected technical effects.

[0209] (3) Compared with other surface modification structures, using the cyclic tetrapeptide-24-aminocyclohexane carboxylate provided by the present invention as a surface modification structure is more conducive to improving the soothing and anti-inflammatory effects of the obtained artificially synthesized exosomes, and has unexpected technical effects.

[0210] (4) Compared with other surface modification structures, the present invention uses cyclic tetrapeptide-24-aminocyclohexane carboxylate for surface modification, and the resulting synthetic exosomes have better stability in terms of soothing and anti-inflammatory effects. The possible reason is that, compared with other surface modification structures, the connection stability of the membrane structure formed by the specific surface modification structure (cyclic tetrapeptide-24-aminocyclohexane carboxylate) provided by the present invention and the specific component provided by the present invention is better, and it is less likely to break and fall off during stable placement.

[0211] Experiment Example 3: Human Trial of Soothing Efficacy

[0212] The soothing efficacy of the aqueous extract from Example 1A was evaluated using the following procedures:

[0213] 1. Subject Information

[0214] Thirty-one participants were enrolled, with 31 valid participants (0 males and 31 females), aged 23 to 59 years, with a mean age of 48 years. All enrolled participants met the following inclusion and exclusion criteria:

[0215] 1.1 Subject inclusion criteria

[0216] 1.1.1 Healthy men and women aged 18-60;

[0217] 1.1.2 Problems such as dull and dry facial skin;

[0218] 1.1.3 People with sensitive skin (through a sensitive skin questionnaire and a lactic acid stinging score ≥3).

[0219] 1.1.4 The patient has not participated in any other clinical studies in the past two months, and the test site has not undergone any skin treatments, cosmetic procedures, or other processes that could affect the test results;

[0220] 1.1.5 Those who can understand the experimental process, voluntarily participate in the experiment, and sign a written informed consent form.

[0221] 1.2 Exclusion criteria for subjects

[0222] 1.2.1 Pregnant or breastfeeding women, or those planning to conceive in the near future;

[0223] 1.2.2 Individuals with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, or severe acne; or individuals with other chronic systemic diseases;

[0224] 1.2.3 Individuals who have taken orally or used topical anti-inflammatory drugs such as corticosteroids within the past month;

[0225] 1.2.4 Individuals with severe allergies;

[0226] 1.2.5 Individuals who have used retinoid preparations or undergone cosmetic treatments such as chemical peels, lasers, or pulsed light on the test site within the past 3 months;

[0227] 1.2.6 Those who are unavoidably exposed to sunlight for extended periods;

[0228] 1.2.7 Individuals who have participated in other clinical trials within the past two months;

[0229] 1.2.8 Other clinical assessments deemed unsuitable for participation in the trial.

[0230] 2. Testing instruments and materials

[0231] Tewameter™ HEX probe for testing skin stratum corneum moisture loss (Courage+Khazaka, Germany); Mexameter MX18 probe for testing skin melanin and hemoglobin (Courage+Khazaka, Germany).

[0232] 3. Test Environment

[0233] Temperature 21±1℃; humidity 50±10%.

[0234] 4. Testing Methods

[0235] 4.1 Sample usage method and frequency

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

[0237] 4.2 Test Items

[0238] 4.2.1 Instrument Testing

[0239] See Table 12.

[0240] Table 12:

[0241] ;

[0242] 4.3 Testing Process

[0243] 1) Recruit subjects according to requirements and obtain written informed consent. Before enrollment, ask subjects a series of questions about their medical history and health status based on inclusion and exclusion criteria, and select subjects who meet the criteria to participate in this testing project.

[0244] 2) The enrolled subjects cleansed their faces with facial cleanser, rinsed them with water, and dried their faces with lint-free absorbent facial tissues. After cleansing their faces, they entered a constant temperature and humidity room and waited for 30 minutes. During the stabilization period, they were not allowed to eat or drink, their foreheads were exposed, they were kept relaxed, and they were advised to avoid touching the test sites.

[0245] 3) After the rest period, complete the skin testing procedure to test the skin's physiological parameters.

[0246] 4) After using the sample for 7 days, 14 days, and 21 days, follow up to complete the tests in step 3), and complete a self-assessment based on your own situation, and truthfully fill out the questionnaire related to the sample.

[0247] 4.4 Data Analysis

[0248] Statistical analysis software was used to perform statistical analysis on the data. Quantitative data were expressed as mean ± standard deviation and tested for normality. If the data met the requirements of a normal distribution, paired t-tests were used for comparisons before and after the same data point; otherwise, the rank-sum test of two related samples was used. All the above statistical analyses were two-tailed tests, with a significance level of α = 0.05.

[0249] 4.5 Basis for Judging Test Conclusions

[0250] 4.5.2 Claimed soothing effects: Using a self-comparison before and after treatment, skin hemoglobin and transepidermal water loss were observed. If, at any time point after sample use, the test values ​​were better than the baseline values ​​before use, and the results showed a significant difference ( P If the value is less than 0.05, the test sample is considered to have a soothing effect.

[0251] 4.6 Test Results

[0252] See Tables 13 and 14.

[0253] Table 13: Results of Skin Hemoglobin Detection

[0254] ;

[0255] Note: The rate of change represents the rate of change relative to the value before use (baseline value).

[0256] Table 14: Results of transepidermal water loss measurement

[0257] ;

[0258] in conclusion:

[0259] The synthetic exosome composition provided by this invention has excellent soothing effects.

[0260] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. An artificial synthetic exosome, characterized in that, The artificial synthetic exosome comprises a membrane structure, an active ingredient, a polyol and water; the membrane structure comprises phospholipid, a membrane structure regulator, a membrane fluidity stabilizer, a membrane stability enhancer, an emulsifier and a surface modification structure; The phospholipid comprises at least one of stearoyl phosphatidylcholine, myristoyl phosphatidylcholine or phosphatidylcholine; The membrane structure regulator is at least one of dihydro sphingosine or phytosphingosine phosphate; The membrane fluidity stabilizer is at least one of sitosterol or hydrogenated cholesterol; The membrane stability enhancer is squalane; The emulsifier is sodium stearoyl glutamate; The surface modification structure comprises a targeting peptide; the targeting peptide comprises cyclo-tetrapeptide-24 aminocyclohexane formate; The weight ratio of the phospholipid to the membrane structure regulator is (2-8):(0.4-0.8); The weight ratio of the phospholipid to the membrane fluidity stabilizer is (2-8):(0.8-1.2); The weight ratio of the phospholipid to the membrane stability enhancer is (2-8):(0.3-0.6); The weight ratio of the phospholipid to the emulsifier is (2-8):(0.2-0.8); The weight ratio of the phospholipid to the surface modification structure is (2-8):(0.001-0.05); The weight ratio of the phospholipid to the polyol is (2-8):(8-30); The weight ratio of the polyol to the water is (8-30):(56-85.1); The weight ratio of the active ingredient to the water is (1-5):(56-85.1).

2. The artificial synthetic exosome according to claim 1, wherein the active ingredient comprises at least one of PDRN, mixed amino acids, miRNA, siRNA or SOD; and / or The polyol is at least one of 1,3-butanediol, 1,3-propanediol, glycerol, 1,2-hexanediol or ethoxydiglycol.

3. The artificial synthetic exosome according to any one of claims 1-2, wherein the content of the active ingredient is 1 wt%-5 wt%, the content of the phospholipid is 2 wt%-8 wt%, the content of the membrane structure regulator is 0.4 wt%-0.8 wt%, the content of the membrane fluidity stabilizer is 0.8 wt%-1.2 wt%, the content of the membrane stability enhancer is 0.3 wt%-0.6 wt%, the content of the emulsifier is 0.2 wt%-0.8 wt%, the content of the surface modification structure is 0.001 wt%-0.05 wt%, the content of the polyol is 8 wt%-30 wt%, and the content of the water is 56 wt%-85.1 wt%, based on the total weight of the artificial synthetic exosome.

4. A method of producing the synthetic exosome according to any one of claims 1 to 3, characterized by, comprising the following steps: (1) Preparation of an alcohol phase: mixing phospholipid, a membrane structure regulator, a membrane fluidity stabilizer, a membrane stability enhancer, an emulsifier, a surface modification structure and a polyol to obtain an alcohol phase; (2) Preparation of an aqueous phase: mixing an active ingredient and water to obtain an aqueous phase; (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; (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 exosome.

5. The preparation method of claim 4, wherein the pressure of high-pressure homogenization in step (4) is 1500-2000 bar; and / or the number of cycles of high-pressure homogenization in step (4) is 4-6 times; and / or the temperature of high-pressure homogenization in step (4) is 10-25°C.

6. Use of the artificial synthetic exosome of any one of claims 1-3 or the artificial synthetic exosome prepared by the preparation method of any one of claims 4-5 in the preparation of a cosmetic.

7. The use of claim 6, wherein the cosmetic is used for anti-inflammatory or soothing.

8. A cosmetic product, characterized by, The artificial synthetic exosome of any one of claims 1-3 or the artificial synthetic exosome prepared by the preparation method of any one of claims 5-6.

9. The cosmetic of claim 8, wherein the cosmetic is used for anti-inflammatory or soothing.

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