Bionic exosome for anti-inflammatory and repair as well as preparation method and application of bionic exosome

Abstract

The invention relates to a bionic exosome for anti-inflammation and repair as well as a preparation method and application thereof, and belongs to the field of cosmetics. The bionic exosome provided by the invention comprises a membrane structure and an internal load phase, the membrane structure comprises a phospholipid bilayer, a stabilizer and a targeting ligand; the internal load phase comprises a solvent and an active component; the phospholipid bilayer is composed of phospholipid compounds; preferably, at least one of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and the 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine is adopted as the phospholipid compound, and at least one of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and the 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine is adopted as the phospholipid compound; at least one of cholesterol, phytosphingosine and sodium stearoyl glutamate is preferably adopted as a stabilizer, so that the stabilizer of the obtained bionic exosome is improved, and the bionic exosome has the advantages of high encapsulation efficiency, good encapsulation efficiency stability, simplicity in operation, excellent anti-inflammatory and repairing effects and the like.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to a bionic exosome for anti-inflammatory and repairing purposes, and a preparation method and application thereof. Background Art

[0002] In the cosmetics field, skin repair and anti-inflammatory functions have long been a hot topic of research. Exosomes, as crucial mediators of intercellular communication, are widely used in skin repair due to their excellent biocompatibility and delivery capabilities. However, natural exosomes are expensive to prepare, exhibit poor stability, and are difficult to scale up. This has prompted researchers to develop biomimetic exosomes (nanocarriers that artificially mimic the structure of exosomes) as an alternative to natural exosomes.

[0003] In existing technologies, the construction of biomimetic exosomes usually faces the following technical challenges: (1) Insufficient stability: The membrane structure of traditional biomimetic exosomes is easily affected by the external environment (such as temperature and pH), which can lead to leakage of active ingredients and affect product efficacy. For example, the membrane structure composed of a single phospholipid compound lacks sufficient rigidity, and commonly used stabilizers (such as single cholesterol or sphingolipids) are difficult to simultaneously take into account membrane stability and cell compatibility.

[0004] (2) Limited anti-inflammatory and repair effects: The anti-inflammatory mechanism of existing biomimetic exosomes mostly relies on a single component (such as phytosphingosine). However, due to insufficient targeting of the carrier system or low encapsulation efficiency of the active ingredient, it is difficult to effectively inhibit the release of inflammatory factors (such as TNF-α, IL-6) or promote cell migration. In addition, the low loading efficiency of traditional carriers for water-soluble active ingredients limits their repair effects.

[0005] (3) Complex preparation process: The preparation of some biomimetic exosomes requires complex emulsification or freeze-drying processes, which not only increases production costs but may also destroy the biological activity of the active ingredients.

[0006] Therefore, there is still an urgent need for a bionic exosome product that is simple to operate, has good stability, high encapsulation rate, and excellent anti-inflammatory and repair effects. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: In a first aspect, the present invention provides a biomimetic exosome.

[0008] A biomimetic exosome comprising: a membrane structure and an internal load phase; The membrane structure includes a phospholipid bilayer, a stabilizer and a targeting ligand; The internal loading phase includes a solvent and an active ingredient; The phospholipid bilayer is composed of phospholipid compounds; The phospholipid compound includes at least one of distearoylphosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The stabilizer includes at least one of cholesterol, phytosphingosine and sodium stearoyl glutamate.

[0009] In some embodiments, the stabilizer comprises cholesterol, phytosphingosine, and sodium stearoyl glutamate.

[0010] In some embodiments, the phospholipid compounds include distearoylphosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0011] In some embodiments, the solvent comprises water.

[0012] In some embodiments, the active ingredient is a water-soluble active ingredient.

[0013] In some embodiments, the active ingredient comprises a water-soluble amino acid or a protein.

[0014] In some embodiments, the protein comprises collagen.

[0015] In some embodiments, the protein comprises at least one of recombinant type I collagen, recombinant type III collagen, and recombinant type III humanized collagen.

[0016] In some embodiments, the protein has a weight average molecular weight of 5 kDa to 100 kDa. In some embodiments, the protein has a weight average molecular weight of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, 55 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa.

[0017] In some embodiments, the protein is a single molecular weight protein having a single molecular weight selected from 5 kDa to 100 kDa. In some embodiments, the protein is a single molecular weight protein having a single molecular weight of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, 55 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa.

[0018] In some embodiments, the targeting ligand comprises an RGD peptide-modified ceramide.

[0019] In some embodiments, the mass ratio of the phospholipid compound to the stabilizer is 1:1-10:1. In some embodiments, the mass ratio of the phospholipid compound to the stabilizer is 2:1-10:1. In some embodiments, the mass ratio of the phospholipid compound to the stabilizer is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0020] In some embodiments, the mass ratio of cholesterol, phytosphingosine, and sodium stearoyl glutamate is (0.1-100):(0.01-50):(0.1-100). In some embodiments, the mass ratio of cholesterol, phytosphingosine, and sodium stearoyl glutamate is (10-30):(5-20):(5-10). In some embodiments, the mass ratio of cholesterol, phytosphingosine, and sodium stearoyl glutamate is 20:20:10, 30:10:10, or 10:5:5.

[0021] In some embodiments, the mass ratio of the phospholipid compound to the internal support phase is 1:5-1:10. In some embodiments, the mass ratio of the phospholipid compound to the internal support phase is 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0022] In some embodiments, the mass ratio of the solvent to the active ingredient is 10: 1 to 50: 1. In some embodiments, the mass ratio of the solvent to the active ingredient is 24: 1 to 49: 1. In some embodiments, the mass ratio of the solvent to the active ingredient is 10: 1, 15: 1, 20: 1, 21: 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1, 29: 1, 30: 1, 35: 1, 40: 1, 45: 1, 46: 1, 47: 1, 48: 1, 49: 1, or 50: 1.

[0023] In some embodiments, the mass ratio of the phospholipid compound to the targeting ligand is 100: 1-200: 1. In some embodiments, the mass ratio of the phospholipid compound to the targeting ligand is 100: 1, 110: 1, 120: 1, 130: 1, 140: 1, 150: 1, 160: 1, 170: 1, 180: 1, 190: 1 or 200: 1.

[0024] In some embodiments, the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol 2000 to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is (3-800):(1-100). In some embodiments, the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol 2000 to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is 1:9-19:1. In some embodiments, the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1 or 19:1.

[0025] In a second aspect, the present invention provides a biomimetic exosome composition.

[0026] A biomimetic exosome composition, comprising the biomimetic exosomes described in the first aspect.

[0027] In some embodiments, the biomimetic exosome composition further comprises a polyol.

[0028] In some embodiments, the polyol includes at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol. In some embodiments, the polyol includes at least one of 1,3-butanediol and 1,2-hexanediol.

[0029] In a third aspect, the present invention provides a method for preparing the biomimetic exosomes described in the first aspect or the biomimetic exosome composition described in the second aspect.

[0030] A method for preparing the biomimetic exosomes according to the first aspect or the biomimetic exosome composition according to the second aspect, comprising the following steps: S1: Preparation of alcohol phase: mixing the phospholipid compound, targeting ligand, stabilizer and polyol to obtain an alcohol phase; S2: Preparation of internal loading phase: mixing the active ingredient with the solvent to obtain the internal loading phase; S3: Preparation of a mixed solution: adding the internal support phase obtained in step S2 dropwise to the alcohol phase obtained in step S1, stirring, to obtain a mixed solution; S4: The mixed solution obtained in step S3 is treated by a membrane extrusion method to obtain a biomimetic exosome composition containing biomimetic exosomes.

[0031] In some embodiments, the polyol includes at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol. In some embodiments, the polyol includes at least one of 1,3-butanediol and 1,2-hexanediol.

[0032] In some embodiments, the mass ratio of the phospholipid compound to the stabilizer is 1:1-10:1. In some embodiments, the mass ratio of the phospholipid compound to the stabilizer is 2:1-10:1. In some embodiments, the mass ratio of the phospholipid compound to the stabilizer is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0033] In some embodiments, the mass ratio of the phospholipid compound to the polyol is 1:5-1:10. In some embodiments, the mass ratio of the phospholipid compound to the polyol is 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0034] In some embodiments, the weight ratio of cholesterol, phytosphingosine, and sodium stearoyl glutamate is (0.1-100): (0.01-50): (0.1-100). In some embodiments, the weight ratio of cholesterol, phytosphingosine, and sodium stearoyl glutamate is (10-30): (5-20): (5-10). In some embodiments, the weight ratio of cholesterol, phytosphingosine, and sodium stearoyl glutamate is 20:20:10, 30:10:10, or 10:5:5.

[0035] In some embodiments, the mass ratio of the phospholipid compound to the internal support phase is 1:5-1:10. In some embodiments, the mass ratio of the phospholipid compound to the internal support phase is 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0036] In some embodiments, the mass ratio of the solvent to the active ingredient is 10: 1-50: 1. In some embodiments, the mass ratio of the solvent to the active ingredient is 24: 1-49: 1. In some embodiments, the mass ratio of the solvent to the active ingredient is 10: 1, 15: 1, 20: 1, 21: 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1, 29: 1, 30: 1, 35: 1, 40: 1, 45: 1, 46: 1, 47: 1, 48: 1, 49: 1 or 50: 1.

[0037] In some embodiments, the mass ratio of the phospholipid compound to the targeting ligand is 100: 1-200: 1. In some embodiments, the mass ratio of the phospholipid compound to the targeting ligand is 100: 1, 110: 1, 120: 1, 130: 1, 140: 1, 150: 1, 160: 1, 170: 1, 180: 1, 190: 1 or 200: 1.

[0038] In some embodiments, the mass ratio of the distearoylphosphatidylethanolamine-polyethylene glycol 2000 to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is (3-800):(1-100). In some embodiments, the mass ratio of the distearoylphosphatidylethanolamine-polyethylene glycol 2000 to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is 1:9-19:1. In some embodiments, the feeding mass ratio of the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1 or 19:1.

[0039] In some embodiments, the dropping speed of step S3 is 1 ml / min-20 ml / min. In some embodiments, the dropping speed of step S3 is 1 ml / min, 2 ml / min, 3 ml / min, 4 ml / min, 5 ml / min, 6 ml / min, 7 ml / min, 8 ml / min, 9 ml / min, 10 ml / min, 11 ml / min, 12 ml / min, 13 ml / min, 14 ml / min, 15 ml / min, 16 ml / min, 17 ml / min, 18 ml / min, 19 ml / min, or 20 ml / min.

[0040] In some embodiments, the filter membrane extrusion method in step S4 uses a polycarbonate membrane, a nitrocellulose membrane, or a mixed cellulose ester membrane as the filter membrane.

[0041] In some embodiments, the pore size of the filter membrane in the filter membrane extrusion method in step S4 is 50 nm to 150 nm. In some embodiments, the pore size of the filter membrane in the filter membrane extrusion method in step S4 is 50 nm to 100 nm.

[0042] In some embodiments, the pressure of the membrane extrusion method in step S4 is 0.2 MPa-10.0 MPa. In some embodiments, the pressure of the membrane extrusion method in step S4 is 1.0 MPa-5.0 MPa. In some embodiments, the pressure of the membrane extrusion method in step S4 is 0.2 MPa, 0.5 MPa, 1.0 MPa, 1.5 MPa, 2.0 MPa, 2.5 MPa, 3.0 MPa, 3.5 MPa, 4.0 MPa, 4.5 MPa, 5.0 MPa, 5.5 MPa, 6.0 MPa, 6.5 MPa, 7.0 MPa, 7.5 MPa, 8.0 MPa, 8.5 MPa, 9.0 MPa, 9.5 MPa or 10.0 MPa.

[0043] In some embodiments, the number of extrusions in the membrane extrusion method of step S4 is 3 to 20 times. In some embodiments, the number of extrusions in the membrane extrusion method of step S4 is 5 to 15 times. In some embodiments, the number of extrusions in the membrane extrusion method of step S4 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.

[0044] In a fourth aspect, the present invention provides an application of the bionic exosomes described in the first aspect, the bionic exosome composition described in the second aspect, or the bionic exosomes or the bionic exosome composition prepared by the preparation method described in the third aspect.

[0045] A use of the bionic exosomes of the first aspect, the bionic exosome composition of the second aspect, or the bionic exosome composition prepared by the preparation method of the third aspect in the preparation of cosmetics.

[0046] In some embodiments, the cosmetic is used for anti-inflammatory and / or repair purposes.

[0047] In a fifth aspect, the present invention provides a cosmetic.

[0048] A cosmetic comprising the biomimetic exosomes described in the first aspect, the biomimetic exosome composition described in the second aspect, or the biomimetic exosomes or the biomimetic exosome composition prepared by the preparation method described in the third aspect.

[0049] In some embodiments, the cosmetic is used for anti-inflammatory and / or repair purposes.

[0050] Beneficial effects Compared with the prior art, a certain embodiment of the present invention has at least one of the following beneficial effects: (1) The biomimetic exosomes or biomimetic exosome compositions provided by the present invention have the advantages of high encapsulation efficiency, good encapsulation efficiency stability, simple operation, and excellent anti-inflammatory and repair effects.

[0051] (2) Compared with other stabilizers or stabilizer combinations, the stabilizer combination (cholesterol + phytosphingosine + sodium stearoyl glutamate) provided by the present invention is more conducive to improving the encapsulation rate stability of the obtained biomimetic exosome composition, and has unexpected technical effects.

[0052] (3) Compared with other phospholipid compounds or phospholipid compound combinations, the phospholipid compound combination provided by the present invention (distearoylphosphatidylethanolamine-polyethylene glycol 2000+1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) is more conducive to improving the encapsulation rate stability of the obtained biomimetic exosome composition, and has unexpected technical effects.

[0053] Terminology In the description of the present invention, “plurality” means two or more, unless otherwise clearly defined.

[0054] 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.

[0055] The term "wt%" means percentage by mass.

[0056] The term "rpm" means revolutions per minute.

[0057] The term "v / v" means a volume ratio.

[0058] The term "single molecular weight protein" refers to a protein with uniform molecular mass, whose molecular structure, amino acid sequence and modification pattern are highly consistent. Therefore, it appears as a single molecular weight band or peak in analytical methods such as electrophoresis and mass spectrometry.

[0059] The term "single molecular weight" refers to the fact that protein molecules exhibit uniform molecular mass values ​​in their physicochemical properties, that is, the masses of each molecule in the protein population are completely consistent or have very small differences (usually the error range is within the accuracy of mass spectrometry detection, such as ±0.1%~1%).

[0060] 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.

[0061] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram and naming convention for the well plate lines of the cell scratch migration experiment.

[0063] Figure 2 This is a photo of cell scratches before treatment with the biomimetic exosome composition of Example 1 in Experimental Example 3.

[0064] Figure 3 This is a photo of cell scratches 48 hours after treatment with the biomimetic exosome composition of Example 1 in Experimental Example 3. DETAILED DESCRIPTION

[0065] 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.

[0066] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0067] 1. The sources of some of the reagents used in the embodiments or comparative examples of the present invention are as follows: Collagen: Yuelaixin ® Recombinant type III collagen, 55 kDa (single molecular weight), model: SFA01; Zhejiang Zhuji Juyuan Biotechnology Co., Ltd.

[0068] RGD peptide modified ceramide: take Nifutai ® Ceramide (Shandong Huazhen Biotechnology Group Co., Ltd.) was dried under reduced pressure at room temperature or freeze-dried to obtain a powder.

[0069] 2. Encapsulation efficiency test Accurately measure 0.5 g of the sample to be tested and place it in an ultrafiltration centrifuge tube (molecular weight cutoff: 10 kDa). Centrifuge at 10,000 r / min for 30 min. Transfer the filtrate to a 5 mL volumetric flask, dissolve it in methanol, dilute to the mark, and shake well. Filter through a 0.45 μm microporous membrane and inject into a liquid chromatograph. Calculate the active ingredient content in the supernatant using the external standard method based on the main peak area. Separately, add methanol to the sample to be tested. Ultrasonic demulsification is performed at a sample:methanol ratio of 1:4 (v / v) for 30 min. Filter through a 0.45 μm organic filter membrane and inject into a liquid chromatograph. Calculate the active ingredient content in the sample to be tested using the external standard method based on the main peak area.

[0070] Encapsulation efficiency % = (active ingredient content in the sample to be tested - free active ingredient content in the supernatant) / active ingredient content in the sample to be tested × 100% 3. In vitro fibroblast inflammatory factor content test 1. Definition / Abbreviation DPBS: Dulbecco's phosphate buffered saline; FBS: fetal bovine serum; DMSO: dimethyl sulfoxide; LPS: lipopolysaccharide; DXM: dexamethasone.

[0071] 2. Principle When the 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, based on an in vitro evaluation model for anti-inflammatory efficacy, employed an LPS-stimulated fibroblast model to assess anti-inflammatory efficacy. The anti-inflammatory efficacy of the test sample was evaluated by measuring changes in the levels of related inflammatory factors after the sample acted on the fibroblasts.

[0072] 3. Procedure 3.1 Equipment and consumables: CO2 incubator, biological safety cabinet, PCR instrument, fluorescence quantitative PCR instrument, autoclave, microplate reader, cell counter, 12-well cell culture plate, pipette.

[0073] 3.2 Solution reagents: (1) Complete culture medium: containing 89 wt% DMEM medium (low glucose, containing pyruvate) (Gibco; Catalog No.: 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 No.: 15140-122); (2) LPS solution: Weigh a certain amount of LPS (lipopolysaccharide) and dissolve it in phosphate buffer to make a 1 mg / mL stock solution. Filter and sterilize it with a 0.22 μm filter and store it at -20°C to avoid repeated freezing.

[0074] (3) DXM stock solution: Weigh a certain amount of dexamethasone and dissolve it in DMSO (dimethyl sulfoxide) to make a 500 mg / ml stock solution. Filter and sterilize with a 0.22 μm filter and store at -20°C to avoid repeated freezing.

[0075] (4) Positive control: Take a certain amount of DXM stock solution and dilute it with complete culture medium to a 400 μg / mL working solution for immediate use.

[0076] 4. Experimental Design

[0077] 5. Experimental methods (1) Sample processing Water-soluble samples are diluted with cell culture medium to form a multi-step concentration gradient. Oil-soluble samples are first diluted with DMSO or another organic solvent, then diluted with cell culture medium to form a multi-step concentration gradient. Check for precipitation. Samples are first diluted to a high concentration, sterilized with a sterilizing filter, and then diluted to the working concentration.

[0078] (2) Experimental steps Cell subculture Perform cell recovery, culture and passaging procedures on cells.

[0079] Cell proliferation rate assay Cytotoxicity experiment: According to the toxicity results, select the appropriate sample concentration to measure the content of inflammatory factors in cells.

[0080] Cell seeding plate Select cells in the logarithmic growth phase, harvest them after trypsinization, prepare a cell suspension, and count them using a cell counter. Dilute the cells with cell culture medium to an appropriate seeding density (70%–80% confluency 24 hours after seeding) and seed them into 12-well culture plates with 1 mL per well. After seeding, incubate the plates in a CO2 incubator for 24 ± 2 hours.

[0081] Cell loading Discard the culture medium in the cell culture plate and proceed with dosing. Add complete culture medium to the blank wells, complete culture medium containing 1 μg / mL LPS and varying concentrations of the test substance to the test substance wells, complete culture medium containing 10 μg / mL LPS or complete culture medium containing 10 μg / mL LPS test substance solvent to the model control wells, and complete culture medium containing 10 μg / mL LPS and 400 μg / mL DXM to the positive control wells. Add 1 mL of complete culture medium to each well of the 12-well plate. Incubate in a CO2 incubator for 4 h ± 15 min.

[0082] Inflammatory factor content detection RNA was extracted according to the kit instructions, its concentration was detected and quantified, and reverse transcribed into DNA for qPCR reaction.

[0083] 6. Result calculation Calculated according to the formula ∆CT = CT (target gene) - CT (reference gene) ∆∆CT = ∆CT (target gene) - ∆CT (NC average) Fold expression=2^ -(∆∆CT) IV. Cell Scratch Migration Assay 1. Definition / Abbreviation DMEM: high glucose medium; DPBS: Dulbecco's phosphate buffered saline; FBS: fetal bovine serum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, also known as: thiazolyl blue; DMSO: dimethyl sulfoxide.

[0084] 2. Principle The wound healing assay is a simple method for measuring cell migration and repair. Similar to an in vitro wound healing model, a micropipette tip or other hard object is used to scratch a line in the center of a monolayer of adherent cells cultured in a dish or plate, creating a blank area called a "scratch." Cells at the edge of the scratch gradually move into the blank area, healing the wound. This, to a certain extent, mimics the in vivo cell migration process and is a relatively simple in vitro method for studying cell migration. The wound healing assay is a simple, low-cost method for measuring cell movement and can be used to examine the invasive and metastatic abilities of adherent cells.

[0085] 3. Procedure 3.1 Equipment and consumables: CO2 incubator, biological safety cabinet, inverted phase contrast microscope, autoclave, microplate reader, cell counter, 96-well cell culture plate, pipette.

[0086] 3.2 Solution reagents: (1) Complete culture medium: 89 wt% DMEM + 10 wt% FBS + 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).

[0087] (2) 5 mg / ml MTT stock solution: Dissolve MTT in DPBS to a concentration of 5 mg / mL. Sterilize the solution with a 0.22 μm syringe filter and store at -20°C in the dark.

[0088] (3) 0.5 mg / ml MTT working solution: Dilute 5 mg / ml MTT stock solution with complete culture medium to 1 mg / ml MTT working solution, and prepare it for use immediately.

[0089] 3.3 Experimental methods (1) Sample processing Water-soluble samples are diluted with cell culture medium to form a multi-step concentration gradient. Oil-soluble samples are first diluted with DMSO or another organic solvent, then diluted with cell culture medium to form a multi-step concentration gradient. Check for precipitation. Samples are first diluted to a high concentration, sterilized with a sterilizing filter, and then diluted to the working concentration.

[0090] (2) Experimental steps Cell subculture The cells were revived and cultured according to the recovery, passaging and freezing procedures of ZKJ-XB-OI-007 cells.

[0091] Cell proliferation rate assay Cytotoxicity experiments were performed according to the ZKJ-XB-OI-000 protocol, and the sample concentration with cell viability ≥ 80% was selected for the scratch test.

[0092] Cell seeding plate Before seeding cells in 6-well plates Figure 1 As shown, use a marker to draw straight parallel lines on the back of the cell plate with a spacing of about 0.5 cm.

[0093] Cells in the logarithmic growth phase were digested and centrifuged with 0.25% trypsin. A cell suspension of appropriate density was prepared in complete culture medium for each cell type, with a confluency of ≥98% after 24 hours. The prepared cell suspension was seeded into a 6-well culture plate according to the experimental requirements, with 2 ml added to each well. Experimental requirements: Three parallel fields of view were photographed for each sample concentration. A solvent control or blank control was also prepared for each experiment. The cells were cultured in a 37°C, 5% CO2 incubator for 24 ± 2 hours.

[0094] Cell scratch When the cell confluence reaches more than 98%, use a sterile pipette tip to draw straight lines perpendicular to the bottom of the 6-well plate (usually 3 lines). The direction of the lines should be perpendicular to the straight lines drawn with the marker. After the lines are drawn, discard the original culture medium and wash the cells with DPBS 3 times. Remove the drawn cells and add samples according to the experimental groups. Add serum-free culture medium containing samples of different concentrations to the experimental group, add serum-free culture medium containing solvent to the solvent control group, and add serum-free culture medium to the blank control group. Add 2 ml to each well and continue to culture in a 37°C, 5% CO2 incubator.

[0095] According to the experimental requirements, select the appropriate time points for taking pictures (0h, 24h, 48h, 72h), and use an inverted microscope to randomly select 3 fields of view in each experimental group hole to take pictures (the scratch size of the 3 fields of view should be similar). Be careful to ensure that the conditions for each picture are consistent (consistent field of view and magnification at each time point). Image naming rules (see Figure 1 ): Sample number - experimental group abbreviation - field of view (well number of the 6-well plate + number of rows of scratches + number of points divided by the marked line on the scratch) - scratch time point.

[0096] Result Analysis The images were analyzed and processed using Image J software. The software operation steps are as follows: ①Open Image J (image processing software) v1.51 official version software.

[0097] ②Open the picture: Select [File], click [Open], and then select the picture to be analyzed.

[0098] ③Convert the image to grayscale: Select [Image], click [Type], and then click [8-bit].

[0099] ④ Enhance contrast: Click [Process], then select [Enhance Contrast] - [Saturated pixels], select 0.3, the default is Normalize, and finally click [OK].

[0100] ⑤ Smooth + find edges, threshold setting: Smooth edges: Process→Smooth (Ctrl+Shift+S); Find edges: Process→Find Edges.

[0101] ⑥Threshold setting: [Image] → Adjust → Threshold (Ctrl+Shift+T); Set and select: Select red, adjust the threshold to 0-50 (adjust according to the image to clearly define the scratch and cell edges), click [Apply], select the Magic Wand tool in the [Toolbar], and click on the scratch area.

[0102] ⑦Click [Analyze] → [Measure] to measure the area.

[0103] ⑧Export original data: Select [File] → Save As The Image J analysis results were exported and archived as raw data, where the “Area” data was the scratch area for subsequent calculations.

[0104] 4. Result calculation Calculation of cell migration rate The scratch area was analyzed using Imagej software, and the cell migration ability was expressed as migration rate; Cell migration rate (%) = (scratch area at 0 h - scratch area at each time point) / scratch area at 0 h × 100%.

[0105] Example 1-Example 3: Preparation of biomimetic exosome composition Recipe: See Table 1.

[0106] Table 1: Biomimetic exosome composition formula

[0107]

[0108] Preparation method: S1: Preparation of alcohol phase: mixing the phospholipid compound, targeting ligand, stabilizer and polyol to obtain an alcohol phase; S2: Preparation of internal loading phase: mixing the active ingredient with the solvent to obtain the internal loading phase; S3: Preparation of a mixed solution: Add the internal load phase obtained in step S2 dropwise to the alcohol phase obtained in step S1 at a rate of 3 ml / min, and stir to obtain a mixed solution; S4: The mixed solution obtained in step S3 was treated by a membrane extrusion method (3.0 MPa, polycarbonate membrane, pore size 100 nm, extrusion times 10 times) to finally obtain the biomimetic exosome composition.

[0109] Comparative Examples 1-7: Investigation of Stabilizers The selection of stabilizers was investigated based on the formulation and preparation method of Example 2, as follows: Comparative Example 1: The only difference from Example 2 is that phytosphingosine and sodium stearoyl glutamate are replaced by cholesterol, that is, the stabilizer in Comparative Example 1 is only cholesterol, and the other conditions are the same as Example 2.

[0110] Comparative Example 2: The only difference from Example 2 is that cholesterol and sodium stearoyl glutamate are replaced by phytosphingosine, that is, the stabilizer in Comparative Example 2 is only phytosphingosine, and the other conditions are the same as Example 2.

[0111] Comparative Example 3: The only difference from Example 2 is that cholesterol and phytosphingosine are replaced by sodium stearoyl glutamate, that is, the stabilizer in Comparative Example 3 is only sodium stearoyl glutamate, and the other conditions are the same as Example 2.

[0112] Comparative Example 4: The only difference from Example 2 is that cholesterol is replaced by sodium cholate, and the other conditions are the same as Example 2.

[0113] Comparative Example 5: The only difference from Example 2 is that sodium stearoyl glutamate is replaced by cholesterol, that is, the stabilizers in Comparative Example 5 are only cholesterol and phytosphingosine, and the other conditions are the same as Example 2.

[0114] Comparative Example 6: The only difference from Example 2 is that phytosphingosine is replaced by cholesterol, that is, the stabilizers in Comparative Example 6 are only cholesterol and sodium stearoyl glutamate, and the other conditions are the same as Example 2.

[0115] Comparative Example 7: The only difference from Example 2 is that cholesterol is replaced by phytosphingosine, that is, the stabilizers in Comparative Example 7 are only phytosphingosine and sodium stearoyl glutamate, and the other conditions are the same as Example 2.

[0116] Comparative Examples 8-10: Investigation of Phospholipid Compounds The selection of phospholipid compounds was investigated based on the formulation and preparation method of Example 2, as follows: Comparative Example 8: The only difference from Example 2 is that distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is replaced by soybean lecithin, and the other conditions are the same as Example 2.

[0117] Comparative Example 9: The only difference from Example 2 is that distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is replaced by hydrogenated soy lecithin, and the other conditions are the same as Example 2.

[0118] Comparative Example 10: The only difference from Example 2 is that 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is replaced by soybean lecithin, and the other conditions are the same as Example 2.

[0119] Test Example 1: Encapsulation Efficiency Stability Investigation Accelerated stability study: The biomimetic exosome compositions obtained in the above examples and comparative examples were placed under accelerated conditions (25°C ± 2°C / 60% RH) for 6 months to test the stability of the encapsulation efficiency during the accelerated conditions. The results are shown in Table 2.

[0120] Table 2: Stability encapsulation efficiency under accelerated conditions

[0121] in conclusion: (1) Compared with other stabilizers or stabilizer combinations, the stabilizer combination provided by the present invention (cholesterol + phytosphingosine + sodium stearoyl glutamate, see Examples 1-3) is more conducive to improving the encapsulation rate stability of the obtained biomimetic exosome composition, and has unexpected technical effects.

[0122] (2) Compared with other phospholipid compounds or phospholipid compound combinations, the phospholipid compound combination provided by the present invention (distearoylphosphatidylethanolamine-polyethylene glycol 2000+1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, see Examples 1-3) is more conducive to improving the encapsulation rate stability of the obtained biomimetic exosome composition, and has unexpected technical effects.

[0123] Test Example 2: In vitro fibroblast inflammatory factor inhibition test The biomimetic exosome compositions obtained in the above examples were respectively taken to perform in vitro fibroblast inflammatory factor content test at a test concentration of 0.1 wt %, and the inhibition rate was calculated. The results are shown in Table 3.

[0124] Table 3: Inhibition rate of inflammatory factors in fibroblasts in vitro

[0125] Conclusion: The biomimetic exosome composition provided by the present invention has good anti-inflammatory effect.

[0126] Experimental Example 3: Cell scratch migration assay The biomimetic exosome compositions obtained in the above examples were used to perform cell scratch migration experiments. The results are shown in Table 4.

[0127] Table 4: Results of cell scratch migration assay

[0128] Conclusion: From the results in Table 4, it can be seen that the biomimetic exosome composition provided by the present invention has excellent repair effect.

[0129] 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 biomimetic exosome, characterized in that: include: membrane structure and internal loading phase; The membrane structure includes a phospholipid bilayer, a stabilizer and a targeting ligand; The internal loading phase includes a solvent and an active ingredient; The phospholipid bilayer is composed of phospholipid compounds; The phospholipid compound includes at least one of distearoylphosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The stabilizer includes at least one of cholesterol, phytosphingosine and sodium stearoyl glutamate.

2. The biomimetic exosome according to claim 1, wherein the stabilizer comprises cholesterol, phytosphingosine and sodium stearoyl glutamate; and / or The phospholipid compounds include distearoylphosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The solvent includes water; The active ingredient is a water-soluble active ingredient; and / or The active ingredient includes water-soluble amino acids or proteins; and / or The protein comprises collagen; and / or The weight average molecular weight of the protein is 5kDa-100kDa; and / or The protein is a single molecular weight protein, wherein the single molecular weight of the single molecular weight protein is selected from 5kDa-100kDa; and / or The targeting ligand includes RGD peptide-modified ceramide.

3. The biomimetic exosome according to any one of claims 1-2, wherein the mass ratio of the phospholipid compound to the stabilizer is 1:1-10:1 or 2:1-10:1; and / or The mass ratio of cholesterol, phytosphingosine and sodium stearoyl glutamate is (0.1-100): (0.01-50): (0.1-100) or (10-30): (5-20): (5-10); and / or The mass ratio of the phospholipid compound to the internal load phase is 1:5-1:10; and / or The mass ratio of the solvent to the active ingredient is 10:1-50:1 or 24:1-49:1; and / or The mass ratio of the phospholipid compound to the targeting ligand is 100:1-200:1; and / or The mass ratio of the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is (3-800): (1-100) or 1:9-19:

1.

4. A biomimetic exosome composition, characterized in that: Comprising the biomimetic exosomes according to any one of claims 1 to 3; and / or The biomimetic exosome composition further comprises a polyol; and / or The polyol includes at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol.

5. A method for preparing the biomimetic exosomes according to any one of claims 1 to 3 or the biomimetic exosome composition according to claim 4, characterized in that: The following steps are involved: S1: Preparation of alcohol phase: mixing the phospholipid compound, targeting ligand, stabilizer and polyol to obtain an alcohol phase; S2: Preparation of internal loading phase: mixing the active ingredient with the solvent to obtain the internal loading phase; S3: Preparation of a mixed solution: adding the internal support phase obtained in step S2 dropwise to the alcohol phase obtained in step S1, stirring to obtain a mixed solution; S4: The mixed solution obtained in step S3 is treated by a membrane extrusion method to obtain a biomimetic exosome composition containing biomimetic exosomes.

6. The preparation method according to claim 5, wherein the polyol comprises at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol; and / or The mass ratio of the phospholipid compound to the stabilizer is 1:1-10:1 or 2:1-10:1; and / or The mass ratio of the phospholipid compound to the polyol is 1:5-1:10; and / or The mass ratio of cholesterol, phytosphingosine and sodium stearoyl glutamate is (0.1-100): (0.01-50): (0.1-100) or (10-30): (5-20): (5-10); and / or The mass ratio of the phospholipid compound to the internal load phase is 1:5-1:10; and / or The mass ratio of the solvent to the active ingredient is 10:1-50:1 or 24:1-49:1; and / or The mass ratio of the phospholipid compound to the targeting ligand is 100:1-200:1; and / or The feeding mass ratio of the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is (3-800): (1-100) or 1:9-19:

1.

7. The preparation method according to any one of claims 5 to 6, The dropping speed of step S3 is 1 ml / min-20 ml / min; and / or The membrane extrusion method in step S4 uses a polycarbonate membrane, a nitrocellulose membrane or a mixed cellulose ester membrane as the filter membrane; and / or The pore size of the filter membrane in the filter membrane extrusion method in step S4 is 50nm-150nm or 50nm-100nm; and / or The pressure of the membrane extrusion method in step S4 is 0.2 MPa-10.0 MPa or 1.0 MPa-5.0 MPa; and / or The number of extrusions in the membrane extrusion method of step S4 is 3 to 20 times or 5 to 15 times.

8. Use of the biomimetic exosomes according to any one of claims 1 to 3, the biomimetic exosome composition according to claim 4, or the biomimetic exosomes or biomimetic exosome composition prepared by the preparation method according to any one of claims 5 to 7 in the preparation of cosmetics.

9. A cosmetic, characterized in that: Including the biomimetic exosomes according to any one of claims 1-3, the biomimetic exosome composition according to claim 4, and the biomimetic exosomes or biomimetic exosome composition prepared by the preparation method according to any one of claims 5-7.

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