An engineered bionic exosome with anti-aging effect and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses an engineered bionic exosome with anti-aging efficacy, a preparation method and application thereof, comprising the following steps: (S1) preparing a bionic cell membrane modified targeting ligand: adding collagen, mitochondrial targeting polypeptide, condensing agent, and 4-dimethylaminopyridine to an organic solvent, activating under a protective atmosphere, and then adding lysine to the above system for reaction to obtain a bionic cell membrane modified targeting ligand; (S2) embedding the bionic cell membrane modified targeting ligand into an engineered exosome bionic membrane, and encapsulating active substances to form an engineered bionic exosome. The exosome of the present invention has better skin permeability, mitochondrial targeting effect, and cell endocytosis effect, has a better protective effect on mitochondria, and can achieve efficient delivery of active substances.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an engineered bionic exosome with anti-aging efficacy, and a preparation method and application thereof. Background Art

[0002] Exosomes are nanoscale vesicles (30-100nm) secreted by most cells. They have a phospholipid bimolecular structure and contain substances such as DNA, RNA, proteins, and a large number of active ingredients. They are mainly involved in intercellular communication and material transport and are natural nanocarrier structures. As an emerging nanocarrier, exosomes can penetrate intercellular spaces and target the target site of action. However, there are still many problems that have not been effectively solved in the application of exosomes in the delivery of active substances, such as the diverse risks of cell sources, the difficulty of purification and acquisition, the harsh storage conditions (below -18°C), the carrying of genetic material, and the weak loading capacity.

[0003] Tea polyphenols is a general term for polyphenols in tea, among which flavanols (catechins) are the most important, accounting for about 60%-80% of the total polyphenols; catechins are mainly composed of several monomers such as EGC, DLC, EC, EGCG, GCG, ECG, etc. Epigallocatechin gallate (EGCG) has excellent antioxidant effects, and its ability to scavenge active free radicals is significantly higher than that of vitamin C and vitamin E. It can be used as an antioxidant and anti-aging ingredient. However, due to the large polarity of EGCG and the presence of multiple phenolic hydroxyl groups in its molecular structure, EGCG has problems such as poor stability and low transdermal bioavailability when actually used in cosmetics, resulting in low bioavailability. In order to improve the stability and transdermal permeability of EGCG, nanocarriers are often used in the prior art to encapsulate EGCG, but there is still a problem of extremely low transdermal absorption and inability to effectively penetrate the stratum corneum of the skin to exert its efficacy.

[0004] The decline of mitochondrial function is considered to be an important part of the aging mechanism. With the increase of age, the function of mitochondria gradually declines, which is manifested as reduced energy production, increased oxidative stress, etc. These changes lead to a decline in cell function, which in turn causes the functional decline of tissues and organs, and eventually manifests as aging. Therefore, by activating mitochondrial function, anti-aging can be effectively achieved. In the prior art, the anti-aging effect of EGCG is based on its powerful antioxidant effect, and the anti-aging effect of EGCG in activating mitochondrial function has not been achieved. Therefore, how to target EGCG to mitochondria to exert its effect is also a technical problem that needs to be solved. Summary of the invention

[0005] In order to overcome the above-mentioned prior art, the primary purpose of the present invention is to provide a method for preparing engineered bionic exosomes with anti-aging efficacy.

[0006] Another object of the present invention is to provide engineered bionic exosomes with anti-aging efficacy prepared by the above method.

[0007] The present invention also provides the use of engineered bionic exosomes with anti-aging efficacy prepared by the above method.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The engineered bionic exosomes with anti-aging effects of the present invention are formed in two steps: first, a bionic cell membrane modified targeting ligand is prepared; and then an active substance (EGCG) is encapsulated by exosome engineering bionics. The bionic cell membrane modified targeting ligand is obtained by modifying collagen with a mitochondrial targeting polypeptide.

[0010] Specifically, a method for preparing engineered bionic exosomes with anti-aging efficacy comprises the following steps:

[0011] (S1) preparing a bionic cell membrane modified targeting ligand: adding collagen, a mitochondrial targeting polypeptide, a condensing agent, and 4-dimethylaminopyridine to an organic solvent, activating the mixture under a protective atmosphere, and then adding lysine to the above system for reaction to obtain a bionic cell membrane modified targeting ligand;

[0012] (S2) The engineered bionic exosomes are formed by embedding the bionic cell membrane-modified targeting ligand into the engineered exosome bionic membrane and encapsulating the active substances.

[0013] Preferably, after the reaction (S1) is completed, a purification step is further included: the reaction mixture is transferred to a dialysis bag (molecular weight cutoff is 1500-12000 Da), and dialyzed in deionized water for 48-96 hours to remove unreacted collagen, arginine and active polypeptide raw materials, and the deionized water is replaced every 6 hours. The liquid in the dialysis bag is centrifuged at high speed (8000-10000 rpm) for 10-30 minutes, and the supernatant is freeze-dried for 24-48 hours to obtain a refined bionic cell membrane modified targeting ligand.

[0014] Preferably, the activation conditions in S1 are: activation at -10-10°C for 1-3h; and the reaction conditions are: reaction at 25-50°C for 24-72h.

[0015] Preferably, the mitochondrial targeting polypeptide is at least one of glutathione (Glu-Cys-Gly, GSH), lysine-leucine-alanine tripeptide (Lys-Leu-Ala, LLA), hexapeptide-2 (Gly-Arg-Ala-Asp-Ser-Pro, GAAASP), SS peptide (Cys-D-Arg-Dmt-Lys-PHe-NH2, CADLP), and SS-31 peptide (D-Arg-dimethylTyr-Lys-Phe-NH2); the molecular weight of the collagen is 3000 Da -150000 Da.

[0016] Preferably, the molecular weight of the collagen is 50000Da-100000Da; the molar ratio of the collagen to the mitochondrial targeting polypeptide is 1:0.5-2.5; the molar ratio of the collagen to 4-dimethylaminopyridine is 1:0.5-2.5; and the molar ratio of the collagen to lysine is 1:0.1-1.0.

[0017] Preferably, the condensing agent is at least one of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC); the molar ratio of the collagen to the condensing agent is 1:0.5-2.5;

[0018] The organic solvent is at least one of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, ethanol, tetrahydrofuran, dichloromethane and ethyl acetate; the mass ratio of the collagen to the organic solvent is 1:5-100.

[0019] Preferably, the preparation of the engineered bionic exosomes with anti-aging efficacy comprises the following steps:

[0020] (1) adding phospholipids, biomimetic cell membrane modified targeting ligands, and biomimetic cell membrane stabilizers into a polyol solvent, stirring evenly, and obtaining an alcohol phase;

[0021] (2) adding the active substance to the yeast / Acetobacter xylinum / black tea fermentation product and stirring evenly to obtain an aqueous phase;

[0022] (3) uniformly adding the aqueous phase to the alcohol phase, and after the addition is completed, continuing to stir to obtain a mixed solution;

[0023] (4) The mixed solution is subjected to membrane extrusion treatment to obtain engineered biomimetic exosomes;

[0024] The added amount of the bionic cell membrane modified targeting ligand is 0.02%-0.30% of the weight of the exosomes.

[0025] Preferably, in terms of mass percentage, phospholipids are 2-5%; bionic cell membrane modified targeting ligands are 0.1%-0.2%; bionic cell membrane stabilizers are 0.1-0.3%; polyol solvents are 10-30%; active substances are 1-7%; yeast / acetobacter xylinum / black tea fermentation products are 57.5-86.8%; and the active substances are tea polyphenols.

[0026] Preferably, the tea polyphenols include one or more of epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin gallate (GC), epigallocatechin gallate (CG) and epigallocatechin (EGC).

[0027] Preferably, the phospholipid in step (1) is at least one of soybean lecithin, hydrogenated soybean lecithin, enzymatically hydrolyzed lecithin, dipalmitoylphosphatidylcholine, palmitoylphosphatidylglycerol and egg yolk lecithin.

[0028] Preferably, the bionic cell membrane stabilizer is at least one of cholesterol and sodium cholate.

[0029] Preferably, the polyol solvent is at least one of 1,3-butylene glycol, 1,3-propylene glycol, glycerol, 1,2-hexanediol, and ethoxydiglycol. The solvent is green, safe, non-toxic, and has low skin irritation, thus avoiding skin sensitive stress reactions.

[0030] Preferably, the stirring temperature in step (1) is 25-60° C.; the stirring speed is 100-500 rpm; and the stirring time is 0.5-6 h.

[0031] Preferably, the stirring temperature in step (2) is 25-60° C.; the stirring speed is 100-500 rpm; and the stirring time is 10-20 min.

[0032] Preferably, in step (3), the dropping speed is 2-10 mL / min; the stirring temperature is 25-60° C.; the stirring speed is 100-500 rpm; and the stirring time is 2-4 h.

[0033] Preferably, the filter membrane in step (4) is a polycarbonate membrane, the pressure is 1.0-7.0 MPa, and the extrusion time is 10-30 min.

[0034] Preferably, the stirring conditions of steps (1) and (3) are a rotation speed of 300±150 rpm and a time of 3±1h; the stirring conditions of step (2) are a rotation speed of 300±100 rpm and a time of 30±20min; the dropping speed of step (3) is 5±3 ml / min; and the extrusion treatment conditions of step (4) are a pressure of 4.0±2.0MPa and a treatment time of 20±5min.

[0035] In the above reaction steps, inert gas protection is used, and the inert gas used is at least one of nitrogen, helium and argon gases commonly used in the art.

[0036] The stirring method in steps (1) to (3) is magnetic stirring or mechanical stirring.

[0037] The engineered bionic exosomes with anti-aging effects prepared by the above method can be used in cosmetics or in the preparation of external skin medicines.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) The present invention uses mitochondrial targeting active polypeptides to modify collagen to prepare bionic cell membrane modified targeting ligands, and then embeds the bionic cell membrane modified targeting ligands into the engineered exosome bionic membrane to encapsulate active substances to prepare engineered bionic exosomes, which can effectively target and deliver active substances to mitochondria to play a protective role. Compared with collagen (not modified with mitochondrial targeting polypeptides) and non-mitochondrial targeting polypeptide modified collagen on the market, the engineered bionic exosomes in the present invention have better skin permeability, mitochondrial targeting effect and cell endocytosis effect, and have a better protective effect on mitochondria, which can achieve efficient delivery of active substances.

[0040] (2) On the one hand, the present invention can give the product the properties of encapsulating active substances and targeted delivery that exosomes have, and on the other hand, it can solve the problems of difficult preservation, unknown safety, and difficulty in obtaining exosomes.

[0041] (3) Compared with free EGCG (not encapsulated by engineered bionic exosomes), the engineered bionic exosomes of the present invention have better skin permeability and mitochondrial targeting, have better protective effects on mitochondria, and can achieve anti-aging from the source. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a photo of the exosome sample prepared in Example 1.

[0043] Figure 2 This is a graph showing the mitochondrial membrane potential test results of the samples of Example 1 and Comparative Example 1 using the JC-1 method.

[0044] Figure 3 These are fluorescent photographs of mouse skin penetration thickness of samples in Example 1 and Comparative Examples 1, 2, and 10.

[0045] Figure 4 The graph is a mouse skin penetration test result graph of Example 1 and Comparative Examples 1, 2, and 10.

[0046] Figure 5These are fluorescent photos of cell endocytosis experiments of samples in Example 1 and Comparative Examples 1 and 10.

[0047] Figure 6 The figure is the result of the cell endocytosis test of the samples of Example 1 and Comparative Examples 1 and 10. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. In the examples, the specific conditions are indicated, and the conditions recommended by the manufacturer are carried out according to the conventional conditions. The reagents or instruments used that do not indicate the manufacturer are conventional products that can be purchased commercially. The reagents used in the examples can be purchased conventionally from the market unless otherwise specified.

[0049] In the embodiments of the present invention, the phospholipids used can be purchased directly from Shanghai Taiwei Pharmaceutical Co., Ltd. The collagen, glutathione, lysine-leucine-alanine tripeptide, hexapeptide-2 and SS peptide used can be purchased directly from Guangdong Shanyan Biotechnology Co., Ltd. Among them, type III collagen macromolecules can be obtained by enzymatic hydrolysis of type III collagen with a small molecular weight after α-chymotrypsin, and α-chymotrypsin is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The yeast / acetobacter xylinum / black tea fermentation products used can be purchased directly from Guangzhou Xiyuan Biotechnology Co., Ltd.

[0050] Example 1

[0051] (1) Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 100k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine source, Mn=100000, 100 g, 1.0 mmol), glutathione (0.461 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (1000 mL) and activated at 0°C for 2 h. Under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GSH-COL 100k (90.1 g, yield 89.7%).

[0052] (2) Preparation of engineered bionic exosomes: 3.5% phospholipids and 0.15% GSH-COL were added to 100k , 0.15% cholesterol by mass was added to 20% 1,3-butanediol by mass, and stirred (speed 300 rpm) for 3h to obtain the alcohol phase; 5% EGCG by mass was added to 71.2% yeast / acetobacterium xylinum / black tea fermentation product by mass, and stirred (speed 300 rpm) for 30min to obtain the aqueous phase; the aqueous phase was uniformly added to the alcohol phase at a dropping speed of 5 ml / min. After the addition was completed, stirring was continued (speed 300 rpm) for 3h. The above mixed solution was subjected to membrane extrusion (pressure of 4.0MPa) for 20min to obtain engineered bionic exosomes (code: EGCG True Tea V1). The appearance of the sample is as follows Figure 1 shown.

[0053] (Example 2-4 Different types of active peptides)

[0054] Example 2

[0055] (1) Biomimetic cell membrane modification and targeted embedding of ligand LLA-COL 100k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine source, Mn=100000, 100 g, 1.0 mmol), lysine-leucine-alanine tripeptide (0.550 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (1000 mL) and activated at 0°C for 2 h; under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand LLA-COL 100k (87.6 g, yield 87.2%).

[0056] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% LLA-COL 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V2) were obtained.

[0057] Example 3

[0058] (1) Biomimetic cell membrane modification and targeted embedding of ligand GAAASP-COL 100k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine source, Mn=100000, 100 g, 1.0 mmol), GAAASP (0.903 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (1000 mL) and activated at 0°C for 2 h. Under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GAAASP-COL 100k (87.2 g, yield 86.6%).

[0059] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% by mass of GAAASP-COL 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V3) were prepared.

[0060] Example 4

[0061] (1) Biomimetic cell membrane modification and targeted embedding of ligand CADLP-COL 100k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine, Mn=100000, 100 g, 1.0 mmol), CADLP (1.224 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (1000 mL) and activated at 0°C for 2 h. Lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand CADLP-COL100k (83.3 g, yield 82.5%).

[0062] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with CADLP-COL with a mass percentage of 0.15% 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V4) were obtained.

[0063] (Example 5-7 Collagen with different molecular weights)

[0064] Example 5

[0065] (1) Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 50k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine, Mn=50000, 50 g, 1.0 mmol), glutathione (0.461 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (500 mL) and activated at 0°C for 2 h. Under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GSH-COL 50k (40.5 g, yield 80.2%).

[0066] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% by mass of GSH-COL 50k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V5) were prepared.

[0067] Example 6

[0068] (1) Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 10kPreparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine, Mn=10000, 10 g, 1.0 mmol), glutathione (0.461 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (100 mL) and activated at 0°C for 2 h. Under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GSH-COL 10k (8.38 g, yield 80.2%).

[0069] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% by mass of GSH-COL 10k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V6) were prepared.

[0070] Example 7

[0071] (1) Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 3k Preparation (taking the preparation of 1.0 mmol as an example): Type III enzymatic hydrolyzed collagen (porcine, Mn=3000, 3 g, 1.0 mmol), glutathione (0.461 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (300 mL) and activated at 0°C for 2 h. Under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 3000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GSH-COL 3k (2.01 g, yield 81.8%).

[0072] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% by mass of GSH-COL 3k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V7) were prepared.

[0073] (Examples 8-11 Different amounts of added ligands for biomimetic cell membrane modification and targeted embedding)

[0074] Example 8

[0075] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k Replaced with 0.02% GSH-COL 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V8) were obtained.

[0076] Example 9

[0077] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k Replaced with 0.10% by mass of GSH-COL 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V9) were prepared.

[0078] Example 10

[0079] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k Replaced with 0.20% GSH-COL 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V10) were prepared.

[0080] Embodiment 11

[0081] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k Replaced with 0.30% by mass of GSH-COL 100k , the other conditions remained unchanged, and engineered bionic exosomes (code: EGCG True Tea V11) were prepared.

[0082] (Examples 12-14 Different amounts of EGCG added)

[0083] Example 12

[0084] The 5% by mass EGCG in Example 1 was replaced with 1% by mass EGCG, and the other conditions remained unchanged to prepare engineered bionic exosomes (code: EGCG True Tea V12).

[0085] Example 13

[0086] The 5% by mass EGCG in Example 1 was replaced with 3% by mass EGCG, and the other conditions remained unchanged to prepare engineered bionic exosomes (code: EGCG True Tea V13).

[0087] Embodiment 14

[0088] The 5% by mass EGCG in Example 1 was replaced with 7% by mass EGCG, and the other conditions remained unchanged to prepare engineered bionic exosomes (code: EGCG True Tea V14).

[0089] (Examples 15-16 have different preparation process parameters)

[0090] Embodiment 15

[0091] Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 100k The preparation steps are the same as in Example 1.

[0092] The mass percentage of phospholipids was 3.5%, the mass percentage of GSH-COL was 0.15%. 100k , 0.15% cholesterol by mass was added to 20% 1,3-butanediol by mass, stirred (speed 450 rpm) for 2h to obtain the alcohol phase; 5% EGCG by mass was added to 71.2% yeast / acetobacterium xylinum / black tea fermentation product by mass, stirred (speed 150 rpm) for 15min to obtain the aqueous phase; the aqueous phase was uniformly added to the alcohol phase at a drop rate of 2ml / min. After the drop addition was completed, the stirring was continued (speed 300 rpm) for 3h. The above mixed solution was squeezed through a filter membrane (pressure of 2.0MPa) for 15min to obtain the engineered bionic exosomes (code: EGCG True Tea V15).

[0093] Example 16

[0094] Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 100k The preparation steps are the same as in Example 1.

[0095] The mass percentage of phospholipids was 3.5%, the mass percentage of GSH-COL was 0.15%. 100k, 0.15% cholesterol by mass was added to 20% 1,3-butanediol by mass, stirred (speed 150 rpm) for 4h to obtain the alcohol phase; 5% EGCG by mass was added to 71.2% yeast / acetobacterium xylinum / black tea fermentation product by mass, stirred (speed 350 rpm) for 10min to obtain the aqueous phase; the aqueous phase was uniformly added to the alcohol phase at a drop rate of 8ml / min. After the drop was completed, it was continued to stir (speed 300 rpm) for 3h. The above mixed solution was squeezed through a filter membrane (pressure of 6.0MPa) for 25min to obtain the engineered bionic exosomes (code: EGCG True Tea V16).

[0096] Comparative Example 1 (Collagen original protein without targeted modification)

[0097] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k The EGCG composition (No.: EGCG-A1) was prepared by replacing the type III collagen (porcine source, Mn=100000) with 0.15% by mass and keeping other conditions unchanged.

[0098] Comparative Example 2 (without addition of biomimetic cell membrane modified targeted embedded ligand)

[0099] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k The EGCG composition (No.: EGCG-A2) was prepared by replacing the mixture with 0.15% by mass of 1,3-butanediol while keeping other conditions unchanged.

[0100] Comparative Example 3 (Collagen and GSH were not modified by reaction and were directly added to the composition)

[0101] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k The EGCG composition (No.: EGCG-A3) was prepared by replacing 0.1495% by mass of type III collagen (porcine source, Mn=100000) and 0.0005% by mass of glutathione with other conditions remaining unchanged.

[0102] Comparative Example 4 (Collagen Molecular Weight Too Large)

[0103] (1) Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 500kPreparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine source, Mn=500000, 500 g, 1.0 mmol), glutathione (0.461 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (5000 mL) and activated at 0°C for 2 h; under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GSH-COL 500k .

[0104] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% by mass of GSH-COL 500k , and the other conditions remained unchanged to obtain engineered bionic exosomes (code: EGCG-A4).

[0105] Comparative Example 5 (Collagen Molecular Weight Too Small, Collagen Tripeptide)

[0106] (1) Biomimetic cell membrane modification and targeted embedding of ligand GSH-COL 0.28k Preparation (taking the preparation of 1.0 mmol as an example): Type III enzymatically hydrolyzed collagen (porcine source, Mn=280, 0.28 g, 1.0 mmol), glutathione (0.461 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (2.8 mL) and activated at 0°C for 2 h; under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 500Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand GSH-COL 0.28k .

[0107] (2) Preparation of engineered bionic exosomes: 0.15% by mass of GSH-COL in Example 1 100k Replaced with 0.15% by mass of GSH-COL 0.28k , and the other conditions remained unchanged to obtain engineered bionic exosomes (code: EGCG-A5).

[0108] Comparative Example 6 (the amount of biomimetic cell membrane modified targeted embedded ligand added is too low)

[0109] The mass percentage of GSH-COL in Example 1 is 0.15%. 100k Replaced with 0.01% GSH-COL 100k , and the other conditions remained unchanged to obtain engineered bionic exosomes (code: EGCG-A6).

[0110] Comparative Example 7 (Excessive EGCG Addition)

[0111] The 5% by mass EGCG in Example 1 was replaced with 10% by mass EGCG, and the other conditions remained unchanged to prepare engineered bionic exosomes (No.: EGCG-A7).

[0112] Comparative Example 8 (Non-targeted active polypeptide modified collagen)

[0113] (1) Modified collagen PAL-COL 100k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine, Mn=100000, 100 g, 1.0 mmol), palmitoyl pentapeptide-4 (1.203 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (1000 mL) and activated at 0°C for 2 h; under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand PAL-COL 100k .

[0114] (2) Add 0.15% by mass of GSH-COL in Example 1 100k Replace with 0.15% PAL-COL 100k, and the other conditions remained unchanged to obtain an EGCG composition (No.: EGCG-A8).

[0115] Comparative Example 9 (Non-targeted active polypeptide modified collagen)

[0116] (1) Modified collagen ACH-COL 100k Preparation (taking the preparation of 1.0 mmol as an example): Type III collagen (porcine source, Mn=100000, 100 g, 1.0 mmol), acetyl hexapeptide-8 (1.305 g, 1.5 mmol), DCC (0.309 g, 1.5 mmol) and 4-dimethylaminopyridine (0.183 g, 1.5 mmol) were added to N,N-dimethylformamide (1000 mL) and activated at 0°C for 2 h; under argon protection, lysine (0.088 g, 0.6 mmol) was added to the reaction system and reacted at 40°C for 48 hours; the reaction mixture was transferred to a dialysis bag (molecular weight cutoff of 10000Da), dialyzed with deionized water for 72 hours, and the deionized water was replaced every 6 hours; the liquid in the dialysis bag was centrifuged at high speed (speed of 9000rpm) for 20 minutes, and the supernatant was freeze-dried for 36 hours to obtain the bionic cell membrane modified targeted embedded ligand ACH-COL 100k .

[0117] (2) Add 0.15% by mass of GSH-COL in Example 1 100k Replace with 0.15% ACH-COL 100k , and the other conditions remained unchanged to obtain an EGCG composition (No.: EGCG-A9).

[0118] Comparative Example 10 (EGCG aqueous solution)

[0119] 5% by mass of EGCG was dissolved in 95% by mass of deionized water to prepare an EGCG aqueous solution.

[0120] Test Example 1 Particle Size Characterization

[0121] The samples of Examples 1-16 and Comparative Examples 1-7 were diluted 100 times with deionized water, and the particle sizes of the samples were characterized by a Malvern Nano-ZS90 dynamic light scattering particle size analyzer. The test angle was 90° and the test temperature was 25°C. Three parallel experiments were conducted for each group of experiments, and the arithmetic mean of the experimental results was taken.

[0122] Test Example 2 Encapsulation Efficiency Characterization

[0123] Examples 1-16 and Comparative Examples 1-7, take 200 μL sample was ultrafiltrated and centrifuged (9000 rpm, 30 min) and 5 μ The filtrate was tested by high performance liquid chromatography (HPLC, Shimadzu, Japan) to determine the EGCG content in the filtrate, i.e., the free EGCG content in the test sample. Another test sample was added with a mixed solution of methanol and deionized water, and ultrasonic demulsification was performed for 30 min at a ratio of sample: methanol: deionized water = 1:4:5 (v / v). μ After filtering with organic filter membrane, take 5 μ The EGCG content of the sample solution was determined by high performance liquid chromatography (HPLC, Shimadzu, Japan) to obtain the total EGCG content in the test sample. The encapsulation efficiency (EE) of the test sample was calculated according to formula (1).

[0124] Formula (1)

[0125] C1 represents the free EGCG content; C0 represents the total EGCG content in the sample after ultrasonic demulsification of a mixed solution of methanol and deionized water.

[0126] Table 1 Particle size and encapsulation efficiency test results of samples of Examples 1-16 and Comparative Examples 1-7

[0127]

[0128] By comparing Examples 1-4, it can be seen that the mitochondrial targeting active peptides in the bionic cell membrane modified targeted embedded ligand are different, which ultimately affects the particle size and encapsulation efficiency of the product. In Examples 1-4, the mitochondrial targeting active peptides are glutathione (GSH), lysine-leucine-alanine tripeptide (LLA), hexapeptide-2 (GAAASP) and SS peptide (CADLP). The results show that the sample in Example 1 has the smallest particle size (60.8nm) and the highest encapsulation efficiency (91.5%).

[0129] Comparison of Example 1, Example 5-7, Comparative Example 4 and Comparative Example 5 shows that the molecular weight of collagen in the biomimetic cell membrane modified targeted embedded ligand is different, which ultimately affects the particle size and encapsulation efficiency of the product. In Example 1, Example 5-7, Comparative Example 4 and Comparative Example 5, the molecular weight of collagen in the biomimetic cell membrane modified targeted embedded ligand is 100000Da, 50000Da, 10000Da, 3000Da, 500000Da and 280Da, respectively. The results show that as the molecular weight of collagen increases, the particle size of the final sample gradually increases, and the encapsulation first increases and then decreases. When the molecular weight of collagen is 100000Da (Example 1), the encapsulation efficiency of the final sample is the highest. When the molecular weight of collagen is 500000Da (Comparative Example 4) and 280Da Comparative Example 5, the encapsulation efficiency of the final sample is too low.

[0130] Comparing Example 1, Example 8-11, Comparative Example 2 and Comparative Example 6, it can be seen that the addition amount of the bionic cell membrane modified targeted embedded ligand is different, which ultimately affects the particle size and encapsulation rate of the product. In Example 1, Example 8-11, Comparative Example 2 and Comparative Example 6, the addition amount of the bionic cell membrane modified targeted embedded ligand is 0.15%, 0.02%, 0.10%, 0.20%, 0.30%, 0%, and 0.01%, respectively. The results show that as the addition amount of the bionic cell membrane modified targeted embedded ligand gradually increases, the particle size of the final sample gradually increases, and the encapsulation first increases and then decreases. When the addition amount of the bionic cell membrane modified targeted embedded ligand is 0.15% (Example 1), the encapsulation rate of the final sample is the highest. When the addition amount of the bionic cell membrane modified targeted embedded ligand is 0.01% (Comparative Example 6) or no bionic cell membrane modified targeted embedded ligand is added (Comparative Example 2), the encapsulation rate of the final sample is too low.

[0131] By comparing Example 1, Examples 12-14, and Comparative Example 7, it can be seen that the different amounts of EGCG added ultimately affect the particle size and encapsulation efficiency of the product. In Example 1, Examples 12-14, and Comparative Example 7, the amounts of EGCG added were 5%, 1%, 3%, 7%, and 10%, respectively. The results show that as the amount of EGCG added gradually increases, the particle size of the final sample gradually increases, and the encapsulation efficiency first increases and then decreases. When the amount of EGCG added is 5% (Example 1), the encapsulation efficiency of the final sample is the highest. When the amount of EGCG added is 10% (Comparative Example 7), the encapsulation efficiency of the final sample is too low.

[0132] By comparing Example 1 and Examples 15-16, it can be seen that the preparation process parameters of engineered bionic exosomes are different, which ultimately affects the particle size and encapsulation efficiency of the product. The results show that the sample finally prepared by the process in Example 1 has the highest encapsulation efficiency.

[0133] By comparing Example 1, Comparative Example 1 and Comparative Example 3, it can be seen that compared with collagen not modified with mitochondrial targeting polypeptide (Comparative Example 1) and direct mixing of GSH and collagen (Comparative Example 3), in Example 1, GSH-modified collagen was first used to prepare bionic cell membrane-modified targeting embedded ligand, and then EGCG was encapsulated by bionic exosome technology, and the encapsulation rate of the sample finally obtained was the highest.

[0134] Test Example 3 ATP Content

[0135] Mitochondria are important regulators of cell energy and metabolism, and play a vital role in maintaining cell growth and survival. The core function of mitochondria is to provide energy to cells by synthesizing ATP through oxidative phosphorylation. 95% of ATP in the human body is provided by mitochondria, so by detecting the amount of ATP generated in cells, the function and activity of mitochondria in cells can be evaluated.

[0136] The ATP content in this test example is detected using an ATP detection kit, which can be purchased directly from Shanghai Biyuntian Biotechnology Co., Ltd.

[0137] The specific experimental method is as follows: (1) Preparation of ATP standard solution: melt the reagent to be used in an ice bath, dilute the ATP standard solution with ATP detection lysis buffer to a certain concentration gradient (0.1nM, 0.3nM, 1nM, 3nM, 10nM, 30nM, 100nM). (2) Sample determination preparation: digest human skin keratinocytes (HaCaT cells), make a cell suspension, and inoculate it into a 6-well plate, 5.0×10 per well. 5 -10.0×10 5 The cells were cultured in DMEM medium for 24 hours, and then the blank control group and the experimental group were exposed to UVA light (light dose of 6-10 J / cm 2 After the illumination, the culture medium was replaced and 50 μ L different test samples (experimental group) or PBS solution (blank group and control group), continue to culture for 24h. Remove the cell culture medium and wash with PBS three times. Collect the cells by centrifugation and wash with PBS once. After absorbing PBS, flick the cells appropriately. Add 200 μL of PBS according to the number of cells in each well. μ L of lysis buffer, lyse the cells, centrifuge at 4°C for 5 min (12,000 rpm), and take the supernatant for testing. (3) Determination of ATP concentration: Add 100 μL of lysis buffer to the test tube. μ L of ATP detection working solution, place at room temperature for 3-5 minutes to consume all the background ATP, thereby reducing the background. μ L of the sample to be tested or ATP standard solution, mix with a micropipette, and measure the RLU value with a chemiluminescence instrument. Draw an ATP standard curve with the ATP content of the ATP standard solution as the horizontal axis and the RLU value as the vertical axis. The ATP content of the sample to be tested can be calculated based on the ATP standard curve.

[0138] Test Example 4 Mitochondrial membrane potential

[0139] Under normal circumstances, the potential of the inner mitochondrial membrane is high and maintained at a negative potential, while the potential of the outer membrane is low and maintained at a positive potential. +) The formation of a transmembrane gradient will lead to a decrease in the mitochondrial membrane potential, which is positive outside and negative inside, i.e. depolarization. A large number of studies have shown that a decrease in mitochondrial membrane potential is related to autophagy, apoptosis or necrosis. Mitochondrial membrane potential dysfunction, or even subtle abnormal changes, may greatly affect the biological activity within the cell.

[0140] Therefore, mitochondrial membrane potential is one of the important indicators for evaluating the normal function of mitochondria. It not only reflects the integrity of mitochondrial function, but also has an important influence on the synthesis of TP (adenosine triphosphate), ion transport, regulation of cell apoptosis and antioxidant effect. It also reflects the health status of cells and is of great significance to the survival and development of cells.

[0141] In this test, the mitochondrial membrane potential was tested using the mitochondrial membrane potential detection kit (JC-1), which can be purchased directly from Shanghai Biyuntian Biotechnology Co., Ltd. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence; in abnormal mitochondrial membrane potential decreases, JC-1 cannot aggregate in the mitochondrial matrix, but is in a monomeric state, showing green fluorescence.

[0142] ① Preparation of JC-1 staining working solution:

[0143] Take an appropriate amount of JC-1 (200×) and dilute JC-1 at a ratio of 8 mL of ultrapure water per 50 μL of JC-1 (200×). Vortex vigorously to fully dissolve and mix JC-1. Then add 2 mL of JC-1 staining buffer (5X) and mix to obtain the JC-1 staining working solution.

[0144] ② Sample processing

[0145] The processing of samples in this test example is the same as that of samples in test example 3.

[0146] ③ JC-1 staining and detection

[0147] Add 0.5 mL of JC-1 staining working solution to the cells in the test group or the positive control group and mix well. Incubate in a cell culture incubator at 37°C for 20 min; discard the supernatant and wash twice with JC-1 staining buffer (1×); add 1 mL / well of staining buffer (1×) to resuspend the cells, centrifuge and discard the supernatant; add 1 mL / well of staining buffer (1×) to resuspend the cells, centrifuge and discard the supernatant; resuspend the cells with an appropriate amount of JC-1 staining buffer (1×) and observe with a laser confocal microscope (CLSM, Leica SP8, Leica Microsystems, USA). The excitation wavelength is 525 nm; the emission wavelength is 590 nm.

[0148] Table 2 ATP content and mitochondrial membrane potential test results of samples

[0149]

[0150] By comparing Examples 1-4, it can be seen that the mitochondrial targeting active peptides in the bionic cell membrane modified targeted embedded ligand are different, which ultimately affects the protective effect of the product on mitochondria. In Examples 1-4, the mitochondrial targeting active peptides are glutathione (GSH), lysine-leucine-alanine tripeptide (LLA), hexapeptide-2 (GAAASP) and SS peptide (CADLP). The results show that after the UV-damaged cells were co-incubated with the sample of Example 1, the ATP content in the mitochondria was the highest, at 0.346nM, which could restore the cells to 98.57% of the level of normal healthy cells; in addition, the fluorescence intensity of JC-1 aggregates in its mitochondria was the largest; the fluorescence intensity of JC-1 monomers was the smallest, and was equivalent to that of normal healthy cells.

[0151] By comparing Example 1, Examples 5-7, Comparative Example 4 and Comparative Example 5, it can be seen that the molecular weight of collagen in the bionic cell membrane modified targeted embedded ligand is different, which ultimately affects the protective effect of the product on mitochondria. The results show that when the molecular weight of collagen is 100,000 Da (Example 1), the engineered bionic exosomes are finally prepared. After the UV-damaged cells are co-incubated with the sample of Example 1, the ATP content in the mitochondria is the highest, and the fluorescence intensity of the JC-1 aggregates in the mitochondria is the highest; the fluorescence intensity of the JC-1 monomer is the lowest, which is equivalent to that of normal healthy cells.

[0152] By comparing Example 1, Examples 8-11, Comparative Example 2 and Comparative Example 6, it can be seen that the addition amount of the bionic cell membrane modified targeted embedded ligand is different, which ultimately affects the protective effect of the product on mitochondria. The results show that when the addition amount of the bionic cell membrane modified targeted embedded ligand is 0.15% (Example 1), the engineered bionic exosomes are finally prepared. After the UV-damaged cells are co-incubated with the sample of Example 1, the ATP content in the mitochondria is the highest, and the fluorescence intensity of the JC-1 aggregates in the mitochondria is the highest; the fluorescence intensity of the JC-1 monomer is the lowest, which is equivalent to that of normal healthy cells.

[0153] By comparing Example 1, Examples 12-14, and Comparative Example 7, it can be seen that the addition amount of EGCG is different, which ultimately affects the protective effect of the product on mitochondria. The results show that when the addition amount of EGCG is 5% (Example 1), the engineered bionic exosomes are finally prepared. After the UV-damaged cells are co-incubated with the sample of Example 1, the ATP content in the mitochondria is the highest, and the fluorescence intensity of the JC-1 aggregates in the mitochondria is the highest; the fluorescence intensity of the JC-1 monomer is the lowest, which is equivalent to that of normal healthy cells.

[0154] By comparing Example 1 and Examples 15-16, it can be seen that the preparation process parameters of engineered bionic exosomes are different, which ultimately affects the protective effect of the product on mitochondria. The results show that the engineered bionic exosomes finally prepared by the process in Example 1 have the best protective effect on mitochondria, and can achieve better anti-aging effects from the source.

[0155] Comparison of Example 1, Comparative Examples 1 and 3 shows that compared with collagen not modified with mitochondrial targeting polypeptide (Comparative Example 1) and physical mixture of GSH and collagen (Comparative Example 3), in Example 1, GSH-modified collagen was first used to prepare bionic cell membrane modified targeting embedded ligand, and then EGCG was encapsulated by bionic exosome technology to finally prepare engineered bionic exosomes. After the UV-damaged cells were co-incubated with the sample of Example 1, the ATP content in the mitochondria was the highest. And combined with Figure 2 It can be seen that the fluorescence intensity of JC-1 aggregates in mitochondria is the highest (red fluorescence), while the fluorescence intensity of JC-1 monomers is the lowest (green fluorescence), which is comparable to that of normal healthy cells.

[0156] Comparison of Example 1, Comparative Examples 8, 9, and 10 shows that compared with the use of non-mitochondrial targeting polypeptides palmitoyl pentapeptide-4 (Comparative Example 8) and acetyl hexapeptide-8 (Comparative Example 9) and EGCG aqueous solution (Comparative Example 10), the use of mitochondrial targeting polypeptide modified collagen to prepare bionic cell membrane modified targeted embedded ligands in Example 1, and then the EGCG is encapsulated by bionic exosome technology, and the engineered bionic exosomes finally prepared have the best protective effect on mitochondria.

[0157] Test Example 6 Mouse Transdermal Absorption Test

[0158] This test example uses live mice for transdermal absorption, which is different from the conventional in vitro transdermal permeation test (vertical diffusion cell). The in vitro transdermal permeation test uses ex vivo nude mouse skin as a model, while this application uses the back skin of live mice as a test model, which can more realistically and reliably simulate the actual transdermal absorption of human skin.

[0159] ① Treatment of live mice: After feeding for one week, shave the hair on the back of the mice with a hair trimmer and rinse with saline. Mark a dosing area with a diameter of about 2 cm on the back skin of the mice, take 0.3 mL of the samples of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 10, and evenly apply them to the marked skin position for 30 minutes, 1 hour, 3 hours and 6 hours respectively.

[0160] ② Mouse skin processing: Remove the skin from the back of the mouse and remove the subcutaneous fat. Embed it with OCT and freeze-slice it. Scan and analyze the slices using a fluorescence microscope.

[0161] The results of the dialysis absorption test of the samples are as follows: Figure 3 and Figure 4 As shown. The results show that with the increase of the action time, the skin fluorescence thickness in the test groups of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 10 gradually increases; and compared with the use of collagen without mitochondrial targeting polypeptide modification as the biomimetic membrane embedding wall material (Comparative Example 1), no collagen added as ligand embedding (Comparative Example 2) and EGCG aqueous solution (Comparative Example 10), the experimental mice used the sample of Example 1 at 30min, 1h, 3h and 6h action time periods, and the fluorescence thickness of the mouse skin was the largest, indicating that its skin permeability was the best. Therefore, in Example 1, the mitochondrial targeting polypeptide GSH-modified collagen was used to prepare the biomimetic cell membrane modified targeting embedding ligand, and then EGCG was encapsulated by the biomimetic exosome technology, and the engineered biomimetic exosomes finally obtained had better skin permeability.

[0162] Test Example 7 Cell endocytosis test

[0163] Receptor-mediated endocytosis Specific proteins on the surface of exosomes can bind to corresponding receptors on the surface of target cells to form receptor-ligand complexes, triggering intracellular signal transduction, promoting cell membrane invagination, and then initiating the endocytosis process. For example, membrane proteins on the surface of exosomes are recognized by receptors on the surface of target cells and activate target cells, triggering endocytosis. Cell membrane invagination and wrapping of cell membranes have fluidity. When the sample approaches the target cell, the cell membrane invaginates and gradually wraps the sample to form a small vesicle containing the sample. This process is similar to the cell "swallowing" the sample into the cell using the cell membrane. Laser confocal microscopy is used to perform fluorescence imaging of the drug-endocytosed cells under specific excitation waves.

[0164] Exponentially growing HACAT cells (density 5×10 4 / well) were inoculated in a six-well plate with a cover glass in advance, and the culture medium was DMEM solution (2 mL), and it was placed in an incubator at 37°C, saturated humidity and 5% CO2 for 24 hours. The culture medium was removed, and 2 mL of the sample solutions of Example 1, Comparative Example 1 and Comparative Example 10 (aqueous solution with a mass percentage of 10%) were added to the wells, respectively, and incubated in an incubator at 37°C, saturated humidity and 5% CO2 for 30 minutes, 1 hour, 2 hours and 4 hours, respectively. The culture medium was removed, and the cells were rinsed twice with PBS solution and fixed with 4% paraformaldehyde for 0.5 hours. The paraformaldehyde solution was removed and the cells were rinsed twice with PBS solution. Incubate with DAPI (4',6-diamidino-2-phenylindole) for 30 minutes, rinse the cells three times with PBS solution, and place them on a cover glass for sealing. The coverslip was placed under a laser confocal microscope (CLSM, LeicaSP8, Leica Microsystems, USA) for observation with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0165] like Figure 5 and Figure 6 As shown, the results show that with the increase of the action time, the fluorescence intensity in the test groups of Example 1, Comparative Example 1 and Comparative Example 10 gradually increases, indicating that more samples enter the cells and cell nuclei through endocytosis. Compared with the use of collagen without mitochondrial targeting polypeptide modification as the biomimetic membrane embedding wall material (Comparative Example 1) and the EGCG aqueous solution (Comparative Example 10), the fluorescence intensity in the cells of the sample of Example 1 and HACAT cells was the largest during the co-incubation time of 30min, 1h, 2h and 4h, indicating that the more the number of engineered bionic exosomes in Example 1 that enter the cell and reach the cell nucleus, the stronger its mitochondrial targeting. Therefore, in Example 1, the mitochondrial targeting polypeptide GSH-modified collagen was used to prepare the bionic cell membrane modified targeted embedding ligand, and then the EGCG was encapsulated by the bionic exosome technology, and the engineered bionic exosomes finally obtained had better targeting, which can more effectively achieve the anti-aging effect from the source.

[0166] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing engineered bionic exosomes with anti-aging effects, characterized in that: The steps include: S1: preparing biomimetic cell membrane modified targeting ligand: adding collagen, mitochondrial targeting polypeptide, condensation agent and 4-dimethylaminopyridine into an organic solvent, activating under a protective atmosphere, and then adding lysine for reaction to obtain biomimetic cell membrane modified targeting ligand; The preparation of S2 engineered bionic exosomes includes the following steps: (1) adding phospholipids, biomimetic cell membrane modified targeting ligands, and biomimetic cell membrane stabilizers into a polyol solvent, stirring evenly, and obtaining an alcohol phase; (2) adding the active substance to the yeast / Acetobacter xylinum / black tea fermentation product and stirring evenly to obtain an aqueous phase; (3) uniformly adding the aqueous phase to the alcohol phase, and after the addition is completed, continuing to stir to obtain a mixed solution; (4) The mixed solution is subjected to membrane extrusion treatment to obtain engineered biomimetic exosomes; The mitochondrial targeting polypeptide described in S1 is at least one of glutathione, lysine-leucine-alanine tripeptide, hexapeptide-2 and SS-31 peptide; the molecular weight of the collagen is 3000 Da -150000 Da; In S2, the bionic cell membrane stabilizer is at least one of cholesterol and sodium cholate; the active substance is tea polyphenols; In terms of mass percentage, phospholipids are 2%-5%; bionic cell membrane modified targeting ligands are 0.02%-0.30%; bionic cell membrane stabilizers are 0.1%-0.3%; polyol solvents are 10%-30%; active substances are 1%-7%; and yeast / acetobacter xylinum / black tea fermentation products are 57.5%-86.8%.

2. The preparation method according to claim 1, characterized in that: The molecular weight of the collagen is 50000Da-100000Da; the molar ratio of the collagen to the mitochondrial targeting polypeptide is 1:0.5-2.5; the molar ratio of the collagen to 4-dimethylaminopyridine is 1:0.5-2.5; and the molar ratio of the collagen to lysine is 1:0.1-1.

0.

3. The preparation method according to claim 2, characterized in that: The condensing agent is at least one of dicyclohexylcarbodiimide, diisopropylcarbodiimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the molar ratio of the collagen to the condensing agent is 1:0.5-2.5; The organic solvent is at least one of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, ethanol, tetrahydrofuran, dichloromethane and ethyl acetate; the mass ratio of the collagen to the organic solvent is 1:5-100.

4. The preparation method according to any one of claims 1 to 3, characterized in that The bionic cell membrane modified targeting ligand is 0.1%-0.2%.

5. The preparation method according to claim 4, characterized in that: The phospholipid in step (1) is at least one of soybean lecithin, hydrogenated soybean lecithin, enzymatic lecithin, dipalmitoyl phosphatidylcholine, palmitoyl phosphatidylglycerol and egg yolk lecithin; the polyol solvent is at least one of 1,3-butanediol, 1,3-propylene glycol, glycerol, 1,2-hexanediol and ethoxydiglycol.

6. The preparation method according to claim 5, characterized in that: The tea polyphenols include one or more of epigallocatechin gallate, epicatechin, catechin gallate, catechin gallate and epigallocatechin.

7. The preparation method according to claim 6, characterized in that: The stirring temperature in step (1) is 25-60°C; the stirring speed is 100-500 rpm; and the stirring time is 0.5-6h; The stirring temperature in step (2) is 25-60°C; the stirring speed is 100-500 rpm; and the stirring time is 10-50 min; The dropping speed in step (3) is 2-10 mL / min; the stirring temperature is 25-60°C; the stirring speed is 100-500 rpm; and the stirring time is 2-4 h; The filter membrane in step (4) is a polycarbonate membrane, the pressure is 1.0-7.0 MPa, and the extrusion time is 10-30 min.

8. The preparation method according to claim 7, characterized in that: The stirring conditions of steps (1) and (3) are a rotation speed of 300±150 rpm and a time of 3±1h; the stirring conditions of step (2) are a rotation speed of 300±100 rpm and a time of 30min; the dropping speed of step (3) is 5±3 ml / min; the extrusion treatment conditions of step (4) are a pressure of 4.0±2.0MPa and a treatment time of 20±5min; The activation conditions in S1 are: activation at -10-10°C for 1-3h; the reaction conditions are: reaction at 25-50°C for 24-72h.

9. Engineered bionic exosomes with anti-aging efficacy obtained by the method according to any one of claims 1 to 8.

10. Use of the engineered bionic exosomes with anti-aging efficacy according to claim 9 in the preparation of cosmetics, or in the preparation of topical skin medicines.

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