Exosome with high permeability and application thereof

Abstract

The present application relates to an exosome with high permeability and application thereof, the surface of the exosome is connected with dioleoyl phosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine and hirudin. The exosome constructed by the present application can improve the permeability of the skin, can carry active components directly to the skin epidermis and dermis, and accumulate in the epidermis and dermis, continuously provide nutrition for the skin, and fully exert the skin care and beauty effect.

Description

Technical Field

[0001] This invention relates to the field of active component permeation technology, and in particular to a highly permeable exosome and its applications. Background Technology

[0002] Trauma treatment is divided into three phases: the inflammatory phase, the proliferative phase, and the scar formation phase. During wound healing, the extracellular matrix (ECM) is the core target area. The ECM is a network structure composed of macromolecules secreted by cells, filled with exosomes that transmit substances and signals. Newly generated skin cells deposit and proliferate on the ECM, and protein degradation pathways during wound formation and healing also degrade the ECM, promoting scar formation.

[0003] For repairing injuries where the skin barrier is intact but the subcutaneous and intradermal tissues are damaged, effective therapeutic components need to be delivered through the stratum corneum and epidermis to the dermis and subcutaneous tissues to promote repair. The skin consists of the epidermis, dermis, subcutaneous tissue, and appendages; effective ingredients must reach the basal layer of the epidermis and the dermis to achieve optimal wound healing and repair. The skin barrier is mainly composed of the lipophilic stratum corneum and the hydrophilic active epidermis. The outermost stratum corneum, in particular, is a "brick wall structure" composed of 10 to 20 layers of flattened dead cells and intercellular matrix, effectively preventing the penetration of most external substances. When the skin barrier is intact, effective ingredients are absorbed transdermally via three main pathways: transcellular pathway, transintercellular matrix pathway, and transdermal appendage pathway (hair follicles, sweat glands, and sebaceous glands). Currently, the absorption of effective components for subcutaneous injury repair through direct application is very limited; the vast majority cannot penetrate the stratum corneum, making it difficult to achieve full efficacy.

[0004] Therefore, there is an urgent need for methods to improve the penetration rate of high-efficacy components. Based on the different locations where different functional ingredients exert their effects, this approach can not only promote the penetration and absorption of effective ingredients, enabling them to be precisely positioned and quickly exert their skincare effects, but also improve the stability of effective ingredients and increase their duration of action in the extracellular matrix, thereby improving the utilization rate of effective ingredients.

[0005] In order to enable active ingredients to penetrate deep into the skin and exert their effects, existing methods for enhancing penetration include (1) nanotechnology: processing ingredients into microstructures, which is not suitable for most raw materials; (2) solvent-based penetration: extracting extracellular lipids with sulfoxide, urea and free fatty acids, which can cause skin irritation or toxicity; (3) physical penetration enhancement: ion or ultrasound introduction, which is equipment-dependent and can damage the skin barrier; (4) biological penetration enhancement: liposomes, penetration-enhancing peptides, etc., alter lipid metabolism, but the formulations are immature, have poor stability, and cannot be stored at room temperature or used daily.

[0006] Therefore, the ability to achieve effective ingredient penetration and extracellular matrix retention through simple application is crucial for improving the efficacy of wound treatment and intradermal injury repair. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a highly permeable exosome and its application. The exosome constructed by this invention can improve skin permeability, deliver the carried active ingredients directly to the epidermis and dermis, and accumulate in the epidermis and dermis to continuously provide nutrition to the skin, thereby fully exerting the skin care and beauty effects.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an exosome with high permeability, wherein the surface of the exosome is connected to dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine and hirudin.

[0010] In this invention, dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and hirudin are linked. The high lipophilicity of DOPS and DOPE allows them to be miscible with lipids in the intercellular spaces of the stratum corneum and penetrate the stratum corneum, thus exhibiting excellent permeation-enhancing properties. After hirudin enters the dermis and activates the PAR-1 receptor, it induces endothelial contraction, increases the permeability of local capillaries, and enhances the permeability from the dermis into the capillaries to reach the surrounding tissues.

[0011] Preferably, the dioleoylphosphatidylserine accounts for 1%-10% of the total phospholipid content of the exosomes. The 1%-10% can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0012] Preferably, the 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine accounts for 1%-10% of the total phospholipid content of the exosomes. The 1%-10% can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0013] Preferably, the hirudin accounts for 1%-10% of the total protein content of the exosomes. The 1%-10% can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0014] Preferably, the dioleoylphosphatidylserine forms a conjugate with cholesterol-PEG-NH2 lipid and inserts into the exosomal phospholipid bilayer.

[0015] In this invention, the carboxyl group of DOPS is activated by EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). The carboxyl group (–) forms an amide bond with cholesterol-PEG-NH2 lipid, which constitutes a DOPS-cholesterol lipid conjugate. This conjugate spontaneously fuses with exosomes through emulsification to form a solid-supported lipid bilayer (SLB) inserted into the exosomal phospholipid bilayer.

[0016] Preferably, the 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine forms a conjugate with cholesterol-NHS lipids and inserts into the exosomal phospholipid bilayer.

[0017] DOPE forms a carbamate bond with cholesterol-NHS ester, constituting a DOPE-cholesterol lipid conjugate that spontaneously fuses with exosomes to form a solid-supported lipid bilayer (SLB) that is inserted into the exosomal phospholipid bilayer.

[0018] Preferably, the hirudin forms a hirudin-cholesterol conjugate with cholesterol-NHS ester and is inserted into the exosome phospholipid bilayer.

[0019] The connection between hirudin and exosomes is that hirudin and cholesterol-NHS ester form a hirudin-cholesterol conjugate, which emulsifies and spontaneously fuses with exosomes to form a solid-supported lipid bilayer (SLB) inserted into the exosome phospholipid bilayer.

[0020] Preferably, the exosomes are derived from plants.

[0021] The exosomes selected in this invention are derived from plants. Exosomes extracted from plants have higher safety and fewer sensitizing components, reducing the risk of skin allergies and greatly improving safety.

[0022] Preferably, the plant includes any one or a combination of at least two of the following: potato, ginger, motherwort, tomato, lemon, broccoli, gynostemma pentaphyllum, cucumber, orange, ginseng, gromwell root, centella asiatica, senna, astragalus, or yam.

[0023] In a second aspect, the present invention provides an active ingredient delivery system, the delivery system comprising the highly permeable exosomes described in the first aspect and the active ingredient loaded thereon.

[0024] Preferably, the active ingredient is located inside the exosome.

[0025] In this invention, exosomes are small membrane vesicles containing complex RNA and proteins. This invention uses these membrane vesicles to encapsulate active ingredients, thereby achieving the delivery of active ingredients.

[0026] Preferably, the loading of the active ingredient in the delivery system is 1-20%. This 1-20% can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0027] Preferably, the active ingredient includes any one or a combination of at least two of wound repair agents, moisturizers, or anti-wrinkle agents.

[0028] Preferably, the wound repair agent comprises any one or a combination of at least two of the following: collagen, chitosan, hyaluronic acid, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, astragalus polysaccharide, shikonin, asiaticoside, senna extract, or total glycosides of Gynostemma pentaphyllum.

[0029] Preferably, the moisturizer comprises any one of ceramide, lactic acid, sodium lactate, sodium pyrrolidone carboxylate, hyaluronic acid, or hydroxypropyl tetrahydropyranotriol, or a combination of at least two of these.

[0030] Preferably, the anti-wrinkle agent comprises any one of the following, or a combination of at least two: proanthocyanidins, retinoic acid, vitamin E, vitamin C, 6-furfurylaminopurine, 3-pyridinecarboxamide (nicotinamide), fruit acid, tranexamic acid, arbutin, caffeine, thiohistidine trimethylammonium salt (ergothioneine), or coenzyme Q10.

[0031] Thirdly, the present invention provides the use of the highly permeable exosomes according to the first aspect or the active ingredient delivery system according to the second aspect in the preparation of pharmaceuticals or cosmetics.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] 1. The exosomes constructed in this invention connect dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine and hirudin, which can significantly improve skin permeability, deliver the active ingredients directly to the epidermis and dermis, and accumulate in the epidermis and dermis to continuously provide nutrition to the skin and fully exert the skin care and beauty effects.

[0034] 2. The exosomes constructed in this invention are derived from plants. Exosomes extracted from plants have high safety and fewer sensitizing components, reducing the risk of skin allergies and greatly improving safety. Detailed Implementation

[0035] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0036] The reagents used in the following examples were sourced from the following sources:

[0037] Potato exosomes: After cleaning the potatoes, rinse them three times with pure water, soak them for 1 hour, peel them, and then soak them in a 70% ethanol aqueous solution for 5 minutes. Repeat this sterilization process three times. Place them in a sterilization oven with a HEPA filter system and blow them at 4°C and an air velocity of 8 m / s until no visible water droplets remain on the surface. At 4°C, using pre-sterilized tools, shred 1 kg of potatoes into small particles of 3 mm × 3 mm × 5 mm, add 350 g of sodium chloride, mix well, and then transfer the mixture into 50 mL centrifuge tubes. Purge with nitrogen and let stand for 2 hours, then centrifuge at 4°C, 2000 g for 30 min. Collect the supernatant and desalt it using a dialysis bag until the sodium chloride content is <0.01% by mass to obtain the dialysate. Filter the dialysate sequentially through 0.8 μm, 0.45 μm, and 0.22 μm mPVDF cup filters. Centrifuge the filtrate at 4°C, 100,000 g for 2 hours to precipitate potato exosomes, which are then resuspended in 20 mL of PBS solution.

[0038] Motherwort exosomes: Fresh motherwort leaves were thoroughly cleaned, ultrasonically rinsed with distilled water until clear and colorless, soaked for 1 hour, and then disinfected by soaking in 70% ethanol aqueous solution for 5 minutes. This process was repeated three times. The leaves were then removed and dried in a sterilizing oven with a HEPA filtration system at 4°C and an air velocity of 8 m / s until the surface was dry and no visible water droplets were visible. 1 kg of motherwort leaves were gently mixed with 350 g of sodium chloride and sealed at 4°C for 12 hours to obtain exudate. This exudate was desalted using a dialysis bag until the sodium chloride content was <0.01% by mass, yielding dialysate. The dialysate was filtered sequentially through 0.8 μm, 0.45 μm, and 0.22 μm mPVDF cup filters to obtain filtrate. The filtrate was centrifuged at 2000 g for 30 minutes at 4°C, and the supernatant was collected. The supernatant was transferred to a new centrifuge tube and centrifuged at 100,000 g for 120 minutes at 4°C, and the precipitate was collected. The exosome resuspension was obtained by resuspending the precipitate in 20 mL of PBS.

[0039] Preparation Example 1

[0040] This preparation example prepares highly permeable exosomes.

[0041] The potato exosomes used were surface-modified with 5% dioleoylphosphatidylserine, 5% 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and 5% hirudin.

[0042] The synthesis of DOPS-cholesterol lipid conjugates is briefly described as follows: (1) Dissolve 10 μmol DOPS (dioleoylphosphatidylserine, Avanti Research #840035) in 200 μL of anhydrous DMF (N,N-dimethylformamide, Sigma-Aldrich, #L091000); (2) Add 400 μL of MES buffer (0.1 M, pH 5.5, Sigma-Aldrich, #M8250) to the DOPS solution and disperse it by probe-type ultrasonication to form a turbid emulsion (4℃ 50W 5min); (3) Add 50 μmol EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, Sigma-Aldrich, #39391) and 100 μmol NHS (N-hydroxysuccinyl ... μmol Cholesterol–PEG–NH2 (purchased from Xi’an Qiyue Biotechnology Co., Ltd., #Q-0259862) was dissolved in 100 μL DMF and slowly added dropwise to the DOPS–NHS ester system. The pH was adjusted to 7.2 with 1 M HEPES (Sigma-Aldrich, #H3375). (5) The reaction was carried out at 20°C in the dark under nitrogen protection for 4 h to form amide bonds. (6) Tris buffer was added to quench unreacted NHS esters and byproducts were removed with molecular sieves (Millipore, #UFC5003, MWCO 3kDa). (7) The DOPS-cholesterol lipid conjugate was collected by ultracentrifugation at 4°C, 100,000 g for 2 hours and reconstituted with pure water as needed.

[0043] The synthesis of DOPE-cholesterol lipid conjugates is briefly described as follows: (1) Dissolve 10 μmol DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, Avanti Research, #850725P) in 200 μL of anhydrous DMF; (2) Add the DOPE solution to 0.5 mL of 0.1 M HEPES buffer (pH 8.2) and gently vortex to form a turbid emulsion; (3) Dissolve 12 μmol Cholesteryl–NHS (Xi'an Qiyue Biotechnology Co., Ltd., #Q-0357717.) in 100 μL of anhydrous DMF and slowly add it dropwise to the DOPE solution; (4) React at 20℃ in the dark under nitrogen protection for 4 h to form carbamate bonds; (5) Add 50 μL of 1 M glycine solution (pH 8.0) and continue incubation at room temperature for 30 min to block the remaining active esters and terminate the reaction; (6) Add C18 to the solution. Filtered using a Sep-Pak column (Waters, #WAT051910), washed three times with 4 mL of pure water (3 × 4 mL), eluted with 4 mL of 80% methanol aqueous solution, dried under nitrogen at 20°C in the dark, and the DOPE-cholesterol lipid conjugate was collected and reconstituted with pure water as needed.

[0044] The steps for synthesizing hirudin-cholesterol conjugate are briefly described as follows: (1) Dissolve 1 mg of hirudin (Hirudin Sigma-Aldrich, #94581) in 1 mL of 0.1 M HEPES buffer (pH 8.2) and mix well; (2) Dissolve 12 μmol Cholesterol-NHS in 100 μL of anhydrous DMF and slowly add it dropwise to the hirudin solution; (3) React at 4°C in the dark for 4 h under nitrogen protection to form amide bonds; (4) Add 50 μL of 1 M glycine solution (pH 8.0) and continue incubation at 4°C for 60 min to block the remaining active ester and terminate the reaction; (5) Add molecular sieve (Millipore, #UFC5010, MWCO 10kDa) and centrifuge at 14000 g for 15 min at 4°C, and discard the filtrate; (6) Add 100 μL of pure water, centrifuge again, and repeat washing 5 times to obtain a final volume of 500 μL. μL, freeze-dried under nitrogen protection, collect hirudin-cholesterol conjugate, and reconstitute with pure water as needed.

[0045] The aforementioned 1 mL of 0.5 mM DOPS-cholesterol lipid conjugate, 1 mL of 0.5 mM DOPE-cholesterol lipid conjugate, and 1 mL of 0.15 mM hirudin-cholesterol lipid conjugate were mixed with 3 mL of 2×10 12A potato exosome solution of 1 exosome / mL was mixed and emulsified to spontaneously fuse and form a solid-supported lipid bilayer (SLB) inserted into the exosome phospholipid bilayer. Unbound raw materials and other impurities were removed using a 10 kDa molecular sieve. The modified, highly permeable exosomes were collected by ultracentrifugation at 4°C, 100,000 g, for 2 hours.

[0046] Complete lysis was performed using 2 mL of Roche cOmplete™ Lysis-M lysis buffer (ROCHE, #04719964001). 1 mL of this lysate was extracted with 3 mL of methyl tert-butyl ether-methanol solution (2:1, v / v) using the Bligh & Dyer method. 0.5 mL of the remaining 1 mL was retained for storage, and the other 0.5 mL was used to analyze the ratio of hirudin (ELISA, Shanghai Yaji Biotechnology Co., Ltd., Hirudin ELISA kit, #YS05547B) to total exosome protein (Bradford protein assay kit, Shanghai Beyotime Biotechnology Co., Ltd., P0006).

[0047] The phospholipid composition of the samples was analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The proportions of DOPS (Avanti, #850725P) and DOPE (Avanti, #850725P) as standards were calculated for calibration. Based on the above conditions and mass ratios, DOPS and DOPE each accounted for 5% of the total exosome phospholipids, and hirudin accounted for 5% of the total exosome protein.

[0048] Preparation Example 2

[0049] This preparation example prepares highly permeable exosomes.

[0050] The potato exosomes used were surface-modified with 1% dioleoylphosphatidylserine, 10% 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and 1% hirudin. DOPS-cholesterol lipid conjugate, DOPE-cholesterol lipid conjugate, and hirudin-cholesterol lipid conjugate were prepared using the method of Example 1.

[0051] Mix 1 mL of 0.1 mM DOPS-cholesterol lipid conjugate, 1 mL of 1 mM DOPE-cholesterol lipid conjugate, 1 mL of 0.03 mM hirudin-cholesterol lipid conjugate, and 3 mL of 2×10 12The potato exosome solution was mixed at a concentration of 1 / mL, and subsequent steps were the same as in Preparation Example 1. DOPS accounted for 1% of the total phospholipids in the exosomes, DOPE accounted for 10% of the total phospholipids in the exosomes, and hirudin accounted for 5% of the total protein in the exosomes.

[0052] Preparation Example 3

[0053] This preparation example prepares highly permeable exosomes.

[0054] The exosomes of Leonurus japonicus used were surface-modified with 10% dioleoylphosphatidylserine, 1% 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and 5% hirudin. DOPS-cholesterol lipid conjugate, DOPE-cholesterol lipid conjugate, and hirudin-cholesterol lipid conjugate were prepared using the method of Example 1.

[0055] Mix 1 mL of 1 mM DOPS-cholesterol lipid conjugate, 1 mL of 0.1 mM DOPE-cholesterol lipid conjugate, 1 mL of 0.3 mM hirudin-cholesterol lipid conjugate, and 3 mL of 2 × 10⁻⁶ mol / L solution. 12 The Leonurus japonicus exosome solution was mixed at a concentration of 1 / mL, and subsequent steps were the same as in Preparation Example 1. Based on the above conditions and mass ratios, DOPS accounted for 10% of the total exosome phospholipids, DOPE accounted for 1% of the total exosome phospholipids, and hirudin accounted for 5% of the total exosome protein.

[0056] Preparation Example 4

[0057] This preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is the loading of dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and hirudin. Dioleoylphosphatidylserine accounts for 0.5% of the total phospholipid content of the exosomes, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine accounts for 0.5% of the total phospholipid content of the exosomes, and hirudin accounts for 15% of the total protein content of the exosomes. The rest is the same as Preparation Example 1.

[0058] Preparation Example 5

[0059] This preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is the loading of dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and hirudin. Dioleoylphosphatidylserine accounts for 15% of the total phospholipid content of the exosomes, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine accounts for 15% of the total phospholipid content of the exosomes, and hirudin accounts for 0.5% of the total protein content of the exosomes. The rest is the same as Preparation Example 1.

[0060] Preparation Example 6

[0061] This preparation example prepares highly permeable exosomes. The only difference between this example and Preparation Example 1 is that the exosomes are ginger exosomes; otherwise, they are identical to Preparation Example 1.

[0062] Comparative Preparation Example 1

[0063] This comparative preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is that the surface of the potato exosomes is not modified with hirudin, and the loading of hirudin is allocated to dioleoylphosphatidylserine and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine according to the proportion in Preparation Example 1. All other aspects are the same as in Preparation Example 1.

[0064] Comparative Preparation Example 2

[0065] This comparative preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is that the surface of the potato exosomes is not modified with dioleoylphosphatidylserine, and the loading of dioleoylphosphatidylserine is allocated to hirudin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine according to the proportion in Preparation Example 1. All other aspects are the same as in Preparation Example 1.

[0066] Comparative preparation example 3

[0067] This comparative preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is that the surface of the potato exosomes was not modified with 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and the loading of 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine was allocated to hirudin and dioleoylphosphatidylserine according to the proportion in Preparation Example 1. All other aspects were the same as in Preparation Example 1.

[0068] Comparative preparation example 4

[0069] This comparative preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is that the surface of the potato exosomes was modified with 15% dioleoylphosphatidylserine. All other aspects are the same as those in Preparation Example 1.

[0070] Comparative preparation example 5

[0071] This comparative preparation example prepared highly permeable exosomes. The only difference between this example and Preparation Example 1 is that the surface of the potato exosomes was modified with 15% 1,2-dioleoyl-sn-glycerol-3-phosphocholine. All other aspects are the same as those in Preparation Example 1.

[0072] Example 1

[0073] This embodiment prepares an active ingredient delivery system.

[0074] In this embodiment, the highly permeable exosomes prepared in Preparation Example 1 were mixed with hydroxypropyltetrahydropyranotriol (Bosein), and the loading of hydroxypropyltetrahydropyranotriol in the exosomes was 5%.

[0075] Prepare a buffer solution containing 300 mM sucrose, 250 mM glucose, 0.1% K2-EDTA, 0.1% PBS, and 200 nM trehalose. Resuspend the exosomes prepared in Example 1 in this buffer solution to a final volume of 10 μL. 11 4.3 g of hydroxypropyltetrahydropyranotriol was added to 10 mL of exosome solution at 35°C and mixed at low speed at 4°C until no solids were detected. The ThermoFisher Neon NxT electroporator was set to 250 V, 1000 μF, and a square wave pulse of 3 ms once. Exosomes were extracted using exosome extraction reagent (SBI# EXOquick-TC). After lysis, the hydroxypropyltetrahydropyranotriol released from exosome lysis was quantified using HPLC-ELSD. If the content was less than 5%, 20% was removed, and the remaining exosome solution was pulsed once at 1 ms with a square wave. This process and detection were repeated until the content slightly exceeded 5%, and the dilution was calculated to 5%. The loaded high-permeability exosomes were collected by ultracentrifugation at 4°C, 100,000 g, for 2 hours.

[0076] Example 2

[0077] This embodiment prepares an active ingredient delivery system.

[0078] In this embodiment, the highly permeable exosomes prepared in Preparation Example 2 were mixed with hydroxypropyl tetrahydropyranotriol, and the loading of hydroxypropyl tetrahydropyranotriol in the exosomes was 20%.

[0079] Using the method of Example 1, 10 were obtained. 11 10 g of hydroxypropyl tetrahydropyranotriol was added to 10 mL of exosome buffer at 35°C. The electroporator parameters were set to 250 V, 1000 μF as the reference, and 5 square wave pulses of 2 ms each. The quantification method was the same as before. If the content was less than 20%, 20% was removed, and the remaining exosome solution was pulsed once with a square wave of 1 ms each. This process and detection were repeated until the content was slightly higher than 20%, and the dilution was calculated to 20%. The loaded high-permeability exosomes were collected by ultracentrifugation at 4°C, 100,000 g, for 2 hours.

[0080] Example 3

[0081] This embodiment prepares an active ingredient delivery system.

[0082] In this embodiment, the highly permeable exosomes prepared in Preparation Example 3 were mixed with hydroxypropyl tetrahydropyranotriol, and the loading of hydroxypropyl tetrahydropyranotriol in the exosomes was 1%.

[0083] Using the method of Example 1, 10 were obtained. 11 1 g of hydroxypropyl tetrahydropyranotriol was added to 10 mL of exosome buffer at 35°C. The electroporator parameters were set to 250 V, 1000 μF, and a square wave pulse of 2 ms once. The quantification method was the same as before. If the content was less than 1%, 20% was removed, and the remaining exosome solution was pulsed once with a square wave of 1 ms once. This process and detection were repeated until the content was slightly higher than 1%, and the dilution was calculated to 1%. The loaded high-permeability exosomes were collected by ultracentrifugation at 4°C, 100,000 g, for 2 hours.

[0084] Examples 4-6

[0085] The active ingredient delivery system prepared in this embodiment differs from that in Example 1 only in that it uses the exosomes prepared in Examples 4-6; otherwise, it is the same as in Example 1.

[0086] Example 7

[0087] The active ingredient delivery system prepared in this embodiment differs from that in Example 1 only in that the loading amount of hydroxypropyl tetrahydropyranotriol is 0.5%, while the rest is the same as in Example 1.

[0088] Example 8

[0089] The active ingredient delivery system prepared in this embodiment differs from that in Example 1 only in that the loading of hydroxypropyl tetrahydropyranotriol is 25%, while the rest is the same as in Example 1.

[0090] Comparative Examples 1-5

[0091] The active ingredient delivery system prepared in this comparative example differs from Example 1 only in that it uses the exosomes prepared in Comparative Preparation Examples 1-5; otherwise, it is identical to Example 1.

[0092] Test Example 1

[0093] This test case investigates the permeability and retention rate of active ingredients.

[0094] According to the national standard GB / T 27818-2011 "In vitro tests for skin absorption of chemicals", fresh isolated porcine back skin was cleaned of hair and subcutaneous tissue and cut into 2.5cm × 2.5cm pieces for use. Skin permeability was studied using a Franz transverse diffusion cell. The porcine back skin was fixed between two diffusion cells. The receiving solution was isotonic PBS, and the test solutions were the solutions from the above-mentioned examples and comparative examples, respectively, at a concentration of 10... 12The sample was divided into three 3 mL vials for each 10 mL sample, and the transdermal test was performed in a 37°C circulating water bath. Samples were taken at 4 h, 8 h, and 24 h after the start of the test. Quantification was achieved using high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) with standards. The content of each component in the receiving solution, stratum corneum, epidermis, and dermis was detected, and the cumulative permeability or retention rate (%) was calculated.

[0095] The active ingredient delivery systems prepared in the above examples and comparative examples were supplemented with negative groups 1 and 2, which were 5% and 25% hydroxypropyl tetrahydropyranotriol PBS solutions, respectively, filtered through a 0.22 μm filter membrane as test samples.

[0096] Remove the pigskin from the diffusion chamber, absorb excess moisture from the skin surface with filter paper, and then repeatedly peel and stick transparent tape onto one side of the stratum corneum 20 times to completely adhere the stratum corneum to the tape. Place the transparent tape in a glass bottle, add a measured amount of purified water, and ultrasonically extract the active components from the stratum corneum. Filter the extract through a 0.22 μm filter membrane and use it as the test sample. Take a blank pig back skin that was not used for the transdermal test, cut it into pieces of the same size as the transdermal test sample, and treat it in the same way. Use the extract as a blank control for the stratum corneum sample to eliminate the influence of inherent components in the stratum corneum. Detect and calculate the cumulative retention rate of the effective components in the stratum corneum.

[0097] Pig back skin with the keratin layer removed was cut into small pieces and placed in a glass bottle. A measured amount of purified water was added, and the active components were extracted by ultrasonication. The extract was filtered through a 0.22 μm filter membrane and used as the test sample. Separately, blank pig back skin not used for the transdermal test was taken, cut into pieces of the same size as the transdermal test sample, and treated in the same way. The extract was used as a blank control for the epidermal and dermal samples to eliminate the influence of inherent components in the epidermis and dermis. The cumulative retention rate of the active ingredients in the epidermis and dermis was detected and calculated. The formulas for calculating the permeability and retention rate are as follows. The permeability results are shown in Table 1, and the retention rate results are shown in Table 2.

[0098] Cumulative transmittance (%) = Total transmittance in the receiving liquid / Total amount in the diffusion cell of the test liquid × 100%.

[0099] Cumulative retention rate (%) = Total amount retained in the skin / Total amount in the diffusion cell of the test solution × 100%.

[0100] Table 1

[0101]

[0102] Table 2

[0103]

[0104] The experimental results above show that:

[0105] (1) As can be seen from the comparison of Examples 1-3, the higher the loading concentration of the active ingredient, the more active ingredients penetrate through the skin and remain in the dermis. However, there is no significant linear or multiple correlation between loading concentration and penetration rate or retention rate.

[0106] (2) A comparison of Examples 1 and 4-5 shows that the loading amounts of dioleoylphosphatidylserine (DOPS), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and hirudin on the exosomes are outside the scope of this invention, leading to decreased permeability and retention. The concentrations of DOPS and DOPE have a more significant impact on permeability. Low epidermal permeability means a reduced number of exosomes that have passed through the epidermal layer, preventing hirudin from reaching the dermis to promote retention. Therefore, Example 4 exhibits lower permeability and retention. Significantly increasing the concentrations of DOPS and DOPE did not result in better permeability, but the lack of hirudin significantly reduced the retention rate of exosomes after reaching the dermis. Therefore, Example 5 exhibits a poorer retention rate.

[0107] (3) By comparing Example 1 and Example 6, it can be seen that using other plant exosomes with the same modification scheme can maintain high permeability and retention rate.

[0108] (4) A comparison between Example 1 and Examples 7-8 shows that excessive loading of active ingredients will lead to an increase in the proportion of exosome breakage, resulting in a decrease in permeability and a significant decrease in retention. Insufficient loading will result in a low loading success rate and poor uniformity, meaning that some exosomes fail to load the effective ingredients, resulting in low stability of the permeation and retention effects.

[0109] (5) By comparing Example 1 with Comparative Examples 1-3, it can be seen that dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine and hirudin have a synergistic effect. The absence of any one of them will reduce the permeation and retention effects of exosomes.

[0110] (6) It can be seen from the comparison between Example 1 and Comparative Examples 4-5 that using DOPS or DOPE alone will reduce the permeation and retention of exosomes.

[0111] Test Example 2

[0112] This test case explores the effects of loading different active ingredients.

[0113] To investigate the effectiveness of the active ingredient delivery system of the present invention in delivering different active components, the active ingredient delivery system was prepared using the method of Example 1, and different active components were loaded onto it. The permeability and retention rate of different active components were detected using the method of Test Example 1. The specific results are shown in Table 3.

[0114] Table 3

[0115]

[0116] The results above demonstrate that the modified exosomes significantly enhance penetration and retention. Even for components with good permeability in aqueous solutions, such as nicotinamide and caffeine, the modified exosomes significantly promote penetration and increase retention. This proves that the modified exosomes are versatile in increasing the transdermal penetration and intradermal retention of active ingredients.

[0117] In summary, this invention, by loading the active ingredients into exosomes and then modifying the exosomes to enhance permeability, not only promotes the transdermal absorption of the active ingredients but also increases their retention in the epidermis and dermis. This allows the active ingredients to fully exert their physiological effects within the skin, achieving better wound treatment and intradermal damage repair.

[0118] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An exosome, characterized in that, The surface of the exosomes is linked to dioleoylphosphatidylserine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and hirudin; The dioleoylphosphatidylserine accounts for 1%-10% of the total phospholipid content of the exosomes; The 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine accounts for 1%-10% of the total phospholipid content of the exosomes; Hirudin accounts for 1%-10% of the total protein content of exosomes; The dioleoylphosphatidylserine forms a conjugate with cholesterol-PEG-NH2 and inserts into the exosomal phospholipid bilayer; The 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine forms a conjugate with cholesterol-NHS ester and inserts into the exosomal phospholipid bilayer; The hirudin forms a hirudin-cholesterol conjugate with cholesterol-NHS ester and inserts into the exosome phospholipid bilayer; The exosomes are derived from any one of potatoes, ginger, or motherwort.

2. An active ingredient delivery system, characterized in that, The delivery system includes the exosomes of claim 1 and the active components loaded thereon.

3. The active ingredient delivery system according to claim 2, characterized in that, The active ingredient is located inside the exosome.

4. The active ingredient delivery system according to claim 3, characterized in that, The loading of the active ingredient in the delivery system is 1-20%.

5. The active ingredient delivery system according to claim 3, characterized in that, The active ingredients include any one or a combination of at least two of the following: wound repair agents, moisturizers, or anti-wrinkle agents.

6. The active ingredient delivery system according to claim 5, characterized in that, The wound repair agent includes any one or a combination of at least two of the following: hyaluronic acid, recombinant human epidermal growth factor, shikonin, or asiaticoside. The moisturizer includes any one or a combination of at least two of the following: ceramide, lactic acid, sodium lactate, sodium pyrrolidone carboxylate, dipotassium glycyrrhizate, hyaluronic acid, or hydroxypropyl tetrahydropyranotriol. The anti-wrinkle agent includes any one of the following, or a combination of at least two: proanthocyanidins, retinoic acid, vitamin E, vitamin C, 6-furfurylaminopurine, 3-pyridinecarboxamide, fruit acid, tranexamic acid, arbutin, caffeine, or trimethylammonium thiohistidine.

7. The use of the exosome according to claim 1 or the active ingredient delivery system according to any one of claims 2-6 in the preparation of pharmaceuticals or cosmetics.

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