A transdermal delivery combination for promoting skin repair

Abstract

The application discloses a transdermal delivery combination for promoting skin repair, comprising: a composition containing sponge bone needles for pretreating skin; an exosome vesicle freeze-dried powder; the exosome vesicle freeze-dried powder is obtained by membrane fusion of exosomes and liposomes, and then freeze-drying; a reconstitution medium; and the exosome vesicle freeze-dried powder is mixed with the reconstitution medium at 5-40 DEG C and then applied to the skin. The application firstly pretreats the skin by using the composition containing sponge bone needles, reduces the skin barrier resistance and stimulates the skin repair response, and then reconstitutes the exosome vesicle and applies it to the skin surface, so as to promote the transdermal utilization rate of the exosome content. The exosome vesicle provided by the application has good in-vitro storage stability, and the activity retention rate is more than 80% after storage at 0-5 DEG C for 180 days and reconstitution.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a transdermal delivery combination for promoting skin repair. Background Technology

[0002] Exosomes are extracellular vesicles with a diameter between 30 and 150 nanometers. They possess a lipid bilayer structure and act as naturally released "cellular messengers," encapsulating various bioactive substances such as proteins, lipids, and nucleic acids (e.g., DNA, mRNA, microRNA). These tiny vesicles can transmit information between cells, regulating many physiological and pathological processes. Exosomes can effectively penetrate the skin barrier and act on deep skin cells, showing great promise for applications in skin repair and care, such as scar repair, wound healing, and the treatment of inflammatory skin diseases.

[0003] However, since the bioactivity of exosomes is easily inactivated at room temperature in vitro, they are usually stored at -80°C and therefore often administered to animals via injection. This invention is proposed to improve bioavailability and enable use and storage under routine conditions. Summary of the Invention

[0004] This invention provides a transdermal delivery combination for promoting skin repair, aiming to improve the in vitro storage stability and transdermal utilization of exosomes, thereby enabling them to exert a good skin repair effect.

[0005] This invention is achieved through the following scheme:

[0006] This invention provides a transdermal delivery assembly for promoting skin repair, comprising:

[0007] (1) A composition containing sponge bone needles for pretreatment of skin;

[0008] (2) Lyophilized exosome vesicle powder; the lyophilized exosome vesicle powder is obtained by fusion of exosomes and liposomes through a membrane and then freeze-drying.

[0009] (3) Reconstitution medium; after mixing with the exosome vesicle lyophilized powder at 5~40℃, it is applied to the skin.

[0010] This invention employs a composition containing sponge-like bone needles to pretreat the skin, creating numerous micron-sized physical channels in the stratum corneum. This step significantly reduces skin barrier resistance, paving the way for subsequent exosome penetration, and the resulting micro-damage activates the skin's initial repair response. Exosomes are then fused with liposomes via a membrane to form exosome vesicles, which are then freeze-dried to obtain lyophilized powder. This process improves the in vitro room-temperature storage stability of exosomes and maintains their bioactivity. When used, the exosome vesicles are mixed with a reconstitution medium, allowing them to regain their biological morphology and thus penetrate the skin percutaneously.

[0011] In some embodiments, the method for preparing the exosome vesicles includes the following steps: mixing an exosome suspension with a liposome suspension and subjecting them to freeze-thaw cycles, then extruding the mixture through a polycarbonate membrane with a pore size of 200 nm to obtain an exosome vesicle dispersion, and then freeze-drying it to obtain an exosome vesicle lyophilized powder.

[0012] In some embodiments, the liposomes in the liposome suspension are composed of phospholipids and cholesterol; the phospholipids include functional phospholipids; the functional phospholipids contain polyethylene glycol segments and / or amino acid sequences containing matrix metalloproteinases (MMPs) that can recognize and cleave sites.

[0013] Constructing liposomes using functional phospholipids containing polyethylene glycol (PEG) segments is beneficial for maintaining the physical morphology and biological activity of exosomes during freeze-thaw cycles and freeze-drying. Specifically, PEG forms a protective layer for exosome vesicles, preventing aggregation and fusion of exosome vesicles during freeze-drying and reconstitution through steric hindrance. Simultaneously, PEG can inhibit ice crystal growth, reducing mechanical damage to the exosome lipid bilayer caused by ice crystals formed during freezing. Furthermore, PEG's low glass transition temperature and flexible molecular chains help form an amorphous glassy matrix, "fixing" the exosome vesicles within it and minimizing physical and chemical changes.

[0014] Skin inflammation and micro-damage induced by pretreatment with sponge needles trigger a skin repair response, promoting the secretion of matrix metalloproteinases (MMPs). MMPs can recognize specific amino acid sequences and cleave them at designated sites. This invention utilizes phospholipids containing amino acid sequences that MMPs can recognize and cleave to construct liposomes and form exosome vesicles. When these exosome vesicles are applied to the repair-responsive skin, the MMPs recognize and cleave the sites, altering the hydrophilicity and hydrophobicity of the exosomes, causing structural instability and rupture, thus accelerating the release of exosome contents for skin repair.

[0015] In some embodiments, the exosome vesicle lyophilized powder retains more than 83% of its activity after reconstitution after being stored at room temperature for 30 days; and retains more than 80% of its activity after reconstitution after being stored at 0-5°C for 180 days.

[0016] In some embodiments, the functional phospholipid is at least one of distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), dilinoleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG), and dimyristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG).

[0017] In some embodiments, the functional phospholipid includes DC-PT-PE, which is prepared as follows: 1. A matrix metalloproteinase-responsive polypeptide Boc-PT-OH, N-hydroxysuccinimide, and a carbodiimide condensing agent are reacted; the resulting reaction solution is purified and dried to obtain Boc-PT-NHS; 2. Boc-PT-NHS is mixed with PE-type phospholipids and triethylamine and reacted; 3. The resulting reaction solution is purified and dried to obtain DC-PT-PE.

[0018] In some embodiments, the functional phospholipid includes PEG-PT-PE, and the preparation method of PEG-PT-PE is as follows: 1. A matrix metalloproteinase-responsive polypeptide Boc-PT-OH, N-hydroxysuccinimide, and a carbodiimide condensing agent are reacted; the resulting reaction solution is purified and dried to obtain Boc-PT-NHS; 2. Boc-PT-NHS is mixed with PE-type phospholipids and triethylamine and reacted; 3. The resulting reaction solution is purified and dried to obtain Boc-PT-PE; 4. Boc-PT-PE is deamination protected to obtain H2N-PT-PE; 5. H2N-PT-PE is mixed with polyethylene glycol activated ester PEG-NHS and triethylamine and reacted; 6. The resulting reaction solution is purified and dried to obtain PEG-PT-PE.

[0019] In some embodiments, the activated glycoacid ester is obtained by reacting a glycoacid compound DC with N-hydroxysuccinimide and a carbodiimide condensing agent for 5, and then purifying and drying the resulting reaction solution; the glycoacid compound is at least one of gluconic acid, D(+)-glucuronic acid C and D-xyloic acid.

[0020] In some embodiments, the carbodiimide condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0021] In some embodiments, the PE phospholipid is at least one of phosphatidylethanolamine (PE), dilinoleoyl phosphatidylethanolamine (DOPE), distearate phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), and dimyristoyl phosphatidylethanolamine (DMPE).

[0022] In some embodiments, the PEG-NHS is O-[(N-succinimide)succinyl-aminoethyl]-O'-methyl polyethylene glycol with a number average molecular weight of 500~1000 g / mol.

[0023] In some embodiments, the amino acid sequence of the matrix metalloproteinase-responsive polypeptide Boc-PT-OH from the N-terminus to the C-terminus is: Boc-Pro-Leu-X-Gly-[Ile / Leu]-Ala-OH, where X is one of Gly, Ala, Leu, Ile, and Val; Boc is a tert-butoxycarbonyl group bonded to an imidazoleamine group on a proline residue; [Ile / Leu] refers to Ile or Leu; and OH refers to the carboxyl group on the Ala residue.

[0024] The matrix metalloproteinase-responsive polypeptide Boc-PT-OH was prepared by solid-phase synthesis.

[0025] Functional phospholipid DC-PT-PE possesses a glycosidic unit (DC), an enzyme-responsive polypeptide unit (PT), and a phospholipid unit (PE). The glycosidic unit (DC) has abundant hydroxyl structures, enabling it to form a thick and stable hydration layer, thereby improving the stability of exosome vesicles in the aqueous phase. After liposomes constructed from DC-PT-PE form exosome vesicles, the exosome vesicles, in response to MMPs (microenzymes), lose their glycosidic units (DC), resulting in a significant decrease in hydrophilicity, instability of the hydration layer, and changes in the permeability of the liposomes, even leading to their disintegration and accelerating the release of exosome contents.

[0026] Liposomes constructed using PEG-PT-PE, a functional phospholipid containing polyethylene glycol (PEG) segments and enzyme-responsive PT units, are beneficial for maintaining the physical morphology and biological activity of exosomes during freeze-thaw cycles and freeze-drying. The PEG segments, with their long hydrophilic chains, provide steric hindrance in the aqueous phase, improving the dispersibility of exosome vesicles and thus enhancing their stability in aqueous solution. After the liposomes constructed using PEG-PT-PE form exosome vesicles, the PEG segments are removed upon enzyme response by the exosome MMPs, significantly reducing hydrophilicity, destabilizing the hydration layer, altering the permeability of the liposomes, and even causing their disintegration, thus accelerating the release of exosome contents.

[0027] In some embodiments, the phospholipids include neutral phospholipids; the neutral phospholipids are selected from PE phospholipids or PC phospholipids; the PC phospholipids are at least one of dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and distearylphosphatidylcholine (DSPC); the PE phospholipids are at least one of phosphatidylethanolamine (PE), dilinoleoylphosphatidylethanolamine (DOPE), distearylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), and dimyristoylphosphatidylethanolamine (DMPE).

[0028] In some embodiments, the liposome suspension is prepared as follows: phospholipids and cholesterol are mixed in a solvent, the solvent is evaporated by rotary evaporation, PBS buffer is added for hydration, ultrasonic treatment is performed, and the liposome suspension is obtained by extrusion through a polycarbonate membrane with a pore size of 200 nm.

[0029] In some embodiments, the liposome suspension is prepared as follows: phospholipids, cholesterol, and a lyophilization protectant are mixed in a solvent, the solvent is evaporated by rotary evaporation, PBS buffer is added for hydration, the mixture is sonicated, and then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain the liposome suspension; the lyophilization protectant includes one or more of polyethylene glycol, polyols, and disaccharides; the polyol is mannitol and / or sorbitol; the disaccharide is trehalose and / or sucrose.

[0030] In some embodiments, the exosomes in the exosome suspension are derived from at least one of mesenchymal stem cells, adipose stem cells, and fibroblasts.

[0031] In some embodiments, the composition containing sponge spicules comprises sponge spicules and an oily matrix.

[0032] In some embodiments, the oily matrix is ​​at least one of mineral oil, vegetable oil, and lanolin.

[0033] In some embodiments, the composition containing sponge spicules has a mass percentage of 1% to 4% for the sponge spicules.

[0034] In some embodiments, the pretreatment involves applying the composition containing sponge bone needles to the skin, massaging for 1 to 5 minutes, and then wiping it off.

[0035] In some embodiments, the composition containing sponge needles is a paste, cream, or ointment.

[0036] In some embodiments, the resolution medium is a pH buffer solution or a pH buffer solution containing a competing cosolvent; the competing cosolvent includes at least one of sucrose, trehalose, maltitol, mannitol, erythritol, and sorbitol.

[0037] In some embodiments, the pH buffer solution is a PBS buffer or a Tris-HCl buffer; the concentration of the competing cosolvent in the reconstitution medium is 0-5 g / L.

[0038] Competitive cosolvents are beneficial to improving the stability of exosome vesicles after reconstitution. Specifically, competitive cosolvents are all small molecule compounds with excellent hydrophilicity. After dissolving in water, they form a thick hydration layer, which makes their affinity for exosome vesicles weak and creates a repulsive force (steric hindrance). This makes them more likely to remain in the bulk solution, resulting in a lower concentration of competitive cosolvents in the region near the exosome vesicles. In other words, the concentration of water molecules in the region near the exosome vesicles is relatively higher, thereby stabilizing the hydration layer of the exosome vesicles, achieving a stabilizing effect, and preventing them from agglomerating.

[0039] Beneficial effects:

[0040] This invention provides a transdermal delivery assembly for promoting skin repair. First, the skin is pretreated with a composition containing sponge-like puncture needles to reduce skin barrier resistance and stimulate a skin repair response. Then, exosome vesicles are reconstituted and applied to the skin surface, thereby promoting the transdermal utilization of the exosome contents. The exosome vesicles provided by this invention exhibit good in vitro storage stability, maintaining over 80% activity after reconstitution after 180 days of storage at 0–5°C. Attached Figure Description

[0041] Figure 1 High-performance liquid chromatography and mass spectra of the enzyme-responsive peptide Boc-Pro-Leu-Ile-Gly-Leu-Ala-OH.

[0042] Figure 2 High-performance liquid chromatography and mass spectra of the enzyme-responsive peptide Boc-Pro-Leu-Ala-Gly-Ile-Ala-OH.

[0043] Figure 3 High performance liquid chromatogram and mass spectrum of DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE.

[0044] Figure 4 PEG 750 Mass spectrum of Pro-Leu-Ile-Gly-Leu-Ala-DOPE.

[0045] Figure 5 The image shows the fluorescence resonance energy transfer results of the exosome vesicles prepared in Example 1.

[0046] Figure 6 A pathological section of mouse skin from the back. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] In the specific implementation, the sponge spicules used are bee sponge spicules, purchased from Shaanxi Xintianyu Biotechnology Co., Ltd.

[0049] The extraction of exosomes, taking human adipose-derived mesenchymal stem cell exosomes as an example, includes the following steps:

[0050] Serum-free culture medium from the first three generations of human adipose-derived mesenchymal stem cells was collected and subjected to gradient centrifugation at 4°C, as follows: 300g × 10 min, supernatant collected; 2000g × 20 min, supernatant collected; 12000g × 40 min, supernatant collected. The supernatant was filtered through a 0.22 μm filter and centrifuged at 100000g × 40 min, the supernatant was discarded, the pellet was resuspended in 1×PBS, centrifuged at 200000g × 120 min, the supernatant was discarded, and the pellet was resuspended in 1×PBS to obtain an exosome suspension. The suspension was then processed using Pierce chromatography. The BCA protein assay kit (purchased from Thermo Fisher Scientific, Waltham, Massachusetts, USA) is used to quantify the total protein concentration of exosome suspensions. The particle size distribution of the exosome suspensions was obtained by dynamic light scattering (DLS). The protein concentration in the exosome suspension was 142 ng / µL, and the average particle size of the exosomes was 124 nm.

[0051] In the following specific embodiments, the exosome suspensions used are all human adipose-derived mesenchymal stem cell exosome suspensions with a protein concentration of 142 ng / uL.

[0052] The preparation of matrix metalloproteinase-responsive peptide (hereinafter referred to as enzyme-responsive peptide) Boc-PT-OH, taking Boc-Pro-Leu-Ile-Gly-Leu-Ala-OH as an example, includes the following steps:

[0053] (1) Weigh 2g of Fmoc-Ala-Wang resin (substitution degree of 0.5mmol / g, 200 mesh), put it into a peptide synthesis tube, add 20mL of DMF and soak for 30min, then connect the peptide synthesis tube to a vacuum pump to dry the solution to obtain activated resin; add 10mL of piperidine / DMF (20 / 80 v / v) solution to the activated resin for deprotection, stir and react under N2 blowing at 25℃ for 10min, filter the solid and wash the resin three times with 20mL of dichloromethane (DCM), methanol and N,N-dimethylformamide (DMF) to obtain deprotected resin;

[0054] (2) After activating Fmoc-Leu-OH, it was added to the above deprotected resin. After reacting at 25°C for 3 hours with N2 blowing, the solid was filtered and the resin was washed three times with 20 mmol DCM, methanol and DMF to obtain Fmoc-Leu-Ala- resin. Activation refers to reacting 5 mmol Fmoc-Leu-OH with 6 mmol benzotriazole. N ,N ,N',N' Tetramethylurea hexafluorophosphate (HBTU) and 6 mmol of 1-hydroxybenzotriazole (HOBT) were mixed and completely dissolved in 30 mL of DMF. Then, 10 mmol of diisopropylethylamine (DIEA) was added and the mixture was activated at 25 °C in the dark for 5 min.

[0055] Following steps (1) and (2), resin deprotection, amino acid activation, and bonding were performed sequentially to bond Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, and Boc-Pro-OH to the resin, resulting in Boc-Pro-Leu-Ile-Gly-Leu-Ala-resin.

[0056] (3) Add 95wt% TFA aqueous solution to Boc-Pro-Leu-Ile-Gly-Leu-Ala- resin for cleavage, stir in an ice-water bath for 3 hours, filter to remove resin using a sintered glass funnel, concentrate the filtrate at 35°C, then add 35 times the volume of ice-cold anhydrous diethyl ether to precipitate the peptide, let stand at 4°C for 40 minutes, centrifuge at 4000 r / min for 3 minutes to obtain the precipitate NH2-Pro-Leu-Ile-Gly-Leu-Ala-OH;

[0057] (4) After dissolving the precipitate NH2-Pro-Leu-Ile-Gly-Leu-Ala-OH in DMF, N,N-diisopropylethylamine (DIPEA) and (Boc)2O were added and reacted at 0℃ for 3h. After the reaction was completed by HPLC analysis, the solvent was removed by vacuum distillation. Then the solid was dissolved in ethyl acetate and washed successively with 1M HCl solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic phase was dried to obtain the crude product.

[0058] (5) The crude product was dissolved in ultrapure water containing 0.1% trifluoroacetic acid at a concentration of 0.1±0.1 g / L. After filtration through a 0.45 μm filter membrane, it was separated and identified by liquid chromatography-mass spectrometry. The mobile phase A was 0.1% trifluoroacetic acid-water, and the mobile phase B was 0.1% trifluoroacetic acid-acetonitrile. The chromatographic column was a silica gel alkyl bonded phase C18 column (4.6 mm × 300 mm). A binary mobile phase gradient elution system was used for gradient elution. That is, within 30 min, the content of mobile phase B in the eluent increased linearly from 0% to 80%. The flow rate was 1 mL / min, the detection wavelength was 215 nm, and the determination was carried out at 25 °C. The mass spectrometry conditions were as follows: Ion source: ESI; Sheath gas flow rate: 20 psi; Auxiliary gas flow rate: 8 psi; Sweep gas flow rate: 5 psi; Spray voltage: 4.5 kV; Capillary temperature: 275 °C; Capillary voltage: 35 V; Sleeve lens voltage: 110 V.

[0059] The liquid chromatography and mass spectra (ESI-MS) of the enzyme-responsive peptide Boc-Pro-Leu-Ile-Gly-Leu-Ala-OH are as follows: Figure 1 As shown in the results, the purity of the finished polypeptide is 98.58% (M+H). + Its m / z is 666.81, and its relative component is 665.8 Da.

[0060] In some specific embodiments, the enzyme-responsive peptide is Boc-Pro-Leu-Ala-Gly-Ile-Ala-OH, and its preparation method is similar to that described above. The liquid chromatography and mass spectrometry (ESI-MS) chromatograms of the enzyme response Boc-Pro-Leu-Ala-Gly-Ile-Ala-OH are shown below. Figure 2 As shown in the results, the purity of the finished polypeptide is 99.07% (M+H). + Its m / z is 624.82, and its relative component is 623.8 Da.

[0061] The preparation of DC-PT-PE, taking DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE as an example, includes the following steps: Boc-Pro-Leu-Ile-Gly-Leu-Ala-OH, N-hydroxysuccinimide (NHS), and EDC hydrochloride are mixed in dichloromethane and stirred at room temperature for 12 hours. The resulting reaction solution is washed with saturated sodium chloride solution and dried to obtain the enzyme-responsive peptide active ester Boc-Pro-Leu-Ile-Gly-Leu-Ala-NHS; Boc-Pro-Leu-Ile-Gly-Leu-Ala-NHS is mixed with DOPE and triethylamine in dichloromethane and reacted at 5°C for 30 minutes, then reacted at room temperature for 48 hours. The resulting reaction solution is washed with saturated sodium chloride solution. After washing and drying, Boc-Pro-Leu-Ile-Gly-Leu-Ala-DOPE was obtained. Boc-Pro-Leu-Ile-Gly-Leu-Ala-DOPE was dissolved in dichloromethane, TFA was added dropwise, and the mixture was stirred in an ice bath for 3 hours. The mixture was then precipitated with diethyl ether to obtain H2N-Pro-Leu-Ile-Gly-Leu-Ala-DOPE. H2N-Pro-Leu-Ile-Gly-Leu-Ala-DOPE was mixed with gluconate activated ester and triethylamine in dichloromethane and reacted at 5°C for 30 minutes, followed by reaction at room temperature for 48 hours. The resulting reaction solution was washed and dried with saturated sodium chloride solution to obtain DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE.

[0062] The present invention also uses the same method to prepare DC2-Pro-Leu-Ala-Gly-Ile-Ala-DMPE (the corresponding sugar acid compound is D(+)-glucuronic acid C, and the corresponding phospholipid is dimyristoyl phosphatidylethanolamine) and DC3-Pro-Leu-Ile-Gly-Leu-Ala-PE (the corresponding sugar acid compound is D-xyloic acid, and the corresponding phospholipid is phosphatidylethanolamine).

[0063] Boc-Pro-Leu-Ile-Gly-Leu-Ala-OH, NHS, and EDC hydrochloride were fed in a ratio of n(COOH):n(NHS):n(EDC) = 1:5:2; Boc-Pro-Leu-Ile-Gly-Leu-Ala-NHS, DOPE, and triethylamine were fed in a ratio of n(NHS):n(DOPE):n(triethylamine) = 1:1.1:2; Boc-Pro-Leu-Ile-Gly-Leu-Ala-DOPE and dichloromethane were used to neutralize TFA in a ratio of 1g:5mL:5 The following reaction mixtures were added: H2N-Pro-Leu-Ile-Gly-Leu-Ala-DOPE, gluconate activated ester, and triethylamine were added at a ratio of n(NHS):n(NH2):n(triethylamine) = 1:1.1:2; gluconate activated ester was prepared by mixing gluconic acid with NHS and EDC hydrochloride in dichloromethane and stirring at room temperature for 12 hours, and the resulting reaction solution was washed with saturated sodium chloride solution and dried; gluconic acid, NHS, and EDC hydrochloride were added at a ratio of n(COOH):n(NHS):n(EDC) = 1:5:2.

[0064] DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE was separated and identified using liquid chromatography-mass spectrometry (LC-MS). The LC chromatogram and ESI-MS chromatogram are attached. Figure 3 As shown, the purity is 97.87%, (M+H) + Its m / z is 1487.91 and its relative molecular weight is 1486.9 Da.

[0065] Preparation of PEG-PT-PE, using PEG 750 Taking H2N-Pro-Leu-Ile-Gly-Leu-Ala-DOPE as an example, the steps include: mixing H2N-Pro-Leu-Ile-Gly-Leu-Ala-DOPE with PEG 750 The activated ester and triethylamine were mixed in dichloromethane and reacted at 5°C for 30 min, then at room temperature for 48 h. The resulting reaction solution was washed with saturated sodium chloride solution and dried to obtain PEG. 750 -Pro-Leu-Ile-Gly-Leu-Ala-DOPE. H2N-Pro-Leu-Ile-Gly-Leu-Ala-DOPE, PEG 750 The activated ester and triethylamine were fed in a ratio of n(NHS):n(NH2):n(triethylamine) = 1:1.1:2; PEG 750 The activated ester was purchased from Shanghai Sigma-Aldrich Trading Co., Ltd.

[0066] PEG was analyzed using a mass spectrometer.750 Identification was performed using Pro-Leu-Ile-Gly-Leu-Ala-DOPE, and the mass spectrum (ESI-MS) is attached. Figure 4 As shown, (M+H) + Its m / z is 2041.76 and its relative molecular weight is 2040.7 Da.

[0067] The present invention also uses the same method to prepare PEG. 750 -Pro-Leu-Ala-Gly-Ile-Ala-DMPE (corresponding phospholipid is dimyristoyl phosphatidylethanolamine) and PEG 750 -Pro-Leu-Ile-Gly-Leu-Ala-PE (the corresponding phospholipid is phosphatidylethanolamine).

[0068] Example 1

[0069] (1) Apply the composition containing sponge bone needles to the skin and massage with your fingertips for 3 minutes, then wipe it off with a wet wipe;

[0070] (2) Mix the exosome vesicle freeze-dried powder and the reconstitution medium at 25°C and let stand for 3 minutes, then apply it to the skin; the exosome vesicle freeze-dried powder is an exosome vesicle obtained by membrane fusion of exosomes and liposomes, and then freeze-dried.

[0071] The composition containing sponge spicules comprises, by weight percentage, the following components: 3% peak sponge spicules, 10% jojoba oil, 2% cyclopentamethoxydimethylsiloxane and 10% triglycerides decanoate, with the balance being petrolatum.

[0072] The preparation method of exosome vesicle lyophilized powder is as follows:

[0073] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform, and the solvent was evaporated by rotary evaporation at 50°C. Then, 1×PBS solution was added and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension. The ratio of phospholipids to cholesterol was 1:0.3. The phospholipids included DMPC and DOPE in a molar ratio of 5:3. The average particle size of the obtained liposomes was 122 nm, and the zeta potential was -10.11 mV.

[0074] S2, Preparation of exosome vesicles: After adding a lyophilization protectant to the liposome suspension and stirring until homogeneous, add the exosome suspension to obtain a precursor solution; then perform three freeze-thaw cycles, each cycle consisting of freezing at -80°C for 30 seconds and thawing at room temperature for 5 minutes. The vesicles are then extruded through a 200nm polycarbonate membrane to obtain an exosome vesicle dispersion. The lyophilization protectants include mannitol, trehalose, and PEG.2000 Mannitol, trehalose, and PEG in the precursor solution 2000 The concentrations were 0.02 g / mL, 0.05 g / mL, and 0.005 g / mL, respectively; the phospholipid concentration in the catalytic solution was 4.05 mg / mL, and the protein concentration was 0.08 mg / mL.

[0075] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0076] Fluorescence spectroscopy was used to investigate the membrane fusion of exosomes and liposomes based on the principle of fluorescence resonance energy transfer (FRET). Liposomes and exosomes were labeled with DiO and DiI, respectively (liposome to DiO mass ratio 100:1, exosome to DiI mass ratio 100:1). The mixture was vortexed several times at room temperature to ensure complete insertion of the DiO and DiI fluorescent dyes into the liposomes and exosomes, respectively. The DiO-labeled liposomes and DiI-labeled exosomes were subjected to freeze-thaw cycles as described above to obtain exosome vesicles. The DiO-labeled liposome suspension, the DiI-labeled exosome suspension, and the dispersion of the fused exosome vesicles were placed in 96-well plates, and the fluorescence emission spectra at 450 nm excitation wavelength were measured using a microplate reader. The results are shown in the appendix. Figure 5 As shown in the figure, when only DiO-labeled liposomes are present, the fluorescence emission spectrum shows only a single DiO emission peak at 510 nm; when only DiI-labeled exosomes are present, the fluorescence emission spectrum is a flat line with no obvious emission peak. When exosomes are fused with liposomes, both DiO and DiI fluorescence emission peaks appear in the fluorescence emission spectrum, indicating the occurrence of the FRET effect and demonstrating successful fusion of the liposome and exosome membranes.

[0077] The reconstitution medium was PBS buffer at pH 7.4.

[0078] The ratio of exosome vesicle lyophilized powder to reconstitution medium was 0.5 g: 4 mL.

[0079] Example 2

[0080] The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows:

[0081] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform, and the solvent was evaporated by rotary evaporation at 50°C. Then, 1×PBS solution was added, and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min, and the liposome suspension was obtained by extrusion through a polycarbonate membrane with a pore size of 200 nm. The ratio of phospholipids to cholesterol was 1:0.3. The phospholipids included DMPC, DOPE, and DOPE-PEG in a molar ratio of 5:3:2. 2000 The average particle size of the obtained liposomes was 141 nm, and the zeta potential was -7.54 mV.

[0082] S2, Preparation of exosome vesicles: After adding the lyophilization protectant to the liposome suspension and stirring evenly, the exosome suspension was added to obtain the precursor solution; then, three freeze-thaw cycles were performed, each cycle consisting of freezing at -80℃ for 30 seconds and thawing at room temperature for 5 minutes, followed by extrusion through a polycarbonate membrane with a pore size of 200 nm to obtain the exosome vesicle dispersion; the lyophilization protectant included mannitol and trehalose; the concentrations of mannitol and trehalose in the precursor solution were 0.02 g / mL and 0.05 g / mL, respectively; the phospholipid concentration in the precursor solution was 4.25 mg / mL, and the protein concentration was 0.08 mg / mL;

[0083] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0084] Example 3

[0085] The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows:

[0086] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform. After evaporating the solvent by rotary evaporation at 50°C, 1×PBS solution was added and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension. The ratio of phospholipids to cholesterol was 1:0.3. The phospholipids included DMPC, DOPE, and PEG in a molar ratio of 5:3:2. 750 -Pro-Leu-Ile-Gly-Leu-Ala-DOPE; the average particle size of the obtained liposomes was 132 nm, and the zeta potential was -8.65 mV;

[0087] S2, Preparation of exosome vesicles: A lyophilization protectant was added to the liposome suspension and stirred until homogeneous. Then, an exosome suspension was added to obtain a precursor solution. Three freeze-thaw cycles were then performed, each cycle consisting of freezing at -80°C for 30 seconds and thawing at room temperature for 5 minutes. The vesicles were then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain an exosome vesicle dispersion. The lyophilization protectant included mannitol and trehalose. The concentrations of mannitol and trehalose in the precursor solution were 0.02 g / mL and 0.05 g / mL, respectively. The phospholipid concentration in the precursor solution was 4.16 mg / mL, and the protein concentration was 0.08 mg / mL.

[0088] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0089] Example 4

[0090] The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows:

[0091] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform, and the solvent was evaporated by rotary evaporation at 50°C. 1×PBS solution was added, and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min, and the liposome suspension was obtained by extrusion through a polycarbonate membrane with a pore size of 200 nm. The ratio of phospholipids to cholesterol was 1:0.3. The phospholipids included DMPC, DOPE, and DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE in a molar ratio of 5:3:2. The average particle size of the obtained liposomes was 127 nm, and the zeta potential was -10.05 mV.

[0092] S2, Preparation of exosome vesicles: After adding the lyophilization protectant to the liposome suspension and stirring until homogeneous, the exosome suspension is added to obtain the precursor solution; then, three freeze-thaw cycles are performed, each cycle consisting of freezing at -80°C for 30 seconds and thawing at room temperature for 5 minutes. The vesicles are then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain the exosome vesicle dispersion; the lyophilization protectants include trehalose and PEG. 2000 The concentrations of mannitol and trehalose in the precursor solution were 0.05 g / mL and 0.005 g / mL, respectively; the concentration of phospholipids in the precursor solution was 4.51 mg / mL, and the concentration of protein was 0.09 mg / mL.

[0093] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0094] Example 5

[0095] The difference from Example 4 is that the reconstitution medium is PBS buffer (pH=7.4) with a sorbitol concentration of 1 g / L.

[0096] Example 6

[0097] The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows:

[0098] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform. After evaporating the solvent by rotary evaporation at 50°C, 1×PBS solution was added and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension. The ratio of phospholipids to cholesterol was 1:0.3. The phospholipids included DMPC, DOPE, and PEG in a molar ratio of 5:3:1:1. 750 -Pro-Leu-Ile-Gly-Leu-Ala-DOPE, DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE; the average particle size of the obtained liposomes was 125 nm, and the zeta potential was -9.25 mV;

[0099] S2, Preparation of exosome vesicles: Exosome suspension was added to liposome suspension to obtain precursor solution; then, three freeze-thaw cycles were performed, each cycle consisting of freezing at -80℃ for 30s and thawing at room temperature for 5min, followed by extrusion through a polycarbonate membrane with a pore size of 200nm to obtain exosome vesicle dispersion; the phospholipid concentration in the precursor solution was 4.48mg / mL, and the protein concentration was 0.08mg / mL;

[0100] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0101] Example 7

[0102] The difference from Example 6 is that the reconstitution medium is PBS buffer (pH=7.4) with a sorbitol concentration of 1 g / L.

[0103] Example 8

[0104] (1) Apply the composition containing sponge bone needles to the skin and massage with your fingertips for 2 minutes, then wipe it off with a wet wipe;

[0105] (2) Mix the exosome vesicle freeze-dried powder and the reconstitution medium at 35°C and let stand for 5 minutes, then apply it to the skin; the exosome vesicle freeze-dried powder is an exosome vesicle obtained by membrane fusion of exosomes and liposomes, and then freeze-dried.

[0106] The composition containing sponge spicules comprises, by weight percentage, the following components: 4% peak sponge spicules, 2% sweet almond oil, 5% grape seed oil, 2% cyclopentamethoxysiloxane and 15% triglycerides of decanoate, with the balance being lanolin.

[0107] The preparation method of exosome vesicle lyophilized powder is as follows:

[0108] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform. After evaporating the solvent by rotary evaporation at 50°C, 1×PBS solution was added and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension. The ratio of phospholipids to cholesterol was 1:0.52. The phospholipids included DMPC, DOPE, and PEG in a molar ratio of 5:3:1:1. 750 -Pro-Leu-Ala-Gly-Ile-Ala-DMPE, DC2-Pro-Leu-Ala-Gly-Ile-Ala-DMPE; the average particle size of the obtained liposomes was 109 nm, and the zeta potential was -17.2 mV;

[0109] S2, Preparation of exosome vesicles: Exosome suspension was added to liposome suspension to obtain precursor solution; then, three freeze-thaw cycles were performed, each cycle consisting of freezing at -80℃ for 30s and thawing at room temperature for 5min, followed by extrusion through a polycarbonate membrane with a pore size of 200nm to obtain exosome vesicle dispersion; the phospholipid concentration in the precursor solution was 5.14mg / mL, and the protein concentration was 0.11mg / mL;

[0110] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0111] The reconstitution medium was PBS buffer (pH=7.4) with a maltitol concentration of 2 g / L.

[0112] The ratio of exosome vesicle lyophilized powder to reconstitution medium was 0.5 g: 4 mL.

[0113] Example 9

[0114] (1) Apply the composition containing sponge bone needles to the skin and massage with your fingertips for 5 minutes, then wipe it off with a wet wipe;

[0115] (2) Mix the exosome vesicle freeze-dried powder and the reconstitution medium at 8°C and let stand for 5 minutes, then apply it to the skin; the exosome vesicle freeze-dried powder is an exosome vesicle obtained by membrane fusion of exosomes and liposomes, and then freeze-dried.

[0116] The composition containing sponge spicules comprises, by weight percentage, the following components: 1% peak sponge spicules, 8% grape seed oil, 5% cyclopentamethoxydimethylsiloxane and 12% triglycerides decanoate, with the balance being lanolin.

[0117] The preparation method of exosome vesicle lyophilized powder is as follows:

[0118] S1, Preparation of liposomes: Phospholipids and cholesterol were dissolved in chloroform. After evaporating the solvent by rotary evaporation at 50°C, 1×PBS solution was added and the mixture was stirred and hydrated at 50°C for 30 min. The mixture was then sonicated at 200 W for 20 min and extruded through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension. The ratio of phospholipids to cholesterol was 1:0.47. The phospholipids included DMPC, DOPE, and PEG in a molar ratio of 5:3:1:1. 750 -Pro-Leu-Ile-Gly-Leu-Ala-PE, DC3-Pro-Leu-Ile-Gly-Leu-Ala-PE; the average particle size of the obtained liposomes was 121 nm, and the zeta potential was -9.47 mV;

[0119] S2, Preparation of exosome vesicles: Exosome suspension was added to liposome suspension to obtain precursor solution; then, three freeze-thaw cycles were performed, each cycle consisting of freezing at -80℃ for 30s and thawing at room temperature for 5min, followed by extrusion through a polycarbonate membrane with a pore size of 200nm to obtain exosome vesicle dispersion; the phospholipid concentration in the precursor solution was 5.01mg / mL, and the protein concentration was 0.09mg / mL;

[0120] S3, Preparation of lyophilized powder: The exosome vesicle dispersion was freeze-dried to obtain exosome vesicle lyophilized powder; the freeze-drying conditions were as follows: pre-freezing at -10℃ for 30 min, freezing at -50℃ for 4 h, and then drying at -20℃ and 12 Pa vacuum for 15 h.

[0121] The reconstitution medium was PBS buffer (pH=7.4) with an erythritol concentration of 3 g / L.

[0122] The ratio of exosome vesicle lyophilized powder to reconstitution medium was 0.5 g: 4 mL.

[0123] Test Example 1

[0124] Exosome vesicle lyophilized powders (Examples 1-7 and Comparative Example 2) were stored at 4±1℃ and 25±1℃ for several periods (0d, 15d, 30d, 90d, 180d) and then reconstituted. Alternatively, the exosome vesicle dispersion of Comparative Example 3 was stored at -80℃ (0d, 15d, 30d, 90d, 180d), and the Zeta potential of the exosome vesicles was measured. The reconstitution procedure was as follows: the exosome vesicle lyophilized powder was mixed with the corresponding reconstitution medium of each example at a ratio of 0.5g:4mL, and incubated at 37℃ for 0.5h before sampling and testing. The in vitro storage stability of exosome vesicles was evaluated by the Zeta potential retention rate. The Zeta potential retention rate = Zeta potential after storage (reconstitution) ÷ Zeta potential of the exosome vesicle dispersion (before freeze-drying) × 100%. The Zeta potential retention rate results for each experimental group are summarized in Table 1.

[0125] Table 1

[0126] ;

[0127] Over time, the absolute value of the zeta potential of exosome vesicles typically decreases gradually. This change is a typical indicator of decreased stability, reflecting particle aggregation and precipitation. Table 1 shows that the zeta potential retention rates of the various examples are ranked as follows: Example 7 > Example 6 > Example 3 ≈ Example 2 ≈ Example 5 > Example 4 > Example 1 > Comparative Example 3 > Comparative Example 2. This indicates that hybridization with liposomes increases the in vitro storage stability of the exosome vesicle lyophilized powder. Adding a competing co-solvent to the reconstitution medium is beneficial for improving the activity retention of the exosome vesicle lyophilized powder after reconstitution. Example 3 uses PEG-PT-PE to construct liposomes, which, compared to Example 4 using DC-PT-PE, results in a higher activity retention rate after hybridization with liposomes. This is because the PEG segment can simultaneously perform hydration and steric hindrance dispersion functions compared to the DC unit. Examples 6 and 7, which simultaneously add PEG-PT-PE and DC-PT-PE to construct liposomes, can perform lyophilization protection of exosome vesicles, and the effect is better than Examples 1-3, which added additional lyophilization protectants.

[0128] Test Example 2

[0129] In vitro transdermal experiments were conducted using a vertical diffusion cell system, with the abdominal skin of hairless mice as a model (subcutaneous fat and vascular tissue removed). The prepared skin sample was fixed between the supply and receiving chambers, with the skin layer facing the supply chamber. A 1 g / 1 mL sponge spicule PBS suspension was added to the supply chamber (comparative example 1 used an equal volume of PBS solution), and the mixture was stirred for 5 min. The supply chamber was then cleaned, the solution was changed, and the skin sample was rinsed with PBS. After the solution change, the test sample was added to the supply chamber, and PBS solution was added to the receiving chamber. The test sample was prepared by mixing the lyophilized exosome vesicle powder (stored at 4±1℃ for 180 days) prepared in each specific embodiment with its corresponding reconstitution medium at a ratio of 0.5 g: 4 mL, and incubating at 37℃ for 0.5 h. During the experiment, the liquid in the supply chamber was intermittently and gently stirred. The receiving liquid was collected after 8 hours and analyzed using Pierce chromatography. The BCA protein assay kit was used to determine the total protein concentration and calculate the cumulative permeation per unit area. All experiments were performed in triplicate, and results are expressed as the arithmetic mean. The cumulative permeation per unit area, Q, was calculated as (c × V) / A, where c is the total protein concentration in the receiving solution, V is the volume of the receiving solution, and A is the effective permeation area. The cumulative permeation per unit area results for each experimental group are summarized in Table 2.

[0130] Table 2

[0131] ;

[0132] As shown in Table 2, the cumulative permeation per unit area is ranked as follows: Example 7 > Example 6 > Example 5 > Example 4 > Example 3 > Example 2 > Example 1 > Comparative Example 2 ≈ Comparative Example 1. Comparative Example 1, which did not include sponge bone in the skin sample pretreatment, had the worst cumulative permeation per unit area, indicating that the method provided by this invention can promote the transdermal absorption of exosome active substances. As shown in Test Example 1, the activity retention rate of the reconstituted exosome vesicles in Example 7 is higher than that in Example 6, therefore the transdermal permeation of active substances is also higher. The activity retention rate of the reconstituted exosome vesicles in Example 4 is lower than that in Example 3, but the cumulative permeation per unit area is higher than that in Example 3. This is because Example 4 has a more sensitive enzyme response, which is determined by the molecular structure of the functional phospholipid. Because the PEG segment gives the phospholipid greater hydration steric hindrance, it makes it difficult for enzymes to enter the molecule, thus resulting in weaker enzyme response. The cumulative permeation per unit area of ​​Example 6 is greater than that of Examples 3 and 4. This is because the compounding of PEG-PT-PE and DC-PT-PE functional phospholipids results in high stability of exosome vesicles during in vitro storage, thus leading to a higher permeation per dermal layer after reconstitution.

[0133] Test Example 4

[0134] Enzyme solution (a mixed solution containing 10 μg / mL LMMP-1, 10 μg / mL LMMP-2, 10 μg / mL LMMP-3, and 10 μg / mL LMMP-9) was added to the exosome vesicle dispersions prepared in each specific embodiment at a volume ratio of 1:1. After incubation at 37°C for several hours, the Zeta potential was measured, and the Zeta potential change rate was calculated as: Zeta potential after enzymatic hydrolysis ÷ Zeta potential of exosome vesicle dispersion × 100%. The results are listed in Table 3.

[0135] Table 3

[0136] ;

[0137] The rate of change of Zeta potential at this point reflects the enzyme responsiveness of each exosome vesicle; the stronger the enzyme responsiveness, the greater the rate of change of Zeta potential over a certain period. As shown in Table 3, the phospholipids in Example 2 do not contain enzyme-responsive units; therefore, their potential change can be used as a blank control. The potential change is due to the spontaneous aggregation of exosome vesicles over time and the hydrolysis of proteins carried by the enzymes within the exosomes themselves. In Example 3, the PEG chain segment causes greater hydration steric hindrance to the phospholipids, making it difficult for enzymes to enter the molecule; therefore, the enzyme responsiveness of the exosome vesicles is weaker than in Examples 4 and 6. Comprehensive analysis of Examples 1-3 shows that the exosome vesicles prepared in Example 6 or 7 achieve a good balance of in vitro storage stability, transdermal permeability, and enzyme responsiveness.

[0138] Test Example 5

[0139] The skin repair effects of each specific embodiment of the present invention were evaluated using a mouse model of specific dermatitis. Female C57BL / 6J mice, aged 6-8 weeks and weighing 18-20g, were selected. The hair on the back was trimmed short with electric clippers. An appropriate amount of 8% sodium sulfide solution was evenly applied to the trimmed area to remove residual hair. After 2-3 minutes, the area was washed with warm water and dried. The hair removal area was approximately 3cm × 2cm. A clean mouse model was defined as smooth skin on the back of the mouse, without any damage or residual hair. A 0.5wt% dinitrofluorobenzene solution was continuously applied to the hair removal area for 15 days to establish the mouse model of specific dermatitis. The model mice were divided into four groups of five mice each, corresponding to Example 1, Example 7, Comparative Example 1, and the positive control group, respectively. A blank control group of five mice was also included, without any modeling treatment. Mice in Example 7 and the control group were treated with the drug as described in Example 7. Specifically, the drug treatment began on day 5 after successful modeling. The dosage of the compound containing sponge spicules was 0.05 g / g, and the dosage of exosome vesicle recombinant solution was 0.01 mL / g. The treatment of Example 7 and Comparative Example 1 was the same as that of Example 1. The positive group was given an equal volume of PBS solution daily. After 7 days of continuous drug treatment, the skin on the back of the mice was fixed with 4% paraformaldehyde, paraffin-embedded, stained with hematoxylin and eosin (HE), and the skin repair effect was analyzed. The results are shown in the appendix. Figure 6 .

[0140] As shown in the attached figures, the epidermis of the blank group was intact and clearly structured, with a visible keratinized layer and neatly arranged collagen fibers in the dermis, showing no inflammatory cell infiltration. This indicates that the drug treatment administered to mice using the method provided by this invention does not cause irritation or damage to the mouse skin. In contrast, the structure of the damaged skin tissue in the positive group was unclear, with missing epidermis, large areas of eosinophilic necrotic material, thickened stratum corneum, and extensive proliferation of connective tissue, fibroblasts, and fibroblasts, accompanied by infiltration of lymphocytes and granulocytes. Compared to the positive group, the stratum corneum of groups 1, 7, and 1 (Comparative Example 1) was significantly thinner, lymphocyte infiltration was reduced, and epidermal thickening was significantly alleviated. Furthermore, the repair effects of 1 and 7 (Comparative Example 1) were significantly better than those of 1 (Comparative Example 1).

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A transdermal delivery assembly for promoting skin repair, characterized in that, include: (1) A composition containing sponge bone needles for pretreatment of skin; (2) Lyophilized exosome vesicle powder; the lyophilized exosome vesicle powder is obtained by fusion of exosomes and liposomes through a membrane and then freeze-drying. (3) Reconstitution medium; after mixing with the exosome vesicle lyophilized powder at 5~40℃, it is applied to the skin; The liposomes are composed of phospholipids and cholesterol; the phospholipids include functional phospholipids; the functional phospholipids include DC-PT-PE and / or PEG-PT-PE; The preparation method of DC-PT-PE is as follows:

1. A matrix metalloproteinase-responsive polypeptide Boc-PT-OH, N-hydroxysuccinimide, and a carbodiimide condensing agent are reacted. The resulting reaction solution is purified and dried to obtain Boc-PT-NHS.

2. Boc-PT-NHS is mixed with PE-type phospholipids and triethylamine and reacted. The resulting reaction solution is purified and dried to obtain Boc-PT-PE.

3. Boc-PT-PE is deaminated and protected to obtain H2N-PT-PE.

4. H2N-PT-PE is mixed with a sugar-acid activated ester and triethylamine and reacted. The resulting reaction solution is purified and dried to obtain DC-PT-PE. The preparation method of PEG-PT-PE is as follows:

1. React Boc-PT-OH, a matrix metalloproteinase-responsive peptide, N-hydroxysuccinimide, and a carbodiimide condensing agent. The resulting reaction solution is purified and dried to obtain Boc-PT-NHS.

2. React Boc-PT-NHS with PE-type phospholipids and triethylamine. The resulting reaction solution is purified and dried to obtain Boc-PT-PE.

3. Deamination protection of Boc-PT-PE yields H2N-PT-PE.

4. React H2N-PT-PE with polyethylene glycol activated ester PEG-NHS and triethylamine. The resulting reaction solution is purified and dried to obtain PEG-PT-PE. The PE-type phospholipids are at least one of phosphatidylethanolamine, dilinoleoylphosphatidylethanolamine, distearate phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, and dimyristoylphosphatidylethanolamine; the glycoacid activated ester is obtained by reacting a glycoacid compound DC with N-hydroxysuccinimide and a carbodiimide condensing agent for 5, and then purifying and drying the resulting reaction solution; the glycoacid compound is at least one of gluconic acid, D(+)-glucuronic acid C, and D-xyloic acid; the amino acid sequence of the matrix metalloproteinase-responsive polypeptide Boc-PT-OH from the N-terminus to the C-terminus is: Boc-Pro-Leu-X-Gly-[Ile / Leu]-Ala-OH, where X is one of Ala and Ile; Boc is a tert-butyloxycarbonyl group bonded to an imidazoleamine bond in a proline residue; [Ile / Leu] refers to Ile or Leu; OH refers to the carboxyl group on the Ala residue.

2. The transdermal delivery assembly for promoting skin repair according to claim 1, characterized in that, The method for preparing the exosome vesicles includes the following steps: mixing an exosome suspension with a liposome suspension and subjecting them to freeze-thaw cycles, then extruding the mixture through a polycarbonate membrane with a pore size of 200 nm to obtain an exosome vesicle dispersion, and then freeze-drying it to obtain an exosome vesicle lyophilized powder.

3. The transdermal delivery assembly for promoting skin repair according to claim 1, characterized in that, The carbodiimide condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide; And / or, the number-average molecular weight of the PEG-NHS is 500~1000 g / mol.

4. The transdermal delivery assembly for promoting skin repair according to claim 1, characterized in that, The phospholipids also include neutral phospholipids; the neutral phospholipids are selected from PE-type phospholipids or PC-type phospholipids; the PC-type phospholipids are at least one of dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine and distearylphosphatidylcholine; the PE-type phospholipids are at least one of phosphatidylethanolamine, dilinoleoylphosphatidylethanolamine, distearylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylethanolamine.

5. The transdermal delivery assembly for promoting skin repair according to claim 2, characterized in that, The liposome suspension is prepared as follows: phospholipids and cholesterol are mixed in a solvent, the solvent is evaporated by rotary evaporation, PBS buffer is added for hydration, ultrasonic treatment is performed, and the liposome suspension is obtained by extrusion through a polycarbonate membrane with a pore size of 200 nm. And / or, the liposome suspension is prepared as follows: phospholipids, cholesterol, and a lyophilization protectant are mixed in a solvent, the solvent is evaporated by rotary evaporation, PBS buffer is added for hydration, the mixture is sonicated, and then extruded through a polycarbonate membrane with a pore size of 200 nm to obtain the liposome suspension; the lyophilization protectant includes one or more of polyethylene glycol, polyols, and disaccharides; the polyol is mannitol and / or sorbitol; the disaccharide is trehalose and / or sucrose; And / or, the exosomes in the exosome suspension are derived from at least one of mesenchymal stem cells, adipose stem cells, and fibroblasts.

6. The transdermal delivery assembly for promoting skin repair according to claim 1, characterized in that, The composition containing sponge spicules comprises: sponge spicules and an oily matrix; And / or, the oily matrix is ​​at least one of mineral oil, vegetable oil and lanolin; And / or, the composition containing sponge spicules has a mass percentage of 1% to 4% for the sponge spicules; And / or, the pretreatment refers to applying the composition containing sponge bone needles to the skin and massaging for 1 to 5 minutes, and then wiping it off; And / or, the composition containing sponge needles is a paste, cream, or ointment.

7. The transdermal delivery assembly for promoting skin repair according to claim 1, wherein the resolution medium is a pH buffer solution or a pH buffer solution containing a competing cosolvent; the competing cosolvent includes at least one selected from sucrose, trehalose, maltitol, mannitol, erythritol, and sorbitol.

8. The transdermal delivery assembly for promoting skin repair according to claim 7, characterized in that, The pH buffer solution is PBS buffer or Tris-HCl buffer; the concentration of the competing cosolvent in the reconstitution medium is 0~5 g / L.

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