Method for promoting skin repair by using sponge spicule to assist exosome transdermal delivery

Abstract

The invention discloses a method for promoting skin repair by assisting exosome transdermal delivery through a sponge spicule. The method comprises the following steps: pretreating skin by using a composition containing the sponge spicule; mixing the exosome vesicle freeze-dried powder with a redissolving medium at 5-40 DEG C, and then applying the mixture to the skin; the exosome vesicle freeze-dried powder is obtained by performing membrane fusion on exosome and liposome to obtain exosome vesicles and then performing freeze drying. According to the invention, the skin is pretreated by using the composition containing the sponge bone needle, the skin barrier resistance is reduced, the skin repair response is stimulated, and then the exosome vesicles are re-dissolved and then applied to the skin surface, so that the transdermal utilization rate of exosome contents is promoted. The exosome vesicle provided by the invention has good in-vitro storage stability, and after the exosome vesicle is stored at 0-5 DEG C for 180 days, the activity retention rate after redissolution is 80% or above.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to a method for assisting exosome transdermal delivery to promote skin repair by spongy bone needles. BACKGROUND

[0002] Exosomes are extracellular vesicles with a diameter of 30-150 nanometers, have a lipid bilayer membrane structure, are "cell messengers" naturally released by cells, and encapsulate various bioactive substances such as proteins, lipids, nucleic acids (such as DNA, mRNA, microRNA), etc. These tiny vesicles can transmit information between cells and regulate many physiological and pathological processes. Exosomes can effectively penetrate the skin barrier and act on deep skin cells, and have broad application prospects in skin repair and care, such as scar repair, wound healing, and treatment of inflammatory skin diseases, etc.

[0003] However, the biological activity of exosomes is easily inactivated at room temperature in vitro, so they are usually stored at -80℃, and thus are usually injected into animals. In order to improve the bioavailability and enable daily use and storage, the present application is proposed. SUMMARY

[0004] The present application provides a method for assisting exosome transdermal delivery to promote skin repair by spongy bone needles, aiming to improve the in vitro storage stability and transdermal utilization rate of exosomes, so as to make them play a good effect in skin repair.

[0005] The present application is realized by the following scheme: The present application provides a method for assisting exosome transdermal delivery to promote skin repair by spongy bone needles, comprising the following steps: (1) Pretreating the skin with a composition containing spongy bone needles; (2) Mixing exosome vesicle freeze-dried powder and a reconstitution medium at 5-40℃, and then applying it to the skin; the exosome vesicle freeze-dried powder is obtained by membrane fusion of exosomes and liposomes, and then freeze-drying.

[0006] The present application pretreats the skin with a composition containing spongy bone needles to create a large number of micron-sized physical channels on the stratum corneum of the skin, which significantly reduces the skin barrier resistance and paves the way for the subsequent penetration of exosomes, and the micro-damage caused by it can activate the initial repair response of the skin. The exosomes and liposomes are fused by membrane fusion to form exosome vesicles, and then freeze-dried to obtain freeze-dried powder, which is beneficial to improve the in vitro room temperature storage stability of exosomes and maintain their biological activity. When used, the exosome vesicles can recover their biological form, so as to be able to penetrate the skin.

[0007] In some embodiments, the method for preparing the exosome vesicles comprises the following steps: mixing an exosome suspension with a liposome suspension and performing freeze-thaw cycles, then extruding through a polycarbonate membrane with a pore size of 200 nm to obtain an exosome vesicle dispersion, and then freeze-drying to obtain an exosome vesicle lyophilized powder.

[0008] 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 contain amino acid sequences recognizable and cleavable by matrix metalloproteinases MMPs.

[0009] The use of functional phospholipids containing polyethylene glycol segments to construct liposomes is conducive to maintaining the physical form and biological activity of exosomes during freeze-thaw cycles and freeze-drying. Specifically, polyethylene glycol forms a protective layer on the surface of exosome vesicles, inhibiting their aggregation and fusion during freeze-drying and reconstitution through steric hindrance effects; at the same time, polyethylene glycol can inhibit ice crystal growth, reducing the mechanical damage to the exosome lipid bilayer caused by ice crystals formed during freezing; in addition, the low glass transition temperature and flexible molecular chain of polyethylene glycol can help form an amorphous glassy matrix, "fixing" the exosome vesicles therein and minimizing physical and chemical changes.

[0010] Skin inflammation and micro-injuries induced by spongy bone needle pretreatment can trigger a repair response in the skin, promoting the secretion of matrix metalloproteinases that can recognize specific amino acid sequences and cleave at the cleavage site. The present application uses phospholipids containing amino acid sequences recognizable and cleavable by matrix metalloproteinases (MMPs) to construct liposomes and form exosome vesicles. When the exosome vesicles are applied to the repair-responsive skin, MMPs recognize and cleave the site, causing changes in the hydrophilic and hydrophobic properties of the exosome vesicles, leading to structural instability and thus rupture, accelerating the release of exosome contents to repair the skin.

[0011] In some embodiments, the exosome vesicle lyophilized powder is stored at room temperature for 30 days, and the activity retention rate after reconstitution is 83% or higher; stored at 0-5°C for 180 days, and the activity retention rate after reconstitution is 80% or higher.

[0012] In some embodiments, the functional phospholipids are at least one of distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG), dilinoleyl phosphatidylethanolamine-polyethylene glycol (DOPE-PEG), and dimyristoyl phosphatidylethanolamine-polyethylene glycol (DMPE-PEG).

[0013] In some embodiments, the functional phospholipid comprises DC-PT-PE, which is prepared by the following method: reacting matrix metalloproteinase-responsive polypeptide Boc-PT-OH, N-hydroxysuccinimide and a carbodiimide-based condensing agent in reaction 1, purifying and drying the reaction solution to obtain Boc-PT-NHS; mixing Boc-PT-NHS with PE-based phospholipid and triethylamine to perform reaction 2, purifying and drying the reaction solution to obtain Boc-PT-PE; removing the Boc protecting group from Boc-PT-PE to obtain H2N-PT-PE; mixing H2N-PT-PE with a sugar acid activated ester and triethylamine to perform reaction 3, purifying and drying the reaction solution to obtain DC-PT-PE.

[0014] In some embodiments, the functional phospholipid comprises PEG-PT-PE, which is prepared by the following method: reacting matrix metalloproteinase-responsive polypeptide Boc-PT-OH, N-hydroxysuccinimide and a carbodiimide-based condensing agent in reaction 1, purifying and drying the reaction solution to obtain Boc-PT-NHS; mixing Boc-PT-NHS with PE-based phospholipid and triethylamine to perform reaction 2, purifying and drying the reaction solution to obtain Boc-PT-PE; removing the Boc protecting group from Boc-PT-PE to obtain H2N-PT-PE; mixing H2N-PT-PE with a polyethylene glycol activated ester PEG-NHS and triethylamine to perform reaction 4, purifying and drying the reaction solution to obtain PEG-PT-PE.

[0015] In some embodiments, the sugar acid activated ester is obtained by mixing sugar acid compound DC with N-hydroxysuccinimide and a carbodiimide-based condensing agent in reaction 5, purifying and drying the reaction solution to obtain; the sugar acid compound is at least one of gluconic acid, D(+)-glucuronic acid C and D-xylose acid.

[0016] In some embodiments, the carbodiimide-based condensing agent is 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl) carbodiimide or 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride.

[0017] In some embodiments, the PE-based phospholipid is at least one of phosphatidylethanolamine (PE), dilinoleyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE) and dimyristoyl phosphatidylethanolamine (DMPE).

[0018] In some embodiments, the PEG-NHS is O-[(N-succinimidyl) succinyl-aminoethyl]-O'-methyl polyethylene glycol with a number average molecular weight of 500-1000 g / mol.

[0019] In some embodiments, the matrix metalloproteinase-responsive polypeptide Boc-PT-OH has an amino acid sequence from N-terminus to C-terminus as follows: Boc-Pro-Leu-X-Gly-[Ile / Leu]-Ala-OH, wherein X is one of Gly, Ala, Leu, Ile and Val; Boc is tert-butyloxycarbonyl, which is bonded to the imidazole amine in the proline residue; [Ile / Leu] means Ile or Leu; and OH means carboxyl on the Ala residue.

[0020] The matrix metalloproteinase-responsive polypeptide Boc-PT-OH is prepared by solid-phase synthesis.

[0021] The functional phospholipid DC-PT-PE has a sugar unit DC, an enzyme-responsive polypeptide unit PT and a phospholipid unit PE. The sugar unit DC has a rich hydroxyl structure, which can form a thick and stable hydration layer, thereby improving the stability of the exosome vesicle in the aqueous phase. After the liposome constructed by DC-PT-PE forms an exosome vesicle, when the exosome vesicle responds to MMPs, the sugar unit DC is removed, thereby greatly reducing the hydrophilicity, destabilizing the hydration layer, causing changes in the permeability of the liposome or even disintegration, and accelerating the release of the contents of the exosome.

[0022] The functional phospholipid PEG-PT-PE containing a polyethylene glycol segment PEG and an enzyme-responsive unit PT is used to construct a liposome, which is beneficial to maintain the physical form and biological activity of the exosome during freeze-thaw cycles and freeze-drying. The polyethylene glycol segment PEG has a long chain of hydrophilic molecules, which can provide steric hindrance to improve the dispersibility of the exosome vesicle in the aqueous phase, thereby improving the stability of the exosome vesicle in the aqueous phase. After the liposome constructed by PEG-PT-PE forms an exosome vesicle, when the exosome vesicle responds to MMPs, the PEG segment is removed, thereby greatly reducing the hydrophilicity, destabilizing the hydration layer, causing changes in the permeability of the liposome or even disintegration, and accelerating the release of the contents of the exosome.

[0023] In some embodiments, the phospholipid comprises neutral phospholipid; the neutral phospholipid is selected from the group consisting of PE-type phospholipid or PC-type phospholipid; the PC-type phospholipid is at least one of dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC); the PE-type phospholipid is at least one of phosphatidylethanolamine (PE), dilinoleylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE) and dimyristoylphosphatidylethanolamine (DMPE).

[0024] In some embodiments, the liposome suspension preparation method is as follows: mixing phospholipid and cholesterol in a solvent, evaporating the solvent by rotary evaporation, adding PBS buffer for hydration, ultrasonic treatment, and extruding through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension; In some embodiments, the liposome suspension preparation method is as follows: mixing phospholipid, cholesterol and lyoprotectant in a solvent, evaporating the solvent by rotary evaporation, adding PBS buffer for hydration, ultrasonic treatment, and extruding through a polycarbonate membrane with a pore size of 200 nm to obtain a liposome suspension; the lyoprotectant comprises one or more of polyethylene glycol, polyhydric alcohol and disaccharide; the polyhydric alcohol is mannitol and / or sorbitol; the disaccharide is trehalose and / or sucrose.

[0025] In some embodiments, the exosome suspension is derived from at least one of mesenchymal stem cells, adipose stem cells and fibroblasts.

[0026] In some embodiments, the composition containing spongin spicules comprises: spongin spicules and an oily base.

[0027] In some embodiments, the oily base is at least one of mineral oil, vegetable oil and lanolin.

[0028] In some embodiments, the mass percentage of spongin spicules in the composition containing spongin spicules is 1% to 4%.

[0029] In some embodiments, the pretreatment refers to applying the composition containing spongin spicules to the skin and massaging for 1 to 5 minutes, and then wiping off.

[0030] In some embodiments, the composition containing spongin spicules is a paste, cream or ointment.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Beneficial effects: This invention provides a method for promoting skin repair through transdermal delivery of exosomes using sponge-like spicules. First, the skin is pretreated with a composition containing sponge-like spicules 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

[0035] Figure 1 High-performance liquid chromatography and mass spectra of the enzyme-responsive peptide Boc-Pro-Leu-Ile-Gly-Leu-Ala-OH.

[0036] Figure 2 High-performance liquid chromatography and mass spectra of the enzyme-responsive peptide Boc-Pro-Leu-Ala-Gly-Ile-Ala-OH.

[0037] Figure 3 High performance liquid chromatogram and mass spectrum of DC1-Pro-Leu-Ile-Gly-Leu-Ala-DOPE.

[0038] Figure 4 PEG 750 Mass spectrum of Pro-Leu-Ile-Gly-Leu-Ala-DOPE.

[0039] Figure 5The image shows the fluorescence resonance energy transfer results of the exosome vesicles prepared in Example 1.

[0040] Figure 6 A pathological section of mouse skin from the back. Detailed Implementation

[0041] 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.

[0042] In the specific implementation, the sponge spicules used are bee sponge spicules, purchased from Shaanxi Xintianyu Biotechnology Co., Ltd.

[0043] The extraction of exosomes, taking human adipose-derived mesenchymal stem cell exosomes as an example, includes the following steps: 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 using the following program: 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, and the precipitate was resuspended in 1×PBS. The precipitate was then centrifuged at 200000g × 120 min, the supernatant was discarded, and the precipitate was resuspended in 1×PBS to obtain an exosome suspension. The total protein concentration of the exosome suspension was quantified using the Pierce™ BCA protein assay kit (purchased from Thermo Fisher Scientific, Waltham, Massachusetts, USA). The particle size distribution of the exosome suspension 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.

[0044] 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.

[0045] 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: (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; (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 washed with 20 mL DCM, methanol and DMF three times to obtain Fmoc-Leu-Ala- resin. Activation means mixing 5 mmol Fmoc-Leu-OH with 6 mmol benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and 6 mmol 1-hydroxybenzotriazole (HOBT), adding 30 mL DMF to completely dissolve, then adding 10 mmol diisopropylethylamine (DIEA), and activating at 25°C in the dark for 5 minutes. 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.

[0046] (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; (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.

[0047] (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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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).

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] Example 1 A method for promoting skin repair through transdermal delivery of exosomes using sponge-like bone needles includes the following steps: (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; (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.

[0058] 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.

[0059] The preparation method of exosome vesicle lyophilized powder is as follows: 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. 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. 2000Mannitol, 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. 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.

[0060] 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.

[0061] The reconstitution medium was PBS buffer at pH 7.4.

[0062] The ratio of exosome vesicle lyophilized powder to reconstitution medium was 0.5 g: 4 mL.

[0063] Example 2 The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows: 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. 2000The average particle size of the obtained liposomes was 141 nm, and the zeta potential was -7.54 mV. 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; 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.

[0064] Example 3 The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows: 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; 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. 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.

[0065] Example 4 The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows: 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. 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. 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.

[0066] Example 5 The difference from Example 4 is that the reconstitution medium is PBS buffer (pH=7.4) with a sorbitol concentration of 1 g / L.

[0067] Example 6 The difference from Example 1 is that the preparation method of the exosome vesicle lyophilized powder is as follows: 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; 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; 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.

[0068] Example 7 The difference from Example 6 is that the reconstitution medium is PBS buffer (pH=7.4) with a sorbitol concentration of 1 g / L.

[0069] Example 8 A method for promoting skin repair through transdermal delivery of exosomes using sponge-like bone needles includes the following steps: (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; (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.

[0070] 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.

[0071] The preparation method of exosome vesicle lyophilized powder is as follows: 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; 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; 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.

[0072] The reconstitution medium was PBS buffer (pH=7.4) with a maltitol concentration of 2 g / L.

[0073] The ratio of exosome vesicle lyophilized powder to reconstitution medium was 0.5 g: 4 mL.

[0074] Example 9 A method for promoting skin repair through transdermal delivery of exosomes using sponge-like bone needles includes the following steps: (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; (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.

[0075] 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.

[0076] The preparation method of exosome vesicle lyophilized powder is as follows: 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; 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; 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.

[0077] The reconstitution medium was PBS buffer (pH=7.4) with an erythritol concentration of 3 g / L.

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

[0079] Test Example 1 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.

[0080] Table 1

[0081] 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.

[0082] Test Example 2 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 replaced, and the skin sample rinsed with PBS. After the solution replacement, 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 according to 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. After 8 hours, the receiving liquid was collected, and the total protein concentration was determined using the Pierce™ BCA protein assay kit. The cumulative permeation per unit area was calculated. All experiments were performed in triplicate, and the results are expressed as an arithmetic mean. The cumulative permeability per unit area, Q, is calculated as Q = (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 permeability per unit area results for each experimental group are summarized in Table 2.

[0083] Table 2

[0084] 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.

[0085] Test Example 4 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.

[0086] Table 3

[0087] 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.

[0088] Test Example 5 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 .

[0089] 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).

[0090] 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 method for promoting skin repair through transdermal delivery of exosomes assisted by sponge-like bone needles, characterized in that, Includes the following steps: (1) Pretreatment of skin with a composition containing sponge bone needles; (2) Mix the exosome vesicle lyophilized powder and reconstitution medium at 5~40℃, and then apply it to the skin; The exosome vesicle lyophilized powder is obtained by fusion of exosomes and liposomes through a membrane, followed by freeze-drying.

2. The method for promoting skin repair via transdermal exosome delivery assisted by sponge needles 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 method for promoting skin repair via transdermal exosome delivery assisted by sponge needles according to claim 2, characterized in that, 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 that can be recognized and cleaved by matrix metalloproteinases (MMPs).

4. The method for promoting skin repair by transdermal delivery of exosomes assisted by sponge needles according to claim 2 or 3, characterized in that, The functional phospholipid is at least one of distearylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, dilinoleoylphosphatidylethanolamine-polyethylene glycol, and dimyristoylphosphatidylethanolamine-polyethylene glycol; And / or, the functional phospholipid includes DC-PT-PE, and the preparation method of DC-PT-PE is as follows:

1. React Boc-PT-OH, N-hydroxysuccinimide, and a carbodiimide condensing agent; the resulting reaction solution is purified and dried to obtain Boc-PT-NHS; 2. Mix Boc-PT-NHS with PE-type phospholipids and triethylamine; 3. Mix H2N-PT-PE with a gluconic acid activated ester and triethylamine; 4. The resulting reaction solution is purified and dried to obtain DC-PT-PE. And / or, the functional phospholipid includes PEG-PT-PE, and the preparation method of PEG-PT-PE is as follows:

1. React Boc-PT-OH, N-hydroxysuccinimide, and a carbodiimide condensing agent to obtain Boc-PT-NHS after purification and drying; 2. Mix Boc-PT-NHS with PE phospholipids and triethylamine to obtain Boc-PT-PE after purification and drying; 3. Remove the Boc protecting group from Boc-PT-PE to obtain H2N-PT-PE; 4. Mix H2N-PT-PE with polyethylene glycol activated ester PEG-NHS and triethylamine to obtain PEG-PT-PE after purification and drying.

5. The method for promoting skin repair by transdermal delivery of exosomes assisted by sponge needles according to claim 4, characterized in that, The activated ester of sugar acid is obtained by reacting a sugar acid compound DC with N-hydroxysuccinimide and a carbodiimide condensing agent for 5, and then purifying and drying the resulting reaction solution; the sugar acid compound is at least one of gluconic acid, D(+)-glucuronic acid C and D-xyloic acid. And / or, the carbodiimide condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide; And / or, the PE phospholipid is at least one of phosphatidylethanolamine, dilinoleylphosphatidylethanolamine, distearylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylethanolamine; And / or, the number-average molecular weight of the PEG-NHS is 500~1000 g / mol; And / or, 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 an Ala residue.

6. The method for promoting skin repair via transdermal exosome delivery assisted by sponge spicules according to claim 3, characterized in that, The phospholipids 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.

7. The method for promoting skin repair via transdermal exosome delivery assisted by sponge bone needles according to claim 3, 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.

8. The method for promoting skin repair by exosome transdermal delivery assisted by sponge needles 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.

9. The method for promoting skin repair by exosome transdermal delivery assisted by sponge spicules according to claim 1, wherein the reconstitution 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.

10. The method for promoting skin repair through transdermal delivery of exosomes assisted by sponge needles according to claim 9, 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.

Images