CN116725975A - Extracellular vesicle carrying active protein and preparation method and application thereof - Google Patents
Extracellular vesicle carrying active protein and preparation method and application thereof Download PDFInfo
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- CN116725975A CN116725975A CN202310812220.3A CN202310812220A CN116725975A CN 116725975 A CN116725975 A CN 116725975A CN 202310812220 A CN202310812220 A CN 202310812220A CN 116725975 A CN116725975 A CN 116725975A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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Abstract
The invention discloses an extracellular vesicle carrying active proteins, a preparation method and application thereof, and particularly relates to the technical field of pharmaceutical preparations. The extracellular vesicles carrying the active proteins are prepared by constructing an extracellular vesicle bionic nano system carrying the active proteins and derived from grapes. The invention designs and prepares the grape-derived extracellular vesicles carrying the polypeptide macromolecular drugs, and avoids the reduction of the drug activity caused by complex preparation processes, materials and organic solvents. The material source is natural edible grape, and compared with a nanometer drug delivery system prepared by using a synthetic polymer material, the grape-derived extracellular vesicle oral administration has better safety. The grape-derived extracellular vesicles have good stability in the gastrointestinal tract and can promote intestinal absorption of drugs. Can improve the trans-intestinal transfer efficiency of macromolecular drugs. The grape-derived extracellular vesicles have better oral absorption effects than the milk-derived extracellular vesicles.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to an extracellular vesicle carrying active proteins, a preparation method and application thereof.
Background
Extracellular vesicles (extracellular vesicles, EVs) are biological nanoparticles with a bilayer lipid membrane structure and multiple biomolecules, a new mechanism of intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Extracellular vesicles have great application potential in the field of drug carriers by virtue of the advantages of relatively small molecular structure, natural molecular transport property, good biocompatibility and the like. To date, there have been many studies on the delivery of proteins, RNAs or other small molecule drugs via extracellular vesicles for the purpose of treating diseases.
Oral administration has the advantages of convenient administration, controllable dosage, good patient compliance and the like, and is considered to be the optimal administration mode. However, most polypeptide protein drugs have poor stability and low permeability, and are difficult to overcome multiple absorption barriers of the gastrointestinal tract, so that the oral bioavailability is extremely low, and the clinical transformation is difficult to realize. How to keep the activity of the medicine in the gastrointestinal tract and the body and break through the physiological barrier to reach the focus part is a key scientific problem affecting the oral effectiveness of the medicine, and is also a bottleneck problem to be solved in oral medicine delivery. Although the stability of the medicine in the gastrointestinal tract transportation process can be improved by adopting a new preparation method, the medicine is difficult to exit from the basal side and enter the blood circulation due to the barrier effect of the intestinal epithelial cell layer and the mucus layer covered on the intestinal epithelial cell layer, and the improvement of the oral bioavailability is very little.
At present, the methods for loading extracellular vesicles can be divided into two types, namely, the methods are operated on parent cells, namely, the parent cells are transfected, the methods are complicated and time-consuming to operate, and the methods can only be used on cultured cells, have low drug loading rate and are difficult to be used for extracellular vesicles of special sources such as blood plasma, milk, plants and the like; the other category is to load the medicine on the purified extracellular vesicles, which specifically comprises electroporation, co-incubation, repeated freeze thawing, extrusion, vortex oscillation and the like, however, the medicine loading rate is low, and the clinical application requirements are difficult to meet.
Disclosure of Invention
Therefore, the invention provides an extracellular vesicle carrying active proteins, and a preparation method and application thereof, so as to solve the problems that the existing extracellular vesicle has low drug loading rate, is difficult to meet clinical requirements and the like.
The intestinal mucosa is used as an important absorption part, can efficiently absorb the nutrient components of food, and is regulated by various natural mechanisms. Extracellular Vesicles (EVs) derived from vegetables, fruits and dairy products can maintain good stability in gastrointestinal tract, and have good mucous-penetrating and intestinal cell targeting ability.
According to the invention, grape pulp is obtained, gradient centrifugation is carried out after homogenization to obtain grape-derived Extracellular Vesicles (EVs), and polypeptide macromolecular drugs such as insulin, liraglutide, cable Ma Lutai and the like are loaded into the grape-derived Extracellular Vesicles (EVs) through probe ultrasound to obtain the food-derived membrane bionic oral nanoparticle.
The nanoparticles can rapidly pass through mucus under the action of an EVs membrane with hydrophilic surface, and the targeting effect of EVs is utilized to promote the cell uptake; after the nano particles enter the cells, the nano particles are then discharged from the basal side to enter the blood circulation under the mediation of the EVs membrane to exert the drug effect, so that the oral bioavailability of the drug is improved.
In order to achieve the above object, the present invention provides the following technical solutions:
according to the extracellular vesicle carrying active protein provided by the first aspect of the invention, the extracellular vesicle carrying active protein is obtained by constructing the extracellular vesicle carrying active protein taking grape as a source, and the extracellular vesicle bionic nano system is provided.
Further, the active protein is one or more of protein polypeptide drugs, nucleic acid drugs and chemical drugs.
The protein polypeptide drugs include, but are not limited to: insulin, octreotide leuprorelin acetate, calcitonin, thymopentin, luteinizing hormone releasing hormone, tikeke peptide acetate, buserelin, exenatide, glucagon-like peptide-1, triptorelin acetate, leukocyte growth factor, erythrocyte growth factor, macrophage growth factor, tumor necrosis factor, epidermal growth factor, interleukins, angiostatin, bovine serum albumin, ovalbumin, parathyroid hormone, growth hormone, somatostatin, interferons, monoclonal antibodies and vaccines; as one of the embodiments of the present invention, insulin is preferable as an active ingredient.
Such nucleic acid agents include, but are not limited to, small interfering ribonucleic acids and plasmid DNA;
further, the content of the active protein is 0.1% -90% (w/w); preferably 1% to 80% (w/w).
According to a second aspect of the present invention there is provided a method of preparing an extracellular vesicle carrying an active protein comprising:
step one, preparation of grape-derived extracellular vesicles
Mixing pulp of grape at high speed, homogenizing, filtering with a screen, and collecting filtrate, wherein the size of the screen is 80-500 meshes;
taking filtrate, and carrying out gradient centrifugation; removing cells and larger particles, and continuing to centrifuge the supernatant to obtain a precipitate;
adding phosphate buffer solution into the precipitate to form dispersion liquid, adding sucrose gradient solution into the dispersion liquid, and centrifuging at low temperature and high speed to obtain grape-derived extracellular vesicles Gra-EVs;
step two, preparation of extracellular vesicles carrying active proteins
Planting active protein into Gra-EVs; extracellular vesicles carrying active proteins are obtained.
Further, in the first step, the condition of gradient centrifugation is 1000×g centrifugation for 10min,3000×g centrifugation for 20min, and 10,000×g centrifugation for 40min; the conditions for continued centrifugation were 150,000Xg, 90min,4 ℃.
Further, in the first step, the conditions of low-temperature high-speed centrifugation were 150,000Xg, 120min,4 ℃.
Further, in the second step, the method for loading the active protein into the Gra-EVs is a probe ultrasonic method, an incubation method, a saponin permeation method, an electroporation method, an extrusion method or a cyclic freeze thawing method; as an example, the probe ultrasonic condition is 1-500 w ultrasonic power of the probe for 1 s-3600 s; wherein, the ultrasonic treatment is carried out for 1 to 600 seconds and stopped for 1 to 600 seconds.
According to the application of the extracellular vesicles carrying active proteins in preparing a grape-source extracellular vesicle drug delivery system carrying active protein drugs, the extracellular vesicles carrying active proteins are provided.
Further, the delivery system is an oral delivery system.
The use of an extracellular vesicle carrying an active protein according to the fourth aspect of the present invention for the preparation of a skin care product.
The invention has the following advantages:
the invention promotes the medicine to penetrate through the intestinal mucosa multiple absorption barrier and more to be absorbed into the blood circulation by constructing the grape source membrane bionic oral nano medicine delivery system carrying active protein medicines, and improves the oral absorption bioavailability of the medicine.
Compared with the grape skin and grape seed derived extracellular vesicles, the invention has the advantages that the yield of the grape pulp derived extracellular vesicles is higher, and the production is easier to expand. In addition, when the human eating grape, the grape pulp is mainly taken, and phospholipid components such as phosphatidic acid, phosphatidylethanolamine and the like which are rich in the grape pulp-derived extracellular vesicles can promote the carried polypeptide macromolecular drugs to cross intestinal epithelial cells, so that the absorption efficiency of the drugs can be improved by adopting the grape pulp-derived extracellular vesicles to prepare the polypeptide macromolecular drugs.
The same concentration of grape-derived extracellular vesicles of the invention have higher uptake efficiency on Caco-2 cells, while the same concentration of grape-derived extracellular vesicles has higher uptake efficiency on the intestinal tract, compared to milk-derived extracellular vesicles.
The preparation method has mild preparation conditions, and can avoid the stability reduction or the inactivation of the medicine caused by the preparation process.
The extracellular vesicles carrying the active proteins have good colloid stability in the gastrointestinal tract and can continuously release medicines.
The invention designs and prepares the grape-derived extracellular vesicles carrying the polypeptide macromolecular drugs, and avoids the reduction of the drug activity caused by complex preparation processes, materials and organic solvents. The material source is natural edible grape, and compared with a nanometer drug delivery system prepared by using a synthetic polymer material, the grape-derived extracellular vesicle oral administration has better safety. The grape-derived extracellular vesicles have good stability in the gastrointestinal tract and can promote intestinal absorption of drugs. The absorption effect of the grape-derived extracellular vesicles is better than that of free macromolecular drugs and milk-derived extracellular vesicles.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
FIG. 1 is a trace observation particle size distribution diagram of an insulin-carrying grape-derived extracellular vesicle particle provided in example 1 of the present invention;
FIG. 2 is a scanning image of an extracellular vesicle transmission electron microscope of insulin-carrying grape source according to example 1 of the present invention;
FIG. 3 is a graph showing the cumulative release of insulin-loaded grape-derived extracellular vesicles in simulated gastric fluid and intestinal fluid provided in Experimental example 3 of the present invention;
FIG. 4 is a graph showing the comparison of the absorption of free insulin, extracellular vesicles derived from milk carrying insulin, and extracellular vesicles derived from grape carrying insulin in the intestinal tract in the eversion intestine test according to example 4 of the present invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The present example provides an insulin-carrying grape-derived extracellular vesicle delivery system:
to obtain grape-derived extracellular vesicles (Gra-EVs), 250g of grape (Kyoho grape) pulp was homogenized by high-speed stirring, and the filtrate was collected after filtration through a screen. The filtrate was subjected to gradient centrifugation (1000 Xg centrifugation for 10min,3000 Xg centrifugation for 20min,10,000Xg centrifugation for 40 min) to remove cells and larger particles, and the supernatant was further centrifuged (150,000Xg, 90min,4 ℃) to obtain a precipitate.
10mL of Phosphate Buffer (PBS) was added to the precipitate to form a dispersion, and the dispersion was added to a sucrose gradient (8%/15%/30%/45%), centrifuged at low temperature and high speed (150,000Xg, 120min,4 ℃ C.) to obtain 8%/15% layers, 15%/30%, 30%/45% layers of bands, all labeled Gra-EVs.
Dispersing 1-200 mg insulin and 1-10 mL15%/30% layered Gra-EVs in PBS; ultrasonic power of 50-200 w is used for ultrasonic treatment for 1-10 min (ultrasonic treatment for 1-60 s and stopping for 1-60 s) in ice bath to obtain the insulin-carrying grape-derived extracellular vesicles.
Particle tracing and observing particle size distribution as shown in figure 1; transmission electron microscope scanning is shown in fig. 2.
Example 2
This example provides a grape-derived extracellular vesicle drug delivery system carrying liraglutide:
to obtain grape-derived extracellular vesicles (Gra-EVs), 250g of grape (Kyoho grape) pulp was homogenized by high-speed stirring, and the filtrate was collected after filtration through a screen. The filtrate was subjected to gradient centrifugation (1000 Xg centrifugation for 10min,3000 Xg centrifugation for 20min,10,000Xg centrifugation for 40 min) to remove cells and larger particles, and the supernatant was further centrifuged (150,000Xg, 90min,4 ℃) to obtain a precipitate.
10mL of Phosphate Buffer (PBS) was added to the precipitate to form a dispersion, and the dispersion was added to a sucrose gradient (8%/15%/30%/45%), centrifuged at low temperature and high speed (150,000Xg, 120min,4 ℃ C.) to obtain 8%/15% layers, 15%/30%, 30%/45% layers of bands, all labeled Gra-EVs.
Dispersing 1-200 mg of liraglutide and 1-10 mL of 15%/30% layer band Gra-EVs in PBS; ultrasonic power of 50-200 w is used for ultrasonic treatment for 1-10 min (ultrasonic treatment for 1-60 s and ultrasonic treatment for 1-60 s) in ice bath, and the grape-derived extracellular vesicles carrying liraglutide are obtained.
Example 3
The present example provides a grape-derived extracellular vesicle drug delivery system of carrier cable Ma Lutai:
to obtain grape-derived extracellular vesicles (Gra-EVs), 250g of grape (Kyoho grape) pulp was homogenized by high-speed stirring, and the filtrate was collected after filtration through a screen. The filtrate was subjected to gradient centrifugation (1000 Xg centrifugation for 10min,3000 Xg centrifugation for 20min,10,000Xg centrifugation for 40 min) to remove cells and larger particles, and the supernatant was further centrifuged (150,000Xg, 90min,4 ℃) to obtain a precipitate.
10mL of Phosphate Buffer (PBS) was added to the precipitate to form a dispersion, and the dispersion was added to a sucrose gradient (8%/15%/30%/45%), centrifuged at low temperature and high speed (150,000Xg, 120min,4 ℃ C.) to obtain 8%/15% layers, 15%/30%, 30%/45% layers of bands, all labeled Gra-EVs.
Dispersing 1-200 mg of cord Ma Lutai and 1-10 mL of 15%/30% layered Gra-EVs in PBS; the grape-derived extracellular vesicles of the carrier cable Ma Lutai are obtained by ultrasonic power of 50-200 w of probe in ice bath for 1-10 min (ultrasonic for 1-60 s and stopping for 1-60 s).
The probe ultrasound in examples 1-3 can also be used to load drugs into extracellular vesicles by incubation, saponin permeation, electroporation, extrusion, and cyclic freeze thawing, with no difference in effect.
Experimental example 1
The nano-encapsulation efficiency of the insulin-loaded grape-derived extracellular vesicles obtained in example 1 was measured as follows:
the extracellular vesicles obtained in example 1 were taken into a 100kDa ultrafiltration centrifuge tube and centrifuged (5000 rpm,10 min) to separate nanoparticles from free insulin. Insulin and free insulin in extracellular vesicles were quantitatively analyzed by reverse phase high performance liquid chromatography (RP-HPLC) and the encapsulation efficiency (EE%) of insulin was calculated.
The encapsulation efficiency of the insulin was measured to be 5% -80%.
Experimental example 2
Analysis and detection of extracellular vesicle lipid components derived from insulin-carrying grape obtained in example 1:
the lipid components of the insulin-carrying grape-derived extracellular vesicles were extracted and quantified and analyzed, respectively, using LC-MS/MS and lipidsearich4.2 software, and the results are shown in table 1.
TABLE 1 lipid fraction analysis of insulin-carrying grape-derived extracellular vesicles
Conclusion: the lipid component of grape-derived extracellular vesicles mainly comprises fatty acid, phospholipid, glycolipid, sphingolipid, etc., including cardiolipin, digalactosyl Shan Xian glyceride, fatty acid, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyl diacylglycerol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, sulfatidyl, etc.
Experimental example 3
Cumulative release amount detection of insulin-carrying grape-derived extracellular vesicle insulin obtained in example 1:
artificial gastric juice (SGF) without pepsin and artificial intestinal juice (SIF) without trypsin were formulated according to USP 39-N37. 2mL of insulin-loaded grape-derived extracellular vesicles were added to a dialysis tube with a molecular weight cut-off of 100 kDa. The dialysis tube was placed in SGF without pepsin and dialyzed with stirring at 37℃for 2h, and the release medium was replaced with SIF without enzyme and dialyzed with stirring for 6h. Samples were taken from 200. Mu.L dialysis tubing at the indicated time points, and after addition of Triton-X100 to disrupt the nanoparticles, the supernatant was centrifuged and assayed for insulin concentration by RP-HPLC to calculate the cumulative release of insulin. The results are shown in FIG. 3.
Conclusion: experimental results show that the insulin-carrying grape-derived extracellular vesicles can slowly release the drug in simulated gastric fluid and intestinal fluid for 8 hours, and about 60% of the drug is released.
Experimental example 4
Intestinal absorption Capacity detection of insulin-carrying grape-derived extracellular vesicle insulin obtained in example 1:
in order to verify that the insulin-carrying grape-derived extracellular vesicles have better intestinal tract absorption capacity, an eversion intestine experiment is adopted to compare the intestinal tract absorption conditions of free insulin, the insulin-carrying milk-derived extracellular vesicles and the insulin-carrying grape-derived extracellular vesicles. SD rats (males, 220-250g,7 weeks) were fasted overnight and allowed to drink freely. After anesthesia with chloral hydrate, dissecting along the ventral midline, cutting off the abdominal cavity along the ventral midline, cutting off about 10cm jejunum from the lower 15cm of the pylorus of the stomach, placing in Krebs-Ringer nutrient solution pre-cooled at 4deg.C, washing off the contents and stripping off the residual mesentery and adipose tissue. Mucous membrane is made by round head glass rodThe surface turns to the outside and the serosa faces inwards. After ligating one end of the intestinal segment, a certain amount of Krebs-Ringer nutrient solution is injected into the intestinal cavity, and the other end is sleeved into a silicone tube with the outer diameter of 5 mm. The intestinal sections were placed vertically in 40mL Krebs-Ringer nutrient solution at 37℃and gas (95% O) was introduced 2 And 5% CO 2 ). 200. Mu.L of the solution was collected from the serosal side of the intestinal lumen at the indicated time points (15, 30, 45, 60 and 90 min) while the same volume of Krebs-Ringer nutrient solution was supplemented and the permeation of the drug was examined by HPLC. After spotting, the diameter and length of the intestinal segment were measured and the Papp value of insulin was calculated. The results are shown in FIG. 4.
Conclusion: the higher efficiency of trans-membrane transport of grape-derived extracellular vesicles in the gut at the same drug concentration compared to free insulin, insulin-loaded milk-derived extracellular vesicles suggests that grape-derived extracellular vesicles contribute to their overcoming of intestinal mucosal absorption barriers.
Experimental example 5
The grape-derived extracellular vesicles of liraglutide obtained in example 2 were tested for nanocapsule as follows:
the extracellular vesicles obtained in example 2 were taken into a 100kDa ultrafiltration centrifuge tube and centrifuged (5000 rpm,10 min) to separate nanoparticles from free insulin. Insulin and free insulin in extracellular vesicles were quantitatively analyzed by reverse phase high performance liquid chromatography (RP-HPLC) and the encapsulation efficiency (EE%) of insulin was calculated.
The encapsulation rate of the liraglutide is measured to be 5% -80%.
Experimental example 6
The grape-derived extracellular vesicles of the somalunin obtained in example 3 were tested for nano encapsulation as follows:
the extracellular vesicles obtained in example 3 were taken into a 100kDa ultrafiltration centrifuge tube and centrifuged (5000 rpm,10 min) to separate nanoparticles from free insulin. Insulin and free insulin in extracellular vesicles were quantitatively analyzed by reverse phase high performance liquid chromatography (RP-HPLC) and the encapsulation efficiency (EE%) of insulin was calculated.
The encapsulation efficiency of the measured somalundin is 5% -80%.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. An extracellular vesicle carrying active proteins is characterized in that the extracellular vesicle is obtained by constructing the extracellular vesicle carrying active proteins which take the extracellular vesicle of grape as a source, and the extracellular vesicle bionic nano-system is provided.
2. The extracellular vesicle carrying an active protein of claim 1, wherein the active protein is one or more of a protein polypeptide drug, a nucleic acid drug, and a chemical drug.
3. An extracellular vesicle loaded with an active protein according to claim 2, wherein the active protein content is 0.1% to 90% (w/w).
4. A method for preparing an extracellular vesicle carrying an active protein, comprising:
step one, preparation of grape-derived extracellular vesicles
Mixing grape pulp at high speed, homogenizing, filtering with a screen, and collecting filtrate;
taking filtrate, and carrying out gradient centrifugation; removing cells and larger particles, and continuing to centrifuge the supernatant to obtain a precipitate;
adding phosphate buffer solution into the precipitate to form dispersion liquid, adding sucrose gradient solution into the dispersion liquid, and centrifuging at low temperature and high speed to obtain grape-derived extracellular vesicles Gra-EVs;
step two, preparation of extracellular vesicles carrying active proteins
Loading the active protein into Gra-EVs to obtain extracellular vesicles carrying the active protein.
5. The method according to claim 4, wherein in the first step, the condition of gradient centrifugation is 1000 Xg for 10min,3000 Xg for 20min, and 10,000 Xg for 40min; the conditions for continued centrifugation were 150,000Xg, 90min,4 ℃.
6. The method according to claim 4, wherein the low-temperature high-speed centrifugation is performed at 150,000Xg, 120min, and 4 ℃.
7. The method for preparing extracellular vesicles loaded with active protein according to claim 4, wherein in the second step, the method for loading the active protein into the Gra-EVs is probe ultrasound method, incubation method, saponin permeation method, electroporation method, extrusion method or cyclic freeze thawing method.
8. Use of extracellular vesicles carrying active proteins in the preparation of a grape-derived extracellular vesicle delivery system carrying active protein drugs.
9. The use of claim 8, wherein the delivery system is an oral delivery system.
10. Use of an extracellular vesicle loaded with an active protein for the preparation of a skin care product.
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