A method of extracting nanolipidic ectosomes from mammalian milk

Abstract

This invention provides a method for extracting nanolipid exovesicles from mammalian milk, belonging to the field of biomedical technology. The method provided by this invention employs a three-step process—pretreatment, deep removal of impurities such as proteins, and polyethylene glycol precipitation and purification—to efficiently remove highly concentrated specific impurities such as lipids, casein, and whey proteins from milk. The entire method eliminates the need for an ultracentrifuge, requiring only a conventional high-speed centrifuge and a centrifugal filter column, resulting in low equipment requirements, simple operation, and significantly shorter overall time compared to traditional ultracentrifugation methods. The exosomes prepared by this method can be stably stored for at least 3 months, maintaining stable particle size and concentration, and without degradation of marker proteins, demonstrating significant clinical value and market potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for extracting nanolipid exovesicles from mammalian milk. Background Technology

[0002] Exosomes, also known as nanolipid exovesicles, are tiny vesicle structures with a diameter of approximately 30 to 150 nanometers secreted by cells. Exosomes are widely distributed in various body fluids and biological secretions of animals, carrying bioactive molecules such as proteins, lipids, messenger RNA, and microRNAs from their origin. They play important roles in intercellular signal transduction, tissue homeostasis maintenance, and immune response regulation. In recent years, with the deepening of research on exosomes, their biological functions in promoting tissue repair, anti-inflammation, anti-oxidation, and immune regulation have been gradually revealed, leading to widespread attention on their application value in clinical treatment, biopharmaceutical development, and cosmetic skincare.

[0003] Currently, the main source of exosomes is cell culture supernatant. This method requires obtaining a large amount of cell culture supernatant through a complex cell culture system, followed by separation and purification using methods such as ultracentrifugation, size exclusion chromatography, and immunoaffinity capture. However, the preparation of exosomes from cell culture supernatant has many limitations: cell culture is costly, time-consuming, and difficult to scale up; furthermore, purification methods such as ultracentrifugation require expensive ultracentrifuge equipment, are cumbersome and time-consuming, and cannot meet the needs of industrial applications.

[0004] Therefore, finding more economical, efficient, and readily available sources of exosome raw materials, and developing specific purification methods adapted to these sources, has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting nanolipid exovesicles from mammalian milk, which solves the problems of cumbersome operation, long time consumption, reliance on high-end equipment such as ultracentrifuges, low purity of the obtained product, serious contamination by impurities and proteins, and low yield in the existing technology for extracting nanolipid exovesicles (exosomes) from mammalian milk.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing nanolipid exovesicles, comprising the following steps: S1: Centrifuge mammalian milk, discard the lower lipid and precipitate, and collect the upper whey; S2: Mix the whey with a protein precipitation reagent until the whey becomes translucent to obtain pretreated whey; S3: Mix the pretreated whey with the flocculant and let it stand to obtain a precipitated mixture; S4: Centrifuge the precipitate mixture and collect the supernatant to obtain a protein-free supernatant; S5: The protein-free supernatant is brought into contact with a centrifugal filter column for filtration, and the filtrate is collected to obtain the filtered filtrate. S6: Mix the filtered filtrate with the polyethylene glycol precipitate mother liquor, let stand, centrifuge, discard the supernatant, collect the precipitate, and obtain the crude precipitate of nanolipid exovesicles. S7: The coarse precipitate of the nanolipid exovesicles is mixed with phosphate buffer and resuspended. After centrifugation, the supernatant is collected to obtain the nanolipid exovesicles. The precipitant contains acetic acid solution.

[0008] Preferably, the mammalian milk is selected from cow's milk, sheep's milk, human milk, or horse milk.

[0009] Preferably, in S1, the centrifugation temperature is 2-8℃, the centrifugation force is 8000g-12000g, and the centrifugation time is 10-30min.

[0010] Preferably, the final concentration of the acetic acid solution in the flocculant is 0.005 M to 0.02 M.

[0011] Preferably, in step S3, the settling temperature is 2–8°C and the settling time is 5–15 min.

[0012] Preferably, in S4, the centrifugal force is 2000g to 5000g, and the centrifugation time is 5 to 15min.

[0013] Preferably, the pore size of the centrifugal filter column is 0.22 μm or 0.45 μm.

[0014] Preferably, the polyethylene glycol precipitation mother liquor contains polyethylene glycol and sodium chloride, wherein the polyethylene glycol is selected from polyethylene glycol 6000 or polyethylene glycol 8000, the final concentration of polyethylene glycol in the polyethylene glycol precipitation mother liquor is 10% to 20% by weight / volume, the final concentration of sodium chloride in the polyethylene glycol precipitation mother liquor is 0.2 M to 1.0 M, and the pH of the polyethylene glycol precipitation mother liquor is 7.0 to 7.5.

[0015] Preferably, in step S6, the volume ratio of the filtered filtrate to the polyethylene glycol precipitate mother liquor is 1:0.5 to 1:2, the settling time is 0.5 to 2 hours, the centrifugal force is 8000g to 12000g, and the centrifugation time is 30 to 90 minutes.

[0016] Preferably, in S7, the centrifugal force is 10000g to 15000g, and the centrifugation time is 1 to 5min.

[0017] The beneficial effects of this invention are: This invention provides a method for preparing nanolipid exovesicles specifically adapted for mammalian milk. This method employs a three-step process—pretreatment, deep removal of impurities such as proteins, and polyethylene glycol precipitation and purification—to efficiently remove highly concentrated lipids, casein, whey protein, and other specific impurities from milk. The entire method eliminates the need for an ultracentrifuge, requiring only a conventional high-speed centrifuge and a centrifugal filter column. It has low equipment requirements, is easy to operate, and has a significantly shorter overall processing time than traditional ultracentrifugation methods. Using this method to process mammalian milk, 1.5–2.010 nanolipid exovesicles can be extracted per milliliter of milk. 9 The method yielded exosomes with a significantly higher yield than traditional methods. The main peak particle size of the obtained exosomes was located between 30 and 150 nm, exhibiting a typical cup-shaped lipid bilayer structure. They highly expressed specific marker proteins such as CD9, CD63, CD81, and TSG101, but did not express the negative marker Calnexin. The exosomes prepared by this method were highly pure and free from contamination by other proteins. The exosomes prepared by this method can be stably stored at -80°C for at least 3 months, maintaining stable particle size and concentration, and without degradation of marker proteins.

[0018] The mammalian milk-derived nanolipid exovesicles prepared by this invention possess high purity and high bioactivity, fully preserving the inherent biological functions of exosomes, such as promoting tissue repair, anti-inflammation, immunomodulation, antioxidation, and promoting skin cell proliferation. This product can be derived from widely available natural raw materials such as cow's milk, sheep's milk, human milk, and mare's milk, making it inexpensive and suitable for large-scale industrial production. It provides a high-quality and stable bioactive raw material for various downstream applications. These nanolipid exovesicles can be widely used in biomedicine, biopharmaceuticals, clinical treatment, and cosmetic skincare. Specifically, they can be used to prepare biopharmaceuticals, functional biopharmaceuticals, or cosmetic skincare products that promote tissue repair, anti-inflammation, immunomodulation, antioxidation, or promote skin cell proliferation. They can also serve as carriers for targeted drug delivery systems. This application expands the raw material sources and application scenarios of exosomes, possessing significant clinical value and market potential. Attached Figure Description

[0019] Figure 1 Results of NTA assay for bovine milk exosomes; Figure 2 Electron micrograph of exosomes (bar=100 nM); Figure 3 This is a graph showing the results of exosome protein electrophoresis. Detailed Implementation

[0020] This invention provides a method for preparing nanolipid exovesicles, comprising the following steps: S1: Centrifuge mammalian milk, discard the lower lipid and precipitate, and collect the upper whey; S2: Mix the whey with a protein precipitation reagent until the whey becomes translucent to obtain pretreated whey; S3: Mix the pretreated whey with the flocculant and let it stand to obtain a precipitated mixture; S4: Centrifuge the precipitate mixture and collect the supernatant to obtain a protein-free supernatant; S5: The protein-free supernatant is brought into contact with a centrifugal filter column for filtration, and the filtrate is collected to obtain the filtered filtrate. S6: Mix the filtered filtrate with the polyethylene glycol precipitate mother liquor, let stand, centrifuge, discard the supernatant, collect the precipitate, and obtain the crude precipitate of nanolipid exovesicles. S7: The coarse precipitate of the nanolipid exovesicles is mixed with phosphate buffer and resuspended. After centrifugation, the supernatant is collected to obtain the nanolipid exovesicles. The precipitant contains acetic acid solution.

[0021] In this invention, mammalian milk refers to the natural liquid secreted by the mammary glands of mammals after childbirth, rich in proteins, fats, lactose, and various bioactive components, and naturally contains abundant nanolipid vesicles. Common mammalian milks that can be used in this invention include, but are not limited to, cow's milk, sheep's milk, human milk, mare's milk, camel's milk, buffalo milk, and yak milk. The centrifugation operation refers to the process of using the centrifugal force generated by a centrifuge to separate solid particles or denser components in the liquid by sedimentation. The purpose of centrifugation in step S1 of this invention is to remove most of the lipids (such as milk fat) and insoluble impurities (such as some casein micelles) from the milk, thereby obtaining a relatively clear upper whey layer. Whey is the liquid portion remaining after removing fat and casein precipitates from the milk, containing whey protein, lactose, minerals, and the target product, nanolipid vesicles. The discarded lower lipids and precipitates mainly include the milk fat layer and precipitated casein, cell debris, etc. The protein precipitation reagent refers to a chemical reagent that can denature or aggregate soluble proteins in a solution, thereby precipitating them. Examples include trichloroacetic acid, ammonium acetate, ammonium sulfate, ethanol, acetone, or a mixed solution of organic acids. In this invention, after mixing the protein precipitation reagent with whey, the whey gradually changes from turbid to translucent, indicating that some soluble impurities have been removed. The flocculant refers to a substance that assists in protein precipitation or promotes precipitate aggregation. In this invention, the flocculant contains an acetic acid solution. Acetic acid, also known as glacial acetic acid, is a weak organic acid. Its aqueous solution can adjust the pH and neutralize the charge on the protein surface, promoting the aggregation and precipitation of protein molecules. The acetic acid solution can be any concentration solution obtained by diluting glacial acetic acid with water, and its function is to further improve the removal efficiency of impurities based on the protein precipitation reagent. Settling refers to placing the mixed liquid under undisturbed conditions for a period of time to allow the precipitate to fully form and aggregate. The centrifugal filter column is a device that combines centrifugal action with a filter medium. It typically consists of a tubular column and an internal filter membrane. Under centrifugal force, the liquid passes through the filter membrane while retaining solid particles. The centrifugal filter column used in this invention can be a commercially available microporous filter device, such as the BeyoGold™ microporous filter column from Beyotime or the Sigma-Aldrich Ultrafree® centrifugal filter, and its membrane pore size can be selected as needed. The polyethylene glycol precipitate mother liquor refers to a concentrated solution containing polyethylene glycol (PEG). PEG is a non-ionic, water-soluble polymer that can induce precipitation of nanoparticles (such as exosomes) in solution through steric hindrance. Phosphate-buffered saline (PBS) is an isotonic buffer solution composed of dihydrogen phosphate and dihydrogen phosphate, commonly used for the resuspension and preservation of biological samples, with its pH typically maintained between 7.2 and 7.4. Resuspension refers to the operation of redispersing the centrifuged solid particles in a liquid medium to form a homogeneous suspension.

[0022] Preferably, the mammalian milk is selected from cow's milk, sheep's milk, human milk, or horse milk.

[0023] In this invention, cow's milk refers to the milk secreted by dairy cows after calving. It is widely available, inexpensive, and produced in large quantities, making it a preferred raw material for industrial production. Goat's milk includes goat milk and sheep milk; its fat globules are smaller in diameter, and its exosome content may be higher. Human milk refers to human breast milk, which is rich in bioactive components and suitable for preparing human biological agents. Horse milk has a lower yield but a unique nutritional composition. In addition, camel milk, buffalo milk, donkey milk, etc., can also be selected according to actual needs. The nanolipid vesicles extracted from different milk sources may have slight differences in particle size distribution, marker protein expression levels, and biological functions, but all can be used to obtain high-purity products using the method of this invention.

[0024] Preferably, in S1, the centrifugation temperature is 2-8℃, the centrifugation force is 8000g-12000g, and the centrifugation time is 10-30min.

[0025] In this invention, the centrifugation temperature is controlled within a low-temperature range (e.g., 2–8°C) to maximize the protection of the integrity and bioactivity of the nanolipid exovesicles, avoiding protein degradation or vesicle rupture caused by high temperatures. Specifically, the centrifugation temperature can be selected from 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C, preferably 4°C. The centrifugal force refers to the relative centrifugal force, measured in g, and its value depends on the centrifuge speed and rotor radius. A centrifugal force range of 8000g to 12000g is sufficient to precipitate fat and most insoluble proteins in the milk, while preventing premature sedimentation of exosomes due to excessive centrifugal force. Specifically, the centrifugal force can be 8000g, 8500g, 9000g, 9500g, 10000g, 10500g, 11000g, 11500g, or 12000g, preferably 10000g. The centrifugation time is 10 to 30 minutes; too short a time results in incomplete precipitation, while too long a time increases precipitation time. Specifically, the centrifugation time can be 10 min, 15 min, 20 min, 25 min, or 30 min, preferably 20 min. By combining these conditions, lipids and insoluble impurities in the milk can be effectively removed, resulting in a clearer whey.

[0026] Preferably, the final concentration of the acetic acid solution in the flocculant is 0.005 M to 0.02 M.

[0027] In this invention, the final concentration refers to the molar concentration of acetic acid in the final mixture after the flocculant is added to the pretreated whey. An acetic acid concentration range of 0.005 M to 0.02 M effectively promotes the precipitation of impurity proteins without damaging the vesicle structure of the nanolipid exovesicles. Specifically, the final concentration of the acetic acid solution can be 0.005 M, 0.008 M, 0.01 M, 0.012 M, 0.015 M, 0.018 M, or 0.02 M, preferably 0.01 M. The acetic acid solution can be obtained by diluting it with glacial acetic acid proportionally; for example, adding approximately 0.057 mL of glacial acetic acid to every 100 mL of whey can achieve a final concentration of approximately 0.01 M. This concentration of acetic acid provides a weakly acidic environment, which is beneficial for the isoelectric point precipitation of proteins, while not causing exosome membrane rupture.

[0028] Preferably, in step S3, the settling temperature is 2–8°C and the settling time is 5–15 min.

[0029] In this invention, the settling temperature is preferably low (2-8°C) to avoid inactivation of exosomes during precipitation. Specific temperatures can be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C, preferably 4°C. The settling time refers to the duration the mixture is left undisturbed after the addition of the flocculant. A time range of 5 to 15 minutes allows soluble proteins to fully precipitate into a bean curd-like precipitate; too short a time results in incomplete precipitation, while too long a time may increase non-specific co-precipitation. Specifically, the settling time can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, preferably 10 min. Under these conditions, obvious flocculent or bean curd-like precipitates can be observed in the mixture, indicating that the proteins have been effectively removed.

[0030] Preferably, in S4, the centrifugal force is 2000g to 5000g, and the centrifugation time is 5 to 15min.

[0031] In this invention, the purpose of centrifugation in step S4 is to separate the protein precipitate formed in step S3 from the supernatant. A centrifugal force of 2000g to 5000g is suitable for removing larger protein aggregates without settling smaller exosomes. Specifically, the centrifugal force can be 2000g, 2500g, 3000g, 3500g, 4000g, 4500g, or 5000g, preferably 3000g. A centrifugation time of 5 to 15 minutes allows the precipitate to be fully compacted. Specifically, the centrifugation time can be 5 minutes, 7 minutes, 10 minutes, 12 minutes, or 15 minutes, preferably 10 minutes. The supernatant collected after centrifugation is the protein-free supernatant, which contains the target nanolipid exovesicles, while the precipitate is the removed contaminating protein.

[0032] Preferably, the pore size of the centrifugal filter column is 0.22 μm or 0.45 μm.

[0033] In this invention, the pore size of the centrifugal filter column determines the maximum size of particles that can pass through the filter membrane. A 0.22 μm pore size filter membrane can retain the vast majority of bacteria and larger particle fragments, making it suitable for sterilization filtration; a 0.45 μm pore size filter membrane allows slightly larger particles to pass through while still removing micron-sized protein aggregates. Both pore sizes can be used in this invention to further remove residual small protein precipitates. The specific choice depends on the sample turbidity and subsequent application requirements: a 0.22 μm pore size can be used if higher purity is required; a 0.45 μm pore size can be used if less exosome loss is desired. The centrifugal filter column is typically pre-washed before use, then the sample is added to the column tube, and under appropriate centrifugal force, the liquid passes through the filter membrane into the collection tube, while the retained material remains on the filter membrane. The centrifugal filter column used in this invention can be any commercially available similar product, such as BeyoGold™ microporous filter column (part number FFT022 or FFT045) or Sigma-Aldrich Ultrafree® centrifugal filter.

[0034] Preferably, the polyethylene glycol precipitation mother liquor contains polyethylene glycol and sodium chloride, wherein the polyethylene glycol is selected from polyethylene glycol 6000 or polyethylene glycol 8000, the final concentration of polyethylene glycol in the polyethylene glycol precipitation mother liquor is 10% to 20% by weight / volume, the final concentration of sodium chloride in the polyethylene glycol precipitation mother liquor is 0.2 M to 1.0 M, and the pH of the polyethylene glycol precipitation mother liquor is 7.0 to 7.5.

[0035] In this invention, the polyethylene glycol (PEG) precipitation mother liquor is a concentrated precipitant solution. PEG is a polymer of ethylene oxide; its physical properties and applications vary depending on its molecular weight. PEG 6000 has an average molecular weight of approximately 6000 Daltons, and PEG 8000 has an average molecular weight of approximately 8000 Daltons. Both are white, waxy solids, readily soluble in water, and exhibit low toxicity and good biocompatibility. They are commonly used to precipitate nanoparticles such as viruses and exosomes, and their precipitation efficiency is closely related to the PEG concentration, molecular weight, and ionic strength of the solution. In this invention, the final concentration of PEG in the precipitation mother liquor can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (weight-to-volume ratio, i.e., grams of PEG per 100 ml of solution), preferably 16%. The addition of sodium chloride increases the ionic strength, promotes the interaction between PEG and water, and thus enhances the precipitation effect. The final concentration of sodium chloride in the precipitate mother liquor can be 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1.0 M, preferably 0.5 M. Adjusting the pH to a neutral range of 7.0–7.5 is beneficial for maintaining the structural stability of exosomes. Specific pH values ​​can be 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, preferably 7.2–7.4. Common acid-base adjusters such as hydrochloric acid or sodium hydroxide can be used for pH adjustment. To prepare the precipitate mother liquor, the weighed PEG and sodium chloride are dissolved in distilled water or a buffer solution, and the pH is adjusted to a fixed volume before use.

[0036] Preferably, in step S6, the volume ratio of the filtered filtrate to the polyethylene glycol precipitate mother liquor is 1:0.5 to 1:2, the settling time is 0.5 to 2 hours, the centrifugal force is 8000g to 12000g, and the centrifugation time is 30 to 90 minutes.

[0037] In this invention, the volume ratio of the filtrate to the polyethylene glycol (PEG) precipitate affects the precipitation efficiency of exosomes. A volume ratio ranging from 1:0.5 to 1:2 covers different proportions, from filtrate volume being twice that of the precipitate to the precipitate volume being twice that of the filtrate. Specifically, the volume ratio can be 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, or 1:2, preferably 1:1 (equal volume mixing). The settling time after mixing is 0.5 to 2 hours, during which time PEG molecules cause the exosomes to gradually aggregate and settle through steric hindrance. The specific settling time can be 0.5 hours, 1 hour, 1.5 hours, or 2 hours, preferably more than 1 hour (e.g., 1.5 hours). The settling is carried out at low temperatures (2–8°C) to protect the exosome activity. The subsequent centrifugation step uses a centrifugal force of 8000 g to 12000 g, which effectively settles the aggregated exosomes without damaging their structure. The specific centrifugal force can be 8000g, 9000g, 10000g, 11000g, or 12000g, preferably 10000g. A centrifugation time of 30 to 90 minutes ensures sufficient precipitation. Specific centrifugation times can be 30, 40, 50, 60, 70, 80, or 90 minutes, preferably 60 minutes. After centrifugation, discard the supernatant; the precipitate at the bottom of the tube is the coarse precipitate of the nanolipid exovesicles. To remove as much residual supernatant as possible, a short centrifugation (e.g., 2 minutes) can be performed after discarding the supernatant, followed by pipetting to remove any remaining liquid from the tube wall.

[0038] Preferably, in S7, the centrifugal force is 10000g to 15000g, and the centrifugation time is 1 to 5min.

[0039] In this invention, the centrifugation in step S7 is to remove any trace residual impurities that may remain after resuspension. A centrifugal force of 10,000 g to 15,000 g, higher than the previous step, is used to settle any small amounts of large particles or aggregates, while keeping the exosomes in the supernatant. Specific centrifugal forces can be 10,000 g, 11,000 g, 12,000 g, 13,000 g, 14,000 g, or 15,000 g, preferably 12,000 g. The centrifugation time is 1 to 5 minutes; a shorter time allows for the sedimentation of minute impurities while retaining the exosomes in the supernatant. Specific centrifugation times can be 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes, preferably 2 minutes. The supernatant after centrifugation is the purified nanolipid in vitro vesicle. This product can be used immediately or aliquoted and stored at -80°C for extended periods; the particle size and activity remain stable for at least 3 months after freezing. Phosphate-buffered saline (PBS) can be commercially available sterile PBS or can be prepared at home. It is usually composed of sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, with a pH of 7.2-7.4. When resuspending, the volume of PBS can be adjusted as needed to obtain the desired concentration of exosome suspension.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1 Preparation and identification of milk-derived nanolipid exovesicles (exosomes) Experimental preparation: Take 30 mL of fresh milk and place it on ice for later use; prepare a high-speed centrifuge, vortex shaker, 50 mL centrifuge tubes, 1.5 mL centrifuge tubes, sterile 1×PBS buffer, protein precipitation reagent, precipitation aid, exosome precipitation agent combination, and 50 mL centrifuge filter column.

[0042] Sample pretreatment: Add 30 mL of milk to a 50 mL centrifuge tube, centrifuge at 4 °C and 10000 × g (9500 rpm) for 20 min, discard the lower lipid layer and precipitate, and carefully transfer the whey to a new 50 mL centrifuge tube.

[0043] Deep removal of impurities: Add 5 mL of protein precipitation reagent to whey and vortex until the whey is translucent; add 2 mL of precipitant, mix gently, and let stand at 2–8℃ for 10 min until a distinct bean curd-like precipitate appears; centrifuge at 3000×g for 10 min and collect the supernatant; transfer the supernatant to a 50 mL centrifugal filter column, centrifuge at 3000×g for 2 min, and collect the filtrate.

[0044] Enrichment and purification of exosomes: Add 8 mL of exosome precipitant mixture to the filtrate, vortex for 1 min until well mixed, and let stand at 4℃ for 1.5 h; centrifuge at 4℃, 10000×g for 60 min, discard the supernatant, and retain the precipitate at the bottom of the tube; centrifuge briefly for 2 min, and aspirate the residual supernatant; resuspend the precipitate in 500 μL of sterile 1×PBS buffer, transfer to a 1.5 mL centrifuge tube, and centrifuge at 4℃, 12000×g for 2 min; take the supernatant, which is the purified milk-derived nanolipid exovesicles (exosomes), aliquot into 200 μL / tube, and store at -80℃.

[0045] Test results: NTA detection: Particle concentration was 8.22 × 10⁻⁶. 10 The particle size was 88.0 nm, with the main peak between 30-150 nm. Figure 1 ); TEM observation: Typical "cup-shaped" exosome morphology is visible, with an intact lipid bilayer structure and no obvious lipid or protein impurities. Figure 2 The images are electron micrographs of exosomes (bar=100nM). Transmission electron microscopy observations confirmed that each exosome sample had a lipid bilayer structure or cup morphology, with a diameter of approximately 30-150 nm.

[0046] Western blot (WB) test: CD63, TSG101, and CD9 were all positive; Calnexin was negative. Figure 3 This proves that it is a high-purity exosome.

[0047] The purified nanolipid exosomes were aliquoted and stored at -80°C for long-term preservation while maintaining high bioactivity, meeting the needs of various downstream experiments and applications. Stability validation: Purified milk-derived nanolipid exosomes (exosomes) were aliquoted into sterile centrifuge tubes and stored at -80°C. Samples were taken at 0 days (fresh), 1 month, and 3 months of storage. After thawing, nanoparticle tracking analysis (NTA) was used to detect particle size distribution and particle concentration, and Western blotting was used to detect the expression of exosome-specific markers CD9, CD63, and TSG101. The results are shown in Table 1 below: Table 1. Detection results at different storage times

[0048] The results showed that after being frozen at -80℃ for 3 months, the average particle size of the exosomes increased slightly (from 92.1 nm to 94.2 nm), but remained within the typical range of 30–150 nm, with no obvious aggregation or rupture observed; the particle concentration remained stable (approximately 96.9% of the pre-freezing concentration was retained after 3 months); the expression of CD9, CD63, and TSG101 marker proteins was positive, with no degradation or loss. These data fully demonstrate that the milk-derived nanolipid exovesicles prepared in this invention can be stably stored at -80℃ for at least 3 months, maintaining intact particle size characteristics and high bioactivity, thus meeting the needs of downstream experiments and industrial applications.

[0049] The obtained bovine milk-derived exosomes were diluted with PBS to a concentration of 1×10⁻⁶. 9 A solution of exosomes / mL was prepared; an acute inflammation model was established in mice, and the mice were randomly divided into a model group and an exosome treatment group, with 10 mice in each group.

[0050] Experimental procedure: Mice in the exosome treatment group were injected intraperitoneally with 100 μL of bovine milk exosome solution, while mice in the model group were injected with an equal volume of PBS. The treatment was carried out for 3 consecutive days. The levels of inflammatory factors TNF-α and IL-6 in the serum of mice were detected.

[0051] The levels of inflammatory cytokines TNF-α and IL-6 in the serum of mice in each group were detected by ELISA. Compared with the model group, the levels of TNF-α and IL-6 in the serum of mice treated with exosomes were significantly reduced. The serum TNF-α level in the model group was 124.5±18.7 pg / mL, while that in the exosome-treated group was 68.3±10.2 pg / mL (p<0.01); the serum IL-6 level in the model group was 286.2±32.5 pg / mL, while that in the exosome-treated group was 145.7±21.8 pg / mL (p<0.01). The reduction rates in the exosome-treated group compared with the model group were approximately 45.1% (TNF-α) and 49.1% (IL-6), respectively. Data are expressed as mean ± standard deviation (mean±SD), and independent samples t-tests were used for comparison. Statistical analysis was performed using GraphPad Prism software.

[0052] The above results indicate that milk-derived nanolipid exovesicles (exosomes) can significantly inhibit the production of pro-inflammatory factors TNF-α and IL-6 in the serum of mice with acute inflammation, and have significant anti-inflammatory biological functions, which can be applied to the development of therapeutic drugs for inflammatory diseases.

[0053] The method described in this embodiment, ultracentrifugation (modified according to the standard protocol of Thery et al., 2006), and a commercially available total exosome extraction kit (Thermo Fisher 4484453, operated according to the instructions) were used to process fresh milk samples of the same source and volume (30 mL) in parallel. Comparative metrics included: exosome yield (particles / mL milk), purity (total protein / particles, SDS-PAGE impurity bands), particle size distribution (NTA), total processing time, and equipment requirements. Each experiment was repeated three times, and results are expressed as mean ± standard deviation.

[0054] Brief description of the comparison method: The method of this invention: follow the steps described above (pretreatment → deep impurity removal → PEG precipitation → resuspension purification), without the need for an ultracentrifuge.

[0055] Ultracentrifugation: Milk was centrifuged at 2000×g for 20 min at 4°C to remove fat and cell debris; the supernatant was collected and centrifuged at 10000×g for 30 min at 4°C to remove large particles such as casein; the supernatant was ultracentrifuged at 100000×g for 90 min at 4°C to precipitate exosomes; after resuspending in PBS, it was washed again by ultracentrifugation at 100000×g for 90 min; finally, it was resuspended in 500 μL PBS.

[0056] Kit method: Using Thermo Fisher Total Exosome Isolation Reagent (from other body fluids), follow the instructions: centrifuge milk at 4°C, 2000×g for 20 min to remove fat; mix the supernatant with the reagent at a ratio of 2:1 and incubate at 4°C overnight; centrifuge at 4°C, 10000×g for 60 min to precipitate exosomes; resuspend in PBS to obtain the final product.

[0057] The comparison results are shown in Table 2: Table 2 Comparison results of different methods

[0058] SDS-PAGE purity verification An equal amount of exosomal protein (10 μg) was subjected to SDS-PAGE and Coomassie Brilliant Blue staining. The results showed that the sample obtained by the method of this invention had fewer bands, concentrated in the 35–100 kDa range, with no significant contamination from other proteins; samples obtained by ultracentrifugation showed multiple bands of other proteins (especially in the 15–25 kDa region, corresponding to casein residue); the kit-based sample had the most abundant bands of other proteins and severe background. These results are consistent with the above protein quantification, proving that the purification effect of the method of this invention is optimal.

[0059] Therefore, compared with existing technologies (ultracentrifugation method, commercially available reagent kit method), the method of the present invention has significant advantages in terms of yield (increased by about 1.5 to 3 times), purity (reduced by about 5 to 9 times in terms of protein impurities), operation time (shortened by about 40% to 85%), and equipment threshold (no need for an ultracentrifuge).

[0060] Example 2 The difference from Example 1 is that fresh cow's milk was replaced with sheep's milk to prepare sheep's milk-derived nanolipid exovesicles (exosomes).

[0061] Experiments to promote skin cell proliferation Experimental preparation: Sheep milk-derived nanolipid exovesicles (exosomes) were prepared using the method provided in this invention and diluted with cell culture medium to obtain exosome solutions of different concentrations; human skin fibroblasts were taken, seeded in 96-well plates, and cultured until the cell confluence reached 70%-80%.

[0062] Experimental groups: A blank control group (no exosomes) and a low-concentration exosome group (1×10⁻⁶) were set up. 8 (5 × 10⁶ cells / mL), medium concentration exosome group (5 × 10⁶ cells / mL 8 (number / mL), high concentration exosome group (1×10) 9 (number per mL), with 3 replicates per group.

[0063] Experimental procedure: Add the corresponding concentration of exosome solution to each well and continue culturing for 48 h; use the CCK-8 assay to detect cell proliferation rate.

[0064] Results: The cell proliferation rates of the low, medium, and high concentration exosome groups were all significantly higher than those of the blank control group (p<0.01), and this trend was concentration-dependent. CCK-8 assay results showed that, compared with the blank control group (cell proliferation rate of 100%), the low concentration exosome group (1×10⁻⁶) showed significantly higher cell proliferation rates. 8 The cell proliferation rate was increased to approximately 120%–125% in the medium concentration group (5×10⁶ cells / mL). 8 The concentration of (ci / mL) was increased to approximately 135%–145%, and the high concentration group (1×102) increased to approximately 135%–145%. 9 The cell proliferation rate of exosomes (cells / mL) was increased to approximately 155%–170%. The cell proliferation rates of the low, medium, and high concentration exosome groups were significantly higher than those of the blank control group (p<0.01), and this was concentration-dependent—the cell proliferation rate gradually increased with the increase of exosome concentration.

[0065] The above results indicate that the sheep milk-derived nanolipid exovesicles (exosomes) prepared in this invention have significant bioactivity in promoting the proliferation of human skin fibroblasts and can be applied to the development of skin repair products and functional biological agents.

[0066] As demonstrated by the above embodiments, this invention provides a method for efficiently extracting high-purity nanolipid exovesicles from mammalian milk. Systematic characterization and functional verification of nanolipid exovesicles derived from cow's milk and sheep's milk confirmed that the exosomes prepared by the method of this invention possess typical morphological structures and marker protein expression characteristics, uniform particle size distribution, high purity, and no significant contamination from other proteins. Cell experiments showed that the sheep milk-derived exosomes prepared by this invention significantly promoted the proliferation of human skin fibroblasts in a concentration-dependent manner. Animal experiments showed that cow's milk-derived exosomes significantly reduced the levels of pro-inflammatory factors tumor necrosis factor-α and interleukin-6 in the serum of mice with acute inflammation, exhibiting a clear anti-inflammatory biological function. Furthermore, a parallel comparison of the method of this invention with existing ultracentrifugation and commercially available reagent kits showed that the method of this invention has significant advantages in terms of exosome yield, purity, operation time, and equipment requirements, yielding the product with the lowest impurity content and the most uniform particle size distribution. In summary, this invention successfully establishes a simple, efficient, low-cost method for preparing milk-derived nanolipid exovesicles without the need for an ultracentrifuge. The obtained product has high purity and high bioactivity, which can meet the needs of various downstream applications.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing nanolipid exovesicles, characterized in that, Includes the following steps: S1: Centrifuge mammalian milk, discard the lower lipid and precipitate, and collect the upper whey; S2: Mix the whey with a protein precipitation reagent until the whey becomes translucent to obtain pretreated whey; S3: Mix the pretreated whey with the flocculant and let it stand to obtain a precipitated mixture; S4: Centrifuge the precipitate mixture and collect the supernatant to obtain a protein-free supernatant; S5: The protein-free supernatant is brought into contact with a centrifugal filter column for filtration, and the filtrate is collected to obtain the filtered filtrate. S6: Mix the filtered filtrate with the polyethylene glycol precipitate mother liquor, let stand, centrifuge, discard the supernatant, collect the precipitate, and obtain the crude precipitate of nanolipid exovesicles. S7: The coarse precipitate of the nanolipid exovesicles is mixed with phosphate buffer and resuspended. After centrifugation, the supernatant is collected to obtain the nanolipid exovesicles. The precipitant contains acetic acid solution.

2. The method according to claim 1, characterized in that, The mammalian milk is selected from cow's milk, sheep's milk, human milk, or horse milk.

3. The method according to claim 1, characterized in that, In S1, the centrifugation temperature is 2-8℃, the centrifugation force is 8000g-12000g, and the centrifugation time is 10-30min.

4. The method according to claim 1, characterized in that, The final concentration of the acetic acid solution in the flocculant is 0.005 M to 0.02 M.

5. The method according to claim 1, characterized in that, In S3, the settling temperature is 2-8°C and the settling time is 5-15 minutes.

6. The method according to claim 1, characterized in that, In S4, the centrifugal force is 2000g to 5000g, and the centrifugation time is 5 to 15min.

7. The method according to claim 1, characterized in that, The centrifugal filter column has a pore size of 0.22 μm or 0.45 μm.

8. The method according to claim 1, characterized in that, The polyethylene glycol (PEG) precipitation mother liquor contains PEG and sodium chloride. The PEG is selected from PEG 6000 or PEG 8000. The final concentration of PEG in the PEG precipitation mother liquor is 10% to 20% by weight / volume. The final concentration of sodium chloride in the PEG precipitation mother liquor is 0.2 M to 1.0 M. The pH of the PEG precipitation mother liquor is 7.0 to 7.

5.

9. The method according to claim 1, characterized in that, In step S6, the volume ratio of the filtered filtrate to the polyethylene glycol precipitate mother liquor is 1:0.5 to 1:2, the settling time is 0.5 to 2 hours, the centrifugal force is 8000g to 12000g, and the centrifugation time is 30 to 90 minutes.

10. The method according to claim 1, characterized in that, In S7, the centrifugal force is 10000g to 15000g, and the centrifugation time is 1 to 5min.

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