Preparation method of extracellular vesicles for improving drug delivery capacity

Abstract

The invention relates to a preparation method of extracellular vesicles for improving drug delivery capacity, which comprises the following steps: freezing milk under a first temperature condition to enable adjacent proteins to interact and gather, so as to obtain frozen milk liquid; unfreezing the frozen milk liquid under a second temperature condition so as to layer the milk liquid to obtain unfrozen milk liquid; centrifuging to remove one or more of cell debris, fat and protein aggregate in the layered unfrozen milk to obtain intermediate layer whey; the intermediate layer whey is filtered and centrifuged, and the extracellular vesicles are obtained. Compared with the oral characteristics of mEVs prepared by three methods of low-temperature freezing, acetic acid treatment and continuous super-centrifugation, the invention firstly proposes that the purity of the mEVs extracted by the low-temperature freezing method is higher, the enrichment degree of mEVs related proteins is higher, the total yield is highest, and the research result can provide more personalized choices for the application of the mEVs as an oral administration system in the future.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing extracellular vesicles for improving drug delivery capability. Background Art

[0002] Oral administration is the preferred route of administration. Due to the influence of the gastrointestinal environment (the intestinal barrier plays an important role in nutrient absorption and preventing harmful substances from entering the human body), it is difficult for drugs to be completely absorbed by the human body through the gastrointestinal wall, and oral administration faces huge challenges. For a long time, researchers have explored the use of functionalized nano drug delivery systems to improve the bioavailability of drugs after oral administration.

[0003] Existing technologies Extracellular vesicles isolated from tumor cell lines, lymphocytes and stem cells have been used for targeted drug delivery. However, the yield of extracellular vesicles produced by cell lines is low, so it is difficult to reach the level required for commercial drug production. At the same time, the protein components of extracellular vesicles produced by cell lines may cause immune responses when administered systemically. Extracellular vesicles or extracellular vesicles (EVs) are nanoscale vesicles secreted by cells, containing a variety of immunomodulatory proteins and microRNAs, and play an important role in a variety of physiological metabolic processes. Extracellular vesicles contain a variety of biomolecules, such as lipids, metabolites, cytoplasm, nucleic acids and cell surface proteins. Extracellular vesicles have attracted much attention due to their potential application value in laboratory research and clinical treatment. The ability of extracellular vesicles to carry and deliver some important signaling molecules forms a new intercellular information transmission system, which is closely related to the maintenance of health and the occurrence of diseases. Therefore, extracellular vesicles have long been considered as drug delivery systems for small molecules, nucleic acids and other biological payloads. The combined advantages of cell delivery and nanotechnology give extracellular vesicles better targeting ability, biosafety and higher drug delivery efficiency. However, the limited extracellular vesicles and the lengthy isolation process still hinder the application of extracellular vesicles. It is well known that extracellular vesicles can be found in most natural body fluids, including blood, urine, saliva, and breast milk. Cow's milk is rich in sources, low in cost, can be prepared in large quantities, and has a strong ability to cross biological barriers. At the same time, milk-derived extracellular vesicles have good biocompatibility (good transmembrane transport ability) and low immunogenicity. They can play a regulatory role in the intestine and other parts or after being transported to the circulatory system, overcome the intestinal epithelial cell barrier and the vascular endothelial barrier and enter the blood circulation. That is, the ability of milk-derived extracellular vesicles to cross the gastrointestinal barrier has broad prospects in drug delivery and disease treatment research.

[0004] Cow's milk is an ideal choice for isolating extracellular vesicles. However, the composition of milk is complex, and milk-derived extracellular vesicles obtained by different extraction methods may affect their oral administration. Studies have shown that extracellular vesicles (mEVs) derived from breast milk and cow's milk can tolerate the gastrointestinal digestive environment (surviving under the strong acidic conditions in the stomach and the regulation of digestion in the intestine), that is, they can still remain active in the presence of gastric juice and a variety of proteases, and can still maintain integrity after entering the intestine. On the other hand, as a carrier derived from daily food, the biosafety and acceptability of extracellular vesicles through the oral route can be guaranteed. Studies by Arntz et al. have shown that oral administration of milk-derived extracellular vesicles themselves can improve diseases such as spontaneous and induced rheumatoid arthritis (Arntz OJ, Pieters BC, Oliveira MC, Broeren MG, Bennink MB, de Vries M, van Lent PL, Koenders MI, van den Berg WB, van der Kraan PM et al. Oral administration of bovine milkderived extracellular vesicles attenuates arthritis in two mouse models. Mol Nutr Food Res. 2015; 59: 1701-1712), which indicates that it has great potential in oral drug delivery applications. In terms of drug carriers, Geonhee Han et al. observed that mEV / TNF-α siRNA can reach colitis lesions through the gastrointestinal tract, with excellent stability, and successfully reversed the imbalance of inflammatory cytokines caused by colitis (Han G, Kim H, Jang H, Kim ES, Kim SH, Yang Y. Oral TNF-α siRNA delivery via milk-derived exosomes for effective treatment of inflammatory bowel disease. Bioact Mater. 2024; 34: 138-149). Ashish K. et al. proposed the possibility of oral drug delivery by loading paclitaxel (PAC) with milk-derived extracellular vesicles to avoid the severe toxicity associated with currently available intravenous PAC therapy. Yi H et al. reported that milk-derived extracellular vesicles have a relieving and protective effect on colon inflammation in the oral administration route. Therefore, compared with extracellular vesicles from other sources, researchers have paid more and more attention to the application of milk-derived extracellular vesicles in drug delivery, especially oral administration, in recent years.

[0005] However, despite the rich source of cow's milk, it contains a large amount of impurities such as free protein and fat, which affects the extraction and purification of extracellular vesicles, thereby limiting the further application of milk-derived extracellular vesicles. Ultracentrifugation is the most classic method for isolating extracellular vesicles, with high yield, but cumbersome operation. Bao, H. et al. used a combination of classic gradient centrifugation and ultracentrifugation to isolate extracellular vesicles from mesenchymal stem cells and regulatory T cells, forming the core of the "pseudocell" system (Bao H, Tian Y, Wang H, Ye T, Wang S, Zhao J, Qiu Y, Li J, Pan C, Ma G et al. Exosome-loaded degradable polymeric microcapsules for the treatment of vitreoretinal diseases. Nat Biomed Eng. 2023). Although the separation methods of extracellular vesicles are constantly developing, the purity and yield of extracellular vesicles separated by the emerging gel filtration chromatography and immunocapture have always been low, and the existing separation methods are expensive. The above reasons restrict the further application and clinical transformation of extracellular vesicles. Some studies have shown that after co-incubation with tumor cells, porous silicon nanoparticles can promote extracellular secretion (exocytosis) through autophagy, thereby increasing the yield of extracellular vesicles, but the high complexity of this method limits its application. For the separation methods of milk-derived extracellular vesicles, most of them are currently the same as the methods for extracting extracellular vesicles from other sources, using ultracentrifugation, acid precipitation, and immunocapture methods for purification.

[0006] Ultracentrifugation uses the difference in sedimentation coefficients between extracellular vesicles and substances such as proteins, fats and cell fragments in milk to obtain precipitates with different sedimentation coefficients under certain conditions such as speed and centrifugation time, thereby separating and purifying extracellular vesicles from milk. Although ultracentrifugation is still the gold standard for extracting exosomes at this stage and still occupies a dominant position in related research, the extraction process of this method requires repeated centrifugation operations, which is cumbersome and requires high instrumentation. In addition, the extracted target substances are prone to aggregation and have low purity. Density gradient centrifugation combines ultracentrifugal force with the continuous density stratification formed by sucrose solution to achieve separation by using the density difference of substances. Although this method can improve the separation purity, the operation steps will become more complicated. Acid precipitation is based on ultracentrifugation, using the principle that acid can denature free proteins in milk to simplify the extraction process. However, the use of acid may affect the integrity of the membrane, resulting in problems such as reduced extracellular vesicle yield in milk. Immunocapture is based on some specific proteins on the membrane surface, and the purpose of separation and purification is achieved by combining these proteins with various specific antibodies on the carrier. Although the product obtained by this extraction method that relies on immune affinity is of high purity, the extraction cost also increases, making it difficult to be used for mass production of extracellular vesicles.

[0007] The Chinese patent application with publication number CN109468265A discloses a method for extracting milk exosomes, the method comprising the following steps: (1) taking a milk sample, centrifuging at 2000g, 4°C, 30min, and collecting supernatant A; (2) centrifuging the collected supernatant A at 12000g, 4°C, 30min, and collecting supernatant B; (3) further adjusting the pH of the collected supernatant B to 5.5-6.0; (4) adding rennet to the pH-adjusted supernatant B under 36-37°C water bath conditions to precipitate milk protein and obtain supernatant C; (5) freeze-drying the subcomponent of supernatant C that can pass through a 0.22μM membrane into powder, namely milk exosomes. The milk sample in step (1) can be a fresh milk sample or a milk sample thawed by freezing and storing at -80°C. Compared with the traditional centrifugation-ultra-high-speed centrifugation method, this method reduces the steps of the ultra-high-speed centrifugation method, saves the cost of consumables used in extraction, and increases the sample extraction volume.

[0008] In summary, a fast and efficient method for preparing extracellular vesicles for improving drug delivery has not yet been developed. The extraction of extracellular vesicles is the cornerstone of their subsequent research and application. Therefore, it is urgent to develop an efficient and convenient extraction method with strong drug delivery ability to provide support for the application and development of milk extracellular vesicles.

[0009] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0010] The present invention aims to propose a milder way to denature the protein in milk, so as to obtain safer and more friendly milk-derived extracellular vesicles on a simple basis. Based on this, the present invention uses a low-temperature environment, i.e., a low-temperature freezing method, to denature the protein. The water in milk can be divided into bound water and free water according to the degree of binding, wherein the bound water and the active groups on the protein molecules are firmly bound together, while the free water is not bound by anything and can move freely. In a low-temperature environment, when the milk is frozen, the free water freezes first, while the bound water is difficult to freeze. When the temperature drops to a certain level, part of the bound water is separated from the protein and then frozen to form ice crystals, resulting in interaction between adjacent proteins, causing them to condense and thus causing irreversible denaturation of the protein in the milk. In the process of ice crystals advancing into the sample, the protein is enriched at the ice-water interface. When the protein contacts the ice surface, the low temperature weakens the interaction between water and the hydrophobic group, and the hydrophobic part embedded in the protein is easily exposed on the protein surface, which promotes the unfolding of the protein and makes the protein unstable. In addition, the formation of ice crystals in a low temperature environment causes the fat, vitamins, minerals and other substances excluded from the milk to move to the remaining water, making the unfrozen components a concentrated solution, thereby separating the exocysts from other components.

[0011] In view of the shortcomings of the prior art, the first aspect of the present invention provides a method for preparing extracellular vesicles for improving drug delivery capacity, comprising: freezing milk under a first temperature condition so that adjacent proteins interact and aggregate to obtain frozen milk; thawing the frozen milk under a second temperature condition so that the milk is stratified to obtain thawed milk; removing one or more of cell debris, fat and protein aggregates in the stratified thawed milk by centrifugation to obtain an intermediate layer of whey; filtering and centrifuging the intermediate layer of whey to obtain extracellular vesicles, wherein the first temperature condition and the second temperature condition are set in the following manner, that is, so that the extracellular vesicles tend to accumulate in the liver through the gastrointestinal tract.

[0012] The prior art, such as the Chinese patent application with publication number CN109468265A, only discloses a method for extracting milk exosomes, and does not involve freezing and thawing of milk at all. By setting the temperature and / or time of the freezing and thawing process, the extracellular vesicles obtained after filtration and centrifugation show different metabolic pathways in vivo from the extracellular vesicles extracted without freezing and thawing. In particular, it is not disclosed that by setting the temperature of the freezing and thawing process, the extracellular vesicles obtained by extraction are more likely to accumulate in the liver through the gastrointestinal tract after oral administration, rather than being more inclined to remain in the intestine, compared with the extracellular vesicles separated by acetic acid precipitation and the extracellular vesicles separated by ultracentrifugation. Further, the inventor unexpectedly found that this property is particularly suitable for applying the extracellular vesicles obtained in this way to the preparation of oral administration carriers, especially the preparation of administration carriers for oral insulin and / or treatment of non-alcoholic fatty hepatitis, so as to improve the cellular transport efficiency of the drug.

[0013] The present invention compares the oral properties of mEVs prepared by three methods: cryogenic freezing, acetic acid treatment and continuous ultracentrifugation. The cryogenic freezing method proposed and utilized for the first time in the present invention extracts mEVs with higher purity, higher enrichment of EVs-related proteins, and the highest total yield (25 times that of Acid-M-EVs and 2 times that of Ultra-M-EVs). In the in vitro Caco-2 cell uptake and transport experiment, the present invention found that Acid-M-EVs had the best cell uptake effect and the lowest cell transport efficiency, while Cryo-M-EVs had the best cell transport efficiency. Similarly, when mice were fed Cryo-M-EVs, Acid-M-EVs and Ultra-M-EVs respectively, Cryo-M-EVs could cross the gastrointestinal barrier and accumulate in the liver, while Acid-M-EVs mainly stayed in the small intestinal epithelium, and Ultra-M-EVs accumulated in both the intestine and the liver. In addition, when Caco-2 cells or mice were treated with mEVs, none of the three mEVs showed side effects, indicating that mEVs have good safety and biocompatibility. In particular, when the present invention uses mEVs as an oral drug delivery system, the extraction method of mEVs can be changed according to the disease to be addressed. Different extraction methods may lead to differences in the protein composition and lipid composition of mEVs.

[0014] According to a preferred embodiment, in the process of freezing the milk in a first temperature environment to obtain frozen milk, the purpose is to obtain milk in which the free proteins contained are aggregated and denatured.

[0015] Preferably, in the process of filtering the middle layer whey to obtain filtered milk, the purpose is to remove larger insoluble impurities.

[0016] According to a preferred embodiment, the first temperature condition can be -80°C to -20°C. Preferably, the first temperature condition can be -20°C.

[0017] According to a preferred embodiment, the milk is placed under the first temperature condition for freezing treatment for 5 to 60 days. Preferably, the milk is placed under the first temperature condition for freezing treatment for 10 to 60 days. Preferably, the milk is placed under the first temperature condition for freezing treatment for 20 to 60 days. More preferably, the milk is placed under the first temperature condition for freezing treatment for 20 to 40 days. Particularly preferably, the milk is placed under the first temperature condition for freezing treatment for 40 days.

[0018] According to a preferred embodiment, the second temperature condition can be 25° C. to 37° C. Preferably, the second temperature condition can be 25° C. to 30° C. Specifically, the second temperature condition can be 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C. or 37° C.

[0019] According to a preferred embodiment, the centrifugation temperature for obtaining the middle layer whey can be 4°C, and the relative centrifugal force can be 10000g to 15000g. Specifically, the relative centrifugal force can be 10000g, 10500g, 11000g, 11500g, 12000g, 12500g, 13000g, 13500g, 14000g, 14500g or 15000g. Preferably, the centrifugation time can be 30min.

[0020] According to a preferred embodiment, the filtering device used for filtering the middle layer whey is a 0.22 μm filter membrane.

[0021] According to a preferred embodiment, the centrifugation temperature for obtaining extracellular vesicles can be 4°C, the relative centrifugal force can be 160000g. Preferably, the centrifugation time can be 70 minutes.

[0022] According to a preferred embodiment, after filtering and centrifuging the middle layer whey, it is resuspended in a phosphate buffer solution. Specifically, the phosphate buffer solution can be a PBS solution.

[0023] Preferably, in the process of centrifuging the filtered milk to obtain the extracellular vesicle precipitate, the purpose is to obtain milk extracellular vesicles with higher purity.

[0024] Preferably, the purpose of resuspending the extracellular vesicle pellet with a solution is to obtain an extracellular vesicle solution with higher purity compared to the ultracentrifugation process.

[0025] The second aspect of the present invention provides an extracellular vesicle, which is prepared by the preparation method provided by the first aspect of the present invention. The extracellular vesicle provided by the present invention has the characteristic of improving drug delivery ability.

[0026] A second aspect of the present invention provides an application of extracellular vesicles in the preparation of drug carriers for enhancing the effect of immunotherapy, wherein the extracellular vesicles are capable of carrying immunomodulators and targeted delivery to immune cells.

[0027] The third aspect of the present invention provides the preparation method provided by the first aspect of the present invention and the use of the extracellular vesicles provided by the second aspect of the present invention in preparing an oral administration carrier.

[0028] The fourth aspect of the present invention provides the use of the preparation method provided by the first aspect of the present invention and the extracellular vesicle provided by the second aspect of the present invention in the preparation of a drug delivery carrier for oral insulin and / or treatment of non-alcoholic fatty hepatitis.

[0029] The fifth aspect of the present invention provides a pharmaceutical composition comprising the extracellular vesicles provided in the second aspect of the present invention. Preferably, the extracellular vesicles are used to prepare oral or injectable drug delivery vehicles, wherein the extracellular vesicles can protect the drug ingredients and control the release of the drug. Preferably, the extracellular vesicles are used to prepare oral drug delivery vehicles, and the extracellular vesicles can effectively encapsulate and deliver the drug to the target site.

[0030] The sixth aspect of the present invention provides a drug delivery system, comprising the extracellular vesicles provided by the second aspect of the present invention, and a drug encapsulated in the extracellular vesicles, for improving the targeting and bioavailability of the drug.

[0031] The seventh aspect of the present invention provides a method for preparing an oral administration carrier, comprising combining a drug with the extracellular vesicles provided by the second aspect of the present invention to improve the bioavailability and targeted delivery efficiency of the drug.

[0032] An eighth aspect of the present invention provides a method for preparing a drug delivery system, comprising the following steps:

[0033] a. preparing extracellular vesicles by the method provided in the first aspect of the present invention;

[0034] b. Encapsulating at least one drug component in the extracellular vesicles to form a drug delivery system for improving the drug delivery efficiency and therapeutic effect.

[0035] The beneficial technical effects of the present invention include at least: the preparation method of extracellular vesicles for improving drug delivery capacity provided by the present invention utilizes the principle that low-temperature freezing can denature proteins, combines low-temperature freezing with the classic ultracentrifugation method, and realizes the simple and efficient extraction of milk extracellular vesicles. The present invention simplifies the process of extracting milk extracellular vesicles by a single ultracentrifugation method, which requires multiple high-speed and ultracentrifugation, and the extracted milk extracellular vesicles are higher in purity than the ultracentrifugation method; on the other hand, compared with the traditional acid precipitation method, the separation method of the present invention is not only simple and easy to operate, but also improves the purity of milk extracellular vesicles on the basis of ensuring safety and reliability. At the same time, the separation method provided by the present invention is economical and convenient, overcoming the problems of some existing methods, such as the expensive extraction reagents used in the immunocapture method, which are not conducive to mass production, and has significant commercial and economic value.

[0036] Inspired by the aggregation of proteins in milk induced by low temperature environment, the present invention proposes a simple, safe and low-cost method (i.e., cryogenic freezing treatment) for isolating milk-derived extracellular vesicles. Cryogenic freezing utilizes the principle that the water in milk is frozen and thawed at low temperature to aggregate and precipitate the proteins in milk. This operation breaks the intermolecular forces of proteins and affects the spatial conformation of proteins. Among them, the effects of different methods such as ultracentrifugation, acid precipitation, and cryogenic freezing on the purity, yield and oral biological function of mEVs have not been evaluated in detail by relevant studies. The research of the present invention confirms that mEVs obtained by different extraction methods have different characteristics and application potentials. Compared with acetic acid treatment and multiple ultracentrifugation methods, the yield of mEVs separated by cryogenic freezing is 25 times and 2 times that of the aforementioned methods, respectively. The purity of extracellular vesicles separated by cryogenic freezing is only slightly lower than that of extracellular vesicles separated by acetic acid precipitation. Interestingly, extracellular vesicles separated by cryogenic freezing and acetic acid precipitation have been shown to have higher cellular uptake efficiency, and extracellular vesicles separated by cryogenic freezing have the best epithelial transport effect. It is worth noting that after oral administration of the three types of milk-derived extracellular vesicles extracted by the three methods, the extracellular vesicles isolated by low-temperature freezing method are more likely to accumulate in the liver through the gastrointestinal tract, while the extracellular vesicles isolated by acetic acid precipitation method and the extracellular vesicles isolated by ultracentrifugation method are more likely to remain in the intestine.

[0037] As a natural intercellular information carrier, EVs, especially mEVs with abundant sources, have great potential in the field of drug delivery. The lack of a unified extraction method selection standard has greatly limited its promotion. Compared with cell culture medium, milk has a more complex composition, so the steps to obtain mEVs from milk are more cumbersome. Based on this, this study proposes that mEVs obtained by different extraction methods lead to differences in their oral properties.

[0038] In summary, the present invention verifies the differences in the oral properties of mEVs obtained by different extraction methods, and the research results can provide more personalized options for the future application of mEVs as oral drug delivery systems. In addition, it is necessary to establish a unified comprehensive evaluation system to provide better quality control and wider application for the selection criteria of mEVs extraction methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a flow chart and result diagram of the separation method and characteristic research of milk-derived extracellular vesicles in fresh milk provided by the present invention;

[0040] Figure 2 The state change results of the fresh milk provided by the present invention after being frozen and centrifuged for different days;

[0041] Figure 3 It is the biomarker identification, purity analysis and yield evaluation of mEVs obtained by the three extraction methods provided by the present invention;

[0042] Figure 4 is the analysis of mEVs lipid carbon chain length and lipid carbon chain unsaturation;

[0043] Figure 5 The results are as follows: Caco-2 cells were incubated with mEVs of different protein concentrations from three extraction methods for 24 h, and Caco-2 cell viability was measured by CCK-8 method (n=6);

[0044] Figure 6 It is the evaluation result of the cell safety, uptake efficiency, and transport efficiency of mEVs obtained by three extraction methods;

[0045] Figure 7 are TEM images of the lower chamber of the three types of mEVs after Transwell experiments (Transwell migration or invasion assay) (scale bar, 200 nm);

[0046] Figure 8 These are the biodistribution results of mEVs obtained by the three extraction methods;

[0047] Fig. 9 It is the quantitative results of the in vitro mean fluorescence intensity of the main organs of mEVs obtained by the three extraction methods 6 hours after oral administration;

[0048] Fig.10 These are the results of in vivo biosafety evaluation after continuous and repeated administration of the three mEVs. DETAILED DESCRIPTION

[0049] The following is a detailed description with reference to the accompanying drawings.

[0050] Sources of reagents and consumables:

[0051] The milk used in this study was fresh milk from Beijing Sanyuan Food Co., Ltd. Annexin V-FITC / PI apoptosis detection kit, cell counting kit-8 (CCK-8) and 6-indoleaminoamine dihydrochloride (DAPI) were purchased from Beijing Solebao Technology Co., Ltd. 1,1'-dioctadecyl-3,3,3',3'-tetramethylpolydicarbonate, 4-chlorobenzenesulfonate was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were provided by Beijing Vicente Biotechnology Co., Ltd. BCA protein concentration determination kit was purchased from Beijing Solebao Technology Co., Ltd. 0.22μm filter membrane was purchased from Sartorius stedim, Germany. Penicillin and streptomycin were purchased from Invitrogen. Phosphate-Buffered Saline (PBS) was purchased from Dalian Meilun Biotechnology Co., Ltd. Anti-CD81 and anti-TSG101 for Western blot were purchased from Abcam, UK, anti-GM130 and anti-Alix were purchased from Cell Signaling Technology, USA, and anti-β-actin, horseradish peroxidase-labeled anti-mouse secondary antibody and anti-rabbit secondary antibody were purchased from Shanghai Bio-Technology Co., Ltd. Caco-2 cells were stored in liquid nitrogen in the laboratory. Polycarbonate ultracentrifuge tubes were purchased from Beckman Coulter, USA.

[0052] Male C57BL / 6J mice (6 weeks old, SPF grade) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Animal experiments were performed in accordance with the standards of the Bioethics and Medical Ethics Committee of Beihang University (Beijing University of Aeronautics and Astronautics Bioscience and Medical Ethics Committee; BM20200165). The water mentioned in all experiments was deionized water (DI) (Millipore milliq grade, 18.2 MΩ).

[0053] The relevant experimental data are expressed as mean ± standard deviation (SD). Student's t test, one-way analysis of variance (ANOVA) and two-way analysis of variance (Two-way ANOVA) were used for statistical analysis of the samples. * P < 0.05 was considered a statistically significant threshold. ** P<0.01, *** P < 0.001, ****P < 0.0001. GraphPad Prism 9 software was used for statistical comparison.

[0054] Protein denaturation refers to the phenomenon that the stable and compact three-dimensional structure of protein in its natural state is destroyed due to the influence of physical or chemical factors, which ultimately leads to changes in the function, physical, chemical and biochemical properties of the protein.

[0055] In the present invention, d is equivalent to day, representing the number of days; h is equivalent to hour, representing the number of hours.

[0056] The present invention compares the oral properties of mEVs prepared by three methods: cryogenic freezing, acetic acid treatment and continuous ultracentrifugation. The cryogenic freezing method proposed and utilized for the first time in the present invention extracts mEVs with higher purity, higher enrichment of EVs-related proteins, and the highest total yield (25 times that of Acid-M-EVs and 2 times that of Ultra-M-EVs). In the in vitro Caco-2 cell uptake and transport experiment, the present invention found that Acid-M-EVs had the best cell uptake effect and the lowest cell transport efficiency, while Cryo-M-EVs had the best cell transport efficiency. Similarly, when mice were fed Cryo-M-EVs, Acid-M-EVs and Ultra-M-EVs respectively, Cryo-M-EVs could cross the gastrointestinal barrier and accumulate in the liver, while Acid-M-EVs mainly stayed in the small intestinal epithelium, and Ultra-M-EVs accumulated in both the intestine and the liver. In addition, none of the three mEVs showed any side effects when Caco-2 cells or mice were treated with mEVs, indicating that mEVs have good safety and biocompatibility.

[0057] In particular, when the present invention uses mEVs as an oral drug delivery system, the extraction method of mEVs can be changed according to the disease to be addressed. Different extraction methods may lead to differences in the protein composition and lipid composition of mEVs. It has been reported that EVs can also form a protein corona, so if the mEVs are too pure (Acid-M-EVs) or not too pure (Ultra-M-EVs), the percentage of relevant proteins involved in cellular transport will be reduced, resulting in increased nonspecific uptake. Similarly, it has been reported that the membrane of mEVs changes its hardness with changes in intracellular and extracellular pH, which determines its fate within the cell. Therefore, the differences in protein composition and lipid composition caused by different extraction methods may be the ultimate reason for the differences in their final oral properties.

[0058] Example 1

[0059] The test method provided in this embodiment is as follows.

[0060] Ultracentrifugation and acid treatment methods for extracting mEVs require the same pretreatment steps without the need for cryogenic freezing.

[0061] Pretreatment: Use a 5804R high-speed centrifuge (Eppendorf) to centrifuge fresh milk at 10,000 g for 30 min at 4°C; then centrifuge the intermediate whey at 10,000 g for 30 min at 4°C. Collect whey from the intermediate layer for use. The upper layer is the fat in the milk, and the lower layer is the protein particles and cell debris present in the milk. The collected whey should be used immediately or frozen and stored at -80°C.

[0062] The present embodiment provides a method for preparing extracellular vesicles for improving drug delivery capability, comprising: freezing milk at a first temperature to allow adjacent proteins to interact and aggregate, thereby obtaining frozen milk; thawing the frozen milk at a second temperature to allow the milk to be separated into layers, thereby obtaining thawed milk; removing one or more of cell debris, fat and protein aggregates in the separated thawed milk by centrifugation, thereby obtaining an intermediate layer of whey; and filtering and centrifuging the intermediate layer of whey, thereby obtaining extracellular vesicles.

[0063] The following are the specific experimental steps of the low-temperature freezing method. Freeze the fresh milk at -20°C for 40 days, thaw it completely at room temperature, and centrifuge it at 10,000g for 30 minutes at 4°C to remove the top layer of fat and the bottom layer of flocculent precipitates (including cell debris, protein aggregates, etc.). First, filter the middle layer of whey with a 0.22μm membrane, and use an Optima XPN-100 ultracentrifuge (Beckman Coulter, USA) to centrifuge at 4°C, with the relative centrifugal force set to 160,000g and the centrifugation time for 70 minutes. After discarding the supernatant, the transparent precipitate obtained is Cryo-M-EVs. mEVs are resuspended in PBS solution.

[0064] The following are the experimental steps of the acid treatment method. Preferably, this embodiment uses acetic acid to treat fresh milk. The fresh milk is first treated according to the pretreatment steps described above. The obtained whey is preheated at 37°C for 30 minutes, acetic acid (whey: acetic acid = 100: 1, v / v) is added to the whey to pH = 4.6, and then heated at 37°C for 30 minutes to perform isoelectric precipitation of casein. The supernatant collected from the 37°C whey is centrifuged at 4°C and 10,000g for 30 minutes. The supernatant is then filtered with a 0.22μm membrane. The filtrate is ultracentrifuged at 4°C and 160,000g for 70 minutes. After discarding the supernatant, the transparent precipitate obtained is Acid-M-EVs. mEVs are resuspended in PBS solution.

[0065] The following are the experimental steps of the ultracentrifugation method. Specifically, fresh milk is first treated according to the pretreatment steps described above. The whey obtained is ultracentrifuged at 4°C and 100,000g for 60 minutes. The intermediate whey is then ultracentrifuged at 4°C and 160,000g for 70 minutes. After discarding the supernatant, the transparent precipitate obtained is Ultra-M-EVs. mEVs are resuspended in PBS solution.

[0066] The characterization experiment of mEVs used transmission electron microscopy (TEM) and dynamic light scattering (DLS) to collect data. Specifically, the morphology of mEVs was observed using a HT7700 (Hitachi Ltd, Tokyo, Japan) transmission electron microscope. Transmission electron microscopy observation was stained with 2% uranyl acetate for 30 seconds. Dynamic light scattering (DLS) was used to measure the zeta potential and particle size of mEVs.

[0067] The results of protein marker analysis of mEVs were obtained by Western blotting (WB) and SDS-PAGE silver staining. Specifically, Western blotting (WB) was used to identify protein markers of the three extraction products. The protein concentration of all samples was quantified by bis-cholic acid (BCA) assay. After spotting, electrophoresis and membrane transfer were performed, and then EV biomarker proteins were detected with anti-CD81 (ImmunoWay Biotechnology, USA), anti-TSG101 (Cell Signaling, USA), anti-CD9 (CellSignaling, USA) and anti-Alix (Abcam, England), and finally incubated with secondary antibodies. SDS-PAGE silver staining showed the total protein of mEVs. After separating proteins of different molecular weights in different samples by SDS-PAGE, most of the proteins in the samples, including lower levels of proteins, were detected by silver staining.

[0068] The steps of proteomic analysis of mEVs included resuspending the three mEVs in pure water and lyophilizing them, performing LC-MS / MS analysis on each sample using a QExactive mass spectrometer (Thermo Scientific, Odense, Denmark), and performing database searches using the data processing software Proteome Discoverer 2.4 (Thermo), searching the databases Bostaurus (Bovine) (proteome: UP000009136, sequence 37505) and the common contaminant database (sequence 124).

[0069] The lipidomics analysis steps of mEVs include: all three types of mEVs are required to have a particle number of 10 in a volume of 100 μl. 10, and prepared for LC-MS / MS analysis; MS / MS spectra were acquired in data-dependent acquisition (DDA) mode under the control of acquisition software (Xcalibur 4.0.27, Thermo), so a QE mass spectrometer was used. The raw data files were formatted in mzXML format by the “msconvert” program in ProteoWizard. The MS1 data with peak detection were first used. The CentWave algorithm in XCMS was used for mass spectrometry peak detection, and the LipidBlast library was used for lipid identification.

[0070] The steps for mEVs particle counting and yield quantification include: resuspending and diluting freshly extracted Cryo-M-EVs, Acid-M-EVs and Ultra-M-EVs in PBS to an appropriate concentration, and counting particles using a flow nanoanalyzer (NanoAnalyzer, NanoFCM); resuspending and diluting freshly extracted Cryo-M-EVs, aAcid-M-EVs and Ultra-M-EVs with pure water to an appropriate concentration, quantifying the protein concentration of each sample using the BCA method, and calculating the protein yield of each mEVs; taking an appropriate amount of sample and freeze-drying it in a 1.5 ml centrifuge tube, and calculating the total yield of each mEVs.

[0071] The steps of cytotoxicity assay (CCK-8 and Annexin V / PI experiment) include: Caco-2 cells were cultured in 96-well plates for 24 hours and incubated with mEVs of different concentrations (0, 10, 25, 50, 100, 200, 250, 300, 400 and 500 μg / ml) for 24 hours, 100 μl of 10% CCK solution was added to each well, incubated at 37°C for 60 minutes, and the effect of mEVs on cell viability was detected by enzyme labeling at 450 nm (TECAN, Zurich, Switzerland). In order to further verify the biosafety of mEVs obtained by different methods, a cell apoptosis experiment was performed in this example. Caco-2 cells were incubated with mEVs (250 μg / ml) for 24 hours, digested and collected by centrifugation. Annexin V / PI fluorescence staining was used to detect the apoptosis of Caco-2 cells after treatment, and then flow cytometer (Beckman Coulter, Brea, CA, US) was used to measure and compare the fluorescence intensity of Annexin V and PI in Caco-2 cells.

[0072] The steps of cell uptake determination (confocal and flow cytometry) include: Caco-2 cells were incubated with Cryo-M-EVs, Ultra-M-EVs and Acid-M-EVs (250 μg / ml) for 24 h, and the phospholipid membrane and cell nucleus of Caco-2 cells were stained with red fluorescent dye DiD and blue fluorescent dye DAPI, respectively; then the uptake of different mEVs by Caco-2 cells was observed using a Zeiss LSM710 laser scanning confocal microscope (Carl ZEISS, Oberkochen, Germany). In order to further confirm the cell uptake of Cryo-M-EVs, Ultra-M-EVs and Acid-M-EVs, flow cytometry was used for quantitative analysis. The mEVs obtained by different methods were labeled with the red fluorescent dye Did. The fluorescence intensity of Caco-2 cells (λ = 633 nm) was detected and observed by flow cytometry, so as to more clearly compare the uptake differences between different experimental groups.

[0073] The in vitro Transwell assay of mEVs used Caco-2 cells to construct an in vitro intestinal model. Specifically, Caco-2 cells were seeded at a density of 2×10 cells / well in a 0.4 μm transwell plate (24-well transwell, Corning Incorporated). Then incubate with fresh culture medium for 20 days (replace fresh culture medium every two days for the first ten days, and replace fresh culture medium every day for the next ten days). The mEVs in this example were labeled with the fluorescent dye Did and added to the upper culture dish. Culture dish samples were collected at 0.25, 1, 2, and 4 hours, respectively. The fluorescence intensity of the mEVs samples was detected using a multimode microplate detection system (Enspire).

[0074] The tissue distribution test steps of mEVs in mice included: 250 μg / ml Ultra-M-EVs, Acid-M-EVs and Cryo-M-EVs were labeled with fluorescent dye DiD, respectively. Each group of mice was fed with mEV sample solution (15 mg / kg), and analyzed by in vivo imaging system (PerkinElmer, Waltham, Masachusetts, USA) at 1h, 2h, 4h, 6h, 8h, 12h, and 24h; 6h later, one mouse was randomly selected from each group for dissection, and IVIS was used to examine the main organs.

[0075] The long-term biosafety and biocompatibility test steps of mEVs were as follows: oral administration was repeated for 14 consecutive days, and blood was drawn for comprehensive blood biochemistry and major blood cell count analysis; the heart, liver, spleen, lung, kidney, and small intestine were dissected, fixed with 10% formalin, embedded in paraffin, and sliced ​​for H&E staining.

[0076] Example 2

[0077] Figure 1 The paper provides the process and results diagram of the isolation method and characteristic study of milk-derived extracellular vesicles in fresh milk. Figure 1 A shows the workflow and instructions for extracting milk-derived extracellular vesicles (Cryo-M-EVs) by cryogenic freezing. Figure 1 B is a TEM (transmission electron microscopy) image of extracellular vesicles isolated by cryostasis. Figure 1 C is the workflow and instructions for acid treatment to extract milk-derived extracellular vesicles (Acid-M-EVs). Figure 1 D is a TEM image of extracellular vesicles isolated by acid treatment. Figure 1 E is the workflow and instructions for extracting milk-derived extracellular vesicles (Ultra-M-EVs) by multiple ultracentrifugation. Figure 1 F is a TEM image of extracellular vesicles separated by multiple ultracentrifugation, scale bar, 200 nm. Figure 1 G is the size distribution of Cryo-M-EVs (red), Acid-M-EVs (orange) and Ultra-M-EVs (pink). Figure 1 H is the potential difference of Cryo-M-EVs (red), Acid-M-EVs (orange), and Ultra-M-EVs (pink), n=3.

[0078] The present embodiment provides a method for preparing extracellular vesicles for improving drug delivery ability, comprising: freezing fresh milk in a -20°C environment for 40 days to obtain main whey, centrifuging the main whey at 4°C and 10,000 g for 30 minutes to obtain whey, filtering the whey using a 0.22 μm filter membrane, and centrifuging the filtered whey at 4°C and 160,000 g for 70 minutes.

[0079] Figure 2 The results show the state changes of fresh milk after being frozen and centrifuged for different days.

[0080] Traditional extracellular vesicles are derived from cell culture media, while mEVs are derived from milk, which has a fairly complex matrix composition. Before separation, components such as cells, proteins, and fats in milk should be removed to ensure the purity and integrity of the final mEVs. In this research work, in order to ensure consistency, this example selected fresh milk as the extraction source. Milk will change during frozen storage. This example controls the freezing time of milk and changes the thawing process (such as Figure 1 A and Figure 2 ). Figure 2The results showed that the freezing and thawing process caused the non-mEVs protein components in milk to precipitate and aggregate, resulting in milk fat separation. Furthermore, whey was directly obtained after high-speed centrifugation, and the whey was filtered and centrifuged in one step to obtain mEVs. In addition, Figure 1 C and Figure 1 As shown in Figure E, this example prepared milk-derived extracellular vesicles (Acid-M-EVs) separated by acid treatment and milk-derived extracellular vesicles (Ultra-M-EVs, separated by two ultracentrifugations) extracted by multiple ultracentrifugation. Compared with the acid treatment method and continuous ultracentrifugation, the process of obtaining mEVs by low-temperature freezing simplifies the extraction steps and reduces the loss caused by the intermediate process ( Figure 1 A).

[0081] In this example, the mEVs obtained by the three extraction methods were basically characterized. TEM showed that the three mEVs had uniform size and cup-shaped morphology ( Figure 1 B. Figure 1 D and Figure 1 F). The hydrodynamic size (apparent particle size) of the three mEVs was measured by dynamic light scattering. The size of Cryo-M-EVs and Ultra-M-EVs was 40nm, while the size of Acid-M-EVs was larger, 80nm. Figure 1 G. Zeta potential shows that all three mEVs have negative surface charges ( Figure 1 H). The particle size distribution of Acid-M-EVs was wider and the particle size was larger, which may be related to the acetic acid treatment. Based on the morphological characteristics and related experimental results, we successfully isolated EVs from milk.

[0082] Figure 3 Biomarker identification, purity analysis and yield evaluation of mEVs obtained by three extraction methods. Figure 3 A is Western blot analysis of mEVs obtained by three extraction methods. Figure 3 B shows the results of silver staining (upper figure) and Western blot (lower figure) of mEVs and their raw materials using three extraction methods. Figure 3 C is a Venn diagram of the number of proteins detected in mEVs by the three extraction methods for proteomic analysis. Figure 3 D is a Venn diagram of the number of lipids detected in mEVs by the three extraction methods for lipidomics analysis. Figure 3 E is a pie chart of lipid classification and proportion in mEVs. Figure 3 F is the heat map of hierarchical clustering analysis of lipid components of mEVs in each group. Figure 3 G is the number of mEVs particles per milliliter obtained by the three extraction methods when the protein concentration was 1 mg / ml (n=3). Figure 3H is the total protein yield of mEVs per milliliter of milk obtained by the three extraction methods (n=3). Figure 3 I is the total yield of mEVs per milliliter of milk by the three extraction methods (n=3). All data are expressed as mean ± standard deviation, ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P<0.0001. The difference was statistically significant using one-way analysis of variance.

[0083] Identification and purity analysis of mEVs. By detecting the removal of non-EV components and the enrichment of EV protein components in milk, as well as the whey and mEVs obtained by the three extraction methods at the same protein amount, the purity of mEVs obtained by different methods can be tested to a certain extent ( Figure 3 A). Band analysis of different samples extracted by silver staining showed that mEVs extracted by the three methods can effectively enrich new protein bands in milk and whey. According to the molecular weight of the protein bands, non-EVs components such as casein and bovine whey protein were effectively reduced. In order to further analyze the purity of mEVs obtained by the three extraction methods and the enrichment of biomarkers, the expression of biomarkers (CD9, CD81, CD63, TSG101, Alix) was characterized by Western blot in this example. Cryo-M-EVs and Acid-M-EVs were enriched in large quantities on the corresponding bands, while Ultra-M-EVs were not ( Figure 3 B).

[0084] Quantitative analysis of protein and lipid enrichment in mEVs. Based on proteomic analysis, a total of 352 proteins were detected in Cryo-M-EVs, 692 proteins were detected in Acid-M-EVs, and 236 proteins were detected in Ultra-M-EVs. mEVs obtained by the three extraction methods can detect EV biomarker proteins, such as CD81, CD9, TSG101, etc., but also whey proteins, such as albumin, casein, lactotransferrin, etc. ( Figure 3 C, Table 1).

[0085] Table 1 Summary of proteomic results of three types of M-EVs

[0086]

[0087] According to the lipidomics results, the pie chart shows that a total of 44 super lipids were detected, of which PC and TAG accounted for the highest proportion ( Figure 3E). A total of 643 lipids were detected in Cryo-M-EVs, 788 lipids were detected in Acid-M-EVs, and only 85 lipids were detected in Ultra-M-EVs. Among them, Acid-M-EVs enriched more PC, PE, Cer and other lipids, while Cryo-M-EVs and Ultra-M-EVs enriched more TAGs ( Figure 3 D. Figure 3 F). In the analysis of lipid content and lipid carbon chain length and unsaturation, the differences between the acid-treated group and the other groups were more significant ( Figure 4 and Figure 5 ). This result may be due to the change in lipid enrichment trend caused by the acetic acid treatment process. Figure 4 Analysis of mEVs lipid carbon chain length and lipid carbon chain unsaturation. Figure 4 A shows the changes in lipid content of three mEVs with different chain lengths (n=3). Figure 4 B shows the changes in lipid content of three mEVs with different numbers of unsaturated bonds (n=3). Figure 5 Caco-2 cells were incubated with mEVs with different protein concentrations from three extraction methods for 24 h, and the CCK-8 method was used to determine the viability of Caco-2 cells (n=6).

[0088] Quantification of mEVs particle number and mEVs yield. In different studies, mEVs are used as delivery vehicles. There are currently multiple methods to quantify mEVs, such as mEVs particle number, protein concentration, and mEVs mass. In order to more comprehensively quantify the purity and yield of mEVs obtained by the three extraction methods in this study, this embodiment adopts multiple methods. Preferably, this embodiment adopts a nanoflow method to calculate the number of EV particles with 1 ml of mEVs sample with a protein concentration of 1 mg / ml. The results show that the number of mEVs in both Cryo-M-EVs and Acid-M-EVs can reach 10 11 / ml, while the number of Ultra-M-EVs was about half of that of Acid-M-EVs ( Figure 3 G). Then, the BCA method was used to calculate the protein concentration of mEVs obtained by the three extraction methods. After calculating the corresponding protein concentration, the mass of each EV after freeze-drying in the aqueous solution was calculated ( Figure 3H). The protein yield and total yield were calculated according to the above steps. The ratio of protein concentration to mass concentration of Cryo-M-EVs and Ultra-M-EVs was approximately 1:2, while that of Acid-M-EVs was approximately 1:3, indicating that the mEVs obtained by acetic acid treatment were of higher purity. However, the protein yield and total yield of Cryo-M-EVs and Ultra-M-EVs were much higher than those of Acid-M-EVs, among which the yield of Cryo-M-EVs was approximately 2 times that of Ultra-M-EVs and approximately 25 times that of Acid-M-EVs ( Figure 3 I). Since the composition of milk is far more complex than that of cell culture medium, the extraction process of mEVs requires cumbersome pretreatment compared to the extraction process of traditional EVs. For example, milk is first subjected to high-speed centrifugation at 5000g to 13000g to remove fat and cell debris. Each of these steps is only to obtain the middle layer whey, which will result in a large amount of loss in the process of repeatedly removing the upper fat and lower sediment. In addition, the ultracentrifugation method requires additional ultracentrifugation steps to purify the whey. In some studies, 4 to 5 centrifugation steps and 3 to 4 filtration steps are often used to improve the purity of the whey. The acid treatment method adds additional chemicals, and the protein precipitation time of this method is short and strong, which also reduces the yield of mEVs. The low-temperature freezing method reduces the total number of centrifugation to two times and does not require the addition of additional chemicals, thereby minimizing extraction losses and maintaining purity.

[0089] Cytotoxicity and cellular uptake of mEVs. In order to evaluate the in vitro toxicity of mEVs obtained by different methods, this example used CCK-8 and Annexin V / PI methods to quantify the apoptosis of Caco-2 cells treated with mEVs. The CCK-8 results showed that there was no significant difference in the viability of Caco-2 cells as the protein concentration of Cryo-M-EVs, Acid-M-EVs, and Ultra-M-EVs increased from 0 μg / ml to 500 μg / ml ( Figure 5 ), indicating that mEVs have good biosafety in a wide concentration range. After 250 μg / ml of mEVs were extracted by the three extraction methods and co-incubated with Caco-2 cells for 24 h, flow cytometry was used to detect the apoptosis induced by mEVs. Figure 6 As shown in the figure, the apoptosis rates of Caco-2 cells after co-culture with Cryo-M-EVs, Acid-M-EVs and Ultra-M-EVs were only 2.17%, 2.59% and 3.35%, respectively. Consistent with the results of the CCK-8 experiment, all Cryo-M-EVs, Acid-M-EVs and Ultra-M-EVs had negligible effects on cell viability.

[0090] Figure 6 Evaluation results of cellular safety, uptake efficiency, and transport efficiency of mEVs obtained by three extraction methods. Figure 6 A shows the apoptosis of Caco-2 cells after 24 hours of co-incubation of mEVs obtained by three extraction methods using flow cytometry. The protein concentration of the mEVs used was 250 μg / ml. Annexin-positive cells showed early apoptosis, while propidium iodide-positive cells showed late apoptosis. Figure 6 B is a confocal laser scanning microscopy image (scale bar, 20 μm). Figure 6 C is the average fluorescence intensity of mEVs obtained by three extraction methods after co-incubation with Caco-2 cells for 4 hours (n=3). Figure 6 D is the flow cytometry quantitative data of mEVs obtained by three extraction methods after co-incubation with Caco-2 cells for 4 hours (n=3). Figure 6 E is a schematic diagram of the in vitro intestinal barrier model. Figure 6 F shows the results of the transport efficiency of mEVs in the in vitro intestinal barrier model obtained by three extraction methods (n=3). Figure 6 G is the effect of mEVs obtained by three extraction methods on the TEER of in vitro intestinal barrier model (n = 3). All data are expressed as mean ± standard deviation; ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical significance was analyzed using one-way analysis of variance and two-way analysis of variance.

[0091] After the in vitro cytotoxicity assay, the cellular uptake of mEVs from the three extraction methods after incubation with Caco-2 cells for 4 h was evaluated by confocal laser scanning microscopy and flow cytometry. Figure 6 B shows the result that Caco-2 cells significantly uptake Cryo-M-EVs, Acid-M-EVs and Ultra-M-EVs.

[0092] In addition, the fluorescence intensity of Caco-2 cells co-incubated with Cryo-M-EVs and Acid-M-EVs was significantly higher than that of the Ultra-M-EVs group ( *** P<0.001)( Figure 6 C), indicating that Cryo-M-EVs and Acid-M-EVs have higher cellular uptake efficiency than Ultra-M-EVs. In particular, flow cytometry results showed that the percentage of DiD+ cell population in Caco-2 cells incubated with Acid-M-EVs and Cryo-M-EVs was higher than that with Ultra-M-EVs ( *** P<0.001)( Figure 6D), indicating that Cryo-M-EVs and Acid-M-EVs have higher uptake efficiency than Ultra-M-EVs. Figure 6 B and Figure 6 C. Overall, Cryo-M-EVs, Acid-M-EVs, and Ultra-M-EVs have good biosafety and good cellular uptake properties, especially Cryo-M-EVs and Acid-M-EVs, which were shown to have higher uptake efficiency.

[0093] Epithelial transport capacity of mEVs. Due to the harsh gastrointestinal environment, protein drugs are difficult to maintain stability and effective penetration when passing through the gastrointestinal tract. In order to evaluate the transepithelial transport capacity of mEVs obtained by different methods, this example constructed an in vitro intestinal barrier model ( Figure 6 E) and incubated with mEVs. Figure 6 As shown in F, after 1 hour of co-culture, more than 30% of Cryo-M-EVs were able to successfully penetrate the cell monolayer. After 4 hours of co-culture, the cumulative penetration efficiency of Cryo-M-EVs reached 60% ( Figure 6 F), which was significantly higher than that of the Acid-M-EVs group ( ** P<0.01). In addition, the transepithelial electrical resistance (TEER) results showed that the process of mEVs passing through the monolayer did not cause cell damage ( Figure 6 G) The above results indicate that mEVs have the ability to cross the gastrointestinal tract without damaging cells. Figure 7 As shown, mEVs in the basolateral medium were observed by transmission electron microscopy and all three types of mEVs remained morphologically intact after completing cellular trafficking.

[0094] Figure 7 TEM images of the lower chamber of 3 mEVs after Transwell experiment (Transwell migration or invasion assay) (scale bar, 200 nm). Interestingly, because the cellular uptake of Cryo-M-EVs group was slightly lower than that of Acid-M-EVs group, its cellular transport efficiency was much higher in Acid-M-EVs group, which is due to the fact that the transport process includes the steps of endocytosis and exocytosis. Although the uptake level showed similar results, this process may be mediated by different pathways or receptors, resulting in differences in the exocytosis process that affect the final transport efficiency. EVs can form different protein coronas in different media, thus affecting their biological functions. During acid treatment, many proteins that may be related to transport are removed, thus affecting its transport efficiency without affecting its nonspecific uptake process.

[0095] Biodistribution of mEVs. Encouraged by the results of the in vitro study, the researchers conducted further studies to evaluate the biodistribution of mEVs in vivo and explore the potential of mEVs as oral drug delivery vehicles. According to the results of fluorescence imaging within 24 hours, there was no significant difference in the distribution of Cryo-M-EVs, Ultra-M-EVs, and Acid-M-EVs ( Figure 8 A).

[0096] Figure 8 The biodistribution results of mEVs obtained by three extraction methods. Figure 8 A shows the changes in the in vivo fluorescence intensity of mEVs obtained by three extraction methods after oral administration (mEVs were labeled with DiD). The experiment was repeated three times independently and similar results were obtained. Figure 8 B is the in vitro fluorescence images of mEVs in major organs obtained by three extraction methods 6 hours after oral administration (n=3). Figure 8 C is the fluorescence images of sections of mEVs from the liver (scale bar, 200 μm; magnified image, scale bar, 200 μm) and small intestine (scale bar, 100 μm; magnified image, scale bar, 100 μm) obtained by three extraction methods 6 hours after oral administration.

[0097] To further demonstrate the tissue distribution and metabolism of mEVs, this example observed fluorescence imaging of mouse ex vivo tissues at different time points after administration. The results showed that there was no significant difference in the biodistribution of samples extracted by different extraction methods in the heart, spleen, lung, and kidney. Furthermore, the fluorescence signal of Cryo-M-EVs was mainly concentrated in the liver and colorectal regions, and rarely appeared in the stomach ( Figure 8 B and Figure 8 C, Fig. 9 ), indicating that Cryo-M-EVs can effectively cross the gastrointestinal barrier and accumulate in the liver. The fluorescence signals of Acid-M-EVs and Ultra-M-EVs 6 hours after administration were mainly concentrated in the stomach, small intestine and colorectal regions ( Figure 8 B and Figure 8 C, Fig. 9). Six hours after oral administration, the fluorescence signals of Acid-M-EVs and Ultra-M-EVs in the liver were significantly weaker than those in the Cryo-M-EVs group. In summary, the data of this study confirmed the accumulation of Cryo-M-EVs in the liver and the longer retention time of Acid-M-EVs and Ultra-M-EVs in the intestine, further emphasizing the potential of Cryo-M-EVs in crossing the gastrointestinal barrier and oral administration to the liver, such as oral insulin or treatment of non-alcoholic steatohepatitis (NASH); as well as the application of Acid-M-EVs and Ultra-M-EVs in the treatment of intestinal-related diseases such as inflammatory bowel disease (IBD), regulation of intestinal flora and intestinal immunity.

[0098] Fig. 9 The quantitative results of the in vitro mean fluorescence intensity of mEVs in the main organs obtained by three extraction methods 6 hours after oral administration. All data are expressed as mean ± standard deviation; ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P<0.0001. The difference was statistically significant using two-way analysis of variance.

[0099] Biocompatibility and long-term safety of mEVs. Finally, this example evaluates the potential systemic toxicity of mEVs as an oral drug delivery system. After oral administration of a high dose of 50 mg / kg of mEVs for 14 days, there was no significant change in the body weight of mice. Fig.10 A. This example comprehensively analyzed blood biochemistry and major blood cell populations. Compared with PBS treatment, serum biochemical indicators (such as aspartate aminotransferase, alanine aminotransferase, and creatinine) treated with mEVs were not much different, indicating that mEVs as a basic delivery system have good biosafety ( Fig.10 B). This example further observed changes in red blood cell, white blood cell, and platelet counts in mice treated with mEVs to assess any potential inflammation associated with the proposed source and extraction method. Encouragingly, repeated treatment with mEVs did not result in elevated toxicity compared to the PBS group when evaluated ( Fig.10 C). In addition, this example also studied the effects of mEVs on the heart, liver, spleen, lungs, kidneys, and intestines by histological analysis. Hematoxylin and eosin (H&E) staining showed that the structure and integrity of the major organs in the oral mEVs group were almost the same as those in the PBS group ( Fig.10 D). The above experimental results show that mEVs prepared by three different extraction methods have long-term safety and good biocompatibility.

[0100] Fig.10These are the results of in vivo biosafety evaluation after continuous and repeated administration of three mEVs. Fig.10 A is the change in body weight of mice during repeated administration (n=6). Fig.10 B is a complete set of blood biochemical tests performed on mice after repeated administration (n=3), ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMY, amylase; TBIL, total bilirubin; BUN, blood urea nitrogen; UA, uric acid; PHOS, phosphorus; CREA, creatinine; GLU, glucose; NA + , sodium ion; K + , potassium ion; TP, total protein. Fig.10 C is the blood cell counts of mice after repeated administration (n=3). Fig.10 D is a representative H&E staining of histological sections of major organs (heart, liver, spleen, lung, kidney, small intestine) after sequential repeated administration (scale bar, 200 μm).

[0101] Example 3

[0102] This example proposes a method for optimizing the freezing temperature for extracting extracellular vesicles from milk using the low-temperature freezing method.

[0103] Specifically, the method includes:

[0104] Step 1, low-temperature freezing treatment of milk: fresh milk is frozen and stored at -80°C for 30 days to allow the free proteins in the milk to aggregate and denature.

[0105] Step 2, thawing of denatured milk: Place the frozen milk in a 25°C environment until it is completely thawed.

[0106] Step 3, high-speed centrifugation of thawed milk: the thawed milk is divided into centrifuge tubes, and after balancing, centrifuged at 4°C and 10,000 g for 30 minutes.

[0107] Step 4, filtering the whey after centrifugation: taking the middle layer whey after centrifugation, and filtering it using a 0.22 μm filter membrane.

[0108] Step 5, ultracentrifugation of the filtered whey: the filtered whey is divided into ultracentrifuge tubes, balanced and centrifuged at 160,000 g for 70 min at 4°C.

[0109] Step 6, washing and resuspending milk extracellular vesicles: The extracellular vesicle pellet obtained by ultracentrifugation was washed three times with ultrapure water and resuspended in PBS.

[0110] Example 4

[0111] This example proposes a method for optimizing the freezing time of extracellular vesicles extracted from milk by low-temperature freezing.

[0112] Specifically, the method includes:

[0113] Step 1, low-temperature freezing treatment of milk: fresh milk is frozen and stored at -20°C for 40 days to allow the free proteins in the milk to aggregate and denature.

[0114] Step 2, thawing of denatured milk: Place the frozen milk in a 25°C environment until it is completely thawed.

[0115] Step 3, high-speed centrifugation of thawed milk: the thawed milk is divided into centrifuge tubes, and after balancing, centrifuged at 4°C and 10,000 g for 30 minutes.

[0116] Step 4, filtering the whey after centrifugation: taking the middle layer whey after centrifugation, and filtering it using a 0.22 μm filter membrane.

[0117] Step 5, ultracentrifugation of the filtered whey: the filtered whey is divided into ultracentrifuge tubes, balanced and centrifuged at 160,000 g for 70 min at 4°C.

[0118] Step 3.6 Washing and resuspending milk extracellular vesicles: The extracellular vesicle pellet obtained by ultracentrifugation was washed three times with ultrapure water and resuspended in PBS.

[0119] Example 5

[0120] This example proposes a method for optimizing the thawing temperature of extracellular vesicles extracted from milk by low-temperature freezing.

[0121] Specifically, the method includes:

[0122] Step 1, low-temperature freezing treatment of milk: fresh milk is frozen and stored at -20°C for 30 days to allow the free proteins contained therein to aggregate and denature.

[0123] Step 2, thawing of denatured milk: Place the frozen milk in a 37°C environment until it is completely thawed.

[0124] Step 3, high-speed centrifugation of thawed milk: the thawed milk is divided into centrifuge tubes, and after balancing, centrifuged at 4°C and 10,000 g for 30 minutes.

[0125] Step 4, filtering treatment of whey after centrifugation: collecting the whey in the middle layer of the tube after centrifugation, and filtering it using a 0.22 μm filter membrane.

[0126] Step 5, ultracentrifugation of the filtered whey: the filtered whey is divided into ultracentrifuge tubes, balanced and centrifuged at 160,000 g for 70 min at 4°C.

[0127] Step 6, washing and resuspending milk exocysts: The exocyst pellet obtained by ultracentrifugation was washed three times with ultrapure water and resuspended in PBS.

[0128] Example 6

[0129] Specifically, the method includes:

[0130] Step 1, low-temperature freezing treatment of milk: fresh milk is frozen and stored at -20°C for 30 days to allow the free proteins in the milk to aggregate and denature.

[0131] Step 2, thawing of denatured milk: Place the frozen milk in a 25°C environment until it is completely thawed.

[0132] Step 3, high-speed centrifugation of thawed milk: the thawed milk is divided into centrifuge tubes, and after balancing, centrifuged at 4°C and 15,000 g for 30 minutes.

[0133] Step 4, filtering the whey after centrifugation: taking the middle layer whey obtained after centrifugation, and filtering it using a 0.22 μm filter membrane.

[0134] Step 5, ultracentrifugation of the filtered whey: the filtered whey is divided into ultracentrifuge tubes, balanced and centrifuged at 160,000 g for 70 min at 4°C.

[0135] Step 6, washing and resuspending the milk exocysts: The exocyst pellet obtained by ultracentrifugation was washed three times with ultrapure water and resuspended in PBS.

[0136] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention. The equivalent replacement of the raw materials selected by the present invention, the addition of auxiliary components, the selection of specific methods, etc., also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description of the present invention and its drawings are illustrative and do not constitute limitations on the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", which means that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept.

Claims

1. A method for preparing extracellular vesicles for improving drug delivery capability, characterized in that: include: placing the milk under a first temperature condition for freezing treatment so that adjacent proteins interact and aggregate, thereby obtaining frozen milk liquid; Placing the frozen milk at a second temperature for thawing so as to separate the milk into layers, thereby obtaining thawed milk; Centrifugally removing one or more of cell debris, fat and protein aggregates in the thawed milk to obtain an intermediate layer of whey; The intermediate layer whey is filtered and centrifuged to obtain extracellular vesicles. Wherein, the first temperature condition and the second temperature condition are set in such a manner that the extracellular vesicles tend to accumulate in the liver through the gastrointestinal tract.

2. The preparation method according to claim 1, characterized in that: The first temperature condition can be -80°C to -20°C.

3. The preparation method according to claim 1 or 2, characterized in that: The milk is placed under the first temperature condition for freezing treatment for 5 to 60 days.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The second temperature condition can be 25°C to 37°C.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The centrifugal temperature for obtaining the intermediate layer whey can be 4° C., and the relative centrifugal force can be 10000 g to 15000 g.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The centrifugation temperature for obtaining the extracellular vesicles can be 4° C., and the relative centrifugal force can be 160,000 g.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The first temperature condition can be -20°C.

8. An extracellular vesicle, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the preparation method according to any one of claims 1 to 7 and the extracellular vesicle according to claim 8 in the preparation of an oral administration carrier.

10. Use of the preparation method according to any one of claims 1 to 7 and the extracellular vesicle according to claim 8 in the preparation of a drug delivery carrier for oral administration of insulin and / or treatment of non-alcoholic steatohepatitis.

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