Lipid composition for preparing extracellular vesicles

Abstract

The invention discloses a lipid composition for preparing extracellular vesicles, which is prepared from the following lipid substances in percentage by mass: 2 to 18 percent of phosphatidylcholine, 2 to 14 percent of phosphatidylethanolamine, 7 to 27 percent of sphingomyelin, 0.5 to 7 percent of ceramide, 1.5 to 2.5 percent of phosphatidylinositol, 30 to 70 percent of cholesterol and 5 to 8 percent of phosphatidylserine, the fatty acid carbon number of the lipid substance satisfies the following conditions: the fatty acid carbon number of phosphatidylcholine is 16-20, the fatty acid carbon number of phosphatidyl ethanolamine is 16-18, the fatty acid carbon number of sphingomyelin is 16-24, the fatty acid carbon number of ceramide is 16-24, the fatty acid carbon number of phosphatidylinositol is 16-20, the fatty acid carbon number of phosphatidylserine is 18-24, and the molar ratio of cholesterol to sphingomyelin is 4-7. The extracellular vesicles are prepared from the lipid composition and used for biomarkers or drug carriers, and efficient oral drug delivery is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a lipid composition for preparing extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are vesicle structures secreted by different types of cells into the extracellular space and are widely present in various biological fluids such as blood, urine, and milk. Since EVs are rich in various active molecules such as nucleic acids, lipids, and proteins, they not only play important roles in intercellular signal transduction, immune regulation, etc., but are also regarded as potential biomarkers and drug carriers.

[0003] Currently, the main applications of using extracellular vesicles for drug delivery are as follows:

[0004] CN 117137882 A discloses a method for preparing drug-loaded ginger extracellular vesicles for the treatment of rheumatoid arthritis, which has the advantages of immune regulatory drug loading and stability.

[0005] CN 117487762 A discloses a method for preparing extracellular vesicles by physically extruding exogenous nucleic acids into extracellular vesicles, realizing nucleic acid quantification and improving its stability.

[0006] CN 117815183 A discloses a drug delivery system based on the combination of extracellular vesicles and multilamellar liposomes, which can directly fuse with cell membranes and enhance drug efficacy.

[0007] CN 110872562 A discloses a modular extracellular vesicle mass production device, which greatly improves the vesicle yield and uniformity through automated culture and separation.

[0008] CN 111304049 A discloses a cyclic separation and purification platform, which uses multi-stage membrane filtration technology to efficiently enrich extracellular vesicles of different sizes and remove impurities.

[0009] CN 116725975 A discloses a method for preparing grape-derived extracellular vesicles carrying active proteins, which is used for oral administration and improves digestive stability and absorption efficiency.

[0010] Among EVs from different biological sources, milk-derived EVs have received extensive attention due to their good biocompatibility, strong anti-digestion ability, and stable lipid bilayer membrane structure. Among them, milk-derived EVs, especially those in breast milk, not only carry a variety of functional lipids, can protect the human intestinal barrier and prevent diseases such as necrotizing enterocolitis, but also their special lipid bilayer membrane can remain stable in the complex digestive tract environment.

[0011] Based on the excellent natural advantages of milk-derived EVs, researchers have extensively explored their applications in fields such as oral drug delivery. However, due to the cumbersome extraction process and limited production scale of milk-derived EVs, it is currently difficult to achieve large-scale preparation under industrial conditions. Therefore, how to mimic and replace natural EVs with artificially synthesized lipid nanoparticles (LNPs) has become the focus of research in recent years. Compared with natural EVs that are difficult to produce on a large scale, artificial lipid nanoparticles are more operable in terms of preparation and quality control, and can endow specific physicochemical properties and biological functions by precisely regulating their lipid components. Therefore, it is highly necessary to study a lipid composition that is similar to breast milk EVs in key parameters such as lipid types, content ratios, and membrane structures, and whose composition and structure are conducive to digestion and absorption by adults and even infants, so as to improve the delivery efficiency of nutrients or active molecules. Summary of the Invention

[0012] Object of the Invention: In order to overcome the deficiencies in the prior art, the present invention provides a lipid composition for preparing extracellular vesicles. According to the phospholipid composition and content of extracellular vesicles in breast milk, artificial lipid nanoparticles are used to mimic and replace breast milk extracellular vesicles to achieve efficient oral drug delivery.

[0013] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows:

[0014] The first object of the present invention is to provide a lipid composition for preparing extracellular vesicles. By mass ratio, the lipid composition contains the following lipid substances:

[0015] Phosphatidylcholine (PC) 2-18%,

[0016] Phosphatidylethanolamine (PE) 2-14%,

[0017] Sphingomyelin (SM) 7-27%,

[0018] Ceramide (Cer) 0.5-7%,

[0019] Phosphatidylinositol (PI) 1.5-2.5%,

[0020] Cholesterol (CHOL) 30-70%, and

[0021] Phosphatidylserine (PS) 5-8%, wherein,

[0022] The fatty acid carbon number of the lipid substances satisfies:

[0023] The fatty acid carbon number of the phosphatidylcholine (PC) is 16-20,

[0024] The fatty acid carbon number of the phosphatidylethanolamine (PE) is 16-18,

[0025] The fatty acid carbon number of the sphingomyelin (SM) is 16-24,

[0026] The fatty acid carbon number of the ceramide (Cer) is 16-24,

[0027] The fatty acid carbon number of the phosphatidylinositol (PI) is 16-20,

[0028] The fatty acid carbon number of the phosphatidylserine (PS) is 18-24, and

[0029] The molar ratio of cholesterol / sphingomyelin (CHOL / SM) is 4-7.

[0030] The second object of the present invention is to provide a method for preparing extracellular vesicles, using the lipid composition as described in any one of the above, including the following steps:

[0031] Dissolve the lipid in chloroform-methanol, and dry it with a rotary evaporator or nitrogen to remove the solvent, forming a uniform lipid film on the bottle wall;

[0032] Hydrate the lipid film with a phosphate buffer solution containing miR-148a, and help the lipid film to fall off by rolling glass beads to form a lipid dispersion;

[0033] The lipid dispersion is extruded using a microextruder to obtain a lipid composition.

[0034] The third object of the present invention is to provide a lipid preparation, comprising the lipid composition as described in any one of the above.

[0035] The fourth object of the present invention is to provide the use of the lipid composition as described in any one of the above or the lipid preparation as described above, and the use is for biomarker or drug carrier.

[0036] Beneficial effects: Compared with the existing research on drug delivery using extracellular vesicles, the lipid composition for preparing extracellular vesicles prepared by the present invention uses a process route that is easy to scale up production to prepare lipid nanoparticles, so as to overcome the bottlenecks such as low yield and complex extraction of natural EVs. Its phospholipid composition and structure meet the requirements of the human body for lipids, are more conducive to the digestion, absorption and metabolism of lipids in the human body, are suitable for adults and even infants and young children to eat, and have the advantages of non-toxicity, high biocompatibility, low immune response to tolerate the gastrointestinal environment, and long half-life. Description of the Drawings

[0037] Figure 1 It is a schematic structural diagram of a lipid composition that simulates the membrane composition of breast milk extracellular vesicles in an embodiment of the present invention.

[0038] Figure 2 Particle size results of lipid compositions and breast milk extracellular vesicles in one embodiment of the present invention.

[0039] Figure 3 Comparison of in vitro digestion particle size results between lipid compositions and breast milk extracellular vesicles in one embodiment of the present invention.

[0040] Figure 4 Changes in the content of lipid compositions and breast milk extracellular vesicles during in vitro digestion in one embodiment of the present invention.

[0041] Figure 5 Changes in the phospholipid composition secreted by the Caco-2 cell monolayer model after treatment with digestion products of different lipid compositions in one embodiment of the present invention.

[0042] Figure 6 Changes in the cellular phospholipid composition of the Caco-2 cell monolayer model after treatment with digestion products of different lipid compositions in one embodiment of the present invention.

[0043] Figure 7 Digested lipid compositions and EVs are absorbed by Caco-2 cells in one embodiment of the present invention.

[0044] Figure 8 Number of differentially expressed genes in the comparison group in one embodiment of the present invention. Specific Embodiments

[0045] In a first aspect, the present invention provides a lipid composition for preparing extracellular vesicles. By mass ratio, the lipid composition comprises the following lipid substances:

[0046] Phosphatidylcholine (PC) 2-18%,

[0047] Phosphatidylethanolamine (PE) 2-14%,

[0048] Sphingomyelin (SM) 7-27%,

[0049] Ceramide (Cer) 0.5-7%,

[0050] Phosphatidylinositol (PI) 1.5-2.5%,

[0051] Cholesterol (CHOL) 30-70%, and

[0052] Phosphatidylserine (PS) 5-8%, wherein,

[0053] The fatty acid carbon number of the lipid substances satisfies:

[0054] The fatty acid carbon number of the phosphatidylcholine (PC) is 16-20,

[0055] The fatty acid carbon number of the phosphatidylethanolamine (PE) is 16-18,

[0056] The fatty acid carbon number of the sphingomyelin (SM) is 16-24,

[0057] The fatty acid carbon number of the ceramide (Cer) is 16-24,

[0058] The fatty acid carbon number of the phosphatidylinositol (PI) is 16-20,

[0059] The fatty acid carbon number of the phosphatidylserine (PS) is 18-24, and

[0060] The molar ratio of cholesterol / sphingomyelin (CHOL / SM) is 4-7.

[0061] Optionally, in some embodiments of the present invention, by mass ratio, the lipid composition comprises the following lipid substances: phosphatidylcholine (PC) 5-14%,

[0062] phosphatidylethanolamine (PE) 4-12%,

[0063] sphingomyelin (SM) 14-21%,

[0064] ceramide (Cer) 2-5%,

[0065] phosphatidylinositol (PI) 1.5-2.1%,

[0066] cholesterol (CHOL) 35-50%, and

[0067] phosphatidylserine (PS) 6-7%.

[0068] Optionally, in some embodiments of the present invention, the fatty acid carbon numbers of the lipid substances satisfy:

[0069] The fatty acid carbon number of the phosphatidylcholine (PC) is 16-18,

[0070] The fatty acid carbon number of the phosphatidylethanolamine (PE) is 18,

[0071] The fatty acid carbon number of the sphingomyelin (SM) is 18-20,

[0072] The fatty acid carbon number of the ceramide (Cer) is 18-20,

[0073] The fatty acid carbon number of the phosphatidylinositol (PI) is 16-18,

[0074] The fatty acid carbon number of the phosphatidylserine (PS) is 18-20,

[0075] The molar ratio of cholesterol / sphingomyelin (CHOL / SM) is 5 to 6.

[0076] Optionally, in some embodiments of the present invention, the average particle size of the lipid composition is 160 - 200 nm.

[0077] Furthermore, in some embodiments of the present invention, the average particle size of the lipid composition is 170 - 190 nm.

[0078] Optionally, in some embodiments of the present invention, the lipid substances are all derived from a milk source of a natural non - brain animal lipid source, preferably the milk fat globule membrane in the milk source, and more preferably the bovine milk fat globule membrane. For the lipid composition for preparing extracellular vesicles in the present invention, the lipid substances are all from the animal milk fat globule membrane, preferably the bovine milk fat globule membrane.

[0079] In a second aspect, the present invention provides a method for preparing extracellular vesicles, using the lipid composition as described in any one of the above, comprising the following steps:

[0080] Dissolve the lipid substances in chloroform - methanol, and use a rotary evaporator or nitrogen to blow dry to remove the solvent, forming a uniform lipid film on the bottle wall;

[0081] Hydrate the lipid film with a phosphate - buffered solution containing miR - 148a, and help the lipid film to fall off by rolling glass beads to form a lipid dispersion;

[0082] The lipid dispersion is extruded using a micro - extruder to obtain the lipid composition.

[0083] Optionally, in some embodiments of the present invention, the extrusion includes obtaining the lipid composition through multiple consecutive extrusion steps through a polycarbonate membrane with a final filter.

[0084] Optionally, in some embodiments of the present invention, the lipid substances are separated from a natural non - brain animal lipid source by a method including fractionation / or extraction.

[0085] In a third aspect, the present invention provides a lipid preparation comprising the lipid composition as described in any one of the above.

[0086] In a fourth aspect, the present invention provides the use of the lipid composition as described in any one of the above or the lipid preparation as described above, and the use is for biomarker or drug carrier.

[0087] Examples

[0088] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0089] Example 1: Preparation of lipid substances

[0090] Collection of breast milk samples: Breast milk samples at different lactation stages (the sample sizes of colostrum, transitional milk, and mature milk are all 6; n = 18) were donated by volunteer mothers in good health and nutrition in Wuxi City, and all were full-term deliveries. The samples were freshly secreted breast milk, collected from 9:00 to 11:00 in the morning, and were all the breast milk from one breast. The collected breast milk samples were stored in an ice box and immediately sent back to the laboratory, and refrigerated in a 4°C refrigerator, and the experiment was completed within 24 hours.

[0091] Isolation of extracellular vesicles: The collected milk samples should be centrifuged immediately (within 4 hours after collection). Use a centrifuge to centrifuge at 2000×g for 20 minutes at 4°C. The upper layer is the fat layer, and the lower layer is the whey phase containing extracellular vesicles. Store the whey phase in a 4°C refrigerator for later use. Add 2M hydrochloric acid to adjust the pH to about 4.6 to reach the isoelectric point of casein, causing casein to precipitate. After centrifuging twice at 10,000×g for 45 minutes at 4°C, filter using a 0.22μm polyethersulfone filter to remove protein and cell components. The filtered whey phase is centrifuged at 100,000×g for 120 minutes at 4°C, pour off the whey phase to obtain EVs, suspend them in 200μL of phosphate buffer solution, and store them at -80°C until further analysis.

[0092] Extraction of milk fat: Transfer 100μL of phosphate buffer solution to a 2mL centrifuge tube, add 1500μL of chloroform:methanol (2:1, v / v), vortex for 3 minutes, add 375μL of NaCl (0.9%, w / w) solution, vortex for 3 minutes, then ultrasonicate in an ice-water bath for 10 minutes, centrifuge at 8000rpm at room temperature for 10 minutes, take the lower chloroform phase, extract twice with half the amount of solvent for the second time, collect the chloroform layer and leave it under gentle nitrogen. The obtained fat is placed in a -80°C refrigerator for freezing and storage until analysis.

[0093] Determination of phospholipid composition: The extracted polar lipids of extracellular vesicles were redissolved in 150 μL of chloroform:methanol (2:1, v / v). After vortex oscillation, they were centrifuged at 10,000 rpm for 10 min at room temperature, and the supernatant was injected. The UPSFC conditions were as follows: The chromatographic column was an Acquity UPC2TM BEH column (1.7 μm, 3 mm × 150 mm, Waters, USA). The separation conditions were: Eluent A was supercritical CO2 (purity ≥ 99.99%), and eluent B was methanol:water (33:1, v / v) containing 20 mM ammonium acetate modifier; Gradient elution program: 0 min, 5% B; 5.5 min, 48% B; 10 min, 48% B. The MSE mode was selected as the mass spectrometry data acquisition mode, and the mass spectrometry conditions were as follows: The flow rate of cone gas (nitrogen) was 700 L / h; the flow rate of desolvation gas (argon) was 50 L / h; the ion source temperature was 100 °C; the desolvation temperature was 400 °C; the cone voltage was 30 eV; the low collision energy was 6 eV; the high collision energy range was 20 - 45 eV; the scanning range was 100 - 1600 m / z.

[0094] Phospholipid data processing: By comparing the obtained m / z values and fragment data with the calculated exact masses, Progenesis QI software equipped with a self-built local database Progenesis MetScope and the Lipidblast database was used to identify polar lipids.

[0095] Table 1 Phospholipid distribution of extracellular vesicles in breast milk

[0096]

[0097]

[0098] Table 2 Phospholipid molecular content of extracellular vesicles in breast milk

[0099]

[0100] The results showed that:

[0101] (1) As shown in Table 1, among all polar lipids, the CHOL content in extracellular vesicles of breast milk was the highest, reaching 0.55 mg / 100 mL, accounting for approximately 50.46%. The most abundant phospholipid was SM, at 0.19 mg / 100 mL, accounting for approximately 17.43%, followed by PC, PE, and PS in turn.

[0102] (2) As shown in Table 2, SM d18:1 / 24:1, PC 18:0 / 18:2, PE 18:0 / 18:2, PS 18:0 / 18:1, Cer d18:1 / 24:1, and PI 16:0 / 18:1 are the most abundant SM, PC, PE, PS, Cer, and PI in breast milk extracellular vesicles, respectively.

[0103] Therefore, the mass ratio of PC in breast milk extracellular vesicles is 2-18%, the mass ratio of PE in the oil composition is 2-14%, the mass ratio of SM in the oil composition is 7-27%, the mass ratio of Cer in the oil composition is 0.5-7%, the mass ratio of PI in the oil composition is 1.5-2.5%, the mass ratio of CHOL in the oil composition is 30-70%, and the mass ratio of PS in the oil composition is 5-8%.

[0104] Example 2: Simulating the preparation of breast milk extracellular vesicles

[0105] Extraction of milk fat globule membrane material: 1 L of fresh cow's milk was left standing at 4 °C for 12 h and then placed in a large-capacity centrifuge tube. It was centrifuged at 10,000×g for 30 min to remove the upper fat and whey phases, and the lower paste was retained. The collected paste was fully suspended with PBS at pH 7.4 to further wash away impurities and water-soluble proteins; it was centrifuged at 4 °C and 10,000×g for 20 min, and the washing was repeated 3 times. After obtaining the preliminary milk fat globule membrane, total lipid extraction was carried out using the Folch method. 510 g of wet-weight milk fat globule membrane precipitate was taken, and chloroform:methanol (2:1, v / v) was added at a ratio of sample:solvent of 1:10 (w / v). It was shaken at room temperature for 30 min, and an NaCl (0.9%, w / w) solution was added to make the system layer, so that the ratio of chloroform-methanol-water was 8:4:3 (v / v / v). After standing and layering, the lower chloroform phase was collected. After extracting 3 times repeatedly and combining the organic phases, finally the solvent was removed in a rotary evaporator at 40 °C to obtain a preliminary milk fat globule membrane extract.

[0106] Enrichment and purification of key lipids: The solid-phase extraction method established in our laboratory was used to purify the polar lipids in milk. 10 mL of n-hexane was used to adjust the solid-phase extraction column to make it balanced. 50 mg of total lipids was dissolved in 1 mL of chloroform:methanol (4:1, v / v) and loaded onto the column. Non-polar lipids were eluted with 5 mL of n-hexane:diethyl ether (50:1, v / v) and 3 mL of n-hexane:diethyl ether (6:1, v / v), and polar lipids were eluted with 1 mL of n-hexane:diethyl ether (1:1, v / v), 6 mL of methanol, and 3 mL of chloroform:methanol:water (3:5:2, v / v / v). The polar lipid eluate was evaporated under a gentle nitrogen stream, and the obtained polar lipids were stored in a -80 °C refrigerator until the next step of treatment.

[0107] To obtain purer single components such as PC, PE, and SM, preparative high-performance liquid chromatography (HPLC) was used for separation. The mobile phase A was 90% acetonitrile and 10% water, containing 0.1% formic acid; the mobile phase B was 50% acetonitrile and 50% water. The initial condition was 90% mobile phase A, and the proportion of the aqueous phase was gradually increased to the final 50% acetonitrile within 30 min. The flow rate was 15 mL / min. The column temperature was 35°C, and the evaporative light scattering detector was used for detection. During the elution process, the components in the corresponding retention time periods were collected according to the detected peak shapes in combination with TLC / MS qualitative analysis. After removing the solvent from the collected fractions in a vacuum drying device, purer single polar lipids such as PC, PE, and SM were obtained.

[0108] Preparation of extracellular vesicles from lipid compositions:

[0109] (1) Selected phospholipids and lipid components such as cholesterol were obtained in a certain proportion to form a lipid composition, which was dissolved in chloroform-methanol and dried by rotary evaporation or nitrogen blowing to remove the solvent, forming a uniform lipid film on the wall of the flask or round-bottom flask.

[0110] Among them, in order to horizontally compare the effects of lipid components with different ratios on the preparation of extracellular vesicles, in this example, breast milk EV was designed as the control group, and a total of 5 groups of lipid compositions were set, namely:

[0111] The lipid composition of lipid composition 1 was: 48.85% CHOL, 17.21% SM, 12.11% PC, 10.19% PE, 6.72% PS, 2.97% Cer, 1.95% PI;

[0112] The lipid composition of lipid composition 2 was: 35.54% CHOL, 20.99% SM, 13.93% PC, 11.76% PE, 6.72% PS, 4.92% Cer, 2.09% PI;

[0113] The lipid composition of lipid composition 3 was: 49.83% CHOL, 14.01% SM, 13.42% PC, 10.45% PE, 6.37% PS, 4.60% Cer, 1.32% PI;

[0114] The lipid composition of lipid composition 4 was: 47.13% CHOL, 18.37% SM, 12.74% PC, 11.85% PE, 6.04% PS, 2.35% Cer, 1.52% PI;

[0115] The lipid composition of lipid composition 5 is as follows: 43.90% CHOL, 16.41% SM, 13.97% PC, 11.91% PE, 6.76% PS, 4.95% Cer, 2.10% PI.

[0116] (2) Subsequently, it was placed in a water bath environment at about 65 °C, 1.5 g of glass beads with a diameter of 3 mm were added, and the dry film (i.e., lipid film) was hydrated with a phosphate buffer solution (10 mM, pH 7.4) containing miR-148a (20 μg / mL), and the rolling of the glass beads was used to help the film fall off to form a lipid dispersion.

[0117] (3) After sufficient hydration to form a uniform dispersion system, lipid vesicles with a uniform particle size were further obtained by microextrusion. Specifically, the extrusion process was carried out at a temperature of 65 °C to ensure that the lipids remained fluid, and polycarbonate membranes were selected as the filter membrane materials. First, a filter membrane with a pore size of 300 nm was used for 35 consecutive extrusions, and then a filter membrane with a pore size of 200 nm was used to repeat 3 times to make the particle size gradually tend to a uniform distribution. The extrusion pressure was 40 psi, and the extrusion speed was 0.5 mL / min. The schematic structural diagram of lipid composition 1 that simulates the membrane composition of breast milk extracellular vesicles is as Figure 1 .

[0118] Comparative test of lipid composition simulating breast milk extracellular vesicles and breast milk extracellular vesicles: Use the Nanosight NS300 system to measure the size distribution of vesicles and lipid compositions (as Figure 2 ). In addition, in addition to the influence of different ratios of lipid components on the preparation of extracellular vesicles, the examples of the present invention further used UPSFC-Q-TOF-MS to perform a full lipidome detection on the prepared lipid vesicles to confirm whether the phospholipid and cholesterol composition information matches that of the target breast milk extracellular vesicles (Table 3) to study the influence of the differences in the phospholipid information of each lipid component on the preparation of extracellular vesicles.

[0119] Among them, CHOL is a sterol molecule with a fixed structure, different from PC, SM, etc. that can carry fatty acid chains of different lengths and different saturations. Cholesterol has a steroid ring structure and a short side chain, with a total of 27 carbon atoms. CHOL and SM can form highly ordered liquid-ordered (Lo) domains in the vesicle membrane, which is determined by the binding between molecules. When we want to know whether they exist in a ratio similar to 1:1 in the membrane structure or compare the intermolecular interaction strength, the molar ratio is a more accurate measurement method. Therefore, we use CHOL / SM (mol%) as an index to distinguish different lipid compositions.

[0120] Table 3 Phospholipid information of lipid composition and breast milk extracellular vesicles

[0121]

[0122] The results showed that:

[0123] (1) The average particle size of the extracted breast milk extracellular vesicles was 189.1 nm, and the average particle size of the lipid composition (lipid composition 1) used for preparing extracellular vesicles was 180.4 nm, with a concentrated distribution; while the particle size uniformity of lipid composition 2 and lipid composition 3 was poor, and the particle sizes were relatively large, being 318.1 nm and 382.2 nm respectively. The average particle sizes of lipid composition 4 and lipid composition 5 were 204.7 nm and 137.6 nm respectively, which were relatively close to the particle size of breast milk extracellular vesicles.

[0124] (2) Lipid composition 1, lipid composition 4, and lipid composition 5 used for preparing extracellular vesicles had a high similarity in lipid composition with the extracted breast milk extracellular vesicles, all containing a high proportion of SM and CHOL, and the molar ratio of CHOL / SM was between 5 and 6. While the molar ratios of CHOL / SM of lipid composition 2 and lipid composition 3 were 3.39 and 7.11 respectively, showing a large difference from the phospholipid composition of breast milk extracellular vesicles.

[0125] Example 3: In vitro digestion experiment simulating the gastrointestinal environment

[0126] An in vitro digestion experiment simulating the gastrointestinal environment was carried out. Since the polar lipids in the membrane of breast milk extracellular vesicles are rich in a large amount of sphingomyelin, and in all current in vitro digestion studies on milk-derived extracellular vesicles, only pancreatic enzymes are added to digest some polar lipids, lacking digestive enzymes for sphingomyelin. Therefore, it is very necessary to add sphingomyelinase in the digestion system simulating the human body's digestion of extracellular vesicles. In this study, the existing in vitro digestion model for full-term infants was optimized by adding sphingomyelinase to evaluate the stability of the lipid composition simulating the membrane composition of breast milk extracellular vesicles during gastrointestinal digestion.

[0127] In vitro digestion experiment: Simulate the digestion process of breast milk in the gastrointestinal tract of full-term newborns at four weeks of age to further improve the in vitro digestion model. Specifically, the lipid composition and breast milk (100 mL) were respectively mixed with 100 mL of simulated gastric juice and placed in a 250 mL reactor, the pH was adjusted to 6.5, the temperature was maintained at 37 °C for 10 min through a circulating water bath device, and rabbit gastric enzyme was added and reacted for 30 min, with a rotation speed of 5 rpm during this period. 15 mL of digestion products were taken out at 0 min, 15 min, and 30 min respectively, 1 drop of concentrated hydrochloric acid was added to inactivate the enzyme, and then they were frozen and stored in a -20 °C refrigerator until analysis.

[0128] Gastric phase digestion simulation: In the gastric digestion stage, 0.1 mol / L HCl or NaOH was used to adjust the pH, and the pH changed from 6.5 to 4.9 over time.

[0129] Intestinal phase digestion simulation: Take the remaining 100 mL of gastric phase digestion product and mix it with 100 mL of simulated intestinal fluid, then put it into a 250 mL reaction kettle. Adjust the pH to 6.2, and maintain the temperature at 37 °C for 10 min through a circulating water bath device. Subsequently, add bile salts, Bacillus cereus sphingomyelinase, and porcine pancreatin and react for 30 min. Use an automatic potentiometric titrator and 0.1 mol / L NaOH to titrate to keep the pH of the intestinal phase at 6.2. Take out 26 mL of digestion product at 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min respectively for subsequent extraction of vesicles.

[0130] Structural characterization of the digestion product: The lipid composition simulating the extracellular vesicle membrane of breast milk and the particle size of the extracellular vesicles of breast milk were measured according to the method in Example 1 above, and the measurement results are as Figure 3 shown.

[0131] Content detection of the digestion product: Use the Nanosight NS300 system to measure the content changes of vesicles and lipid compositions, and the results are as Figure 4 shown.

[0132] The results show that:

[0133] (1) As Figure 3 shown, during the digestion process, the particle size distributions of lipid composition 1, lipid composition 4, lipid composition 5 simulating the extracellular vesicle membrane of breast milk and the extracellular vesicles of breast milk are relatively concentrated and there are no significant fluctuations; while the particle sizes of lipid composition 2, lipid composition 3, and lipid composition 4 have large fluctuations throughout the digestion process, the lipid structure is unstable, ruptures during gastric phase digestion, and then aggregates in the intestinal phase, with low stability.

[0134] (2) As Figure 4 shown, in the optimized static in vitro digestion model for term infants, lipid composition 1, lipid composition 5 simulating the extracellular vesicle membrane of breast milk and the extracellular vesicles of breast milk can all resist gastric digestion; among them, lipid composition 1 and the extracellular vesicles of breast milk show similar partial intestinal digestion characteristics, and their digestion turning points are both between the end point of gastric phase digestion (G30) and 5 min of intestinal phase digestion (I5), with similar digestion characteristics; while lipid composition 2, lipid composition 3, and lipid composition 4 decompose in the gastric phase digestion environment, the content decreases significantly, and continue to hydrolyze in the intestinal phase, approaching the baseline at the digestion end point, with low digestion stability.

[0135] Example 4: Simulation of human intestinal absorption experiment

[0136] Conduct a simulation of human intestinal absorption experiment to evaluate the feasibility of lipid compositions simulating the extracellular vesicle membrane of breast milk in oral drug delivery.

[0137] Cell experiment: Caco-2 cells between passages 20 - 30 were selected. The Caco-2 cell model was used to simulate the intestinal absorption model of infants. Log-phase Caco-2 cells were seeded into 12-well Transwell plates at a concentration of 2×10 4 cells / mL. All volumes were kept constant, with 0.5 mL of cell suspension on the top and 1.5 mL on the basolateral side. The culture medium was changed every two days during the first week and then daily until intestinal cell differentiation was completed after 21 days. After the model was constructed, the gastric digestion end products of vesicles and lipid compositions diluted with serum-free medium were added to the upper chamber and cultured for 24 h. The culture media from the upper and lower chambers were aspirated and stored in a -20°C refrigerator for subsequent lipid extraction. 200 μL of 0.25% EDTA-trypsin was added to the cells and digested for 5 min. Then the cell suspension was stored in a 2 mL centrifuge tube for subsequent detection.

[0138] Lipid extraction and detection: 200 μL of cell suspension was transferred to a 5 mL centrifuge tube, 3 mL of chloroform:methanol (2:1, v / v) was added, and it was vortexed for 3 min. 750 μL of NaCl (0.9%, w / w) solution was added, vortexed for 3 min, then sonicated in an ice-water bath for 10 min, centrifuged at 8000 rpm at room temperature for 10 min, and the lower chloroform phase was taken. The second extraction was performed with half the amount of solvent twice, and the chloroform layer was collected and gently evaporated under nitrogen. The obtained cellular lipids were stored frozen at -80°C until analysis. 1.5 mL of basolateral solution was transferred to a 15 mL centrifuge tube, 10 mL of chloroform:methanol (2:1, v / v) was added, and it was vortexed for 3 min. 2.5 mL of NaCl (0.9%, w / w) solution was added, vortexed for 3 min, then sonicated in an ice-water bath for 10 min, centrifuged at 8000 rpm at room temperature for 10 min, and the lower chloroform phase was taken. The second extraction was performed with half the amount of solvent twice, and the chloroform layer was collected and gently evaporated under nitrogen. The obtained basolateral phospholipids were stored frozen at -80°C until analysis. The secretion amounts of intracellular phospholipids and basolateral phospholipids were determined according to the method in Example 1 above, and the measurement results are as Figure 5 、 Figure 6 shown.

[0139] Cell absorption experiment: The milk-derived extracellular vesicles and lipid compositions were fluorescently labeled according to the method in the PKH-26 kit instructions. Different concentrations of PKH-26 (red)-labeled EVs and lipid compositions were added to Caco-2 cells that had been starved for 12 h, and then after incubation for 12 h, the excess PKH-26-labeled vesicles were washed away with phosphate buffer solution. After labeling the Caco-2 cell nuclei with 4',6'-diamidino-2-phenylindole (DAPI, blue), the uptake of vesicles at different concentrations by the cells was observed under a confocal microscope. The results are as Figure 7as shown

[0140] Cell differential gene analysis: Total RNA in cells co-cultured with vesicles for 24 h was extracted using Trizol reagent, and the RNA concentration was measured using a ultra-micro spectrophotometer. RNA samples with (A260 / 280 = 1.8 - 2.1) had good purity and were kept at -80 °C until analysis.

[0141] Enrich mRNA from total RNA using Oligo dT magnetic beads. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers, followed by the synthesis of the second-strand cDNA. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the library was ready. The library was quantified by Qubit and real-time fluorescence quantitative PCR, and the fragment size distribution was detected by a bioanalyzer. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers. Then, the second-strand cDNA was synthesized using dUTP instead of dTTP. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the directional library was ready. The library was quantified by Qubit and real-time fluorescence quantitative PCR, and the fragment size distribution was detected by a bioanalyzer.

[0142] After the library passed the inspection, different libraries were pooled together according to the requirements of the effective concentration and the target output data volume for Illumina sequencing. The raw data was first processed by the FASTP software. In this step, clean reads were obtained by removing reads containing adapters, reads containing poly-N, and low-quality reads from the raw data. The reference genome and gene model annotation files were downloaded from the genome website. HISAT2 was used to build the index of the reference genome, and HISAT2 was used to align the paired-end clean reads with the reference genome. featureCounts was used to calculate the reads mapped to each gene.

[0143] For samples with biological replicates: The DESeq2 R package was used for differential expression analysis of two conditions / groups. DESeq2 provides statistical procedures for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. For samples without biological replicates: Before differential gene expression analysis, for each sequencing library, the read counts were adjusted by a scaling normalization factor to eliminate the differences in sequencing depth between samples, and then differential expression analysis was performed. The results are as Figure 8 as shown

[0144] The results showed that

[0145] (1) As can be seen from the results of the measurements on the basolateral (BL) side and intracellularly, compared with the blank group, both the EV group and the lipid composition 1 group significantly enhanced the transport of triglycerides and cholesterol to the BL side ( Figure 5 ); while there was no significant difference in the regulation of cellular phospholipids between the lipid composition 2, lipid composition 3, lipid composition 4, and lipid composition 5 groups and the blank group. This indicates that the lipid composition 1 that mimics the extracellular vesicle membrane composition of breast milk is beneficial for lipid transcellular transport, but there is no significant difference in sphingomyelin secretion.

[0146] (2) The polar lipid composition of Caco-2 cells in the lipid composition 1 group was close to that of the EV group; while the cellular lipid content of the lipid composition 2, lipid composition 3, lipid composition 4, and lipid composition 5 groups was close to that of the blank group. This shows that the lipid composition 1 that mimics the extracellular vesicle membrane composition of breast milk is similar to EV and can participate in the process of intracellular lipid homeostasis.

[0147] (3) After fluorescence labeling, good distribution of red fluorescence (PKH-26) was observed in Caco-2 cells in both the lipid composition 1 group and the EV group, and it was distributed near the cell nucleus (DAPI, blue); while no obvious red fluorescence aggregation was seen in the cells of the lipid composition 2, lipid composition 3, lipid composition 4, and lipid composition 5 groups, indicating poor absorption; this shows that the lipid composition (lipid composition 1) that mimics the extracellular vesicle membrane composition of breast milk has better intestinal cell absorption effect and is closer to breast milk EV.

[0148] (4) Compared with the blank group, both the lipid composition 1 and EV can induce a large number of differentially expressed genes (DEGs), which are involved in cell metabolism, membrane transport, and signaling pathways, etc.; while there are only a small number of DEGs between the lipid composition 2, lipid composition 3, lipid composition 4, and lipid composition 5 groups and the blank group, and the information transmission effect is poor.

[0149] (5) Compared with EV, the total number of differentially expressed genes of the lipid composition 1 was 745, among which 289 were up-regulated and 186 were down-regulated, indicating that the lipid composition 1 prepared in the embodiment of the present invention can mimic the transcriptional regulation effect of EV on intestinal epithelial cells and produce a gene expression regulation effect similar to EV in Caco-2 cells.

[0150] (6) The synthesized lipid composition 1 for preparing extracellular vesicles has good membrane fusion and the performance of delivering biological information to intestinal cells, indicating the feasibility and application potential of this lipid composition in oral drug delivery.

[0151] Based on the above experimental results, it can be seen that lipid compositions formed by different lipid substances have significant differences in aspects such as average particle size, in vitro digestion characteristics, changes in the phospholipid composition secreted by the Caco-2 cell monolayer model, changes in the cellular phospholipid composition of the Caco-2 cell monolayer model, and Caco-2 cell absorption characteristics. Considering all the characterization effects, lipid composition 1 has the most significant similarity to breast milk EVs.

[0152] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A lipid composition for preparing extracellular vesicles, characterized in that, By mass ratio, the lipid composition comprises the following lipid substances: Phosphatidylcholine 2-18%, Phosphatidylethanolamine 2-14%, Sphingomyelin 7-27%, Ceramide 0.5-7%, Phosphatidylinositol 1.5-2.5%, Cholesterol 30-70%, and Phosphatidylserine 5-8%, wherein, The fatty acid carbon number of the lipid substances satisfies: The fatty acid carbon number of the phosphatidylcholine is 16-20, The fatty acid carbon number of the phosphatidylethanolamine is 16-18, The fatty acid carbon number of the sphingomyelin is 16-24, The fatty acid carbon number of the ceramide is 16-24, The fatty acid carbon number of the phosphatidylinositol is 16-20, The fatty acid carbon number of the phosphatidylserine is 18-24, and The molar ratio of cholesterol / sphingomyelin is 4-7.

2. The lipid composition according to claim 1, wherein By mass ratio, the lipid composition comprises the following lipid substances: Phosphatidylcholine 5-14%, Phosphatidylethanolamine 4-12%, Sphingomyelin 14-21%, Ceramide 2-5%, Phosphatidylinositol 1.5-2.1%, Cholesterol 35-50%, and Phosphatidylserine 6-7%.

3. The lipid composition according to claim 1, wherein The fatty acid carbon number of the lipid substances satisfies: The fatty acid carbon number of the phosphatidylcholine is 16-18, The fatty acid carbon number of the phosphatidylethanolamine is 18, The fatty acid carbon number of the sphingomyelin is 18-20, The fatty acid carbon number of the ceramide is 18-20, The fatty acid carbon number of the phosphatidylinositol is 16-18, The fatty acid carbon number of the phosphatidylserine is 18-20, The molar ratio of cholesterol / sphingomyelin is 5-6.

4. The lipid composition according to claim 1, wherein The average particle size of the lipid composition is 160-200 nm, Preferably, the average particle size of the lipid composition is 170-190 nm.

5. The lipid composition according to claim 1, characterized in that, The lipid substances are all from a milk source of a natural non-brain animal lipid source, preferably the milk fat globule membrane in the milk source, preferably the bovine milk fat globule membrane.

6. A method for preparing extracellular vesicles, characterized in that, Using the lipid composition according to any one of claims 1-5, comprising the following steps: Dissolve each lipid substance of the lipid composition in chloroform-methanol, and dry it with a rotary evaporator or nitrogen to remove the solvent, forming a uniform lipid film on the bottle wall; Hydrate the lipid film with a phosphate buffer solution containing miR-148a, and help the lipid film to fall off by rolling glass beads to form a lipid dispersion; The lipid dispersion is extruded using a microextruder to obtain the lipid composition.

7. The method according to claim 6, wherein The extrusion includes obtaining the lipid composition through a plurality of consecutive extrusion steps through a polycarbonate membrane with a final filter.

8. The method according to claim 1, characterized in that, The lipid substances are separated from a natural non-brain animal lipid source by a method including fractional separation / or extraction.

9. A lipid preparation comprising the lipid composition according to any one of claims 1-5.

10. The lipid composition according to any one of claims 1-5 or the lipid preparation according to claim 9, for use as a biomarker or a drug carrier.

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